Functional Genomics

Carotenoids are important accessory pigments in plants that are essential for photosynthesis. Phytoene synthase (PSY), a rate-controlling enzyme in the carotenoid biosynthesis pathway, has been widely characterized in rice, maize, and sorghum, but at present there are no reports describing this enzyme in cotton. In this study, GhPSY was identified as a candidate gene for the red plant phenotype via a combined strategy using: (1) molecular marker data for loci closely linked to R1; (2) the whole-genome scaffold sequence from Gossypium raimondii; (3) gene expression patterns in cotton accessions expressing the red plant and green plant phenotypes; and (4) the significant correlation between a single nucleotide polymorphisms (SNP) in GhPSY and leaf phenotypes of progeny in the (Sub16 × T586) F2 segregating population. GhPSY was relatively highly expressed in leaves, and the protein was localized to the plastid where it appeared to be mostly attached to the surface of thylakoid membranes. GhPSY mRNA was expressed at a significantly higher level in T586 and SL1-7-1 red plants than TM-1 and Hai7124 green plants. SNP analysis in the GhPSY locus showed co-segregation with the red and green plant phenotypes in the (Sub16 × T586) F2 segregating population. A phylogenetic analysis showed that GhPSY belongs to the PSY2 subfamily, which is related to photosynthesis in photosynthetic tissues. Using a reverse genetics approach based on Tobacco rattle virus-induced gene silencing, we showed that the knockdown of GhPSY caused a highly uniform bleaching of the red color in newly-emerged leaves in both T586 and SL1-7-1 plants with a red plant phenotype. These findings indicate that GhPSY is important for engineering the carotenoid metabolic pathway in pigment production.

Fiber cell initiation and elongation is critical for cotton fiber development. Previously, three cotton fiber-expressed genes (CFEs), GhCFE1, GhCFE2, and GhCFE3, were isolated and submitted to the genebank; however, the in vivo function of CFEs during cotton fiber development still remains unknown. Here, we characterized a gene which is originated from A-genome species and homeologous to GhCFE1 from D-genomes, so we named it GhCFE1A and designated GhCFE1 as GhCFE1D thereafter. GhCFE1 is preferentially expressed at initiation and rapid elongation stages in the developing fibers, in addition, much higher expression of GhCFE1 was detected at fiber initiation stage in the fiberless cotton mutants than that in TM-1. Interestingly, overexpression of GhCFE1A in cotton not only delayed fiber cell elongation but also significantly reduced the density of lint and fuzz fiber initials and stem trichomes. Further yeast two-hybrid assay showed that GhCFE1A interacts with at least four actin isoforms, and the interaction requires two conserved domains in GhCFE1A with unknown function, DUF761 and DUF4408. Moreover, actin staining and quantitative assay revealed reduced intensity of F-actin at rapid elongation stage in GhCFE1A-overexpressiong fibers. Taken together, the results demonstrated that GhCFE1A interacts with actin cytoskeleton and negatively regulates fiber cell initiation and elongation during cotton fiber development.

