Li-Xia Qin

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.

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.

Arabidopsis drought-induced protein Di19-3 participates in plant response to drought and high salinity stresses.

Di19 (drought-induced protein19) family is a novel type of Cys2/His2 zinc-finger proteins. In this study, Arabidopsis Di19-3 was functionally characterized. The experimental results revealed that AtDi19-3 is a transcriptional activator, and could bind to the TACA(A/G)T sequence. AtDi19-3 expression in plants was remarkably induced by NaCl, mannitol and abscisic acid (ABA). T-DNA insertion mutation of AtDi19-3 results in an increase in plant tolerance to drought and high salinity stresses and ABA, whereas overexpression of AtDi19-3 leads to a drought-, salt- and ABA-sensitive phenotype of the transgenic plants. In the presence of NaCl, mannitol or ABA, rates of seed germination and cotyledon greening in Atdi19-3 mutant were higher, but in AtDi19-3 overexpression transgenic plants were lower than those in wild type. Roots of Atdi19-3 mutant seedlings were longer, but those of AtDi19-3 overexpression transgenic seedlings were shorter than those of wild type. Chlorophyll and proline contents in Atdi19-3 mutant were higher, but in AtDi19-3 overexpression seedlings were lower than those in wild type. Atdi19-3 mutant showed greater drought-tolerance, whereas AtDi19-3 overexpression transgenic plants exhibited more drought-sensitivity than wild type. Furthermore, expression of the genes related to ABA signaling pathway was altered in Atdi19-3 mutant and AtDi19-3 transgenic plants. These data suggest that AtDi19-3 may participate in plant response to drought and salt stresses in an ABA-dependent manner.

Cotton GalT1 encoding a putative glycosyltransferase is involved in regulation of cell wall pectin biosynthesis during plant development.

Arabinogalactan proteins (AGPs), are a group of highly glycosylated proteins that are found throughout the plant kingdom. To date, glycosyltransferases that glycosylate AGP backbone have remained largely unknown. In this study, a gene (GhGalT1) encoding a putative β-1,3-galactosyltransferase (GalT) was identified in cotton. GhGalT1, belonging to CAZy GT31 family, is the type II membrane protein that contains an N-terminal transmembrane domain and a C-terminal galactosyltransferase functional domain. A subcellular localization assay demonstrated that GhGalT1 was localized in the Golgi apparatus. RT-PCR analysis revealed that GhGalT1 was expressed at relatively high levels in hypocotyls, roots, fibers and ovules. Overexpression of GhGalT1 in Arabidopsis promoted plant growth and metabolism. The transgenic seedlings had much longer primary roots, higher chlorophyll content, higher photosynthetic efficiency, the increased biomass, and the enhanced tolerance to exogenous D-arabinose and D-galactose. In addition, gas chromatography (GC) analysis of monosaccharide composition of cell wall fractions showed that pectin was changed in the transgenic plants, compared with that of wild type. Three genes (GAUT8, GAUT9 and xgd1) involved in pectin biosynthesis were dramatically up-regulated in the transgenic lines. These data suggested that GhGalT1 may be involved in regulation of pectin biosynthesis required for plant development.

GhHyPRP4, a cotton gene encoding putative hybrid proline-rich protein, is preferentially expressed in leaves and involved in plant response to cold stress

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.

A cotton mitogen-activated protein kinase (GhMPK6) is involved in ABA-induced CAT1 expression and H2O2 production

The mitogen-activated protein kinase (MAPK) cascade is one of the major and evolutionally conserved signaling pathways and plays a pivotal role in the regulation of stress and developmental signals in plants. Here, we identified one gene, GhMPK6, encoding an MAPK protein in cotton. GFP fluorescence assay demonstrated that GhMAPK6 is a cytoplasm localized protein. Quantitative RT-PCR analysis revealed that mRNA accumulation of GhMPK6 was significantly promoted by abscisic acid (ABA). Overexpression of GhMPK6 gene in the T-DNA insertion mutant atmkk1 (SALK_015914) conferred a wild-type phenotype to the transgenic plants in response to ABA. Under ABA treatment, cotyledon greening/expansion in GhMPK6 transgenic lines and wild type was significantly inhibited, whereas the atmkk1 mutant showed a relatively high cotyledon greening/expansion ratio. Furthermore, CAT1 expression and H(2)O(2) levels in leaves of GhMPK6 transgenic lines and wild type were remarkably higher than those of atmkk1 mutant with ABA treatment. Collectively, our results suggested that GhMPK6 may play an important role in ABA-induced CAT1 expression and H(2)O(2) production.

