(03348) Jian-Xun FENG et al:Analysis of Five Differentially Expressed Gene Familiesin Fast Elongating Cotton Fiber

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e-mail: [email protected]

ISSN
0582-9879 Acta Biochim et Biophysica Sinica 2004, 36(1):51-57 CN 31-1300/Q

Analysis of Five Differentially Expressed Gene Families
in Fast Elongating Cotton Fiber

Jian-Xun FENG, Sheng-Jian JI, Yong-Hui SHI, Yu XU, Gang
WEI, and Yu-Xian ZHU*

( National Laboratory of Protein Engineering and Plant Genetic
Engineering, College of Life Sciences, Peking University, Beijing
100871, China )

Abstract Using the suppression subtractive hybridization
method, we isolated five gene families, including proline-rich proteins (PRPs),
arabinogalactan proteins (AGPs), expansins, tubulins and lipid transfer
proteins (LTPs), from fast elongating cotton fiber cells. Expression profile analysis
using cDNA array technology showed that most of these gene families were highly
expressed during early cotton fiber developmental stages (0–20 day post
anthesis, DPA). Many transcripts accumulated over 50-fold in 10 DPA fiber cells
than in 0 DPA samples. The entire gene family ―AGP, together with 20 individual members in other 4 gene families, are
reported in cotton for the first time. Accumulation of cell wall proteins, wall
loosening enzymes, microtubules and lipid transfer protein may contribute directly
to the elongation and development of fiber cells.

Key words cotton fiber; cDNA array; cell elongation;
arabinogalactan protein; lipid transfer protein

Cotton fibers are long (30–40 mm) and thin (15 μm) unicellular
structures that emerge from certain epidermal cells in the outer integuments of
cotton ovules. Fiber development can be conceptually separated into four
phases: initiation, elongation, secondary wall deposition and maturation [1].
Usually fibers initiate at or just before anthesis and soon enter a rapid
elongation period that lasts until 25 d post anthesis (DPA). Fiber cells
display the most rapid elongating rate around 10 DPA. During secondary wall
deposition stage (20–45 DPA), fibers undergo mainly cellulose biosynthesis,
which results in very high cellulose content (about 90%) in these cells. In the
past decade, great efforts have been made to elucidate molecular mechanisms of
cotton fiber development, many cotton genes have been isolated, and even
transgenic cottons have been obtained [2,3]. However, no single crucial fiber
factor has been discovered, which suggests that regulation of fiber development
may require a number of genes [4].

Proline-rich proteins (PRPs) and arabinogalactan proteins
(AGPs) are two important cell wall structural proteins[5,6]. PRPs are widely
distributed in plants and are encoded by gene families. PRP members showed to
be regulated both temporally and spatially during plant development [7]. In
cotton, several different PRP genes were isolated and analyzed [8,9]. AGPs are
a family of proteoglycans that have been implicated in various aspects of
cellular activities [10,11]. Expansin(EXP), encoded by a highly conserved
multigene family, mediate pH-dependent extension of plant cells by disrupting
hydrogen bonds at the interfaces between individual cell wall polymers
[6,12,13]. Expansins appear to be involved in a variety of plant processes,
such as cell growth and polarity, root hair formation and growth, organogenesis
and fruit ripening [14]. Several expansin genes were reported from cotton fiber
[15,16]. Microtubules play central roles in many important cellular processes
in higher plants [17]. Microtubules consist mainly of α-tubulin (ATub) and β-tubulin (BTub), both of
which are encoded by multigene families in plants [2]. In cotton, nine α-tubulin and seven β-tubulin isotypes were

identified using two-dimensional gels, and the expression
of α-tubulin
genes were studied in fiber cells [18,19].However, only two β-tubulin genes were
isolated and characterized so far [2,20].

Lipid transfer proteins (LTPs) are small cysteine-rich
lipid-binding proteins and are also called non-specific LTPs (nsLTPs) since
they transfer membrane lipids with no specificity. In plants, LTPs are probably
involved in somatic embryogenesis, in defence against pathogens, and in the
formation and reinforcement of waxy cuticle layers in plant surface [21,22].
Several plant LTPs have been identified in cotton fibers [21,23,24]. Despite
recent progress in cotton gene isolation, there is no report concerning the
expression patterns of gene families as a whole.

