ArticlePDF Available

ENOD40 affects phytohormone cross-talk /

January 2003

January 2003

Authors:

Research Institute for Agriculture Fisheries and Food (ILVO)

Thesis (doctoral)--Wageningen Universiteit, 2003. Vita. "Stellingen" ([1] leaf) inserted. Includes bibliographical references.

GFP Fluorescence of a 35S:NtENOD40-GFP Transgenic Line.

…

Short stretches of ENOD40 Sequence Homology in Arabidopsis thaliana genome database.

…

Application of a BA concentration range (0-10-6 M) induces a response similar to the triple response in dark grown wild type and 35S:NtENOD40 Arabidopsis seedlings. The response to BA is not affected by overexpression of NtENOD40. Bar equals 1 cm.

…

Figures - uploaded by Tom Ruttink

Content may be subject to copyright.

Content uploaded by Tom Ruttink

Content may be subject to copyright.

ENOD40

affects phytohormone cross-talk

Tom Ruttink

ENOD40 affects phytohormone cross-talk

Tom Ruttink

Promotor: Prof. dr. T. Bisseling

Hoogleraar in de Moleculaire Biologie

Wageningen Universiteit

Co-promotor: Dr. H. Franssen

Universitair docent

Laboratorium voor Moleculaire Biologie

Wageningen Universiteit

Samenstelling promotiecommissie:

Prof. dr. A. E. Emons, Wageningen Universiteit

Dr. ir. A. R. van der Krol, Wageningen Universiteit

Prof. dr. J. W. Kijne, Universiteit Leiden

Dr. D. Geelen, Universiteit Gent, Belgie

ENOD40 affects phytohormone cross-talk

Tom Ruttink

Proefschrift

Ter verkrijging van de graad van doctor

Op gezag van de rector magnificus

Van Wageningen Universiteit

Prof. dr. ir. L. Speelman

In het openbaar te verdedigen

Op vrijdag 5 december 2003

Des namiddags te vier uur in de Aula

ENOD40 affects phytohormone cross-talk

Ruttink, Tom

Thesis Wageningen University, The Netherlands

With references – with summary in Dutch

ISBN 90-5808-979-7

CONTENTS

Scope 6

Chapter 1 Introduction 11

Chapter 2 ENOD40 and Hormonal Control of Cell Size in Tobacco Bright Yellow-2

Cells 37

Chapter 3 The Two Conserved Regions of ENOD40 transcripts Have Biological

Activity and Are Connected In A Complex Manner 74

Chapter 4 Arabidopsis thaliana: Useful for Functional Analysis of ENOD40? 92

Chapter 5 Concluding Remarks 106

Samenvatting 117

Een woord van dank 122

A word of gratitude 123

Curriculum Vitea 124

List of publications 125

Scope

SCOPE

During the interaction between legumes and rhizobia a completely new organ, the

nodule, is formed on the roots of leguminous plants to host the bacteria. Upon infection

with rhizobia, differentiated cortical cells are triggered to dedifferentiate, divide, and give

rise to the nodule primordium. After the bacteria have reached and entered the

primordium cells, the primordium develops into a nodule and the bacteria differentiate

into their endosymbiotic form, the bacteroids. The developmental process is coordinated

by an intricate network of signaling pathways. In addition to signaling molecules like

Nod-factors released by the bacteria and plant factors like the phytohormones ethylene,

auxin and cytokinin, a specific set of plant genes is induced during the early stages of

nodulation (Mylona et al., 1995). These are known as the early nodulin (ENOD) genes,

and some are thought to have a regulatory role during nodule organogenesis. One of

the earliest activated ENOD genes is ENOD40. The spatio-temporal ENOD40

expression pattern is closely associated with the nodule developmental program (Yang

et al., 1993; Kouchi et al., 1993). Strikingly, ENOD40 is also expressed in tissues not

related to symbiosis indicating that the role of ENOD40 is probably not restricted to

nodulation (Crespi et al., 1994; Papadopoulou et al., 1996; Varkonyi Gasic and White,

2001). ENOD40 genes are present in non-legumes, suggesting that ENOD40 has a

general role in plant development (van de Sande et al., 1996; Kouchi et al., 1999).

However, the precise function of ENOD40 genes is poorly understood. In this thesis, we

explored new ways to elucidate the function of this gene.

In chapter 1, we describe what is known about ENOD40 regulation, structure and

function, based on ENOD40 genes from plant species across the plant kingdom in

search for common features of ENOD40 action. By now, almost 40 different ENOD40

homologs have been identified in species across the plant kingdom. Among these are

monocots such as rice, sorghum, maize and ryegrass; but also dicots like pineapple,

tomato, tobacco and citrus and several leguminous species, showing that ENOD40 is a

common gene. Mainly based on the ENOD40 expression patterns several functions of

ENOD40 have been proposed. These, in combination with the phenotypes induced by

misexpression of ENOD40, have lent support to the hypothesis that ENOD40 genes

could be involved in regulation of plant growth and development. In addition, several

studies have indicated that the function of ENOD40 potentially overlaps with

phytohormone action (Crespi et al., 1994; van de Sande et al., 1996; Charon et al.,

1999). This led to our working hypothesis that ENOD40 could act on the phytohormone

status of a cell, thus participating in regulation of the (cellular) responses to

phytohormones. Therefore, we decided to focus on the development of bioassays

specifically designed to test the effect of ENOD40 on responses to phytohormones. In

this thesis we set up two new and complementary test systems in a dicot, but non-

legume, plant background; a cellular system, using the tobacco BY-2 cell-suspension

and a whole plant system, using Arabidopsis.

An important advantage of the BY-2 cell suspension is the observation that elongation

growth and cell division frequency of cells are regulated by the balance between

cytokinin and auxin in the culture medium (Hasezawa and Syono, 1983). In this thesis

we have adapted their approach to turn the morphological response to phytohormones

into a bioassay for the effect of ENOD40. In this bioassay, elongation growth rate and

cell division frequency are morphological growth parameters that can be quantified in

dose response curves for phytohormones, and it now forms the basis for an

experimental system to test the effect of ENOD40 on phytohormone signaling. In

chapter 2, we describe the experiments that showed that ENOD40 could indeed affect

cellular responses to some phytohormones.

The comparison of ENOD40 transcripts in chapter 1 revealed the presence of two short

yet highly characteristic conserved regions that together make up only 10-20% of the

length of transcripts. ENOD40 transcripts are further characterized by the absence of a

long open reading frame. Therefore, it is not obvious which ENOD40 gene product has

biological activity. A short ORF resides in one of the two conserved regions and the

encoded oligopeptide may have biological activity. Strikingly, in only about 50% of

ENOD40 transcripts, an ORF is present in the second conserved nucleotide region and

therefore this region may be active as RNA.

As we have developed a bioassay for ENOD40 activity, it is possible to use this

bioassay to determine which ENOD40 gene products (oligopeptides encoded by the

short ORF or RNA) have biological activity. Since we studied the effect of ENOD40 by a

reverse genetics approach, we used a set of constructs carrying mutations in the two

conserved regions to test their biological activity in the bioassay. In chapter 3, we

describe experiments that were performed to test whether translation of the small ORF

located in the first conserved region occurs and is required for biological activity of

ENOD40. In order to test conservation of gene function between plant species, we also

tested biological activity of a distantly related ENOD40 transcript in tobacco BY-2 cells.

To come to a detailed description of the molecular mechanism underlying the activity of

ENOD40, it is required to identify the biologically active ENOD40 gene product as well

as direct interactors, but also factors acting further up- or down-stream in the pathway.

As this is not possible in the cellular model system, we searched for effects of ENOD40

in whole plants as this could raise the possibility to develop a suppressor screen for

interactors of ENOD40 in an un-biased genetic approach. We chose Arabidopsis as a

genetic model system and generated stable transgenic lines overexpressing

NtENOD40. Analysis of these lines is described in chapter 4.

In chapter 5 we present our concluding remarks in which we discuss the implications of

the proposed interaction of ENOD40 with phytohormone responses for the role of

ENOD40 on organ or plant level.

REFERENCES

Charon, C., Sousa, C., Crespi, M., and Kondorosi, A. (1999). Alteration of ENOD40

expression modifies Medicago truncatula root nodule development induced by Sinorhizobium

meliloti. Plant Cell 11, 1953-1965.

Cohn, J., Day, R.B., and Stacey, G. (1998). Legume nodule organogenesis. Trends Plant Sci.

3, 105-110.

Crespi, M.D., Jurkevitch, E., Poiret, M., d'Aubenton-Carafa, Y., Petrovics, G., Kondorosi,

E., and Kondorosi, A. (1994). ENOD40, a gene expressed during nodule organogenesis,

codes for a non- translatable RNA involved in plant growth. EMBO J. 13, 5099-5112.

Hasezawa, S., and Syono, K. (1983). Hormonal Control of Elongation of Tobacco Cells Derived

from Protoplasts. Plant Cell. Physiol. 24, 127-132.

Kouchi, H., and Hata, S. (1993). Isolation and characterization of novel nodulin cDNAs

representing genes expressed at early stages of soybean nodule development. Mol. Gen.

Genet. 238, 106-119.

Kouchi, H., Takane, K., So, R.B., Ladha, J.K., and Reddy, P.M. (1999). Rice ENOD40:

isolation and expression analysis in rice and transgenic soybean root nodules. Plant J. 18,

121-129.

Mylona, P., Pawlowski, K., and Bisseling, T. (1995). Symbiotic Nitrogen Fixation. Plant Cell 7,

869-885.

Papadopoulou, K., Roussis, A., and Katinakis, P. (1996). Phaseolus ENOD40 is involved in

symbiotic and non-symbiotic organogenetic processes: Expression during nodule and lateral

root development. Plant Mol. Biol. 30, 403-417.

van de Sande, K., Pawlowski, K., Czaja, I., Wieneke, U., Schell, J., Schmidt, J., Walden, R.,

Matvienko, M., Wellink, J., vanKammen, A., Franssen, H., and Bisseling, T. (1996).

Modification of phytohormone response by a peptide encoded by ENOD40 of legumes and a

nonlegume. Science 273, 370-373.

Varkonyi Gasic, E., and White, D.W.R. (2002). The white clover ENOD40 gene family.

Expression patterns of two types of genes indicate a role in vascular function. Plant Physiol.

129, 1107-1118.

Yang, W.C., Katinakis, P., Hendriks, P., Smolders, A., de Vries, F., Spee, J., van Kammen,

A., Bisseling, T., and Franssen, H. (1993). Characterization of GmENOD40, a gene showing

novel patterns of cell- specific expression during soybean nodule development. Plant J. 3,

573-585.

Chapter 1

Introduction

INTRODUCTION

The symbiotic interaction between legumes and rhizobia leads to the development of a

new organ, the nodule, which is formed on the roots of leguminous plants to host the

bacteria. A set of plant genes is induced during initial stages of nodulation. These genes

are called the early nodulin (ENOD) genes. One of these ENOD genes is ENOD40, and

its expression pattern is closely associated with the nodule developmental program

(Yang et al., 1993; Kouchi et al., 1993). ENOD40 is induced in pericycle cells within

three hours after infection and is also expressed in the dividing cortical cells that give

rise to the nodule primordium. In later stages of nodule development, ENOD40 is

expressed in vascular tissue and in the zone between the meristem and the cells of the

central tissue of the mature nodule in which nitrogen fixation takes place (Compaan et

al., 2001). Overexpression of ENOD40 leads to acceleration of nodulation whereas

silencing impairs nodule development and aberrant nodules are formed, indicating that

ENOD40 has a regulatory role in this process (Charon et al., 1999). The function of

ENOD40 is probably not restricted to nodule development because ENOD40 expression

is also found in tissues that are not related to symbiosis (Asad et al., 1994;

Papadopoulou et al., 1996). ENOD40 genes are also present in non-legumes and

expression patterns are comparable between legume and non-legume plant species

(Kouchi, Takane et al., 1999; Varkonyi Gasic and White, 2002). Moreover,

overexpression affects non-legume plant development (van de Sande et al., 1996).

Thus, it has been suggested that ENOD40 may also have a regulatory role during

different stages of plant development but its precise function is poorly understood.

For the vast majority of genes, the encoded polypeptide is the biologically active

molecule. In this respect, ENOD40 is unusual due to the lack of a long open reading

frame. However, several short ORFs are present in ENOD40 transcripts, so it is possible

that oligopeptides are translated from ENOD40 transcripts and that these are

biologically active. Sequence comparison between ENOD40 transcripts revealed two

regions of high nucleotide sequence similarity named region I and region II. Some of the

short ORFs reside in these regions and therefore are conserved among plant species. It

is likely that region I and II are important for the function of ENOD40, but it remains to be

solved whether the transcripts are active as RNA or that a short conserved oligopeptide

has biological activity. To better understand the molecular mechanism of ENOD40

action and its role in plant development, two important questions remain to be solved

and are the subject of this thesis; what is the function of ENOD40 and which gene

product has biological activity? In this introduction, we describe the current knowledge

on ENOD40 structure and function.

ENOD40 Homologs Are Present in Plant Species Across the Plant Kingdom

We start by comparing the sequence of ENOD40 transcripts to study which nucleotide

regions are likely to be important for biological activity. We wanted to include

representative transcripts of different plant clades across the plant kingdom and

therefore we searched for ENOD40 genes in clades in which no ENOD40 homologs

have yet been described. All known ENOD40 transcripts are relatively short, around

400-800 bases. Overall sequence homology is about 70% in closely related species but

is down to at most 30% in distantly related plant species. The stretches of high

sequence homology in region I and II span about 30-40 bp and 60-100 bp, respectively.

The highest level of nucleotide sequence conservation is found in region II (Kouchi,

Takane et al. 1999; Flemetakis, Kavroulakis et al. 2000; Compaan et al., 2001). Due to

low homology between distantly related transcripts, it is difficult to identify novel

members by cross-hybridization. Large-scale sequencing programs provide genomic

and transcript (EST) sequence information from species across the plant kingdom

(Figure 1.1). The short stretches of conserved nucleotide sequence in ENOD40

transcripts are specific and long enough to be used for sequence homology searches.

Therefore, nucleotide sequence databases are an easily accessible source of

unidentified ENOD40 homologs from distantly related plant species.

From 27 different plant species, in total 35 different genes homologous to ENOD40 can

be identified by means of standard BLAST searches in the Genbank sequence

database. Figure 1.1 shows the occurrence of ENOD40 homologs in angiosperms.

These ENOD40 homologs were present in the Genbank sequence database, as of May

2003 and include NtENOD40-2 and LeENOD40, which are not yet present in the

sequence database, but have been cloned in our laboratory (Matvienko et al., 1996;

Vleghels et al., 2003). In this way we identified novel ENOD40 homologs from Populus,

Prunus, Solanum, Citrus, and Sorghum. These sequences contain both conserved

nucleotide regions. The Antirrhinum majus, Lactuca sativa and Vigna radiata transcripts

only contain the region II sequence. Because these are shorter than all other ENOD40

transcripts, these may be partial cDNA sequences. Most ENOD40 sequences (including

the novel sequences) that are available were identified as an entry in an EST database,

showing that these sequences are transcribed. For all ENOD40 genes for which this

information is available, the cDNA sequence is identical to the corresponding genomic

sequence, indicating that splicing does not occur.

The occurrence of ENOD40 homologs in monocots and different clades within the core

eudicots shows that ENOD40 is an ancient gene that has been maintained in these

plant clades during evolution. For most families for which a substantial number of

sequences (>20.000) are available, ENOD40 homologs can be identified. Because

ENOD40 is a commonly occurring gene, we anticipate that when similar numbers of

sequences will become available for other families, many more ENOD40 homologous

sequences will appear. It must be noted though that for the families Caryophyllales,

Malvales and Brassicales, 52.000, 60.000 and 1.124.000 sequences, respectively, are

already available but none are homologous to ENOD40, even when ENOD40

sequences from closely related plant families (e.g. Sapindales for the Malvales and

Brassicales families) are used for the search. The reason for the absence of ENOD40

homologs in these sets of sequences is as yet unclear. In most non-legume plant

species ENOD40 is not abundantly expressed, so it is possible that ENOD40 is not

represented in these data sets when sequences are mainly obtained from ESTs. The

sequence database contains a complete genome (Arabidopsis thaliana) belonging to the

Brassicales, suggesting that ENOD40 is not present in this plant species. In Citrus

species, which belong to the Sapindales family, ENOD40 homologous sequences can

be found that contain both regions, indicating that at least the ancestor of the

Brassicales family to which Arabidopsis belongs, still contained an ENOD40

homologous sequence.

Several plant species contain multiple copies of ENOD40 in their genomes (Figure 1.1).

As this is found in most plant clades, monocots as well as dicots, this suggests that

ENOD40 belongs to a small gene family in most plant species. Thus, extensive and

targeted searches based on the highly characteristic conserved regions are expected to

reveal additional copies in plant species for which single ENOD40 genes are already

known. Some sequence divergence can be found between ENOD40 transcripts within a

plant species. Strikingly, in a number of cases, ENOD40 transcripts in closely related

species contain a higher percentage sequence similarity between plant species, than

within plant species (Compaan et al., 2003; Varkonyi-Gasic and White, 2002),

suggesting that ENOD40 genes have been duplicated and diverged in an ancestor of

these plant species.

 Figure 1.1

The numbers of nucleotide sequence entries were obtained from

www.ncbi.nlm.gov/taxonomy/browser/wwwtax.cgi in May 2003. The angiosperm phylogenetic tree is

redrawn after "an update of the Angiosperm Phylogeny Group classification for the orders and families of

flowering plants: APG II, Botanical journal of the Linnean Society, 2003, 141, 399-436". Plant species

abbreviations and Genbank Accession Numbers: Am, Antirrhinum majus, snapdragon (AJ559999); Cu,

Citrus unshiu (C95533); Cg, Casuarina glauca (AJ487686); Cs, Citrus sinensis, pineapple (BQ624698);

Gm, Glycine max (1, X69154; 2, D13503; 3, AI431225); Hv, Hordeum vulgare (BQ765935); Ls, Lactuca

sativa, lettuce (BQ854021); Le, Lycopersicon esculentum; Lj, Lotus japonicus (1, AF013594; 2,

AJ271788); Lp, Lolium perenne, ryegrass (1, AF538350; 2, AF538351); Ms, Medicago sativa, alfalfa

(L32806); Medicago truncatula (1, X80264; 2, X80262); Nt, Nicotiana tabacum (X98716); Ob, Oryza

brachyantha (AB024055); Os, Oryza sativa (1, AB024054, (CA755970); 2, AU101849); Pa, Prunus

armeniaca (CB822805); Ps, Pisum sativum (X81064); Pt, Populus tremula x Populus tremaloides

(BU883953); Pv, Phaseolus vulgaris (X86441); Sb, Sorghum bicolor (BE362667); Sr, Sesbania rostrata

(Y12714); St, Solanum tuberosum (AJ276864); Ta, Triticum aestivum (BJ278615); Tr, Trifolium repens (1,

AF426838; 2, AF426839; 3, AF426840); Vr, Vigna radiata (AF061818); Vs, Vicia sativa (X83683); Zm,

Zea mays (1, AI001271+W21740; 2, AI491369).

( - )

( 53 )

( 5 )

( 2 )

( - )

( 4 )

( - )

( 125 )

( - )

( 640 )

( - )

( 3 )

( 4 )

( 1124 )

( 60 )

( 12 )

( 29 )

( 5 )

( - )

( 16 )

( 11 )

( 334 )

( - )

( 141 )

( - )

( 6 )

euasterids II euasterids I eurosids II eurosids I

( - )

( 2 )

( 1 )

( - )

( 1 )

( - )

( 1 )

( - )

( 1 )

( 10 )

( - )

( 1 )

( - )

commelinids

magnoliids

Poaceae

Ehrhartoideae

PACCAD clade

Pooideae

Oryzeae

Panicoideae

Poeae

Triticum

( 1 )

( 821 )

( 1089 )

( 425 )

( Pt )

(Gm(3), Pv, Sr, lj(2), Ms(2), Ps, Mt(2), Tr(3), Vs )

( Cs, Cu )

( Pa )

( Le, St, Nt (2))

( Ls )

( Zm (2), Sb )

( Lp (2) )

( Os (2), Ob )

( Hv, Ta )

( none )

( none)

( Cg )

( Am )

Figure 1.1. Occurrence of ENOD40 Homologs in Angiosperms.

Phylogenetic tree with the total number of nucleotide sequence entries per plant family indicated between

brackets (*103). (-) Indicates less than 500 nucleotide sequence entries. ENOD40 homologs were

obtained from the Genbank database by BLAST homology searches. Plant species containing ENOD40

homologs are listed and the number of copies per plant species is indicated between brackets. This

reveals that in most plant families for which a substantial number of sequences are available, ENOD40

homologs can be identified. Striking exceptions are the Malvales and the Brassicales family. Two different

Citrus species (Cs, Cu) belonging to the closely related Sapindales family do contain ENOD40 homologs

indicating that their common ancestor still contained an ENOD40 homolog.

Common Features of ENOD40 Transcripts

To understand the molecular mechanism of ENOD40 action, it is required to identify the

biologically active gene product. For most genes this is the encoded protein but for

some genes it has been shown that they are biologically active at the RNA level. It has

been suggested that the lack of a long conserved open reading frame (ORF) indicates

that ENOD40 RNA is the active product (Crespi et al., 1994). As ENOD40 transcripts do

contain many short ORFs (Figure 1.2) encoding putative oligopeptides, these might also

have biological activity. The ENOD40 sequences have been identified by the occurrence

of the conserved regions I and II. In case the peptides are the biologically active gene

products it seems probable that ORFs encoding these peptides are conserved. The only

ORFs that are conserved must be located within region I or region II and these ORFs

were analyzed in further detail.

Region I

Nucleotide sequence alignment of region I and amino-acid alignment of ORF I are

presented in Figure 1.4A. The length of ORF I varies slightly in different plant species,

but is usually identical in closely related plant species. In species in which multiple

ENOD40 genes have been identified, the length and sequence of ORF I in different

transcripts is not always identical (Figure 1.4A, Zm, Mt and Tr). Taken together, all

requirements for a functional ORF are present in region I, even in distantly related

species. These include: in all ENOD40 genes (except LjENOD40-2) it contains the first

start codon of the transcript, the ORF encodes a conserved peptide, conservation on

amino-acid level but not necessarily nucleotide level between closely related species,

comparable length of the ORF indicating a conserved stop. Because all requirements for

a functional ORF are met within region I, it is highly likely that this region is translated

and that the ORF I encoded peptide is biologically active.

We identified the [M-X1-4-W-X4-HGS*] sequence as the characteristic peptide motive

encoded by region I. There are six exceptions with regard to the amino-acid sequence of

ORF I: Lolium perenne-1,2 [M-X1-4-W-X4-HSS*] ; Prunus armeniaca [M-X5-W-X5-HGT*] ;

Populus tremula x tremaloidus, [M-X10-QGP*] ; Medicago truncatula-2 [M-X3-W-X3-IYD*],

whereas Casuarina glauca ENOD40 does not encode the conserved peptide due to a

frame shift and the lack of the conserved stop codon in region I (Santi et al., 2003).

However, all these transcripts have significant homology to ENOD40 as they contain a

perfect region II motive.

Several approaches have been undertaken to study translation of ORF I. The

GmENOD40 ORF I is translated in an in vitro assay (Rohrig et al., 2002) and,

translationally fused to GFP, is translated in tobacco protoplasts (van de Sande et al.,

1996). The NtENOD40 ORF I is translated in cowpea protoplasts (Compaan et al.,

2001). Translation of MtENOD40 ORF I was demonstrated in in vitro assays and in root

epidermal and cortical cells of Medicago truncatula using GUS translational reporter

fusions (Sousa et al., 2001). Van de Sande (1996) detected an antigenic determinant in

extracts of soybean nodules with an antibody raised against a synthetic soybean ORF I

peptide. Together, these data support that ORF I is translated in vivo but whether the

peptide encoded by ORF I has biological activity remains to be elucidated.

MEVLWQEGPHGS. MHGKPASHETAMGSAIESC NGFFYFFSVLK.

MGSAIESCNGFFYFFSVL K.

MALRKKHQVHGEGYI LTTLL LQNSTVCLCFTL LALC. MANRQVTKRQWARLLNLVMAFFTFSLY.

MAF F T F S L Y .

20 40 60 80 100 120 140 160 180 200 220 240 260 280 300 320

MEV L WQEGP H GS . MHGKPASHETAMGSAI ESCYGFFYFFSGLKYDCSSCMSME.

MGSAIESCYGFFYFFSGLK YDCSSCMSME.

MALRKKHQVHGEGYI LTTL LQNSTVCLCFTL LALC. MANRQVTKRQWARLLNLVMAFFTFSLV.

MAFFTFSLV. MYKLQKI SKLI CL TDEFSANQVDNKM

20 40 60 80 100 120 140 160 180 200 220 240 260 280 300 320 340 360 380 400 420

MEDE WL EH AH GS.

MNGLNMHMVLERAEAGGGWRAMAEVQLKKLFDSMVHFLAS.

MHMVLE RAE AGGGWRAMA EV QL KK L FD SMVH FL A S.

MVLE RAE AGGGWRAMA EV QL KK L FD SMVH FL A S.

MAEVQLKKLFDSMVHFLAS.

MHKPASHKTALVSSSRSTPPFFSSPVLSFQLYQVVAK. MIDES IDPSLLSQCLFYV .

MCSMK I T L L I NGC F L S

MKI T L L I NGCF L S

20 40 60 80 100 120 140 160 180 200 220 240 260 280 300 320 340 360 380 400 420 440 460

MEDAWLEHLHGS. MCDGLYTYTLVRQCLSHVYSSLQKQAAINELLL I I KKKKKK

MHGLSICMVLES . MDSGNRASQEAFVFRNAFPDLLGGQLRCAQTGKSQYGNGGLH. MCTPVSRNKLL. MNCCLSSKKKKK

MADSIRSRPFLF LYPFLHSSSVI VSGGS. MFVSCVLQSPETSCYK.

20 40 60 80 100 120 140 160 180 200 220 240 260 280 300 320 340 360 380 400 420 440

MEEEAWQECLHGS.

MVGWRAMTSC.

MGPESRVSSEAVHGWLDGGQ.

MAGVSAWVLRVA. MEGNDQLLSCCCSSWSTISLYLSMPRVAGRLLALACPVPEGGQAR.

MPRVAGRLLALACPVPEGGQAR.

20 40 60 80 100 120 140 160 180 200 220 240 260 280 300 320 340 360 380 400 420 440 460 480 500 520 540

MVAGMSAWFLRVA.

MSAWFLRVA. MDGWMTMAS.

MEEAWWQECLHGS.

MPLVAVFPGWLPVPEGPVRRGQRPASQRSAMVSSCRSLLLI YQFKVSLYLL

LLVFL

RSFLSVP SVPVR AEDFS RSLAFWRQFQCWFWNML L.

MVSSCRSLL LI YQFKV SLYLL

LLVFL

RSFLSVP SVPVR AEDFS RSLAFWRQFQCWFWNML L.

MVLESCI SSKAV HGWLDDNGQLST

20 40 60 80 100 120 140 160 180 200 220 240 260 280 300 320 340 360 380 400 420 440 460 480 500 520 540

MALESGSRVGMNRKAERFS FAVPL LT SQVVA.

MNRKAERFSFAVPL LT SQVVA. MVNSGRSPPPFYPF LSIQLYQVEVE. MDASSPCFVFLFLYYVY SETLL .

MARASTWLLRAEAGSG.

MNQWMLQVHVLFFS SFIMC TR KHYYKWLMFI .

MLQVHVLFFS SFIMC TRKHYYK WLMFI .

MCTRKHYYKWLMFI.

MEGAWLEHLH GS.

MFCFSLPLLC VLGNTTI NG.

20 40 60 80 100 120 140 160 180 200 220 240 260 280 300 320 340 360 380

MALESGSRTSGMNRKAELLGRERFSFVVPLLTSQAVG.

MNRKAELLGRERFSFVVPL LTSQAVG. MVNSGRSLPPFLSFSPHSVVSDGNRI IGILCV .

MARASSWLLRAEAGPAG. MESSLLGPCFFLFFMC ILETLL .

MEGAWLEHLHGS. MDTWSPHSWVHVSFFSLCVFWKHYYKWQIFI.

20 40 60 80 100 120 140 160 180 200 220 240 260 280 300 320 340 360 380

MAKLQFKKLFNSMVPFRPPRWSAEVRTKPASHKTAMV KLQQESTLFFSAFPSFQLYQVVAK.

MVPFRPPRWSAEVRTKPASHKTAMVKLQQESTLFFSAFPSFQLYQVVAK.

MVKL QQESTLFFSAFPSFQLYQVVAK.

MEDEWLEHAHGS.

MCTLEIALLLINGCFLS

MNGLNMHMVLESSRRRRMKSNG.

MHMV LE S S R R RR MK S NG.

MVLE S S R R RR MK S N G.

MLVL C VL W K L P YY .

20 40 60 80 100 120 140 160 180 200 220 240 260 280 300 320 340 360 380 400 420 440 460

MAGASSWFLRSGAA. MAVLFLLL LL L KNSSVCPCLSWHNSLDSRI SGWLREV.

MVGAWQEHLHGS.

MVLE I GSSI S S

MRM

EKNGCAVPSPPPSQELFS LSMPFMA.

MRM

EKNGCAVPSPPPSQELFS LSMPFMA.

MNKPASHKSAIVSSDWI LL L SLSNL VSFPCNL I P VVSTFY CSPLQRSLQLY LVEADKKL SRCNLVL WSQMFACVLLGNILSEMLIFTL

MFAC VL L G NIL S EM LIFTL

20 40 60 80 100 120 140 160 180 200 220 240 260 280 300 320 340 360 380 400 420 440 460 480

MHGLSIYIVLESRSMMRMNHKAELLLKKRF

SFMVPFLSSQVVV.

MMRMNHKAELLLKKRF

SFMVPFLSSQVVV.

MRMNHKAELLLKKRFSFMVPFLSSQVVV.

MNHKAELLLKKRFSFMVPFLSSQVVV.

MVPFLSSQVVV.

MVNSSRSLPPFFLSRPSSCIRSESN NQKHLRVMNP. MYTGKTSITDSCLSSKNTFCCLI.

MLGMQNMPHII. MFQYVNPTEICAATIYSNYLLL.

MFVLCTLEKLV.

MSILLRSVLLQYILIIY YCD

MEDAWLEHLHSS.

MNSSLLSRCLFYVHWKN

MLLPNKFVYVGYAEYATYNLNISCNVSICQSY.

20 40 60 80 100 120 140 160 180 200 220 240 260 280 300 320 340 360 380 400 420 440 460 480 500 520 540 560 580 600 620 640

MHGLSIYI VLE SRSMMRMNHKAELLLKKRFSLMVPI LSSQVVV.

MMRMNHKAELLLKKRFSLMVPI LSSQVVV.

MRMNHKAELLLKKRFSL MVPIL SSQVVV.

MNHKAELLLKKRFSLMVPI LSSQVVV.

MVPILSSQVVV.

MVNSSRSLPPFFLSRPSSCIRSESNNQKHLRVMNP. MYTGKTSI TDSCLSSKNT FLSHL I L LA I VL L I SSLVYMLLPNKFVYVGYAEYATYNLN I SCNVSI CQSY.

MLLPNKFVYVGYAEYATYNLNI SCNVSICQSY. MTKPYVST KTEI SVFM.

MFVLCTLEKLV. MLGMQNMPHII . MFQYVNPTEICAATI YSNYL LL . MFPRRQKFLFLCEMKTSYPKNDS

MKTSYPKNDS

MEDA WLE H L HS S. MNSSL L I R CL F YV HWKN . MSIL LRSVL LQYI L I I YYYDEAI CFHEDRNFCFYVR.

20 40 60 80 100 120 140 160 180 200 220 240 260 280 300 320 340 360 380 400 420 440 460 480 500 520 540 560 580 600 620 640 660 680 700 720

MPKQTGKSQNGKGDSFESYI YMAKANVLYLFYVI SVVAVGKTVCTREYNKTKLI.

MAKANVLYLFYVISVVAVGKTVCTREYNKTKLI .

MCVDCV AV I S KS F I A L FC

MIFVMSFLLLHISNSLIMALTVLSDG.

MSFLLLHISNSLIMALTVLSDG.

MAL T VL S DG.

MYCICFMLLVLL QLEKRFVRENII KRSLYRYDVFYKIL L CVLI ALQ.

MLLVLLQLEKRFVRENI I KRSLYRYDVFYKIL LCVLI AL Q.

MDNAKTNRQVTKRQRRLV. MMFFI RFCYVC.

MFFIRFCYVC.

20 40 60 80 100 120 140 160 180 200 220 240 260 280 300 320 340 360

MKQSMGLRKREAI SWSELRTFKCPSSKYNFKF VHTLPI SL LEFQKMQQKRKVFMD.

MGLRKREAISWSELRTFKCPSSK YNFKFVHTL PI SLLEFQKMQQKRKVFMD.

MQQKR K VF MD .

MLAKAWRTGKSRNGNGLRFGVFLGLL LI VVLYI V I SSLL.

MDSVLESFLAFYL LL YFI LLYP VFYNCVLFVSHMQCVVNMAYQKFII E LLRLS

MQCVVNMAYQKFI I ELLRL S

MAYQKFIIELLRLS

MQWDEAIHGS. MPFLQIQF QVCSYFANFSARI PENAAKKESVYGLI I T LI AKDVGKSMANRQVTKRQWTPFWSLSWPFTYCCTLYCYI QSSI I VFYL .

MA NR Q V T K R Q WT P F WS L S WP F T Y C C T L Y C Y I QS S I I V F Y L .

20 40 60 80 100 120 140 160 180 200 220 240 260 280 300 320 340 360 380 400 420 440 460

MQWDEA I HGS .

MLLEFVQKYRQI HVGFSKFNVI I TTSLESPSKHNSSL YNFKFAHT. MANRQVTKRQLWTPFWSLSWLST.

MKQSTGLRKDRQVGVEEF. MCGYMAK GWQTGKSQNGNCGLRFGVFLGFQLNVVPFKYI I I .

MAK GWQTGKSQNGNCGLRFGVFLGFQLNVVPF KYI I I .

MLYPLSI LLYDI HSCVRL HASVYVLMDFGKIVVHSF SSPMLEYI .

MDFGKIVVHSFSSPMLEYI .

20 40 60 80 100 120 140 160 180 200 220 240 260 280 300 320 340 360 380 400 420 440 460 480 500 520 540 560 580

MPYSIMML GFVQKCR.

MMLGFVQKCR.

ML GFVQKCR.MHVGFSKINAI VTI FL ECTSNITAPYT I ASLLI LEPNSVWIYDDENSK.

MDSRLESVLAVNLMLYPINMLLYYPLL CDIRVLF VRRTCKACVLIDLMDLGRMLKTKKTNMRGGQDAFRP L .

