GhEIN3, a cotton (Gossypium hirsutum) homologue of AtEIN3, is involved in regulation of plant salinity tolerance.

Ethylene insensitive 3 (EIN3), a key transcription factor in ethylene signal transduction, play important roles in plant stress signaling pathways. In this study, we isolated and characterized an EIN3-like gene from cotton (Gossypium hirsutum), designated as GhEIN3. GhEIN3 is highly expressed in vegetative tissues, and its expression is induced by 1-aminocyclopropane-1-carboxylic acid (ACC) and NaCl. Ectopic expression of GhEIN3 in Arabidopsis elevated plants' response to ethylene, which exhibit smaller leaves, more root hairs, shorter roots and hypocotyls. The germination rate, survival rate and root length of GhEIN3 transgenic plants were significantly improved compared to wild type under salt stress. GhEIN3 transgenic plants accumulated less H2O2 and malondialdehyde (MDA), while higher superoxide dismutase (SOD) and peroxidase (POD) activities were detected under salt stress. In addition, expression of several genes related to reactive oxygen species (ROS) pathway and ABA signaling pathway was increased in the GhEIN3 transgenic plants under salt stress. In contrast, virus-induced gene silencing (VIGS) of GhEIN3 in cotton enhanced the sensitivity of transgenic plants to salt stress, accumulating higher H2O2 and MDA and lower SOD and POD activities compared to control plants. Collectively, our results revealed that GhEIN3 might be involved in the regulation of plant response to salt stress by regulating ABA and ROS pathway during plant growth and development.

The cotton (Gossypium hirsutum) NAC transcription factor (FSN1) as a positive regulator participates in controlling secondary cell wall biosynthesis and modification of fibers.

Cotton (Gossypium hirsutum) fibers are the highly elongated and thickened single-cell trichomes on the seed epidermis. However, little is known about the molecular base of fiber cell wall thickening in detail. In this study, a cotton NAC transcription factor (GhFSN1) that is specifically expressed in secondary cell wall (SCW) thickening fibers was functionally characterized. The GhFSN1 transgenic cotton plants were generated to study how FSN1 regulates fiber SCW formation. Up-regulation of GhFSN1 expression in cotton resulted in an increase in SCW thickness of fibers but a decrease in fiber length. Transcriptomic analysis revealed that GhFSN1 activates or represses numerous downstream genes. GhFSN1 has the ability to form homodimers, binds to its promoter to activate itself, and might be degraded by the ubiquitin-mediated proteasome pathway. The direct targets of GhFSN1 include the fiber SCW-related GhDUF231L1, GhKNL1, GhMYBL1, GhGUT1 and GhIRX12 genes. GhFSN1 binds directly to a consensus sequence (GhNBS), (C/T)(C/G/T)TN(A/T)(G/T)(A/C/G)(A/G)(A/T/G)(A/T/G)AAG, which exists in the promoters of these SCW-related genes. Our data demonstrate that GhFSN1 acts as a positive regulator in controlling SCW formation of cotton fibers by activating its downstream SCW-related genes. Thus, these findings give us novel insights into comprehensive understanding of GhFSN1 function in fiber development.

Genome-wide identification and characterization of TCP transcription factor genes in upland cotton (Gossypium hirsutum).

TCP proteins are plant-specific transcription factors (TFs), and perform a variety of physiological functions in plant growth and development. In this study, 74 non-redundant TCP genes were identified in upland cotton (Gossypium hirsutum L.) genome. Cotton TCP family can be classified into two classes (class I and class II) that can be further divided into 11 types (groups) based on their motif composition. Quantitative RT-PCR analysis indicated that GhTCPs display different expression patterns in cotton tissues. The majority of these genes are preferentially or specifically expressed in cotton leaves, while some GhTCP genes are highly expressed in initiating fibers and/or elongating fibers of cotton. Yeast two-hybrid results indicated that GhTCPs can interact with each other to form homodimers or heterodimers. In addition, GhTCP14a and GhTCP22 can interact with some transcription factors which are involved in fiber development. These results lay solid foundation for further study on the functions of TCP genes during cotton fiber development.

