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RESEARCH PAPER
ENOD40 affects elongation growth in tobacco
Bright Yellow-2 cells by alteration of ethylene
biosynthesis kinetics
Tom Ruttink
1,
*, Kees Boot
2
, Jan Kijne
2
, Ton Bisseling
1
and Henk Franssen
1,†
1
Laboratory of Molecular Biology, Department of Plant Sciences, Wageningen University, Dreijenlaan 3,
6703 HA, Wageningen, The Netherlands
2
Institute of Biology, Leiden University, Wassenaarseweg 64, 2333 AL, Leiden, The Netherlands
Received 31 March 2006; Accepted 21 June 2006
Abstract
Plant developmental processes are controlled by co-
ordinated action of phytohormones and plant genes
encoding components of developmental response
pathways. ENOD40 was identified as a candidate for
such a plant factor with a regulatory role during nod-
ulation. Although its mode of action is poorly under-
stood, several lines of evidence suggest interaction
with phytohormone response pathways. This hypothe-
sis was investigated by analysing cytokinin-, auxin-,
and ethylene-induced responses on cell growth and
cell division in transgenic 35S:NtENOD40 Bright Yellow-
2 (BY-2) tobacco cell suspensions. It was found that
cell division frequency is controlled by the balance
between cytokinin and auxin in wild-type cells and
that this regulation is not affected in 35S:NtENOD40
lines. Elongation growth, on the other hand, is reduced
upon overexpression of NtENOD40.Analysisofethy-
lene homeostasis shows that ethylene accumulation
is accelerated in 35S:NtENOD40 lines. ENOD40
action can be counteracted by an ethylene perception
blocker, indicating that ethylene is a negative regulator
of elongation growth in 35S:NtENOD40 cells, and that
the NtENOD40-induced response is mediated by alter-
ation of ethylene biosynthesis kinetics.
Key words: BY-2 cells, elongation growth, ENOD40, ethylene.
Introduction
ENOD40 homologues have been identified in plant spe-
cies across the plant kingdom, including monocots like rice
(Kouchi et al., 1999), rye grass, barley (Larsen et al., 2003),
Zea mays (Compaan et al., 2003), and sorghum, and dicots
such as tomato (Vleghels et al., 2003), tobacco (Matvienko
et al., 1996), citrus, and numerous leguminous species.
The highest expression levels of ENOD40 have been found
during legume nodule formation, and therefore its func-
tion has been studied in most detail during this process.
Misregulation of ENOD40 in Medicago truncatula by co-
suppression reduces the number of nodules and nodule
development is arrested, indicating that ENOD40 has a
regulatory role in nodule organogenesis (Crespi et al.,
1994; Charon et al., 1999). Ectopic expression of ENOD40,
on the other hand, induces cortical cell divisions in
Medicago roots and accelerates nodule development
(Charon et al., 1997, 1999). However, ENOD40 expression
alone is not sufficient for nodule primordium formation
(Minami et al., 1996; Mathesius et al., 2000), and inter-
action with other plant factors is probably required for the
initiation of nodule development. Several observations
(Hirsch et al., 1989; Peters and Crist-Estes, 1989; Lee
and LaRue, 1992; Cooper and Long, 1994; Heidstra et al.,
1997) show the involvement of phytohormones, in par-
ticular auxin, cytokinin, and ethylene, and of ENOD40
(Charon et al., 1997) suggesting that, during nodule devel-
opment, cross-talk between ENOD40 and phytohormone
signalling exists. The expression of ENOD40 homologues in
developmental processes in non-leguminous plant species, for
example, during lateral root formation, flower development,
* Present address: Department of Plant Systems Biology, VIB/Gent University, Technologiepark 927, 9052, Gent, Belgium.
y
To whom correspondence should be addressed. E-mail: Henk.Franssen@wur.nl
Journal of Experimental Botany, Vol. 57, No. 12, pp. 3271–3282, 2006
doi:10.1093/jxb/erl089 Advance Access publication 6 September, 2006
ªThe Author [2006]. Published by Oxford University Press [on behalf of the Society for Experimental Biology]. All rights reserved.
For Permissions, please e-mail: journals.permissions@oxfordjournals.org
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and vascular tissue development (Kouchi et al., 1999;
Varkonyi-Gasic and White, 2002; Vleghels et al., 2003),
indicates that the function of ENOD40 is not confined
to nodule development in leguminous species and suggests
that ENOD40 has a general role in plant development. This
notion is supported by the observation that ectopic expres-
sion of ENOD40 affected formation of somatic embryos
of alfalfa under in vitro culture conditions (Crespi et al.,
1994). Also, overexpression of ENOD40 led to reduced
apical dominance in tobacco (van de Sande et al., 1996).
Both observations indicate that phytohormone signalling is
affected by ENOD40. Up to now, the function of ENOD40
and its mode of action have been poorly understood.
Although observations in both legumes and non-legumes
are pointing to a cross-talk between ENOD40 activity and
phytohormone signalling pathways, direct evidence for
such an interaction is lacking. Establishing whether the
function of ENOD40 involves interaction with phyto-
hormone signalling pathways could be an important step
towards unravelling the role of ENOD40 during organo-
genesis. Therefore, a search was made for a system that
would make it possible to test whether cross-talk between
ENOD40 and phytohormone signalling occurs. The to-
bacco Bright Yellow-2 (BY-2) cell suspension was chosen
as a model system as it is convenient for studying phyto-
hormone responses on a cellular level. In BY-2 cells,
elongation growth and cell division are regulated by the
balance between cytokinin and auxin in the culture medium
(Hasezawa and Syono, 1983). Thus, cell elongation growth
and cell division frequency can be used as morphological
markers to study whether overexpression of ENOD40
affects the response of BY-2 cells to phytohormones. It
was found that overexpression of ENOD40 negatively af-
fects cell elongation growth, whereas cytokinin- or auxin-
dependent control of cell division frequency is not affected
in 35S:NtENOD40 transgenic cell lines. It was shown
further that the altered ethylene biosynthesis kinetics ob-
served in ENOD40-overexpressing cells is a primary cause
of the reduction in cell elongation growth.
Materials and methods
Construction of binary vector p35S:NtENOD40
Nicotiana tabacum contains two ENOD40 homologues that are
96% identical at the nucleotide level (Matvienko et al., 1996). The
cauliflower mosaic virus 35S promoter from pMON999 (Monsanto)
was transferred to pCambia 1390 (Cambia, Australia) yielding
p35S:Tnos. A 470 bp PCR fragment corresponding to the Nt-
ENOD40-1 cDNA sequence was then cloned in p35S:Tnos.
