JXB Advance Access originally published online on August 30, 2007
Journal of Experimental Botany 2007 58(12):3227-3238; doi:10.1093/jxb/erm167
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RESEARCH PAPER |
Molecular characterization of cotton GhTUA9 gene specifically expressed in fibre and involved in cell elongation
College of Life Sciences, HuaZhong Normal University, Wuhan 430079, China
To whom correspondence should be addressed. E-mail: xbli{at}mail.ccnu.edu.cn
Received 5 March 2007; Revised 5 June 2007 Accepted 26 June 2007
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Abstract |
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The microtubule cytoskeleton may play an important role in the polarized growth of fibre cells that are single-cell trichomes on the surface of cotton ovules. To investigate whether the high expression levels of
-tubulin genes are correlated with fibre elongation, nine GhTUA genes (cDNAs) encoding -tubulins with 449–451 amino acid residues were isolated and characterized in cotton. The GhTUA genes share high sequence homology at the nucleotide level (62–93% identity) in the coding region and at the amino acid level (89–99% identity), and can be classified into two subgroups. Real-time quantitative RT-PCR analysis revealed that seven out of the nine GhTUA genes are predominantly expressed in developing fibres. Among them, GhTUA9 displays the highest level of expression, revealing its fibre specificity. The GhTUA9 transcripts in fibres reached its peak value between 5–10 DPA, and dramatically declined to undetectable levels as the ovule matured further, suggesting that its expression is developmentally-regulated in fibres. The GhTUA9 gene including the promoter region was isolated from the cotton genome. To demonstrate the specificity of the GhTUA9 promoter, the 5'-flanking region, including the promoter and 5'-untranslated region, was fused with the GUS gene. Histochemical assays demonstrated that the GhTUA9:GUS gene was specifically expressed in elongating fibres. Overexpression of GhTUA9 in fission yeast (Schizosaccharomyces pombe) promoted atypical longitudinal growth of the host cells by 1.4–1.7-fold, indicating that the GhTUA9 gene is involved in cell elongation. Given all the above results, it is proposed that the GhTUA9 gene may play an important role in fibre elongation. Key words: Cotton, expression profiles of GhTUA genes, fibre elongation, fibre-specific, microtubule, overexpression in yeast cells
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Introduction |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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Cotton, including Upland cotton (Gossypium hirsutum) and Sea Island cotton (Gossypium barbadense), is the main source of natural fibres used in the textile industry. Cotton fibres, or seed hairs, are single-celled trichomes differentiated at anthesis from approximately 30% of the ovule epidermal cells, which undergo rapid and synchronous elongation during seed development. Fibre elongation persists about 16 d post anthesis (DPA) before the onset of secondary cell wall cellulose synthesis and enables the unicellular trichome to lengthen from 10 µm to 15 µm up to 2.5–3.0 cm (Basra and Malik, 1984; Tiwari and Wilkins, 1995), rendering cotton fibre to be one of the longest single cells in higher plants. Cotton fibres driven by high turgor (Dhindsa et al., 1975; Ruan and Chourey, 1998; Smart et al., 1998) can achieve peak growth rates of >2 mm d–1 during the rapid elongation stage (John and Keller, 1996; Ji et al., 2002), while they partition approximately 80% of their available carbon into cellulose throughout secondary wall deposition (Basra, 1999; Haigler et al., 2001). Thus, the unicellular fibres provide a unique system for the study of regulatory mechanisms of cell elongation in the absence of cell division.
The rapid fibre elongation is a highly regulated process, associated with the functional expression of many genes. Global analysis of the fibre transcriptome on a cotton cultivated diploid species (Gossypium arboretum) indicated that the fibre-specific/preferential genes consist of three major functional groups: cell wall structure and biogenesis, cytoskeleton, and energy/carbohydrate metabolism (Arpat et al., 2004). An early study proposed that an observed transient closure of plasmodesmata of the fibres at the commencement of the elongation stage may serve to maintain the high turgor driving force for the rapid lengthening of the lint fibres (Ruan et al., 2001). The expression of sucrose synthase (Sus) in cotton is essential for fibre initiation and elongation. Delayed or insufficient Sus expression resulted in delayed initiation and distinctly shortened fibre elongation in a fuzz-like short fibre cotton mutant (Ruan et al., 2005). Furthermore, previous studies indicated that the cotton GhTUB1 and GhACT1 genes are involved in early fibre development (Li et al., 2002, 2005). Although some progress has been made in identifying cotton fibre-specific/preferential and developmental-regulated genes in recent years, little is known in detail so far of how these genes regulate fibre development. Elucidating the genes specifically involved in the elongation stage of fibre cells enables us to understand further the molecular mechanism(s) of fibre development, thereby improving the fibre quality and yield of cotton by genetic manipulation.