Genome-wide identification and characterization of JAZ gene family in upland cotton (Gossypium hirsutum).

Plant JAZ (Jasmonate ZIM-domain) proteins play versatile roles in multiple aspects of plant development and defense. However, little is known about the JAZ family in allotetraploid upland cotton (Gossypium hirsutum) so far. In this study, 30 non-redundant JAZ genes were identified in upland cotton through genome-wide screening. Phylogenetic analysis revealed that the 30 proteins in cotton JAZ family are further divided into five groups (I - V), and members in the same group share highly conserved motif structures. Subcellular localization assay demonstrated that GhJAZ proteins are localized in the cell nucleus. Quantitative RT-PCR analysis indicated that GhJAZs display different expression patterns in cotton tissues, and most of them could be induced by Jasmonic (JA). Furthermore, some GhJAZ genes are preferentially expressed in cotton ovules and fibers, and showed differential expression in ovules of wild type cotton and fiberless mutant (fl) during fiber initiation. GhJAZ proteins could interact with each other to form homodimer or heterodimer, and they also interacted with some JA signaling regulators and the proteins involved in cotton fiber initiation. Collectively, our data suggested that some GhJAZ proteins may play important roles in cotton fiber initiation and development by regulating JA signaling as well as some fiber-related proteins.

A R2R3-MYB transcription factor that is specifically expressed in cotton (Gossypium hirsutum) fibers affects secondary cell wall biosynthesis and deposition in transgenic Arabidopsis.

Secondary cell wall (SCW) is an important industrial raw material for pulping, papermaking, construction, lumbering, textiles and potentially for biofuel production. The process of SCW thickening of cotton fibers lays down the cellulose that will constitute the bulk (up to 96%) of the fiber at maturity. In this study, a gene encoding a MYB-domain protein was identified in cotton (Gossypium hirsutum) and designated as GhMYBL1. Quantitative real-time polymerase chain reaction (RT-PCR) analysis revealed that GhMYBL1 was specifically expressed in cotton fibers at the stage of secondary wall deposition. Further analysis indicated that this protein is a R2R3-MYB transcription factor, and is targeted to the cell nucleus. Overexpression of GhMYBL1 in Arabidopsis affected the formation of SCW in the stem xylem of the transgenic plants. The enhanced SCW thickening also occurred in the interfascicular fibers, xylary fibers and vessels of the GhMYBL1-overexpression transgenic plants. The expression of secondary wall-associated genes, such as CesA4, CesA7, CesA8, PAL1, F5H and 4CL1, were upregulated, and consequently, cellulose and lignin biosynthesis were enhanced in the GhMYBL1 transgenic plants. These data suggested that GhMYBL1 may participate in modulating the process of secondary wall biosynthesis and deposition of cotton fibers.

A cotton (Gossypium hirsutum) gene encoding a NAC transcription factor is involved in negative regulation of plant xylem development.

NAC proteins that compose of one large family of plant specific transcription factors (TF) play the important roles in many biological processes (such as morphogenesis, development, senescence and stress signal transduction). In this study, a gene (designated as GhXND1) encoding a NAC transcription factor was identified in cotton. Sequence analysis indicated that GhXND1 gene contains two introns inserted in its open reading frame (ORF). GhXND1 protein is localized in the cell nucleus, and displays the transactivation activity. GhXND1 transcripts were mainly detected in cotyledons, petals, roots, hypocotyls and stems, but little or no signals of GhXND1 expression were found in the other tissues. Ectopic expression of GhXND1 in Arabidopsis resulted in a reduction in number of xylem vessel cells and cell wall thickness of interfascicular fibers in the transgenic plants, compared with those of wild type. And expression of some cell wall biosynthesis-related genes was down-regulated in the GhXND1 transgenic plants. Collectively, the data presented in this study suggested that GhXND1 may be involved in regulation of plant xylem development.

