Yong Zheng

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.

Two cotton Cys2/His2-type zinc-finger proteins, GhDi19-1 and GhDi19-2, are involved in plant response to salt/drought stress and abscisic acid signaling

Cotton (Gossypium hirsutum) often encounters abiotic stress such as drought and high salinity during its development, and its productivity is significantly limited by those adverse factors. To investigate the molecular adaptation mechanisms of this plant species to abiotic stress, we identified two genes encoding Di19-like Cys2/His2 zinc-finger proteins in cotton. GFP fluorescence assay demonstrated that GhDi19-1 and GhDi19-2 are two nuclear-localized proteins. Quantitative RT-PCR and Northern blot analyses revealed that mRNA accumulation of both GhDi19-1 and GhDi19-2 was significantly promoted by salinity and drought. Expression of GUS gene driven by the GhDi19-1 and GhDi19-2 promoters, respectively, was intensively induced in cotyledons under NaCl and mannitol stresses. Overexpression of GhDi19-1 and GhDi19-2 in Arabidopsis resulted in the seedlings displaying hypersensitivity to high salinity and abscisic acid (ABA). Seed germination and seedling growth of the transgenic Arabidopsis were dramatically inhibited by salinity and ABA, compared with wild type. In addition, expression levels of the ABA-responsive genes ABF3, ABF4,ABI5 and KIN1 were also remarkably altered in the transgenic plants under ABA treatment. Collectively, our results suggested that both GhDi19-1 and GhDi19-2 may be involved in response to salt/drought stress and ABA signaling during early stages of plant development.

A cotton gene encodes a tonoplast aquaporin that is involved in cell tolerance to cold stress.

To enhance the survival probability in cold stress, plant cells often increase their cold- and freezing-tolerance in response to low, nonfreezing temperatures by expressing some cold-related genes. In present study, a cotton gene encoding tonoplast intrinsic protein (TIP) was isolated from a cotton seedling cDNA library, and designated as GhTIP1;1. GFP fluorescent microscopy indicated that GhTIP1;1 protein was localized to the vacuolar membrane. Assay on GhTIP1;1 expression in Xenopus laevis oocytes demonstrated that GhTIP1;1 protein displayed water channel activity and facilitated water transport to the cells. At normal conditions, GhTIP1;1 transcripts were predominantly accumulated in roots and hypocotyls, but less abundance in other tissues of cotton. The GhTIP1;1 expression was dramatically up-regulated in cotyledons, but down-regulated in roots within a few hours after cotton seedlings were cold-treated. Overexpression of GhTIP1;1 in yeast (Schizosaccharomyces pombe) significantly enhanced the cell survival probability, suggesting that the GhTIP1;1 protein is involved in cell freezing-tolerance.

Two Brassica napus genes encoding NAC transcription factors are involved in response to high-salinity stress

AbstractThe NAC protein family is one of the novel classes of plant-specific transcription factors. In this study, two genes (BnNAC2 and BnNAC5) encoding the putative NAC transcription factors were identified in Brassica napus. Sequence analysis revealed that the deduced BnNAC proteins contain conserved N-terminal region (NAC domain) and highly divergent C-terminal domain. Yeast transactivation analysis showed that BnNAC2 could activate reporter gene expression, suggesting that BnNAC2 functions as a transcriptional activator. Quantitative RT-PCR analysis revealed that BnNAC2 was preferentially expressed in flowers, whereas BnNAC5 mRNAs accumulated at the highest level in stems. Further experimental results indicated that the two genes are high-salinity-, drought- and abscisic acid (ABA)-induced. Overexpression of BnNAC2 and BnNAC5 genes in yeast (Schizosaccharomyces pombe) remarkably inhibited the growth rate of the host cells, and enhanced the cells sensitive to high-salinity and osmotic stresses. Complementation test indicated that BnNAC5 could recover the defects such as salt-hypersensitivity and accelerated-leaf senescence of vni2 T-DNA insertion mutant. Several stress-responsive genes including COR15A and RD29A were enhanced in the complemented plants. These results suggest that BnNAC5 may perform the similar function of VNI2 in response to high-salinity stress and regulation of leaf aging.n Key messageBnNAC2 and BnNAC5 are salt-, drought- and ABA-induced genes. Overexpression of BnNAC5 in Arabidopsisvni2 mutant recovered the mutant defects (salt-hypersensitivity and accelerated-leaf senescence) to the phenotype of wild type.

GhMPK17, a Cotton Mitogen-Activated Protein Kinase, Is Involved in Plant Response to High Salinity and Osmotic Stresses and ABA Signaling

Mitogen-activated protein kinase (MAPK) cascades play pivotal roles in mediating biotic and abiotic stress responses. Cotton (Gossypium hirsutum) is the most important textile crop in the world, and often encounters abiotic stress during its growth seasons. In this study, a gene encoding a mitogen-activated protein kinase (MAPK) was isolated from cotton, and designated as GhMPK17. The open reading frame (ORF) of GhMPK17 gene is 1494 bp in length and encodes a protein with 497 amino acids. Quantitative RT-PCR analysis indicated that GhMPK17 expression was up-regulated in cotton under NaCl, mannitol and ABA treatments. The transgenic Arabidopsis plants expressing GhMPK17 gene showed higher seed germination, root elongation and cotyledon greening/expansion rates than those of the wild type on MS medium containing NaCl, mannitol and exogenous ABA, suggesting that overexpression of GhMPK17 in Arabidopsis increased plant ABA-insensitivity, and enhanced plant tolerance to salt and osmotic stresses. Furthermore, overexpression of GhMPK17 in Arabidopsis reduced H2O2 level and altered expression of ABA- and abiotic stress-related genes in the transgenic plants. Collectively, these data suggested that GhMPK17 gene may be involved in plant response to high salinity and osmotic stresses and ABA signaling.