Microtubules are believed to be involved in the establishment of polar-axis determination and the maintenance of directionality of cell expansion in higher plants. They nucleate chiefly from the 
-tubulin small complex and ring complex (Raynaud-Messina and Merdes, 2007), and are composed of heterodimers of highly conserved - and β-tubulin, encoded by multigene families (Cleveland and Sullivan, 1985; Silflow et al., 1987). The members of the tubulin gene family display isotype-specific C-terminal variation, and expose their extreme C-termini from the assembled polymer in order to interact with MT-associated proteins (Littauer et al., 1986) and provide sites for post-translational modifications (MacRae, 1997). A recent study on keratins showed that GCP6, a component of the -tubulin ring complex, plays a role in the attachment of microtubule-organizing centres (MTOCs) to intermediate filaments (IFs) and microtubule organization in polarized epithelia (Oriolo et al., 2007). Similarly, Arabidopsis -tubulin is required for the formation and organization of microtubule arrays in plant development (Pastuglia et al., 2006). Various drug treatments suggest that microtubules are pivotal for the establishment and maintenance of growth polarity as well as for initiating changes in growth directionality in Arabidopsis trichomes (Baskin et al., 1994; Bibikova et al., 1999; Kost et al., 1999; Mathur and Chua, 2000). Katanins may generate new microtubule ends by severing existing microtubules in higher plants (Quarmby, 2000; Burk et al., 2001; Webb et al., 2002; Stoppin-Mellet et al., 2006), whereas CLASP2 plays a role in microtubule stabilization and the regulation of persistent motility in fibroblasts (Drabek et al., 2006). Turgor-driven cell expansion maintains the elongating cell via the actions of highly organized microfibrils of cellulose controlled by cortical microtubules (Giddings and Staehelin, 1991; Delmer and Amor, 1995; Cyr and Palevitz, 1995). Recent study revealed that microtubules functionally associate with cellulose synthase (CESA). Inhibition of microtubule polymerization changed the fine-scale distribution and pattern of moving CESA complexes in the plasma membrane, indicating a relatively direct mechanism for the guidance of cellulose deposition by the microtubule cytoskeleton (Paredez et al., 2006).
The -tubulin genes have been well characterized in a few plants, such as Arabidopsis (Kopczak et al., 1992), and studies have revealed that the accumulation of transcripts of -tubulin genes in Arabidopsis and maize were shown to exhibit tissue-specificity (Ludwig et al., 1988; Carpenter et al., 1992; Uribe et al., 1998). By contrast, although nine -tubulin and seven β-tubulin isotypes were identified on immunoblots of two-dimensional gels in cotton (Dixon et al., 1994; Whittaker and Triplett, 1999), so far in databases, there are only three cotton -tubulin genes (cDNAs) with complete coding regions present, of which none has been well characterized. Here, the isolation of an additional nine cotton -tubulin genes and the molecular characterization of GhTUA9 are reported. Real-time quantitative RT-PCR and northern blot analyses revealed GhTUA9 to be a fibre- and developmental stage-specific gene whose down-regulation coincides with the termination of fibre elongation. In order to gain insights into its functional role in the elongation stage of cotton fibres, the GhTUA9 gene was over-expressed in yeast (Schizosaccharomyces pombe) cells, which were induced to undergo atypical longitudinal elongation.
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Materials and methods |
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Collection of plant materials
Cotton (Gossypium hirsutum, cv. Coker312) seeds were surface-sterilized with 70% ethanol for 30–60 s and 10% H2O2 for 30–60 min, followed by washing with sterile water. The sterilized seeds were germinated on half-strength MS medium under a 12/12 h light/dark cycle at 28 °C. Cotyledons, roots, and hypocotyls were collected from sterile seedlings, and other tissues were derived from cotton plants (G. hirsutum) grown in a greenhouse or field for DNA and RNA isolation.
