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ORIGINAL ARTICLE
Functional divergence of GhCFE5 homoeologs revealed in cotton
fiber and Arabidopsis root cell development
Fenni Lv
1
•Peng Li
1
•Rui Zhang
1
•Nina Li
1
•Wangzhen Guo
1
Received: 12 July 2015 / Revised: 19 December 2015 / Accepted: 29 December 2015 / Published online: 13 January 2016
ÓSpringer-Verlag Berlin Heidelberg 2016
Abstract
Key message In GhCFE5 homoeologs, GhCFE5D
interacted with more actin homologs and stronger
interaction activity than GhCFE5A. GhCFE5D- but not
GhCFE5A-overexpression severely disrupted actin
cytoskeleton organization and significantly suppressed
cell elongation.
Abstract Homoeologous genes are common in polyploid
plants; however, their functional divergence is poorly
elucidated. Allotetraploid Upland cotton (Gossypium hir-
sutum, AADD) is the most widely cultivated cotton;
accounting for more than 90 % of the world’s cotton pro-
duction. Here, we characterized GhCFE5A and GhCFE5D
homoeologs from G. hirsutum acc TM-1. GhCFE5
homoeologs are expressed preferentially in fiber cells; and
a significantly greater accumulation of GhCFE5A mRNA
than GhCFE5D mRNA was found in all tested tissues.
Overexpression of GhCFE5D but not GhCFE5A seriously
inhibits the Arabidopsis hypocotyl and root cell elongation.
Yeast two-hybrid assay and bimolecular fluorescence
complementation (BiFC) analysis showed that compared
with GhCFE5A, GhCFE5D interacts with more actin
homologs and has a stronger interaction activity both from
Arabidopsis and Upland cotton. Interestingly, subcellular
localization showed that GhCFE5 resides on the cortical
endoplasmic reticulum (ER) network and is colocalized
with actin cables. The interaction activities between
GhCFE5 homoeologs and actin differ in their effects on
F-actin structure in transgenic Arabidopsis root cells. The
F-actin changed direction from vertical to lateral, and the
actin cytoskeleton organization was severely disrupted in
GhCFE5D-overexpressing root cells. These data support
the functional divergence of GhCFE5 homoeologs in the
actin cytoskeleton structure and cell elongation, implying
an important role for GhCFE5 in the evolution and selec-
tion of cotton fiber.
Keywords GhCFE5 Homoeologs Functional
divergence Cell elongation Actin cytoskeleton
Gossypium hirsutum
Abbreviations
CaMV Caulifower mosaic virus
DAG Day after germination
DPA Day post anthesis
DUF Domain of unknown function
SNPs Single-nucleotide polymorphisms
NPT-II Neomycin phosphotransferase II
ER Endoplasmic reticulum
GFP Green fluorescent protein
RFP Red fluorescent protein
Introduction
Allopolyploids contain two or more sets of homoeologous
chromosomes, leading to significant advantages in pheno-
typic variation and adaptation of domestication-related
Communicated by D.-B. Zhang.
Electronic supplementary material The online version of this
article (doi:10.1007/s00299-015-1928-0) contains supplementary
material, which is available to authorized users.
&Wangzhen Guo
moelab@njau.edu.cn
1
State Key Laboratory of Crop Genetics and Germplasm
Enhancement, Hybrid Cotton R&D Engineering Research
Center, MOE, Nanjing Agricultural University,
Nanjing 210095, Jiangsu, People’s Republic of China
123
Plant Cell Rep (2016) 35:867–881
DOI 10.1007/s00299-015-1928-0
traits. Cotton (Gossypium spp.) is the world’s most important
fiber crop plant. Upland cotton (Gossypium hirsutum L.,
AADD), the most extensively cultivated cotton species, is an
allotetraploid that originated from the hybridization of an
A-genome-like ancestral African species and a D-genome-
like North American species 1–2 million years ago (Endrizzi
et al. 1985; Wendel 1989; Wendel and Cronn 2003). Among
the diploid progenitors, the AA genome species produce long
and spinnable fibers, while the DD genome progenitor pro-
duces short and unuseful fibers (Applequist et al. 2001).
Interestingly, interspecific hybridization led to the biological
reunion of the two diploid ancestors and induced the evo-
lution of New World allotetraploids with a higher fiber
quality and yield (Jiang et al. 1998), suggesting the effects of
natural and artificial selection and interaction expression
patterns of homoeologous fiber-related genes (Adams et al.
2003; Lee et al. 2007).
Elongation of cotton fiber cells is a complex physio-
logical process, involving cell wall loosening, turgor
pressure, biosynthesis, and transportation of membranes
and cell wall components (Smart et al. 1998; Ruan et al.
2001; Ruan 2007). Microfilaments composed of actin fil-
aments are reported to play an important role in the
transportation of organelles and secretory vesicles carrying
membrane and cell wall components (Mathur and Hul-
skamp 2002; Chen et al. 2003), suggesting their essential
role in cell elongation. Disruption of the actin cytoskeleton
during pollen tube development by overexpression of Rac/
Rop GTPases converts polar growth into isotropic growth
(Chen et al. 2003). In cotton fibers, silencing the expression
of a fiber-specific actin gene, GhACT1, reduces the amount
of actin filaments and inhibits fiber cell elongation (Li et al.
2005). Actin binding proteins, such as GhPLIM1,
GhWLIM5, GhWLIM1a, GhPFN2, and GhADF1, play
important roles in modulating the actin cytoskeleton, and
some are reported to regulate fiber cell elongation (Wang
et al. 2009,2010a,b; Han et al. 2013; Li et al. 2013,2015).
A previous study also demonstrated that inhibition of
F-actin polymerization in Arabidopsis thaliana inhibited
root hair elongation (Miller et al. 1999). In addition,
mutations of the ACT2 and ACT7 genes, which are strongly
expressed in vegetative tissues, resulted in a dramatic
reduction in root hair length (Gilliland et al. 2002,2003).