MLYPINMLLYYPLL CDIRVL FVRRTCKACVLIDL MDLGRMLKTKKTNMRGGQDAFRPL.

MLLYYPLLCDI RVLFVRRTCKACVLI DLMDLGRMLKTKKTNMRGGQDAFRPL .

MDLGRMLKTKKTNMRGGQDAF RP L .

MLKTKKTNMRGGQDAFR PL .

MRGGQDAF RPL .

MQWDEA I HGS .

MLD SP RS MRL . MMMKT AN DV GKR MANRLVTKRQLWTPVWSLSWLSI.

MMKTANDVGKRMANRLVTKRQLWTPVWSLSWLSI.

MKTANDVGKRMANRLVTKRQLWTPVWSLSWLSI.

MANRLVTKRQLWTPVWSLSWLSI.

MKQSMGLKKR.

MSI D AC W ILQDQCDCDNI SGVYEQHNCSI YNCK FAHT.

MMLAKGWRTG.

MLAKGWRTG. MSGFHSLARFTRQTGKPCLPTIGWGIPFAVAYI KGPADAFPHWHLRECKLR.

20 40 60 80 100 120 140 160 180 200 220 240 260 280 300 320 340 360 380 400 420 440 460 480 500 520 540 560 580 600 620 640 660 680 700 720 740 760 780 800

MKQSMGL R KR .

MQQKREVF MD.

MLAKAWRTGKSQNGNGLRFGVFLGFLLIVVLISSLL.

MDSVLE SF L AF YL L L YL Y PV F CNCVL F V NHMHCVV NMAY

MQWDEAIHGS.

MANRQV T K RQ WTPFWSLSWLFTYCCTYIQSSVIVFYL.

20 40 60 80 100 120 140 160 180 200 220 240 260 280 300 320 340 360 380 400

YSLVFVLQFFK LVK.

MAKFEQTGKSQNGNGLCLSLLL AFVFL

20 40 60 80 100 120 140 160 180 200 220 240 260 280 300 320 340 360

MDLERSWKLIWSEHIVNYMLSSNSTVC.

FGHS.

MLSSNSTVC.

Region I Region II

NtENOD40-2

NtENOD40-1

LeENOD40

StENOD40

LsENOD40

AmENOD40

CsENOD40

CuENOD40

SbENOD40

ZmENOD40-2

OsENOD40-2

HvENOD40

TaENOD40

LpENOD40-1

LpENOD40-2

ObENOD40

OsENOD40-1

ZmENOD40-1

MELC WLT T I HGS . MLV C V V SY D L MR K.

MVLEEAWRERGVRGEGAHSSHSLT. MDSI GVSMAM. MSSVFPFPCLFVLLVMTL.

20 40 60 80 100 120 140 160 180 200 220 240 260 280 300 320 340 360 380 400 420 440 460 480 500 520 540 560 580 600 620 640 660

MLVCVVSYNLMRNKRIVQF.

MRNKRIVQF.

MHSPKEERLWLQPGKPASHEKAMDSIGVSMAIYCSSM.

MDSIGVSMAIYCSSM. MSVFPFPCLFVLLVI TL.

MELCWQTSIHG S. MEIK GCERVLTTHTPPLKTVCFGLALASLINKGCVLTFSLEWQKQICI L QRRRGFGYSLANRQVTKRQWTPLGSLWLSIAHLCSSSCCRM.

20 40 60 80 100 120 140 160 180 200 220 240 260 280 300 320 340 360 380 400 420 440 460 480 500 520 540 560 580 600 620

MDSLSLKGPYGYVCIDSI VVLFL CFGRMY. MTRIRLFF QFLQMSR. MPLYHI PCVLVGMLAMLCPYMQ.

MLAMLCPYMQ.

MELC WQKSIHGS. MEEHNSLSGMKQRSYGHILANRQVTKRQWTLSH.

MKQRSYGHILANRQVTKRQWTLSH. MAMY V L IL L.

MALK EA WRILCERVLTLHTPPPTVCLCFSHWFL. MCVSGYACYVVPLYAIKKLW.

20 40 60 80 100 120 140 160 180 200 220 240 260 280 300 320 340 360 380 400 420 440 460 480 500 520 540 560 580 600 620

MALEEAWRGMCERVRVF HPSHTI L CI LQFV .

MCERVRVFHPSHTILCI L QFV. MNDSFVVLLQVECNKQKCSSSFEVVFI HI TQFAAD. MALEEAWRGMCERVRVF HPSHTI L CI L

MCERVRV FHPSH TI LCI L

MLR SH L QL WI A I . MDPI RGSY GY V. MITL SLLFL CLFSFS PESHLGENRTDKSI QGESVRHEILL AKI HPWLLKKHGEECVSGSGFFTPHTQSYAS

MRFCWQKSIHGS. MHLTVCLGLAI GFSSLQNSLCCGRTCSCGSQFETRVSNHRAYREGVFMSRRS. MRF CWQK S I H GS.

20 40 60 80 100 120 140 160 180 200 220 240 260 280 300 320 340 360 380 400 420 440 460 480 500 520 540 560 580 600 620 640 660 680 700 720 740 760

MVLKETWEEV. MSYDLMSNKRIVKF.

MSNKRIVKF.

MDSIRGSLGSLLISLL.

MKLCWQKSIHGS. MGRSVRGSLTPHTLPLPTVCLCLAIGFSNHQGIC. MCLCACLCYEL.

20 40 60 80 100 120 140 160 180 200 220 240 260 280 300 320 340 360 380 400 420 440 460 480 500 520 540 560 580 600 620

MERKECERVLTPHTPPLSTVCLRLALASLI NKGCA.

MKF CWQAS I HGS.

MVLKKHGEKGV. MHSPKDKRLWLHSGKPASHKKAMDSIGVSMV.

MDSIGVSMV. MRNKR RVQF .

20 40 60 80 100 120 140 160 180 200 220 240 260 280 300 320 340 360 380 400 420 440 460 480 500 520 540 560 580 600

MHSPKEERLWLQSGKPASHKKAMDSIGVSMAVYCSSM.

MDSIGVSMAVYCSSM.

MEKKECERVLTLHTPPLSTVCLCLALASLINKGCV.

MATVRQTGKSQKGNGLHWGLYGCVLLIYVVLLAVECNKQG.

20 40 60 80 100 120 140 160 180 200 220 240 260 280 300 320

MDTVLYVISHYFLELHPMIAKECI . MALEEAWRGMCERVRVFHPSHTLLCILQFV.

MCERVRVFHPSHTLLCILQFV. MLRSHLQLWIAI. MDPIRGSYGYV. MIT LSLLFLCLF SFSHVCMVVVLWPYMQ.

MVVVLWPYMQ.

MCVWLLSCGLICNNSI.

MPFLSSQSSTI. MRFCWQKSIHGS. MNDSFVVLLQVECNKQKCSSSFEVVFIHI TQFAAD.

20 40 60 80 100 120 140 160 180 200 220 240 260 280 300 320 340 360 380 400 420 440 460 480 500 520 540 560 580 600 620 640 660 680 700 720 740 760 780 800 820 840 860 880 900 920 940 960

MAMYQ SL YL CS T D T L D .

MYQSLYLCSTDTLD. MMYFDSL DLQL TRV CSCF SFCR.

MYFDSLDLQLTRVCSCFSFCR. MNVCLFVCVKSYYDLMRS.

MKL L CWQK S I H GS . MSRVCVCAS. MVLSS FEKL PTL. MFACLFVLRVI MI L.

MVL K T NME R SV R GY . MANRQVTKRQWIP FWSLNGYVSI T LSM. MSKVGHCYH. MFLFL CCSPF PHECLL VCLC.

20 40 60 80 100 120 140 160 180 200 220 240 260 280 300 320 340 360 380 400 420 440 460 480 500 520 540 560 580 600 620 640 660 680 700 720

MVL K T NME R S V RG Y. MANRQVTKRQWIPFWSLNGYVSI TL SM. MPVFVLVDCYSYFL AVECNNKHKDGVVFL . MMYFNSLNL QLTRVCSCFSFCR.

MYF NS L NL QL T R V CS C F S F C R.

MAMY QS L Y L C ST DT .

MYQS L YL C ST DT . MVLSSFEKLPTL.

MKL L CWE K S I H GS . MSK VG NC Y H.

20 40 60 80 100 120 140 160 180 200 220 240 260 280 300 320 340 360 380 400 420 440 460 480 500 520 540 560 580 600 620 640 660

MVLKIKHGEKCERVN. MISTNYI I LYTL PPFLQKQFALCFSYWLLI S. MYF K F T Q F A A D PR V CF L F QF L H V R.

MERSVRGLISWY. MCFFLSIRSK. MANRQVTKRQWIPFWSLNGYVSITLSGIIIYEV. MVL SS F EK F P TL .

MKL LC WQKS I HGS . MAMYQSLYLVLSSMKYSTSTDTFRLKTCLMFYKCVSVQRLVSVLCS.

MYQSLYLVLSSMKYSTSTDTFRLKTCLMFYKCVSVQRLVSVLCS.

MKYSTSTDTFRLKTCLMFYKCVSVQRLVSVLCS.

MFYKCVSVQRLVSVLCS.

20 40 60 80 100 120 140 160 180 200 220 240 260 280 300 320 340 360 380 400 420 440 460 480 500 520 540 560 580 600 620 640 660 680 700

MCFFLSSRSK. MAMYQSLYLLL SSMQTLQIEGCL VSDT CVS L RRSRL VSV LRRL L .

MYQSL YL LL S SMQTL Q IEGCL VSDT CVS L RRSRL VSV L RRL L.

MQTL Q IE GCLV SDT CVSL RRSR LV SVL RRL L .

MVL S S F E K F P T L .

MKF L C WQK SI HGS.

MVLKNK YGVKCERAN. MA N R QV T K RQ WI P F WS L N GY V S I TL S V I I I Y A DT S D. MYFK FTQF AADPRVCF LFQF LHVR .

20 40 60 80 100 120 140 160 180 200 220 240 260 280 300 320 340 360 380 400 420 440 460 480 500 520 540 560 580 600 620 640 660 680 700

MSLTCQSVRLCF.

MKNLCERVLTMTLHTLQLQQFA.

MTLHTLQLQQFA. MISDHKENMENQKKLVSKE.

MENQKKLVSKE.

MDRIRGPYGYISVCVYDSGVILL.

MNLCWQKSIYD. MSECAIVFLTKITL

WSSRKSRCYHDIVKLTMIPTMHLVLRRKRSFGYRLVNRQVTKRRWTALEVLMAIYLYVCMILV.

MIPTMHLVLRRKRSFGYRLVNRQVTKRRWTALEVLMAIYLYVCMILV.

MHLVLRRKRSFGYRLVNRQVTKRRWTALEVLMAIYLYVCMILV.

MAIYLYVCMILV.

20 40 60 80 100 120 140 160 180 200 220 240 260 280 300 320 340 360 380 400 420 440 460 480 500 520 540 560

MVLKGRMEKL CE RVL T MTL HTL HL QQFA .

MEKLCE RVL T MTL HTL HL QQFA .

MTL HT L HL QQ FA .

MAICL YVFMYI VI NKDVVCSFEKL PTCDVSSHSI AAEKNL VSSSVYVND.

MYIVI NKDVVCS FEKLPT CDVSSHSI AAEKNLVSSS VYVND. MYYCLLCLDLDSMYYLRDAVI DF DRPI SNQLY.

MYYLRDAVIDFDRPISNQLY.

MLFVPL RNY QLV MFQVT QL QLRR I .

MFQV T QL Q L RR I . MIKL VI VLI I HVPL LVAI YVS VFFLCI I VC YVWI.

MDLCWQKS I HG S . MFKFTVSLT DQSVTMI SSNLL. MDRIRGPYGYMFVCVYVYCNKQRCCLFL .

MFV CV YV YC N KQ R CC L F L . MSLYLWLYT. MFGFRFHVLFA. MIFSTTQSK.

20 40 60 80 100 120 140 160 180 200 220 240 260 280 300 320 340 360 380 400 420 440 460 480 500 520 540 560 580 600 620 640 660 680 700 720 740 760 780 800

MKLLCWQKSI HGS. MANRQVTKRQWIPFWDLYGYV SI TLCST DTSDRRHVWCLRL CFI DYYYYSS FLAVE CNNKHKDGVVFL .

MVL K T NME RS V RG L Y .

MERS VR GL Y . MFFFLSSRSK. MAMYQSLYVALI LQI EGMSGVCVCAS.

MYQSLYVALIL QIEGMSGVCVCAS.

MSGV CVCA S.

MVLSSFEKLPTL. MLVCL C YE Y DL .

MDSF L GS L WLC I N HS M. MNVC L F V CV MNMI YDKL

20 40 60 80 100 120 140 160 180 200 220 240 260 280 300 320 340 360 380 400 420 440 460 480 500 520 540 560 580 600 620 640 660 680 700 720 740

MVL K T NME R SV R GL Y .

MERS V R GL Y . MANRQVTKRQR I P FWDLHGYV SI T LC STDT SDRR HVWCLRL CF I D YYYY SSF L AVEC NNKHK DGVVFL . MYACLFVYDL.

MAMY QL L Y V AL I LQI EGMSGV C V CA S.

MYQL L Y VA L I L QI EGMS GVC V CA S .

MSGV CV C AS .

MVLSS FE KL PT L . MLV C L CMI YDL MR N.

MKL L C WQKS I HGS. MFLFFHMNVCLFVCV.

MNVCL FVC V.

20 40 60 80 100 120 140 160 180 200 220 240 260 280 300 320 340 360 380 400 420 440 460 480 500 520 540 560 580 600 620 640 660 680 700 720

MGAETDWHEEPIHGT.

MKSQSMGLNS MDME.

MGLN S MDM E. MAA NY S KA MI .

MILF SI SVQLLQNVL SKN. MKKDFEVQFESF P.

20 40 60 80 100 120 140 160 180 200 220 240 260 280 300 320 340 360 380 400 420 440 460 480 500

MHACKPASHRTATGSVTSLSWLKCSCSMGCNK. MSVSVSLCNEVLM.

MPANRQVTERQRARSRVFPGLNVAVAWDVINKKVCVLSRSASPRFCGECSLSAVI TKCLCLFHY VMKY.

MEI S CQE HS S I QGP .

20 40 60 80 100 120 140 160 180 200 220 240 260 280 300 320 340 360 380 400 420 440 460 480 500 520

MKPFTDDRMFMGSTKHGTRGRGSTPPHCSRFLTLLMLLATASTNWSGTR.

20 40 60 80 100 120 140 160 180 200 220 240 260 280 300 320 340 360 380 400 420 440 460 480 500 520 540

MFMGSTKHGTRGRGSTPPHCSRFLTLLMLLATASTNWSGTR.

MGSTKHGTRGRGSTPPHCSRFLTLLMLLATASTNWSGTR.

MLLATASTNWSGTR.

MEICSWVLQSMGPGEEVRHPHTALDS.

MGPGEEVRHPHTALDS.

MDSAHESFWLFFFFLSFFIVVLCFSVYYLRERNKKIAYSR. MRFGSSLAVVCFPVSANCTSVHLACIFVLLSFRNKSVLCLSTKKKKKK.

MHIRAPIIQK.

Region IIRegion I

PaENOD40

PtENOD40

VrENOD40

GmENOD40-3

GmENOD40-1

LjENOD40

PvENOD40

SrENOD40

PsENOD40

VsENOD40

MsENOD40

CgENOD40

MtENOD40-1

TrENOD40-2

TrENOD40-1

MtENOD40-2

TrENOD40-3

LjENOD40-2

GmENOD40-2

Figure 1.2.

Schematic overview of ENOD40 transcripts across the plant kingdom reveals the absence of long ORFs.

Conserved nucleotide sequences are indicated as shaded boxes. ENOD40 transcripts are aligned at

region I and transcript lengths are on scale. All ORFs with a length of 9 amino acids or longer are

indicated.

Region II

CuENOD40

CsENOD40

AmENOD40

LsENOD40

LeENOD40

NtENOD40-1

NtENOD40-2

StENOD40

MHGKPASHETAMGSAIESCNGFFYFFSVLK.

MGSAIESCNGFFYFFSVLK.

MANRQVTKRQWARLLNLVMAFFTFSLY.

MHGKPASHETAMGSAIESCYGFFYFFSGLKYDCSSCMSME.

MGSAIESCYGFFYFFSGLKYDCSSCMSME.

MANRQVTKRQWARLLNLVMAFFTFSLV.

MAKFEQTGKSQNGNGLCLSLLLAFVFLYSLVFVLQFFKLVK.

MPKQTGKSQNGKGDSFESYIYMAKANVLYLFYVISVVAVGKTVCTREYNKTKLI.

MAKANVLYLFYVISVVAVGKTVCTREYNKTKLI.

MIFVMSFLLLHISNSLIMALTVLSDG.

MSFLLLHISNSLIMALTVLSDG.

MALTVLSDG.

MDNAKTNRQVTKRQRRLV.

MANRQVTKRQLWTPFWSLSWLST.MLLEFVQKYRQIHVGFSKFNVIITTSLESPSKHNSSLYNFKFAHT. MCGYMAKGWQTGKSQNGNCGLRFGVFLGFQLNVVPFKYIII.

MAKGWQTGKSQNGNCGLRFGVFLGFQLNVVPFKYIII.

MKQSMGLRKREAISWSELRFKCPSSKYNFKFVHTLPISLLEFQKMQQKRKVFMD.

MGLRKREAISWSELRFKCPSSKYNFKFVHTLPISLLEFQKMQQKRKVFMD.

MQQKRKVFMD.

MPFLQIQFQVCSYFANFSARIPENAAKKESVYGLIITLIAKDVGKSMANRQVTKRQWTPFWSLSWPFTYCCTLYCYIQSSIIVFYL.

MANRQVTKRQWTPFWSLSWPFTYCCTLYCYIQSSIIVFYL.

MLAKAWRTGKSRNGNGLRFGVFLGLLLIVVLYIVISSLL.

MDSVLESFLAFYLLLYFILLYPVFYNCVLFVSHMQCVVNMAYQKFIIELLRLS.

MLAKAWRTGKSQNGNGLRFGVFLGFLLIVVLISSLL.

MDSVLESFLAFYLLLYLYPVFCNCVLFVNHMHCVVNMAY.

MANRQVTKRQWTPFWSLSWLFTYCCTYIQSSVIVFYL.

MDSRLESVLAVNLMLYPINMLLYYPLLCDIRVLFVRRTCKACVLIDLMDLGRMLKTKKTNMRGGQDAFRPL.

MQQKREVFMD.

MHVGFSKINAIVTIFLECTSNITAPYTIASLLILEPNSVWIYDDENSK.

MMMKTANDVGKRMANRLVTKRQLWTPVWSLSWLSI.

MMKTANDVGKRMANRLVTKRQLWTPVWSLSWLSI.

MKTANDVGKRMANRLVTKRQLWTPVWSLSWLSI.

MANRLVTKRQLWTPVWSLSWLSI.

MMLAKGWRTG.

MLAKGWRTG.

Region II

ZmENOD40-1

OsENOD40-1

ObENOD40

LpENOD40-2

LpENOD40-1

TaENOD40

HvENOD40

OsENOD40-2

ZmENOD40-2

SbENOD40

PtENOD40

PaENOD40

MDSGNRASQEAFVFRNAFPDLLGGQLRCAQTGKSQYGNGGLH.

MADSIRSRPFLFLYPFLHSSSVIVSGGS.

MAKLQFKKLFNSMVPFRPPRWSAEVRTKPASHKTAMVKLQQESTLFFSAFPSFQLYQVVAK.

MVPFRPPRWSAEVRTKPASHKTAMVKLQQESTLFFSAFPSFQLYQVVAK.

MVKLQQESTLFFSAFPSFQLYQVVAK.

MHKPASHKTALVSSSRSTPPFFSSPVLSFQLYQVVAK.

MNGLNMHMVLERAEAGGGWRAMAEVQLKKLFDSMVHFLAS.

MVLERAEAGGGWRAMAEVQLKKLFDSMVHFLAS.

MAEVQLKKLFDSMVHFLAS.

MVNSSRSLPPFFLSRPSSCIRSESNNQKHLRVMNP.

MHGLSIYIVLESRSMMRMNHKAELLLKKRFSLMVPILSSQVVV.

MRMNHKAELLLKKRFSLMVPILSSQVVV.

MHGLSIYIVLESRSMMRMNHKAELLLKKRFSFMVPFLSSQVVV.

MMRMNHKAELLLKKRFSFMVPFLSSQVVV.

MVNSSRSLPPFFLSRPSSCIRSESNNQKHLRVMNP.

MVNSGRSPPPFYPFLSIQLYQVEVE.

MVNSGRSLPPFLSFSPHSVVSDGNRIIGILCV.

MALESGSRVGMNRKAERFSFAVPLLTSQVVA.

MNRKAERFSFAVPLLTSQVVA.

MAANYSKAMI.

MHACKPASHRTATGSVTSLSWLKCSCSMGCNK.

MPANRQVTERQRARSRVFPGLNVAVAWDVINKKVCVLSRSASPRFCGECSLSAVITKCLCLFHYVMKY.

MVSSCRSLLLIYQFKVSLYLLLLVFLRSFLSVPSVPVRAEDFSRSLAFWRQFQCWFWNMIL.

MPLVAVFPGWLPVPEGDVRRGQRPASQRSAMVSSCRSLLLIYQFKVSLYLLLLVFLRSFLSVPSVPVRAEDFSRSLAFWRQFQCWFWNMIL.

MALESGSRTSGMNRKAELLGRERFSFVVPLLTSQAVG.

MNRKAELLGRERFSFVVPLLTSQAVG.

MAVLFLLLLLLKNSSVCPCLSWHNSLDSRISGWLREV.

MNKPASHKSAIVSSDWILLLSLSNLVSFPCNLIPVVSTFYCSPLQRSLQLYLVEADKKLSRCNLVLWSQMFACVLLGNILSEMLIFTL.

MEGNDQLLSCCCSSWSTISLYLSMPRVAGRLLALACPVPEGGQAR.

MPRVAGRLLALACPVPEGGQAR.

Region II

CgENOD40

MsENOD40

MtENOD40-1

VsENOD40

PsENOD40

TrENOD40-2

TrENOD40-1

MtENOD40-2

SrENOD40

TrENOD40-3

PvENOD40

LjENOD40-2

LjENOD40

GmENOD40-2

GmENOD40-1

GmENOD40-3

VrENOD40

MDSAHESFWLFFFFLSFFIVVLCFSVYYLRERNKKIAYSR.

MAMYQSLYLCSTDTLD.

MANRQVTKRQWIPFWSLNGYVSITLSM.

MAMYQSLYLCSTDT.

MANRQVTKRQWIPFWSLNGYVSITLSGIIIYEV.

MAMYQSLYLVLSSMKYSTSTDTFRLKTCLMFYKCVSVQRLVSVLCS.

MAMYQSLYLLLSSMQTLQIEGCLVSDTCVSLRRSRLVSVLRRLL.

MANRQVTKRQWIPFWSLNGYVSITLSVIIIYADTSD.

MANRQVTKRQWIPFWDLYGYVSITLCSTDTSDRRHVWCLRLCFIDYYYYSSFLAVECNNKHKDGVVFL.

MAMYQSLYVALILQIEGMSGVCVCAS.

MDSFLGSLWLCINHSM.

MANRQVTKRQRIPFWDLHGYVSITLCSTDTSDRRHVWCLRLCFIDYYYYSSFLAVECNNKHKDGVVFL.

MAMYQLLYVALILQIEGMSGVCVCAS.

MDRDRGPYGYISVCVYDSGVILL.

MHLVLRRKRSFGYRLVNRQVTKRRWTALEVLMAIYLYVCMILV.

MIPTMHLVLRRKRSFGYRLVNRQVTKRRWTALEVLMAIYLYVCMILV.

MSECAIVFLTKITLWSSRKSRCYHDIVKLTMIPTMHLVLRRKRSFGYRLVNRQVTKRRWTALEVLMAIYLYVCMILV.

MAIYLYVCMILV.

MDSIRGSLGSLLISLL.

MDPIRGSYGYV.

MDSIGVSMAM.

MAMYVLILL.

MDRIRGPYGYMFVCVYVYCNKQRCCLFL.

MAICLYVFMYIVINKDVVCSFEKLPTCDVSSHSIAAEKNLVSSSVYVND.

MDSIGVSMV.

MHSPKDKRLWLHSGKPASHKKAMDSIGVSMV.

MHLTVCLGLAIGFSSLQNSLCCGRTCSCGSQFETRVSNHRAYREGVFMSRRS.

MEIKGCERVLTTHTPPLKTVCFGLALASLINKGCVLTFSLEWQKQICILQRRRGFGYSLANRQVTKRQWTPLGSLWLSIAHLCSSSCCRM.

MHSPKEERLWLQPGKPASHEKAMDSIGVSMAIYCSSM.

MDSIGVSMAIYCSSM.

MEEHNSLSGMKQRSYGHILANRQVTKRQWTLSH.

MKQRSYGHILANRQVTKRQWTLSH.

MDSLSLKGPYGYVCIDSIVVLFLCFGRMY.

MDSIGVSMAVYCSSM.

MHSPKEERLWLQSGKPASHKKAMDSIGVSMAVYCSSM.

MATVRQTGKSQKGNGLHWGLYGCVLLIYVVLLAVECNKQG.

Figure 1.3.

Schematic overview of open reading frames residing in region II of ENOD40 genes across the plant

kingdom. Conserved amino-acid motives are boxed.

MEDA-WLEHLHGS.

MEDE-WLEHAHGS.

MEDA-WLEHLHSS.

MEGA-WLEHLHGS.

MVGA-WQEHLHGS.

MEEEAWQECLHGS.

MEEAWWQECLHGS.

MEVL-WQEGPHGS.

MQ---WDEAIHGS.

FHGS.

MKPFTDDRMFMGS~

MKLLCWQKSIHGS.

MKLLCWEKSIHGS.

MKLLCWQKSIHGS.

MKFLCWQKSIHGS.

MKLLCWQKSIHGS.

MKLLCWEKSIHGS.

MNL-CWQKSIYD-.

MKL-CWQKSIHGS.

MDL-CWQKSIHGS.

MKF-CWQASIHGS.

MRF-CWQKSIHGS.

MEL-CWQTSIHGS.

MEL-CWLTTIHGS.

MEL-CWQKSIHGS.

M-X1-4-W-X4-HGS.

MEISCQEH-SSIQGP.

MGAETDWHEEPIHGT.

Figure 1.4.

(A) Nucleotide sequence alignment of region I and amino-acid sequence alignment of ORF I of ENOD40

genes shows that this region encodes a conserved peptide.

AACCGGCA AGTCA - (X6) - GGCAAT

Figure 1.4.

(B) Nucleotide sequence alignment of region II of ENOD40 genes.

Region II

The length of region II is not strictly defined as sequence similarity gradually decreases

in domains more remote from the conserved core. We have used 50 bp up- and 30 bp

down-stream of the most conserved core (25 bp) of region II for these comparisons

(Figure 1.3, 1.4B). The AACCGGCAAGTCA-(X6)-GGCAAT nucleotide motive represents

the highest level of sequence conservation in region II. In region II no ORFs are found

that are absolutely conserved (Figure 1.3). In about 50% of the ENOD40 transcripts, an

ORF starts in the middle of region II encoding the MANRQVTKRQ (or similar) peptide

motive. In some plant species containing multiple ENOD40 genes, an ORF encoding

this peptide is not present in all transcripts (Tr, Gm, Lj). This ORF is not present in any of

the monocot transcripts. In some ENOD40 transcripts (25%), a start codon is located in

a different reading frame, leading to an other peptide motive: M- - KPASHET (Figure

1.3, e.g. Ob, Pv, Os-2). Four transcripts contain both these ORFs. In about 33% of the

ENOD40 transcripts, there is no ORF present which spans the highly conserved center

of region II (Figure 1.3, e.g. Sr, Lj, Lp, Ta, Gm-1). Finally, the nucleotide sequence

alignment of region II presented in Figure 1.4B, shows that frame-shift inducing single as

well as multiple nucleotide gaps are present which would disrupt a conserved amino-

acid sequence within the ORF. The translation of ORF II peptides has been tested using

reporter fusions. The NtENOD40 ORF II is only efficiently translated in the absence of

the preceding ORF I in cowpea protoplasts (Compaan et al., 2001). Likewise, translation

of MtENOD40 ORF II was demonstrated in in vitro assays (Sousa et al., 2001) and in

root epidermal and cortical cells of Medicago truncatula using GUS translational reporter

fusions but translation efficiency depends on the presence (and translational activity) of

the preceding ORF I. If a peptide from ORF II is translated that would have biological

activity, about 50% of the ENOD40 transcripts encodes a similar region II peptide. Since

these peptides are not absolutely conserved, but the nucleotide sequence is strongly

conserved, this suggests that region II is active at the RNA level.

Symbiotic tissues Non-symbiotic tissues

Nodule Root Vegetative tissues Reproductive tissues Inducers

Plant species

Root pericycle facing

nodule primordum

Dividing cortical cells/

nodule primordium

Nodule vascular tissue

pericycle

Nodule central tissue

Root

Early stages of

adventitious nodal roots

lateral root

Stem

Stem + root procambial

cells

Meristem lateral shoots

Young leaf / stipule

primordia

Mature leaf

Hypocotyl

Cotyledon

Flower

Fruit / green seed pods

Germinated seeds

Pollen

Embryonic tissues ovules

and embryo

Cytokinine (-N)

NOD factor

Author

(proposed function)

Glycine max

(determinate)

***

1a,b,c

phloem

Pisum sativum

(indeterminate)

Yang, 1993, ¥

(Transport)

Glycine max

(determinate)

1a,b,c

stem

, 1993

(morphogenesis /

function of nodule

vascular system,

transport

photosynthate )

Medicago sativa

(indeterminate)

‡

1a,c

§§

, 1994 ,¥

(meristematic tissues,

mitosis or protein

synthesis)

Pisum sativum

(indeterminate)

1a,c

, 1994

Medicago sativa

(indeterminate)

1a,b,c

***

III

, 1994 †,¥

(function in

differentiation process

in relation to

hormonal status of

tissue)

pGm-2

in Vicia sativa

(indeterminate)

***

, 1995

Vicia sativa

(indeterminate)

, 1995

,¥

Vicia sativa

(indeterminate)

, 1995

Phaseolus

vulgaris

(determinate)

***

III

‡‡

, 1996

(

hormonal status of plant

differentiation process

of vascular tissue, also in

non-symbiotic

organogenic processes)

Papadopoulou

Vijn

Roussis

Crespi

Matvienko

Asad

Kouchi

Glycine max

(determinate)

, 1996, ¥

Medicago sativa

(indeterminate)

, 1997, ¥¥

Medicago sativa

pMs-1

symbiotic

not in

cortex

**III -

‡

æ+

ß+

pMs-2

Non- and

symbiotic

**III +

‡‡‡‡

I+

§§§I

æ+

ß+

1998, ¥¥¥

(Induction of E40 (by

Ck or NF) could serve

as amplification

mechanism, triggering

localised hormone

imbalance, leading to

ccd).

Sesbania

rostrata

***IV +

*II +

‡‡‡

I+

, 1998

(establishment of

zones and patterns of

cells with different

susceptibility to

prevailing hormone

concentrations,leading

to different outcomes)

pGm-2 in

A. thaliana

‡‡

, 1999

Oryza sativa

1a,b

§§§§I

pOs in G. max

(determinate)

, 1999

(function in

differentiation of

vascular bundles)

L. japonicus 1/2

, 2000

(organogenesis)

Trifolium repens

Tr-1/2

1c,2

**IV +

1a,2

‡‡‡

II ++

1c,2

§§§II

1c,2

§§§§II

1c,2

Trifolium repens

Tr-3

1c,2

1a,2

‡‡‡

II +

1c,2

§§§II

1c,2

§§§§II

1c,2

pTr-1/2

in N.Tabaccum

§§§III

§§§§III

pTr-3

in N.Tabaccum

§§§III

, 2002

(role at sites of

intensive lateral

transport of solutes)

Lycopersicon

esculentum

1b,2

‡‡‡‡

II +

1b,2

¶¶

1b,2

Gm in A. thal.

Le in M. trunc.

, 2003

(counter-acting effects

of ethylene)

Trifolium repens

Vleghels

Varkonyi-Gasic

Flemetakis

Kouchi

Mirabella

Corich

Mathesius, 1998

Fang,

v. Rhijn

Minami

1. ENOD40 RNA expression; 1a, RNA gel blot; 1b, RT-PCR; 1c, in situ hybridization.

2. ENOD40 promoter driven GUS expression.

++ high levels of expression, + detectable levels of expression, - undetectable levels of expression.

Nodules:

boundary layer and uninfected cells of central tissue, no expression in infected cells in determinate nodules.

not in meristematic cells, in later stages: residual meristematic cells beginning to differentiate, invasion zone, uninfected cells of fixation zone (in stem-borne nodules).

in infected cells of pre-fixation zone (in indeterminate nodules, these are differentiating tissues). In fixation zone low level of expression.

in infected cells of pre-fixation zone. In fixation zone only in uninfected cells.

III

localised in nodule meristem, including pre-fixation zone, and cells on the periphery of the central region.

in meristem, infection zone and vascular bundles.

***

ENOD40 expression in nodule vascular bundle when procambial cells differentiate into vascular tissue.

***

in young not fully developed nodules: exclusively in root pericycle where provascular strands of the nodule will be initiated.

***

III

vascular tissue: differentiating cells, not in meristematic or terminally differentiated cells (Crespi: in infected (++) and uninfected (+

(¥)

) indeterminate nodules).

***

parenchymatous cells surrounding the developing connecting vascular bundles (in stem-borne nodules).

: ENOD40 expression in empty nodules induced by mutant bacteria, or spontaneous nodules, or NOD factor induced nodule primordia.

¥: ENOD40 expression upon Arbuscular Mycorrhizal symbiosis both in uninfected (epidermal and pericycle) and infected cortical (containing immature arbuscules) cells.

¥¥: pMs-1: no GUS in NPA induced pseudonodules, but normally expressed in R.meliloti exo mutant induced empty nodules.

pMs-2: in NPA induced pseudonodules: dividing cortical cells and pericycle, later stage: around central vascular bundle and peripheral area;

normally expressed in R.meliloti exo mutant induced empty nodules.

Lateral roots :

‡ pre-emerging lateral root tips.

‡‡ pericycle cells giving rise to lateral root primordium, at later stages: in developing vascular cylinder.

‡‡‡

parenchymatous cells surrounding central vascular bundle in adventitious root primordia (inducible upon inoculation, comparable to root pericycle facing nodule primordium).

‡‡‡

Tr-1/2: dividing cells of primordium of adventitious root formed in the cortex of the first visible node, remains strong in zone that corresponds to vascular initials

Tr-3: developing root cap, meristematic cells, vascular initials of the nodal root.

‡‡‡‡

in developing lateral root: central cells of lateral root primordia, after emergence: restricted to central vascular tissue at proximal end of lateral root, remained high at the root tip.