Northern blot analysis
Total RNA was extracted from young fibres, ovules, anthers, petals, cotyledons, leaves, and roots of cotton as described previously (Li et al., 2002). RNA samples (20 µg lane–1) from different cotton tissues were separated on 1.2% agarose–formaldehyde gels, and transferred onto Hybond-N nylon membranes by capillary blotting. The fragment of the GhTUA9 3'-untranslated region was used as a gene-specific probe for northern blot analysis as described previously by Li et al. (2002).
Isolation of GhTUA cDNA and DNA clones
To identify the genes that are specifically/preferentially expressed in cotton fibre, over 2000 cDNA clones were randomly selected from the cotton fibre cDNA libraries (Li et al., 2002) for sequencing. Some GhTUA clones with complete or partial sequences were identified. Then, a 600 bp PCR fragment of GhTUA9 from the coding region was labelled with -32P-dCTP and used as a probe to screen cotton fibre cDNA libraries according to standard procedure. 3x106 cDNA clones were screened and over 100 clones were identified. Among them, 30 full length clones were sequenced and analysed. In total, eight unique cDNA clones were obtained.
Cotton genomic libraries were constructed as described earlier by Li et al. (2002). For isolating the GhTUA genes, about 4x106 clones were screened with an -32P-dCTP-labelled probe (0.6 kb GhTUA9 PCR fragment) generated using a random primer method (Prime-a-Gene Labelling System, Promega). In total, three unique GhTUA genes (DNA clones) were obtained.
RT-PCR analysis
The expression of the GhTUA genes in cotton tissues was analysed by real-time quantitative reverse transcriptase (RT)-PCR using the fluorescent intercalating dye SYBR-Green in a detection system (MJ Research, Opticon 2) described earlier by Li et al. (2005). A cotton polyubiquitin gene (GhUBI) was used as a standard control in the RT-PCR. A two-step RT-PCR procedure was performed in all experiments. First, total RNA samples (2 µg per reaction) from leaves, stems, cotyledons, roots, anthers, petals, and fibres were reverse transcribed into cDNAs. Then, the cDNAs were used as templates in real-time PCR with gene-specific primers (Table 1). The amplification of the target genes was monitored every cycle by SYBR-Green fluorescence. The Ct (cycle threshold), defined as the PCR cycle at which a statistically significant increase of reporter fluorescence is first detected, is used as a measure for the starting copy numbers of the target gene. Relative quantitation of the target GhTUA expression level was performed using the comparative Ct method. The relative value for the expression level of each GhTUA gene was calculated by the equation Y=10
Ct/3.7x100% (Ct is the difference of Ct between the control GhUBI products and the target GhTUA products, i.e. Ct=CtGhUBI–CtGhTUA). To achieve optimal amplification, PCR conditions for each primer combination were optimized for annealing temperature and Mg2+ concentration. PCR products were confirmed on an agarose gel. The efficiency of each primer pair was detected by using GhTUA cDNAs as standard templates, and the RT-PCR data were normalized with the relative efficiency of each primer pair.
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Sequence and phylogenetic analysis
Nucleotide and amino acid sequences were analysed using DNAstar (DNAstar Inc). The GhTUA peptide sequences were aligned with the ClustalW program (http://www.ebi.ac.uk), and phylogenetic analysis was employed to investigate the evolutionary relationships among the GhTUA genes. A Neighbor–Joining tree was generated in MEGA3.1 (Kumar et al., 2004). A bootstrap analysis with 1000 replicates was performed to assess the statistical reliability of the tree topology. Maximum likelihood tree was constructed in PHYLIP package (version 3.65) (Felsenstein, 1993). The infile (phylip format) was input for processing SEQBOOT with 1000 replicates, and the resulting outtree was viewed by TreeView.
Construction of the GhTUA9:GUS chimeric gene
A HindIII site and a BamHI site were introduced at the 5'-end and 3'-end of GhTUA9 5'- upstream region (including the putative promoter fragment and untranslated region before initiation codon ATG), respectively, by PCR method. The HindIII/BamHI fragment (1.5 kb) was then subcloned into pGEM-T vector (Promega). Furthermore, plasmid DNA containing the GhTUA9 5'-upstream fragment was digested with HindIII and SalI, and subcloned into the HindIII/SalI sites of pBI101 vector to generate the chimeric GhTUA9:GUS construct.