Liquid BY-2 cultures and BY-2 transformation
Nicotiana tabacum BY-2 cell suspensions were subcultured weekly
by 403dilution 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 millilitres of a 3-d-old BY-2
cell suspension was co-cultivated for 2 d at 25 8C in the dark with
60 ll of log-phase Agrobacterium tumefaciens strain C58C1,
harbouring the binary vector. Cells were washed three times before
plating on culture medium supplemented with 0.8% Daishin agar,
200 lgl
ÿ1
ticarciline/clavuline, and 40 lgl
ÿ1
hygromycin B.
Transgenic calli that appeared after 3–4 weeks, were cultured on fresh
selection plates for 1 more week, and were subsequently transferred
to liquid selection medium. Six independent 35S:NtENOD40 BY-2
cell lines were generated and named lines Nt1 to Nt6.Eachtransgenic
line was derived from a different callus, which means that they can-
not be siblings. Transgenic lines were continuously maintained in
selection medium.
Protoplast isolation
Protoplasts were obtained from 6-d-old suspension cultures using
1% (w/v) cellulase-YC and 0.1% (w/v) pectolyase Y23 in 0.4 M
D-mannitol, pH 5.5 (Nagata et al., 1981). Cells were incubated in the
enzyme solution for 3 h at room temperature, filtered through 63 lm
nylon mesh, washed twice with 0.2 M 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
ÿ1
MS salts
(without vitamins) supplemented with 1 mg l
ÿ1
thiamine-HCl,
100 mg l
ÿ1
myo-inositol, 10 g l
ÿ1
sucrose, 255 mg l
ÿ1
KH
2
PO
4
,
and 0.4 M D-mannitol at pH 5.7. Elongation growth-inducing
PCM contained 0.1 mg l
ÿ1
1-naphthalene-acetic acid (NAA) and
1.0 mg l
ÿ1
benzyl-adenine (BA). Protoplasts were cultured in 3 ml
liquid medium at a density of approximately 10
5
ml
ÿ1
in small
sealed Petri dishes at 25 8C in the dark (Kuss and Cyr, 1992).
Protoplast assay growth parameter measurements
Growth parameter measurements were performed on random photo-
graphs of protoplast-derived cells after 4 d of culture. Viable cells
were selected for measurements using FDA (fluorescein-diacetate)
staining (Fig. 1). Fluorescent images were captured using a cooled
CCD camera mounted on a Leica DMR microscope with a 320
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. The parameters measured
were number of cells per file, cell width, and cell file length. For each
sample, the average cell division frequency was calculated as (total
number of cells/number of cell files)–1. The cell file length was first
expressed in width units, by calculating the length:width ratio for
individual cells and then averaged over 100–150 cells per sample.
Elongation growth was calculated as increase in cell file length during
the culture time as cell file length
end
–cell file length
begin
, where cell
file length
begin
=1 for a spherical protoplast. Values presented in the
graphs represent average values and standard deviations for each
line/condition, calculated over a number of independent repetitions,
as indicated in the text.
In order to determine elongation growth capacity of the six
individual transgenic lines, protoplasts from the wild-type and the
Nt1–Nt6 cell lines were cultured for 4 d in PCM in the presence of
1.0 mg l
ÿ1
BA and 0.1 mg l
ÿ1
NAA. At least five independent ex-
periments were performed. Pairwise comparisons between trans-
genic lines and the wild type were performed using a two-tailed
Student’s ttest. Significance values were adjusted for multiple tests.
RNA gel blot analysis
Total RNA was isolated using the TRIzol method (GibcoBRL). A
16 lg aliquot of total RNA was subjected to electrophoresis on
a 1% agarose gel in 0.01 M NaH
2
PO
4
(pH 7.0) using the glyoxal/
DMSO method. RNA was subsequently transferred to a genescreen
membrane in 203SSC. RNA gel blots were hybridized with radio-
labelled PCR fragments of the respective transcripts in formamide
hybridization buffer overnight at 42 8C. Autoradiograms were obtained
using a Molecular Dynamics phosphorimager (Sunnyvale, CA, USA).
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Reverse transcriptase-mediated PCR
Total RNA was isolated using the TRIzol method (GibcoBRL). After
DNase I (Promega) treatment to remove chromosomal DNA, cDNA
is synthesized from 2.5 lg of total RNA in a volume of 20 ll [10 mM
TRIS-HCl pH 8.8, 50 mM KCl, 5 mM MgCl
2
, 1 mM dNTPs, 1 lg
oligo-dT
(12)
V anchor primer, 20 U RNA guard (Pharmacia), and
200 U MuMLV reverse transcriptase (RT; Stratagene)]. The samples
were incubated for 1 h at 37 8C and subsequently at 95 8C for 5 min
to inactivate the enzyme. The samples were then diluted to 100 ll
and 1 or 2 ll of the cDNA were used for PCR analysis [10 mM
TRIS-HCl pH 8.3, 50 mM KCl, 2.5 mM MgCl
2
, 100 lM dNTPs,
50 ng primer, and 1 U Taq polymerase (Boehringer Mannheim,
USA) in a total volume of 50 ll].
Primer sets were designed for RT-mediated PCR-based transcript
quantification for each of the genes analysed. 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 ubiquitin levels.
The following primers were used for RT-PCR: UBI-f, 59-ATGCA-
GAT(C/T)TTTGTGAAGAC-39;UBI-r, 59-ACCACCACG(G/A)A-
GACGGAG-39;ACS-f, 59-GATTTAATACAAGAATGGG-39;ACS-r,
59-GAACAATGAAAAGAACAAC-39;ACO-f, 59-GGGCTTCTTTGA
GTTGGTG-39;ACO-r, 59-CTCCGCTGCCTCTTTCTC-39. Ampli-
fied 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 cloned cDNAs.
Autoradiograms were obtained by using a Molecular Dynamics
phosphorimager.
RACE-PCR on NtENOD40 transcripts was as follows: cDNA was
synthesized from RNA isolated from the transgenic lines, using the
RACE-T anchor primer 59-CATCTAGAGGATCGAATTC-T
(16)
-39.