‡‡‡‡

expression in lateral root primordium is low, flanked by regions of expression in vascular bundle of root, later stages also in connecting vascular tissue of main root and lateral root.

Stem:

§ adjacent to secundary phloem (procambial region).

§§ vegetative apical shoot meristems: at leaf primordia, particularly at the margins consisting of developing leaflets.

§§§I node, where leaf is attached

§§§II at developing lateral shoot; Tr-1/2: up-regulation confined to vascular tissue at base of lateral shoot, Tr-3 also in other tissues of axillary bud and more mature lateral shoot.

§§§III Tr-1/2: stem vasculature in the internode on the side where the petiole is attached, at the node at the point of petiole attachment; Tr-3: only at base of axillary shoot bud, not in stem vascular tissue.

§§§§I in rice: only in early developmental stages of stem vascular bundles that conjoin the emerging leaf.

In lateral vascular bundles exclusively in the xylem parenchyma cells surrounding the protoxylem.

§§§§II in stolon vascular bundles: in the phloem-cambium region of all vascular bundles and the parenchyma surrounding the xylem vessels in the leaf vascular traces.

Confined to nodes, rather than internodes, not detectable in petiole vascular bundles.

§§§§III parenchyma surrounding xylem vessels, the internal phloem, and the cells between xylem and internal phloem.

Flower:

in pedicels that connect florets with the inflorescence axes, in pedicel vascular tissue after onset of senescence in lower floret whorls.

¶ in stigma coinciding with anthesis, in petals after pollination just preceding petal senescence.

æ absent during somatic embryogenesis: not in callus or somatic embryos.

ß in root cortex and epidermal cells in root elongation zone; treatment induced (inner) cortical cell divisions, leading to small nodule primordia expressing GUS.

† alteration of ENOD40 expression by overexpression or antisense silencing affects regeneration of transgenic explants

Table 1.1 Overview of expression patterns of ENOD40 genes in 12 different plant species shows that

these are conserved across the plant kingdom.

Several Functions for ENOD40 Genes Have Been Proposed

In general, gene function assignment can benefit from the availability of mutants in that

particular gene. However, only a Zea mays line has been described in which a

transposon has been inserted in region II of one of its ENOD40 genes and this line has

no clear growth aberrations. The lack of a phenotype could be due to either a partial loss

of function or to functional redundancy of the two ZmENOD40 genes (Compaan et al.,

2003). In general, overexpression or silencing of ENOD40 seems not to induce very

severe growth aberrations, but nonetheless supports a role for ENOD40 in nodulation

and plant developmental processes possibly through interaction with phytohormone

signaling pathways (Crespi et al., 1994; van de Sande et al., 1996; Charon et al., 1997;

Charon et al., 1999). Assignment of the function of ENOD40 has mainly been based on

spatio-temporal expression patterns in symbiotic as well as non-symbiotic tissues. An

overview of ENOD40 expression patterns of 12 different plant species is presented in

Table 1.1. Here, we will focus on common features of expression in different plant

backgrounds and refer to the original reports for full details. Possible functions of

ENOD40 fall into three groups, namely transport, organogenesis and regulation of

phytohormone status.

Transport

A function of ENOD40 in transport processes has been proposed on the basis of high

expression levels in pericycle or xylem parenchyma cells of vascular bundles of root,

shoot and nodule. The expression in vascular tissue is found in all plant species

analyzed to date and the highest level of expression is usually found in (nodule)

vascular tissues. Yang, (1993), Papadopoulou, (1996), Kouchi, (1999) and Varkonyi-

Gasic (2002) argue for a role of ENOD40 in vascular bundle functioning and underline

that the expression pattern suggests a role in lateral transport of solutes. Although

ENOD40 expression correlates with a function in transport or differentiation of vascular

tissue, there are no experimental data available which could support an effect of

ENOD40 on vascular development or functioning.

Organogenesis

ENOD40 is often transiently expressed during early stages of organogenesis in various

organs. In later stages, ENOD40 is expressed at lower levels in vascular tissue of

developing organs. ENOD40 transcripts are present in developing lateral roots (Corich

et al., 1998; Fang and Hirsch, 1998; Mirabella et al., 1999; Varkonyi-Gasic and White,

2002; Vleghels, 2003), embryonic tissues (Flemetakis et al., 2000) and during early

stages of lateral shoot development (Asad et al., 1994) and nodal root development

(Corich et al., 1998; Varkonyi-Gasic and White, 2002). Therefore, it has been proposed

that it functions in early stages of organogenesis. This function is further supported by

reverse genetic data showing that misregulation of ENOD40 expression interferes with

regulation of Rhizobium induced cortical cell division (Charon et al., 1997), nodule

development (Charon et al., 1999), somatic embryogenesis during regeneration of

transgenic calli (Crespi et al., 1994) and development of adventitious shoots in tobacco

(van de Sande et al., 1996).

Hormone Status

Since phytohormones are signaling molecules in developmental processes like

organogenesis, the third function that was proposed for ENOD40 is a modifier of

hormone status of cells. This function is mainly based on the correlation between the

timing of ENOD40 expression, the effects of misexpression of ENOD40 and

phytohormone action during organogenesis.

It was reported that transformation with anti-sense ENOD40 constructs arrested callus

growth of Medicago sativa explants, whereas calli overexpressing MtENOD40

developed in teratomas. In proliferating explants, these phenotypes can be mimicked by

alteration of the cytokinin/auxin ratio (Crespi et al., 1994). Transgenic tobacco plants

overexpressing a GmENOD40 transcript had an increased number of adventitious

shoots at the base of the main shoot. This suggests reduced apical dominance, raising

the possibility that the transgenic plants were changed in terms of auxin metabolism or

perception (van de Sande et al., 1996). The expression in differentiating vascular tissue

(Yang et al., 1993; Varkonyi-Gasic and White, 2002) could indicate that the function of

ENOD40 is linked to auxin action since vascular tissue patterning and development is

regulated by auxin (Mattson et al., 2003). Constitutive ENOD40 expression in stable

transgenic Medicago truncatula lines induced dedifferentiation and divisions of cortical

cells (Charon et al., 1997). This effect is observed in the absence of Rhizobium, but only

under nitrogen limiting conditions. Transient expression in epidermal and outermost

cortical cells after bombardment induces dedifferentiation and division of inner cortical

cells and expression of MsENOD12, a molecular marker for cortical cell division (Charon

et al., 1997). Both auxin and cytokinin accumulate in cortical cells opposite protoxylem

poles upon inoculation, at the site of cortical cell divisions (Hirsch et al., 1997).

Application of auxin transport inhibitors or cytokinin can trigger pseudo-nodule

development (Cooper and Long, 1994; Hirsch et al., 1989). These observations suggest

that perturbation of the auxin/cytokinin ratio is a developmental cue during nodule

development. Thus, cell division could be triggered by an ENOD40 induced modification

of phytohormone status of the cortical cells.

Several phenotypes are observed in ENOD40 overexpressing plants upon bacterial

inoculation. Primary root growth is slightly enhanced and nodulation kinetics is

accelerated, accompanied by extensive cortical cell divisions in the region close to the

root tip. A considerable increase in the number of persistent infection threads reaching

the inner cortex was observed in these plants at early time points (Charon et al., 1999).

Sym5 and sickle mutants are impaired in nodulation and this is accompanied by either

ethylene hyper- or insensitivity. Their nodulation phenotypes showed that both infection

thread formation and cortical cell division are under control of ethylene (Guinel and

LaRue, 1991; Penmetsa and Cook, 1997). Therefore, it is possible that ENOD40 action

affects either ethylene responses during early stages of nodulation, or that it affects

cytokinin /auxin status, as induction of cortical cell divisions is also under the control of

auxin and cytokinin. The hypothesis that ENOD40 function is linked to ethylene action

would be in line with the finding that transient ENOD40 expression coincides with a

temporal burst of ethylene in germinating seeds and flowers of tomato (Vleghels, 2003).

The proposed function of ENOD40 to modify phytohormone status in the cells in which it

is expressed, is consistent with the observation that ENOD40 expression itself is under

control of ethylene and cytokinin (Fang and Hirsch, 1998; Vleghels, 2003).

CONCLUSION

In this chapter we described the current knowledge on ENOD40 structure and function

to answer two questions; what is the function of ENOD40 and which gene product has

biological activity? In the case of ENOD40, identification of the gene product that has

biological activity is not trivial. The study of the structure of ENOD40 transcripts revealed

that all ENOD40 transcripts lack a long open reading frame that could encode a

polypeptide with biological activity. However, two conserved regions, likely required for

biological activity, are present in ENOD40 transcripts. Of these, one contains a

conserved ORF and the other only in 50% of the cases. This indicates that the

biologically active gene products of ENOD40 may either be a (oligo)peptide and/or RNA.

It has been shown that the conserved peptides are translated in vivo, but no biological

function has yet been assigned to them. On the other hand, the second conserved

region also seems to contain biological activity, but only in some transcripts an ORF is

present in this region and it could be active as RNA. Therefore, these options should be

studied in further detail to better understand the molecular mechanism of ENOD40

action.

ENOD40 expression is transiently induced during early developmental stages of several

organs, suggesting that it has a regulatory role in development. Based on the timing of

ENOD40 expression and the effects of misexpression of ENOD40, it has been proposed

that ENOD40 is involved in early stages of organogenesis, and that it acts by modifying

phytohormone status. Whether and how ENOD40 modifies the hormone status of cells

in early stages of organogenesis is difficult to evaluate in whole plants because the

action of phytohormones leads to complex responses. Furthermore, the effects of

ENOD40 misexpression in various organs are diverse and these phenotypes do not give

further insight in the function of ENOD40. Therefore, the function of ENOD40 and its

mode of action are still poorly understood. To study the function of ENOD40 in further

detail requires a less complex test system than intact plants. Using a cellular system

would be very helpful to reduce the complexity of responses. Since the function of

ENOD40 is closely related to morphological changes in early stages of organogenesis,

the cellular test system should allow to study the effect of ENOD40 on cell division and

growth. In addition, because we want to study the effect of ENOD40 on hormone status

of cells, these cells should also have a marked morphological response to

phytohormones.

Taking these requirements into consideration only one such cellular plant system is

available, namely the tobacco BY-2 cell suspension which is generally recognized as the

HeLa cell of plant biology. The BY-2 cell suspension has several important advantages

for our studies. ENOD40 genes have been isolated from tobacco, so we can use a

tobacco ENOD40 homolog for our studies. The cell suspension is transformable,

allowing a reverse genetics approach. With a simple, yet well described morphology, the

characterization of the parameters cell growth and cell division in this filamentous cell

suspension is straightforward. Furthermore, a specific and strong effect of

phytohormones on cell growth and cell division has already been described. By

exogenously applying phytohormones or blockers of phytohormone action at closely

defined conditions, it is possible to study effects of ENOD40 on hormone action by

monitoring the morphological response.

We decided to explore the possibility to set up a bioassay for ENOD40 function in BY-2

cells using a transgenic approach. If ENOD40 induces a phenotype in the BY-2 cells, it

will be possible to test the function of the two conserved regions in ENOD40 transcripts

using a series of constructs carrying mutations in either of the two regions.

REFERENCES

Asad, S., Fang, Y., Wycoff, K.L., and Hirsch, A.M. (1994). Isolation and characterization of

cDNA and genomic clones of MsENOD40; Transcripts are detected in meristematic cells of

alfalfa. Protoplasma 183, 10-23.

Charon, C., Johansson, C., Kondorosi, E., Kondorosi, A., and Crespi, M. (1997). ENOD40

induces dedifferentiation and division of root cortical cells in legumes. Proc. Natl. Acad. Sci.

USA 94, 8901-8906.

Charon, C., Sousa, C., Crespi, M., and Kondorosi, A. (1999). Alteration of ENOD40

expression modifies Medicago truncatula root nodule development induced by SinoRhizobium

meliloti. Plant Cell 11, 1953-1965.

Compaan, B., Ruttink, T., Albrecht, C., Meeley, R., Bisseling, T., and Franssen, H. (2003).

Identification and characterization of a Zea mays line carrying a transposon tagged ENOD40.

BBA - Gene Structure and Expression, 1629 (1-3) 84-91.

Compaan, B., Yang, W.C., Bisseling, T., and Franssen, H. (2001). ENOD40 expression in the

pericycle precedes cortical cell division in Rhizobium-legume interaction and the highly

conserved internal region of the gene does not encode a peptide. Plant and Soil 230, 1-8.

Cooper, J.B., and Long, S.R. (1994). Morphogenetic Rescue of Rhizobium meliloti Nodulation

Mutants by trans-Zeatin Secretion. Plant Cell 6, 215-225.

Corich, V., Goormachtig, S., Lievens, S., VanMontagu, M., and Holsters, M. (1998). Patterns

of ENOD40 gene expression in stem-borne nodules of S. rostrata. Plant Mol. Biol. 37, 67-76.

Crespi, M.D., Jurkevitch, E., Poiret, M., d'Aubenton-Carafa, Y., Petrovics, G., Kondorosi,

E., and Kondorosi, A. (1994). ENOD40 , a gene expressed during nodule organogenesis,

codes for a non- translatable RNA involved in plant growth. EMBO J. 13, 5099-5112.

Fang, Y.W., and Hirsch, A.M. (1998). Studying early nodulin gene ENOD40 expression and

induction by nodulation factor and cytokinin in transgenic alfalfa. Plant Physiol. 116, 53-68.

Flemetakis, E., Kavroulakis, N., Quaedvlieg, N.E.M., Spaink, H.P., Dimou, M., Roussis, A.,

and Katinakis, P. (2000). Lotus japonicus contains two distinct ENOD40 genes that are

expressed in symbiotic, nonsymbiotic, and embryonic tissues. MPMI 13, 987-994.

Guinel, F.C., and LaRue, T.A. (1991). Light microscopy study of nodule initiation in Pisum

sativum L. cv. Sparkle and its low-nodulating mutant E2 (sym5). Plant Phys. 97, 1206-1211.

Hirsch, A.M., Bhuvaneswari, T.V., Torrey, j.G., and Bisseling, T. (1989). Early nodulin genes

are induced in alfalfa root ougrowths elicited by auxin transport inhibitors. Proc. Natl. Acad.

Sci. USA 86, 1244-1249.

Hirsch, A.M., Fang, Y., Asad, S., and Kapulnik, Y. (1997). The role of phytohormones in plant-

microbe symbioses. Plant and Soil 194, 171-184.

Kouchi, H., and Hata, S. (1993). Isolation and characterization of novel nodulin cDNAs

representing genes expressed at early stages of soybean nodule development. Mol. Gen.

Genet. 238, 106-119.

Kouchi, H., Takane, K., So, R.B., Ladha, J.K., and Reddy, P.M. (1999). Rice ENOD40:

isolation and expression analysis in rice and transgenic soybean root nodules. Plant J. 18,

121-129.

Mathesius, U., Schlaman, H.R.M., Spaink, H.P., Sautter, C., Rolfe, B.G., and Djordjevic,

M.A. (1998). Auxin transport inhibition precedes root nodule formation in white clover roots

and is regulated by flavonoids and derivatives of chitin oligosaccharides. Plant J. 14, 23-34.

Mattsson, J., Ckurshumova, W., and Berleth, T. (2003). Auxin signaling in Arabidopsis Leaf

Vascular Development. Plant Physiol. 131, 1327-1339.

Matvienko, M., Van de Sande, K., Yang, W.C., van Kammen, A., Bisseling, T., and

Franssen, H. (1994). Comparison of soybean and pea ENOD40 cDNA clones representing

genes expressed during both early and late stages of nodule development. Plant Mol. Biol. 26,

487-493.

Matvienko, M., van de Sande, K., Pawlowski, K., van Kammen, A., Franssen, H., and

Bisseling, T. (1996). Nicotiana tabacum SR1 contains two ENOD40 homologs. In: Biology Of

Plant Microbe Interactions, G. Stacey, B. Mullin, and P. M. Gresshoff, eds. Int. Soc. MPMI. St.

Paul USA, 387-391.

Minami, E., Kouchi, H., Cohn, J.R., Ogawa, T., and Stacey, G. (1996). Expression of the early

nodulin, ENOD40 , in soybean roots in response to various lipo-chitin signal molecules. Plant

J. 10, 23-32.

Mirabella, R., Martirani, L., Lamberti, A., Iaccarino, M., and Chiurazzi, M. (1999). The

soybean ENOD40 (2) promoter is active in Arabidopsis thaliana and is temporally and

spatially regulated. Plant Mol. Biol. 39, 177-181.

Papadopoulou, K., Roussis, A., and Katinakis, P. (1996). Phaseolus ENOD40 is involved in

symbiotic and non-symbiotic organogenetic processes: Expression during nodule and lateral

root development. Plant Mol. Biol. 30, 403-417.

Penmetsa, R.V., and Cook, R.V. (1997). A legume ethylene-insensitive mutant hyperinfected

by its Rhizobial symbiont. Science 275, 527-530.

Rohrig, H., Schmidt, J., Miklashevichs, E., Schell, J., and John, M. (2002). Soybean

ENOD40 encodes two peptides that bind to sucrose synthase. Proc. Natl. Acad. Sci. USA 99,

1915-1920.

Roussis, A., Van, D.S.K., Papadopoulou, K., Drenth, J., Bisseling, T., Franssen, H., and

Katinakis, P. (1995). Characterization of the soybean gene GmENOD40-2. J. Exp. Bot. 46,

719-724.

Santi, C., von Groll, U., Ribeiro, A., Chiurazzi, M., Auguy, F., Bogusz, D., Franche, C.,

Pawlowski, K. (2003). Comparison of nodule induction in legume and actinorhizal symbiosis:

the induction of actinorhizal nodules does not involve ENOD40. MPMI 16, 808-816.

Sousa, C., Johansson, C., Charon, C., Manyani, H., Sautter, C., Kondorosi, A., and Crespi,

M. (2001). Translational and structural requirements of the early nodulin gene ENOD40 , a

short-open reading frame-containing RNA, for elicitation of a cell-specific growth response in

the alfalfa root cortex. Mol. Cell. Biol. 21, 354-366.

van de Sande, K., Pawlowski, K., Czaja, I., Wieneke, U., Schell, J., Schmidt, J., Walden, R.,

Matvienko, M., Wellink, J., vanKammen, A., Franssen, H., and Bisseling, T. (1996).

Modification of phytohormone response by a peptide encoded by ENOD40 of legumes and a

nonlegume. Science 273, 370-373.

van Rhijn, P., Fang, Y., Galili, S., Shaul, O., Atzmon, N., Wininger, S., Eshed, Y., Lum, M.,

Li, Y., To, V., Fujushige, N., Kapulnik, Y., and Hirsch, A.M. (1997). Expression of Early

nodulin genes in alfalfa mycorrhizae indicates that signal transduction pathways used in

forming arbuscular mycorrhizae and Rhizobium-induced nodules may be conserved. Proc.

Natl. Acad. Sci. USA 94, 5467-5472.

Varkonyi Gasic, E., and White, D.W.R. (2002). The white clover ENOD40 gene family.

Expression patterns of two types of genes indicate a role in vascular function. Plant Physiol.

129, 1107-1118.

Vijn, I., Martinez, A.F., Yang, W.C., Das, N.L., Van, B.A., Van, K.A., and Bisseling, T.

(1995a). Early nodulin gene expression during Nod factor-induced processes in Vicia sativa.

Plant J. 8, 111-119.

Vijn, I., Yang, W.C., Pallisgard, N., Jensen, E.O., Van, K.A., and Bisseling, T. (1995b).

VsENOD5, VsENOD12 and VsENOD40 expression during Rhizobium-induced nodule

formation on Vicia sativa roots. Plant Mol. Biol. 28, 1111-1119.

Vleghels, I. (2003). Comparative studies on ENOD40 in legumes and non-legumes. Thesis,

Wageningen University and Research Center Wageningen, the Netherlands, pp 99.

Yang, W.C., Katinakis, P., Hendriks, P., Smolders, A., de Vries, F., Spee, J., van Kammen,

A., Bisseling, T., and Franssen, H. (1993). Characterization of GmENOD40, a gene showing

novel patterns of cell-specific expression during soybean nodule development. Plant J. 3, 573-

585.

Chapter 2

ENOD40 and Hormonal Control of Cell Size

in Tobacco Bright Yellow-2 Cells

ABSTRACT

We show that ENOD40 overexpression leads to suppressed elongation growth of BY-2

cells, whereas cell division frequencies are not affected in 35S:NtENOD40 BY-2 cell

suspensions. As auxin, cytokinin and ethylene control elongation growth and cell

division of tobacco BY-2 cell suspensions we used these growth parameters in a

bioassay to investigate interaction between ENOD40 and auxin, cytokinin and ethylene

signaling pathways. Thus, we show that ENOD40 affects cytokinin/auxin dependent

control of cell size. Analysis of ethylene homeostasis shows that ethylene accumulation

is accelerated in 35S:NtENOD40 lines. Furthermore, ENOD40 action can be counter-

acted by ethylene receptor blockers, suggesting that ethylene is a negative regulator of

elongation growth in BY-2 cells and that acceleration of ethylene accumulation is a

primary cause and not a consequence of suppressed elongation growth in

35S:NtENOD40 cells. Our data show that overexpression of NtENOD40 results in a

marked different behavior of phytohormone regulated cellular processes and support the

hypothesis that the regulatory function of ENOD40 in organogenesis depends on cross-

talk with ethylene and cytokinin/auxin signaling pathways. Implications of our findings for

the role of ENOD40 during nodule primordium formation are discussed.

INTRODUCTION

Homologs of ENOD40 genes have been identified in plant species across the plant

kingdom including monocots like rice, maize and sorghum, and dicots such as tomato,

tobacco, citrus and several leguminous species. The highest expression levels of

ENOD40 have been found during legume nodule formation, and therefore its function

has been studied in most detail during this process. However, the presence of ENOD40

homologs in genomes of non-leguminous plant species, and the presence of transcripts

in non-symbiotic tissues indicates that the function of ENOD40 is not confined to nodule

development in leguminous species. Low levels of ENOD40 expression have been

detected during lateral root formation, flower development and vascular tissue

development in several different plant species, indicating that ENOD40 expression is

associated with certain specific stages of organogenesis. This suggests that ENOD40

has a general role in plant development.

During nodulation, ENOD40 expression is induced within 3 hrs after inoculation with

Rhizobium in pericycle cells positioned opposite the protoxylem poles, prior to the onset

of cortical cell divisions which lead to formation of the nodule primordium (Compaan et

al., 2001; Kouchi and Hata, 1993; Mylona et al., 1995; Yang et al., 1993). Misregulation

of ENOD40 by co-suppression reduces the number of nodules and nodule development

is arrested which indicates that ENOD40 has a regulatory role in these processes

(Charon et al., 1999; Crespi et al., 1994). ENOD40 overexpression on the other hand,

induces cortical cell divisions and accelerates nodule development in Medicago (Charon

et al., 1999). However, ENOD40 expression alone is not sufficient for nodule primordium

formation (Mathesius et al., 2000; Minami et al., 1996), and interaction with other plant

factors is probably required for the initiation of nodule morphogenesis. Whereas

Rhizobial Nod-factor signaling sets the process of nodulation in action, the process is

further regulated by the phytohormones ethylene, cytokinin and auxin. ENOD40

expression is induced by Nod-factors or cytokinin in legumes suggesting that these

could be candidate interactors of ENOD40.

The role of phytohormones during early stages of nodule primordium development has

been implicated by experiments showing that either an auxin transport inhibitor or

cytokinin are able to induce cortical cell divisions and eventually nodule-like structures

on roots of legumes as well as non-legumes (Cooper and Long, 1994; Hirsch et al.,

1989). Additionally, both auxin and cytokinin accumulate in cortical cells opposite

protoxylem poles upon inoculation, at the site of cortical cell divisions. These

observations suggest that perturbation of auxin and/or cytokinin flow through the root

acts as a developmental cue during nodule development. Inhibitors of ethylene

biosynthesis have been reported to increase nodule formation and to modify positioning

of cortical cell division in roots (Heidstra et al., 1997; Lee and LaRue, 1992; Peters and

Crist-Estes, 1989). Further, a blocker of ethylene production, AVG, partially mimics the

effect of ENOD40 overexpression in Medicago (Charon et al., 1997). Taken together,

these observations suggest that during nodule development cross-talk between

ENOD40 and phytohormone signaling exists. Ectopic overexpression of ENOD40

affected certain hormonal responses of somatic embryos of alfalfa under in vitro culture

conditions (Charon et al., 1999; Crespi et al., 1994). Also, overexpression of ENOD40

led to reduced apical dominance in tobacco suggesting that phytohormone signaling is

affected by ENOD40 in non-legumes (van de Sande et al., 1996).

To date, the function of ENOD40 and its mode of action are poorly understood. Direct

evidence of an interaction between ENOD40 activity and phytohormone signaling

pathways is lacking. Establishing whether the function of ENOD40 involves interaction

with phytohormone signaling pathways is an important step towards unraveling the role

of ENOD40 during developmental processes to which its expression is associated. We

searched for a bioassay to test the effect of ENOD40 on cellular processes and chose

the tobacco BY-2 cell suspension as a model system. We found that overexpression of

ENOD40 suppresses elongation growth but that cell division frequency is not affected.

An important advantage of the BY-2 cell suspension for our study is the observation that

elongation growth and cell division frequency of cells are regulated by the balance

between cytokinin and auxin in the culture medium (Hasezawa and Syono, 1983). In

addition, we provide evidence that ethylene is a negative regulator of elongation growth

in BY-2 cells. These observations raised the possibility to test whether the morphological

changes induced by ENOD40 are caused through interaction with phytohormone

signaling.

RESULTS

Generation of Stably Transformed Cell Lines Carrying 35S:NtENOD40

To determine whether overexpression of NtENOD40 affects morphology of BY-2 cells,

we generated a set of six 35S:NtENOD40 BY-2 cell lines by Agrobacterium-mediated

transformation (Methods). These lines were named line Nt1 through Nt6. Each

transgenic line was derived from a different callus, which means that they cannot be

siblings. PCR analysis on genomic DNA showed that all lines contain the construct (data

not shown). Expression of the transgenes was detected by RNA gel blot analysis

performed with total RNA isolated from these transgenic lines in three independent

experiments, each time with similar results. One representative set of data is presented

in Figure 2.1D. In the wild type line, NtENOD40 mRNA could not be detected, indicating

a very low expression level of the endogenous NtENOD40 gene. In the six lines that

carry the 35S:NtENOD40 construct NtENOD40 transcripts were expressed at varying

levels but this level was, in all lines except line Nt6, much higher than in the wild type

line. Therefore, we concluded that NtENOD40 is overexpressed in all 35S:NtENOD40

transgenic lines, except in line Nt6. Hybridization with the HPTII probe was performed as

a positive control, and revealed that in all transgenic lines HPTII transcripts could be

detected, conferring resistance to hygromycin.

Further, hybridization with the NtENOD40 probe resulted in two bands on the RNA gel

blot, indicating the presence of two NtENOD40 RNAs with different lengths. To further

characterize the nature of these two RNAs, 3' RACE-PCR was performed on NtENOD40

transcripts of the transgenic lines (Methods). Analysis of nucleotide sequences of 11

cloned RACE-PCR fragments revealed that all sequences that were obtained are 100%

identical to the transgene sequence and that read-through occurs through the NOS

terminator that flanks the NtENOD40 cDNA sequence in the construct resulting in

transcripts of two different lengths (data not shown).

 Figure 2.1

The value for each repetition is the means of 100-150 individual cells per sample. Error bars indicate SD

between independent experiments. An * marks transgenic cell lines with a significant reduction of

elongation growth compared to the wild type (p < 0.001).

(A) Elongation growth rate (cell file length expressed in width units).

(B) Cell division frequency (number of cells per cell file).

(D) The level of transgene expression is determined at the start of protoplast culture by RNA gel blot

analysis. Hybridization with the NtENOD40 probe and HPTII probe show expression of the transgene

transcripts. Hybridization with the Ubiquitine (UBI) probe is performed in order to compare loading of the

separate samples.

Wt Nt1 Nt2 Nt3 Nt4 Nt5 Nt6

Wild type Nt 1 Nt 2 Nt 3 Nt 4 Nt 5 Nt 6

1,0

2,0

3,0

4,0

Wild type Nt 1 Nt 2 Nt 3 Nt 4 Nt 5 Nt 6

** **

1,0

2,0

3,0

4,0

Wild type Nt 1 Nt 2 Nt 3 Nt 4 Nt 5 Nt 6

** **

1,0

2,0

1,2

1,4

1,6

1,8

Wild type

35S:

NtENOD40

Wild type

35S:

NtENOD40

Wild type

35S:

NtENOD4

ELONGATION GROWTH RATE

(cell-file length in width units)

DIVISION FREQUENCY

(number of cells per file)

CELL SIZE

(length in width units)

NtENOD40

HPTII

UBI

line

Figure 2.1. Elongation Growth Rate, Cell Division Frequency, Cell Size and Transgene Expression Levels

of Cultured Cells of Wild Type and 35S:NtENOD40 BY-2 Cell Lines.

Growth parameters of protoplast derived cells are determined after four days of culture in medium

supplemented with 0.1 mg/l NAA and 1.0 mg/l BA. Data are average of 11 (Wild type), 8 (Nt1), 9 (Nt2) and

5 (Nt3-Nt6) independent repetitions.

NtENOD40 Suppresses Elongation Growth But Does Not Affect Cell Division

Frequency of Cultured Protoplasts

It has been shown that a protoplast bioassay provides the most accurate data

concerning elongation growth and cell division frequency in BY-2 cell suspensions.

Therefore, we adapted this protoplast-based bioassay described by Hasezawa (1983),

to quantitatively determine the contribution of cell division frequency and elongation

growth to cell morphology. By preparing protoplasts from the cell suspension, a

population of single cells with a similar size is obtained. This bioassay has two main

advantages in comparison to direct use of suspension-cultured cells. Firstly, the cell

division frequency can be directly scored by counting the number of cells in cell files that

are formed from protoplasts. Under our growth conditions BY-2 cells normally form cell

files. The number of cell files during culture remains similar to the number of protoplasts

at the start of the experiment. This means that the vast majority of the cells remain

attached to each other after division. Therefore, by starting from single cells, the

average number of cells per cell file reflects the number of cell divisions that took place

during the incubation time, and this parameter is from hereon called the ‘cell division

frequency’. For example, cultured protoplasts remain single cells when no cell division

takes place, whereas finding 2 cells per file means that one round of cell division has

occurred during the incubation time.

The second advantage of protoplasts as starting material for our tests concerns

quantification of elongation growth. The width of cells remains similar to the diameter of

protoplasts during culture. This means that no radial expansion growth occurs and the

length (expressed in width units) of the cell files is a parameter for elongation growth of

cells during the incubation period. This parameter is from hereon called the ‘elongation

growth rate’. The average length of individual cells, again expressed in width units, is

from hereon called ‘cell size’. This parameter depends on the relative rates of elongation

growth and cell division and therefore should not be used to indicate growth rates.

Instead, in a population of simultaneously elongating and dividing cells, this parameter

(length per cell) reflects the ratio of the elongation growth rate (length per cell file)

divided by the cell division frequency (number of cells per file).

To study the relation between cell size, elongation growth rate, cell division frequency

and NtENOD40 expression level, protoplasts were obtained from the wild type and

35S:NtENOD40 cell lines (Methods), (Nagata et al., 1992). Cells were subsequently

cultured for four days in protoplast culture medium (PCM) in the presence of 1.0 mg/l BA

and 0.1 mg/l NAA (Methods). We determined elongation growth rate, cell division

frequency and cell size, respectively, for each line in at least 5 independent experiments

(Methods). Data are presented in Figure 2.1A-C. We found that four lines have a

strongly reduced elongation growth rate as compared to the wild type (Nt1 2.27 ± 0.27;

Nt2 2.31 ± 0.25; Nt3 3.09 ± 0.25 and Nt4 2.48 ± 0.65, respectively, versus 3.93 ± 0.52

for the wild type) (Figure 2.1A). The reduction of elongation growth rate is significant

(p<0.001) for the lines Nt1, Nt2, Nt3 and Nt4. These data show that in the lines with the

strongest phenotype (Nt1 and Nt2), elongation growth is reduced by about 60% in

comparison to that of the wild type. The RNA gel blot analysis (Figure 2.1D) revealed

that from the set of six transgenic lines, lines Nt1 and Nt2 express 35S:NtENOD40 at

the highest level. In addition, lines Nt5 and Nt6 have an elongation growth rate that is

similar to wild type (3.70 ± 0.57 and 3.24 ± 0.37 respectively). In line Nt5 only a limited

level of NtENOD40 transcripts can be detected, and line Nt6 contains no detectable

NtENOD40 mRNA. So, a good correlation between NtENOD40 expression level and

reduction of elongation growth is found. Under the used hormonal conditions, the

average number of cells per cell file in the wild type line is 1.15 ± 0.09. This means that

about 15% of the cells has undergone one round of cell division during the culture

period. The average number of cells per cell file in the transgenic lines is Nt1 1.17 ±

0.10; Nt2 1.20 ± 0.07; Nt3 1.17 ± 0.09; Nt4 1.14 ± 0.09; Nt5 1.09 ± 0.08 and Nt6 1.08 ±

0.03 (Figure 2.1B). This shows that division frequency is not affected in the transgenic

lines, indicating that overexpression of NtENOD40 does not alter cell division frequency.

The cell size reflects the ratio of elongation growth rate divided by cell division frequency

and is depicted in Figure 2.1C. The average size of cells in the different lines is: Nt1

1.94 ± 0.16; Nt2 1.92 ± 0.17; Nt3 2.64 ± 0.18; Nt4 2.18 ± 0.50; Nt5 3,44 ± 0,68 and Nt6

3,06 ± 0,33; versus 3.42 ± 0.47 for the wild type). The reduction of cell size is significant

(p<0.001) for the lines Nt1, Nt2, Nt3 and Nt4. These data reveal that overexpression of

NtENOD40 results in reduced cell size in cultured BY-2 cells. Representative

photographs taken after four days of culture of wild type cells and cells of a transgenic

line with a strong phenotype (line Nt1) are presented in Figure 2.4D,E. These pictures

show the clear elongated appearance of wild type cells and reveal that cell size is

smaller in line Nt1. Thus, the protoplast bioassay allows us to differentiate between

effects of ENOD40 on cell division and elongation growth. We show that cell size is

reduced in NtENOD40 overexpressing cell lines by a reduced elongation growth rate,

rather than by increased cell division frequency under these conditions. Two lines (line

Nt1 and Nt2) that have the highest level of NtENOD40 expression and have a strong

phenotype were selected for further analysis.

 Figure 2.2

(A) Wild type elongation growth rate, cell division frequency and cell size at increasing concentrations of

BA, each in the presence of 0.1 mg/l NAA.

(B) Wild type elongation growth rate, cell division frequency and cell size at increasing concentrations of

NAA, each in the presence of 1.0 mg/l BA.