Cocultivation of cotton ovules with Agrobacterium in vitro
Ovules (1–2 DPA) dissected out from bolls were infected with Agrobacterium tumefaciens strain AGL1 containing GhTUA9:GUS fusion genes, and cocultivated in an ovule-medium (Stewart and Hsu, 1977) without any antibiotics (28 °C, dark), using pBI 121 vector as a control. After 2–4 d of cocultivation with Agrobacterium in vitro, histochemical assay of GUS activity were used to detect the transient expression of the GUS gene, controlled under the GhTUA9 promoter, in the fibres.
Histochemical assay of GUS gene expression
Histochemical assays for GUS activity in the transformed ovules were conducted according to a modified protocol. The cocultivated ovules with fibres were incubated for 4–6 h in X-gluc (5-bromo-4-chloro-3-indolylglucuronide) solution, consisting of 0.1 M sodium phosphate (pH 7.0), 10 mM ethylene diaminetetraacetic acid (EDTA), 0.5 mM potassium ferrocyanide, 0.5 mM potassium ferricyanide, and 0.1% X-gluc (Clontech chemical). The stained ovule materials were subsequently cleaned and fixed by rinsing with 100% and 70% ethanol successively, and the samples were examined and photographed directly or under a Nikon microscope (Japan).
Overexpression of GhTUA9 gene in fission yeast
The coding sequence of GhTUA9 gene, amplified from its cDNA by PCR with the proofreading pfu DNA polymerase, was cloned into yeast vector pREP5N with NotI/BamHI sites. Afterwards, the construct was transferred into yeast (S. pombe) cells by electroporation (Bio-Rad MicroPulser Electroporation Apparatus) according to the manufacturer's instructions. Transformants were then selected on plates, containing minimal medium (MM) supplemented with 75 mg l–1 adenine and uracil at 29 °C (Alfa et al., 1993). 5–10 colonies were picked out to grow in liquid MM with 2 µM thiamine, which represses the nmt-1 promoter activity, until mid-log phase in a shaker (220 rev. min–1, 29 °C). Subsequently, the yeast cells were harvested and washed three times with MM containing no thiamine to de-repress the promoter, and then incubated in the same thiamine-free MM for 22 h (220 rev. min–1, 29 °C). The yeast cells were observed and photographed under a Nikon microscope (Japan), and the lengths of 100 cells per transformant (single-colony) were measured for evaluating the cell elongation, using empty pREP5N transformants and untransformed cells as controls.
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Results |
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Isolation and characterization of GhTUA cDNAs
To isolate genes involved in cotton fibre development, over 2000 cDNA clones were randomly sequenced from a fibre cDNA library (Li et al., 2002). Clones, including
-tubulin cDNAs, and which were probably involved in cell elongation were chosen for further study. Using the -tubulin cDNA as probe, eight unique -tubulin cDNAs were isolated (designated as GhTUA5–GhTUA12 genes, accession numbers in Genbank: EF151301–EF151308) from a cotton cDNA library. Sequence analysis predicted that three GhTUA genes (GhTUA5, GhTUA8, GhTUA11) encode a 449 amino acid polypeptide, whereas the others encode an -tubulin protein containing 450 amino acid residues (GhTUA6, GhTUA9, GhTUA10, GhTUA12) or 451 amino acid residues (GhTUA7) with one or two residue insertions at positions 445–447 just two to four amino acid residues upstream the end of the protein C-terminus (Fig. 1). The GhTUA genes share high sequence homology at nucleotide level (62–93% identity) in the coding region and at amino acid level (89–99% identity). There is only 1–11% substitution rate at amino acid level compared with each other. A total of 62 substitution sites were presented in the GhTUA proteins (Fig. 1). Among them, 49 out of 62 substitution locations belong to conservative interchanges (i.e. the property of the substituted residue is synonymous with that of the original), and these alterations may not impact the protein structure and its function. On the other hand, there are 13 positions at which the non-synonymous substitutions occurred among the GhTUAs. The significant interchanges of the charged/uncharged or positive-charged/negative-charged residues were found at these positions. The charged or strongly polar residues were substituted for non-polar residues at positions 35 (gln/thr), 50 (asn/tyr), 139 (asn/ser), 279 (glu/ala), and 295 (asn/ser), whereas non-polar and uncharged residues were replaced by strongly polar or charged residues at positions 29 (gly/glu), 253 (thr/asn), 381 (ser/asn), and 442 (gly/asp). Furthermore, the negatively charged or strong polar residues were substituted for positively charged residues at positions 31 (gln/his), 46 (asp/his), and 256 (glu/arg), while positively charged lysine was replaced by negatively charged glutamine at position 370. These interchanges may alter protein structure and result in functionally distinct GhTUAs. Noticeably, a majority of the substitutions locates at N- and C-terminus of the protein sequences, suggesting that the two termini of the GhTUAs are more vulnerable to sustainable mutations and may contribute to their isotypic specificities. By contrast, peptide sequences in the middle regions are relatively conserved through evolution, implying that these regions may be important for maintaining the fundamental structure of GhTUA as well as its basic function.