The PCR cycles were 94 8C for 5 min; 30 cycles of 94 8C for 20 s,
50 8C for 20 s, 72 8C for 30 s, and a final extension at 72 8C for 5 min
using, in the first run, primers RACE-A, 59-CATCTAGAGGATC-
GAATTC-39, and reverse primer, 59-CGGGATCCTAGTTGGAGT-
GAATTAAGGA-39, and, in the second run, RACE-A primer and
reverse primer, 59-AAGCTTTTGGAGTCTTTCTTGGCCTTT-39.
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).
Dose–response curves
Protoplasts from three lines (WT, Nt1, and Nt2) were cultured for 4 d
in PCM with various concentrations of cytokinin (0.0 mg l
ÿ1
BA;
0.1 mg l
ÿ1
BA; 0.5 mg l
ÿ1
BA; 1.0 mg l
ÿ1
BA; and 2.0 mg l
ÿ1
BA)
and a fixed concentration of auxin (0.1 mg l
ÿ1
NAA), or with vari-
ous concentrations of auxin (0.0 mg l
ÿ1
NAA; 0.05 mg l
ÿ1
NAA;
0.1 mg l
ÿ1
NAA; 0.5 mg l
ÿ1
NAA; and 1.0 mg l
ÿ1
NAA) and a
fixed concentration of cytokinin (1.0 mg l
ÿ1
BA). The auxin/cytokinin
dose–response curve (DRC) experiment was repeated six, three, and
five times for the wild-type, Nt1, and Nt2 lines, respectively.
Significant line (P
L
), dosage (P
D
), and the interaction between line
and dosage (P
L
3
D
) effects were obtained by two-way analysis of
variance (ANOVA) using the SAS glm procedure (windows version
9.1; SAS Inc, North Carolina, USA). The model applied was:
y
ijk
=l+L
i
+D
j
+L3D
ij
+e
ijk
, where y
ijk
is either the elongation growth
or the cell division frequency from line i(i=1, 2, 3), dosage j(j=1,
..., 5), and observation k(k=1, ..., 6). lrepresents the overall mean,
Lis the main effect for lines, Dis the concentration or dosage ef-
fect for BA or NAA, L3Dis the interaction effect, and eis the stocha-
stic error. Differences of the least square means (LSMeans) for the
line3dosage effects, along with associated t-tests and P-values, were
calculated. For the DRC experiment of the wild type, single factor
analysis was performed to estimate the significance of the dosage
effect, using the model y
jk
=l+D
j
+e
jk
. Duncan’s multiple range test
was applied to compare the effects of the different concentrations.
In a similar set-up, the ethylene perception blocker AgNO
3
was ap-
plied at a concentration range from 10
ÿ8
Mto10
ÿ5
M with 10-fold
increments to protoplasts of wild-type, Nt1, and Nt2 lines, cultured
in PCM supplemented with 0.1 mg l
ÿ1
NAA and 1.0 mg l
ÿ1
BA. To
give the appropriate final concentrations of AgNO
3
,30ll of a serial
dilution of AgNO
3
in water was transferred to the culture medium
containing the protoplasts just before sealing the Petri dishes at the
start of the culture period. Three independent sets of experiments
were performed. Regression analysis of each line was performed
using SPSS 9.0 (SPSS, Chicago, IL, USA). The slopes (means 6
standard error) of the linear functions obtained for the lines Nt1,Nt2,
and wild type were 54.500624.000 (P=0.041; R
2
=0.28); 90.500
613.000 (P<0.001; R
2
=0.80), and ÿ2.790628.300 (P=0.923;
R
2
=0.004), respectively.
Ethylene measurements
For each line, protoplasts were divided over six Petri dishes at the
start of the experiment and were cultured in parallel. Each Petri dish
was sampled every 24 h for 7 d. In order not to severely alter
accumulating ethylene levels, gas samples of 1 ml from a total
of 30 ml headspace volume, were taken with a syringe through a
rubber gasket in the lid of the Petri dish without opening the
sealed Petri dishes. Ethylene concentration was determined directly
by standard GC-analysis on a gas chromatograph equipped with
an alumina column and a flame ionization 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.
Distribution of materials
Upon request, all novel materials described in this publication will
be made available in a timely manner for non-commercial re-
search purposes.
Results
Cytokinin–auxin DRCs of BY-2 cells
To create a reference to determine the effect of NtENOD40
overexpression on the phytohormone response of BY-2
cells, cytokinin and auxin DRCs of wild-type BY-2 cells
were made by measuring elongation growth and cell
division as a function of increasing cytokinin or auxin
concentration. It has been shown previously that the most
accurate data concerning cytokinin- and auxin-regulated
elongation growth and cell division in BY-2 cells are
obtained with a bioassay starting from BY-2 protoplasts,
which subsequently divide and elongate (Hasezawa and
Syono, 1983). The reason for using protoplasts in these
assays is that the composition of the BY-2 cell suspension
is heterogeneous with respect to cell file length and number
of cells per file. By preparing protoplasts from the cell sus-
pension, a population of single cells with a similar diameter
is obtained. Analysing growth parameters of these cultured
protoplasts has an advantage over using the cell suspension
directly as it allows the effects on elongation growth and
cell division to be separated. Firstly, under the conditions
of the present study the number of cell files during culture
ENOD40 and ethylene biosynthesis kinetics 3273
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remains similar to the number of protoplasts at the start of
the experiment. So cells remain attached to each other after
division. Hence, by starting from protoplasts, the increase
in the number of cells per cell file directly reflects the
number of cell divisions that took place during the incu-
bation time, and this parameter is from here onwards called
the ‘cell division frequency’. This parameter is expressed as
the average number of cells per cell file–1, i.e. cultured
protoplasts remain single cells when no cell division takes
place, whereas finding two cells per file means that one
round of cell division has occurred during the incubation
time. Secondly, the width of cells remains similar during
culture to the diameter of protoplasts at the start of the
incubation (Fig. 1A, B). This means that no radial ex-
pansion growth occurs and the length of the cell files at the
end of the culture period is a measure of elongation growth.
This parameter is hereafter called the ‘cell file length’ and
is expressed in width units. Elongation growth is calculated
as the increase in cell file length during the culture time.
During the incubation time of 4 d used in these bioas-
says, wild-type cells can become, on average, about four to
five times as long as a protoplast. So, the increase in cell
length is about three to four times the initial size of a
protoplast.