(C), (D) Dose response curves measuring cell division frequency as a function of (C) BA concentration

and (D) NAA concentration.

(E), (F) Dose response curves measuring elongation growth of cell files as a function of (E) BA

concentration and (F) NAA concentration.

(G), (H) Dose response curves measuring cell size as a function of (G) BA concentration and (H) NAA

concentration.

1,0

2,0

3,0

4,0

5,0

0,0 0,10 0,50 1,0 2,0

1,0

2,0

3,0

4,0

5,0

0,0 0,05 0,10 0,5 1,0

0,0 0,10 0,50 1,0 2,0 0,0 0,05 0,10 0,5 1,0

1,0

2,0

3,0

4,0

0,0 0,10 0,50 1,0 2,0

1,0

2,0

3,0

4,0

0,0 0,05 0,10 0,5 1,0

1,0

2,0

3,0

4,0

5,0

0,0 0,10 0,50 1,0 2,0

1,0

2,0

3,0

4,0

5,0

0,0 0,05 0,10 0,5 1,0

Wild type

35S:

NtENOD40

(Nt1)

35S:

NtENOD40

(Nt2)

Wild type

35S:

NtENOD40

(Nt1)

35S:

NtENOD40

(Nt2)

Wild type

35S:

NtENOD40

(Nt1)

35S:

NtENOD40

(Nt2)

Wild type division rate

Wild type cell size

Wild type elongation growth

Wild type

35S:

NtENOD40

(Nt1)

35S:

NtENOD40

(Nt2)

Wild type

35S:

NtENOD40

(Nt1)

35S:

NtENOD40

(Nt2)

Wild type

35S:

NtENOD40

(Nt1)

35S:

NtENOD40

(Nt2)

Wild type division rate

Wild type cell size

Wild type elongation growth

1,0

2,0

1,2

1,4

1,6

1,8

1,0

2,0

1,2

1,4

1,6

1,8

1,0

2,0

1,2

1,4

1,6

1,8

1,0

2,0

1,2

1,4

1,6

1,8

Cytokinin Dose Response Curve

at 0.1 mg/l NAA

ELONGATION GROWTH

(length in width units)

ELONGATION GROWTH

(length in width units)

DIVISION FREQUENCY

(number of cells per file)

DIVISION FREQUENCY

(number of cells per file)

at 1.0 mg/l BA

Auxin Dose Response Curve

[BA] in mg/l

[BA] in mg/l [NAA] in mg/l

[NAA] in mg/l

DIVISION FREQUENCY

(number of cells per file)

DIVISION FREQUENCY

(number of cells per file)

ELONGATION GROWTH RATE

(cell-file length in width units)

ELONGATION GROWTH RATE

(cell-file length in width units)

CELL SIZE

(length in width units)

CELL SIZE

(length in width units)

Figure 2.2. Dose Response Curves for Cytokinin and Auxin in Wild Type and 35S:NtENOD40 BY-2 Cells.

Protoplasts are cultured for four days in medium supplemented with various concentrations of cytokinin or

auxin. Data represent average (± SD) of 4 (Wild type), 3 (Nt1) and 4 (Nt2) independent experiments.

Effect of ENOD40 on Auxin and Cytokinin Responses

To test the effect of increased NtENOD40 transcript levels on hormone responses, we

made use of the effects of exogenously applied auxin and cytokinin on elongation

growth and cell division of BY-2 cells. These have previously been determined by

means of dose response curves for auxin and cytokinin (Hasezawa and Syono, 1983).

Low concentrations of auxin stimulate BY-2 cell elongation, whereas high concentrations

reduce the average cell length by promoting cell division. Increasing the concentration of

cytokinin in the culture medium causes an opposite response since this reduces the cell

division frequency and promotes elongated growth of cells (Hasezawa et al., 1988;

Iwata, 1995; Tamura et al., 1999). In order to study the effect of overexpression of

ENOD40 on elongation growth and cell division in response to auxin and cytokinin, dose

response curves were made for the wild type cell line and transgenic lines Nt1 and Nt2.

For the cytokinin dose response curve, we determined elongation growth rate, cell

division frequency and cell size as a function of cytokinin (BA) concentration at a fixed

concentration of auxin (0.1 mg/l NAA). For the auxin dose response curve, we

determined the same parameters as a function of auxin (NAA) concentration at a fixed

concentration of cytokinin (1.0 mg/l BA). The dose response curves were made four

times, in independent experiments, for the wild type line and line Nt2, and three times for

line Nt1. The results from independent experiments were similar and we calculated the

average value for each parameter (Figure 2.2A-H).

We first describe the response of wild type cells to exogenously applied cytokinin and

auxin. The dose response curve for elongation growth rate as a function of cytokinin

concentration in the presence of 0.1 mg/l NAA (Figure 2.2A) shows that elongation

growth is almost constant although it is possible that an increasing cytokinin

concentration has a slight negative effect on elongation growth. Elongation growth in the

absence of cytokinin is similar to that in the presence of various concentrations of

cytokinin. Therefore, exogenous application of cytokinin is not essential, nor does it

markedly influence elongation growth in the presence of auxin. The auxin dose

response curve for elongation growth (Figure 2.2B) shows that elongation growth is

hardly affected by the concentration of auxin as in the absence of auxin it is similar to

that at the various concentrations of exogenous auxin. A low concentration of auxin only

has a slight positive effect on elongation growth. This shows that elongation growth

neither requires auxin, nor does auxin markedly affect it, when cytokinin is applied to the

medium. Taken together, these data show that either cytokinin or auxin is sufficient to

sustain the growth rates achieved under our culture conditions.

The dose response curve for cell division frequency as a function of cytokinin

concentration (Figure 2.2A) shows that the cell division frequency is reduced at

increasing concentrations of cytokinin in the presence of 0.1 mg/l NAA, and that the cell

division frequency is maximal in the absence of exogenous cytokinin. The dose

response curve for cell division frequency as a function of auxin concentration in the

presence of 1.0 mg/l BA (Figure 2.2B) shows that the cell division frequency increases

at increasing concentrations of auxin. In the absence of auxin, the cell division frequency

is '1 cell per file' which means that cells have not divided during the culture period.

These results show that exogenously applied auxin is essential for cell division in BY-2

cells. Taken together, these observations show that exogenously applied cytokinin has

an inhibitory effect on cell division and that exogenous auxin has a promoting effect on

cell division.

The size of individual cells in these filamentous cell suspensions is controlled by two

processes, elongation growth and cell division, and the ratio of these two parameters

reflects the individual cell size in our samples (file-length / (nr cells/file) = cell-length).

Thus, the effect of external application of cytokinin and/or auxin on the average size of

cells can be determined by calculating the ratio of elongation growth rate over cell

division frequency in a dose response curve. The results are depicted in Figure 2.2A-B.

These data show that cell size increases at increasing concentrations of cytokinin

(Figure 2.2A) in the presence of 0.1 mg/l NAA. Further, the auxin dose response curve

(Figure 2.2B) shows that the cell size decreases at increasing concentrations of auxin in

the presence of 1.0 mg/l BA.

Taken together, the analysis of these three parameters reveals how the cell size can be

regulated by phytohormones in wild type BY-2 cells. Cytokinin has an inhibiting effect on

cell division frequency whereas auxin has a stimulating effect. By quantifying the division

frequency in the two complementary dose response curves, we show that the effect of

increasing the auxin concentration at a fixed cytokinin concentration is a mirror image to

the effect of increasing the cytokinin concentration in the presence of a fixed auxin

concentration. This means that the division frequency of BY-2 cells is dependent on the

cytokinin to auxin balance, within the concentration range tested. Since elongation

growth is almost constant in these dose response curves, these observations further

infer that a high cytokinin to auxin ratio suppresses cell division by which individual cells

become longer. The observation that cell division frequency can be affected by the

cytokinin to auxin balance, without affecting elongation growth, infers that the two

processes are not strictly coupled in wild type cells.

Cell Division and Elongation Growth Rates Are Strictly Coupled in 35S:NtENOD40

Cells

To determine whether overexpression of NtENOD40 affects the response of BY-2 cells

to cytokinin and auxin, dose response curves for 35S:NtENOD40 lines Nt1 and Nt2 are

compared to those of the wild type line (Figure 2.2C-H). Figure 2.2C shows that the

inhibitory effect of cytokinin on cell division is similar in wild type and 35S:NtENOD40

lines. Further, the stimulating effect of auxin on cell division is also similar in wild type

and 35S:NtENOD40 lines, as revealed by the auxin dose response curve depicted in

Figure 2.2D. The promoting effect of auxin on cell division at higher auxin concentrations

is slightly less strong in the 35S:NtENOD40 lines than in the wild type. However, at the

highest auxin to cytokinin ratio (0.1 mg/l NAA and 0 mg/l BA, i.e. a condition that most

strongly induces cell division in the wild type line), on average about 60% of the cells

within the population has divided in the wild type line as well as in the 35S:NtENOD40

lines. This means that under these conditions, wild type and 35S:NtENOD40 cells can

reach similar maximal division frequencies.

The effect of ENOD40 on elongation growth at various cytokinin concentrations is shown

in the cytokinin dose response curve in Figure 2.2E. In the absence of cytokinin, at 0.1

mg/l NAA, elongation growth is reduced in 35S:NtENOD40 cells by about 40% in

comparison to the wild type (Figure 2.2E). So, ENOD40 can reduce elongation growth in

the absence of exogenous application of cytokinin. Addition of cytokinin has only a slight

negative effect on elongation growth of wild type cells since at most a 20% reduction of

elongation growth is induced by 2 mg/l cytokinin in comparison with wild type elongation

growth in the absence of cytokinin. In the presence of 2 mg/l cytokinin, elongation

growth in 35S:NtENOD40 cells is reduced by about 70% of wild type levels. Thus, the

combined effect of ENOD40 and cytokinin is slightly stronger than the sum of the

separate effects of ENOD40 and cytokinin. So, addition of cytokinin to 35S:NtENOD40

cells attenuates elongation growth, suggesting that ENOD40 and cytokinin act

synergistically in inhibition of growth.

The auxin dose response curve (Figure 2.2F) shows that elongation growth of wild type

cells is largely unaffected by application of NAA. Elongation growth of 35S:NtENOD40

cells in the absence of auxin (at 1.0 mg/l BA) is reduced by about 75% of wild type

levels whereas elongation growth is reduced by about 60% upon application of 1.0 mg/l

NAA. This shows that the negative effect of ENOD40 and cytokinin on elongation growth

can be partially counter-acted by auxin in 35S:NtENOD40 cells. So, whereas cytokinin

reduces elongation growth of ENOD40 overexpressing cells, this negative effect can be

counter-acted by auxin. This shows that in 35S:NtENOD40 lines, elongation growth is

dependent on the ratio of exogenously applied cytokinin and auxin.

We showed that cell size is reduced in 35S:NtENOD40 lines by reduction of elongation

growth, rather than by increased division frequency (Figure 2.1). By using these dose

response curves we can determine how ENOD40 affects the control on cell size in

relation to auxin and cytokinin responses by first discriminating between the effects on

elongation growth and cell division frequency and subsequently calculating the ratio

between both parameters at the various concentrations of cytokinin and auxin. Both the

cytokinin dose response curve (Figure 2.2G) and the auxin dose response curve (Figure

2.2H) show that this ratio remains constant for 35S:NtENOD40 cells at all different

cytokinin to auxin ratios tested. These data reveal that in 35S:NtENOD40 lines Nt1 and

Nt2, elongation growth and cell division frequency depend on the cytokinin to auxin ratio

in a similar fashion. Since the size of individual cells is the net result of elongation

growth and cell division frequency, these data show that the processes of elongation

growth and cell division are strictly coupled in 35S:NtENOD40 cells whereas in wild type

cells they are not (Figure 2.2G-H). Thus, 35S:NtENOD40 cell lines have a marked

different behavior with respect to the control of cell size in response to phytohormones.

In wild type, the balance between cytokinin and auxin regulates the cell division

frequency and this regulation is not affected in 35S:NtENOD40 lines. The observation

that in 35S:NtENOD40 lines the cell size remains constant at different division

frequencies, suggests that ENOD40 controls cell size by regulating elongation growth in

accordance with the frequency at which cell division takes place. The observation that

ENOD40 and cytokinin may act synergistically in inhibition of elongation growth and that

auxin can counter-act this effect provides evidence that elongation growth of

35S:NtENOD40 cells is (at least partially) controlled by the auxin to cytokinin balance.

However, the strict control of ENOD40 on cell size is not influenced by exogenous

application of either cytokinin or auxin or different combinations of both, suggesting that

the effect of ENOD40 may rely on other plant factors. One such plant factor may be

ethylene.

ENOD40 And Ethylene Both Influence Elongation Growth

Genetic as well as physiological studies have shown that at least part of the diverse

effects of ethylene on plant growth and development are caused by regulation of cell

elongation growth rather than cell division frequency. Specific cellular phenotypes of

ethylene perception mutants show that ethylene affects leaf expansion by suppressing

cell enlargement rather then division (Kieber et al., 1993; Rodrigues-Pousada et al.,

1993). For example, the ctr1 mutation which leads to constitutive ethylene signaling,

results in a dramatically reduced stature and unexpanded leaves in ctr1 mutants; in

contrast, most ethylene insensitive mutants have a larger rosette than the wild type

(Ecker, 1995), resulting from cell enlargement (Hua et al., 1995). Additionally, ethylene

has been implicated to control the rate of elongation growth in cells in the elongation

zone of the Arabidopsis root (Le et al., 2001). Ethylene can influence expression of

genes encoding enzymes involved in cell wall loosening on one hand and microtubule

organization on the other (Shibaoka, 1994). For example, a role for ethylene has been

suggested in regulating the expression of cell wall peroxidases involved in the control of

cell wall extensibility and cell growth (Ridge and Osborne, 1971).

Although direct evidence for a role of ethylene in the regulation of elongation growth in

BY-2 cells is lacking, several observations do suggest such a role. A number of studies

have used the BY-2 cell suspension to investigate the role of cell wall components and

the cytoskeleton in elongation growth. These studies include cell wall associated

ascorbate oxidase (Kato and Esaka, 2000), as well as peroxidases (Iwata, 1995) and

expansins (Link and Cosgrove, 1998) which have been suggested to have a role in cell

wall loosening and acid growth response. Furthermore, cortical microtubule orientation

and stability as well as actin filament organization have been shown to be major

determinants of directional expansion growth in BY-2 cells (Collings et al., 1998; Iwata,

1995; Kuss and Cyr, 1992). When either of these components is limited or disturbed by

molecular techniques or chemical blockers, a reduction of elongation growth is

observed, rather then severe morphological abnormalities of BY-2 cells. Since the

expression of the genes encoding these structural components is under control of

ethylene, this suggests that ethylene has a central role in regulation of elongation growth

of BY-2 cells. Because the role of ethylene in regulating elongation growth would closely

resemble the effect of overexpression of ENOD40 in BY-2 cells, we hypothesize that

ethylene homeostasis can be affected by overexpression of ENOD40 and that this leads

to altered regulation of elongation growth in our transgenic cell lines.

0.5

1.0

1.5

2.0

2.5

3.0

2345678

Wild type

35S:

NtENOD40

(Nt1)

35S:

NtENOD40

(Nt2)

days of culture

024

Wild type

35S:

NtENOD40

(Nt1)

35S:

NtENOD40

(Nt2)

[Ethylene] in ppm

ACS

ACO

UBI

ACS

ACO

UBI

ACS

ACO

UBI

Figure 2.3. Temporal Ethylene Accumulation Profile and Transcript Profiles of Genes Required for

Ethylene Biosynthesis in Wild Type and 35S:NtENOD40 BY-2 Cells.

(A) Kinetics of ethylene accumulation in the headspace of protoplast derived cells, cultured in the

presence of 0.1 mg/l NAA and 1.0 mg/l BA. The vertical bar at day 4 indicates the typical time point for

quantifying growth parameters of cultured cells.

(B) RT-PCR analysis on ACC synthase (ACS) and ACC oxidase (ACO) transcript levels of Wild type and

35S:NtENOD40 cells on day 0, 2 and 4. Amplification is shown for 3 consecutive PCR cycles; 16, 18 and

20 cycles for UBI; 28, 30 and 32 cycles for ACS; 22, 24 and 26 cycles for ACO, including a control on

genomic DNA contamination (equivalent amount of RNA, without cDNA synthesis) in the fourth lane of

each block.

ENOD40 Affects Ethylene Homeostasis

We tested whether ethylene production kinetics is affected by overexpression of

ENOD40. As a first step we determined whether ethylene production is altered in

transgenic cell lines Nt1 and Nt2, as compared to wild type lines. To this end,

protoplasts are cultured in the presence of 1.0 mg/l BA and 0.1 mg/l NAA since under

those conditions the strongest ENOD40 phenotype is obtained (See Figure 2.1A, 2.2E-

H). The headspace of protoplast cultures was sampled with 24 hrs intervals during 8

days and ethylene concentrations were determined by GC analysis (Methods). The

experiment was performed five times, with similar results. In the wild type culture

ethylene gradually accumulates during up to 6 days of culture to a maximal level of

about 2 ppm (Figure 2.3A). In cultures of the 35S:NtENOD40 lines, ethylene

accumulates to similar maximal levels as in wild type cultures but transgenic lines

already reached maximal levels at day 3. These results show that ethylene production is

accelerated in 35S:NtENOD40 lines while the final level that accumulates is not severely

affected.

To determine whether early ethylene accumulation causes reduced elongation growth in

35S:NtENOD40 lines, the effect of ethylene perception blockers on elongation growth

was tested for the two transgenic lines, Nt1 and Nt2, and the wild type line. Thus, AgNO3

or 1-MCP were applied during culture of BY-2 protoplasts in medium supplemented with

1.0 mg/l BA and 0.1 mg/l NAA. In three independent experiments, elongation growth

rate, cell division frequency and cell size were scored after four days of culture. The

results from independent experiments were similar and we calculated the average value

for each parameter (Figure 2.4). The results show that elongation growth of wild type

cells is similar in the absence and presence of a range of AgNO3 concentrations (Figure

2.4A). Thus, a block of ethylene perception has no effect on elongation growth in wild

type cells. In the absence of ethylene perception blockers, elongation growth of lines Nt1

and Nt2 is about 30% of wild type levels (Figure 2.4A), which is consistent with previous

experiments (Figure 2.1A, 2.2E). By application of 10 µM AgNO3 at the start of the

protoplast culture, elongation growth increases to about 70% (Nt1) and 75% (Nt2) of

wild type levels. Since elongation growth increases to about 60% of wild type levels by

application of 30 ppb 1-MCP to the headspace of transgenic cell cultures (data not

shown), 1-MCP treatment is similar to, though slightly less effective than, AgNO3

treatment in counter-acting the negative effect of ENOD40 on elongation growth.

Further, at all concentrations of ethylene perception blockers tested, the division

frequency of wild type as well as transgenic cells is similar to the division rate in the

absence of ethylene perception blockers indicating that neither AgNO3 (Figure 2.4B) nor

1-MCP (data not shown) severely affects the division frequency. Since ethylene

perception blockers do not affect the division frequency, the increase in elongation

growth leads to an increase in cell size in transgenic cell cultures (Figure 2.4C). This

shows that a blocker of ethylene perception releases the strict control of cell size in

35S:NtENOD40 lines. Representative photographs of a population of wild type cells

(Figure 2.4D) and Nt1 cells show that the cell size of transgenic cell cultures is strongly

reduced in the absence of AgNO3 (Figure 2.4E). In the presence of 10 µM AgNO3 cell

size of Nt1 cells is similar to wild type (Figure 2.4F). Thus, ethylene perception blockers

specifically counter-act the effect of overexpression of ENOD40. Since application of

blockers of ethylene perception lead to increased elongation growth in 35S:NtENOD40

cells, we show that elongation growth of 35S:NtENOD40 cells is suppressed by

ethylene. So, both the timing of ethylene accumulation as well as the studies with

ethylene perception blockers strongly suggest that accelerated ethylene accumulation in

ENOD40 lines is a primary cause for reduced elongation growth of these cells.

1,0

2,0

-8 -7 -6 -5

Wild type

35S:

NtENOD40

(Nt1)

35S:

NtENOD40

(Nt2)

1,0

2,0

3,0

4,0

-8 -7 -6 -5

Wild type

35S:

NtENOD40

(Nt1)

35S:

NtENOD40

(Nt2)

1,0

2,0

3,0

4,0

-8 -7 -6 -5

Wild type

35S:

NtENOD40

(Nt1)

35S:

NtENOD40

(Nt2)

10 10 10 10

1,2

1,4

1,6

1,8

[AgNO

] in M

[AgNO

] in M

[AgNO

] in M

ELONGATION GROWTH RATE

(Cell-file length in width units)

DIVISION FREQUENCY

(number of cells per file)

CELL SIZE

(length in width units)

Figure 2.4. Recovery of Elongation Growth of 35S:NtENOD40 Cells by AgNO3 Treatment.

Cells are cultured in the presence of 0.1 mg/l NAA, 1.0 mg/l BA and various concentrations of AgNO3.

Pictures are taken with a fluorescence microscope after four days of culture. FDA staining facilitates

selection of viable protoplasts for measurements and aids object recognition with the NIH-image software

for quantification of growth parameters. Data are average (±SD) of three independent experiments.

Left panels:

(A) Elongation growth rate in wild type and 35S:NtENOD40 cell lines.

(B) Cell division frequency in wild type and 35S:NtENOD40 cell lines.

Right panels: Representative photographs of populations of:

(D) Wild type cells.

(E) 35S:NtENOD40 (Nt1) cells.

(F) 35S:NtENOD40 (Nt1) cells cultured in the presence of 10 µM AgNO3.

Ethylene Accumulation Is Regulated by ACS But Not ACO Expression

We wanted to know how ethylene biosynthesis is accelerated in 35S:NtENOD40 lines.

Ethylene biosynthesis is known to be regulated at different levels, including control of

gene expression and post-translational regulation of ACC Synthase (ACS) and ACC

Oxidase (ACO) (Wang et al., 2002). Therefore, we tested whether regulation of

expression of these genes encoding enzymes involved in ethylene biosynthesis is

affected by ENOD40 expression. In order to compare ACS and ACO expression levels

and ethylene accumulation during culture of protoplasts, cells of each line (wild type, Nt1

and Nt2) were harvested at day 0, 2 and 4 for RNA extraction and RT-PCR based ACS

and ACO transcript quantification. In many plant species, both the ACC synthase and

the ACC oxidase family comprise a small number of genes. We designed primers to

highly conserved sequences, such that most likely all ACS mRNAs that are expressed in

BY-2 cells can be amplified in a single RT-PCR reaction. Since regions of high

sequence conservation also occur in the ACC oxidase gene families of different plant

species, the same approach was taken for the ACC oxidase gene family. Although the

sequence in between these primers is slightly variable, the distance between the

primers is very similar in different members. Therefore, the RT-PCR products that are

amplified using these primers are detected as a single band on an RT-PCR blot after

hybridization with the respective probes (Methods).

Figure 2.3A shows a time-course of ethylene accumulation for the wild type culture and

for two 35S:NtENOD40 lines, Nt1 and Nt2. In Figure 2.3B, corresponding ACS and ACO

transcript accumulation profiles are shown. In 35S:NtENOD40 cultures, ethylene

accumulates to maximal levels as early as day 2-3, whereas in wild type cultures

ethylene accumulation does not take place before day 5. The maximal level of ethylene

as well as the time at which the maximum is reached is comparable to the former

experiments. In wild type cultures, ACS transcripts gradually accumulate during the 4-

day culture period, and maximal ACS expression levels are found at day 4. In contrast,

in 35S:NtENOD40 lines the maximal ACS transcript level is found at day 0, directly after

protoplast isolation, and gradually decreases during the culture period (Figure 2.3B).

These results show that in 35S:NtENOD40 lines ACS transcripts accumulate at an

earlier time point and this is consistent with the accelerated ethylene production. In wild

type as well as in 35S:NtENOD40 cultures, ACS transcript accumulation precedes

ethylene production. Further, the maximal level of ACS expression, as well as the

maximal level of ethylene accumulation is similar in wild type and 35S:NtENOD40 lines

but the timing is different.

In wild type protoplast cultures, ACO transcripts are present directly after protoplast

culture has started, and their level only slightly increases during the 4-day culture period.

The ACO transcript accumulation profile in both 35S:NtENOD40 lines is similar to that in

wild type (Figure 2.3B). The temporal regulation of ACO transcript accumulation does

not correlate with the timing of ethylene production in the different lines. Thus, these

data indicate that ACO expression levels are probably not rate-limiting for ethylene

production in these cell lines and regulation of ACO transcript levels does not contribute

to regulation of ethylene production in wild type and 35S:NtENOD40 cultures. Since a

tight correlation between ethylene biosynthesis and ACS, but not ACO, transcript

accumulation is found, regulation of ethylene biosynthesis can be largely attributed to

transcriptional regulation of ACS.

It is known that different members of the ACS family are differentially expressed.

Therefore, we wanted to know whether the expression of a specific ACS member is

altered in 35S:NtENOD40 cells. By using a 3' RACE-PCR based approach, we cloned

partial 3' cDNA sequences of ACS genes that are expressed in elongating BY-2 cells

(Methods). In total six different genes were identified. Based on their 3' UTR sequences,

cDNAs could be pooled in three major groups in which members have more than 92%

sequence similarity between clones within the group. One group includes sequences

highly homologous to NtACS. The second group consists of sequences that are highly

homologous to NtACS2. The third group is only represented by NtACCS2. Since we

were not able to design specific primers for each of the different transcripts separately

(except ACCS2), reverse primers were designed on the 3' UTR for the three ACC

synthase cDNA groups. RT-PCR analysis indicated that the expression level of ACCS2

transcripts is much higher than that of any other ACS gene. Furthermore, the ACCS2

expression profile closely reflects the total ACS transcript profile, suggesting that

ACCS2 transcripts predominantly represent ACC synthase expression. Furthermore, the

increase of ACCS2 expression occurs at an earlier time point in 35S:NtENOD40

cultures than in wild type cultures, indicating that overexpression of NtENOD40 affects

ACCS2 expression kinetics.

Next, we wanted to know whether altered regulation of ACCS2 leads to reduced

elongation growth in BY-2 protoplasts. Stably transformed BY-2 cell lines carrying the

full size ACCS2 cDNA driven by the CaMV 35S promoter were generated. Four

independent lines were tested in the bioassay for elongation growth and cell division

during culture in the presence of 1.0 mg/l BA and 0.1 mg/l NAA. These lines showed a

reduction of elongation growth rate by at most about 50 % of wild type levels, whereas

no severe effects on cell division frequency were observed (data not shown). Hence,

their phenotype closely resembles the phenotype of cell lines that overexpress

ENOD40. Further, this observation is consistent with the proposed hypothesis that

ethylene is a negative regulator of elongation growth in BY-2 cells. Taken together, our

data strongly suggest that altered regulation of elongation growth in 35S:NtENOD40

lines is caused by accelerated expression of ACCS2.

DISCUSSION

In this study we showed that overexpression of ENOD40 reduces BY-2 cell size by

suppressing elongation growth, whereas the cell division frequency is unaffected. These

observations offered the possibility to examine the relation between the ENOD40

induced effect and the effect of cytokinin and auxin on elongation growth and cell

division frequency in BY-2 cells. The protoplast bioassay developed by Hasezawa

(1983) was originally used to determine experimental conditions that are optimal for

uniform semi-synchronous elongation growth in single cell populations. In this report we

have used the morphological response to phytohormones in a bioassay to study

ENOD40 activity. In this bioassay, elongation growth rate and cell division frequency are

morphological growth parameters that can be quantified in dose response curves for

phytohormones, and now form the basis for an experimental system to test the effect of

ENOD40 on phytohormone signaling.

Auxin / cytokinin dose response curves showed that in wild type cells auxin induces cell

division, while cytokinin lowers the number of cell divisions. The opposite effects of

cytokinin and auxin explain why the division rate depends on the balance between these

hormones in wild type BY-2 cells. Our observations are consistent with studies reported

by Hasezawa (1983). When auxin is omitted from the culture medium, cell division

ceases and cell differentiation sets in, associated with morphological changes in the

golgi, an accumulation of starch, and an increase in cell length (Miyazawa et al., 1999;

Winicur et al., 1998). BY-2 cells do not contain sufficient endogenous auxins to sustain

their growth, since they are unable to divide in the absence of exogenous 2,4D (Ishida et

al., 1993) or 1-NAA (Figure 2.2D). BY-2 cells are cytokinin autotroph. Specific cytokinins

were shown to transiently accumulate at G2/M and (although to lower levels) at G1/S

phase in synchronized BY-2 cells, indicating a regulatory role at those specific phases of

cell cycle progression (Laureys et al., 1998; Laureys et al., 1999). It has been proposed

that cytokinins modulate the activity of cell cycle regulatory components (Geelen and

Inze, 2001). It is likely that down-regulation of cytokinin activity at specific phases of the

cell cycle is equally important for progression as is transient accumulation. This could

explain the observation that exogenous cytokinin is inhibiting for cell division activity as

increasing concentrations of exogenous cytokinin may interfere with the control of

endogenous cytokinin levels (Laureys et al., 1999). We found that ENOD40 does not

affect phytohormone control on cell division frequency, since in 35S:NtENOD40 cells the

cell division frequency at various cytokinin / auxin ratios is similar to that in wild type BY-

2 cells.

ENOD40 Accelerates Ethylene Biosynthesis and Ethylene Acts As A Negative

Regulator of Elongation Growth in BY-2 Cells

Three lines of evidence in our study indicate that suppressed elongation growth in

35S:NtENOD40 cells is primarily caused by acceleration of ethylene accumulation.

Firstly, we found a temporal correlation between ethylene accumulation and reduced

elongation growth during culture of wild type cells and transgenic cells that overexpress

ENOD40. Wild type cells begin to elongate 1-2 days after subculture and elongate most

rapidly between day 3 and 7. Elongation then ceases and stops completely after day 9

(Hasezawa and Syono, 1983). In wild type cells, maximal ethylene accumulation is

reached at day 5-6, whereas in 35S:NtENOD40 lines similar maximal ethylene levels

occur but this takes place as early as day 3. These data suggest that the duration (and

therefore degree) of elongation growth can be controlled by the timing of ethylene

accumulation, but without necessarily changing the maximal level of ethylene

accumulation.

Secondly, recovery of elongation growth in 35S:NtENOD40 lines can be achieved by

application of AgNO3 or 1-MCP, both blockers of ethylene action at the ethylene

receptor. In the wild type cultures these treatments do not affect elongation growth.

These data strongly suggest that accelerated ethylene production in 35S:NtENOD40

cells is a primary cause and not a consequence of the reduced elongation growth and

this shows that ethylene is a negative regulator of elongation growth. This is consistent

with the absence of an effect of ethylene perception blockers on elongation growth of

wild type protoplasts within 5 days of culture, since ethylene does not accumulate to

high levels in wild type cultures until day 5-6. The observation that elongation growth of

35S:NtENOD40 cells cannot be fully restored to wild type levels indicates that ethylene

action may not be completely blocked and/or that ethylene accumulation is not the only

signal that regulates elongation growth of these cells.

Thirdly, our data showed that the mRNA accumulation profile of a specific member

(NtACCS2) of the ACC synthase family correlates with the respective ethylene

accumulation curves in wild type and in 35S:NtENOD40 cells. Since ACO mRNA levels

are unaffected in 35S:NtENOD40 lines, it is likely that regulation of ethylene production

can be mainly attributed to transcriptional regulation of NtACCS2. Elongation growth is

reduced in transgenic 35S:NtACCS2 lines, whereas the cell division frequency is not

affected. This phenotype strongly resembles the morphological changes induced by

overexpression of ENOD40 and this confirms that ethylene accumulation negatively

regulates elongation growth in BY-2 cells. Together, these data show that ENOD40

provoked effects on elongation growth involve altered transcriptional regulation of

ACCS2.

Coupling of Processes Implies Coupling of Regulating Pathways

The quantitative analysis of the effects of cytokinin, auxin and ethylene on cell division

and cell elongation growth indicate that at least two phytohormone dependent signaling

pathways exist that together control morphology in BY-2 cells. We found that ethylene

acts as a negative regulator of elongation growth, but does not affect the cell division

frequency under these conditions. In contrast, the cytokinin to auxin ratio controls cell

division frequency, independently of elongation growth in the concentration range

tested. Since in wild type cells these processes can be influenced independently of each

other under changing cytokinin/auxin regimes, this indicates that the processes of

elongation growth and cell division are not strictly coupled in wild type BY-2 cells. In

contrast, elongation growth and cell division are not independent in the presence of

ENOD40 since our assays showed that a strict coupling of the elongation growth rate to

the cell division frequency occurs in 35S:NtENOD40 cells. Our results suggest that

ENOD40 and cytokinin act synergistically in suppressing elongation growth and that the

negative effect of ENOD40 on elongation growth is counter-acted by auxin (Figure 2.2).

Since the cytokinin/auxin dependent pathway primarily regulates cell division frequency

and the ethylene dependent pathway regulates elongation growth, we hypothesize that

coupling of the two processes relies on cross-talk between ethylene and cytokinin/auxin

signaling pathways and that this provides a mechanism to regulate cell size. The

observations that the reduction of elongation growth in ENOD40 cells is, at least in part,

dependent on the cytokinin to auxin ratio and that the reduction of elongation growth is

mediated by accelerated ethylene accumulation in 35S:NtENOD40 lines support this

hypothesis.

Coordinated control of cell division and cell growth determine cell shape and patterning

during plant growth and development. Although little is known about the mechanism that

connects these processes during plant development, our studies revealed that ENOD40

and phytohormones are likely involved in this mechanism. Cytokinin and auxin together

control cell division and cell growth rates in the root meristem (Beemster and Baskin,

2000). Additionally, it has been postulated that ethylene levels act as a local 'fine-tuner'

of cell size by controlling the rate of elongation growth in cells in the elongation zone of

the Arabidopsis root (Le et al., 2001). These BY-2 protoplast assays provide a novel

experimental system to validate the function of candidate components that affect

regulation of cell growth and division.

Our studies suggest that transcriptional regulation of a specific ACS is part of the

molecular mechanism that underlies cross-talk between phytohormone signaling

pathways. Many reports have described that ACC Synthase and/or ACC Oxidase

regulate ethylene production in response to diverse environmental or developmental

stimuli (Johnson and Ecker, 1998). ACS and ACO both are multigene families

comprising a small number of genes. Their expression is known to co-localize with

ethylene production, and mRNA expression levels correlate with ethylene production

levels as the same inducers that stimulate ethylene production induce ACS and/or ACO

expression. Each of the members is induced by a small specific array of inducers

including auxin and cytokinin (Johnson and Ecker, 1998; Van Der Straeten and Van

Montagu, 1991; Wang et al., 2002). For example, it was shown that other hormones do

not induce the auxin inducible Vigna radiata ACS6 promoter, but that they greatly

modified the response to auxin (Yoon et al., 1999). Taken together, these observations

suggested that cross-talk between phytohormone and other signaling pathways takes

place at the level of transcriptional regulation of different ACS (or in some cases ACO)

family members. Our data showing that regulation of one ACS gene is specifically

affected in 35S:NtENOD40 lines is consistent with this model. However, ENOD40 is not

simply a trigger of ACS expression, as indicated by the ACS transcript accumulation

curves. Instead, we propose that altered regulation of ACS expression is part of the

mechanism that leads to phytohormone cross-talk. It will be interesting to find out if and

how ENOD40 can attenuate promoter activity of ACCS2, and which other inducers are

able to regulate transcriptional activity of this specific promoter. Such studies may

provide clues on how ENOD40 is integrated with developmental, hormonal or

environmental cues in order to affect ethylene biosynthesis.