The goal of this work was to identify differentially expressed genes at two different stages during cotton fibre development i.e. 0 dpa and 10 dpa. The techniques like microarray, suppression substractive hybridization (SSH) and qRT-PCR were followed using the isogenics representing FL (Fuzzy linted) and Fl (Fuzzy-lintless) linesof G.arboreum. The scanning electron microscopy (SEM) revealed no difference in the fibre initials except for the fuzz development in Fl (Fuzzy-lintless) line which is due to the down-regulation of the some genes necessary for the fibre development. Microarrays identified transcripts which were either down-regulated or up-regulated in the fibre. Most of the genes up-regulated at 0 dpa but at 10 dpa large number of genes were down-regulated and out of total 278 transcripts 239 were showing matches with the Arabidopsis. The network covers broad processes like transcription factors. The transcription factors like AP2-EREBP, C2H2, WRKY were up-regulated at 0 dpa and during 10 dpa transcription factors coding AP2-EREBP, AUX/IAA, bHLH, C2H2, C3H, HB, MYB, NAC, Orphans, PLATZ and WRKY were down-regulated which may be the probale reason for the undergrowth of the fibre in the fuzzy-lintless line. Phytohormone signaling is also one of the important aspect in the fibre developmental process in the Fl (Fuzzy-lintless) line because most of the phytohormones like abscisic acid, auxin, brassinosteroid, ethylene, gibberellin, and salicylic acid were up-regulated at the 0 dpa and same were down-regulated at 10 dpa. This alteration in the regulation at the two stages might have led to the loss of co-ordination in the development process and ceased the fibre growth in the fuzzy-lintless line. In the Fl line, down-regulation of the transcript at 10 dpa coding for the trehalose-6-phosphate synthase (TPS), endo-xyloglucan hydrolase family proteins, UDP-glucose 4-epimerase (UGE), EXPANSINs, Arabinogalactan proteins (AGPs), β-galactosidase 13 (BGAL13), UDP-glucosyl transferase 74B1 (UGT74B1), Tubulins (TUBs) may the possible reason for the undergrowth of the fibre because these elements are necessary for the energy and cell wall metabolism. Fatty acids are essential for the fibre growth but in the Fl line acyl-CoA (ACO) oxidase 4 was down-regulated at the 10 dpa may have resukted in the undergrowth of the fibre due to loss in the VLCFA chains which interact with the other factors for normal fibre growth. The down-regulation of the genes like lipid transfer proteins (LPTs) and the hydrolases family representing from the fatty acid synthesis lead to undergrowth of the fibre in the Fl line at 10 dpa. Phosphatidylinositol (PtdIns) transfer proteins (PITPs) are signaling molecules in lipid metabolism and tip directed growth of the fibre and the down-ragulation of such gene may be the factor for the reduction of the fibre growth in developing fibres of fuzzy-lintless line.Transcripts related to the signaling including the Ca2+ and reactive oxygen species (ROS) which are the leading factors for fibre initation and cotton fibre growth itself is a kind of stress but their down-regulation resulted to the loss of co-ordination during the 0 dpa. Down-regulation of ROS scavenging factor peroxidase 2 (PA2) which has affected the ROS and led to the reduced fibre growth in fuzzy lintless line. Some miscellaneous factors like cellular and signal transcduction coding for MAPK cascade, Plant receptor-like kinases (RLKs) and LRR-family protein were down-regulation at 10 dpa. These factors have been involved in the cellular communication and the development of the fibre and down-regulation at 10 dpa may have resulted to loss of the function of such genes and reduced fibre growth in the Fl (fuzzy-lintless) line. Heat shock proteins (HSPs), which are the involved protein refolding and stabilizing the plant in response to the abiotic stress but such genes which are down-regulated at 10 dpa may be the on eof thereason for the reduction of the fibre growth and it is worth to mention that fibre development is itself a stress. Spermine synthase (SPDS3) was one of the genes down-regulated at both the stages in fuzzy-lintless line. It is considered as the SPDS gene which is responsible for the polyamine biosynthesis and in the Fl line it was down-regulation which may lead reduce fibre growth by modulating the concentration of polyamines. In SSH, based on the putative functions of genes preferentially or specifically expressed in fibre elongation stage in cotton fibre development BLAST results showed that many novel genes were contained in forward library and a few had been previously reported. On the basis of previous reports and the putative functions of the genes which are involved in fibre development and how those genes regulate fibre growth were reported in the database. This include actin depolymerizing factor 2 (ACT2), acyl- oxidase 1 (ACO1), beta-galactosidase (β-galactosidase) from Ricinus communis. There were contigs showing the homology to the beta-glucanase (β-glucanase), β-ketoacyl-Coa synthase family protein, beta-tubulin 19 (β-tubulin 19), elongation factor 1 (EF1), glucose-6-phosphate 1-epimerase, malate dehydrogenase, xyloglucan endotransglucosylase hydrolase protein 32 (XTH32/XET32). Genes like calcineurin b-like protein (CBL), calmodulin binding (CaM), mitogen-activated protein kinase 16 (MAPK/ MPK16), diacylglycerol acyltransferase (ACY) Phospholipase-C (PLC), phosphatidylcholine transferases (PCY), Phospholipase-C (PLC) were also found in the database and attempt was made for some of this genes to validate by the Quantitave real-time PCR which confirmed the chance of the undergrowth of the fibre in the Fl line. The microarray and the quantitave real-time PCR showed a good correlation between the expression levels. Quantitave real-time PCR confirmed the chance of the undergrowth of the fibre in the Fl line which evaluated the expression patterns of 32 genes, including 17 from microarray and 15 from SSH using quantitative RT-PCR.

