Functional Genomics

Cotton is the largest renewal source of textile fiber and a significant oil seed crop. Cotton improvement is hindered by the large and complex tetraploid genomes. Recent studies have shown that development of seed hair (fiber) in cotton share many molecular similarities with the development of leaf trichomes in Arabidopsis. Given the plethora amount of genetic and genomic resources in Arabidopsis, we developed a systematic approach to identify functional homologues of cotton genes in Arabidopsis thaliana. The system is known as cotton full-length cDNA over-expression (cFOX) in A. thaliana. We converted a cotton (Gossypium hirsutum L. cv. TM-1) library of full-length cDNA that are expressed in young ovules and immature fibers into a binary vector and transformed the full cotton cDNAs into A. thaliana in three genetic backgrounds, Col-0 wild-type, the hairless glabrous1 (gl1) mutant, and the hairy triptchon (try) mutant. In an initial screen, we analyzed 608 transgenic lines in Col-0, 866 lines in gl1,and 497 lines in try and identified 46 lines with stably altered phenotypes in two additional generations. In 20 lines, the cotton cDNA inserts were recovered by genomic PCR and cloned. These cotton genes were grouped infunctional categories of phytohormone signaling, lipid metabolism, and transcription factors, many of which are expressed in early stages of fiber development. The observed phenotypes corresponding with the cotton cDNA inserts are consistent with the known functions and expression patterns of these genes. Experiments are underway to validate functions for these cotton genes in Arabidopsis and cotton. The systematic screen will be expanded to identify novel cotton genes, especially related to trichome and fiber cell development. The cFOX system will become a valuable resource for discovering functions of cotton genes, improving functional annotation of cotton genes,and providinga translational repository of cotton genes in Arabidopsis.

Root length and its architecture govern the adaptability of plants to various stress conditions, including drought stress. Genetic variations in root growth, length, and architecture are genotypes dependent. In this study, we compared the drought-induced transcriptome of four genotypes of Gossypium herbaceum that differed in their drought tolerance adaptability. Three different methodologies, namely, microarray, pyrosequencing, and qRT–PCR, were used for transcriptome analysis and validation. The variations in root length and growth were found among four genotypes of G.herbaceum when exposed to mannitol-induced osmotic stress. Under osmotic stress, the drought tolerant genotypes Vagad and GujCot-21 showed a longer root length than did by drought sensitive RAHS-14 and RAHS-IPS-187. Further, the gene expression patterns in the root tissue of all genotypes were analyzed. We obtained a total of 794 differentially expressed genes by microarray and 104928 high-quality reads representing 53195 unigenes from the root transcriptome. More than 10% of these unigenes were novel, the remaining had homologs in public data bases. The Vagad and GujCot-21 respond to water stress by inducing various genes and pathways such as response to stresses, response to water deprivation, and flavonoid pathways. Some key regulatory genes involved in abiotic stress, such as AP2 EREBP, MYB, WRKY, ERF, ERD9, and LEA, were highly expressed in Vagad and GujCot-21. The genes RHD3, NAP1, LBD, and transcription factor WRKY75, known for root development under various stress conditions, were expressed specifically in Vagad and GujCot-21. The genes related to peroxidases, transporters, cell wall-modifying enzymes, and compatible solutes (amino acids, amino sugars, betaine, sugars, or sugar alcohols) were also highly expressed in Vagad and Gujcot-21. Our analysis highlights changes in the expression pattern of genes and depicts a small but highly specific set of drought responsive genes induced in response to drought stress. Some of these genes were very likely to be involved in drought stress signaling and adaptation, such as transmembrane nitrate transporter, alcohol dehydrogenase, pyruvate decarboxylase, sucrose synthase, and LEA. These results might serve as the basis for an in-depth genomics study of Gossypium herbaceum, including a comparative transcriptome analysis and the selection of genes for root traits and drought tolerance.

Abiotic stress associated with insufficient water is a primary limiting factor for most crop plants, including cotton. Plants are able to acclimate to reduced water availability through physiological and biochemical responses to reduce water loss, protect tissues from damage, and repair tissue damage once it has occurred. Acclimation is dependent on changes in gene expression that are controlled by a wide range of regulatory mechanisms functioning at all levels of the gene expression system from chromatin-based epigenetic signaling through ubiquitin-dependent protein lability. Using information from model systems such as Arabidopsis, we are evaluating the functions of a number of stress-responsive regulatory factors in cotton. These include well characterized transcription factors such as ABF and CBF proteins, along with more recently identified factors involved in chromatin remodeling and ubiquitin ligases that regulate the activity and stability of other regulatory proteins. Our results indicate that individual regulatory factors can affect stress responses in specific ways. For example, expression of the ABA-responsive transcription factor ABF3 strongly impacts stomatal responses while expression of the ABA-dependent transcription factor CBF3 does not. These functional differences suggest the possibility of synergistic action. Also, since inappropriate expression of some regulatory factors can negatively affect plant development and productivity, effective use of these genes requires optimization of transgene structure using a variety of transcriptional promoters to optimize expression patterns. Based on these and other results, we anticipate that this research will provide a deeper understanding of the genetic factors that determine stress tolerance traits in cotton and facilitate the translation of basic research findings into applications that can benefit producers throughout the world.