Role of ENOD40 in Nodulation

Our data in BY-2 cells provide experimental evidence that ENOD40 interferes with

cytokinin, auxin and ethylene signaling pathways and suggests that ENOD40 couples

phytohormone dependent processes through an unknown mechanism. Thus, our

studies in a cellular system are in support of the hypothesis that ENOD40 acts in several

developmental processes in order to fine-tune phytohormone signaling (Crespi et al.,

1994; van de Sande et al., 1996). In nodulation, a regulatory role of ENOD40 has been

suggested based on its highly regulated expression pattern (Compaan et al., 2001;

Yang et al., 1993), and on the nodulation phenotypes observed in plants with perturbed

ENOD40 expression (Charon et al., 1999). Based on our BY-2 studies we propose that

during nodulation ENOD40 changes the coupling between cytokinin, auxin and ethylene

signaling pathways. During early stages of nodulation, ENOD40 expression is induced

by Nod factors in pericycle cells opposite the protoxylem poles as early as 3 hrs after

inoculation. Subsequently, expression is found in inner cortical cells during nodule

primordium initiation and development. So, if the function of ENOD40 is to couple

phytohormone dependent processes, what are the processes that have to be coupled in

the cells that express ENOD40? Prior to answering this question, we need to address

what is the function of these phytohormones in the cells that express ENOD40.

The ACO expression pattern suggests that ethylene is predominantly produced opposite

phloem poles, whereas cortical cell divisions are specifically induced opposite

protoxylem poles (Heidstra, Yang et al. 1997). The correlation between the ACO

expression pattern and the location of cortical cell division suggests that ethylene is a

negative regulator of cortical cell division, and that localized ethylene production

provides positional information for the location of the nodule primordium. The causal role

of ethylene distribution in the positioning of cortical cell divisions is supported by

biochemical studies using blockers of ethylene biosynthesis or perception which showed

that when ethylene production or perception were suppressed nodules were also formed

opposite phloem poles (Heidstra, Yang et al. 1997). Genetic evidence is provided by the

ethylene insensitive mutant sickle in which primordia also form opposite phloem poles

(Penmetsa and Cook, 1997).

In both wild type and 35S:NtENOD40 lines, ethylene is produced at a concentration that

does not affect the cell division frequency under the conditions that we used, as shown

by the absence of an effect of AgNO3 application on the cell division frequency, but

instead is involved in the control of elongation growth of cells. The negative effect of

ethylene on root cortical cell division is different, from the role of ethylene in the control

on elongation growth that we observed in BY-2 cells and is likely provoked at higher

concentrations. The ACO expression pattern suggests that ENOD40 expressing cells in

the inner cortical cell layers, opposite protoxylem poles have low ethylene production.

Based on our observations in BY-2 cells, we propose that in these cells, ethylene could

be involved in the regulation of elongation growth.

It was shown that exogenous application of cytokinin could induce nodule formation

(Cooper and Long, 1994). Upon inoculation with Rhizobium, or Nod factor treatment,

auxin transport is inhibited and auxin accumulates transiently in the inner cortical cells

that are located opposite protoxylem poles as shown using auxin reporter lines.

Treatment of roots with auxin transport blockers can induce cell division and pseudo-

nodule formation in the inner cortex (Hirsch et al., 1989). Taken together, these findings

suggested that it is unlikely that either the cytokinin or the auxin level alone determines

the conditions for nodule initiation, but that the ratio of cytokinin to auxin levels are

important for nodule initiation. So, a combination of positively acting factors like uridine

(Smit et al., 1995) and the auxin to cytokinin ratio and negatively acting signals like

ethylene, control induction of cortical cell divisions. Each of these factors has a specific

distribution in the root and this leads to induction of cortical cell divisions at the right

place. It seems plausible that coordinated control of cell division and cell growth is

important during formation of the nodule primordium and that phytohormones play an

important role in these stages of the developmental program. In line with the observation

that ENOD40 couples the phytohormone dependent relative rates of elongation growth

and cell division in BY-2 cells, we propose that ENOD40 couples phytohormone

signaling pathways in order to closely coordinate growth in the dividing cells of the

nodule primordium. ENOD40 expression by itself is not sufficient to induce nodule

primordium formation indicating that ENOD40 is not a key-regulator of the participating

processes. However, ENOD40 function may be required to mediate cross-talk between

phytohormone signaling pathways during development.

METHODS

Construction of Binary Vectors p35S:NtENOD40 and p35S:ACCS2

Nicotiana tabacum contains two ENOD40 homologs that are 96% identical at the nucleotide

level (Matvienko et al., 1996). The Cauliflower Mosaic Virus 35S promoter was used for ectopic

expression of NtENOD40-1. To this end, the 35S promoter from pMON999 (Monsanto) was

transferred to pCambia 1390 (Cambia, Australia) yielding p35S:Tnos. A 470 bp PCR fragment

corresponding to the NtENOD40-1 cDNA sequence was then cloned in p35S:Tnos using primers

5’-GCTCTAGACTAGCTTGTCTCAAGAAC-3’ and

5’-CGGGATCCATGACAATCTTAACAACTCTAT-3’.

The full size ACCS2 cDNA sequence (1568 bp) was cloned from cDNA that was prepared from

total RNA obtained from elongating BY-2 cells and was transferred to p35S:Tnos using primers

5’- GCTCTAGAGGCACGAGGAGAAGATG-3’, and

5’- CGGGATCCGTGGTTAAGACTTGATTATTC-3’.

The resulting constructs were introduced in Agrobacterium tumefaciens strain C58C1.

Liquid BY-2 Cultures and BY-2 Transformation

Nicotiana tabacum BY-2 cell suspensions were sub-cultured weekly by 40 times dilution in fresh

medium (Nagata et al., 1981). BY-2 transformation was performed using a modification of the

procedure reported by Gu and Verma (1997). Five ml of a 3 day-old BY-2 cell suspension was

co-cultivated for 2 days at 25ºC in the dark with 60 µl of log-phase Agrobacterium, harboring the

binary vectors. Cells were then washed three times with fresh medium containing 200 µg/l

ticarciline/clavuline and plated on agar solidified selection plates containing the normal culture

medium supplemented with 0.8 % Daishin Agar, 200 µg/l ticarciline/clavuline and 40 µg/l

Hygromycin B. Individual transgenic calli that appeared after 3-4 weeks, were first cultured on

fresh selection plates for one more week, and were subsequently transferred to liquid selection

medium to initiate suspension cultures from independent transgenic calli. Transgenic lines were

continuously maintained in selection medium.

Protoplast Isolation

Protoplasts were obtained from 6 day-old suspension cultures using 1% Cellulase-YC and 0.1%

Pectolyase Y23 in 0.4 M D-mannitol, pH 5.5 (Nagata et al., 1981). Cells were incubated in the

enzyme solution for 3 hours at RT, filtered through 63 µm nylon mesh, washed two times with

0.4 Osm KCl, purified over a one-step (18% w/v) sucrose gradient and subsequently washed

three times with Protoplast Culture Medium (PCM) containing 4.3 g/l MS salts (without vitamins)

supplemented with 1 mg/l Thiamine-HCl, 100 mg/l Myo-inositol, 10 g/l sucrose, 255 mg/l KH2PO4

and 0.4 M D-mannitol at pH 5.7. Elongation growth inducing PCM contained 0.1 mg/l 1-

Naphthalene-acetic acid (1-NAA) and 1.0 mg/l Benzyl-adenine (BA). For the dose response

curves, protoplasts were cultured in PCM supplemented with various concentration of NAA

and/or BA as indicated in the text and/or graphs. Protoplasts were cultured in 3 ml liquid medium

at a density of approximately 105/ml in small sealed petridishes at 25ºC in the dark (Kuss and

Cyr, 1992).

Protoplast Assay Growth Parameter Measurements

Cell size measurements were performed on random photographs of the protoplast derived cells

after four days of culture. Viable protoplasts were selected for measurements after FDA

(fluorescein-diacetate) staining. Fluorescent images were captured using a cooled CCD camera

mounted on a Leica DMR microscope with a 20x objective. The digital fluorescent images

facilitated computer based morphometric measurements using the NIH-IMAGE program

(http:/rsb.info.nih.gov/nih-image) in which objects can be contoured by applying the

invert/threshold option. Cell-file length, cell width, and number of cells/file were scored for 100-

150 cells per sample. The mean cell-file length as well as the mean division frequency was then

calculated for each protoplast sample. Subsequently, the average elongation growth rate and

cell division frequency for each line was determined as the average over values from

independent repetitions at different dates. Also, the standard deviation between measurements

at different dates is determined for each line. Significance is tested using a two-tailed students T

test.

RNA Gel Blot Analysis

Total RNA was isolated using the TRIzol method (GibcoBRL). 16 µg of total RNA was subjected

to electrophoresis on a 1% agarose gel in 0.01 M NaH2PO4 (pH 7.0) using the glyoxal/DMSO

method. RNA was subsequently transferred to a genescreen membrane in 20xSSC. RNA gel

blots were hybridized with radiolabeled PCR fragments of the respective transcripts in

formamide hybridization buffer O/N at 42ºC. Autoradiograms were obtained using a Molecular

Dynamics Phosphorimager (Sunnyvale, CA).

Reverse Transcriptase-Mediated PCR

Total RNA was isolated using the TRIzol method (GibcoBRL). After DNaseI (Promega)

treatment to remove chromosomal DNA that could disturb the PCR reactions, cDNA is

synthesized from 2.5 µg of total RNA in a volume of 20 µl (10 mM Tris-HCl pH 8.8, 50 mM KCl, 5

mM MgCl2, 1 mM dNTPs, 1 µg oligodT(12)V anchor primer, 20 U RNA guard (Pharmacia) and

200 U MuMLV reverse transcriptase (Stratagene). The samples were incubated for 1 hr at 37ºC.

To inactivate the reverse transcriptase enzyme the samples were incubated at 95ºC for 5

minutes. The RT samples were then diluted to 100 µl and 1 or 2 µl of the cDNA were used for

PCR analysis (10 mM Tris-HCl pH 8.3, 50 mM KCl, 2,5 mM MgCl2, 100 µM dNTPs, 50 ng primer

and 1 U Taq polymerase (Boehringer Mannheim, USA) in a total volume of 50 µl.

RACE-PCR on ACS transcripts was as follows: cDNA is synthesized from RNA isolated from the

wild type BY-2 cell suspension, using the RACE-T anchor primer 5'-

CATCTAGAGGATCGAATTC-T(16)-3'. The first PCR cycles were: 94°C for 5 min; 3 cycles of

94°C for 1 min, 54°C for 1 min, 72°C for 1 min then 28 cycles of 94°C for 1 min, 50°C for 1 min

and 72°C for 1 min and a final extension at 72°C for 5 min, run with primers: RACE-A: 5'-

CATCTAGAGGATCGAATTC-3' and 5'- GTTGTTCTTTTCATTGTTC-3'. The second PCR is run

with primers RACE-A and ACS-3'race: 5'-GGTTGGTTTAGAGTTTGTTT-3', 94°C for 5 min; 30

cycles of 94°C for 1 min, 50°C for 1 min, 72°C for 1 min and then a final extension at 72°C for 5

min. After the second PCR, the total RACE-PCR product mixture was purified using a PCR

purification kit (Boehringer) and was cloned in pGEM-T (Promega). From a total set of 28 cloned

cDNAs, 18 representative clones were sequenced in both directions.

RACE-PCR on NtENOD40 transcripts of RNA isolated from the transgenic lines was identical to

RACE-PCR on ACS, except primers and PCR cycles. First run; RACE-A primer, reverse primer:

5'-CGGGATCCTAGTTGGAGTGAATTAAGGA-3', second run; RACE-A primer, reverse primer:

5'-AAGCTTTTGGAGTCTTTCTTGGCCTTT-3'. Both PCR cycles were as follows: 94°C for 5

min; 30 cycles of 94°C for 20 sec, 50°C for 20 sec, 72°C for 30 sec and a final extension at 72°C

for 5 min.

Specific primers are designed for reverse transcriptase mediated (RT) PCR based transcript

quantification for each of the analyzed genes. Specificities of the primer sets were verified by

sequencing the RT-PCR products. The number of PCR cycles was adapted to the linear range

of the PCR amplification reaction for each gene, corresponding to the relative expression levels.

All samples were normalized on ubiquitine levels. Primers that were used for RT-PCR are: UBI-f:

5'-ATGCAGAT(C/T)TTTGTGAAGAC-3'; UBI-r: 5'-ACCACCACG(G/A)AGACGGAG-3', General

ACS primers: ACS-f: 5'-GATTTAATACAAGAATGGG-3' / ACS-r: 5'-

GAACAATGAAAAGAACAAC-3'; NtACOf: 5'-GGGCTTCTTTGAGTTGGTG-3' / NtACOr: 5'-

CTCCGCTGCCTCTTTCTC-3'. Primer combinations for specific ACS members: ACS-3'race /

ACCS2-r : 5'-AAAGAAAAAGAAACATTACAAG-3'; ACS-3'race / ACS2-r: 5'-

TCCCATTTTGATACACTTTAC-3'; ACS-3'race / ACS1-r: 5'-TTCTTTTCCTTTATCTTCTTC-3'.

Amplified DNA fragments were run on a 1% agarose gel, alkaline blotted to Hybond-N+

membrane (Amersham Pharmacia) and hybridized to radiolabelled PCR fragments of the

corresponding cDNA clones. Autoradiograms were obtained by using a Molecular Dynamics

Phosphorimager (Sunnyvale, CA).

Ethylene Measurements

For each line, protoplasts were divided over six petridishes at the start of the experiment and

were cultured in parallel. Each petridish was sampled every 24 hrs during 8 days in 5

independent experiments. A gas sample of 1 ml from a total of 30 ml headspace volume was

used for analysis on a gas chromatograph. In order not to severely alter accumulating ethylene

levels, gas samples were taken with a syringe through a rubber gasket in the lid of the petridish

without opening the sealed petridishes. Ethylene concentration was determined by standard GC-

analysis on a gas chromatograph equipped with an alumina column and a flame ionisation

detector (Gilissen and Hoekstra, 1984). Ethylene accumulation at each time point was

determined as the average ethylene concentration in the headspace of these six cultures.

1 mg of 1-MCP was freshly dissolved in 10 ml of water in a sealed 600 ml bottle with a rubber

gasket to give a stock concentration of 1000 ppb. At the start of the culture period, 1 ml volumes

of serial dilutions of 1-MCP were transferred to the headspace of sealed petridishes containing

the protoplasts by injection through a rubber gasket in the lid of the petridish, to give the

appropriate final concentration of 1-MCP in the headspace. AgNO3 was freshly dissolved in

water to a stock concentration of 1 mM. To give the appropriate final concentrations of AgNO3,

30 µl of a serial dilution was transferred to the culture medium containing the protoplasts just

before sealing the petridishes at the start of the culture period.

Accession Numbers

The accession numbers are NtENOD40-1 (X98716); NtACS1 (X65982); NtACS2 (AJ005002)

and NtACCS2 (X98492).

ACKNOWLEDGMENTS

We thank Danny Geelen (University of Gent, Belgium) for kindly supplying the BY-2 cell

suspension. We gratefully acknowledge Gerard van der Krogt (Dutch Cancer Institute)

for his valuable contribution to the BY-2 transformations and the excellent technical

assistance of Ciska Braam and Maelle Lorvellec. We are thankful to Mark Hink and Jan-

Willem Borst (Wageningen University Microscopy Center) for use of the microscope

facilities. T.R. is supported by the Netherlands Organization for Scientific Research

(NWO 805.49.004).

REFERENCES

Asad, S., Fang, Y., Wycoff, K.L., and Hirsch, A.M. (1994). Isolation and characterization of

cDNA and genomic clones of MsENOD40; Transcripts are detected in meristematic cells of

alfalfa. Protoplasma 183, 10-23.

Bauer, P., Rated, P., Crespi, M.D., Schultze, M., and Kondorosi, A. (1996). Nod factors and

cytokinins induce similar cortical cell division, amyloplast deposition and MsENOD12A

expression patterns in alfalfa roots. Plant J. 10, 91-105.

Beemster, G.T., and Baskin, T.I. (2000). Stunted plant 1 mediates effects of cytokinin, but not

of auxin, on cell division and expansion in the root of Arabidopsis. Plant Physiol. 124, 1718-

1727.

Charon, C., Johansson, C., Kondorosi, E., Kondorosi, A., and Crespi, M. (1997). ENOD40

induces dedifferentiation and division of root cortical cells in legumes. Proc. Natl. Acad. Sci.

USA 94, 8901-8906.

Charon, C., Sousa, C., Crespi, M., and Kondorosi, A. (1999). Alteration of ENOD40

expression modifies Medicago truncatula root nodule development induced by SinoRhizobium

meliloti. Plant Cell 11, 1953-1965.

Chen, J.-G., Shimomura, S., Sitbon, F., Sandberg, G., and Jones, A.M. (2001). The role of

auxin-binding protein 1 in the expansion of tobacco leaf cells. Plant J. 28, 607-617.

Collings, D.A., Asada, T., Allen, N.S., and Shibaoka, H. (1998). Plasma membrane-

associated actin in Bright Yellow-2 tobacco cells. Evidence for interaction with microtubules.

Plant Physiol. 118, 917-928.

Compaan, B., Yang, W.C., Bisseling, T., and Franssen, H. (2001). ENOD40 expression in the

pericycle precedes cortical cell division in Rhizobium-legume interaction and the highly

conserved internal region of the gene does not encode a peptide. Plant and Soil 230, 1-8.

Cooper, J.B., and Long, S.R. (1994). Morphogenetic Rescue of Rhizobium meliloti Nodulation

Mutants by trans-Zeatin Secretion. Plant Cell 6, 215-225.

Crespi, M.D., Jurkevitch, E., Poiret, M., d'Aubenton-Carafa, Y., Petrovics, G., Kondorosi,

E., and Kondorosi, A. (1994). ENOD40, a gene expressed during nodule organogenesis,

codes for a non- translatable RNA involved in plant growth. EMBO J. 13, 5099-5112.

Ecker, J.R. (1995). The ethylene signal transduction pathway in plants. Science 268, 667-675.

Geelen, D.N.V., and Inze, D. (2001). A Bright Future for the Bright Yellow-2 Cell Culture. Plant

Physiol. 127, 1375-1379.

Gilissen, L.J.W., and Hoekstra, F.A. (1984). Pollination-induced corolla wilting in Petunia

hybrida: rapid transfer through the style of a wilting-inducing substance. Plant Physiol. 75,

496-498.

Gu, X., and Verma, D.P.S. (1997). Dynamics of phragmoplastin in living cells during cell plate

formation and uncoupling of cell elongation from the plane of cell division. Plant Cell 9, 157-69

Hasezawa, S., Hogetsu, T., and Syono, K. (1988). Rearrangement of cortical microtubules in

elongating cells derived from tobacco protoplasts - A time-coarse observation by

immunofluorescence microscopy. Plant Physiol. 133, 46-51.

Hasezawa, S., and Syono, K. (1983). Hormonal Control of Elongation of Tobacco Cells Derived

from Protoplasts. Plant Cell Physiol. 24 (1), 127-132.

Heidstra, R., Yang, W.C., Yalcin, Y., Peck, S., Emons, A.M., van Kammen, A., and

Bisseling, T. (1997). Ethylene provides positional information on cortical cell division but is

not involved in Nod factor-induced root hair tip growth in Rhizobium-legume interaction.

Development 124, 1781-1787.

Herbert, R.J., Vilhar, B., Evett, C., Orchard, C.B., Rogers, H.J., Davies, M.S., and Francis,

D. (2001). Ethylene induces cell death at particular stages of the cell cycle in the tobacco

TBY-2 cell line. J. Exp. Bot. 52, 1615-1623.

Hirsch, A.M., Bhuvaneswari, T.V., Torrey, j.G., and Bisseling, T. (1989). Early nodulin genes

are induced in alfalfa root ougrowths elicited by auxin transport inhibitors. Proc. Natl. Acad.

Sci. USA 86, 1244-1249.

Hua, J., Chang, C., Sun, Q., and Meyerowitz, E.M. (1995). Ethylene insensitivity conferred by

Arabidopsis ERS gene. Science 269, 1712-1714.

Ishida, S., Takahashi, Y., and Nagata, T. (1993). Isolation of cDNA of an auxin-regulated gene

encoding a G protein beta subunit-like protein from tobacco BY-2 cells. Proc. Natl. Acad. Sci.

USA 90, 11152-11156.

Iwata, K. (1995). Effects of reduction of auxin and destruction of microtubules on cell wall

proteins and cell morphology of the BY-2 line of tobacco cells. J. Plant Res. 108, 469-476.

Johnson, P.R., and Ecker, J.R. (1998). The ethylene gas signal transduction pathway: a

molecular perspective. Annu. Rev. Genet. 32, 227-254.

Kato, N., and Esaka, M. (2000). Expansion of transgenic tobacco protoplasts expressing

pumpkin ascorbate oxidase is more rapid than that of wild-type protoplasts. Planta 210, 1018-

1022.

Kieber, J.J., Rothenberg, M., Roman, G., Feldmann, K.A., and Ecker, J.R. (1993). CTR1, a

negative regulator of the ethylene response pathway in Arabidopsis, encodes a member of the

raf family of protein kinases. Cell 72, 427-441.

Kouchi, H., and Hata, S. (1993). Isolation and characterization of novel nodulin cDNAs

representing genes expressed at early stages of soybean nodule development. Mol. Gen.

Genet. 238, 106-119.

Kuss, W.C.L., and Cyr, R.J. (1992). Tobacco protoplasts differentiate into elongate cells without

new microtubule depolymerization. Protoplasma 168, 64-72.

Laureys, F., Dewitte, W., Witters, E., Van Montagu, M., Inze, D., and Van Onckelen, H.

(1998). Zeatin is indispensable for the G2-M transition in tobacco BY-2 cells. FEBS Lett. 426,

29-32.

Laureys, F., Smets, R., Lenjou, M., Van Bockstaele, D., Inze, D., and Van Onckelen, H.

(1999). A low content in zeatin type cytokinins is not restrictive for the occurrence of G1/S

transition in tobacco BY-2 cells. FEBS Lett. 460, 123-128.

Le, J., Vandenbussche, F., Van Der Straeten, D., and Verbelen, J. (2001). In the early

response of Arabidopsis roots to ethylene, cell elongation is up- and down-regulated and

uncoupled from differentiation. Plant Physiol. 125, 519-22.

Lee, K.H., and LaRue, T.A. (1992). Exogenous ethylene inhibits nodulation of Pisum sativum L.

cv Sparkle. Plant Physiol. 100, 1759-1763.

Link, B.M., and Cosgrove, D.J. (1998). Acid-growth response and alpha-expansins in

suspension cultures of Bright Yellow-2 tobacco. Plant Physiol. 118, 907-916.

Mathesius, U., Charon, C., Rolfe, B.G., Kondorosi, A., and Crespi, M. (2000). Temporal and

spatial order of events during the induction of cortical cell divisions in white clover by

Rhizobium leguminosarum bv. trifolii inoculation or localized cytokinin addition. MPMI 13, 617-

628.

Mattsson, J., Ckurshumova, W., and Berleth, T. (2003). Auxin signaling in Arabidopsis Leaf

Vascular Development. Plant Physiol. 131, 1327-1339.

Matvienko, M., van de Sande, K., Pawlowski, K., van Kammen, A., Franssen, H., and

Bisseling, T. (1996). Nicotiana tabacum SR1 contains two ENOD40 homologs. In: Biology Of

Plant Microbe Interactions, G. Stacey, B. Mullin, and P. M. Gresshoff, eds. Int. Soc. MPMI. St.

Paul USA, 387-391.

Minami, E., Kouchi, H., Cohn, J.R., Ogawa, T., and Stacey, G. (1996). Expression of the early

nodulin, ENOD40, in soybean roots in response to various lipo-chitin signal molecules. Plant

J. 10, 23-32.

Miyazawa, Y., Sakai, A., Miyagishima, S.-y., Takano, H., Kawano, S., and Kuroiwa, T.

(1999). Auxin and Cytokinin Have Opposite Effects on Amyloplast Development and the

Expression of Starch Synthesis Genes in Cultured Bright Yellow-2 Tobacco Cells. Plant

Physiol. 121, 461-469.

Mylona, P., Pawlowski, K., and Bisseling, T. (1995). Symbiotic Nitrogen Fixation. Plant Cell 7,

869-885.

Nagata, T., Nemoto, Y., and Hasezawa, S. (1992). Tobacco BY-2 cell line as the He-la cell in

the cell biology of higher plants. Int. Rev. Cytol. 132, 1-30.

Nagata, T., Okada, K., Tabeke, I., and Matsui, C. (1981). Delivery of tobacco mosaic virus

RNA into plant protoplasts mediated by reverse-phase evaporation vesicles (liposomes). Mol.

Gen. Genet. 184, 161-165.

Penmetsa, R.V., and Cook, R.V. (1997). A legume ethylene-insensitive mutant hyperinfected

by its Rhizobial symbiont. Science 275, 527-530.

Peters, N.K., and Crist-Estes, D.K. (1989). Nodule formation is stimulated by the ethylene

inhibitor aminoethoxyvinylglycine. Plant Physiol. 91, 690-693.

Redig, P., Shaul, O., Inze, D., van Montagu, M., and van Onckelen, H. (1996). Levels of

endogenous cytokinins, indole-3-acetic acid and abscisic acid during the cell cycle of

synchronized tobacco BY-2 cells. FEBS Lett. 391, 175-180.

Ridge, I., and Osborne, D.J. (1971). Role of peroxidase when hydroxyproline-rich protein in

plant cell walls is increased by ethylene. Nat. New. Biol. 229, 205-208.

Rodrigues-Pousada, R.A., De Rycke, R., Dedonder, A., Van Caeneghem, W., Engler, G.,

Van Montagu, M., and Van Der Straeten, D. (1993). The Arabidopsis 1-Aminocyclopropane-

1-Carboxylate Synthase Gene 1 Is Expressed during Early Development. Plant Cell 5, 897-

911.

Tamura, K., Liu, H., and Takahashi, H. (1999). Auxin induction of cell cycle regulated activity of

tobacco telomerase. J. Biol. Chem. 274, 20997-21002.

van de Sande, K., Pawlowski, K., Czaja, I., Wieneke, U., Schell, J., Schmidt, J., Walden, R.,

Matvienko, M., Wellink, J., van Kammen, A., Franssen, H., and Bisseling, T. (1996).

Modification of phytohormone response by a peptide encoded by ENOD40 of legumes and a

nonlegume. Science 273, 370-373.

Van Der Straeten, D., and Van Montagu, M. (1991). The molecular basis of ethylene

biosynthesis, mode of action, and effects in higher plants. Subcell. Biochem. 17, 279-326.

Shibaoka, H. (1994). Plant hormone-induced changes in the orientation of corical microtubules:

Alterations in the cross-linking between microtubules and the plasma membrane. Annu. Rev.

Plant Physiol. Plant Mol. Biol. 45, 527-544.

Smit, G., Koster, C.C., Schripsema, J., Spaink, H.P., van Brussel, A.A., Kijne, J.W. (1995).

Uridine, a cell division factor in pea roots. Plant Mol. Biol. 29, 869-873.

Wang, K.L., Li, H., and Ecker, J.R. (2002). Ethylene biosynthesis and signaling networks. Plant

Cell 14, 131-151.

Winicur, Z.M., Zhang, G.F., and Staehelin, L.A. (1998). Auxin Deprivation Induces

Synchronous Golgi Differentiation in Suspension-Cultured Tobacco BY-2 Cells. Plant Physiol.

117, 501-513.

Yang, W.C., Katinakis, P., Hendriks, P., Smolders, A., de Vries, F., Spee, J., van Kammen,

A., Bisseling, T., and Franssen, H. (1993). Characterization of GmENOD40, a gene showing

novel patterns of cell- specific expression during soybean nodule development. Plant J. 3,

573-585.

Yoon, I.S., Park, D.H., Mori, H., Imaseki, H., and Kang, B.G. (1999). Characterization of an

auxin-inducible 1-aminocyclopropane-1- carboxylate synthase gene, VR-ACS6, of mungbean

(Vigna radiata (L.) Wilczek) and hormonal interactions on the promoter activity in transgenic

tobacco. Plant Cell. Physiol. 40, 431-438.

Chapter 3

The Two Conserved Regions of ENOD40 Transcripts Have

Biological Activity and Are Connected In A Complex Manner

INTRODUCTION

A remarkable feature of ENOD40 genes is the absence of a long open reading frame,

but instead several small ORFs are in general present in ENOD40 transcripts. In chapter

1 we described that two regions are highly conserved in all ENOD40 transcripts, which

argues for biological importance of these regions. From the short ORFs that are present

in different ENOD40 transcripts, only the ORF within the first conserved region (ORF I)

is conserved and encodes an oligopeptide of 10-13 amino-acids. In region II, an ORF

can be found that contains the MANRQVTKRQ peptide motive and we refer to this ORF

as ORF II. However, in only about 50% of the ENOD40 transcripts this ORF is present.

Further, it is striking that this region is more conserved at nucleotide level than at amino-

acid level (chapter 1). Despite that in some ENOD40 sequences an ORF II exists, it

seems more likely that region II is active at the RNA level. As a consequence, it is still

under debate which ENOD40 gene product - protein or RNA - has biological activity. If

ENOD40 activity is of proteinaceous nature, the peptide encoded by the conserved ORF

I is the best candidate. So, is this small ORF translated in vivo?

For several ENOD40 transcripts it has been shown that ORF I is translated in in vitro

translation assays (Rohrig et al., 2002) and also in vivo by making use of reporter

fusions (Charon et al., 1997; Compaan et al., 2001). Van de Sande (1996) detected an

antigenic determinant in extracts of 15-day old soybean nodules using an antibody

raised against a synthetic GmENOD40 peptide which suggests that the putative peptide

encoded by ORF I indeed is produced in planta. Additionally, some other ORFs in

Medicago truncatula and soybean ENOD40 transcripts could be translated. Because the

peptides that are derived from these ORFs are not conserved, it is unlikely that these

are required for biological activity of ENOD40 genes. Thus, how many ORFs in ENOD40

transcripts are translated in planta, and whether they have activity in vivo remains to be

elucidated. It has been suggested that the ORF I derived oligopeptides are highly

instable in plant tissues since degradation of corresponding synthetic peptides in plant

extracts occurs in less than 1 minute (Sousa et al., 2001). Thus, using synthetic

peptides may not allow an analysis of their activity and a bioassay system to test activity

of ENOD40 constructs would provide a useful alternative.

A bioassay system has already been described to study which ENOD40 gene product

has activity (Charon et al., 1997; Sousa et al., 2001). This system is based on ballistic

targeting of ENOD40 (or -derivative) constructs in root epidermal and outermost cortical

cells of Medicago roots. Transient expression of ENOD40 leads to induction of inner

cortical cell division. Since cell divisions are induced at a spatially separated site from

the cells that are targeted with the construct, the effect of ENOD40 is provoked in a non-

cell autonomous manner, suggesting that the ENOD40 product is transported or that a

secondary signal is required (Charon et al., 1997). Mutating the translation start or stop

of ORF I in the complete transcript reduced activity, whereas replacing the nucleotide

sequence of this ORF with an altered nucleotide sequence in a construct retaining the

same amino-acids did not affect activity of the transcript. This confirms that the

translation product rather than the primary nucleotide sequence is important for

biological activity. Likewise, a point mutation of the start codon of ORF II of MtENOD40

abolishing its translation reduced the activity of MtENOD40, suggesting that translation

products of both regions are biologically active. This is striking, since this ORF in region

II is not absolutely conserved among plant species (chapter 1).

These experiments further showed that transient expression of truncated constructs

encompassing either region I with the small ORF I or a 3’ sequence carrying region II

evoked a response similar to that evoked by the complete transcript in Medicago sativa

roots. Mutating the start codon of ORF I in a partial transcript spanning only region I,

abolished the cell division inducing activity. This suggests that translation of this short

ORF is required for activity in the absence of the downstream region. Similarly, mutating

the start codon of ORF II in a construct spanning only the 3’ region reduced activity of

this transcript. Taken together, these data suggest that in the absence of the other

region, either of the two regions is sufficient for biological activity of ENOD40 and that

the encoded peptides of ORF I and II are important for biological function. However,

mutation of the start codon of ORF I in MtENOD40 resulted in significantly reduced

activity in the complete transcript, although the activity of the 3’ region was expected to

be retained in this derivative. Thus, interaction between the two regions may be complex

since this indicates that the activity of a complete ENOD40 transcript is primarily caused

by the peptide residing in region I and that in the presence of an ineffective ORF I,

region II is no longer able to induce an effect, although region II by itself it is functional.

We have developed a novel bioassay based on the effect of overexpression of ENOD40

in BY-2 cells (chapter 2). The effect of ENOD40 on cell elongation growth and cell

division has been tested in dose response curves in which different cell division

frequencies can be induced by changing the hormone concentrations in the medium.

These analyses showed that the strict coupling between these two processes in

35S:NtENOD40 lines was primarily provoked by the negative effect of ENOD40 on cell

elongation growth. To study the effect of ENOD40-derived transcripts on this strict

coupling would require the performance of these dose response curves for several

individual lines for different constructs. As this would be too laborious and the effect of

ENOD40 is primarily caused by reduction of elongation growth, we decided to test the

effect of ENOD40-derived transcripts at a specific concentration of cytokinin and auxin,

which induces strong elongation growth. Since the negative effect of ENOD40 on BY-2

cell elongation growth is enhanced by cytokinin and can be counter-acted by auxin, the

most pronounced effect of ENOD40 on elongation growth is obtained under culture

conditions with a high cytokinin to auxin ratio. Under these conditions, overexpression of

ENOD40 also alters the timing of ethylene biosynthesis. Activities of ENOD40 gene

products could be scored by analyses of either of these effects. However, the temporal

ethylene production curve has a broad maximum. Therefore, characterization of the

ethylene accumulation profile is not very suitable for quantification, whereas quantifying

elongation growth provides the least laborious bioassay to study ENOD40 activity.