Cotton fibers supply the main raw material to textile industry, fiber initiation and elongation are the key determinants of fiber output and quality in cotton. Although transcriptome and functional genomics have made great progress, the mechanism of fiber development is still not well understood. With Gossypium arboreum, G. stocksii and their somatic hybrid as our research materials, the cDNA-AFLP was used to analyze their fiber development transcriptome. At present, a total of 598 differentially expressed genes were isolated. This project intends to screen and clone the genes involved in fiber development by bioinformatic and expression analyses firstly. Then construct the expression vector VIGS and transform into G. hirsutum. Functional candidate genes will be validated by analyzing transgenic cotton fiber development and endogenous gene expression. For further analysis of gene function and regulatory mechanism, CRISPR/Cas system and overexpression strategies will be applied in cotton and Arabidopsis. The fibers, root hairs and trichomes of transgenic plants will be analyzed in detail. Ultimately, we hope to explore the key genes for cotton fiber quality improvement, and elucidate the regulatory mechanism of fiber development preliminarily.

Screening and functionally charactering a promoter expressed in specific tissue or organ and different developmental stages is an important field in plant molecular biology. Cotton fibers are single-celled seed trichomes originated from the epidermis of the outmost integument of ovules. Fiber cell development includes four discrete but partially overlapping stages: initiation, elongation, secondary cell-wall accumulation, and maturation. In order to express target gene only in fiber cell or certain developmental stage of fiber cell, study on the gene and its promoter specifically or preferentially expressed in fiber cell is a major area of molecular biology in cotton. The GhLIM6 gene was expressed preferentially in cotton fiber and pollen. It played important roles in the quality and yield of fibers. Overexpressing GhLIM6 can promote the elongation of fiber. On the contrary, suppressing GhLIM6 can inhibit the elongation of fiber. Either overexpression or suppression of the GhLIM6 in transgenic cotton, the pollen fertility was impacted. The percentage of setting boll and the yield of per plant were lower compared with wild type. For further study the regulatory mechanism of the gene in cotton fiber development and screen the appropriate promoter expressed in fiber, we have cloned GhLIM6 promoter (L6p) and constructed plant expression vectors in which the reporter gene GUS was controlled of L6p through 5'-end deletion mutants. Through transgenic tomato plants and cotton plants, we clarified that L6p specifically expressed in pollen and fiber. The 1.7 kb fragment of L6p has been cloned from upland cotton genome by genomic DNA walking (YADE walking). There are many cis-acting regulatory elements in the fragment such as pollen-specific expression elements AGAAA, light regulatory elements (G-Box and I-box), hormone response cis-acting elements (GA-responsive elements GARE, ABA-responsive element ABRE), low-temperature-responsive element and so on. In transgenic tomato plants, GUS activity was highest in pollen and seed hair. Weak GUS signal also be detected in fruit vascular and endosperm. The result was confirmed by quantitative real-time RT-PCR. These revealed that L6p expressed preferentially in pollen and seed hairs. In transgenic cotton plants, the GUS activity presented in pollen and fiber cell while the other detected samples did not display any GUS signal. The highest expression level of GUS was in pollen and the moderate level was in fiber cell. At various developmental stages of fiber cell, the GUS activity was detected after 10 DPA. The higher activity was in the fiber of 15 DPA. The result from quantitative real-time RT-PCR was similar with that from GUS stain except for a certain transcript of GUS was detected in ovules after 10 DPA. This might result from fiber cells contained in the ovule sample. Furthermore, GUS stain showed there was no significant difference between 1.5 kb and 0.5 kb sequence of L6p. It suggested that the 0.5 kb sequence is a core sequence of L6p. These results indicate that GhLIM6 promoter expressed specifically in cotton pollen and fiber.