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Isolation and characterization of the three GhTUA genes
Cotton genomic DNA libraries were screened using PCR fragments of the cotton GhTUA9 gene as probe. Three positive clones were isolated from the genomic libraries, and two of them were identified to contain the complete GhTUA6 and GhTUA9 genes, respectively, while the remaining one clone represented a novel cotton
-tubulin gene (designated as GhTUA13) encoding a predicted 551-amino acid polypeptide. The isolated GhTUA9 gene is approximately 4.7 kb in length (including a 2.6 kb 5' upstream region, a 1.8 kb transcribed region, and a 0.3 kb 3' downstream sequence), the isolated GhTUA6 gene is 5.1 kb in length (including a 3 kb 5' upstream region, a 1.8 kb transcribed region, and a 0.3 kb 3' downstream sequence), whereas the isolated GhTUA13 only contains a 1.9 kb transcribed region and a longer 3' downstream sequence (>2 kb). Furthermore, sequence comparison between its cDNA and genomic clone revealed that the GhTUA9 gene contains only one intron splitting its open reading frame into two exons. The intron resides between codons 31–32 (encoding residues gln31 and pro32, respectively), and is 459 bp in length (Fig. 2). The GhTUA6 gene, however, contains three introns located between condons 31/32, at codon 110, and between condons 233/234, respectively. By contrast, GhTUA13 gene contains two introns which are positioned in condons 31/32 and condons 233/234, respectively (Fig. 2). By comparing the cotton GhTUA6/9/13 genes with Arabidopsis TUA2/4/6 genes (Kopczak et al., 1992), it was found that the first intron position, which is between codons 31/32, is exactly identical for all these genes, suggesting it is relatively conserved. On the other hand, the other two introns (the second and third introns) are absent in the GhTUA9 as well as Arabidopsis TUA6, and the second intron is deleted in the GhTUA13 (Fig. 2) as well as Arabidopsis TUA4 (Kopczak et al., 1992), implying that they are probably non-essential for the regulation of GhTUA expression.
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Phylogenetic relationships of GhTUAs
The evolutionary relationships of the 12 cotton GhTUA proteins were determined by two different tree-building methods. As shown in Fig. 3, although both trees were essentially identical, there were slight variations between tree A and B. The 12 GhTUAs available could be divided into two subgroups in tree A. GhTUA5, GhTUA8, and GhTUA11 form one subgroup, while the remaining nine GhTUAs are another sister subgroup. Likewise in tree B, the GhTUA5, GhTUA8, and GhTUA11 were located at one end, whereas the rest of the GhTUAs resides on the other end of the tree. GhTUA8, however, occupies a distinct branch on tree B that is basal to the clade containing the other 11 GhTUAs, unlike that in tree A. This result suggests that GhTUA5, GhTUA8, and GhTUA11 may have diverged relatively early from the other GhTUAs during evolution. By contrast, one protein pair (GhTUA2/9) forms a distinct clade on both trees A and B, indicating that divergence in the protein pair occurred relatively recently. Furthermore, GhTUA12 occupies, on both trees, a distinct branch that is basal to the GhTUA2/9 clade. Similarly, the three proteins (GhTUA1/7/13) form a distinct clade on both trees, although there is slight difference between trees A and B. However, trees A and B differ with respect to the placements of GhTUA4, GhTUA6, and GhTUA10. On the one hand in tree A, GhTUA4 is basal to the GhTUA2/9/12 clade, and GhTUA10 is a sister subgroup to the GhTUA1/7/13 clade, while GhTUA6 forms a distinct branch that is basal to the clades GhTUA2/9/12/4 and GhTUA10/1/7/13. On the other hand in tree B, GhTUA4 and GhTUA10 occupy a distinct branch, and GhTUA6 is basal to the clade, resulting in an independent subgroup, from which the three GhTUAs are more closely related to each other than to the other GhTUAs.