DRCs were made using protoplasts prepared from the
wild-type cell line (see Materials and methods). For the
cytokinin DRC, elongation growth and cell division fre-
quency were determined as a function of cytokinin (BA)
concentration at a fixed concentration of auxin (0.1 mg l
ÿ1
NAA). For the auxin DRC, the same parameters were
determined as a function of auxin (NAA) concentration at a
fixed concentration of cytokinin (1.0 mg l
ÿ1
BA) (see
Materials and methods). Although data for the wild-
type line were obtained in parallel with that of the trans-
genic lines (see below), the results for the wild-type line are
discussed first to illustrate the action of cytokinin and auxin
in wild-type BY-2 cells. The DRCs were made six times,
in independent experiments, for the wild-type line. The re-
sults from independent experiments were similar and the
average value (6standard deviation) was calculated for
each parameter (Fig. 2A, B). The cytokinin DRC for elong-
ation growth (Fig. 2A) shows that elongation growth is
maximal when cells are grown in the absence of exogenous
cytokinin. Application of increasing concentrations of
Fig. 1. Representative photographs of protoplasts and protoplast-derived
cells of wild-type and 35S:NtENOD40 transgenic lines. (A) Wild-type
protoplasts directly after protoplast isolation. (B) Wild-type cells after 4 d
of culture in elongation growth-inducing medium. (C) 35S:NtENOD40
(Nt1) cells after 4 d of culture showing a reduction in elongation growth.
(D) 35S:NtENOD40 (Nt1) cells cultured for 4 d in the presence of 10 lM
AgNO
3
showing a recovery of elongation growth. The pictures were
taken with a fluorescence microscope after FDA staining of cells. This fac-
ilitated selection of viable protoplasts for measurements and aided object
recognition with the NIH-image software for quantification of growth
parameters. Scale bars=100 lm.
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cytokinin gradually reduces cell file length of wild-type
cells, suggesting that addition of cytokinin has a mild
negative effect on elongation growth (one-way ANOVA,
P
D
=0.0985). Application of 2 mg l
ÿ1
BA can provoke a
reduction of cell file length of 0.78 units (corresponding to a
21% reduction of elongation growth) compared with the
cell file length reached when cells are grown in the absence
of exogenous cytokinin. Duncan’s multiple range test
indicated that this difference is significant at the 0.05 level.
Thus, exogenous cytokinin is not essential for elongation
growth when auxin is present and exogenous application
of cytokinin has a mild negative effect on elongation
growth. The auxin DRC for cell file length (Fig. 2B) shows
that cell file length is not affected (one-way ANOVA,
P
D
=0.8991) by the concentration of auxin as, in the absence
of auxin, it is similar to that at the various concentrations
of exogenous auxin. This shows that elongation growth
neither requires auxin, nor does auxin markedly affect it,
when cytokinin is applied to the medium. These data show
that the presence of either cytokinin or auxin is sufficient
to sustain the growth rates achieved under the culture
conditions of the present study and that only addition of
cytokinin has a mild negative effect on elongation growth.
The cytokinin DRC for cell division frequency (Fig. 2A)
shows that the cell division frequency is reduced from
0.78 to 0.12 at increasing concentrations of cytokinin, and
that the cell division frequency is highest in the absence
of exogenous cytokinin. The auxin DRC for cell division
frequency (Fig. 2B) shows that the cell division frequency
increases from 0.00 to 0.64 at increasing concentrations
of auxin. One-way ANOVA analysis indicated that the
cell division responses provoked by cytokinin and auxin
are highly significant (P
D
<0.0001). In the absence of
auxin, all cell files still consist of a single cell after 4 d of
culture, 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 divi-
sion and that exogenous auxin has a stimulating effect on
cell division, whereas elongation growth is only mildly
reduced by exogenous application of cytokinin in wild-type
BY-2 cells.
Generation of stably transformed BY-2 cell lines
carrying 35S:NtENOD40
To determine whether overexpression of NtENOD40 af-
fects responses to phytohormones in BY-2 cells, a set of
six independent 35S:NtENOD40 BY-2 cell lines, called
lines Nt1 to Nt6, was generated by Agrobacterium-mediated
transformation (see Materials and methods). Expression of
the transgenes was determined by RNA gel blot analysis
(Fig. 3A). 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 35S:NtENOD40
lines, NtENOD40 transcripts were expressed at various
levels. In all lines except line Nt6, this level was much
higher than in the wild-type line, with the highest expres-
sion in lines Nt1 and Nt2 and the lowest expression in Nt5
and Nt6. Further, hybridization with the NtENOD40 probe
resulted in two bands on the RNA gel blot. To characterize
the nature of these two RNAs further, 39-RACE-PCR was
performed on NtENOD40 transcripts of the transgenic lines.
Analysis of nucleotide sequences of 11 cloned RACE-
PCR fragments revealed that all sequences are fully iden-
tical to the transgene sequence and that read-through occurs
at the NOS terminator that flanks the NtENOD40 cDNA
sequence in the construct, resulting in transcripts of two
different lengths (data not shown).
Fig. 2. Dose–response curves for cytokinin and auxin in wild-type BY-2 cells. (A) Wild-type elongation growth and cell division frequency at
increasing concentrations of BA, each in the presence of 0.1 mg l
ÿ1
NAA. (B) Wild-type elongation growth and cell division frequency at increasing
concentrations of NAA, each in the presence of 1.0 mg l
ÿ1
BA. Data represent average (6standard deviation) of six independent repetitions.
ENOD40 and ethylene biosynthesis kinetics 3275
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Cytokinin–auxin DRCs of 35S:NtENOD40 transgenic
BY-2 cells
To select lines in which the effect of NtENOD40 over-
expression on hormone responses can be studied, first the
effect of ENOD40 overexpression was determined on
elongation growth and division frequency of cells grown
in the presence of 1.0 mg l
ÿ1
BA and 0.1 mg l
ÿ1
NAA.
These conditions represent the intersection of the two sets
of conditions used in the DRCs (Fig. 2). To this end,
protoplasts were obtained from the wild-type and Nt1–Nt6
cell lines and subsequently cultured for 4 d in PCM (see
Materials and methods). Representative photographs taken
directly after protoplast isolation, as well as after 4 d of
culture of wild-type cells and cells of a transgenic line with
a strong phenotype (line Nt1) are presented in Fig. 1A–C.