Cotton KNL1, encoding a class II KNOX transcription factor, is involved in regulation of fibre development.

In this study, the GhKNL1 (KNOTTED1-LIKE) gene, encoding a classical class II KNOX protein was identified in cotton (Gossypium hirsutum). GhKNL1 was preferentially expressed in developing fibres at the stage of secondary cell wall (SCW) biosynthesis. GhKNL1 was localized in the cell nucleus, and could interact with GhOFP4, as well as AtOFP1, AtOFP4, and AtMYB75. However, GhKNL1 lacked transcriptional activation activity. Dominant repression of GhKNL1 affected fibre development of cotton. The expression levels of genes related to fibre elongation and SCW biosynthesis were altered in transgenic fibres of cotton. As a result, transgenic cotton plants produced aberrant, shrunken, and collapsed fibre cells. Length and cell-wall thickness of fibres of transgenic cotton plants were significantly reduced compared with the wild type. Furthermore, overexpression and dominant repression of GhKNL1 in Arabidopsis resulted in a reduction in interfascicular fibre cell-wall thickening of basal stems of transgenic plants. Complementation revealed that GhKNL1 rescued the defective phenotype of Arabidopsis knat7 mutant in some extent. These data suggest that GhKNL1, as a transcription factor, participates in regulating fibre development of cotton.

Molecular characterization of cotton C-repeat/dehydration-responsive element binding factor genes that are involved in response to cold stress.

Low temperature, drought and salinity are major abiotic stresses that influence survival, productivity and geographical distribution of many important crops across the globe. The C-repeat/dehydration-responsive element binding transcription factors (CBF/DREB) are important proteins involved in response to abiotic stresses in plants. In this study, twenty-one CBF genes were identified in cotton (Gossypium hirsutum) by bioinformatic approach. The twenty-one CBF genes (named as GhCBF1--GhCBF21) were characterized to encode proteins that share high similarity with those plant cold stress-related CBF proteins, which contain the classic AP2 domain of 58 amino acid residues. Phylogenetic analysis revealed that the isolated cotton CBF genes can be classified into 4 groups: GhCBF I, GhCBF II, GhCBF III and GhCBF IV. RT-PCR analysis indicated that GhCBF genes were up-regulated in cotton plants under cold stress. Furthermore, four GhCBF genes were up-regulated in cotton under salinity and drought treatments. Our data provided valuable information for further exploring the roles of the CBF genes in cotton development and in response to cold stress.

Cotton photosynthesis-related PSAK1 protein is involved in plant response to aphid attack.

It is believed that hundreds of genes, including photosynthesis-related genes, are typically involved in plant response to aphid feeding. Up to now, however, it is little known on the relationship between the photosynthesis-related genes and plant response to herbivores. In this study, we identified a cotton photosynthesis-related gene (GhPSAK1) which belongs to PSI-PSAK family and encodes a putative protein of 162 amino acids. RT-PCR analysis revealed that GhPSAK1 transcripts in leaves were increased at 12-24 h, but decreased at 48-72 h after cotton aphid attack or wounding induction. Choice assay and no-choice assay demonstrated that overexpression of GhPSAK1 in Arabidopsis improved plant tolerance to green peach aphids (Myzus persicae). The defense response genes related to salicylic acid signaling pathway were enhanced in the GhPSAK1 overexpressing transgenic plants. In addition, the callose amount in transgenic Arabidopsis leaves was more than that of wild type. Contents of the soluble sugars and total amino acids were also altered in leaves of transgenic Arabidopsis plants. Activities of superoxide dismutase and peroxidase in transgenic leaves were higher than those of wild type. These results suggested that GhPSAK1 may be involved in regulation of cotton response and tolerance to aphid attack.

Cotton PRP5 gene encoding a proline-rich protein is involved in fiber development.