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Seven out of the nine GhTUA genes are predominantly expressed in fibres
To identify GhTUA genes that are preferentially expressed in cotton fibres, the expression patterns of the isolated GhTUA genes were analysed by real-time quantitative SYBR-Green RT-PCR using gene-specific primers (Table 1), as described in the Materials and methods. The cotton polyubiquitin gene (GhUBI gene; XB Li, unpublished data) was chosen as a standard control to normalize differences in RNA template concentrations. Seven out of the nine GhTUA genes are expressed at high levels in fibre cells, except GhTUA12 and GhTUA13 (Fig. 4). Of the seven GhTUAs, the expression of the GhTUA9 gene is exceptionally high and specific to fibres, and its expression level reaches a relative value of 711.9 as compared to about 45–113 in other tissue types. Similarly, GhTUA6, GhTUA8, and GhTUA10 mRNAs are also largely accumulated in fibres (relative values of 542.9, 26.5, and 73.2, respectively), but exist, if at all, at very low levels in the other tissues such as leaves, roots, anthers, petals, ovules, cotyledons, and hypocotyls examined, indicating that they are specifically/preferentially expressed in fibre cells. Both GhTUA5 and GhTUA11 genes are expressed predominantly in fibres and at relatively high levels in hypocotyls, but moderately to weakly in other tissues. The GhTUA7 gene, however, exhibited a different expression pattern from the GhTUA6/8/9/10 and GhTUA5/11 genes. It is expressed strongly in both fibres and anthers, and at a relatively high level in hypocotyls. Furthermore, the expression levels of GhTUA5, GhTUA7, GhTUA8, GhTUA10, and GhTUA11 in fibres are at least 10–20-fold less, compared with those of GhTUA9 and GhTUA6. By contrast, the GhTUA12 gene is expressed at an extremely low level in fibres (less than 1 relative value) if compared with the other GhTUAs, and its transcripts mainly accumulated in hypocotyls and roots. The GhTUA13 gene shows a constitutive expression pattern with a relative high level in anthers. Overall, the real-time quantitative RT-PCR results revealed that the isolated
-tubulin genes in cotton are the predominant isoforms in fibre cells, except GhTUA12/13 (Fig. 4).
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Northern blot analysis, using the 3'-untranslated region (3'-UTRs) of GhTUA9 gene as a probe, demonstrated that the accumulation of the GhTUA9 transcript was specific to fibres. The GhTUA9 transcript in fibre cells reached its highest level during 5–10 d post anthesis (DPA), and its signal dramatically declined to undetectable levels as the ovule matured further. By 15 DPA, hardly any transcript was detectable. In addition, no or very little signal was visible in ovules, cotyledons, hypocotyls, anthers, petals, leaves, and roots (Fig. 5). This result further confirmed that the GhTUA9 gene expression is fibre-specific and developmentally regulated in cotton fibre cells.
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GhTUA9:GUS chimera is specifically expressed in cotton fibres
To characterize the precise expression pattern of GhTUA genes in cotton fibres, GhTUA9 was chosen for further study, for it is representative to all the known GhTUAs expressed specifically in fibres, owing to the exceptionally high accumulation level of its transcript, relative to the other GhTUA genes (Fig. 4). The 1.7 kb GhTUA9 5'-flanking region (including the putative promoter fragment and 5'-untranslated region) was subcloned upstream the GUS reporter gene in pBI101 vector, giving rise to the GhTUA9:GUS chimeric gene. The GhTUA9:GUS construct was introduced into Agrobacterium tumefaciens cells, which then infected cotton ovules (see Materials and methods). During a period of 2–4 d of ovule culture in vitro in the dark, fibre cells can elongate normally, as they did in cotton plants in vivo. Real-time quantitative RT-PCR analysis indicated that the GUS gene driven by the GhTUA9 promoter was transiently expressed in transformed fibres, but no GUS products were detected in non-transformed fibres as a negative control (data not shown). Histochemical assay of GUS activity further demonstrated that the GhTUA9:GUS chimera was expressed specifically in the transformed fibres after 2–4 d of cocultivation with agrobacteria in vitro. Of the 30 transformed ovules examined, strong GUS activity was detected exclusively in the most of fibres, while no or weak GUS staining was observed in ovules (Fig. 6A, B). By contrast, the non-transformed fibres exhibited only weak GUS activity under the same staining conditions (Fig. 6C).