Abscisic acid (ABA) is one of the important classical phytohormones in plant kingdom. However, the role of ABA in the growth and development of cotton fiber is largely unknown to date though ABA was extracted firstly from cotton. To illuminate the role of ABA and related genes in cotton fibers, the ABA 8'-hydroxylase gene (GhABAH) was selected for creating transgenic cotton plants. GhABAH is a key enzyme involved in ABA catabolism. It expressed higher in root than in stem and leaf. At various developmental stages of fiber cell, there was a high expression level at the early stage of fiber elongation (6 DPA). With the increase of fiber elongation rate, the expression level was reduced gradually and reached the bottom at 12 DPA, which was a rapid elongation stage of fiber cell. Then with the decrease of fiber elongation rate, the expression level of GhABAH was increased and reached the top at 18 DPA in which the secondary cell wall started to deposit. These results revealed that the expression pattern of GhABAH is depended on the development of fiber cell. The content of ABA was detected and the phenotypic variation of transgenic cotton plants was characterized either in transgenic or wild type plants. Compared with wild type, the endogenous ABA content of transgenic cotton was reduced by 63.1%, and the transgenic plant were slender, branches were shorter, leaves and bolls were smaller, leaves were wilting, stomata can't close normally, blooming time was delayed, petals and sepals were smaller, pollen grain not spilled out, pollen sterility were reduced. Meanwhile, lateral root number was increased by 200% under the condition of 200 mM NaCl in seedling. On the contrary, suppressing GhABAH expression level led endogenous ABA level to increase by 54.4%, and increased ABA sensitivity in seedlings. These results indicated that modified the GhABAH expression level can affect plant endogenous ABA levels and the development of cotton plants. Over-expressing GhABAH inhibited fiber cell initiation and elongation in transgenic cotton. Mature fiber length of transgenic cotton was 2.64±0.03 cm. Compared with wild type, which fiber length was 2.97±0.02 cm, transgenic fiber was shortened by 11.1%, the secondary wall was thicker, the micronaire increased by 31%, the fiber strength reduced by 20%. At the same time, the transgenic cotton fibers were more resistant to ABA than that of wild type. However, suppressing GhABAH expression didn’t display obvious effect on the mature fiber length. Only the secondary wall was thicker significantly than wild type fibers. These results revealed that change of GhABAH expression level not only affected fiber differentiation, initiation and elongation, but also influenced the formation of the secondary wall, and proved that the gene play an important role in regulating the growth and development of cotton fiber.

Twenty first century is the era where modern genomic approaches like gene identification, their characterization and expression under environmental stresses explore the functional genomics in crop plants. In this regard cDNA library construction, functional annotation and homology studies is a powerful approach for studying a large number of genes (sequences) for those crops whose genome is not completely sequenced. No more information is available on Gossypium arboreum root genes and functions. Therefore study was aimed to develop drought stressed Gossypium arboreum (FDH-786) cDNA library to study the functions and homology of clones expressed in roots under dehydration Biochemical attributes were performed both in cotton (FDH-786) roots and leaf tissue it was found that proline, total soluble protein, total soluble sugars and total free amino acids are significantly higher in drought stressed roots and leaves as compared to irrigated plant tissues. Biochemical assays were performed in roots to confirm significant accumulation of biological molecules (Proteins, sugars and amino acids) that escort to search drought tolerant functional EST in roots of G. arboreum which are kinases, transcription factors and binding proteins playing an important role in drought induction mechanism in roots. A cDNA library from drought stressed roots has been constructed. Ten thousands clones were randomly picked and PCR amplified. The inserts size was found in a range of 200-1000bp. Seven hundred eleven (711) clones (submitted to NCBI GenBank JK757087-JK757798) were sequenced and annotated. Twenty four percent (24%) clones didn't show significant homology to GenBank non-redundant database. It showed the potential of cotton (G.arboreum) genome for the identification of new genes under abiotic stresses. Twenty seven percent (27%) clones sequences show similarity with Gossypium species, seven percent (7%) show significant homology with Populus trichochorpa sequences, six percent (6%) with Oryza, five percent (5%) with Zea mays, four percent (4%) with Glycine max, three percent (3%) with Medicago and Niocotiana species , two percent (2%) with Arabidopsis, Atriplex and Ricinus species, while forteen percent (14%) have significant homology with other plant species. 