Genome sequencing and annotation of ancestral diploid
species, the A-genome species Gossypium arboreum (A2)
and the D-genome species Gossypium raimondii (D5), have
revealed a number of previously unknown gene families
(Paterson et al. 2012; Li et al. 2014), including a large set
of gene families with DUF. The names of DUF protein
families are based on the order of their addition to the
protein-family (Pfam) database that began in 1997 (Bate-
man et al. 2010), and the DUF numbering scheme now
extends to DUF4662. Yamamoto and Baird (1998) isolated
three cotton fiber-expressed genes, namely GhCFE1,
GhCFE2, and GhCFE3, with uncharacterized biological
information of the encoded proteins which contain two
DUFs: DUF761 and DUF4408. In a recent study, we
revealed that overexpressing GhCFE1A, homoeologous to
GhCFE1, inhibits cotton fiber cell initiation and elongation
(Lv et al. 2015).
In the present study, we cloned two novel CFE genes
that are GhCFE5 homoeologs, in G. hirsutum acc. TM-1,
and employed A. thaliana, a popular model organism in
plant biology and genetics, to test the function of
GhCFE5A (originating from the A subgenome of G. hir-
sutum acc. TM-1) and GhCFE5D (originating from the D
subgenome of G. hirsutum acc. TM-1) in cell elongation.
Further, the relationship between GhCFE5 homoeologs and
actin proteins were individually elucidated. The results
suggest significant functional divergence in GhCFE5
homoeologs in actin cytoskeleton and cell elongation;
implying that GhCFE5 plays an important role during the
evolution and selection of cotton fiber development traits.
Materials and methods
Plant materials
The tetraploid cotton species G. hirsutum acc. TM-1 and G.
barbadense cv. Hai7124 were field-cultivated in Nanjing,
China, using normal cotton farming practices. The diploid
cotton species G. herbaceum and G. raimondii, were bred
in a greenhouse with normal management procedures.
Developing ovules and fibers from TM-1 were excised
from flower buds or bolls and ovules fertilized on selected
days post anthesis (0, 5, 10, 20 DPA). Roots, stems, and
leaves were collected from seedlings with four true leaves
cultured in a growth chamber. All materials were imme-
diately frozen in liquid nitrogen and stored at -70 °C
before RNA and DNA extraction. Wild-type Arabidopsis
thaliana Columbia 0 (Col-0) and transgenic A. thaliana
plants were grown in a growth chamber at 22–24 °C under
long-day (16 h of light and 8 h of dark) conditions.
Gene cloning and sequences analysis
The two full-length GhCFE5 cDNA sequences were
amplified from fibers of TM-1 using CFE5-full-F and
CFE5-full-R primers (Table S1). These primers were also
used to obtain the genomic sequences of CFE5 in TM-1,
Hai7124, G. herbaceum and G. raimondii. All the PCR
fragments were sequenced after they were cloned into
T-vectors. At least ten clones for each of the tetraploid
species and five clones for each of the diploid species were
picked randomly and sequenced, with a minimum of three
868 Plant Cell Rep (2016) 35:867–881
123
clones used to determine the gene sequence in each cotton
species. The gene structures were analyzed using the online
software Gene structure display server (GSDS, http://gsds.
cbi.pku.edu.cn). The conserved protein domains were
searched in the National Center for Biotechnology Infor-
mation (NCBI) database. The protein theoretical isoelectric
point (pI) and molecular weight (Mw) were obtained with
the Compute pI/Mw tool (http://web.expasy.org/compute_
pi/). For sequence alignment analysis, the genomic
sequences of CFE5 were aligned using ClustalX software
(Thompson et al. 1997). Unrooted phylogenetic trees were
constructed by the neighbor-joining method with MEGA
5.0 software (Tamura et al. 2011) using the genomic
sequences generated earlier. Bootstrap values were calcu-
lated from 1000 replicates.
Genomic mapping and phylogenetic analysis
Using the [(TM-1 9Hai7124) 9TM-1] BC
1
interspecific
mapping population, GhCFE5A and GhCFE5D were
mapped. A pair of primers consisting of amplified regions
with At or Dt subgenome polymorphisms between TM-1
and Hai7124 was used to survey 138 individuals of the BC
1
mapping population. The polymorphic loci were integrated
in the backbone map (Guo et al. 2008) using JoinMap
version 3.0 (Van Ooijen and Voorrips 2001).
The Hidden Markov Model (HMM) profiles of the
conserved DUF, DUF761 (PF05553) and DUF4408
(PF14364), were obtained from the Pfam website (http://
pfam.sanger.ac.uk/, Finn et al. 2006), which was employed
as a query to identify all possible DUF761- and DUF4408-
containing genes using HMMER (V3.0) software (Finn
et al. 2011). To validate the HMM search, domain analysis
using blastp program in the NCBI non-redundant (nr)
protein database, and Pfam domain analysis were per-
formed individually. The available genomic databases of
three cotton species, G. raimondii, G. arboreum, and G.
hirsutum acc. TM-1, were downloaded from http://www.
phytozome.net/,http://cgp.genomics.org.cn, and http://
mascotton.njau.edu.cn/, respectively, and A. thaliana from
The Arabidopsis Information Resource (TAIR: http://www.
arabidopsis.org). The amino acid sequence alignment of
these genes was performed using the ClustalW application
of the software MEGA 5.0 (Tamura et al. 2011). Unrooted
phylogenetic trees were constructed by the neighbor-join-
ing method with MEGA 5.0 software using the DUF761
and DUF4408 conserved domain amino acid sequences.
Bootstrap values were calculated from 1000 replicates.
Quantitative RT-PCR analysis
Total RNA was extracted from cotton tissues according to
the cetyl trimethylammonium bromide (CTAB)-sour
phenol extraction method (Jiang and Zhang 2003). Ara-
bidopsis total RNA was extracted using TRIzol Reagent
(Invitrogen). 2 lg of total RNA per reaction from each
tissue was reverse-transcribed using an oligo(dT)
18
primer
with M-MLV reverse transcriptase (Promega, Madison,
WI, USA; Cat# M1701) according to the manufacturer’s
recommendations. Real–time quantitative PCR (qRT-PCR)
analysis was performed using a Light Cycler Fast Start
DNA Master SYBR Green I kit (Roche, Basel, Switzer-
land) in an ABI7500 sequence detection system according
to the manufacturer’s protocol (Applied Biosystems, http://
www.appliedbiosystems.com). Amplification of Histon3
was used to normalize the amount of gene-specific RT-
PCR product. The gene-specific primers used in the study
are listed in Supplementary Table S1. The relative
expression levels of GhCFE5A and GhCFE5D were cal-
culated based on the C
T
value from thrice-repeated real-
time PCR reactions for each sample using the comparative
DC
T
method (Livak and Schmittgen 2001).