By preparing protoplasts from the BY-2 cell suspension, a population of single cells with

a similar size is obtained. Since the vast majority of the cells remain attached to each

other after division, the average number of cells per cell file reflects the number of cell

divisions that took place during the incubation time, and this parameter is from hereon

called the ‘cell division frequency’. On the other hand, the width of cells remains similar

to the diameter of protoplasts during culture. This means that no radial expansion

growth occurs and the length (expressed in width units) of the cell files is a parameter

for elongation growth of cells during the incubation period. This parameter is from

hereon called the ‘elongation growth rate’. To determine whether the peptide encoded

by ORF I and/or the nucleotide sequence of region II, or the peptide encoded by ORF II

have biological activity, we constructed a series of NtENOD40-derived transcripts. In

order to assay biological activity of these transcripts, we studied their effects in the BY-2

protoplast bioassay.

RESULTS

Six independent transgenic BY-2 cell lines carrying construct 35S:NtENOD40 (Figure

3.1) were generated by Agrobacterium-mediated transformation and were named Nt1 to

Nt6. Expression of the transgenes was detected by RNA gel blot analysis performed

with total RNA isolated from these transgenic lines. In the wild type line, NtENOD40

mRNA could not be detected, indicating a very low expression level of the endogenous

NtENOD40 gene. This analysis further showed that NtENOD40 is overexpressed in all

35S:NtENOD40 transgenic lines, except in line Nt6. In all transgenic lines HPTII

transcripts can be detected (Figure 3.2A).

The protoplast bioassay data for these lines were obtained within the same set of

experiments as for the lines described in this chapter, but have also been described in

chapter 2. In short, in four out of six lines elongation growth was reduced by about 40-

60% in comparison to that of the wild type, whereas the cell division frequency was

unaffected. Elongation growth is most strongly reduced in cell lines containing a high

expression level of 35S:NtENOD40, whereas elongation growth is not affected in lines

containing low or undetectable levels of expression. So, a good correlation between

35S:NtENOD40 expression levels and reduction of elongation growth was found. To

exclude that introduction of vector sequences could provoke a similar response, the

control vector 35S:Tnos (Figure 3.1) was introduced in BY-2 cells. Three independent

lines (named EV1, EV2 and EV3) were obtained. RNA gel blot analysis showed that

35S:HPTII was expressed to a high level in these transgenic lines (Figure 3.2B). A

reduction of elongation growth of 15-25% in comparison to the wild type is observed in

these lines. Although the negative effect on elongation growth of one of the lines (EV3)

is statistically significant (p<0.05), the effect is not very strong. The division frequency of

these lines is similar to that of the wild type (Table 3.1). Thus, neither elongation growth

nor cell division frequency was severely affected in transgenic lines carrying a control

vector.

ORF I Is Translated But the Encoded Oligopeptide Is Not Essential for a Reduction

of Elongation Growth

Two approaches were taken in order to test whether ORF I is functional and necessary

for the reduction of elongation growth. We first determined whether ORF I is translated

upon expression in stable transgenic BY-2 cells by using an NtENOD40-GFP reporter

fusion. To this end, the GFP coding sequence without its own ATG was cloned in frame

in the middle of ORF I, replacing the last six amino-acids. A schematic representation is

given in Figure 3.1. Figure 3.3 shows the GFP fluorescence that was detected in a

stable transgenic BY-2 cell line carrying the 35S:NtENOD40-GFP construct showing that

ORF I is indeed translated. These data confirmed the results obtained by Compaan

(2001).

Secondly, we tested whether the peptide has biological activity. We approached this by

testing whether translation of ORF I is required for inducing a response in the protoplast

assay. To this end, the ATG start codon of ORF I of NtENOD40 was exchanged by

AAG. Five independent lines carrying construct 35S:NtENOD40-AAG (Figure 3.1) were

obtained. These lines were named 2, 6, 7, 8, and 49. RNA gel blot analysis showed that

the lines express 35S:NtENOD40-AAG, but at different levels, and that HPTII transcripts

were detected in all transgenic lines (Figure 3.2C). Cells from these lines were tested for

their ability to elongate and divide in the protoplast bioassay. The results showed that in

these lines elongation growth is reduced to various degrees. In the lines with the

strongest phenotype (2 and 6), elongation growth was reduced by about 50% of the wild

type elongation growth. The elongation growth in 35S:NtENOD40 lines is reduced by

40-60%. Thus, the degree of the elongation growth reduction induced by this transcript

is comparable to that induced by the intact transcript. Thus, these results show that

translation of ORF I is not required for the reduction of elongation growth. The average

cell division frequency in wild type cells is 1.15. This means that on average about 15%

of the cells in the population has undergone one round of cell division during the culture

period. Strikingly, in some of the lines (2 and 6) only about 5-6 ± 4% of the cells divided

during the culture time in comparison to 15 ± 9% in the wild type line, suggesting that

cell division frequency is reduced in some of the 35S:NtENOD40-AAG lines whereas

cell division frequency was not reduced in 35S:NtENOD40 lines. This suggests that

when the function of one region is tested by mutation in a construct containing both

regions the observed reduction of cell division frequency is a gain of function since this

effects was not observed using the intact construct. Since an additional effect is

observed when translation of ORF I is disrupted, this indicates that the ORF I encoded

peptide does have a biological function. These observations also indicate that the

activity of the two regions are interdependent and that regulation of the activities of

these two regions is complex.

ATG

AAG

box 2

ATG

AAG

ATG

KQWDEAIHGS

box 2

ATG

box1

20 40 60 80 100 120 140 160 180 200 220 240 260 280 300 320 340 360 380 400 420 440 460

20 40 60 80 100 120 140 160 180 200 220 240 260 280 300 320 340 360 380 400 420 440 460 480 500 520 540 560 580 600 620 640 660

20 40 60 80 100 120 140 160 180 200 220 240 260 280 300 320 340 360 380 400 420 440 460 480 500 520 540 560 580 600 620 640 660 680 700 720

20 40 60 80 100 120 140 160 180 200 220 240 260 280 300 320 340 360 380 400 420 440 460 480 500 520 540 560 580 600 620 640 660

MQWDEAIHGS.

MKQSMGLRKREAISWSELRTFKCPSSKYNFKFVHTLPISLLEFQKMQQKRKVFMD.

MGLRKREAISWSELRTFKCPSSKYNFKFVHTLPISLLEFQKMQQKRKVFMD.

MQQKRKVFMD.

MPFLQIQFQVCSYFANFSARIPENAAKKESVYGLIITLIAKDVGKSMANRQVTKRQWTPFWSLSWPFTYCCTLYCYIQSSIIVFYL.

MANRQVTKRQWTPFWSLSWPFTYCCTLYCYIQSSIIVFYL.

MLAKAWRTGKSRNGNGLRFGVFLGLLLIVVLYIVISSLL.

MDSVLESFLAFYLLLYFILLYPVFYNCVLFVSHMQCVVNMAYQKFIIELLRLS.

MAYQKFIIELLRLS.

MQCVVNMAYQKFIIELLRLS.

MKLLCWEKSIHGS.

MVLKTNMERSVRGY.

MKLLCWQKSIHGS.

MAMYQSLYLCSTDTLD.

MYQSLYLCSTDTLD.

MSRVCVCAS.

MANRQVTKRQWIPFWSLNGYVSITLSM.

MAMYQSLYLCSTDT.

MYQSLYLCSTDT.

MPVFVLVDCYSYFLAVECNNKHKDGVVFL.

MVLSSFEKLPTL.

MMYFDSLDLQLTRVCSCFSFCR.

MMYFNSLNLQLTRVCSCFSFCR.

MYFDSLDLQLTRVCSCFSFCR.

MYFNSLNLQLTRVCSCFSFCR.

MSKVGNCYH.

MSKVGHCYH. MFLFLCCSPFPHECLLVCLC.

MFACLFVLRVIMIL.

MNVCLFVCVKSYYDLMRS.

MELCWLTTIHGS.

MVLEEAWRERGVRGEGAHSSHSLT. MDSIGVSMAM.

MLVCVVSYDLMRK.

MSSVFPFPCLFVLLVMTL.

GmENOD40-1

NtENOD40-1

MtENOD40-1

MsENOD40

35S:Tnos

35S:

NtENOD40

ATG

35S:

NtENOD40-GFP

35S:

NtENOD40

AAG

35S:

NtENOD40

ATG, RRII

35S:

NtENOD40

AAG, RRII

35S:

GmENOD40

e35S promoter

HindIII

HindIII BamHI

BamHI

Xba

BamHIXba

BamHIXba SstI

BamHI

Xba

box1

box 2

MQWDEAIHGS

KQWDEAIHGS

MELCWLTTIHGS

MQWD-GFP GFP

MQWDEAIHGS

Tnos

Figure 3.1.

(A) Comparison of ORFs in MtENOD40-1, MsENOD40, NtENOD40-1 and GmENOD40-1 transcripts

shows the absence of a conserved ORF in region II of the GmENOD40 -1 transcript.

(B) Constructs used for overexpression of ENOD40 (-derived) transcripts in BY-2 cells.

Wt123456

***

1.0

2.0

1.6

1.2

1.8

1.4

Wt784962

1.0

2.0

1.6

1.2

1.8

1.4

Wt 1 13 15 18 20

1.0

2.0

1.6

1.2

1.8

1.4

Wt4 12212228 Wt1 1114

1.0

2.0

1.6

1.2

1.8

1.4

12 3

***

GmENOD4

NtENOD40

HPTII

UBI

Nt ENOD40

HPTII

UBI

line

NtENOD40

HPTII

UBI

DIVISION FREQUENCY

(number of cells per file)

DIVISION FREQUENCY

(number of cells per file)

DIVISION FREQUENCY

(number of cells per file) DIVISION FREQUENCY

(number of cells per file)

ELONGATION GROWTH

(length in width units)

ELONGATION GROWTH

(length in width units)

ELONGATION GROWTH

(length in width units)

ELONGATION GROWTH

(length in width units)

A 35S:

NtENOD40

(ATG) B 35S:Tnos

C 35S:

NtENOD40

(AAG) D 35S:

GmENOD40

E 35S:

NtENOD40

(ATG, RRII) F 35S:

NtENOD40

(AAG, RRII)

Figure 3.2. Elongation Growth Rate (white bars) and Cell Division Frequency (black bars) of Cultured

Cells of Wild Type (Wt) and Transgenic BY-2 Lines.

Figure 3.2. Elongation Growth Rate (white bars) and Cell Division Frequency (black bars) of Cultured

Cells of Wild Type (Wt) and Transgenic BY-2 Lines.

(A) Six independent transgenic cell lines carrying 35S:NtENOD40 (ATG region I).

(B) Three independent transgenic cell lines carrying the 35S:Tnos control vector.

(D) Five independent transgenic cell lines carrying 35S:GmENOD40.

(E) Five independent transgenic cell lines carrying 35S:NtENOD40 (ATG region I, RRII).

(F) Three independent transgenic cell lines carrying 35S:NtENOD40 (AAG region I, RRII).

Data are average ± SD of at least three independent experiments. * Indicates significance in reduction of

elongation growth rate or division frequency compared to wild type ( * = p<0.05, ** = p<0.01, *** =

p<0.001). The level of transgene expression (NtENOD40, GmENOD40 and HPTII) is determined at the

start of protoplast culture by RNA gel blot analysis. All lines express the HPTII transcript, which confers

resistance against hygromycin. Ubiquitine (UBI) hybridization is included to compare loading of separate

samples on the RNA gel blot. The RNA gel blot analysis was repeated three times in independent

experiments with similar results, one representative set of data is shown.

Figure 3.3. GFP Fluorescence of a 35S:NtENOD40-GFP Transgenic Line.

Confocal image showing fluorescence of a GFP reporter translationally fused to the ORF of region I

indicating that this ORF is actively translated upon expression in stably transformed BY-2 cell

suspensions.

Construct Line Elongation Growth Rate(Egr) % Increase File-length Cell Division Frequency n

Wild type Wt 3.93 ± 0.52 100 % 1.15 ± 0.09 11

p35S:Tnos Ev1 3.49 ± 0.22 84 % 1.08 ± 0.05 3

Ev2 3.36 ± 0.35 80 % 1.06 ± 0.05 3

Ev3 3.18 ± 0.26 75 % 1.15 ± 0.12 3

p35S:NtENOD40 Nt1 2.27 ± 0.27 43 % 1.17 ± 0.10 8

ATG region I Nt2 2.31 ± 0.25 44 % 1.20 ± 0.07 9

Nt3 3.09 ± 0.25 70 % 1.17 ± 0.09 5

Nt4 2.48 ± 0.65 50 % 1.14 ± 0.09 5

Nt5 3.69 ± 0.57 91 % 1.08 ± 0.08 5

Nt6 3.24 ± 0.37 75 % 1.08 ± 0.03 5

p35S:GmENOD40 Gm1 2.70 ± 0.27 58 % 1.06 ± 0.06 4

Gm13 3.13 ± 0.70 72 % 1.22 ± 0.12 4

Gm15 2.20 ± 0.18 40 % 1.04 ± 0.03 4

Gm18 2.42 ± 0.63 48 % 1.17 ± 0.13 4

Gm20 1.98 ± 0.26 33 % 1.04 ± 0.03 4

p35S:NtENOD40 2 2.58 ± 0.85 53 % 1.05 ± 0.06 4

AAG region I 6 2.51 ± 0.22 51 % 1.06 ± 0.03 4

7 3.27 ± 0.41 77 % 1.12 ± 0.09 4

8 2.86 ± 0.72 64 % 1.11 ± 0.05 3

49 3.00 ± 0.76 66 % 1.13 ± 0.07 3

Table 3.1. Elongation Growth Rate, % Increase File-length and Cell Division Frequency of wild type and

transgenic lines carrying constructs for overexpression of ENOD40–derived transcripts. Data are means ±

SD of (n) number of repetitions in independent experiments. % Increase File-length from start of

protoplast culture is calculated as (Egr(line) –1)/(Egr(Wt) –1) x 100%.

Next, we decided to test whether the ORF I encoded peptide has biological activity, but

in the absence of the region II sequence. In order not to affect the length of the

transcripts, we did not delete the region II sequence from the transcripts but instead,

reversed the downstream region of the transcript (Reversed Region II). Like in the

previous experiment, we also substituted the ATG codon by AAG in a second construct,

thus disrupting the translation start of ORF I. For each construct several independent

transgenic lines were generated, and these are named 35S:NtENOD40 ATG, RRII (lines

4, 12, 21, 22 and 28) and 35S:NtENOD40 AAG, RRII (lines 1, 11 and 14). The

expression level of NtENOD40 RRII and HPTII transcripts was determined by RNA gel

blot analysis (Figure 3.2E-F). In none of the transgenic lines NtENOD40 RRII transcripts

could be detected whereas HPTII transcripts were detected in all transgenic lines. Since

we did not obtain any lines in which the NtENOD40 RRII transcripts were present at a

detectable level, we were not able to study the effect of these constructs.

Overexpression of GmENOD40-1 Reduces Elongation Growth of BY-2 cells

The activity of an NtENOD40 transcript with a disrupted translation start in ORF I

suggests that translation of ORF I is not required to reduce elongation growth and

therefore that the downstream region II contains activity. The NtENOD40 transcript

contains an ORF II and so we can not exclude that the encoded peptide has biological

activity. To test this, we used a heterologous transcript that contains the highly

conserved region II but a corresponding ORF II is not present in this transcript. For this

purpose, we chose the GmENOD40-1 transcript. Five independent lines carrying

construct 35S:GmENOD40-1 (Figure 3.1) were obtained and were named Gm1, Gm13,

Gm15, Gm18 and Gm20. The levels of GmENOD40 and HPTII transcripts were

analyzed by RNA gel blot analysis (Figure 3.2D). This showed that these lines express

35S:GmENOD40 but at different levels and HPTII is expressed at a high level in all

transgenic lines. The effects of overexpression of GmENOD40 on elongation growth

rate and cell division frequency were tested for each individual line (Figure 3.2D, Table

3.1). Lines Gm1, Gm15 and Gm20 contained the highest level of 35S:GmENOD40

expression, and each line had a strong reduction (35-60%) of elongation growth as

compared to that of the wild type. These lines also showed a lower cell division

frequency as compared to that of the wild type (around 4-6 ± 4% of the cells divided

compared to 15 ± 9% in the wild type). Thus, elongation growth was reduced to a similar

degree as that observed in the 35S:NtENOD40 and 35S:NtENOD40-AAG lines, showing

that this heterologous transcript has comparable activity in the protoplast elongation

growth response. In addition, in the lines in which GmENOD40 transcripts are

expressed at a high level (Gm1, Gm15 and Gm20), cell division frequency is reduced in

comparison to the wild type line. This difference is statistically significant (p< 0.05, p<

0.01, p< 0.01 respectively) and is similar to that observed in some of the

35S:NtENOD40-AAG lines (Table 3.1).

DISCUSSION

We studied which ENOD40 gene product is biologically active, in order to reveal a

molecular mechanism of ENOD40 action. Here, we have used BY-2 cells

overexpressing ENOD40 derived constructs to study whether the peptide encoded by

ORF I is biologically active. We demonstrated that ORF I is translated in vivo by using a

GFP reporter gene fusion, and this supports the notion that this peptide can be required

for biological activity of ENOD40. Subsequently, we determined whether translation of

ORF I is required for biological activity of the ENOD40 transcript by disrupting the

translation start of ORF I. Strikingly, transgenic lines overexpressing the NtENOD40

transcript with a disrupted translation start in ORF I show a reduction of elongation

growth comparable to that in cell lines overexpressing the intact NtENOD40 transcript.

This indicates that the ORF I peptide is not required for the reduction of elongation

growth and suggests that this activity may reside in the nucleotide sequence of region I

or in region II. Our data are in disagreement with some of the results reported by Sousa

(2001). In their assay, activity of a MtENOD40 as well as a MsENOD40 transcript was

lost upon disruption of the translation start of ORF I, indicating that region II is not active

in the presence of a disrupted ORF I and that the ORF I peptide is required for biological

activity. Our results show that in NtENOD40, which contains ORF I and II, region II still

contains biological activity when translation of ORF I is disrupted.

Strikingly, cell division frequency was reduced in some of the 35S:NtENOD40-AAG

lines, whereas this effect was not induced by the intact NtENOD40 transcript, indicating

that the peptide encoded by ORF I does have some biological activity. To clarify this

effect, we have attempted to test biological activity of the ORF I encoded peptide in the

absence of the region II sequence by reversing the downstream region. Unfortunately,

we could not detect expression of the NtENOD40-RRII transcript by RNA gel blot

analysis in any of the lines carrying these constructs. It is unlikely that this is due to the

integration-site in the genome (so-called position-effect), since this generally affects

flanking genes in a similar way and we were able to detect substantial levels of HPTII

transcripts in all these lines. Since high expression of both transgenes (HPTII and

ENOD40) is found in the majority of the other lines carrying 35S:NtENOD40 or

35S:GmENOD40 constructs, we assume that the absence of detectable levels of

NtENOD40 RRII transcripts is the result of either post-transcriptional silencing or

severely reduced transcript stability in these lines.

The reduction of elongation growth in 35S:GmENOD40 lines is comparable to that

observed in 35S:NtENOD40 lines, indicating that reduction of elongation growth can

also be induced by overexpression of a heterologous ENOD40 transcript. Both

sequences contain the two conserved regions of ENOD40 genes but have a low overall

homology (~ 25%). Since these transcripts have a similar effect their activity likely

resides in shared sequences. Therefore, these results indicate that the biologically

active sequence is contained in either region I or region II, or a combination of both and

suggests that the function of ENOD40 genes is conserved among plant species. The

results using the NtENOD40-AAG transcript suggest that translation of ORF I is

probably not required for reduction of elongation growth and that this activity resides in

region II. Thus, since region II, but not ORF II, is present in the GmENOD40 transcript, it

is unlikely that the peptide encoded by ORF II has this biological activity and it would be

more likely that region II is active at the RNA level. Strikingly, 35S:GmENOD40 lines

most closely resemble 35S:NtENOD40-AAG lines, since both cell division and cell

elongation growth are reduced in some lines expressing these constructs, whereas only

elongation growth is affected by the intact NtENOD40 construct.

The data using the 35S:NtENOD40-AAG construct showed that, when the function of

one region is tested by mutagenesis in a construct containing both regions, this induced

additional effects in comparison to the effect of the intact construct, indicating that the

activity of the two regions is interdependent. Sousa (2002) tested the function of the two

regions separately using truncated constructs carrying only one of the two regions.

These data showed that the biological activity of either of the two regions is similar to the

complete construct, which suggested that each region has biological activity on its own.

In a second set of experiments, mutating the ATG of ORF I, or mutating the ATG of ORF

II in the complete construct, reduced the biological activity of these constructs. This

shows that in both cases the effect of either region depends on the function of the other

region, even though deletions spanning either region I or region II are active. Thus, our

data as well as Sousa’s data lead to a similar conclusion; testing the function of one

region by mutagenesis in constructs containing both regions showed that the two

regions influence each other, inferring that the functions of the two regions are somehow

coupled.

There are two main reasons why it will be difficult to use these bioassays to further study

the interaction between the two regions. The data using the 35S:NtENOD40-AAG

construct indicate that the activity of the two regions is interdependent and may be

regulated by a complex mechanism. The semi-quantitative nature of these bioassays is

probably not sufficient to study the complexity of the combined effects of the two

regions.

METHODS

Construction of Binary Vectors

Control Vector. The Cauliflower Mosaic Virus p35S promoter was used for ectopic expression

of the different ENOD40 sequences. To this end, the CaMV p35S promoter from pMON999

(Monsanto) was HindIII:BamHI transferred to pCambia1390 (Cambia, Australia) yielding

p35S:Tnos. All cDNA sequences that are used for expression in these studies were cloned

between the XbaI and BamHI restriction sites in the new MCS. The p35S:Tnos vector carries the

hygromycin selection marker under control of a second CaMV 35S promoter in reverse

orientation to the cloned inserts. The p35S:Tnos construct is used as the control vector for

transformation.

p35S:NtENOD40. The endogenous full size NtENOD40-1 cDNA sequence (Genbank accession

number X98716, 470 bp) was cloned in p35S:Tnos after adding XbaI and BamHI linkers to the

NtENOD40 sequence by means of a PCR reaction using primers NtENOD40-1: 5'-

GCTCTAGACTAGCTTGTCTCAAGAAC-3' and NtENOD40-8: 5'-

CGGGATCCATGACAATCTTAACAACTCTAT-3'.

p35S:NtENOD40-GFP. The NtENOD40:ORFI:GFP fusion construct was obtained from

Compaan (2001) and was XbaI:BamHI transferred to p35S:Tnos.

p35S:NtENOD40-AAG. The ATG codon that marks the start of ORF I in NtENOD40 was

mutagenized using a PCR based cloning method. To do so, we first amplified an NtENOD40

DNA fragment by PCR using primers NtENOD40-1, and NtENOD40-AAG: 5'-

CATGCCATGGATTGCTTCATCCCACTGCTTTTT-3' which has a single base mismatch to

introduce an A at the T70 position. This fragment contains bases 1-90 and is digested with XbaI

and NcoI. It was fused in a three-point ligation to an NcoI:BamHI PCR fragment containing

bases 91-470 of NtENOD40-1 and the XbaI:BamHI digested pBSK cloning vector. The construct

was XbaI:BamHI transferred to p35S:Tnos after sequencing.

p35S:GmENOD40-1. A Glycine max ENOD40-1 cDNA clone (Genbank accession number

X69154.1, 656 bp) was available as pKP1 in pMEX. Using primers GmENOD40-1 f: 5'-

GCTCTAGACTAAACCAATCTATCAAGTCC-3' and GmENOD40-1 r: 5'-

CGGGATCCAAAGGACTCTGGAAACTTTTC-3', we introduced XbaI:BamHI linkers by PCR

amplification of the fragment, which was first cloned in pBSK and then transferred to p35S:Tnos.

p35S:NtENOD40 ATG, RR II, p35S:NtENOD0 AAG, RR II. Constructs containing a region II

sequence in the reverse orientation were generated using the NtENOD40-ATG as well as the

NtENOD40-AAG constructs as template. We generated fragments containing the first 117 bases

with either an ATG or AAG codon marking the start of ORF I, with primers NtENOD40-1 and

nt1asregionIISstI: 5'-GGGGCGAGCTCATTGCCTCTCTTTTCCTAA-3'. Both fragments were

fused to the second fragment containing bases 117-470, encompassing the conserved region II

and further 3' sequence of NtENOD40, which were generated with primers

nt2asregionIIBamHI:5'-CGGGATCCTAGTTGGAGTGAATTAAGGA-3' and nt3asregionIISstI: 5'-

GGGGCGAGCTCATGACAATCTTAACAACTCTAT-3'. The orientation of the second fragment is

thereby reversed. The construct was XbaI:BamHI transferred to p35S:Tnos after sequencing. All

binary vectors were sequenced in both directions before Agrobacterium tumefaciens

transformation.

BY-2 Cell Suspension Culture, Protoplast Isolation and BY-2 Transformation

Liquid BY-2 culture and BY-2 transformation were as described in chapter 2. Transgenic lines

were obtained from individual calli, which means that they can not be siblings. Transgenicity of

each individual line was determined by PCR analysis of genomic DNA and by RNA gel blot

analyses. The lines are continuously maintained in selection medium. Protoplast isolation and

growth parameter measurements in the Protoplast Bioassay were performed as described

previously (Chapter 2). Significance was tested using a two-tailed students T-test.

RNA Gel Blot Analysis

Total RNA was isolated using the TRIzol method (GibcoBRL) according to the manufacturers

protocol. 16 µg of total RNA was subjected to electrophoresis on a 1% agarose gel in 0.01 M

NaH2PO4 (pH 7.0) using the glyoxal/DMSO method (Sambrook et al, 1989). RNA was

subsequently transferred to a genescreen (Dupont) membrane in 20xSSC. RNA gel blots were

hybridized with radiolabeled PCR fragments of the respective transcripts in formamide

hybridization buffer O/N at 42ºC. Autoradiograms were obtained using a Molecular Dynamics

Storm 840 Phosphorimager.

Confocal Microscopy

Confocal microscopy was performed on a Zeiss LSM 510 confocal microscope using standard

GFP filter settings.

REFERENCES

Charon, C., Johansson, C., Kondorosi, E., Kondorosi, A., and Crespi, M. (1997). ENOD40

induces dedifferentiation and division of root cortical cells in legumes. Proc. Natl. Acad. Sci.

USA 94, 8901-8906.

Compaan, B., Yang, W.C., Bisseling, T., and Franssen, H. (2001). ENOD40 expression in the

pericycle precedes cortical cell division in Rhizobium-legume interaction and the highly

conserved internal region of the gene does not encode a peptide. Plant and Soil 230, 1-8.

Fütterer, J., and Hohn, T. (1996). Translation in plants - rules and exceptions. Plant Mol. Biol.

32, 159-189.

Lindsey, K., Casson, S., and Chilley, P. (2002). Peptides: new signalling molecules in plants.

TRENDS in Plant Sci. 7, 78-83.

Minami, E., Kouchi, H., Cohn, J.R., Ogawa, T., and Stacey, G. (1996). Expression of the early

nodulin, ENOD40, in soybean roots in response to various lipo-chitin signal molecules. Plant

J. 10, 23-32.

Rohrig, H., Schmidt, J., Miklashevichs, E., Schell, J., and John, M. (2002). Soybean

ENOD40 encodes two peptides that bind to sucrose synthase. Proc. Natl. Acad. Sci. USA 99,

1915-1920.

Sambrook, J., Fritsch, E. F., Maniatis, T. (1989). Molecular Cloning a Laboratory Manual. Cold

Spring Harbor Laboratory Press, USA.

Sousa, C., Johansson, C., Charon, C., Manyani, H., Sautter, C., Kondorosi, A., and Crespi,

M. (2001). Translational and structural requirements of the early nodulin gene ENOD40 , a

short-open reading frame-containing RNA, for elicitation of a cell-specific growth response in

the alfalfa root cortex. Mol. Cell. Biol. 21, 354-366.

van de Sande, K., Pawlowski, K., Czaja, I., Wieneke, U., Schell, J., Schmidt, J., Walden, R.,

Matvienko, M., Wellink, J., van Kammen, A., Franssen, H., and Bisseling, T. (1996).

Modification of phytohormone response by a peptide encoded by ENOD40 of legumes and a

nonlegume. Science 273, 370-373.

Chapter 4

Arabidopsis thaliana:

Useful for Functional Analysis of ENOD40?

Tom Ruttink, Maelle Lorvellec, Ton Bisseling, Henk Franssen.

Submitted to: Proceedings of Molecular Plant Microbe Interactions, 2003

ABSTRACT

In search of a molecular mechanism of ENOD40 action, we aimed to identify interactors

of ENOD40 through a genetic approach. In order to facilitate a genetic screen, we

searched for a phenotype in whole plants and chose Arabidopsis thaliana as a model

system. In this report, we describe the generation and analysis of Arabidopsis thaliana

transgenic lines that overexpress the tobacco homolog of ENOD40. Several

independent transgenic lines were generated and were analyzed for growth aberrations.

Interaction of ENOD40 with cytokinin and ethylene signaling pathways has previously

been suggested and we also tested the response to exogenous application of ACC and

BA in our transgenic lines.

INTRODUCTION

To unravel the molecular mechanism underlying the function and activity of ENOD40, it

is required to identify either direct interactors, or factors acting up- or down-stream of

ENOD40. Studies in BY-2 cell suspensions (chapter 2) support the notion that the

function of ENOD40 depends on interaction with phytohormone signaling. Yet, it is

difficult in this system to resolve the mechanism through which this interaction is

established. As cell suspensions allow molecular biological, biochemical or

pharmacological approaches, these can be used to test known candidate components of

a signaling pathway, but this system does not provide the possibility to screen

genetically for unknown interactors. Therefore, we search for phenotypes that are

induced by overexpression of ENOD40 in whole plants since this provides the possibility

to develop a genetic screen through which unknown interactors of ENOD40 can be

identified in an un-biased manner. The effect of overexpression of ENOD40 has been

reported in Medicago sativa, Medicago truncatula and in tobacco. In the latter

overexpression of ENOD40 leads to reduced apical dominance (van de Sande et al.,

1996). In Medicago sativa no transgenic plants could be generated (Crespi et al., 1994)

and in Medicago truncatula a subtle effect on the timing of nodule formation is observed

(Charon et al., 1999). Although these data show that overexpression of ENOD40 leads

to impaired plant development, we decided not to use these plants for further studies,

because tobacco is not suitable for genetic screens, and the phenotypes observed in the

other two plant species are not suitable for suppressor screens.

The promoter of soybean ENOD40 is active at similar sites in Arabidopsis and legumes

(Mirabella et al., 1999). Moreover, the promoter is responsive to AVG and cytokinin in

Arabidopsis similar to legumes, indicating that factors involved in the regulation of

expression of ENOD40 are conserved among legumes and Arabidopsis (Vleghels,

2003). Therefore, we chose to constitutively express ENOD40 in Arabidopsis as it

represents a plant model species with the established advantages of the fully available

genomic sequence information. Due to its relatively small genome size and short

generation time, genetic screens are relatively easy to perform in Arabidopsis as

compared to other plant species. In addition, many Arabidopsis mutants (among these,

mutants in hormonal production or signaling) are available that may become important in

further studies on ENOD40. Relatively weak phenotypic changes or for example cell-

type specific changes which require many steps of practical handling to visualize may

well be very informative on the function of ENOD40 in whole plants, but can not be used

in a large screen. Therefore, these were not included in this study. Instead, we use

assays that test the response to the phytohormones ethylene and cytokinin to analyze

whether these responses are affected by ENOD40 in Arabidopsis, since we found that in

tobacco cell suspensions ENOD40 affects elongation growth in response to cytokinin

and that alteration of ethylene biosynthesis is involved (chapter 2).

RESULTS

Arabidopsis ENOD40 Homologs Can Not be Identified

Preferentially, we would like to overexpress an Arabidopsis homolog of ENOD40 in

Arabidopsis. As an Arabidopsis ENOD40 homolog has not been described, we first

searched the Genbank sequence database for the presence of ENOD40 homologous

sequences. Sequence comparison between ENOD40 genes, reveals two regions of high

sequence similarity named region I and region II (Compaan et al., 2001; Flemetakis et

al., 2000; Kouchi et al., 1999). The M-X1-4-W-X4-HGS* peptide motive present in the

open reading frame of region I and the AACCGGCAAGTCA-(X6)-GGCAAT nucleotide

sequence motive in region II are characteristic to ENOD40 sequences known to date.

We examined the Genbank sequence databases for the presence of homologous

sequences through blastn alignment searches (Figure 4.1). Several sequences with

homology to region II of ENOD40 can be identified in the Arabidopsis genome.

However, these do not span the entire conserved sequence of region II. In addition, the

small conserved ORFs that are present upstream of region II in all known ENOD40

genes can not be recognized in Arabidopsis. Thus, in the Arabidopsis genome both

motives do not occur within a relatively short (<1kb) sequence range and in the correct

orientation. These results can not simply be explained by a partial gene deletion or

rearrangement. Therefore, we conclude that the Arabidopsis genome does not contain

an ENOD40 homolog. In Citrus species, which are part of the Sapindales family,

ENOD40 homologous sequences can be found that contain both regions, indicating that

the ancestor of the Brassicales family to which Arabidopsis belongs, still contained an

ENOD40 homologous sequence. These results do not exclude that a component able to

recognize the secondary structure of ENOD40 gene products is present in Arabidopsis.

Studies in tobacco BY-2 cells showed that an ENOD40 sequence from a leguminous

species and tobacco, respectively, contain similar biological activity, indicating that

ENOD40 cDNA sequences are active in a heterologous plant background (chapter 3).

Thus, we chose to use the NtENOD40 cDNA sequence for constitutive overexpression

in Arabidopsis.

Figure 4.1. Short stretches of ENOD40 Sequence Homology in Arabidopsis thaliana genome database.

BLASTn searches with the highly conserved nucleotide sequence of region II of ENOD40 reveal the

presence of sequences with homology to this region in the Arabidopsis thaliana genome. However, these

sequences do not span the entire conserved sequence of region II and short ORFs with similarity to

region I of ENOD40 genes can not be identified upstream of these sequences. This indicates that an

ENOD40 homolog is not present in the Arabidopsis thaliana genome. Genbank accession numbers and

corresponding BAC clones are indicated.

Generation of Transgenic Lines

Since the effect of overexpression of ENOD40 is not yet known in Arabidopsis, we aim

to analyze plant morphology in different developmental stages in all organs that can

later be useful in a genetic screen. For this purpose it is important that ENOD40 is

expressed in all tissues under study. Thus, we chose the CaMV 35S promoter to

constitutively drive the expression of the tobacco full-size NtENOD40-1 cDNA sequence.

As a first step, we generated transgenic plants by Agrobacterium-mediated

transformation and out of 32 primary transformants three independent homozygous

transgenic lines were selected and were named line 1, 2 and 3 (Figure 4.2). Genomic

DNA gel blot analysis showed that line 1 probably contains 2 copies; lines 2 and 3 one

copy. Segregation analyses of T1, T2 and T3 generations (3:1) showed that for all three

lines, the transgenes segregate as single insertion loci. From the selected lines RNA

was isolated and inspected for the expression of the transgene by RNA gel blot

analyses (Figure 4.2). All lines showed a high level of expression of the introduced

gene.

