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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.

Jasmonate ZIM-domain (JAZ) proteins and DELLA proteins are key negative regulators of jasmonates (JAs) and gibberellin (GA) signaling, respectively. In this study, we found JA and GA synergistically promote fiber cell initiation. We characterized the cellular function of a JAZ protein (GhJAZ3), and a DELLA protein (GhSLR1) of cotton (Gossypium hirsutum). GhJAZ3 is specifically expressed in elongating fibers, while GhSLR1 is expressed in different tissues and at a relatively higher level in 3 DPA ovules. GhSLR1 and GhJAZ3 proteins are localized in the cell nucleus. Yeast two-hybrid analysis indicated that GhSLR1, GhJAZ3 and GhDEL65 could interact with each other, and GhSLR1 could also interact with GhBZR1. Overexpression of GhJAZ3 in Arabidopsis increased hypocotyl and root length, leaf trichome length, and plant height, but decreased the number of leaf trichome, while overexpression of GhSLR1 in Arabidopsis decreased hypocotyl length, leaf trichome length and density. Expression of several leaf trichome initiation determinators (GL3, GL2, TTG2 and MYB23) was down-regulated in GhJAZ3 or GhSLR1 transgenic Arabidopsis, while expression of the cell elongation related genes (EXP1, EXP8, EXPL2 and XTH4) was altered in the GhJAZ3 and GhSLR1 transgenic Arabidopsis. Taken together, these results demonstrate that GhJAZ3 and GhSLR1 function in jasmonate and gibberellin mediated epidermal cell differentiation and elongation.

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.

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.

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.

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.

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.

Plant-specific NAC transcription factors comprise a large family with diverse roles in plant development and stress regulation. In this study, 73 NAC genes from cotton (Gossypium hirsutum) EST database were identified by bioinformatic approach. Analysis of conserved amino acid residues and phylogeny reconstruction using the NAC conserved domain suggested that the Arabidopsis classification into four major groups is applicable to cotton NAC family. Among them, seven NAC genes, named as GhNAC7–GhNAC13, were characterized to encode NAC proteins that share high similarity with those plant abiotic stress-related NACs. Quantitative RT-PCR analysis indicated that the seven GhNAC genes were preferentially expressed in roots, and regulated in cotton plants under cold, abscisic acid, drought and/or high salt conditions. Our results in this comprehensive study of cotton NAC gene family provide valuable information for further exploring the roles of the NAC genes in cotton development and in response to abiotic stress.

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.

In this study, the cDNA coding for a hybrid proline-rich protein (HyPRP) was isolated from cotton cDNA libraries and designated GhHyPRP3. Analysis of the deduced amino acid sequence revealed that it contained an N-terminal signal peptide, a central proline-rich domain, and a C-terminal cysteine-rich domain highly homologous to other hybrid proline-rich group B proteins. RNA gel blot analysis showed that GhHyPRP3 mRNA was most abundant in petals and 10 DPA ovules indicating that expression of GhHyPRP3 was petal-preferential and ovule developmentally regulated. In addition, GhHyPRP3 transcription in roots was up-regulated by salt stress, cold stress, and osmotic stress, but down-regulated by GA3. A promoter-GUS reporter revealed that the GhHyPRP3 promoter directed gene expression in root–shoot junction, roots, and petals of transgenic Arabidopsis plants. Subcellular localization results showed that GhHyPRP3 was localized to the plasma membrane. Transgenic lines overexpressing GhHyPRP3 had a higher germination rate under cold temperature and high salinity stress conditions compared with wild type. Overall, GhHyPRP3 may function in flower and ovule development and participate in the defense responses to low temperature and salt stress.