Plant transformation and morphological
characterization of transgenic Arabidopsis plants
GhCFE5A and GhCFE5D plant expression vectors were
constructed using a pair of primers with a BamHI restric-
tion site at the 50terminus of each of OE-GhCFE5-F and
OE-GhCFE5-R (Table S1). The amplified BamHI-
GhCFE5A-BamHI and BamHI-GhCFE5D-BamHI products
were inserted into pBI121 vectors at the BamHI site under
the 35S promoter. The successful insertion of the gene
products was confirmed by digestion with BamHI followed
by sequencing and transformation into A. thaliana
Columbia 0 (Col-0) by Agrobacterium tumefaciens strain
GV3101 (Clough and Bent 1998). Full-mature seeds were
collected and screened on MS medium containing
25 mg/L kanamycin. The germinated seedlings were
transplanted into pots with a soil mixture and placed in a
growth chamber for further growth. PCR was performed to
verify the transgenic status of the screened plants.
Seeds of T3 wild-type and transgenic Arabidopsis plants
were surface-sterilized with ethanol and sodium
hypochlorite, and then sown in a row on MS medium
and subjected to stratification treatment at 4 °C in darkness
for 2 days. The imbibed seeds were then exposed to light
for 12 h. The first set of trials was wrapped with two layers
of aluminum foil and placed vertically into a growth
chamber at 22–24 °C. The hypocotyl length of over 30
etiolated seedlings from transgenic lines and wild type was
measured after 3 days, and the length of hypocotyl cells
from the middle region (over 50 cells) was analyzed using
a confocal microscope (Zeiss, LSM780). The second set of
trials without aluminum foil was placed vertically into the
growth chamber at 22–24 °C under long-day (16 h of light
Plant Cell Rep (2016) 35:867–881 869
123
and 8 h of dark) conditions. The primary root length of
over 30 seedlings from transgenic lines and wild type was
measured after 7 days. The root cell length was observed
using the propidium iodide staining method: Cell outlines
were stained with 50 lg/mL propidium iodide for 2 min
and observed under a confocal laser-scanning microscope
(Zeiss, LSM780) with excitation at 543 nm.
Yeast two hybridization
To test interactions between the proteins in vitro,
GhCFE5A and GhCFE5D were cloned into pGBKT7
vectors (Clontech, CA, USA), while AtACT2 (Genbank No.
AY096381), AtACT7 (Genbank No. AY063980), AtACT8
(Genbank No. AY063089), GhACTa (Genbank No.
KF018243), GhACTb (Genbank No. KF018244), GhACT2
(Genbank No. AY305724), and GhACT4 (Genbank No.
AY305726) were cloned into pGADT7 vectors (Clone-
tech). The yeast two-hybrid assay was performed according
to the manufacturer’s instructions. Co-transformed Sac-
charomyces cerevisiae AH109 yeast cells were grown on
SD/-T-L medium with X-a-Gal and incubated at 28 °C for
3d. Positive colonies (the blue colonies) were subsequently
transferred to the selective and stringent SD/-L–T-H
medium, supplemented with 5 mM 3-AT medium or SD/-
L-T-H-A medium.
Co-localization assay and live cell imaging analysis
The ORF of GhCFE5A and GhCFE5D was amplified and
cloned into pGWB5 vector to obtain the CFE5A-GFP and
CFE5D-GFP constructs, respectively. To achieve this,
Topo cloning and a subsequent recombination reaction
were used, as described in a previous study (Liu et al.
2014). The binary vectors were transiently co-expressed
in leaves of Nicotiana benthamiana with ABD2-mCherry
or RFP-HDEL via agroinfiltration (Lv et al. 2015). Ima-
ges were taken on a confocal microscope (Zeiss,
LSM780).
Bimolecular fluorescence complementation (BiFC)
assay
The ORF of GhCFE5A and GhCFE5D was amplified and
cloned into PacI/SpeI sites of the p2YN vector (containing
amino acids 1–158 of YFP) to form the CFE5A-YFP
N
and
CFE5D-YFP
N
plasmids. The CDS of GhACTs was indi-
vidually amplified and cloned into the PacI/SpeI sites of
the vector p2YC (containing amino acids 159–239 of YFP)
to form the ACT-YFP
C
plasmid. Subsequently, the BiFC
constructs were transferred into Agrobacterium tumefa-
ciens strain EHA105 and transiently expressed in tobacco
(Nicotiana benthamiana) via agroinfiltration (Waadt and
Kudla 2008). The fluorescence of YFP was assayed using a
confocal laser-scanning microscope (Zeiss, LSM780).
Fluorescent staining and microscopic observation
In order to investigate F-actin activity in transgenic Ara-
bidopsis, we carefully dissected roots from wild-type and
transgenic Arabidopsis seedlings at 5 DAG. F-actin stain-
ing with Alexa Fluor
Ò
488 phalloidin (Molecular Probes,
Invitrogen) was performed according to the manufacturer’s
instructions and referencing Lv et al. (2015). After briefly
rinsing in phosphate buffered saline (PBS), the roots were
mounted onto glass slides and observed using a confocal
microscope. During image acquisition, all settings,
including excitation wavelength and emission filters
(488 nm/band-pass 505–530 nm for Alexa-Fluor 488),
were fixed.
Results
Isolation of CFE5 homologs in G. hirsutum
By randomly sequencing clones from a cDNA library
constructed using ovules (1 and 3 DPA) and fibers (5–25
DPA) from G. hirsutum acc. 7235, which is a germplasm
line with elite fiber quality (Wang et al. 2010a, b), a 1285
nucleotide-long cDNA clone was obtained. Based on the
sequence of the clone, we designed primer pairs and iso-
lated two full-length cDNAs from G. hirsutum (AADD)
acc. TM-1 fibers. Sequence analysis showed that the two
cDNAs shared similar nucleotide sequences and 96.36 %
of the deduced amino acid sequences were identical.