These pictures show the typical elongated appearance
of wild-type cells after 4 d of culture, whereas cells are
markedly smaller in line Nt1. Wild-type and transgenic
cells are of similar size during propagation of the cell
suspension culture and so the size of isolated protoplasts
is equal. Cell file length and cell division frequency were
determined for each line in at least five independent
experiments (see Materials and methods). The results
from independent experiments were similar and the aver-
age value (6standard deviation) was calculated for each
parameter (Fig. 3B, C). It was found that four lines, Nt1,
Nt2,Nt3, and Nt4, have a strongly reduced cell file length as
compared with the wild type (Fig. 3B). Elongation growth
in these lines ranged from 1.66 units (Nt1) to 0.84 units
(Nt3), corresponding to 56% and 29%, respectively, of the
elongation growth of the wild-type line, and is statistically
significant (two-tailed Student’s t-test, P<0.001) for these
four lines. The elongation growth of lines Nt5 and Nt6 is
similar to the wild type (Fig. 3B). The cell division
frequency in the wild-type line is 0.1560.09. This means
that about 15% of the cells have divided once. The cell
division frequency in the transgenic lines is similar to
that of the wild type (Fig. 3C). This shows that di-
vision frequency is not affected in the transgenic lines.
Taken together, these data show that the lines with the
highest expression level (Nt1 and Nt2) display the strongest
phenotype and that in the lines with the lowest level of
NtENOD40 expression no phenotypical change is ob-
served. This indicates that the observed suppression of
elongation growth most likely is correlated to overexpres-
sion of NtENOD40.
To analyse the effect of NtENOD40 overexpression on
cell division frequency and elongation growth in more
detail and to test for a possible effect of ENOD40 on auxin
or cytokinin responses, DRCs were made for the transgenic
lines Nt1 and Nt2, the two lines that have the highest level
of NtENOD40 expression and a strong phenotype, and
are compared with those of the wild-type line (Fig. 4A–D).
As in the wild-type line, the cell division frequency in
both transgenic lines shows a strong dose-dependent
response to both BA and NAA. The division frequency
drops from 0.42 to 0.15 for Nt1 cells and from 0.70 to 0.18
for Nt2 cells grown in the presence of 0.1 mg l
ÿ1
NAA at
increasing concentrations of BA. For the wild type the
division frequency decreases from 0.78 to 0.12 under these
Fig. 3. Transgene expression levels, elongation growth, and division
frequency of cultured cells of wild-type (Wt) and 35S:NtENOD40 BY-2
cell lines. Growth parameters of protoplast-derived cells were determined
after 4 d of culture in medium supplemented with 0.1 mg l
ÿ1
NAA and
1.0 mg l
ÿ1
BA. Data represent average (6standard deviation) of 11 (wild
type), eight (Nt1), nine (Nt2), and five (Nt3–Nt6) independent repetitions.
An asterisk marks transgenic cell lines with a significant reduction of
elongation growth compared with the wild type (P<0.001). (A) The level
of transgene expression was determined at the start of protoplast culture
by RNA gel blot analysis. Hybridization with the NtENOD40 probe and
HPTII probes shows expression of the transgene transcripts. Hybridiza-
tion with the ubiquitin (UBI) probe was performed in order to compare
loading of the separate samples. RNA gel blot analysis was performed in
three independent experiments, each time with similar results. One
representative set of data is presented. (B) Elongation growth in wild-type
and 35S:NtENOD40 cell lines. (C) Division frequency in wild-type and
35S:NtENOD40 cell lines.
3276 Ruttink et al.
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conditions. In the presence of 1.0 mg l
ÿ1
BA and at
increasing concentrations of NAA the division frequency
increases from 0.00 to 0.30 for Nt1 cells and from 0.00
to 0.40 for Nt2 cells, while for the wild type the division
frequency increases from 0.00 to 0.64. Two-way ANOVA
analysis indicated a highly significant dosage effect for
BA (P
D
<0.0001) as well as for NAA (P
D
<0.0001) in
both transgenic lines. Although division frequencies of line
Nt1, but not of line Nt2, appear slightly lower than that of
the wild type, they are only significantly different between
line Nt1 and the wild type under conditions that most
strongly induce cell division (at 1.0 mg l
ÿ1
BA and 1.0 mg
l
ÿ1
NAA, P=0.0012; at 0.1 mg l
ÿ1
NAA and 0 mg l
ÿ1
BA,
P=0.0104). Thus, these results indicate that both the in-
hibitory effect of cytokinin (Fig. 4A) and the stimulating
effect of auxin (Fig. 4B) on cell division are similar in
wild-type and Nt1 and Nt2 lines and that overexpression
of ENOD40 does not alter the response to these hormones.
Next, the effect of ENOD40 overexpression on elong-
ation growth was examined in the cytokinin–auxin DRCs
(Fig. 4C, D). In all conditions tested, a strong reduction of
elongation growth in Nt1 and Nt2 cells was observed when
compared with that of wild-type cells cultured in the same
respective conditions. This reduction of elongation growth
is observed throughout both DRCs, and ranges from at
least 1.86 units and 1.66 units (50% and 45% of elongation
growth of corresponding wild-type cells), respectively, for
Nt1 and Nt2 cells cultured at 0 mg l
ÿ1
BA and 0.1 mg l
ÿ1
NAA, to a maximal 1.96 units (Nt1) and 2.24 units (Nt2)
(66% and 76%, respectively), for cells cultured at 1.0 mg
l
ÿ1
BA and 0 mg l
ÿ1
NAA. These data reveal that sup-
pression of elongation growth in 35S:NtENOD40 does
not require exogenous cytokinin when cells are cultured
in the presence of 0.1 mg l
ÿ1
NAA, nor does it require
exogenous auxin when 1.0 mg l
ÿ1
BA is applied. Two-way
ANOVA analysis indicated that elongation growth of
Nt1 as well as Nt2 cells differs significantly (P
L
<0.0001)
from that of wild-type cells throughout both DRCs.
Next, it was investigated whether, in addition to the
strong negative effect of ENOD40 overexpression on
Fig. 4. Dose–response curves for cytokinin and auxin in wild-type and 35S:NtENOD40 BY-2 cells. Protoplasts were cultured for 4 d in medium
supplemented with various concentrations of cytokinin or auxin. Data represent the average (6standard deviation) of six (wild type), three (Nt1), and five
(Nt2) independent repetitions. (A) Dose–response curves measuring division frequency as a function of BA concentration, each in the presence of 0.1 mg
l
ÿ1
NAA. (B) Dose–response curves measuring division frequency as a function of NAA concentration, each in the presence of 1.0 mg l
ÿ1
BA.