Proline-rich proteins contribute to cell wall structure of specific cell types and are involved in plant growth and development. In this study, a fiber-specific gene, GhPRP5, encoding a proline-rich protein was functionally characterized in cotton. GhPRP5 promoter directed GUS expression only in trichomes of both transgenic Arabidopsis and tobacco plants. The transgenic Arabidopsis plants with overexpressing GhPRP5 displayed reduced cell growth, resulting in smaller cell size and consequently plant dwarfs, in comparison with wild type plants. In contrast, knock-down of GhPRP5 expression by RNA interference in cotton enhanced fiber development. The fiber length of transgenic cotton plants was longer than that of wild type. In addition, some genes involved in fiber elongation and wall biosynthesis of cotton were up-regulated or down-regulated in the transgenic cotton plants owing to suppression of GhPRP5. Collectively, these data suggested that GhPRP5 protein as a negative regulator participates in modulating fiber development of cotton.

A fasciclin-like arabinogalactan protein, GhFLA1, is involved in fiber initiation and elongation of cotton.

Arabinogalactan proteins (AGPs) are involved in many aspects of plant development. In this study, biochemical and genetic approaches demonstrated that AGPs are abundant in developing fibers and may be involved in fiber initiation and elongation. To further investigate the role of AGPs during fiber development, a fasciclin-like arabinogalactan protein gene (GhFLA1) was identified in cotton (Gossypium hirsutum). Overexpression of GhFLA1 in cotton promoted fiber elongation, leading to an increase in fiber length. In contrast, suppression of GhFLA1 expression in cotton slowed down fiber initiation and elongation. As a result, the mature fibers of the transgenic plants were significantly shorter than those of the wild type. In addition, expression levels of GhFLAs and the genes related to primary cell wall biosynthesis were remarkably enhanced in the GhFLA1 overexpression transgenic fibers, whereas the transcripts of these genes were dramatically reduced in the fibers of GhFLA1 RNA interference plants. An immunostaining assay indicated that both AGP composition and primary cell wall composition were changed in the transgenic fibers. The levels of glucose, arabinose, and galactose were also altered in the primary cell wall of the transgenic fibers compared with those of the wild type. Together, our results suggested that GhFLA1 may function in fiber initiation and elongation by affecting AGP composition and the integrity of the primary cell wall matrix.

Cotton BCP genes encoding putative blue copper-binding proteins are functionally expressed in fiber development and involved in response to high-salinity and heavy metal stresses.

Copper is vitally required for plants at low concentrations but extremely toxic for plants at elevated concentrations. Plants have evolved a series of mechanisms to prevent the consequences of the excess or deficit of copper. These mechanisms require copper-interacting proteins involved in copper trafficking. Blue copper-binding proteins (BCPs) are a class of copper proteins containing one blue copper-binding domain binding a single type I copper. To investigate the role of BCPs in plant development and in response to stresses, we isolated nine cDNAs encoding the putative blue copper-binding proteins (GhBCPs) from cotton (Gossypium hirsutum). Meanwhile, four corresponding genes (including GhBCP1-GhBCP4), which contain a single intron inserted in their conserved position, were isolated from cotton genome. Quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) analysis indicated that the nine GhBCP genes are differentially expressed in cotton tissues. Among them, GhBCP1 and GhBCP4 were predominantly expressed in fibers, while the transcripts of GhBCP2 and GhBCP3 were accumulated at relatively high levels in fibers. These four genes were strongly expressed in early fiber elongation, but dramatically declined with further fiber development. In addition, these GhBCP genes were upregulated in fibers by Cu(2+) , Zn(2+) , high-salinity and drought stresses, but downregulated in fibers by Al(3+) treatment. Overexpression of GhBCP1 and GhBCP4 in yeast (Schizosaccharomyces pombe) significantly increased the cell growth rate under Cu(2+) , Zn(2+) and high-salinity stresses. These results suggested that these GhBCPs may participate in the regulation of fiber development and in response to high-salinity and heavy metal stresses in cotton.