Bioinformatics analysis predicted that both encoded pro-
teins contain two conserved DUF, DUF761 and DUF4408,
and they share over 75 % similarity with GhCFE1A
(Genbank No. DQ073045) and the three CFEs isolated by
Yamamoto and Baird (1998), which also contain the two
conserved DUF domains. Therefore, the two cDNAs iso-
lated in this study were denoted as GhCFE5 homologs.
In G. hirsutum, the A and D genomes of the diploid
progenitors became homoeologous subgenomes. Due to
this complex genomic background, the two identified genes
may represent paralogs (i.e. duplicated genes originating
from the same subgenome) or homoeologs (i.e. orthologous
genes originating from different subgenomes coexisting in
the hybridized allotetraploid genome), therefore further
identification is required.
Genomic origins of two GhCFE5 homologs
To investigate the genomic origins of the two GhCFE5
homologs, we cloned and sequenced CFE5 genomic
870 Plant Cell Rep (2016) 35:867–881
123
sequences from G. hirsutum acc. TM-1(AADD) and G.
barbadense cv. Hai7124 (AADD), and from close relatives
of the progenitor species, G. herbaceum (A1) and G. rai-
mondii (D5) (GenBank Nos: KR997837-KR997842; Sup-
plementary Fig. S1). These CFE5 genes contain one intron
spliced at the same site (the 202nd amino acid) and are
classified into two groups, A- and D-genome like, desig-
nated CFE5A and CFE5D, respectively (Fig. 1a, b). The
GhCFE5 homolog that exhibited the highest similarity to
the G. herbaceum CFE5 gene (CFE5A A1) was named
GhCFE5A (CFE5A TM-1). The other showed a high
sequence similarity to the G. raimondii CFE5 gene
(CFE5D D5) and was thus named GhCFE5D (CFE5D TM-
1). GhCFE5A is homoeologous to GhCFE5D.
Twenty-eight SNPs were found in the coding regions of
the six CFE5 homologs from the four cotton species,
accompanied by one insertion/deletion at the 30terminus,
which resulted in relatively polymorphic proteins with non-
synonymous mutations, frameshift mutations, and differ-
ences in protein sequence length (Supplementary Figs. 1,
1c; Table 1). The mutations, particularly the variations
between acidic amino acids and basic amino acids (K/Q
and K/E identified by red boxes in Fig. 1c), led to changes
in value of Mw and pI (pI
CFE5A
[7.0, pI
CFE5D
\7.0,
Table 1) of the proteins.
Based on SNPs in CFE5 sequences between the two
mapping parents, TM-1 and Hai7124 (Guo et al. 2007),
GhCFE5A was mapped to chromosome A10 (chromosome
10) and GhCFE5D was confirmed to be present on the
homeologous chromosome, D10 (chromosome 20, Sup-
plementary Fig. 2). The location of GhCFE was consistent
with the reference genome sequence of the genetic standard
Upland cotton line, TM-1, released recently (Zhang et al.
2015).
Fig. 1 Sequence analysis of homologous CFE5 genes and proteins in
diploid and allotetraploid cotton. aGene structure analysis of CFE5
genomic sequences from G. herbaceum (A1), G. raimondii (D5), G.
barbadense (AADD, Hai7124) and G. hirsutum (AADD, TM-1). The
exons and introns are represented by different characters. bPhyloge-
netic analysis using nucleic acid sequences of CFE5 genes from
different cotton species. ‘‘CFE5A A1’’, genomic CFE5 sequence in
G. herbaceum (A1); ‘‘CFE5D D5’’, genomic CFE5 sequence in G.
raimondii (D5); ‘‘CFE5A TM-1’’ and ‘‘CFE5D TM-1’’, A and D
subgenome genomic CFE5 sequences in G. hirsutum acc. TM-
1(AADD), respectively; ‘‘CFE5A Hai71240’ and ‘‘CFE5D
Hai7124’’, A and D subgenome genomic CFE5 sequences in G.
barbadense cv. Hai7124 (AADD), respectively. cMultiple sequence
alignment analysis of homologous CFE5 proteins. The amino acids
labeled by red boxes cause the changes in pI values (color
figure online)
Plant Cell Rep (2016) 35:867–881 871
123
Phylogenetic characterization of CFE5 homologs
Motif analysis indicated that CFE5 and previously reported
CFE1 proteins contain two conserved DUFs; DUF761and
DUF4408. To identify CFE family genes in three sequenced
cotton species, G. arboreum,G. raimondii, and G. hirsutum
acc. TM-1, as well as in Arabidopsis, an HMM profile search
was performed. As a result, a set of genome-level CFE can-
didate genes were extracted; 7 in G. arboreum,8inG. rai-
mondii, 9 homoeologs in G. hirsutum acc. TM-1, and 3 in
Arabidopsis. To examine the phylogenetic relationships
between these CFE proteins, an unrooted tree was con-
structed from alignments of their protein sequences using
MEGA 5.0 software (Tamura et al. 2011). As shown in Fig. 2,
CFE5 (Cotonn_A_03844, Gorai.011G022100.1,
Gh_A10G0197 and Gh_D10G2624) exhibited the highest
similarity to CFE1 (Cotonn_A_25581, Gorai.009G172800.1,
Gh_A05G1404 and Gh_D05G1569, with over 75 % identity)
in the three cotton species, and At1g61260 (with 47 %
identity) in Arabidopsis. In a previous study, overexpression
of GhCFE1A was found to inhibit cotton fiber cell initiation
and elongation (Lv et al. 2015). Since CFE5 and CFE1 dis-
played the highest sequence similarity, it is likely that they
have a similar function in fiber development, although this is
still to be confirmed.
Different expression patterns of GhCFE5A
and GhCFE5D
To examine the transcript levels of two CFE5 homoeolo-
gous loci in tetraploid cotton, we designed gene-specific
primers based on SNPs between sequences of GhCFE5A
and GhCFE5D. The specificity of the primers was further
confirmed by PCR using genomic DNA from A1, D5, TM-
1, and Hai7124 as templates (Fig. 3a). Overall expression
levels of GhCFE5A were higher than those of GhCFE5D
across all tissues tested (Fig. 3b). GhCFE5A was primarily
expressed in the ovules and fibers, and the transcript levels
were higher in ovules than those in corresponding fibers. A
high expression of GhCFE5D was detected in elongating
fibers, and the transcript was most abundant in fibers at 10
DPA, the rapid elongation stage of fiber cell development.