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Overexpression of GhTUA9 promotes the cell elongation of fission yeast
To investigate whether the cotton TUA9 isoform plays a role in cell elongation, the coding sequence of the GhTUA9 gene was cloned into yeast vector pREP5N, and the construct transferred into yeast (S. pombe) cells. Morphological studies indicated that transformed cells with the GhTUA9 gene grown in induction medium were remarkably longer than those harbouring the same vector but grown in uninduced conditions (Fig. 7A, B). By contrast, the transformed cells harbouring the empty pREP5N vector displayed normal length, even though grown in the same induction medium (Fig. 7C, D), as non-transformed cells did (data not shown). Measurement and statistical analysis of six transformed yeast cell lines and three controls, which were randomly selected, revealed that the length of the GhTUA9 transformed cells was 1.4–1.7-fold greater than that of the control cells (Fig. 8). On the other hand, statistical analysis demonstrated that there was no significant difference in the rate of cell growth and the percentage of cell division between the transformed yeast lines and controls (data not shown). The results suggest that overexpression of the GhTUA9 gene stimulated the longitudinal growth of the host cells.
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Discussion |
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In this study, nine additional GhTUA genes have been documented and their sequences have been deposited in GenBank. The 12 cotton
-tubulins deduced from all the known GhTUA genes have diverged into two subclasses through evolution (Fig. 2), similar to the six Arabidopsis -tubulins (Kopczak et al., 1992). Although plant -tubulins are generally quite conserved, evolution has given rise to significant variations among GhTUAs. In GhTUAs, there are a total of 13 non-conservative substitutions (Fig. 1), while only five such positions exist among Arabidopsis -tubulins. The non-conservative replacements of charged residues, located on the surface of the -tubulin molecule, may be involved in functional non-equivalency of the -tubulin isovariants.
Previous studies revealed that the intron positions are not conserved in the plant -tubulin gene family, unlike in the plant β-tubulin gene family. The six Arabidopsis TUA genes contain 1–4 introns that define six distinct intron sites (Kopczak et al., 1992), whereas all nine of the Arabidopsis TUB genes, as well as the cotton GhTUB1 gene (Li et al., 2002), contain two introns at precisely conserved positions (Snustad et al., 1992). In Arabidopsis, the TUA6 gene contains only one intron located between codons 31 and 32, while the TUA4 and TUA2 genes contain an additional one (second) or two (second and third) introns, besides the first intron at the same position as that of TUA6 gene. By contrast, TUA1/3/5 genes contain four introns, three of which are at very different locations, except their second intron, which resides at the same position as that of TUA2 gene. On the whole, the intron distribution in the Arabidopsis TUA genes strikingly supports that these genes originate from two ancestral genes, one of which gives rise to TUA2, TUA4, and TUA6, whereas the other descends into TUA1, TUA3, and TUA5 (Kopczak et al., 1992). The maize -tubulin genes, however, belong to three gene subfamilies that may have evolved from three different ancestors (Villemur et al., 1992). On the other hand, cotton GhTUA9 contains a unique and relatively large intron, similar to the Arabidopsis TUA6 gene, while cotton GhTUA6 contains three introns, like the Arabidopsis TUA2 gene, and GhTUA13 contains two introns (Fig. 2), as does Arabidopsis TUA4. Overall, analysis of phylogenetic relationship among the known GhTUAs revealed that all these -tubulin isoforms may be categorized into two subgroups, implying that the cotton TUA genes may have evolved from two progenitors in a similar fashion to the Arabidopsis TUA genes.
