Geng-Qing Huang

A Fasciclin-Like Arabinogalactan Protein, GhFLA1, Is Involved in Fiber Initiation and Elongation of Cotton

Cotton fiber initiation and elongation may be affected by an arabinogalactan protein that alters the integrity of the primary cell wall matrix. 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.

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.

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.

Seven cotton genes encoding putative NAC domain proteins are preferentially expressed in roots and in responses to abiotic stress during root development

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.

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.

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.

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.

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 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.