In this work, five gene families that are preferential to
cotton fiber were identified from a subtractive cDNA library, and their RNA
levels during the early cotton fiber development stages were studied .

Materials and Methods

Plant materials

Upland cotton and its fuzzless-lintless mutant (fl)
were field grown during the summer of 2001. The mutant was originally
discovered in an upland cotton (Gossypium hirsutum L. cv. Xuzhou 142) field
in China [25]. Immediately after harvest, developing ovules were excised from
each boll and fiber cells were carefully scraped from the epidermis of the
ovules. All harvested plant materials were frozen in liquid nitrogen and stored
at –80 ℃ before use.

cDNA array preparations

Suppression subtractive hybridization was carried out
using cDNAs prepared from 10 DPA cotton fiber and fl mutant ovule as
tester and driver, respectively. The subtracted cDNAs were inserted directly
into the T/A cloning vector to transform E. coli DH5α cells, which
produced the resultant subtractive cDNA library[4]. The clones with different
gene families were selected from the subtractive library and cultured in 200 μl of LB-Amp medium in
96- well plates at 37 ℃. The cDNA
inserts were amplified by PCR in a 96-well PTC200 peltier thermal cycler (MJ
Research, USA) following the procedure described previously [4]. All PCR
products were analyzed by agarose gel electrophoresis and quantified using a
spectrophotometer. Then DNA (0.3 μg) samples were printed from PCR plates onto the nylon
membranes (Roche, Germany) using the Biomek2000 laboratory automation
workstation (Beckman Coulter, USA). The cotton ubiquitin was also printed onto
each membrane as the internal controls. Distilled water, PCR primers and vector
DNA were used as the negative controls.

Hybridization of cDNA arrays, image acquisition and
analysis

RNA isolated from 0, 5, 10 and 20 DPA cotton fibers were
used to prepare probes for expression pattern analysis. Images were acquired by
scanning the membranes with a Typhoon 9210 scanner (Molecular Dynamics, USA).
Data analysis was performed using ArrayVision 6.0 software (Imaging Research,
USA). The radioactive intensity of each spot was quantified as volume values
and the levels of the local background were subtracted to obtain the subtracted
volume values designated as sVOL. Ubiquitin cDNA was used as the internal
control, its subtracted volume value was termed as sRef. Normalization among
all images was performed by dividing sVOL of each spot by the sRef value in the
same image, resulting in a normalized volume value (nVOL) for each spot. nVOL
values were comparable among all obtained images. The nVOL values of each gene
at 5, 10 and 20 DPA were divided by that at 0 DPA to obtain the fold of
increase in the gene expression level.

Semi-quantitative RT-PCR

Total RNA was isolated from wild type cotton fibers.
First-strand cDNA was synthesized from 5 μg total RNA using SUPERSCRIPT first-strand synthesis
system for RTPCR (Gibco, New York, USA). One tenth of the synthesized
first-strand cDNA was used as templates in 50-μL PCR with gene-specific RT-PCR primers designed
according to the cDNA sequence and synthesized commercially (TaKaRa, Dalian,
China). Parallel reactions using cotton ubiquitin primers served to normalize
the amount of template added. According to the transcription level of these
family genes, some clones were chosen to verify the result of microarray by
RT-PCR. All RT-PCR primers used in this study are listed in Table1.

Table 1 The sequence
and characterization of RT-PCR primers

 

Results

Isolation of five gene families

From a pool of subtractive cDNA fragments that were
either expressed only in cotton fiber or displayed in significantly higher
levels (>2-fold) in fibers compared to mutant

ovules, five large gene families, including PRPs, AGPs,
expansins, tubulins and LTPs were identified. PRPs, expansins, tubulins and
LTPs showed high sequence identities

with their homologs reported previously in cotton fiber
and other plants (data not shown). AGPs shared 58％–59% identities in the deduced amino acid level (data not shown). Among
the 32 independent cDNAs (6 PRPs, 5 AGPs, 4 expansins, 6 tubulins, 11 LTPs)
belonging to these families, 20 are potential new members. Because no AGP gene
families have been reported in cotton previously, all sequences of these cDNAs
are deposited in the EST division of GenBank with accession numbers from
CB350396 to CB350561 and can be accessed individually from the supplementary
material of Ji et al. [4].