Moreover, higher expression levels were observed in fibers
than in the corresponding ovules. These very distinct
expression patterns indicated that the homoeologous genes
have evolved and are regulated differently at the molecular
level during fiber development.
Ectopic expression of GhCFE5D greatly inhibits
Arabidopsis cell elongation
In order to gain further insight into the function of the two
GhCFE5 homoeologs, GhCFE5A and GhCFE5D were
individually transformed into Arabidopsis driven by CaMV
35S promoter. In total, 12 and 10 independently trans-
formed lines were obtained, respectively, in which the
presence of the transgene was verified by PCR detection of
the 35S promoter-GhCFE5A/Dfragment and NPT-II,a
selectable marker gene that encodes neomycin phospho-
transferase II (Supplementary Fig. 3). Although the tran-
script levels of GhCFE5A and GhCFE5D were variable
among the multiple independently derived transgenic
plants, they were highly expressed in these transgenic lines,
especially in lines 7 (35S::GhCFE5A #7) and 1
(35S::GhCFE5D #1, Fig. 4a). Three lines with different
transcript levels of GhCFE5A and GhCFE5D, respectively
(35S::GhCFE5A lines 7, 9, and 12 and 35S::GhCFE5D
lines 1, 3, and 5) were subsequently chosen for detailed
analyses.
T3 homozygous 35S::GhCFE5A and 35S::GhCFE5D
transgenic lines were grown on MS medium, together
with wild-type (WT) plants. In 35S::GhCFE5D transgenic
plants, the hypocotyl length of etiolated seedlings at 3
DAG and the root length of normal seedlings at 7 DAG
were dramatically reduced compared with WT (Figs. 4b, c,
5a, b), especially in line 1. However, the cell numbers of
hypocotyls and roots in the 35S::GhCFE5D line 1 and the
wild type were similar. In a previous study, comparison of
Table 1 Characteristics of
CFE5 homoeologs from diploid
and allotetraploid cotton
Nomenclature
a
Genomic sequence (bp) Length (aa) CDS (bp) Mw (kDa) pI
CFE5A_A1 1728 332 999 37.89 7.05
CFE5A_TM-1 1720 330 993 37.60 7.77
CFE5A_Hai7124 1720 330 993 37.63 7.77
CFE5D_D5 1734 330 993 37.62 6.29
CFE5D_TM-1 1734 330 993 37.63 6.29
CFE5D_Hai7124 1734 330 993 37.63 6.29
a
‘‘CFE5A A1’’, CFE5 in G. herbaceum (A1); ‘‘CFE5D D5’’, CFE5 in G. raimondii (D5); ‘‘CFE5A TM-
1’’ and ‘‘CFE5D TM-1’’, A and D subgenome sequence of CFE5 in G. hirsutum acc. TM-1(AADD),
respectively; ‘‘CFE5A Hai7124’’ and ‘‘CFE5D Hai7124’’, A and D subgenome sequence of CFE5 in G.
barbadense cv. Hai7124 (AADD), respectively
872 Plant Cell Rep (2016) 35:867–881
123
cell length in different regions of etiolated hypocotyls in
the MDP40 RNAi line and WT showed that the difference
in cell length in the middle region was significant (Wang
et al. 2012). Therefore, to determine whether the shorter
length of hypocotyls and roots in the 35S::GhCFE5D lines
was due to the reduction in cell size in the longitudinal
Fig. 2 Phylogenetic
relationship of CFE proteins
from three sequenced cotton
species and Arabidopsis. Seven
candidate CFE genes in G.
arboreum were downloaded
from http://cgp.genomics.org.
cn;8inG. raimondii from
http://www.phytozome.net/;9
homoeologous pairs in G. hir-
sutum acc. TM-1 from http://
mascotton.njau.edu.cn/; and 3 in
A. thalina from http://www.ara
bidopsis.org. The Neighbor-
Joining tree was generated from
the amino acid sequences men-
tioned above, and the numbers
next to each node give bootstrap
values from 1000 replicates.
Filled circle shows CFE1
homologs in three cotton spe-
cies, filled diamond shows
CFE5 homologs in three cotton
species
Fig. 3 Expression patterns of
GhCFE5A and GhCFE5D.
aDetection of specific-
subgenome primers for
GhCFE5A and GhCFE5D.
bExpression patterns of
GhCFE5A and GhCFE5D in
different tissues. Tissues used
were root, stem, leaf, 0 DPA
ovules, and fibers of 5, 10, 20
DPA. Error bars represent
standard deviation of triplicate
experiments, and His3 was used
as an internal control in qRT-
PCR
Plant Cell Rep (2016) 35:867–881 873
123
Fig. 4 Overexpression of GhCFE5A and GhCFE5D inhibits hypo-
cotyl elongation to different degrees. aqRT-PCR analysis of the
expression levels of GhCFE5A and GhCFE5D in seedlings from
wild-type (WT) plants, 35S::GhCFE5A transgenic plants (lines 3, 7,
8, 9 and 12) and 35S::GhCFE5D transgenic plants (lines 1, 2, 3, 5 and
9). Error bars represent standard deviation of triplicate experiments,
and His3 was used as an internal control. bPhenotype analysis of the
hypocotyl from WT, 35S::GhCFE5A lines 7, 9, and 12, and
35S::GhCFE5D lines 1, 3, and 5 transgenic etiolated seedlings at 3
DAG. Scale bar 2 mm. cAverage hypocotyl lengths of the wild-type,
35S::GhCFE5A lines 7, 9, and 12, and 35S::GhCFE5D lines 1, 3, and
5 transgenic etiolated seedlings at 3 DAG. Error bars indicate
standard deviation of over 30 etiolated seedlings. (*P\0.05,
**P\0.01, by Student’s ttest). dConfocal images of the hypocotyl
cells from WT, 35S::GhCFE5A line 7 and 35S::GhCFE5D line 1
transgenic etiolated seedlings at 3 DAG. These images show a similar
region in the middle of the hypocotyls. Scale bar 50 lm. eHypocotyl
cell lengths were measured from confocal images. The values were
averaged over 50 cells from transgenic and the wild-type Arabidopsis
plants. (*P\0.05, **P\0.01, by Student’s ttest)
874 Plant Cell Rep (2016) 35:867–881
123
direction, we measured the middle region cells of hypo-
cotyls and the differentiation zone cells of roots at 3 DAG
and 7 DAG in 35S::GhCFE5D line 1 and WT plants,
respectively. On average, the hypocotyl and root cells of
35S::GhCFE5D plants exhibited 52 and 68 % reductions in
longitudinal length per cell, respectively (Figs. 4d, e, 5c,
d). The root length of normal seedlings, the hypocotyl
length of etiolated seedlings, and the cell size in the middle
region of hypocotyls and the differentiation zone of roots
were found to be reduced in GhCFE5A-overexpressing
lines as compared with WT plants, however,
35S::GhCFE5A line 9 displayed no statistically significant
difference (Figs. 4,5). These differences were not greater
than those between the 35S::GhCFE5D transgenic plants
and WT plants, suggesting that GhCFE5D has a more
powerful effect on cell elongation.