Genome-wide functional analysis of cotton (Gossypium hirsutum) in response to drought.

Cotton is one of the most important crops for its natural textile fibers in the world. However, it often suffered from drought stress during its growth and development, resulting in a drastic reduction in cotton productivity. Therefore, study on molecular mechanism of cotton drought-tolerance is very important for increasing cotton production. To investigate molecular mechanism of cotton drought-resistance, we employed RNA-Seq technology to identify differentially expressed genes in the leaves of two different cultivars (drought-resistant cultivar J-13 and drought-sensitive cultivar Lu-6) of cotton. The results indicated that there are about 13.38% to 18.75% of all the unigenes differentially expressed in drought-resistant sample and drought-sensitive control, and the number of differentially expressed genes was increased along with prolonged drought treatment. DEG (differentially expression gene) analysis showed that the normal biophysical profiles of cotton (cultivar J-13) were affected by drought stress, and some cellular metabolic processes (including photosynthesis) were inhibited in cotton under drought conditions. Furthermore, the experimental data revealed that there were significant differences in expression levels of the genes related to abscisic acid signaling, ethylene signaling and jasmonic acid signaling pathways between drought-resistant cultivar J-13 and drought-sensitive cultivar Lu-6, implying that these signaling pathways may participate in cotton response and tolerance to drought stress.

A cotton gene encoding a plasma membrane aquaporin is involved in seedling development and in response to drought stress

Cotton (Gossypium hirsutum), the most important textile crop worldwide, often encounters abiotic stress such as drought and waterlog during its growth season (summer), and its productivity is significantly limited by adverse factors. To investigate the molecular adaptation mechanisms of this plant species to abiotic stress, a gene encoding the plasma membrane intrinsic protein (PIP) was isolated in cotton, and designated as GhPIP2;7. Quantitative reverse transcriptase polymerase chain reaction analysis indicated that GhPIP2;7 was preferentially expressed in cotyledons and leaves, and its expression was up-regulated in leaves after drought treatments. Strong expression of GUS gene driven by GhPIP2;7 promoter was detected in leaves of 5- to 10-day-old transgenic Arabidopsis seedlings, but GUS activity gradually became weak as the seedlings further developed. GhPIP2;7 promoter activity was also remarkably induced by mannitol treatment. Furthermore, yeast cells over-expressing GhPIP2;7 displayed relatively higher drought tolerance, compared with controls. Over-expression of GhPIP2;7 in Arabidopsis enhanced plant tolerance to drought stress. Collectively, these data suggested that GhPIP2;7 gene may be involved in leaf development and in response to drought stress.

Three cotton homeobox genes are preferentially expressed during early seedling development and in response to phytohormone signaling

Homeodomain-leucine zipper (HD-Zip) proteins are transcription factors unique to plants. In this study, three cDNAs (designated as GhHB2, GhHB3 and GhHB4) encoding HD-Zip proteins were isolated from cotton cDNA library. GhHB2 gene encodes a protein of 300 amino acids, GhHB3 gene encodes a peptide with 254 amino acids, and GhHB4 gene encodes a protein of 281 amino acids. The deduced proteins, which contain the homeodomain and leucine-rich zipper motif, share relatively high similarities with the other plant HD-Zip proteins. Quantitative RT-PCR analysis indicated that GhHB3 and GhHB4 were preferentially expressed in hypocotyls and cotyledons, whereas GhHB2 gene was predominantly expressed in young stems, at relatively high levels in hypocotyls. Expressions of all the three genes were up-regulated in roots, hypocotyls and cotyledons after GA3 treatments. Additionally, GhHB4 expression was enhanced by 6-BA treatment. A GhHB2 promoter fragment was isolated from cotton by Genome-Walking PCR. Expression of GUS gene controlled under GhHB2 promoter was examined in the transgenic Arabidopsis plants. Strong GUS staining was detected in cotyledon, veins of the emerging leaves and shoot apices of 5- to 15-day-old transgenic seedlings, but GUS activity became more and more weak as the seedlings further developed. In addition, the promoter activity was induced by exogenous GA, indicating that GhHB2 promoter is very active during early seedling development, and may be GA-inducible. The results suggested that the three HB genes may function in early seedling development of cotton and in response to gibberellin signaling.

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 GASL genes encoding putative gibberellin-regulated proteins are involved in response to GA signaling in fiber development

GAST (GA-stimulated transcript)-like genes have been reported as targets of GA regulation in some plant species. In this study, we isolated seven GAST-like cDNAs from cotton (Gossypium hirsutum) cDNA libraries (designated as GhGASL1–GhGASL7). Meanwhile, the genomic DNA clones corresponding to the seven GhGASL genes were isolated by using PCR amplification technique. Analysis of gene structure revealed that four genes (GhGASL1/3/5/6) contain two exons and one intron, while the rest have four exons and three introns. All of the deduced GhGASL proteins contain a putative signal peptide in the N-terminus and a conservative cysteine-rich C-terminal domain. Quantitative RT-PCR analysis indicated that the seven GhGASL genes are differentially expressed in cotton tissues. Among them, GhGASL1/4/7 were predominantly expressed in cotyledons, while the transcripts of GhGASL2/5 were preferentially accumulated at hypocotyls. GhGASL3 mRNA was largely accumulated in fibers, while GhGASL6 transcripts were mainly detected in ovules. Furthermore, GhGASL2/3/5 displayed a relatively high expression levels during early fiber elongation stages, and were regulated by GA. These data suggested that GhGASL genes may be involved in fiber elongation and in response to GA signaling during fiber development.