(C) Dose–response curves measuring elongation growth as a function of BA concentration, each in the presence of 0.1 mg l
ÿ1
NAA. (D) Dose–
response curves measuring elongation growth as a function of NAA concentration, each in the presence of 1.0 mg l
ÿ1
BA.
ENOD40 and ethylene biosynthesis kinetics 3277
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elongation growth, cytokinin or auxin affect elongation
growth in the transgenic lines. Two-way ANOVA analysis
revealed a significant line effect (P
L
<0.0001), but did not
show a significant auxin-dose effect (P
D
=0.3038), indicat-
ing that auxin does not affect elongation growth in any of
the lines, nor did it show a significant interaction between
line and dosage effects (P
L
3
D
=0.9421), indicating that the
response to exogenous auxin is not different between the
three lines.
By contrast, application of increasing concentrations of
cytokinin gradually reduces cell file length of both trans-
genic lines (Fig. 4C), suggesting that addition of cytokinin
has a mild negative effect on elongation growth, similar
to that observed in the wild-type line. (However, two-way
ANOVA with P
D
<0.0001 gave P-values for the differ-
ences of the LSMeans of the line3dosage effects which
were only slightly significant between the different cyto-
kinin concentrations within one line.) So overexpres-
sion of ENOD40 and exogenous cytokinin both negatively
affect elongation growth in a similar manner. Two-way
ANOVA analysis revealed no significant interaction effects
between cytokinin dosage and line (P
L
3
D
=0.9968), indic-
ating that overexpression of ENOD40 does not signific-
antly affect the response to exogenous cytokinin. Therefore,
it seems unlikely that cytokinin and ENOD40 interact in
elongation growth reduction.
ENOD40 affects ethylene homeostasis
It has previously been shown that ethylene is involved in
the regulation of root and hypocotyl growth. Analysis of
the triple response during ethylene treatments or in mutants
of ethylene synthesis or perception pathways has revealed
that ethylene can act as a negative regulator of elongation
growth in Arabidopsis seedlings (Kieber et al., 1993; Le
et al., 2001). Based on this and on the observation that
elongation growth is strongly reduced in NtENOD40-
overexpressing BY-2 cells, it was tested whether ethylene
could be involved in the mechanism that alters the
elongation growth response in 35S:NtENOD40 lines. The
effect was compared of an ethylene perception blocker
on cell division and elongation growth in the lines Nt1
and Nt2, and the wild-type line. Thus, AgNO
3
was applied
during culture of protoplasts in PCM supplemented with
1.0 mg l
ÿ1
BA and 0.1 mg l
ÿ1
NAA. In three independent
experiments, cell file length and cell division frequency
were scored after 4 d of culture. The results from inde-
pendent experiments were similar and the average value
(6standard deviation) was calculated for each parameter
(Fig. 5A, B). The results show that cell file length of wild-
type cells is similar in the absence and presence of a range
of AgNO
3
concentrations (Fig. 5A). Thus, a block of
ethylene perception has no significant effect (one-way
ANOVA, P
D
=0.9420) on elongation growth in wild-type
cells. In the absence of ethylene perception blockers, cell
file length of the transgenic lines is reduced by 1.47 units
(Nt1) and 1.64 units (Nt2) compared with that of the wild
type, in agreement with previous experiments (Figs 3B,
4C). This suppression of elongation growth corresponds
to 56% (Nt1) and 62% (Nt2) of elongation growth of the
wild-type cells cultured in the absence of AgNO
3
.By
application of 10 lM AgNO
3
at the start of the protoplast
culture, cell file length of the lines Nt1 and Nt2 is only
reduced by 0.92 units (34%) and 0.68 units (25%), res-
pectively, compared with that of the wild type (Fig. 5A).
A representative photograph taken after 4 d of culture of
cells of line Nt1 in the presence of 10 lM AgNO
3
is
presented in Fig. 1D. This shows the elongated appearance
of these Nt1 cells, indicating that a block of ethylene per-
ception restores the growth defect caused by overexpres-
sion of NtENOD40. Two-way ANOVA analysis revealed
a significant (P
LxD
=0.019) interaction between line and
dosage, indicating that the differential response of these
Fig. 5. Recovery of elongation growth of 35S:NtENOD40 cells by
AgNO
3
treatment. Cells were cultured for 4 d in the presence of 0.1 mg
l
ÿ1
NAA, 1.0 mg l
ÿ1
BA, and various concentrations of AgNO
3
. Data
are average (6standard deviation) of three independent repetitions.
(A) Elongation growth in wild-type and 35S:NtENOD40 cell lines.
(B) Division frequency in wild-type and 35S:NtENOD40 cell lines.
3278 Ruttink et al.
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lines to AgNO
3
depends on the dosage. Indeed, further
investigation by regression analysis showed a positive
linear relationship between AgNO
3
dosage and elongation
growth for both transgenic lines (Nt1 P
regr
=0.041; Nt2
P
regr
<0.001), whereas this is not the case for the wild-type
line (P
regr
=0.932). As in the wild type, the division fre-
quency of the transgenic cells is, at all concentrations of
AgNO
3
tested, similar to the division frequency in the
absence of AgNO
3
(Fig. 5B). Two-way ANOVA analysis
indicated neither a significant dose effect (P
D
=0.5383) nor
a line effect (P
L
=0.8239), indicating that neither AgNO
3
,
nor NtENOD40 overexpression affects the division fre-
quency. Taken together, these observations show that an
ethylene perception blocker counteracts ENOD40 action
and, therefore, indicate that the suppressing effect of
ENOD40 on elongation growth is, at least in part, mediated
by ethylene.
Ethylene-mediated responses can be regulated at the
level of ethylene production and/or sensitivity. To discrim-
inate between these two possibilities, ethylene production
kinetics of wild-type, Nt1, and Nt2 lines cultured in the
presence of 1.0 mg l
ÿ1
BA and 0.1 mg l
ÿ1
NAA were
compared. The headspace of protoplast cultures was
sampled at 24 h intervals for 7 d and ethylene concen-
trations were determined by gas chromatography (see
Materials and methods). The experiment was performed
five times with similar results. One representative experi-
ment is presented in Fig. 6A. In the wild-type culture,
ethylene gradually accumulates up to 6 d of culture to a
level of about 2 lll
ÿ1
. After this time, production ceases. In
cultures of the Nt1 and Nt2 lines, ethylene accumulates to
similar maximal levels as in wild-type cultures, but trans-
genic lines already reached a maximal level (2 lll
ÿ1
)
around day 3. These results show that ethylene production
is accelerated in 35S:NtENOD40 lines while the maximal
levels are similar in wild-type and Nt1 and Nt2 lines.