GhCFE5A and GhCFE5D interacts differently
with actin homologs
In a previous study, we constructed a yeast two-hybrid
library of cotton ovules and fibers (0 DPA ovules; 5, 10, 15,
and 20 DPA fibers), and found that GhCFE1A could
interact with the protein candidates (GhACTa, GhACTb,
GhACT2 and GhACT4) in vivo and in vitro (Lv et al.
2015). By investigating phylogenetic relationships (Fig. 2),
we found that GhCFE5A and GhCFE5D proteins had high
similarity to GhCFE1A. Since the transgenic Arabidopsis
plants exhibited shorter hypocotyl cells and root cells in the
present study, we used GhCFE5A and GhCFE5D proteins
as baits to test their interaction with potential protein
candidates, including the vegetative actin proteins,
AtACT2, AtACT7, and AtACT8. Yeast two hybridization
Fig. 5 Serious suppression of
root cell elongation by
overexpression of GhCFE5D.
aPhenotype analysis of the
wild-type, 35S::GhCFE5A
lines, 7, 9, and 12, and
35S::GhCFE5D lines,1,3,and
5, transgenic seedlings at 7
DAG. Scale bar 5 mm.
bMorphometric analysis of root
growth of the transgenic
Arabidopsis plants for 7 days.
Error bars indicate standard
deviation of over 30 seedlings.
(*P\0.05, **P\0.01, by
Student’s ttest). cConfocal
images of primary root cells
stained with propidium iodide at
7 DAG. WT, wild type;
35S::GhCFE5A line 7 and
35S::GhCFE5D line 1,
transgenic plants. These images
were taken at a similar position
in differentiation zone of each
root. Scale bar 50 lm.
dMorphometric analysis of the
transgenic Arabidopsis plant
root cell length. The values
were averaged over 50 cells for
transgenic and the wild-type
Arabidopsis plants. (*P\0.05,
**P\0.01, by Student’s ttest)
Plant Cell Rep (2016) 35:867–881 875
123
results showed that GhCFE5A did not interact with Ara-
bidopsis vegetative actins, except AtACT7, while yeast
cells co-transformed with BD-GhCFE5D and AD-
AtACT2, AD-AtACT7 or AD-AtACT8, could grow on the
selective medium SD/-Leu-Trp-His with 5 mM 3-AT, even
though the growth of yeast cells co-transformed with BD-
GhCFE5D and AD-AtACT8 was weak (Fig. 6a). Addi-
tionally, the positive control group (BD-p53 and AD-T-
antigen) could grow on the selective medium whereas the
negative control groups (BD-Lam and AD-T-antigen, BD-
GhCFE5A and AD, and BD-GhCFE5D and AD) could not
grow. In addition, we tested the interactions of GhCFE5
homoeologs with actin proteins which predominantly
expressed in cotton fiber cells. As shown in Fig. 6b, on
restrictive medium (SD/-Leu-Trp-His-Ade), GhCFE5A
interacted with GhACTa and GhACTb, but the intensity
was weaker than that between GhCFE5D and GhACTa or
GhACTb. Meanwhile, co-transforments of GhCFE5D and
GhACT4 exhibited slight growth on SD/-Leu-Trp-His-Ade
medium. Moreover, BiFC assay confirmed that CFE5A and
CFE5D could differentially interact with ACTs in Nico-
tiana benthamiana leaf cells (Fig. 6c). These results indi-
cate that the binding activity of GhCFE5D with
Arabidopsis vegetative actin proteins or Upland cotton
actin homologs predominantly expressed in fiber cells is
stronger than that of GhCFE5A.
To test whether GhCFE5A and GhCFE5D, like
GhCFE1A (Lv et al. 2015), decorate the ER network and
co-localize with the actin cytoskeleton, we constructed
binary vectors encoding GhCFE5A and GhCFE5D with
GFP tags and transiently co-transformed them into
N. benthamiana leaf epidermal cells with ABD2-mCherry
or RFP-HDEL. Confocal microscopy showed that the
green fluorescent signal of CFE5A-GFP and CFE5D-GFP
overlapped with the red fluorescence of RFP-HDEL, indi-
cating that both GhCFE5A and GhCFE5D are located on
the ER network (Fig. 6d). In addition, CFE5A-GFP and
CFE5D-GFP co-localized with actin bundles, visualized by
transiently expressing ABD2-mCherry in pavement cells
(Fig. 6e). This indicates that GhCFE5 functions as a
dynamic linker between the actin cytoskeleton and the ER
network.
To investigate the influence of different interactions
between GhCFE5 homoeologs and actin on F-actin struc-
ture in developing cells, we stained WT and transgenic
Arabidopsis root cells with phalloidin conjugated to a
green fluorescent signal at 5 DAG. The thin actin filaments
and thick arrays or cables in the wild-type existed longi-
tudinally or obliquely and formed a complicated net-like
structure (Fig. 7). This was also true of the F-actin orga-
nization in GhCFE5A-overexpressing root cells, whereas,
in most cases, the F-actin in GhCFE5D-overexpressing
root cells composed thin filaments without thick arrays or
cables and existed in a lateral orientation. Importantly,
changes in the structure of the actin cytoskeleton could
explain the short root phenotype in GhCFE5D-overex-
pressing Arabidopsis seedlings.