Expression analyses by cDNA array

We studied the expression patterns of these gene families
during early cotton fiber development. The printed cDNA arrays were hybridized
to [33P]-labeled
probes prepared from 0, 5, 10 and 20 DPA wild-type cotton RNA,respectively (Fig.
1). Most cDNAs showed very low expression levels in 0 DPA cotton fiber and were
usually activated after 5 DPA. A major portion of these cDNAs reached peak
levels around 10 DPA with decreases observed thereafter, which indicated their
potential importance in the fiber elongation phase. The semi-quantitative
RT-PCR result shown in Fig. 2 was almost similar to the microarray result of
these gene families. So the result of microarray is reliable.

Fig. 1 Microarray analysis showing different expression
patterns for the five gene families during early fiber development

[33P]-labeled cDNA probes
were prepared from RNAs that were isolated from 0, 5, 10 and 20 DPA cotton
fibers. P1–P6: PRP1 to PRP6; A1–A5: AGP1 to AGP5; E2–E5: EXP2 to EXP5; BT1, 4,
8, 9: BTub1, 4, 8, 9; AT2, 4: ATub2, 4; L1–L8 and L10–L12: LTP1– LTP8 and LTP10
–LTP12. Cotton ubiquitin gene (U) was used as the positive control, and PCR
primers (P), ddH2O (H)
and vector DNA (V) were used as negative controls. Their locations were shown
in the key.

Fig. 2 Differential accumulation of different members of
4 gene families as verified by RT-PCR

UBQ, ubiquitin.

Expression profiling of the gene families

Data obtained from the cDNA arrays were quantified and
expressed as number of folds increased at a specific date in comparison with
the intensities of 0 DPA ovules. Five gene families were analyzed and plotted
in Fig. 3. The levels of PRP4 and 5 were 240 and 256-fold higher in 10 DPA
fiber cells than in 0 DPA ovules. A sharp decline was visible for both
messengers when quantified at 20 DPA [Fig. 3(A)]. The other four PRPs were
demonstrated to only have moderate changes in expression and their levels were
accumulated less than 20 folds during all phases of fiber development.

Different from that of PRPs, mRNAs of all AGPs
accumulated to significantly high levels during early fiber elongation period
and soon reached a plateau after 10 DPA[Fig. 3(B)]. The amounts of AGP2 and
AGP4 increased to 400- and 423-fold higher in 10 DPA fiber cells than in 0 DPA
ovules respectively. Other AGPs were found to be at least 150- to 200-fold more
abundant in 10 DPA fiber cells. Fig. 3(C) showed the expression levels changes
with the development of fiber elongation of the five cotton expansins (EXPs)
discovered in the current work. EXP5 accumulated to over 100-fold in 10 DPA
fibers than in 0 DPA ovules. EXP2 increased steadily in a relative low speed
throughout the whole experimental period [Fig. 3(C)]. Fig. 3(D) analyzed the
expression patterns for two gene families, including β-tubulins (BTubs) and α-tubulins (ATubs). BTub8
was noticeable because its transcripts increased 59- and 305-fold at 10 and 20
DPA compared with that at 0 DPA respectively, indicating it might be involved
in middle and/or later stages of fiber development. Other tubulins usually
reached the highest levels at around 10 DPA, and leveled out during 10–20 DPA.

LTP is a large gene family consisting of 11 members
identified in the subtractive library. Among these 11 LTPs, the mRNA level of
LTP6 accumulated 80-fold higher in 10 DPA than in 0 DPA ovules, while the mRNA
levels of other 10 LTPs never increased higher than 35-fold. Usually, the
expression of LTPs reached highest expression levels at around 10 DPA and then
decreased steadily during 10–20 DPA [Fig. 3(E)]. LTP11 reached its highest
level at 5 DPA.