NtENOD40

1 2 3

NtENOD40

1 2 3

NtENOD4

HPTII

UBI

Figure 4.2. Genomic DNA and RNA Gel Blot Analysis of Wild type (Ws) and Three Independent

Transgenic Lines Carrying the 35S:NtENOD40 Construct.

Genomic DNA was HindIII:BamHI digested, electroforesed and blotted. The probe hybridizes to the

HindIII:BamHI 35S:NtENOD40 fragment in all lines, and both one upstream and one downstream

fragment for each unique insertion site. Line 1 probably contains two copies; line 2 and 3 one copy.

Segregation analysis of T1, T2 and T3 generations (3:1), revealed that for all three lines, transgenes

segregate as single insertion loci. RNA gel blot analysis reveals high levels of expression for the

NtENOD40 transgene and the HPTII transgene conferring resistance against hygromycin. Hybridization

with the Ubiquitin (UBI) probe confirms equal loading of RNA on the gel.

Overexpression of NtENOD40 Does Not Lead to Growth Aberrations

To find out whether overexpression of NtENOD40 interferes with Arabidopsis growth, we

inspected plants of the T3 and T4 generation for abnormalities in growth. To this end T3

and T4 plants from homozygous lines were germinated on agar-solidified 1/2MS medium

containing 1% sucrose. Ten-day old plantlets were transferred to soil and grown in the

greenhouse. Plants were screened for germination time, rosette size; number, size and

shape of leaves, flowering time, number and length of inflorescence shoots or number of

seeds. No parameters could be identified that were affected in our 35S:NtENOD40 lines.

Phytohormone Responses Are Not Affected in 35S:NtENOD40 Lines

As ethylene and cytokinin responses are affected by overexpression of ENOD40 in BY-2

cells (chapter 2), we set out to test whether ENOD40 affects the response of

Arabidopsis to the phytohormones cytokinin and ethylene. To this end we tested the

effect of exogenous application of phytohormones on growth and development in

Arabidopsis in a dose response curve for each hormone. Wild type (Ws) and

35S:NtENOD40 plants were grown in the dark in the presence of a range of ACC

concentrations and plants were photographed after 5 days (Figure 4.3). Ethylene or

ACC (a direct precursor of ethylene) treatment induces the triple response in dark-grown

seedlings (Guzman and Ecker, 1990). Hypocotyl length of wild type and 35S:NtENOD40

plants were measured and the length was plotted as a function of the ACC

concentration (Figure 4.4). In the absence of ACC hypocotyl length is similar in wild type

and 35S:NtENOD40 plants of all three independent transgenic lines, indicating that

overexpression of ENOD40 does not affect hypocotyl growth. Further, hypocotyl length

of 35S:NtENOD40 lines and wild type reduced similarly in response to ACC treatment.

Comparison of the other hallmarks of the triple response by eye, including reduction of

root growth and hypocotyl length, thickening of the hypocotyl, and enhanced apical hook

curvature, indicates that wild-type plants and transgenic plants respond similarly to ACC.

These data indicate that these responses to ethylene are not affected by overexpression

of NtENOD40 in Arabidopsis.

A linear relation exists between the ethylene concentration and the length of the root

epidermal cell in which a new root hair just emerges, the length of the root elongation

zone and the length of the primary root (Le et al., 2001). Further, ethylene regulates root

hair development, by determining the number and position of root hairs (Tanimoto et al.,

1995). To find out whether overexpression of NtENOD40 affects these processes, the

length of the elongation zone, defined by the distance between the root tip and the first

bulging root hair, is analyzed in 5-d old plants grown in the light. Neither the length of the

elongation zone, nor the number, distance between nor length of the root hairs was

affected in our transgenic lines (data not shown). Since ethylene signaling controls these

processes, these data indicate that ethylene signaling is not severely affected by

overexpression of ENOD40 under these conditions. Taken together with the normal

triple response to ACC, we have no indication that responses to ethylene are affected in

these 35S:NtENOD40 lines.

There are two ways to test the effect of exogenous application of cytokinin; BA treatment

while plants are growing in the light or in the dark respectively. BA treatment in the light

results in reduced root growth, while hypocotyl length is not affected (Cary et al., 1995).

BA treatment in the dark induces a response similar to the triple response in

Arabidopsis. This response can be counter-acted by application of ethylene receptor

blockers, showing that this response is (at least partially) ethylene dependent. Both

responses were tested in the three different 35S:NtENOD40 lines. To this end, wild type

and transgenic plants were grown on MS plates supplemented with different

concentrations of BA. After 5 days of growth plants were subjected to visual inspection.

Photographs of representative plants show that plants overexpressing NtENOD40

respond similarly to the added BA as wild type plants, both in light (Figure 4.5) and dark

(Figure 4.7) grown plants. Hypocotyl length of dark grown wild type plants and plants

overexpressing ENOD40 were measured and plotted as a function of BA concentration

(Figure 4.6). This shows that hypocotyl length of plants of 35S:NtENOD40 lines was

similar to wild type plants at all tested BA concentrations. We did not measure the

reduction in root length (dark and light grown seedlings) in 35S:NtENOD40 plants and

wild type plants as visible inspection indicated that differences would be marginal. Also,

the other responses to BA treatment in the dark, thickening of the hypocotyl and

enhanced apical hook curvature, do not seem to be affected in 35S:NtENOD40 lines.

Thus, ENOD40 does not affect the response to cytokinin in our transgenic lines.

100

Ws 35S:

NtENOD40-

1 35S:

NtENOD40-

2 35S:

NtENOD40-

0 ACC

0.1 ACC

1 ACC

10 ACC

Figure 4.3. Application of an ACC concentration range (0-10-5M) induces the triple response in dark

grown wild type and 35S:NtENOD40 Arabidopsis seedlings. The reduction of root and hypocotyl growth,

enhanced hypocotyl thickening and enhanced apical hook curvature is similar in wild type and NtENOD40

expressing lines. Bar equals 1 cm.

0 0,1 1 10

100

35S:

NtENOD40

-1

35S:

NtENOD40

-2

35S:

NtENOD40

-3

Hypocotyl length of dark grown Ws and 35S:

NtENOD

seedlings

relative hypocotyl length

% Ws

ACC concentration in uM

Figure 4.4. Quantification of hypocotyl length in a dose response curve shows that the response to ACC

is not affected by overexpression of NtENOD40.

101

35S:

NtENOD40-

335S:

NtENOD40-

2Ws 35S:

NtENOD40-

0 BA

0.01 BA

0.1 BA

1.0 BA

Figure 4.5. Application of a BA concentration range (0-10-6M) induces a response similar to the triple

response in dark grown wild type and 35S:NtENOD40 Arabidopsis seedlings. The response to BA is not

affected by overexpression of NtENOD40. Bar equals 1 cm.

102

100

0 0,01 0,1 1

BA concentration in uM

relative hypocotyl length

% Ws

Hypocotyl length of dark grown Ws and 35S:

NtENOD40

seedlings

35S:

NtENOD40-

35S:

NtENOD40-

35S:

NtENOD40-

Figure 4.6. Quantification of hypocotyl length in a dose response curve shows that the response to BA is

not affected by overexpression of NtENOD40.

Ws 35S:

NtENOD40-

1 35S:

NtENOD40-

2 35S:

NtENOD40-

0 BA

0.01 BA

0.1 BA

1.0 BA

Figure 4.7. Photographs of representative plants show that BA treatment in the light reduces root growth

of wild type and 35S:NtENOD40 seedlings to a similar extent. Bar equals 1 cm.

103

CONCLUSIONS

Here we describe the generation of transgenic Arabidopsis plants that express

NtENOD40 under the control of the CaMV 35S promoter. We anticipated that these

plants could facilitate the performance of genetic screens to identify interactors of

ENOD40 in Arabidopsis, once these plants display a clear, easily recognizable growth

aberration. However, all analyzed parameters failed to reveal an effect of ENOD40 on

Arabidopsis growth and development. Neither did ENOD40 affect the response to the

phytohormones cytokinin and ethylene in Arabidopsis, whereas this is the case in

tobacco BY-2 cells overexpressing NtENOD40. The lack of a clear phenotype as a

result of overexpression of NtENOD40 suggests that this gene does not have a critical

function in the Arabidopsis life cycle. The finding that database searches do not reveal

the presence of an ENOD40 homolog in the genome of Arabidopsis, while the presence

of ENOD40 homolog in a close relative such as citrus is detectable in this way, suggests

that this gene is not present in the genome and therefore the gene products of the

NtENOD40 gene might not be recognized. It does not seem to be feasible to unravel the

molecular mechanism underlying the function of ENOD40 by a genetic approach using

Arabidopsis, due to the absence of a clear phenotype as a result of overexpression of

ENOD40. With the development of plant model systems containing ENOD40, like

M.truncatula, L. japonicus, rice and tomato, a forward genetic approach might come

within reach soon.

METHODS

Generation of Transgenic Lines

The full-size NtENOD40-1 cDNA (Genbank accession number X98716) was cloned in

pCambia1390 (Cambia, Australia) behind the pMON999 (Monsanto) derived CaMV 35S

promoter as described previously (chapter 3). Transgenic Arabidopsis Ws lines were generated

using the dipped plant method, essentially as described by Weigel and Glazebrook (2002) using

Agrobacterium tumefaciens strain AGLO. Primary transgenic seeds were selected on

104

hygromycin selection medium and propagated on soil. Plants were grown in the greenhouse

with 16h photoperiod. The T1 segregation ratio was used to identify lines carrying single

insertions of the transgene (3:1). For each generated line, 10 hygromycin resistant plants were

transferred to soil for propagation. The T2 seeds were again selected on hygromycin, and the

segregation ratio was used to identify homozygous lines and were again propagated in the

greenhouse. 10-d old homozygous T3 plants were used for genomic DNA and RNA gel blot

analyses.

Genomic DNA and RNA Gel Blot Analysis

Genomic DNA was isolated according to standard protocols. 10 µg of genomic DNA was

HindIII:BamHI digested, electroforesed on a 0.8 % agarose gel and blotted onto a Hybond-N+

membrane (Amersham Pharmacia). Blotting, hybridization and washing were performed

according to the manufacturer's recommendations. RNA was isolated using the TRIzol method

(GibcoBRL). 22 µg of total RNA was run on a 1% agarose gel in 0.01 M NaH2PO4 (pH 7.0) using

the glyoxal/DMSO method (Sambrook et al., 1989). RNA was subsequently transferred to a

genescreen membrane (Dupont) in 20xSSC. Probes were hybridized in formamide hybridization

buffer O/N at 42ºC. Blots were washed till 1xSSC at 60ºC. Autoradiograms were obtained by

exposure to a phosphorscreen and scanned using a Storm 840 Phosphor-imager (Molecular

Dynamics).

Phytohormone Response Assays

Seeds are surface sterilized, and sown (20 on a line, 60 plants per plate) on agar-solidified MS

medium with 0,5 % sucrose. Seeds are kept at 4ºC for four days to enhance synchronous

germination. Then, plates are transferred to 25ºC, either in the light (BA treatment) or in the dark

(ACC and BA treatment) for five days. Plants are photographed, and mean hypocotyl length is

determined for at least 20 plants per transgenic line per treatment using NIH image software

according to Weigel and Glazebrook (2002). For each transgenic line, progeny of three different

homozygous T3 plants are tested and compared to three different seed batches of the control

Ws line. Relative hypocotyl length is calculated as percentage of the mean hypocotyl length in

the Ws control treatment.

105

ACKNOWLEDGMENTS

This work was supported by a research grant from the Netherlands Organization of

Scientific Research (NWO 805.49.004). We gratefully acknowledge Bert Essenstam for

technical assistance.

REFERENCES

Cary, A.J., Liu, W., and Howell, S.H. (1995). Cytokinin action is coupled to ethylene in its

effects on the inhibition of root and hypocotyl elongation in Arabidopsis thaliana seedlings.

Plant Physiol. 107, 1075-1082.

Charon, C., Sousa, C., Crespi, M., and Kondorosi, A. (1999). Alteration of ENOD40

expression modifies Medicago truncatula root nodule development induced by SinoRhizobium

meliloti. Plant Cell 11, 1953-1965.

Compaan, B., Yang, W.C., Bisseling, T., and Franssen, H. (2001). ENOD40 expression in the

pericycle precedes cortical cell division in Rhizobium-legume interaction and the highly

conserved internal region of the gene does not encode a peptide. Plant and Soil 230, 1-8.

Crespi, M.D., Jurkevitch, E., Poiret, M., d'Aubenton-Carafa, Y., Petrovics, G., Kondorosi,

E., and Kondorosi, A. (1994). ENOD40, a gene expressed during nodule organogenesis,

codes for a non-translatable RNA involved in plant growth. EMBO J. 13, 5099-5112.

Flemetakis, E., Kavroulakis, N., Quaedvlieg, N.E.M., Spaink, H.P., Dimou, M., Roussis, A.,

and Katinakis, P. (2000). Lotus japonicus contains two distinct ENOD40 genes that are

expressed in symbiotic, nonsymbiotic, and embryonic tissues. MPMI 13, 987-994.

Guzman, P., and Ecker, J.R. (1990). Exploiting the triple response of Arabidopsis to identify

ethylene-related mutants. Plant Cell 2, 513-523.

Kouchi, H., Takane, K., So, R.B., Ladha, J.K., and Reddy, P.M. (1999). Rice ENOD40:

isolation and expression analysis in rice and transgenic soybean root nodules. Plant J. 18,

121-129.

Le, J., Vandenbussche, F., Van Der Straeten, D., and Verbelen, J. (2001). In the early

response of Arabidopsis roots to ethylene, cell elongation is up- and down-regulated and

uncoupled from differentiation. Plant Physiol. 125, 519-522.

Mirabella, R., Martirani, L., Lamberti, A., Iaccarino, M., and Chiurazzi, M. (1999). The

soybean ENOD40(2) promoter is active in Arabidopsis thaliana and is temporally and spatially

regulated. Plant Mol. Biol. 39, 177-181.

Sambrook, J., Fritsch, E. F., Maniatis, T. (1989). Molecular Cloning a Laboratory Manual. Cold

Spring Harbor Laboratory Press, USA.

Tanimoto, M., Roberts, K., and Dolan, L. (1995). Ethylene is a positive regulator of root hair

development in Arabidopsis thaliana. Plant J. 8, 943-948.

van de Sande, K., Pawlowski, K., Czaja, I., Wieneke, U., Schell, J., Schmidt, J., Walden, R.,

Matvienko, M., Wellink, J., van Kammen, A., Franssen, H., and Bisseling, T. (1996).

Modification of phytohormone response by a peptide encoded by ENOD40 of legumes and a

nonlegume. Science 273, 370-373.

Vleghels, I. (2003). Comparative studies on ENOD40 in legumes and non-legumes. Thesis,

Wageningen University and Research Center Wageningen, the Netherlands, pp 99.

Weigel, D., and Glazebrook, J. (2002). Arabidopsis, a laboratory manual. Cold Spring Harbor

Laboratory Press. Cold Spring Harbor, New York.

106

Chapter 5

Concluding Remarks

107

ENOD40 affects hormone responses in BY-2 cells.

The Rhizobium-legume interaction results in the formation of a completely new organ on

the roots of leguminous plants to host the bacteria. A complex network involving signal

molecules like Nod-factors, the phytohormones ethylene, auxin and cytokinin and some

plant genes, coordinates this developmental process (Mylona et al., 1995). One of the

plant genes that are highly induced during early stages of nodulation is ENOD40 (Yang

et al., 1993; Kouchi et al., 1993). Upon overexpression of ENOD40, nodule formation is

accelerated, whereas silencing of ENOD40 arrested nodulation at different stages of

nodule development (Charon et al., 1999), indicating that the control on developmental

progression is disturbed and that ENOD40 has a regulatory role during development. In

addition, ENOD40 is also present in non-legumes (Figure 1.1). The expression patterns

in different plant species show that ENOD40 is transiently expressed in various

developmental programs as we have summarized in chapter 1. Several putative

functions have been suggested, one of which is that ENOD40 modifies the

phytohormone status of cells. To date, the function of the ENOD40 genes and their

mode of action are poorly understood. In this thesis, we have explored the possibility to

set up bioassays to study ENOD40 function in further detail.

We studied the effect of ENOD40 on phytohormone signaling by developing a bioassay

based on the morphological response of suspension cultured tobacco BY-2 cells to the

phytohormones auxin, cytokinin and ethylene. We found that ENOD40 overexpression

leads to suppressed elongation growth of BY-2 cells, whereas cell division frequencies

are not affected in 35S:NtENOD40 BY-2 cell suspensions. In BY-2 cells, the cell division

frequency is controlled by the cytokinin to auxin ratio (Hasezawa and Syono, 1983). By

testing our 35S:NtENOD40 lines at different cytokinin/auxin ratios in dose response

curves, we showed that this control on cell division is not affected by ENOD40.

Elongation growth is independent of the cytokinin to auxin ratio in wild type cells. In

contrast, in the presence of ENOD40, elongation growth is reduced in both a cytokinin

and an auxin dependent manner. That elongation growth in 35S:NtENOD40 lines is not

reduced to a similar degree under various cytokinin/auxin ratios shows that ENOD40 is

108

not simply a blocker of elongation growth in BY-2 cells. Instead, cytokinin and ENOD40

act synergistically whereas the effect of ENOD40 is counter-acted by auxin. This shows

that in the cell elongation growth response, but not in the cell division response, cross-

talk between ENOD40 and both cytokinin and auxin occurs. Close examination of the

relative rates of both processes revealed that ENOD40 expression causes the

elongation growth rate to become rather strictly coupled to the cell division frequency.

Furthermore, analysis of ethylene homeostasis showed that the cytokinin/auxin

dependent effect of ENOD40 on elongation growth is mediated by ethylene.

Ethylene biosynthesis is accelerated in 35S:NtENOD40 lines, which is correlated with

accelerated accumulation of ACS transcripts. The observation that constitutive

expression of ENOD40 does not lead to constitutive ethylene production indicates that

ENOD40 is not a direct inducer of ethylene biosynthesis. The observation that the

ENOD40 induced response can be counter-acted by ethylene receptor blockers leads to

several important conclusions. (i) It suggests that ethylene is a negative regulator of

elongation growth in BY-2 cells. This effect of ethylene had not been described in BY-2

cells, but is in line with the effect of ethylene on several plant processes, like in the

control of elongation growth of cells in the elongation zone of the Arabidopsis root (Le et

al., 2001). It is also consistent with the reduced size of an ethylene overproducing

mutant (eto) or the constitutive ethylene response mutation (ctr1). Further, most

ethylene insensitive mutants have larger leaves than the wild type (Ecker, 1995),

resulting from cell enlargement (Hua et al., 1995). (ii) It shows that acceleration of

ethylene accumulation is a primary cause and not a consequence of suppressed

elongation growth of 35S:NtENOD40 cells. Since ENOD40 is probably not an inducer of

ethylene biosynthesis per sé, it seems more likely that altered regulation of ethylene

biosynthesis is subject to, or part of, the mechanism that couples elongation growth

rates to cell division frequency. (iii) This indicates that ethylene perception is important

for the coupling between these processes, since application of Ag+ leads to a loss of the

strict coupling of cell division and cell elongation. In conclusion, our data show that

overexpression of NtENOD40 results in coupling between phytohormone regulated

cellular processes. The observation that -specifically in the presence of ENOD40 -, the

cytokinin and auxin dependent elongation growth response is mediated by ethylene

109

signaling, supports the hypothesis that ENOD40 affects phytohormone signaling (Crespi

et al., 1994; van de Sande et al., 1996; Charon et al., 1999). These data indicate that

the function of ENOD40 is to facilitate cross-talk between ethylene and cytokinin/auxin

signaling pathways.

Cross-talk Between ENOD40 and Phytohormones

It is generally accepted that phytohormones act as key-regulators of developmental

processes such as cell division, cell growth and cell differentiation and that coordinated

control of these processes determines cell shape, size and patterning in developing

organs. It has become clear that there is a significant amount of cross-talk between the

phytohormone signaling pathways and that this could, at least in part, account for the

observed diversity of cellular responses during development (Lindsey et al., 2002). In

genetic screens for components of phytohormone signaling pathways, often mutations

affecting responses to multiple hormones are isolated and these are beginning to give

insight in the molecular mechanism that underlies phytohormone cross-talk (Swarup et

al., 2002). Signaling of one hormone can be affected by a second hormone at any of the

following steps: hormone level, perception, inhibition or stimulation of signal transduction

processes, regulation of transcription, post-translational modification or interaction at the

response level (Coenen and Lomax, 1997).

We showed that cytokinin and auxin act antagonistically in the regulation of cell division

in wild type BY-2 cells. Several studies have shown that auxin and cytokinin regulate

tobacco pith cell proliferation by synergistically regulating the expression of the cell cycle

regulatory components cdc2 and cycD3. Additionally, cytokinin regulates cdc2 catalytic

activity (John et al., 1993). A similar mechanism has been suggested to control pericycle

cell division leading to lateral root formation, although here cytokinin and auxin act

antagonistically, indicating that the interactions between auxin and cytokinin in this

mechanism differ in a tissue or species-specific manner (Coenen and Lomax, 1997).

Thus, it is likely that auxin/cytokinin cross-talk underlies regulation of BY-2 cell division.

Since ENOD40 does not affect the cell division frequency in BY-2 cells, it is not likely

110

that ENOD40 is part of this mechanism. Instead, the observation that altered regulation

of ACS transcription, and consequently altered ethylene biosynthesis kinetics, mediates

the auxin/cytokinin dependent elongation growth response in tobacco BY-2

35S:NtENOD40 cells, suggests that in the presence of ENOD40, cross-talk between

auxin and ethylene but also cytokinin and ethylene takes place. This mechanism is

consistent with that proposed for cytokinin/ethylene and auxin/ethylene cross-talk in

several plant developmental processes (Swarup et al., 2002). Auxin and ethylene have

been described to interact at the level of ethylene biosynthesis since auxin up-regulates

the expression of ACC synthase in Arabidopsis (Abel et al., 1995), tomato (Abel and

Theologis, 1996) and lupine (Beckman et al., 2000). Cytokinin treated dark-grown

Arabidopsis seedlings display the triple response whereas this effect is not observed

when plants are grown in the presence of ethylene-synthesis or -perception blockers,

indicating that cytokinin and ethylene pathways interact at the level of ethylene

biosynthesis. Both transcriptional and post-translational regulation seem to be involved

(Cary et al., 1995; Vogel et al., 1998). Our data show that, like in intact plants, in BY-2

cell suspensions similar mechanisms leading to phytohormone cross-talk operate and

control cell morphology. A remarkable result is that cytokinin/ethylene and/or

auxin/ethylene cross-talk -resulting in altered regulation of the elongation growth

response- requires ENOD40, suggesting that ENOD40 is part of this pathway(s). How

and at what level ENOD40 acts in the pathway(s) remains to be clarified.

Which gene product is important for the function of ENOD40?

Further insight in the molecular mechanism of the mode of action of ENOD40 requires

the identification of the biologically active gene product. In the case of ENOD40 this is

not trivial since a long open reading frame that could encode a biologically active

peptide is lacking in ENOD40 transcripts. Instead, as described in chapter 1, all

ENOD40 genes contain two short conserved regions. Strikingly, in region I a conserved

small ORF resides encoding a remarkably small peptide of 10-13 amino-acids, whereas

in region II in only about 50% of the transcripts a conserved ORF can be found,

111

suggesting that this region may be active as RNA. We have used our protoplast

bioassay to study the activity of the two regions (chapter 3). We showed that the ORF in

region I is translated in BY-2 cells, as revealed by a GFP reporter fusion. These

observations are consistent with previous reports (Compaan et al., 2001; Sousa et al.,

2001; van de Sande et al., 1996) and support the hypothesis that the peptide encoded

by ORF I has biological activity. However, translation of this oligopeptide was not

required for the ENOD40 induced elongation growth phenotype in the protoplast

bioassay (chapter 3), indicating that biological activity may reside in the RNA sequence

of region I and/or II. Strikingly, disruption of the translation start of ORF I led to a

reduction of the cell division frequency that was not observed in lines expressing the

intact construct. The observation that a disruption of translation of ORF I induces an

additional phenotype in comparison to the effect of the intact construct, indicates that the

encoded peptide has biological activity but also that the function of both conserved RNA

regions may be interconnected. Since the semi-quantitative nature of our assay system

is not sufficient to unravel the complex effects of both regions, the role of the two

conserved regions and the nature of the ENOD40 encoded biologically active

component remain to be elucidated.

Sousa (2001) described a bioassay for ENOD40 activity by scoring for cortical cell

division induced upon ballistic targeting of ENOD40-derived constructs in Medicago

roots. In this bioassay, both the peptide encoded by region I, as well as a peptide

encoded in region II have biological activity, and lead to the same response. The effect

of the MtENOD40 region II in alfalfa roots depends on the presence, correct size and

sequence of the region I encoded peptide, even though deletions spanning either region

I or region II are active, indicating a complex level of regulation acting on the MtENOD40

RNA (Sousa et al., 2001). Although the responses induced by the two regions differ in

the two different assays, both our data and the data described by Sousa (2001) suggest

that the function of the two conserved regions may be connected in a complex manner.

Rohrig (2002) showed that two partially overlapping ORFs located in region I were

translated from a GmENOD40-1 transcript in vitro and that two corresponding synthetic

112

peptides bind sucrose synthase in vitro. The enhanced expression of sucrose synthase

during nodule development indicates that sucrose synthase may have an important

function in phloem unloading of sucrose, which is translocated from shoots to nodules.

Binding of the two ENOD40 peptides may either regulate enzyme activity or may direct

this enzyme to specific intracellular sites. The role of binding between the two ENOD40

peptides to sucrose synthase for the function of ENOD40 in phytohormone cross-talk as

we have observed in BY-2 cells is unclear. Two structural requirements in ENOD40

transcripts, namely the presence and conservation of the second short ORF partially

overlapping with ORF I and the presence of a cysteine in the middle of the ORF I

encoded peptide, which is required for binding, are only present in legume ENOD40

transcripts. This suggests that the interaction between ENOD40 peptides and sucrose

synthase can only occur in legumes. The expression patterns of ENOD40 and sucrose

synthase overlap at least partially in mature soybean nodules (Yang et al, 1993; Komina

et al., 2002). So, it is possible that ENOD40 encoded peptides and sucrose synthase

interact during nodulation.

In a two-hybrid screen, Vleghels (2003) identified a laminin-like protein (p40) with

unknown function, as an interactor of ENOD40. In situ hybridization showed that the

expression patterns of p40 and ENOD40 partially overlap in pea nodules, yet in an other

region as where ENOD40 and sucrose synthase overlap. As a first step to show that

these peptides are part of the molecular mechanism of ENOD40 action, binding of

ENOD40 peptides either with sucrose synthase or with p40 should be confirmed in

planta.

We have explored the possibility to use Arabidopsis thaliana as a genetic model system

to identify ENOD40 interacting components (chapter 4). Sequence homology searches

did not reveal an ENOD40 homolog in the Arabidopsis genome. Since we showed that

ENOD40 transcripts are biologically active in a heterologous plant background (chapter

3), we proceeded by using the tobacco ENOD40 (NtENOD40) homolog to generate

transgenic plants overexpressing NtENOD40. Unfortunately, in neither of the transgenic

lines that were obtained a phenotype was observed, indicating that NtENOD40 does not

affect Arabidopsis growth and development. This can be explained in several ways,

113

most likely Arabidopsis does not contain a gene with significant homology to ENOD40,

the gene products of the NtENOD40 transgene are not recognized and therefore are not

functional in the Arabidopsis background. Thus, it does not seem to be feasible to

unravel the molecular mechanism underlying the function of ENOD40 by a genetic

approach using Arabidopsis, due to the absence of a clear phenotype as a result of

overexpression of NtENOD40.

Is ENOD40 a mediator of phytohormone cross-talk in other processes and/or

other plant species?

A major challenge now is to confirm that interaction between ENOD40 and

phytohormone signaling pathways provides the basis for the biological function of

ENOD40 in organogenesis. An experimental system in intact plants that may well be

exploited to study the function of ENOD40 in further detail would be nodule formation.

ENOD40 is expressed in the nodule primordium where cell division and cell growth take

place and in Zone II which, in indeterminate nodules, is the zone between the

meristematic cells and the differentiated cells of the central tissue of the mature nodule.

Especially in these tissues where different cellular processes occur, it is tempting to

assume that the coupling between cellular processes is important for development and

that ENOD40 is required for the cross-talk between regulating pathways. To determine

whether the coupling of these processes occurs via cross-talk between phytohormone

pathways, one first needs to visualize what the cellular effects of the phytohormones

auxin, cytokinin and ethylene in these tissues are. Subsequently, the effect of

misexpression of ENOD40 on (the coupling of) these processes should be tested. For

this purpose, the investigation of the spatial and temporal correlation between

phytohormone distribution and cellular responses in developing nodules in a plant

background in which phytohormone distribution, synthesis or perception and/or ENOD40

expression is disturbed seems necessary, yet requires a complex experimental set-up.

Whether the ENOD40 induced responses in BY-2 cells provide insight in ENOD40

function in other plant species, depends on whether its function is conserved across the

114

plant kingdom. The function of ENOD40 can be thought of at two levels. ENOD40

function may be thought of in terms of a molecular mechanism, or alternatively in terms

of a physiological or morphological response. We would like to note that the level of

sequence homology between transcripts and the observation that a ENOD40 transcript

is biologically active in a heterologous plant background, suggests that ENOD40 gene

function is at a molecular level similar in different plant species. However, this can only

be confirmed by knowledge of the molecular mechanism of ENOD40 action. On the

other hand, if ENOD40 mediates cross-talk between phytohormone pathways, the

induced cellular responses may either be similar or opposing in different tissues or plant

species depending on additional up- or downstream signaling events, even though the

molecular mechanisms are the same.

In conclusion, the observation that ENOD40 affects phytohormone responses in BY-2

cells is consistent with the spatio-temporal expression patterns, which correlates with

developmental stages involving the activity of phytohormones. We propose that coupling

between cell division, cell growth and cell differentiation may be achieved by transient

and local expression of plant factors like ENOD40 that are involved in phytohormone

cross-talk pathways. The coupling of hormone responses may be crucial to many

developmental programs, and this may explain the complexity of ENOD40 expression

patterns and the variety of tissues with which ENOD40 expression is associated in

different plant species.

115

REFERENCES

Abel, S., Nguyen, M.D., Chow, W., and Theologis, A. (1995). ACS4, a primary indoleacetic

acid-responsive gene encoding 1-aminocyclopropane-1-carboxylate synthase in Arabidopsis

thaliana. J. Biol. Chem. 270, 19093-19099.

Abel, S., and Theologis, A. (1996). Early genes and auxin action. Plant Physiol. 111, 9-17.

Beckman, E.P., Saibo, N.J.M., Di Cataldo, A., Regalado, A.P., Ricardo, C.P., and

Rodrigues-Pousada, C. (2000). Differential expression of four genes encoding 1-

aminocyclopropane-1-carboxylate synthase in Lupinus albus during germination and in

response to indole-3-acetic acid and wounding. Planta 211.

Cary, A.J., Liu, W., and Howell, S.H. (1995). Cytokinin action is coupled to ethylene in its

effects on the inhibition of root and hypocotyl elongation in Arabidopsis thaliana seedlings.

Plant Physiol. 107, 1075-1082.

Charon, C., Sousa, C., Crespi, M., and Kondorosi, A. (1999). Alteration of ENOD40

expression modifies Medicago truncatula root nodule development induced by Sinorhizobium

meliloti. Plant Cell 11, 1953-1965.

Compaan, B., Yang, W.C., Bisseling, T., and Franssen, H. (2001). ENOD40 expression in the

pericycle precedes cortical cell division in Rhizobium-legume interaction and the highly

conserved internal region of the gene does not encode a peptide. Plant and Soil 230, 1-8.

Coenen, C., and Lomax, T.L. (1997). Auxin-cytokinin interactions in higher plants: old problems

and new tools. Trends Plant Sci. 2, 351-356.

Crespi, M.D., Jurkevitch, E., Poiret, M., d'Aubenton-Carafa, Y., Petrovics, G., Kondorosi,

E., and Kondorosi, A. (1994). ENOD40 , a gene expressed during nodule organogenesis,

codes for a non- translatable RNA involved in plant growth. EMBO J. 13, 5099-5112.

Ecker, J.R. (1995). The ethylene signal transduction pathway in plants. Science 268, 667-675.

Hasezawa, S., and Syono, K. (1983). Hormonal Control of Elongation of Tobacco Cells Derived

from Protoplasts. Plant Cell. Physiol. 24, 127-132.

Hua, J., Chang, C., Sun, Q., and Meyerowitz, E.M. (1995). Ethylene insensitivity conferred by

Arabidopsis ERS gene. Science 269, 1712-1714.

John, P.C.L., Zhang, K., Dong, C., Diederich, L., and Wightman, F. (1993). P34-cdc2 related

proteins in control of cell cycle progression, the switch between division and differentiation in

tissue development and stimulation of division by auxin and cytokinin. Aust. J. Plant Physiol.

20, 503-526.

Komina, O., Zhou, Y., Sareth, G., and Chollet, R. (2002). In vivo and in vitro phosphorylation

of membrane and soluble forms of soybean nodule sucrose synthase. Plant Physiol. 129,

1644-1673.

Kouchi, H., and Hata, S. (1993). Isolation and characterization of novel nodulin cDNAs

representing genes expressed at early stages of soybean nodule development. Mol. Gen.

Genet. 238, 106-119.

Le, J., Vandenbussche, F., Van Der Straeten, D., and Verbelen, J. (2001). In the early

response of Arabidopsis roots to ethylene, cell elongation is up- and down-regulated and

uncoupled from differentiation. Plant Physiol. 125, 519-522.

Lindsey, K., Casson, S., and Chilley, P. (2002). Peptides: new signalling molecules in plants.

Trends Plant Sci. 7, 78-83.

Mirabella, R., Martirani, L., Lamberti, A., Iaccarino, M., and Chiurazzi, M. (1999). The

soybean ENOD40(2) promoter is active in Arabidopsis thaliana and is temporally and spatially

regulated. Plant Mol. Biol. 39, 177-181.

Mylona, P., Pawlowski, K., and Bisseling, T. (1995). Symbiotic Nitrogen Fixation. Plant Cell 7,

869-885.

116

Rohrig, H., Schmidt, J., Miklashevichs, E., Schell, J., and John, M. (2002). Soybean

ENOD40 encodes two peptides that bind to sucrose synthase. Proc. Natl. Acad. Sci. USA 99,

1915-1920.