Phosphorylation of serine residue modulates cotton Di19-1 and Di19-2 activities for responding to high salinity stress and abscisic acid signaling

Di19 (drought-induced protein 19) family is a novel type of Cys2/His2 zinc-finger proteins. In this study, we demonstrated that cotton Di19-1 and Di19-2 (GhDi19-1/-2) proteins could be phosphorylated in vitro by the calcium-dependent protein kinase (CDPK). Mutation of Ser to Ala in N-terminus of GhDi19-1/-2 led to the altered subcellular localization of the two proteins, but the constitutively activated form (Ser was mutated to Asp) of GhDi19-1/-2 still showed the nuclear localization. GhDi19-1/-2 overexpression transgenic Arabidopsis seedlings displayed the hypersensitivity to high salinity and abscisic acid (ABA). However, Ser site-mutated GhDi19-1(S116A) and GhDi19-2(S114A), and Ser and Thr double sites-mutated GhDi19-1(S/T-A/A) and GhDi19-2(S/T-A/A) transgenic Arabidopsis did not show the salt- and ABA-hypersensitive phenotypes. In contrast, overexpression of Thr site-mutated GhDi19-1(T114A) and GhDi19-2(T112A) in Arabidopsis still resulted in salt- and ABA-hypersensitivity phenotypes, like GhDi19-1/-2 transgenic lines. Overexpression of GhDi19-1/-2 and their constitutively activated forms in Atcpk11 background could recover the salt- and ABA-insensitive phenotype of the mutant. Thus, our results demonstrated that Ser phosphorylation (not Thr phosphorylation) is crucial for functionally activating GhDi19-1/-2 in response to salt stress and ABA signaling during early plant development, and GhDi19-1/-2 proteins may be downstream targets of CDPKs in ABA signal pathway.

Cotton GhHyPRP3 encoding a hybrid proline-rich protein is stress inducible and its overexpression in Arabidopsis enhances germination under cold temperature and high salinity stress conditions

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.

Two cotton fiber‐associated glycosyltransferases, GhGT43A1 and GhGT43C1, function in hemicellulose glucuronoxylan biosynthesis during plant development

Xylan is the major hemicellulosic constituent in dicot secondary cell walls. Cell wall composition of cotton fiber changes dynamically throughout development. Not only the amounts but also the molecular sizes of the hemicellulosic polysaccharides show substantial changes during cotton fiber development. However, none of the genes encoding glycosyltransferases (GTs) responsible for synthesizing xylan have been isolated and characterized in cotton fiber. In this study, we applied a bioinformatics approach and identified two putative GTs from cotton, designated GhGT43A1 and GhGT43C1, which belong to the CAZy GT43 family and are closely related to Arabidopsis IRX9 and IRX14, respectively. We show that GhGT43A1 is highly and preferentially expressed in 15 and 20 days post-anthesis (dpa) cotton fiber, whereas GhGT43C1 is ubiquitously expressed in most organs, with especially high expression in 15 dpa fiber and hypocotyl. Complementation analysis demonstrates that GhG43A1 and GhGT43C1 are orthologs of Arabidopsis IRX9 and IRX14, respectively. Furthermore, we show that overexpression of GhGT43A1 or GhGT43C1 in Arabidopsis results in increased xylan content. We also show that overexpression of GhGT43A1 or GhGT43C1 leads to more cellulose deposition. These findings suggest that GhGT43A1 and GhGT43C1 likely participate in xylan synthesis during fiber development.

The cotton β-galactosyltransferase 1 (GalT1) that galactosylates arabinogalactan-proteins participates in controlling fiber development

Arabinogalactan proteins (AGPs) are highly glycosylated proteins that play pivotal roles in diverse developmental processes in plants. Type-II AG glycans, mostly O-linked to the hydroxyproline residues of the protein backbone, account for up to 95% w/w of the AGP, but their functions are still largely unclear. Cotton fibers are extremely elongated single-cell trichomes on the seed epidermis; however, little is known of the molecular basis governing the regulation of fiber cell development. Here, we characterized the role of a CAZy glycosyltransferase 31 (GT31) family member, GhGalT1, in cotton fiber development. The fiber length of the transgenic cotton overexpressing GhGalT1 was shorter than that of the wild type, whereas in the GhGalT1-silenced lines there was a notable increase in fiber length compared with wild type. The carbohydrate moieties of AGPs were altered in fibers of GhGalT1 transgenic cotton. The galactose: arabinose ratio of AG glycans was higher in GhGalT1 overexpression fibers, but was lower in GhGalT1-silenced lines, compared with that in the wild type. Overexpression of GhGalT1 upregulates transcript levels of a broad range of cell wall-related genes, especially the fasciclin-like AGP (FLA) backbone genes. An enzyme activity assay demonstrated that GhGalT1 is a β-1,3-galactosyltransferase (β-1,3-GalT) involved in biosynthesis of the β-1,3-galactan backbone of the type-II AG glycans of AGPs. We also show that GhGalT1 can form homo- and heterodimers with other cotton GT31 family members to facilitate AG glycan assembly of AGPs. Thus, our data demonstrate that GhGalT1 influences cotton fiber development via controlling the glycosylation of AGPs, especially FLAs.