All the cDNAs reported in the current work were under the
same designations as specified in Ji et al.[4]. Several redundant
tubulins were excluded from this figure while the original designations were
followed so that sequence data is easily tracked down.

 

Fig. 3 Individual members of each gene family accumulated
to different levels during the fiber elongation period

Transcript levels for the members of gene families are shown. (A) PRP. (B)
AGP. (C) Expansin. (D) Tubulin. (E) LTP. The values for the axis of relative
expression level referring to the folds of the increase of expression level at
5, 10 and 20 DPA compared with that at 0 DPA.

Discussion

The elongation or directional expansion of plant cells
occurs by two main processes: diffuse growth and tip growth [26]. During growth
and elongation, plant cells undergo many cellular changes such as modification
of cell wall composition and organization. Structural proteins including PRPs
and AGPs are known to play crucial roles in restructuring the cell wall [27].
PRPs may function by regulating actin polymerization and promoting membrane
protrusions [28,29] and a particular PRP from Arabidopsis was needed for
root hair initiation by forming new cell wall materials [7]. AGPs are
presumably involved in the molecular interaction and cellular signaling at the
cell surface [11] and are also known to be associated with cell expansion
during root and pollen tube growth [30–32]. SOS5, an Arabidopsis AGP
homolog was required for normal cell expansion [33] and fucosylated AGPs were
required for elongation of Arabidopsis root cells [34]. An AGP from
tobacco stimulated pollen tube elongation both in vivo and in vitro [35].
The finding that many members of these two gene families accumulated to very
high levels in early developing cotton fiber cells [Fig. 3(A) and (B)] may be a
further indication of their importance during cell elongation processes.

Cell wall expansion requires moving apart of the
cellulose microfibrils in the cell wall. Expansins are such a family of
proteins needed for disrupting the non-covalent

bonds between the cellulose microfibrils and cross-linked
glycans [36]. It has been shown that different expansins can be expressed in
different developmental stages [26], so that the maximum expression of expansin
genes could be required for the initiation of expansion growth [37,38]. In this
work, we found that the accumulation rates of expansin, especially that of
EXP5, were in full accordance with the rate of fiber expansion [Fig. 3(C)]. We
suggest that cells may produce large amounts of wall-loosening enzymes,
including expansins, to facilitate the extremely high rate of cell wall
expansion during the early cotton fiber growth phase.

Microtubules are key components of the eukaryotic
cytoskeletons and are also suggested to play important roles in plant cell
expansion for both tip growth and diffuse growth mechanisms [34]. Extensive
mutant studies and drug-treatments have shown the importance of microtubules in
the elongation of specialized single-cell tissues such as trichomes, root hairs
and pollen tubes [39–42]. Ji et al. [20] found that the over-expression
of a cotton β– tubulin induced the elongation of fission yeast cells. Li et al.
[2] reported that preferential accumulation of a cotton β-tubulin gene improved
longitudinal growth potential of a developing fiber cell. Accumulation of
various tubulin transcripts will undoubtedly contribute to the rapidly
elongation of fiber cells.

Although the precise biological functions of LTPs have
not been clearly confirmed yet, they are probably involved in the formation and
reinforcement of surface layers of plant cells as reported previously [22]. It
was shown that extracellular wax matrix was involved in many important
cell-signaling pathways such as epidermal cell differentiation and cell
adhesion [43–45]. The existence of fiberspecific LTPs also suggested their
roles in the fiber development. Although some individual members of the gene
families studied in this work have been reported previously [8,15,19,23], the
results in this paper constitute the most complete characterization of these
families in the

elongation of cotton fibers so far. We believe that
elucidation of the molecular functions of genes preferentially involved in the
early fiber growth phase may help decipher

the mechanisms controlling cell elongation.

Acknowledgements

We thank Prof. M.-H. Liu for her assistance in preparing
the cDNA array and scanning the phosphor image.

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Received: August 27, 2003 Accepted: October 22, 2003

This work was supported by a grant from the Chinese Ministry of Science
and Technology (No. J99-A-03)

* Corresponding author: Tel, 86-10-62751193; Fax, 86-10-62754427; E-mail, [email protected]