Discussion
Allopolyploidization is a widespread biological process
and a predominant factor in the diversification and suc-
cess of many flowering plant species (Abbott et al. 2013;
Lashermes et al. 2014). Many important crops, such as
wheat, oilseed, and cotton, are allopolyploids containing
two or more sets of chromosomes resulting from inter-
specific or intergeneric hybridization and chromosome
doubling. The merging of two or more divergent genomes
and the presence of these parental genomes in duplicate
can set the stage for selection, adaption, and domestica-
tion (Doyle et al. 2008; Leitch and Leitch 2008). Here,
we isolated two GhCFE5 homoeologs from Upland cot-
ton, TM-1 (allotetraploid AADD), investigated their
expression in various cotton tissues, and detected their
possible functional divergence in cotton fiber develop-
ment via protein interaction analysis and the phenotypic
detection of root and hypocotyl cells in GhCFE5-over-
expressing Arabidopsis. Further, the functional role of
these homoeologs in cotton evolution and domestication
was explored.
c
Fig. 6 GhCFE5A and GhCFE5D interact differently with actin
proteins from Arabidopsis or Upland cotton. aYeast two-hybrid
assay on interaction between GhCFE5 homoeologs and Arabidopsis
vegetable actin proteins. Left column, cotransformants grown on X-a-
Gal synthetic complete medium without Leu and Trp (SD-Leu-Trp).
GhCFE5A and GhCFE5D were cloned into the yeast two-hybrid
vector pGBKT7, while AtACT2, AtACT7, and AtACT8 were cloned
into pGADT7. The interaction between pGBKT7-53 and pGADT7-T
was used as the positive control. The interactions between pGBKT7-
Lam and pGADT7-T, BD (pGBKT7)-CFE5A, or BD-CFE5D and AD
(pGADT7) were used as negative controls. Blue colonies represent
positive interactions in the presence of X-a-Gal. Right column,
positive selection of yeast two-hybrid interactions with minimal
selection (SD-Leu-Trp-His) medium supplemented with 5 mM 3-AT.
bYeast two-hybrid assay on interactions between GhCFE5 homoe-
ologs and GhACTs (GhACTa, GhACTb, GhACT2, and GhACT4).
Positive interactions were detected with restrictive (SD-Leu-Trp-His-
Ade) medium. cBiFC assay of GhCFE5 homoeologs and GhACTs in
tobacco (Nicotiana benthamiana) via agroinfiltration. Fluorescence
signal of YFP could be detected as an indicator of protein–protein
interactions. Scale bars 20 lm. dCFE5A-GFP and CFE5D-GFP co-
expressed with an ER luminal marker, RFP-HDEL in tobacco
(Nicotiana benthamiana) leaf epidermal cells. Scale bar 20 lm.
eCFE5A-GFP and CFE5D-GFP co-expressed with an actin filament
marker, ABD2-mCherry, in tobacco leaf epidermal cells. Scale bar
20 lm (color figure online)
876 Plant Cell Rep (2016) 35:867–881
123
Plant Cell Rep (2016) 35:867–881 877
123
Divergence in the expression of GhCFE5
homoeologs
Polyploidization induces nonadditive expression of
homoeologous genes (Chen 2010). The non-additive
expression of homoeologous genes in polyploidy is possi-
bly regulated through mechanisms of DNA methylation
(Chen et al. 2008), chromatin modifications (Ha et al.
2011), RNA-mediated pathways (Ha et al. 2009; Kenan-
Eichler et al. 2011; Ng et al. 2012), and alternative splicing
(Zhou et al. 2011). In GhMYB2 homoeologs, GhMYB2D
mRNA accumulates more than GhMYB2A during fiber
initiation and is reported to be targeted by miR828 and
miR858 (Guan et al. 2014).The expression of the TaEXPA1
homoeologs, TaEXPA1-A,TaEXPA1-Band TaEXPA1-D,
varied at different stages and in different organs, and the
expression divergence is related to epigenetic modifica-
tions (Hu et al. 2013). In our study, based on the sequences
identity between CFE5 genes from G. herbaceum and G.
raimondii and chromosome mapping analysis, we were
able to determine the genomic origin of the two GhCFE5
homoeologs (GhCFE5A and GhCFE5D). In addition, a
higher accumulation of GhCFE5A than GhCFE5D mRNA
was detected across all tissues tested; the transcripts of
GhCFE5A were primarily observed in the ovules and fibers
and its levels were higher in ovules than those in corre-
sponding fibers, while, GhCFE5D mRNA was dramatically
detected in 10 DPA fibers compared with other tested tis-
sues, indicating that GhCFE5A plays an important role in
ovule and fiber development and GhCFE5D functions on
fiber rapid elongation. However, the promoter sequence
analysis of GhCFE5A and GhCFE5D in TM-1 showed that
there were no key cis-elements related to the expression
difference detected (data not shown). Therefore, the dif-
ferent mechanisms of GhCFE5 homoeolog expression
remain to be elucidated.
Functional divergence of GhCFE5 homoeologs
Duplicate genes can function following three evolutionary
paths: the transformation of one copy into a nonfunctional
pseudogene (Lynch and Conery 2003), subfunctionaliza-
tion (Lynch and Force 2000), and neofunctionalization
(Adams et al. 2003). In the study, sequence analysis
revealed that there are non-synonymous mutations and
frameshift mutations within the CFE5 homologs, which
change M
w
and pI values, leading to GhCFE5A being a
basic peptide and GhCFE5D an acidic peptide. These dif-
ferent features between the two GhCFE5 homoeologs may
affect protein function and lead to functional divergence in
Arabidopsis root cells and cotton fiber development. Like
the function of GhCFE1A in cotton (Lv et al. 2015),
overexpression of full-length GhCFE5A and GhCFE5D in
Arabidopsis thaliana induced significant suppression of the
length of the hypocotyls and roots, and the reduction of cell
elongation was more obvious in GhCFE5D-overexpressing
plants than in GhCFE5A transgenic seedlings. Exception-
ally, there was no statistically significant difference
between 35S::GhCFE5A line 9 and WT plants. It might be
related to that GhCFE5A has a less powerful effect on
hypocotyl and root development, which was also confirmed
by yeast two-hybrid assay and actin cytoskeleton
Fig. 7 F-actin organization in the differentiation zone of living Arabidopsis roots. F-actin structure was observed by confocal microscopy in 5
DAG wild-type (WT) and GhCFE5A or GhCFE5D-overexpressing roots
878 Plant Cell Rep (2016) 35:867–881
123
observation. Taken together, there exists the functional
divergence of the GhCFE5 homoeologs, and overexpres-
sion of GhCFE5D in cotton may lead to significant short
fibers while suppression of GhCFE5D and GhCFE1A or all
CFEs would produce improved long fibers. Further cotton
transformation studies would provide more information on
this speculation.