Basic helix-loop-helix transcription factors (TFs), namely MYC2, MYC3, and MYC4, interact with Jasmonate Zim-domain proteins and are their direct targets. These TFs have been shown to function synergistically to control Arabidopsis growth and development. Our results showed similar MYC2, MYC3, and MYC4 expression patterns during Arabidopsis seed development, which remained relatively high during seed mid-maturation. MYC2, MYC3, and MYC4 acted redundantly in seed size, weight control, and in regulating seed storage protein accumulation. Triple mutants produced the largest seeds and single and double mutants' seeds were much larger than those of wild type. The weight of triple mutants' seeds was significantly higher than that of wild-type seeds, which was accompanied by an increase in seed storage protein contents. Triple mutants' seeds presented a marked decrease in 2S amounts relative to those in wild-type seeds. Liquid chromatography tandem mass spectra sequencing results indicated that both the relative abundance and the peptide number of CRA1 and CRU3 were greatly increased in triple mutants compared to wild type. The expression of 2S1-2S5 decreased and that of CRA1 and CRU3 increased in triple mutants relative to those in wild types during seed development, which might have contributed to the low 2S and high 12S contents in triple mutants. Our results contribute to understanding the function of MYC2, MYC3, and MYC4 on seed development, and provide promising targets for genetic manipulations of protein-producing crops to improve the quantity and quality of seed storage proteins.

Arabinogalactan proteins (AGPs), a superfamily of highly glycosylated hydroxyproline-rich glycoproteins, are widely implicated in plant growth and development. A gene (including its cDNA), designated GhAGP31, encoding a non-classical AGP protein was isolated from cotton (Gossypium hirsutum). The deduced GhAGP31 protein contains the conserved features of non-classical AGPs: a putative signal peptide, N-terminal histidine-rich stretch, middle repetitive proline-rich domain and a cysteine-containing 'PAC' domain. GFP fluorescence assay demonstrated that GhAGP31 protein was localised on cell walls. GhAGP31 transcripts were mainly detected in roots, hypocotyls and ovules, but little or almost none were detected in other tissues. In particular, expression of GhAGP31 was developmentally regulated in roots. Further study demonstrated that GhAGP31 expression in cotton roots was remarkably up-regulated by cold stress. Expression of the GUS gene driven by the GhAGP31 promoter was also dramatically enhanced in roots of transgenic Arabidopsis seedlings under cold treatment. Additionally, overexpression of GhAGP31 in yeast and Arabidopsis significantly improved the freezing tolerance of yeast cells and cold tolerance of Arabidopsis seedlings. These data imply that GhAGP31 protein may be involved in the response to cold stress during early root development of cotton.

Plant hybrid proline-rich proteins (HyPRPs) usually consist of an N-terminal signal peptide, a central proline-rich domain, and a conserved eight-cysteine motif C-terminal domain. In this study, one gene (designated as GhHyPRP4) encoding putative HyPRP was isolated from cotton cDNA library. Northern blot and quantitative reverse transcriptase-polymerase chain reaction analyses revealed that GhHyPRP4 was preferentially expressed in leaves. Under cold stress, GhHyPRP4 expression was significantly up-regulated in leaves of cotton seedlings. Using the genome walking approach, a promoter fragment of GhHyPRP4 gene was isolated from cotton genome. GUS (β-glucuronidase) gene driven by GhHyPRP4 promoter was specifically expressed in leaves and cotyledons of the transgenic Arabidopsis thaliana. Furthermore, GUS expression in leaves was remarkably induced by cold stress. Overexpression of GhHyPRP4 in yeast (Schizosaccharomyces pombe) significantly enhanced the cell survival rate upon treatment under -20°C for 60 h. These data suggested that GhHyPRP4 may be involved in plant response to cold stress during seedling development of cotton.

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.

Arabinogalactan proteins (AGPs) are a large family of highly glycosylated of hydroxyproline-rich glycoproteins that play important roles in plant growth, development, and signal transduction. A cDNA encoding a putative classical AGP named GhH6L was isolated from cotton fiber cDNA libraries, and the deduced protein contains 17 copies of repetitive motif of X-Y-proline-proline-proline (where X is serine or alanine and Y is threonine or serine). Northern blotting analysis and quantitative RT-PCR results showed that it was preferentially expressed in 10 days post-anthesis (dpa) fibers and was also developmentally regulated. A promoter fragment was isolated from cotton (Gossypium hirsutum) by genome walking PCR. Expression of beta-glucuronidase (GUS) gene under the GhH6L promoter was examined in the transgenic Arabidopsis plants; only petiole and pedicel were stained, no staining was detected in other tissues. Subcellular localization indicated that GhH6L was localized to the plasma membrane and in the cytoplasm. These data further our understanding of GhH6L as well as shed light on functional insight to GhH6L in cotton.

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.

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.

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.

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.

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.

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.