Three cotton genes preferentially expressed in flower tissues encode actin-depolymerizing factors which are involved in F-actin dynamics in cells.

To investigate whether the high expression levels of actin-depolymerizing factor genes are related to pollen development, three GhADF genes (cDNAs) were isolated and characterized in cotton. Among them, GhADF6 and GhADF8 were preferentially expressed in petals, whereas GhADF7 displayed the highest level of expression in anthers, revealing its anther specificity. The GhADF7 transcripts in anthers reached its peak value at flowering, suggesting that its expression is developmentally-regulated in anthers. The GhADF7 gene including the promoter region was isolated from the cotton genome. To demonstrate the specificity of the GhADF7 promoter, the 5'-flanking region, including the promoter and 5'-untranslated region, was fused with the GUS gene. Histochemical assays demonstrated that the GhADF7:GUS gene was specifically expressed in pollen grains. When pollen grains germinated, very strong GUS staining was detected in the elongating pollen tube. Furthermore, overexpression of GhADF7 gene in Arabidopsis thaliana reduced the viable pollen grains and, consequently, transgenic plants were partially male-sterile. Overexpression of GhADF7 in fission yeast (Schizosaccharomyces pombe) altered the balance of actin depolymerization and polymerization, leading to the defective cytokinesis and multinucleate formation in the cells. Given all the above results together, it is proposed that the GhADF7 gene may play an important role in pollen development and germination.

Characterization of 19 novel cotton FLA genes and their expression profiling in fiber development and in response to phytohormones and salt stress.

Fasciclin-like arabinogalactan proteins (FLAs), a subclass of arabinogalactan proteins (AGPs), are usually involved in cell development in plants. To investigate the expression profiling as well as the role of FLA genes in fiber development, 19 GhFLA genes (cDNAs) were isolated from cotton (Gossypium hirsutum). Among them, 15 are predicted to be glycosylphosphatidylinositol anchored to the plasma membranes. The isolated cotton FLAs could be divided into four groups. Real-time quantitative reverse transcriptase polymerase chain reaction results indicated that the GhFLA genes are differentially expressed in cotton tissues. Three genes (GhFLA1/2/4) were specifically or predominantly expressed in 10 days post-anthesis fibers, and the transcripts of the other four genes (GhFLA6/14/15/18) were accumulated at relatively high levels in cotton fibers. Furthermore, expressions of the GhFLA genes are regulated in fiber development and in response to phytohormones and NaCl. The identification of cotton FLAs will facilitate the study of their roles in cotton fiber development and cell wall biogenesis.

Molecular characterization of cotton GhTUA9 gene specifically expressed in fibre and involved in cell elongation.

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 alpha-tubulin genes are correlated with fibre elongation, nine GhTUA genes (cDNAs) encoding alpha-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.

[Cloning of GhAQP1 gene and its specific expression during Ovule development in cotton].

Plant aquaporins, belonging to the MIP superfamily, are a series of transmembrane proteins that facilitate water transport through cell membranes. In this study, a cDNA clone encoding the PIP1-like protein was isolated from cotton (Gossypium hirsutum) cDNA libraries, and designated as GhAQP1 (Fig.1). We also isolated the GhAQP1 gene from cotton genome by PCR. The gene is 2,096 bp in length, including an open reading frame (ORF) and 5'-/3'-untranslated regions (UTR). It contains two introns in its ORF. The first intron is inserted between codons 209 and 210 in the fifth transmembrane helix, and another is located between codons 256 and 257 in the sixth transmembrane helix of GhAQP1, respectively (Figs.2 and 3). Northern blot analysis showed that GhAQP1 gene is expressed specifically in 6-15 DPA ovule, and reaches a peak in 9 DPA ovule (Figs.4 and 5), suggesting that its expression is ovule-specific and developmentally regulated in cotton.