The yeast two-hybrid assay showed that GhCFE5D
interacts with all tested Arabidopsis actins (AtACT2,
AtACT7 and AtACT8) and three cotton actin isoforms
(GhACTa, GhACTb and GhACT4), while GhCFE5A only
interacts with AtACT7 and two cotton actin isoforms
(GhACTa and GhACTb), which was further confirmed by
BiFC assay. In our previous study, GhCFE1A was
observed to interact with all tested actins (GhACTa,
GhACTb, GhACT2 and GhACT4) (Lv et al. 2015). Thus
the actins and CFEs appeared to have evolved class-
specific, protein–protein interactions that are essential for
the normal regulation of plant growth and development. In
other words, different CFE isoforms bind to special actin
isoforms. A possible interpretation is that the differences in
the gene and protein structures are important for their
functions in vivo. Future experiments will be designed to
test these possibilities.
Compared with GhCFE5A, the greater number of iso-
forms and stronger binding activity of GhCFE5D with
actins ultimately caused clear defects in the organization of
the actin cytoskeleton and induced the loss of thick actin
cables and laterally orientated thin actin filaments in
GhCFE5D-overexpressing plant root cells. It is well known
that the actin cytoskeleton is essential for the development
of plant structures such as root hairs (Kandasamy et al.
2009), leaf trichomes (Li et al. 2003), pollen tubes (Chen
et al. 2003), and cotton fibers (Li et al. 2005). In the actin
cytoskeleton network, F-actin cables are generally con-
sidered to play an important role in accelerating the
transport of vesicles carrying cell growth materials to the
growth sites (Mathur and Hulskamp 2002). In addition, co-
localization analysis indicated that as GhCFE1A per-
formed, GhCFE5 linked the ER and actin cytoskeleton and
regulated cell elongation. Hence, it is probable that CFEs
might be a set of proteins that mediate the interplay
between ER network and actin cytoskeleton with func-
tional divergence, and overexpression of GhCFE5D, but
not GhCFE5A, may affect this bridging function, decrease
the formation of thick actin cables, change the orientation
of actin filaments and finally lead to impairment of cell
elongation. Moreover, in cotton, fiber develops well in
A-genome species but poorly in D-genome species such as
G. raimondii. This may be related to the preferential sup-
pression of one of the homoeologous transcripts, such as
GhCFE5D, that is associated with poor fiber traits.
GhCFE5 might play an important role
in the evolution and selection of fiber traits
Gossypium hirsutum was domesticated from perennial trees
into annual crops. There are seven races of G. hirsutum:
‘yucatanense’, ‘punctatum’, ‘palmeri’, ‘latifolium’, ‘mar-
iegalante’, ‘morrilli’, and ‘richmondii’, which are recog-
nized as semi-domesticated cotton (Hutchinson 1951).
Seed trichomes from these wild G. hirsutum species are
short (typically, 1.5 cm), coarse, and brown fibers. Con-
versely, modern G. hirsutum cultivars (Upland cotton) with
high-yield properties, such as long, strong, and fine white
fibers, are the most important domesticated fiber plant in
the world and make up more than 90 % of global cotton
production. During the phenotypic evolution of single-
celled epidermal trichomes, strong directional selection
dramatically rewired the transcriptome of developing cot-
ton fibers, affecting more than 5000 genes, and with a more
evenly balanced usage of the duplicated copies arising
from genome doubling (Yoo and Wendel 2014).
In the present study, GhCFE5 homoeologs were
expressed preferentially in fiber cells; however, higher
expression levels of GhCFE5A were observed than those of
GhCFE5D across all tissues tested in the cultivated tetra-
ploid cotton TM-1. Further, GhCFE5D interacted with
more actin proteins from both Arabidopsis and Upland
cotton and with a stronger interaction activity than
GhCFE5A, in addition, GhCFE5D-overexpressing Ara-
bidopsis plants had severely disrupted actin cytoskeleton
organization and a significant reduction in root cell length,
indicating that GhCFE5D has a stronger influence than
GhCFE5A in negatively regulating cell elongation.
Through transcriptome profiling analysis of developing
cotton fibers from multiple accessions of wild and
domesticated cottons (Yoo and Wendel 2014), we also
found GhCFE5 is predominately expressed during fiber
elongation, with a higher accumulation of GhCFE5D
transcripts in wild G. hirsutum var. yucatanense (TX2095)
and G. hirsutum var. palmeri (TX665) than TM-1 at
10DPA (P\0.05) (Data not shown). Taken together, these
results suggest that GhCFE5 might play an important role
during the evolution and selection of cotton fiber by bal-
ancing the expression levels of the duplicated copies to
bind actins at the appropriate level for increasing lint yield
and improving fiber quality in domesticated cotton.
Author contribution statement W. Guo conceptualized
the research program and coordinated the project; most
experiments and data analyses were performed by F. Lv; P.
Li contributed to analysis of yeast two hybridization and
BiFC; R. Zhang constructed 35S::GhCFE5A and
35S::GhCFE5D vectors and transformed into Arabidopsis;
Plant Cell Rep (2016) 35:867–881 879
123
N. Li assisted in analysis of transgenic plants; F. Lv drafted
the manuscript; W. Guo revised the manuscript.
Acknowledgments This program was financially supported in part
by National Natural Science Foundation of China (31471539), a
project funded by the Priority Academic Program Development of
Jiangsu Higher Education Institutions (010-809001), and Jiangsu
Collaborative Innovation Center for Modern Crop Production (No.
10).
Compliance with ethical standards
Conflict of interest The authors have declared that no competing
interests exist.
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