Ethylene accumulation is regulated by ACS but
not ACO expression
The next step was to find out how ethylene biosynthesis
is accelerated in Nt1 and Nt2 lines. Ethylene biosynthesis
can be regulated at different levels, including transcrip-
tional control of gene expression and post-translational
regulation of ACC synthase (ACS) and ACC oxidase
(ACO) (Wang et al., 2002). Therefore, expression kinetics
of these genes in wild-type and transgenic cell lines were
analysed until day 4, the typical time-point for scoring
growth parameters in these studies. In order to compare
ACS and ACO expression levels and ethylene accumulation
during culture of protoplasts, protoplasts were cultured in
the presence of 0.1 mg l
ÿ1
NAA and 1.0 mg l
ÿ1
BA and
cells of each line (wild type, Nt1, and Nt2) were harvested
on days 0, 2, and 4 for RNA extraction. RT-PCR-based
ACS and ACO transcript quantification was performed
using primers targeted to highly conserved sequences,
such that most likely all ACS, respectively ACO, transcripts
that are expressed in BY-2 cells can be amplified in a single
RT-PCR reaction (see Materials and methods).
ACS and ACO transcript expression profiles (Fig. 6B)
are correlated to a time-course of ethylene accumulation
(Fig. 6A). In wild-type cultures, ACS transcripts gradually
accumulate during the 4 d culture period, and maximal ACS
expression levels are found on day 4. By contrast, in
35S:NtENOD40 lines the maximal ACS transcript level is
found on day 0, directly after protoplast isolation, and
Fig. 6. Temporal ethylene accumulation profile and transcript profiles
of genes required for ethylene biosynthesis in wild-type and 35S:Nt
ENOD40 cell lines. (A) Kinetics of ethylene accumulation in the
headspace of protoplast-derived cells, cultured in the presence of 0.1 mg
l
ÿ1
NAA and 1.0 mg l
ÿ1
BA. Data are average ethylene concentrations of
six replicate samples cultured in parallel for each condition. The vertical
line 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 days 0, 2, and 4. Amplification is shown for
three 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 4th lane of each block.
ENOD40 and ethylene biosynthesis kinetics 3279
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gradually decreases during the culture period (Fig. 6B).
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 cultures, as well as in 35S:NtENOD40 cultures,
ACS expression profiles correlate with the timing of
ethylene production. In wild-type cultures, ACO transcripts
are present directly after protoplast culture has started, and
their level only slightly increases during the 4 d culture
period. The ACO transcript accumulation profiles in both
35S:NtENOD40 lines are similar to that in the wild type
(Fig. 6B). The temporal regulation of ACO transcript ac-
cumulation does not correlate with the timing of ethylene
production in the different lines. 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.
Discussion
In this report, the interaction of ENOD40 with hormone
signalling pathways was studied by analysing hormone-
induced responses in tobacco BY-2 cell lines. For this
purpose, the BY-2 protoplast assay described by Hasezawa
and Syono (1983) was adapted and the dependence of cell
elongation growth and cell division on cytokinin, auxin,
and ethylene in wild-type and transgenic 35S:NtENOD40
BY-2 cells was investigated.
It was found, in agreement with other studies in BY-2
cells (Hasezawa and Syono, 1983; Hasezawa et al., 1988;
Nagata et al., 1992), that cell division frequency is con-
trolled by the cytokinin:auxin ratio. Exogenous cytokinin
has an inhibitory effect, whereas auxin stimulates cell
division (Fig. 2). The response to different cytokinin or
auxin concentrations is comparable in wild-type and
35S:NtENOD40 lines (Fig. 4), suggesting that ENOD40
overexpression in BY-2 cells does not affect cytokinin-
or auxin-dependent control on cell division. In addition,
evidence is shown that, under the conditions of the present
study, alterations of ethylene production or perception
do not affect cell division frequency. Similar cell division
frequencies are observed in wild-type and transgenic lines
(Figs 3–5), despite the differences in their ethylene pro-
duction levels during early days of protoplast culture (Fig.
6). Also, no effect of ethylene perception blockers on
cell division frequency was found (Fig. 5). Therefore it
is concluded that ethylene is not involved in the control of
cell division under the conditions of the present study.
Elongation growth is not affected by exogenous auxin
(Fig. 2), nor is this process sensitive to application of
ethylene perception blockers in wild-type BY-2 cells (Fig.
5). Thus, elongation growth does not seem to be limited
by either of these two hormones in wild-type cells. By
contrast, cytokinin has a mild negative effect as addition of
cytokinin reduces elongation growth of wild-type cells in
a dose-dependent manner (Fig. 2).
Upon overexpression of NtENOD40, elongation growth
is strongly reduced (Figs 3, 4). Analysis of ethylene
homeostasis showed that ENOD40-provoked phenotypic
responses are mediated by ethylene. Ethylene biosynthesis
is accelerated in 35S:NtENOD40 lines, under conditions
where ENOD40 reduces elongation growth. Constitutive
expression of ENOD40 does not lead to constitutive
ethylene production, but rather accelerates a transient
accumulation of ethylene during cell growth (Fig. 6). In
addition, it was observed that the ENOD40-induced re-
sponse is counteracted by an ethylene perception blocker
(Fig. 5), but this recovery appears not to be complete. This
could imply that the complete block of ethylene percep-
tion requires a higher AgNO
3
concentration that might be
toxic to BY-2 cells or that ethylene is not the only factor
that is responsible for reduced elongation growth in
35S:NtENOD40 cells. As cytokinin also negatively af-
fects elongation growth, an effect of NtENOD40 overexpre-
ssion on the endogenous cytokinin concentration cannot
be excluded as a factor by which overexpression of Nt-
ENOD40 affects elongation growth. However, an increase
in the endogenous cytokinin concentration is expected to
suppress elongation growth (Fig. 4A) and cell division
(Fig. 4C) simultaneously. Since cell division frequencies
are not significantly different between wild-type and trans-
genic lines, it appears unlikely that endogenous cytokinin
concentrations are markedly affected by overexpression
of NtENOD40. Hence, if besides ethylene a second factor
is responsible for reduced elongation growth, the nature of
this factor remains unknown.