27% homology with cotton confirms the authenticity of the cotton cDNA library. It revealed a collection of valuable drought stress (response to water deprevation) tolerant EST that include stress proteins (late embryogenesis abundant, heat shock protein, dehydrin, ERD six like1) Transcription Factor (Cys2/His2-type zinc-finger proteins, WRKY, B-box type zinc finger family, NAC domain containing protein, MADS-box protein, Homeodomain leucine zipper, Basic Helix-Loop-Helix), Kinases (diacylglycerol kinase, Shaggy like kinases, Histidine kinase, leucine rich repeat receptor, RAN GTPase activating protein kinase, like auxin resistant), Binding protein (ATP-Binding Cassette, TGF-β receptor interacting protein, Acyl-COA binding protein, RAS-related GTP-binding nuclear protein) Transferases (glutathione transferase), flavin mononucleotide. All these dehydration responsive factors are involved in the regulation of abscisic acid pathway interaction either directly or indirectly. Ultimately these kinases, protein, transporters and osmo-regulators contribute toward the activation of late responsive genes in plants.

Salinity and drought has adverse affects on the plant development and ultimately reduces the production. Very few reports are available for the genes induced by salt stress in Gossypium arboreum. Abiotic stress responsive genes were screened from cotton leaves by differential display and gene expression study was compared between control and salt stressed conditions in cotton variety FDH-171. By using 107 primers combination, 25 gene fragments were found to be up regulated in response to salt stress. Out of 25 induced fragments, 12 were rejected as false positive on reamplification and in quality control assay. The remaining fragments were selected for cloning and transformation. Sequence analysis on GenBank Databases(JG294129-JG294141) revealed that five fragments whose sizes ranging from 300-600bp have significant homology to well-known proteins (Protein kinase, proton gradient regulation, Yeast cadmium factor and expressed protein). Real-time PCR study confirmed the over expression of the identified transcripts in salt stressed samples as compared to control. Three transcripts showing good protein homology were further characterized as full length gene and submitted to NCBI GenBank database. Proton gradient regulator (JQ861978), Proteinase inhibitor (JX088126)and Yeast cadmium factor (JG294141) were characterized. Expression analysis using real time PCR reveled significant up regulation under salt stress using real time PCR. Cotton variety FDH-786 was selected for expression profiling in root under drought stress condition. Physiological, water relations & biochemical assays were performed to analyze the preliminary evaluation for drought tolerance. In case of physiological and gas exchange parameters, relative water contents were significantly higher in control plants while cell membrane thermostability was found to be higher in drought stress plants which allows accumulation of ions, electrolytes and Osmolytes in drought stressed plants. Keeping an eye on gas exchange parameters it was found that transpiration rate, Stomatal conductance and instantaneous water use efficiency is significantly higher in drought stressed plants which shows their efficient utilization of carbon dioxide and water under drought stress. With regard to biochemical attributes both in roots and leaf tissue it was found that proline, total soluble protein, total soluble sugars and total free amino acids are significantly higher in drought stressed roots and leaves as compared to control plant tissues. Seven hundred eleven (711) clones (submitted to NCBI GenBank JK757087-JK757798) were sequenced and annotated. Twenty four percent (24%) clones didn't show significant homology to GenBank non-redundant database. It showed the potential of cotton (G. arboreum) genome for the identification of new genes under abiotic stresses. Twenty (20) clones were found to be differentially expressed on cDNA microarray platform. EST sequences of potential candidates for drought genes were BLAST to NCBI GenBank for their homology search against nucleotide, EST and protein data bases, using BLASTX and BLASTN. Out of twenty, ten (10) ESTs were novel (didn’t showed any homology) to NCBI GenBank nucleotide, EST and protein data bases. Seven (07) have shown homology in all the three databases. Ten (10) have shown homology to only EST database, seven (07) to EST and protein database and four (04) to nucleotide and protein database. Functionally annotation differentially expressed drought stressed clones clustered, into novel (10), response to abiotic stress and stimulus (total 06 having 03 novel), signal transduction (total 02 having 01 novel), transport (total 02 having 01 novel), developmental processes (total 03 having 01 novel), cell organization & biogenesis (total 02 having 01 novel), electron transport (01), DNA or RNA binding (03), transcription factor activity (total 03 having 02 novel), hydrolase activity (total 03 having 02 novel), transporter activity (03 having 01 novel) and kinase activity (01). These novel 10 differentially expressed transcripts were found to be significantly up regulated using real time PCR studies. These potential abiotic stress transcripts searched from cotton varieties can further characterized and used in molecular breeding to develop the plants with better architecture that can tolerate well under abiotic stresses.