The data presented here provides evidence for predominant expression of seven out of the nine GhTUA genes in cotton fibres. A lot of specialized -tubulin isotypes may have developed differentially functions to meet the different requirements in fibre development. The high accumulation of GhTUA products coincides with the rapid elongation of the fibre cells, suggesting that microtubule cytoskeleton plays an important role in fibre elongation. Furthermore, these results revealed that the accumulation level of GhTUA9 transcripts in young fibres was much higher than that in other tissues of cotton plants (Figs 4, 5), suggesting that this gene is specifically expressed in developing fibres. The GhTUA9 gene exhibited an expression peak at the very early stage of fibre development at around 5–10 DPA, which coincides with the elongation stage and the primary cell wall synthesis of fibres. Moreover, upon further development of the fibre cells, its expression level declined dramatically to an almost undetectable level at 15 DPA, pointing to a sharp and strong down-regulation of the GhTUA9 gene expression right at the onset of secondary cell wall cellulose synthesis and the termination of fibre elongation. This implies that the expression level of the GhTUA9 gene may be an important factor in determining the physical length of cotton fibre cells, primarily achieved through the elongation stage. Similarly, a previous study has revealed that cotton Tua1/5 transcripts are abundant during fibre cell elongation (Whittaker and Triplett, 1999). Furthermore, previous studies indicated that GhTUB1 and GhACT1 genes are preferentially expressed in the early stage of fibre development (Li et al., 2002, 2005). In addition, genes involved in osmoregulation and cell expansion during fibre development are also up-regulated significantly (Orford and Timmis, 1998; Smart et al., 1998; Ruan et al., 2001). These results suggest the existence of cellular mechanisms imposing strict developmental controls on genes, such as GhTUA9, involved in fibre cell elongation of cotton.
Microtubules have been shown to play an important role in the polarized growth of plant cells. A previous study indicated that microtubules regulate the directionality and stability of apical growth through interactions with the cellular machinery that maintains the [Ca2+]c gradient at the tip in root hairs of Arabidopsis thaliana (Bibikova et al., 1999). During trichome branching, microtubules reorient with respect to the longitudinal growth axis, leading to a change in growth orientation in Arabidopsis trichomes (Mathur and Chua, 2000). It is believed that highly dynamic microtubule cytoskeleton maintained by significant amounts of tubulins is essential for cell elongation in trichomes and other cell types. Reduction of the -tubulin level severely affected root elongation and root hair development in Arabidopsis TUA6/AS mutants, and resulted in aberrant microtubule structures in these cells (Bao et al., 2001). In cotton fibre, high levels of /β-tubulin proteins, including the GhTUA9 isoform, may be required to maintain the highly dynamic polymers of microtubule arrays for rapid fibre elongation. Furthermore, these experimental results indicated that overexpression of the GhTUA9 gene in fission yeast induced the longitudinal growth of the host cells, suggesting that this gene may function in the polarized growth of fibre cells.
To investigate its fibre specificity, the GhTUA9 gene, including its promoter, was isolated. The 5' upstream sequence was cloned into the upstream of the GUS reporter gene, and transient expression of the GUS gene was detected in fibres. GUS assay showed that the GhTUA9 promoter is very active in developing fibres. This result is consistent with the GhTUA9 expression pattern revealed by real-time quantitative RT-PCR and northern blot analysis, indicating that the isolated GhTUA9 promoter contains all the cis regulatory elements required for fibre-specific and developmental-regulated expression of the gene, as the other fibre-specific genes in cotton (John and Crow, 1992; John and Keller, 1996; Rinehart et al., 1996; Li et al., 2002, 2005; Ruan et al., 2003). Thus, the isolated GhTUA9 promoter can be used to isolate transcriptional factors that recognize the promoter sequence and to direct target gene expression in fibre cells for genetic modification of cotton.
In brief, GhTUA9 expression exhibits the highest level at the early stage of fibre elongation, and drops to an undetectable level at about 16 DPA with the onset of secondary wall synthesis, indicating that the GhTUA9 expression is down-regulated coincidently with the termination of fibre elongation. Furthermore, overexpression of this gene in fission yeast promotes atypical longitudinal growth of the host cells. All these suggest that the GhTUA9 is a developmental-regulated gene that may participate in the regulation of the concerted and rapid fibre elongation. Thus, the results of this study contribute to the understanding of the gene role in fibre development and provide novel insights into the functional specificity of GhTUA isoforms.
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This work was supported by the National Natural Sciences Foundation of China (Grant No. 30470930) and the National Program for Basic Research (973) of China (Grant No. 2004CB117304).
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* Present address: Department of Immunology, Medical Sciences Building, University of Toronto, 1 King's College Circle, Toronto, Ontario M5S 1A8, Canada.
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