Nevertheless, the observations made in the present study
that ENOD40-provoked responses are mediated by ethylene
and that these responses can be counteracted by addition
of an ethylene perception blocker, show that acceleration of
ethylene accumulation is a primary cause rather than a con-
sequence of the reduced cell elongation in the transgenic
lines. Therefore, it seems likely that altered regulation of
ethylene biosynthesis is part of the mechanism that affects
elongation growth in NtENOD40-overexpressing BY-2
cells. As accelerated accumulation of ACS transcript levels
is correlated with accelerated ethylene accumulation in
ENOD40-overexpressing BY-2 cells (Fig. 6), ACS could
be a primary inducer of the ethylene accumulation. This
wouldbeconsistentwithACS as the rate-limiting step of
ethylene biosynthesis (Yang and Hoffman, 1984) and
a well-known primary target of ethylene biosynthesis re-
gulation (Yang and Hoffman, 1984; Theologis, 1992).
However, since ethylene accumulation is measured in-
directly by analysing the gas phase above the cell culture
it cannot be excluded that the cells have been exposed to
a higher ethylene concentration before ACS expression is
up-regulated. Since ethylene can induce ACS expression, in
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the latter case, ACS may act as a secondary inducer in a
positive feedback mechanism.
Although cytokinin and ENOD40 both negatively affect
elongation growth, providing the possibility that their
signalling pathways share common downstream compo-
nents, no indication was found that interaction between
these factors indeed occurs. This is based on the observa-
tions that ENOD40-provoked growth suppression does
not require exogenous cytokinin, nor is cytokinin dose-
dependent growth suppression significantly different in the
transgenic and wild-type lines (Fig. 4C).
A major question now, is whether the observations made
in this cellular model system can be extrapolated to the
whole-plant level. Ectopic expression of GmENOD40 in
Arabidopsis did not lead to severe changes in overall plant
architecture, but led to a decrease in cell size of epidermal
internode and leaf mesophyll cells (Guzzo et al., 2005).
In addition, a subpopulation of protoplasts isolated from
Arabidopsis cell suspension culture displayed reduced
expansion growth after either transient expression of
GmENOD40 or direct administration of GmENOD40 pe-
ptides (Guzzo et al., 2005). Thus, these data suggest that the
phenotypic effect of ectopic expression of ENOD40 may
depend on the cell type and/or environmental or develop-
mental context. Upon inoculation of leguminous plants
with rhizobia, ENOD40 is highly induced in cortical cells,
several hours prior to the first cell division leading to nodule
primordium formation, suggesting that ENOD40
is involved in the control over cortical cell division. In
Medicago plants ectopically expressing MtENOD40, over-
expression of MtENOD40 leads to proliferation of cells
in the upper region of the root (Charon et al., 1997). How-
ever, upon inoculation of plants with the symbiont
Sinorhizobium meliloti, cell proliferation is induced in the
region close to the root tip (Charon et al., 1999), showing
that overexpression of ENOD40 does not lead to cell
division per se and that ENOD40 expression alone is not
sufficient for nodule primordium formation. Likewise, in
BY-2 cells overexpression of ENOD40 did not lead to
an increase in cell division frequency. Thus, the pheno-
typic response to ectopic expression of ENOD40 may be
dependent on the cellular context and/or on local action
of other plant factors, such as phytohormones.
Since ENOD40 is highly induced and acts as a positive
regulator during nodulation, the observation that ENOD40
overexpression leads to an acceleration of ethylene bio-
synthesis in BY-2 cells appears counter-intuitive to the
function of ethylene as a negative regulator of nodulation
during legume–Rhizobium interaction. It has been demon-
strated that ethylene acts at a multitude of steps in the
nodulation pathway, including nodule formation (Peters
and Crist-Estes, 1989; Lee and LaRue, 1992); infection
thread formation (Penmetsa and Cook, 1997); root hair de-
formation, early gene expression, and calcium spiking in
response to Nod factor (Oldroyd et al., 2001), and regulation
of the maintenance of nodule meristems in Sesbania rost-
rata (Fernandez-Lo´pez et al., 1998). In addition, ethylene
has a possible role in defining the position at which nodule
primordia are initiated (Heidstra et al., 1997). Together,
these observations have led to the hypothesis (Oldroyd
et al., 2001) that ethylene could function as a dynamic
regulator in the nodulation process by acting at an early
point in Nod factor signalling at, or upstream of, calcium
spiking in response to Nod factor, resulting in multiple
ethylene-regulated developmental effects downstream in the
Nod factor-dependent pathway. Alternatively, ethylene
could inhibit several components of the nodulation pathway
directly and independently (Oldroyd et al.,2001).Although
the underlying mechanism remains unclear, the present
studies in BY-2 cells suggest appropriate timing of ethylene
biosynthesis as a critical factor for cellular responses, and
indicate that ENOD40 may participate in the control over
this timing. In the absence of studies describing the effect
of either endogenous or ectopic expression of ENOD40 on
ethylene biosynthesis, the existence of these interactions in
whole-plant systems, or indeed during nodule development,
still needs to be confirmed. Due to the diversity of possible
responses induced by alterations of ethylene levels in whole-
plant systems, the phenotypic outcome of such interactions
is difficult to predict, and may be dependent on the cellular
context. Nevertheless, it is conceivable that local expression
of ENOD40 during early steps of the legume–Rhizobium
interaction (Kouchi and Hata, 1993; Yang et al., 1993;
Compaan et al., 2001) would provide the plant with a means
to attenuate, in a dynamic manner, ethylene production, in
keeping with the proposed role of ethylene as a dynamic
regulator of cellular responses (Oldroyd et al., 2001) during
nodulation. Therefore, effects of ENOD40 on the ethylene
biosynthesis pathway could be considered as an important
component of the complex regulatory pathway controlling
nodule development.
Acknowledgements
We thank Danny Geelen for kindly supplying the BY-2 cell
suspension. We gratefully acknowledge Gerard van der Krogt for
his valuable contribution to the BY-2 transformations, Ve´ronique
Storme for support with data analysis, and the excellent technical
assistance of Ciska Braam and Maelle Lorvellec. We are thankful to
Mark Hink and Jan-Willem Borst (Wageningen University Micro-
scopy Center) for use of the microscope facilities. This work was
supported by the Netherlands Organization for Scientific Research
(NWO 805.49.004).
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