Sousa, C., Johansson, C., Charon, C., Manyani, H., Sautter, C., Kondorosi, A., and Crespi,

M. (2001). Translational and structural requirements of the early nodulin gene ENOD40, a

short-open reading frame-containing RNA, for elicitation of a cell-specific growth response in

the alfalfa root cortex. Mol. Cell. Biol. 21, 354-366.

Swarup, R., Parry, G., Graham, N., Allen, T., and Bennett, M. (2002). Auxin cross-talk:

integration of signaling pathways to control plant development. Plant Mol. Biol. 49, 411-426.

van de Sande, K., Pawlowski, K., Czaja, I., Wieneke, U., Schell, J., Schmidt, J., Walden, R.,

Matvienko, M., Wellink, J., van Kammen, A., Franssen, H., and Bisseling, T. (1996).

Modification of phytohormone response by a peptide encoded by ENOD40 of legumes and a

nonlegume. Science 273, 370-373.

Vleghels. (2003). Comparative studies on ENOD40 in legumes and non-legumes. Thesis,

Wageningen University and Research Center Wageningen, the Netherlands, pp 99.

Vogel, J.P., Woeste, K.E., Theologis, A., Kieber, J.J. (1998) Recessive and dominant

mutations in the ethylene biosynthetic gene ACS5 of Arabidopsis confer cytokinin insensitivity

and ethylene overproduction, respectively. Proc. Natl. Acad. Sci. USA 95, 4766-4771.

Yang, W.C., Katinakis, P., Hendriks, P., Smolders, A., de Vries, F., Spee, J., van Kammen,

A., Bisseling, T., and Franssen, H. (1993). Characterization of GmENOD40, a gene showing

novel patterns of cell- specific expression during soybean nodule development. Plant J. 3,

573-585.

117

SAMENVATTING

De interactie tussen Rhizobium bacterien en vlinderbloemige planten leidt tot de

ontwikkeling van een nieuw orgaan, de wortelknol, op de wortels van leguminose

planten. Een set van signaal moleculen zoals Nod-factoren en de planten hormonen

ethyleen, auxine en cytokinine alsmede plant genen die mogelijk coderen voor een

groeifactor, coördineren gezamenlijk dit ontwikkelingsproces. Een van de genen die

sterk geïnduceerd wordt tijdens de vroege stadia van het proces is het ENOD40 gen.

Als gevolg van overexpressie van ENOD40 wordt het nodulatie proces versneld, terwijl

het uitschakelen van ENOD40 door middel van co-suppressie tot knolletjes leidt die

verstoord zijn in hun ontwikkeling (Charon et al., 1999). Deze observaties duiden erop

dat ENOD40 een regulerende rol zou kunnen hebben bij dit proces. Het ENOD40 gen is

niet alleen aanwezig in vlinderbloemigen maar komt ook voor in de genomen van niet-

vlinderbloemigen. In hoofdstuk 1 hebben we een overzicht gegeven van het voorkomen

van ENOD40 genen in het plantenrijk en van de opbouw van de transcripten. Opvallend

is dat geen van de ENOD40 genen een lang open leesraam bevat dat zou kunnen

coderen voor een eiwit. Wel zijn er twee sterk geconserveerde regio’s aanwezig. Regio I

bestaat uit een kort open leesraam dat codeert voor een opmerkelijk klein peptide van

slechts 10-13 aminozuren terwijl er in regio II in slechts 50% van de transcripten een

geconserveerd leesraam aanwezig is. Dit suggereert dat de tweede regio actief zou

kunnen zijn op het RNA niveau. De sterke mate van conservering duidt erop dat de twee

regio’s belangrijk zouden kunnen zijn voor de biologische activiteit van ENOD40, maar

een functie van de twee regio’s of het (potentieële) peptide is nog niet bekend. De

ENOD40 expressie patronen in verschillende plantensoorten laten zien dat ENOD40

transient tot expressie komt in verscheidene ontwikkelingsprocessen. Er zijn een aantal

mogelijke functies voor het ENOD40 gen gepostuleerd in deze processen, een ervan is

dat het ENOD40 gen de respons op fytohormonen, of de gevoeligheid of de

concentraties van fytohormonen in cellen zou kunnen beïnvloeden. In deze thesis

beschrijven we de bio-assays die we hebben ontwikkeld om de functie van het ENOD40

gen in meer detail te kunnen bestuderen.

118

In hoofdstuk 2 hebben we het effect van ENOD40 op fytohormoon signalering

bestudeerd door een bioassay te ontwikkelen die gebaseerd is op de morfologische

respons van BY-2 tabakscelcultures op de fytohormonen auxine, cytokinine en ethyleen.

We hebben gevonden dat in transgene 35S:NtENOD40 bevattende lijnen een

verhoogde expressie van ENOD40 de elongatiegroei van deze cellen onderdrukt, terwijl

de delingsfrequentie niet beïnvloed wordt. Deze delingsfrequentie staat onder controle

van auxine en cytokinine en wel via hun concentratie ratio. Door de delingsfrequentie

van de transgene 35S:NtENOD40 bevattende lijnen te bepalen bij varierende auxine tot

cytokinine ratio’s konden we aantonen dat de controle op de delingsfrequentie niet

beïnvloed wordt door de verhoogde expressie van ENOD40. Elongatiegroei is

onafhankelijk van de ratio tussen cytokinine en auxine in wild type cellen waar ENOD40

niet of nauwelijks tot expressie komt. In tegenstelling tot de wild type cellen, is in cellen

die ENOD40 tot overexpressie brengen de elongatiegroei gereduceerd in zowel een

cytokinine- als een auxine-afhankelijke manier. Dat de elongatiegroei niet onder alle

omstandigheden in dezelfde mate gereduceerd is, laat zien dat ENOD40 niet simpelweg

een blokker is van elongatiegroei. Deze proeven tonen verder aan dat ENOD40 en

cytokinine synergistisch werken en dat het effect van ENOD40 kan worden

tegengewerkt door auxine. Deze experimenten suggereren dan ook dat bij de controle

op elongatiegroei, maar niet bij de controle op de delingsfrequentie, er cross-talk plaats

vindt tussen ENOD40 en zowel auxine als cytokinine signalering. Analyse van de

relatieve snelheden van de elongatiegroei en de delingsfrequentie laten zien dat in de

transgene 35S:NtENOD40 lijnen, de elongatiegroei sterk gekoppeld wordt aan de

delingsfrequentie met als resultaat dat de grootte van cellen constant wordt. Analyse

van de ethyleen homeostase laat zien dat het cytokinine- en auxine-afhankelijke effect

op de elongatiegroei gemediëerd wordt door ethyleen. Ethyleen biosynthese vindt

versneld plaats in de transgene ENOD40 lijnen en is gecorreleerd met een versnelde

accumulatie van ACC synthase transcripten, die coderen voor een enzym betrokken bij

ethyleen productie. De observatie dat constitutieve expressie van ENOD40 niet leidt tot

constitutieve ethyleen productie suggereert dat ENOD40 niet een directe inducer is van

ethyleen biosynthese. Daarnaast leidt de observatie dat het effect van ENOD40 kan

worden tegengewerkt door toevoeging van een blokker van de ethyleen receptor tot een

119

aantal belangrijke conclusies. In de eerste plaats suggereert het dat ethyleen een

negatieve regulator is van elongatiegroei in BY-2 cellen. Dit effect van ethyleen was nog

niet beschreven in BY-2 cellen maar is vergelijkbaar met het effect van ethyleen in een

aantal andere processen, zoals in de controle op elongatiegroei van cellen in de

elongatiezone van Arabidopsis wortels. Ten tweede laten deze experimenten zien dat

een versnelde accumulatie van ethyleen een primaire oorzaak en niet een gevolg is van

de gereduceerde elongatiegroei van de tabakscellen. Omdat ENOD40 waarschijnlijk

geen directe inducer van ethyleen biosynthese is, is het waarschijnlijker dat de

veranderde regulatie van ethyleen biosynthese onderhevig is aan, of onderdeel is van,

het mechanisme dat de snelheid van elongatiegroei aan de delingsfrequentie koppelt.

Omdat de toevoeging van de ethyleen perceptie blokker er toe leidt dat de koppeling

tussen deze twee processen verloren gaat, suggereren deze observaties dat perceptie

van ethyleen belangrijk is voor de koppeling tussen elongatiegroei en delingsfrequentie.

Concluderend kunnen we stellen dat overexpressie van ENOD40 resulteert in de

koppeling tussen fytohormoon gereguleerde processen. De observatie dat de door

ENOD40 geïnduceerde cytokinine- en auxine-afhankelijke elongatiegroei respons

gemediëerd wordt door ethyleen, ondersteunt de hypothese dat ENOD40 de respons

van cellen op fytohormonen kan beïnvloeden. Onze data wijzen er daarom op dat

ENOD40 de cross-talk tussen ethyleen en auxine/cytokinine signaal transductie routes

faciliteert.

Om meer gedetailleerd inzicht te krijgen in het mechanisme van de ENOD40 activiteit, is

het nodig de biologisch actieve moleculen te identificeren. In het geval van ENOD40 is

dit verre van triviaal omdat een lang open leesraam dat voor een biologische actief eiwit

zou kunnen coderen niet aanwezig is in de ENOD40 transcripten.

In hoofdstuk 3 hebben we de functie van de twee geconserveerde regio’s in the

ENOD40 transcripten onderzocht door de activiteit van verschillende transcripten te

toetsen in de bio-assay die is beschreven in hoofdstuk 2. Experimenten waarbij we het

ENOD40 peptide aan de GFP reporter fuseren, tonen aan dat het korte leesraam van

regio I daadwerkelijk vertaald wordt. Deze data komen overeen met eerdere

experimenten (Compaan et al., 2001; Sousa et al., 2001; Van de Sande et al., 1996) en

120

ze ondersteunen de hypothese dat het kleine peptide biologische activiteit zou kunnen

hebben. Echter, translatie van dit peptide is niet nodig voor de reductie van

elongatiegroei in de protoplasten-assay, en dit suggereert dat de biologische activiteit in

de nucleotide sequentie van regio I en/of II zou kunnen liggen. Een opvallend resultaat

is dat een verstoring van de translatie van regio I leidt tot een afname van de

delingsactiviteit, terwijl dit niet werd gevonden in de lijnen die het intacte construct tot

overexpressie brengen. De observatie dat verstoring van translatie van ORF I een

additioneel fenotype induceert ten opzichte van het effect van het intacte transcript,

duidt erop dat het peptide toch activiteit heeft maar ook dat de functie van de twee

regio’s met elkaar in verband staat. Omdat de semi-kwantitatieve aard van deze bio-

assays waarschijnlijk niet voldoende is om de complexe effecten van de twee regio’s te

ontrafelen, zijn andere experimentele systemen nodig om de rol van de twee

geconserveerde regio’s en de aard van de biologisch active component die door

ENOD40 gecodeerd wordt, op te helderen.

In hoofdstuk 4 hebben we onderzocht of model plant Arabidopsis thaliana te gebruiken

is om de met ENOD40 interacterende componenten te identificeren omdat Arabidopsis

grote voordelen biedt met betrekking tot het uitvoeren van genetische screens. Hoewel

uit het overzicht van het voorkomen van ENOD40 genen in het plantenrijk (hoofdstuk 1)

blijkt dat ENOD40 een algemeen voorkomend gen is, hebben sequentie homologie

analyses geen ENOD40 homoloog in het Arabidopsis genoom opgeleverd. Omdat we in

hoofdstuk 3 hebben laten zien dat ENOD40 homologen ook in een heterologe plant

achtergrond actief kunnen zijn, hebben we een tabaks homoloog van ENOD40

(NtENOD40) gebruikt om transgene lijnen te genereren die NtENOD40 tot

overexpressie brengen. Om een genetische screen uit te kunnen voeren is het allereerst

nodig een fenotype ten gevolge van ENOD40 misexpressie in Arabidopsis te

identificeren. We hebben de transgene lijnen op morfologische afwijkingen getoetst

onder normale groei condities, alsook na behandeling met fytohormonen. Echter, in

geen van de transgene lijnen die gegenereerd zijn, werd een fenotype geobserveerd,

hetgeen suggereert dat NtENOD40 geen effect heeft op Arabidopsis groei en

ontwikkeling. De meest waarschijnlijke verklaring is dat, omdat Arabidopsis geen gen

121

met sterke homologie met NtENOD40 heeft, de gen producten van het NtENOD40

transgen niet herkend worden en daarom niet functioneel zijn in de Arabidopsis

achtergrond. Helaas zullen daarom de transgene Arabidopsis lijnen die we gegenereerd

hebben waarschijnlijk niet gebruikt kunnen worden om het werkingsmechanisme van

ENOD40 op te helderen en zullen andere model planten, zoals bijvoorbeeld Medicago

die het ENOD40 gen wel in zijn genoom heeft, gebruikt moeten worden voor een

dergelijke screen.

De belangrijkste resultaten van deze thesis hebben we verkregen uit de bio-assays

gebaseerd op een cellulair test systeem en deze duiden erop dat het koppelen van

fytohormoon signaal transductie routes de functie van ENOD40 is. Hoewel de

fytohormonen cytokinine, auxine en ethyleen zeer vergelijkbare effecten hebben in het

cellulaire systeem als in hele planten, is hiermee nog niet de functie van ENOD40 in

intacte planten bewezen. De grote uitdaging die nu volgt is aantonen of de interactie

tussen ENOD40 en fytohormoon signaal transductie routes ook daadwerkelijk de basis

vormt voor de functie van ENOD40 genen in hele planten. Een proces dat hiertoe zou

kunnen worden aangewend is uiteraard de knolvorming in vlinderbloemige planten.

Verschillende fytohormonen spelen een belangrijke regulerende rol in cellulaire

processen tijdens de knolontwikkeling. ENOD40 komt tot expressie in het

knolprimordium waar celdeling en celgroei plaatsvinden en in Zone II welke, in knollen

van het ‘indeterminate’ type, de zone is tussen cellen van de meristematische zone en

de gedifferentieerde cellen van het centrale weefsel. Juist in deze weefsels waar

verschillende cellulaire processen plaatsvinden is het aannemelijk dat de koppeling

tussen cellulaire processen belangrijk is voor de voortgang van de ontwikkeling van het

orgaan, en dat ENOD40 benodigd is voor de communicatie tussen de regulerende

factoren.

122

Een Woord van Dank

Als ik de ingredienten die mijn leven de laatste jaren zo mooi hebben gemaakt elk apart zou

moeten noemen zou het een lange lijst worden die ieder geen eer meer aan zou doen. Ik zou

moeten beginnen met de mij zeer aan het hart liggende collega’s van de vakgroep Molbi, waar

de grens tussen collega’s en vrienden vervaagt, ik zou de polo, de koekjes, de cola en mijn

trouwe radiootje willen noemen en natuurlijk mijn familie die me altijd door dik en dun gesteund

heeft en kunnen eindigen met een hele schare vrienden die ik in Wageningen heb mogen leren

kennen. Maar net zo goed zou ik kunnen eindigen met het bedanken van de leden van de

fotoclub die mij opnieuw hebben leren kijken of meneer Nikon voor het maken van geweldige

camera’s.

Dat ik het promotie onderzoek dat in dit boekje beschreven is heb kunnen uitvoeren, heb ik in

ieder geval te danken aan de enorme inzet van een aantal mensen die ik hier wel speciaal wil

noemen. Henk, veel heb ik aan jou te danken, je enthousiasme en liefde voor het vak zijn

waardevol en aanstekelijk. Ik ben er trots op bij jou te zijn gepromoveerd. Ton, het was kort

maar krachtig, helderheid en snelheid; ik heb ongelofelijk veel van je geleerd en hoop het nog

vaker mee te mogen maken. Bert en Ingrid, vrienden voor het leven, samen met jullie en Jan en

Xi had ik het niet beter kunnen treffen in de “ENOD40” groep. “Mijn studenten” Silvia, Ciska en

Maelle, het was geweldig om jullie te zien groeien en echt goed te worden in wat je doet.

Gerard, jij hebt de BY-2 transformaties die de kern van dit onderzoek vormen in ons lab

opgezet, fantastisch werk! Bert en Tinka, jullie hebben me ontzettend veel werk uit handen

genomen bij het verzorgen en oogsten van de arabidopsis planten, bedankt! Verder iedereen

met wie ik op Molbi heb samengewerkt: ik hoop dat we elkaar nog vaak en op onverwachte

plekken tegen mogen komen en dat er meer dan alleen werk is om over te praten. Elf jaar

Wageningen waren fantastisch maar lang genoeg. Ik mag mezelf gelukkig prijzen nooit alleen te

zijn geweest, met zoveel vrienden om me heen heb ik me geen moment verveeld. Te veel

herinneringen om op te noemen, genoeg verhalen voor heel wat nostalgische avondjes, maar ik

leef liever vandaag dan gisteren dus laten we er meteen nog een flinke stapel nieuwe verhalen

bij gaan maken. Laat het gezegd zijn.

Bedankt, het was een mooie tijd. tommie.

123

A word of gratitude

If I would have to list all ingredients that have made my life so valuable during the recent years, I

would end up with a list so long it would not do justice to each of them. I’d have to start with the

dear colleagues of the Molbi department where the difference between colleagues and friends

has disappeared, I’d have to mention the polo, the cookies, the cola and my faithful radio, of

coarse I’m grateful to my dearly beloved family who have always supported me in an amazing

way and I could end with a whole bunch of friends with whom I’ve had the chance to meet. But

just as well I could end by thanking the members of the photography club who have taught me to

look at the world in new and different ways or Nikon for producing wonderful cameras, or…

This thesis is all about the research that I have performed in the last six years and it would never

have been possible without the extraordinary input of a number of people. Henk, I owe a lot to

you, your enthusiasm and love for the work are very valuable and encouraging. I’m proud to

have had you as a mentor. Ton, clarity, speed and a number of eye-openers, I’ve learned an

incredibly from our discussions and hope to do it more often. Bert and Ingrid, friends for life,

together with Jan-the-pun-Ho and Xi, I could not have been more lucky than to work with you in

the ‘ENOD40’ group. As for my students Silvia, Ciska and Maelle; it was great to see you grow

and become really good at what you do. Gerard, you introduced the BY-2 cell transformation in

our lab which turned out to be so crucial, thank you for helping me generate the core of my

research. Bert and Tinka, by taking care of the arabidopsis plants you took a load of work off my

hands, thanks! And to everybody with whom I’ve worked at the Molbi department: I hope that we

meet again and again at unexpected places and that we have more to discuss than just work.

Eleven years of Wageningen were fantastic but long enough. Too many memories to remember,

enough stories to fill a lifetime of nostalgic evenings. But as I’d rather live today than yesterday,

lets just start now with living new adventures. Let it be said. Those who know, know.

Thanks, ‘had a great time. tommie.

124

Curriculum Vitae

Tom Ruttink werd geboren op 15 januari 1974 te Utrecht. In september 1992 haalde hij

het VWO diploma aan het Montessori Lyceum Herman Jordan te Zeist. Tussen 1992 en

1997 studeerde hij Moleculaire Wetenschappen aan de Wageningen Universiteit. In

september 1997 behaalde hij het ingenieursdiploma met afstudeervakken

Plantenfysiologie (onder begeleiding van Dr. W. van Leeuwen en Dr. A.R. van der Krol),

waarvoor de C.T. de Wit scriptieprijs werd uitgereikt, Moleculaire Fysica (onder

begeleiding van Dr. L. Meulenkamp en Dr. H. van As) en Moleculaire Plantenfysiologie

aan de University of Guelph, Canada (onder begeleiding van Prof. J. Strommer). Vanaf

februari 1998 was hij Onderzoeker in Opleiding (OIO) verbonden aan de Wageningen

Universiteit. Het in dit proefschrift beschreven onderzoek is uitgevoerd onder leiding van

Dr. H. Franssen en Prof. Dr. T. Bisseling bij de leerstoelgroep Moleculaire Biologie en is

gefinanciëerd door de Nederlandse Organisatie voor Wetenschappelijk Onderzoek

(NWO 805 49 004). Per december 2003 is hij werkzaam als Post-doc aan het

Vlaanderen Instituut voor Biotechnologie (VIB) in de onderzoeksgroepen van Prof. Dr.

D. Inzé en Prof. Dr. W. Boerjan.

125

List of publications

van der Weerd, L., Ruttink T., van Dusschoten, D., Vergeldt, FJ., de Jager, PA., and

van As, H. (1998). Plant growth studies using low field NMR. In: Spatially resolved

magnetic resonance. P. Blumler, B. Blumich, R. Botto, and E. Fukushima, editors.

Wiley-VCH, Weinheim. 473 - 479.

van Leeuwen, W., Hagendoorn, MJM., Ruttink, T., van Poecke, R., van der Plas,

LH., and van der Krol, AR. (2000). The Use of the Luciferase Reporter System for in

Planta Gene Expression Studies. Plant Mol. Biol. Reporter 18 (2),143

van Leeuwen W., Ruttink T., Borst-Vrenssen AW., van der Plas, LH., van der Krol,

A.R. (2001). Characterization of position-induced spatial and temporal regulation of

transgene promoter activity in plants. J. Exp. Bot. 52 (358) 949-959.

van der Weerd, L., Claessens, MM., Ruttink, T., Vergeldt, FJ., Schaafsma, TJ., Van

As, H. (2001). Quantitative NMR microscopy of osmotic stress responses in maize

and pearl millet. J. Exp. Bot. 52 (365) 2333-2343.

Vleghels, I., Ruttink, T., Compaan, B., and Franssen, H. (2001) Root nodule

formation in legumes; a molecular chat-box. In: Recent Research Developments in

Plant Physiology, Vol. 2 187-199.

Compaan, B., Ruttink, T., Albrecht, C., Meeley, R., Bisseling, T., Franssen, H.

(2003). Identification of a Zea mays line carrying a transposon tagged ENOD40. BBA -

Gene Structure and Expression, 1629 (1-3) 84-91.

126

The research described in this thesis was carried out at the Laboratory of Molecular

Biology, Wageningen University, The Netherlands. This work was supported by a

research grant from the Netherlands Organization for Scientific Research (NWO

805.49.004).

127

Conserved structured domains in plant non-coding RNA enod40, their evolution and recruitment of sequences from transposable elements

Article

Full-text available

Oct 2023

Plant long noncoding RNA enod40 is involved in the regulation of symbiotic associations with bacteria, in particular, in nitrogen-fixing root nodules of legumes, and with fungi in phosphate-acquiring arbuscular mycorrhizae formed by various plants. The presence of enod40 genes in plants that do not form such symbioses indicates its other roles in cell physiology. The molecular mechanisms of enod40 RNA function are poorly understood. Enod40 RNAs form several structured domains, conserved to different extents. Due to relatively low sequence similarity, identification of enod40 sequences in plant genomes is not straightforward, and many enod40 genes remain unannotated even in complete genomes. Here, we used comparative structure analysis and sequence similarity searches in order to locate enod40 genes and determine enod40 RNA structures in nitrogen-fixing clade plants and in grasses. The structures combine conserved features with considerable diversity of structural elements, including insertions of structured domain modules originating from transposable elements. Remarkably, these insertions contain sequences similar to tandem repeats and several stem-loops are homologous to microRNA precursors.

Identification and Expression of Early Nodulin in Sugarcane Transcriptome Revealed by in Silico Analysis

Conference Paper

Sep 2010

Nodulin genes have been defined as plant genes that are induced during nodule formation in legumes. Many studies, however, revealed a number of nodulins in non-legumes, including monocot plants, suggesting that these genes play additional roles besides nodulation. The presence and expression profile of the early nodulin genes (Annexin, DMI3, NIN, NORK, CCS52A, and ENOD8) was evaluated in the sugarcane transcriptome (237,954 ESTs) using in silico procedures. 129 sugarcane clusters were identified (out of 1,476 transcripts) and their expression profile was evaluated. Higher expression was observed in libraries of flowers, roots and normalized mix of tissues, confirming their multi-function character besides the plant-bacteria endophytic interaction in sugarcane. The multiple alignments revealed high homology among sugarcane sequences and respective proteins from other plants, mainly monocots, revealing a relatively conserved genetic structure among species, probably regarding ancient genetic processes.

Identification of conserved secondary structures and expansion segments in ENOD40 RNAs reveals new ENOD40 homologues in plants

Article

Full-text available

Feb 2007
NUCLEIC ACIDS RES

enod40 is a plant gene that participates in the regulation of symbiotic interaction between leguminous plants and bacteria or fungi. Furthermore, it has been suggested to play a general role in non-symbiotic plant development. Although enod40 seems to have multiple functions, being present in many land plants, the molecular mechanisms of its activity are unclear; they may be determined though, by short peptides and/or RNA structures encoded in the enod40 genes. We utilized conserved RNA structures in enod40 sequences to search nucleotide sequence databases and identified a number of new enod40 homologues in plant species that belong to known, but also, to yet unknown enod40-containing plant families. RNA secondary structure predictions and comparative sequence analysis of enod40 RNAs allowed us to determine the most conserved structural features, present in all known enod40 genes. Remarkably, the topology and evolution of one of the conserved structural domains are similar to those of the expansion segments found in structural RNAs such as rRNAs, RNase P and SRP RNAs. Surprisingly, the enod40 RNA structural elements are much more stronger conserved than the encoded peptides. This finding suggests that some general functions of enod40 gene could be determined by the encoded RNA structure, whereas short peptides may be responsible for more diverse functions found only in certain plant families.

Tobacco BY-2 Cell Line as the “HeLa” Cell in the Cell Biology of Higher Plants

Article

Full-text available

Dec 1992
INT REV CYTOL

This chapter highlights tobacco BY-2 (TBY-2) cell line. TBY-2 derived from the seedlings of N. tabacum L. cv. Bright Yellow 2 grows fast and multiplies 80- to 100-fold in 1 week. After the stationary phase, cells of TBY-2 are transferred to a medium containing aphidicolin for 24 hr and then released from treatment; high synchrony is obtained starting from the S phase. TBY-2 cells are propagated in the modified medium of Linsmaier and Skoog, in which KH2PO4 and thiamine HCl are increased to 370 and 1 mg/liter, respectively, and sucrose and 2,4-D are supplemented to 3% and 0.2 mg/liter, respectively. The preparation of protoplasts from TBY-2 cells, from which the isolation of organelles is easy, has been established. Using the synchrony system, the change in the cell cycle progression of TBY-2 cells successfully followed the change in cytoskeletons. Biochemical and molecular biological studies can also be done on TBY-2 cells, as mass culture of this material is readily feasible.

ENOD40 induces dedifferentiation and division of root cortical cells in legumes

Article

Full-text available

Aug 1997
P NATL ACAD SCI USA

Under nitrogen-limiting conditions Rhizobium meliloti can establish symbiosis with Medicago plants to form nitrogen-fixing root nodules. Nodule organogenesis starts with the dedifferentiation and division of root cortical cells. In these cells the early nodulin gene enod40, which encodes an unusually small peptide (12 or 13 amino acids), is induced from the beginning of this process. Herein we show that enod40 expression evokes root nodule initiation. (i) Nitrogen-deprived transgenic Medicago truncatula plants overexpressing enod40 exhibit extensive cortical cell division in their roots in the absence of Rhizobium. (ii) Bombardment of Medicago roots with an enod40-expressing DNA cassette induces dedifferentiation and division of cortical cells and the expression of another early nodulin gene, Msenod12A. Moreover, transient expression of either the enod40 region spanning the oligopeptide sequence or only the downstream region without this sequence induces these responses. Our results suggest that the cell-specific growth response elicited by enod40 is involved in the initiation of root nodule organogenesis.

ASC4, a Primary Indoleacetic Acid-responsive Gene Encoding 1-Aminocyclopropane-1-carboxylate Synthase in Arabidopsis thaliana

Article

Full-text available

Aug 1995

1-Aminocyclopropane-1-carboxylic acid (ACC) synthase is the key regulatory enzyme in the biosynthetic pathway of the plant hormone ethylene. The enzyme is encoded by a divergent multigene family in Arabidopsis thaliana, comprising at least five genes, ACS1-5 (Liang, X., Abel, S., Keller, J. A., Shen, N. F., and Theologis, A.(1992) Proc. Natl. Acad. Sci. U. S. A. 89, 11046-11050). In etiolated seedlings, ACS4 is specifically induced by indoleacetic acid (IAA). The response to IAA is rapid (within 25 min) and insensitive to protein synthesis inhibition, suggesting that the ACS4 gene expression is a primary response to IAA. The ACS4 mRNA accumulation displays a biphasic dose-response curve which is optimal at 10 μM of IAA. However, IAA concentrations as low as 100 nM are sufficient to enhance the basal level of ACS4 mRNA. The expression of ACS4 is defective in the Arabidopsis auxin-resistant mutant lines axr1-12, axr2-1, and aux1-7. ACS4 mRNA levels are severely reduced in axr1-12 and axr2-1 but are only 1.5-fold lower in aux1-7. IAA inducibility is abolished in axr2-1. The ACS4 gene was isolated and structurally characterized. The promoter contains four sequence motifs reminiscent of functionally defined auxin-responsive cis-elements in the early auxin-inducible genes PS-IAA4/5 from pea and GH3 from soybean. Conceptual translation of the coding region predicts a protein with a molecular mass of 53,795 Da and a theoretical isoelectric point of 8.2. The ACS4 polypeptide contains the 11 invariant amino acid residues conserved between aminotransferases and ACC synthases from various plant species. An ACS4 cDNA was generated by reverse transcriptase-polymerase chain reaction, and the authenticity was confirmed by expression of ACC synthase activity in Escherichia coli.

Ethylene biosynthesis and signaling networks

Article

Jan 2002

Article

Jan 1993

Progress through the plant cell cycle involves changes of phosphorylation state and catalytic activity in p34cdc2 -like protein. The plant protein has protein kinase activity, binds p13suc1, copurifies with cyclin B-like protein and its catalytic activation at mitosis is shown, by a metaphase arresting mutant, to be tightly coupled to cyclin accumulation and breakdown. These properties correlate with findings in other laboratories that some plant cdc2 genes can complement the yeast cdc2 gene. Activation of p34cdc2 at mitosis is proposed to trigger preprophase band breakdown since these two events continue in cells inhibited with okadaic acid although completion of chromosome condensation and spindle formation, which normally precede breakdown, do not occur and are therefore not triggers. In plant development the level of this key division protein relative to other proteins declines in cells entering division to less than one-tenth of that in dividing cells and resumption of division follows restoration of the level in all tissues that have been tested, including cotyledon, stem pith and root. Hormonal induction of division revealed tissue-specific differences in effects of individual hormones on p34cdc2-like protein accumulation and its catalytic activation. In tobacco pith, auxin could induce synthesis of p34cdc2-like protein but cytokinin was necessary for its activation and for cell cycle activity. Within the cell cycle, hormone-dependent control points were identified at late G1 and at mitotic initiation by measurement of nuclear DNA content and the timing of activation of p34cdc2 and cell division, in Nicotiana suspension culture cells restimulated with hormone. Auxin was required at both control points, but cytokinin was required only at mitotic initiation. A direct effect of auxin on cdc2 gene expression is indicated by an increase of cdc2 mRNA levels within 10 min of exposing root tissue to 3-indolylacetic acid (IAA).

Rearrangement of Cortical Microtubules in Elongating Cells derived from Tobacco Protoplasts — A Time-course Observation by Immunofluorescence Microscopy —

Article

Aug 1988
J PLANT PHYSIOL

Protoplasts derived from a tobacco cell culture (BY-2), Nicotiana tabacum L. cv. Bright Yellow, could be elongated by cultivation in a medium containing α-naphthaleneacetic acid and benzyladenine (Hasezawa and Syono, 1983). The transition of cortical microtubules (MTs) in the elongating cells were closely observed by immunofluorescence microscopy. The arrays of MTs were random in the protoplasts, but they were gradually rearranged with cell elongation to form lines at right angles to the elongating axis. After 10 days of culture, the MT arrays became sparse and began to slant towards the axis. Finally, MT arrays collapsed and the cells lost dividing abilities. The significance of this system in the study of the cytoskeleton of higher plant cells will be discussed.

Molecular cloning: A laboratory manual, second ed

Article

Jan 1989

Legume nodule organogensis

Article

Mar 1998
TRENDS PLANT SCI

Bacteria of the family Rhizobiaceae and leguminous plants have the ability to establish a symbiosis in which the bacteria fix dinitrogen within a novel plant organ, the root nodule. The development of the root nodule involves signal exchange between the bacterial symbionts and their plant hosts. Flavonoids produced by the plant trigger the induction of bacterial nodulation genes that encode proteins involved in production and transport of specific lipo-chitin signal molecules that initiate nodule organogenesis. Several discoveries regarding the molecular mechanisms of nodule organogenesis have recently been made, with implications for understanding Nod-signal action, and bringing into focus possibilities such as the identification of Nod-signal receptors and investigations into putative conserved lipo-chitin developmental regulators in both legumes and non-legumes.

Plant Hormone-Induced Changes in the Orientation of Cortical Microtubules: Alterations in the Cross-Linking Between Microtubules and the Plasma Membrane

Article

Nov 2003
Annu Rev Plant Physiol Plant Mol Biol

H Shibaoka

Nod factors and cytokinins induce similar cortical cell division, amyloplast deposition and MsEnod12A expression patterns in alfalfa roots

Article

Feb 2003
PLANT J

Under nitrogen limitation, Rhizobium meliloti Nod factors induce cell divisions in the inner cortex of alfalfa roots in a still unknown way. These cell division clusters subsequently develop into symbiotically nitrogen-fixing nodules. To study the involvement of plant signals in nodule initiation transgenic alfalfa carrying the promoter of the early nodulin gene MsEnod12A fused to the reporter gene gusA were generated. In untreated plants, low level GUS staining was only found in lateral root primordia and in front of the root apices. After inoculation with R. meliloti or after treatment with purified Nod factors, GUS activity was first induced in the cell division foci of the inner cortex. The GUS staining patterns in nodules and roots were in agreement with the activation of the endogenous MsEnod12A gene as revealed by reverse transcription-PCR analysis, rendering the MsEnod12A-gusA fusion a valuable novel marker for studying the onset of nodule and lateral root developmental processes. Treatment of roots with purified Nod factors and cytokinins induced similar patterns of cortical cell division, GUS staining and amyloplast accumulation while upon application of auxin transport inhibitors and auxins these patterns were different. Like the Nod factor responses, the cytokinin responses required photosynthesis and limiting combined nitrogen supply. Thus, cytokinins and Nod factors may share elements of their signal transduction pathways to the inner root cortex. A model on the possible involvement of cytokinins in coordinating plant metabolism with nodule initiation is proposed.

ENOD40 affects phytohormone cross-talk /

Abstract and Figures

Recommended publications

Changes in cotton leaf lipid and surface wax synthesis with phytohormone treatment and stage of plan...

Hormonal control of organogenesis on leaf explants of Browallia /

Phytohormonal regulation of 3-Hydroxy-3-methylglutaryl Coenzyme A Reductase in plant cell cultures [...

Biochemische Untersuchungen über die Wirkung endogener Phytohormone auf Differenzierungsprozesse bei...