Agricultural production is often influenced by non-biological stresses, wherein salt stress is just one of the major non-biological stresses of agricultural production. As the most important source of textile fiber, cotton not only has strong salt tolerance, which makes it one of the superior crops adapting to saline-alkali soil, but also has high economic benefits. In this research, methylation-sensitive amplification polymorphism (MSAP) method based on high performance capillary electrophoresis (HPCE) was used to analyze DNA methylation level in four cotton varieties, including two salt-tolerant varieties CCRI 35 and CCRI 07, and two salt-sensitive varieties CCRI 12 and Xinyan 96-48, so as to discuss the molecular mechanism of salt tolerance in cotton. The results showed that all kinds of variation of DNA methylation happened in the four cotton varieties under salt stress, including hypermethylation, hypomethylation and other patterns; hypermethylation happened at a significantly higher rate than that of hypomethylation in the two salt-tolerant cotton varieties, CCRI 35 and CCRI 07. However, in CCRI 12 hypomethylation happened at a significantly higher rate than that of hypermethylation, whereas no significant difference was detected between different variation types in Xinyan 96-48. DNA methylation level significantly increased under salt stress in salt-tolerant varieties CCRI 35 and CCRI 07, whereas there was no significant difference in salt-sensitive varieties CCRI 12 and Xinyan 96-48. Our results suggested that salt-tolerant cotton might have the potential mechanism of increasing methylation level when responding to salt stress, which might partly explain salt stress tolerance in cotton. Preliminary cloning and analysis of methylated DNA sequences showed that hypermethylation happened in some MYB transcription factor related genes, which was further validated by qRT-PCR.

NAC proteins constitute one of the largest families of plant specific transcription factors (TF). They play the important roles in many biological processes such as morphogenesis, senescence, development and stress signal transduction pathways. AtXND1 (designated for XYLEM NAC Domain 1), one of the NAC family members, has been supposed as a negative regulator of secondary cell wall synthesis. In this work, a homolog of AtXND1 gene, designated GhXND1, was isolated in cotton. GhXND1 has three exons and two introns within genomic DNA sequence and encoded a polypeptide with 195 amino acids which contains the conserved features of NAC family: NAC domain and C-terminal transactivation region (TAR). GFP fluorescence assay demonstrated that GhXND1 protein is localized in cell nucleus. The C-terminus of GhXND1 has transactivation activity in yeast. GhXND1 transcripts were mainly detected in cotyleton, petal, root, hypocotyl and stem, but little or no signals of GhXND1 expression were found in other tissues. Additionally, compared with wild type (WT), overexpression of GhXND1 in Arabidopsis resulted in less number of xylem vessel cells and thinner interfascicular fibers in transgenic plants, and up-regulating the expression of KNAT7 gene and down-regulating the expression of cell wall relative genes. Overall, the data presented in this study suggested that GhXND1 may be involved in regulation of xylem development.