We aim to identify regulatory processes that confer the higher fiber quality in Gossypium barbadense with the aim of transferring those traits in a targeted manner to higher yielding and more adaptable G. hirsutum. We characterized the progression of fiber development in elite cultivars of G. hirsutum and G. barbadense growing in parallel in a highly temperature-controlled greenhouse to reveal experimentally convenient commonalities and differences in the comparative stages of fiber development. The quality of the fiber was analyzed to reveal the expected differences, e.g. longer and stronger fiber in G. barbadense. We then analyzed comparative changes in cell wall composition in the two species using a comprehensive ELISA-based approach, finding a difference that is related to the composition of the cotton fiber middle lamella. We are in the process of performing transcriptomics using RNA-Seq technology in parallel with metabolomics (using the services of a commercial firm) for 10 to 28 DPA fiber of each species. By using several high throughput technologies in parallel on the same well-characterized fiber samples, we are establishing a strong basis for identifying differences that may control the higher fiber quality in G. barbadense. We thank Utku Avci, Lissete Betancur, Virginia L. Brown, Z. Jeffrey Chen, Mary V. Duke, Richard E. Glick, Xueying Guan, Michael G. Hahn, Gyoungju Nah, Sivakumar Pattathil, Brian E. Scheffler, and Bir Singh for their contributions to this research project. For research support, we thank Cotton Inc., Cary, NC and the National Science Foundation (Grant Number IOS1025947).

Cotton is the leading fiber crop in the world, producing the most prevalent natural fibers used in textile industry. Cotton fibers are single cells derived from outer epidermis of ovules. Their growth and development consist of four overlapping stages: fiber initiation, cell elongation, secondary cell wall deposition, and maturation. Fiber cells from G. hirsutum are 1000 to 3000 times longer than they are wide. With highly elongated structure composed almost of cellulose, cotton fibers are considered as an ideal model for studies of plant cell elongation and cell wall biogenesis. Brassinosteroids (BRs) are a kind of steroidal hormone in plant kingdom. Exogenous application of BRs increased fiber length in field experiments or in ovule culture condition. Contrarily, brassinazole (BRZ), a specific inhibitor of BR biosynthesis, repressed cotton fiber initiation and elongation. However, the role of endogenous BRs and the mechanism of BRs activity are largely unknown in the growth and development of cotton fiber cell. To illuminate the effect of endogenous BRs on fiber cell development, we have cloned GhDWF4 gene, a DWF4 (steroid C-22 hydroxylase) homologue from upland cotton and generated a lot of transgenic cotton lines over-expressing GhDWF4 or suppressing GhDWF4 expression. In the transgenic fiber cell over-expressed GhDWF4 gene, the endogenous BRs content increased. In 12-DPA transgenic fiber cell, for example, 6-Deoxocathasterone, 6-deoxotyphasterol, and 6-Deoxocastasterone increased by 53 fold, 3 fold, and 4 fold, respectively. In 20-DPA transgenic fiber cell, 6-Deoxocathasterone, 6-deoxoteasterone, 6-Deoxotyphasterol, and 6-Deoxocastasterone increased by 46 times, 22 times, 25 times, and 7 times, respectively. Meanwhile, the changes have not detected for the intermediates involved in the early C-6 oxidation pathway between transgenic fiber and control. The fiber length of CaMV35S::GhDWF4 increased by 9.8%-17.14% and the fiber length of pFBP7::GhDWF4 increased by 4.5%-7.7% compared with control. Furthermore, the strength of GhDWF4-increased fibers improved while micronaire decreased. The fiber strength of pFBP7::GhDWF4 and CaMV35S::GhDWF4 increased 2.8% and 9.8%, respectively. These result revealed that enhanced endogenous BRs level not only promoted fiber cell elongation but also improved the quality of cotton fiber. To understand the molecular mechanism of BRs promoting fiber elongation, we performed digital gene expression analysis used 12-DPA fibers. The results indicated that large numbers of important genes related to fiber elongation were up-regulated, such as aquaporins and cellulose biosynthases. It is suggested that increased BRs level promotes many physiological processes involved in fiber cell elongation. In addition to fiber length increased, the growth and development of transgenic plants were modified. The plants overexpressing GhDWF4 grew faster and displayed larger leaf size, longer stalks, and bigger flowers and balls. GhDWF4 repressed plants displayed dwarf, deep green and small leaves, and shorter internodes. The growth of a few lines was seriously suppressed, displaying severely dwarf and very slow growth. The height of 2-year grown plant was still shorter than 20cm, and the leaf stalk length and internode length were extremely short. GhDWF4–repressed plants displayed sterile phenotype for flower bud abortion.

Water-deficit stress is an increasing concern in cotton production and can result in severe yield losses with dire economic consequences. To that end, it is important to understand drought resistance mechanisms at the gene expression level to enable the development of cotton cultivars that can provide more dependable production under water-deficit stress. For the identification of differentially expressed transcripts under water-deficit stress, an RNA-Seq experiment was performed using field grown cotton plants (Gossypium hirsutum cv. Siokra L-23) that were subjected to different water supplies- well watered and naturally rain fed, respectively. In total, nearly 300 million trimmed Illumina sequence reads were obtained almost equally from leaf and root tissues of well watered and water-deficit treated plants. These reads were computationally mapped to the annotated genes of the draft D-genome sequence. Analyses on differentially expressed genes are under way. These data will help efforts to understand the complex responses governing transcriptomic regulatory mechanisms and identify candidate genes that should benefit advanced plant breeding programs for the cotton and other crops.

Epicuticular wax and proline contents were studied in five varieties of Gossypium arboreum, namely FDH-170, FDH-300, FDH-306, FDH-786 and Ravi. Variety FDH-786, being the most drought tolerant, exhibited 9- and 5.75-fold higher levels in wax and proline contents respectively when compared with non-stressed plants. A small alpha-crystalline heat shock protein gene (GHSP26) was isolated and characterized using RACE and genomic DNA PCR. Segments of 1026 bp cDNA sequences and 1108 bp genomic (exon & intron) were obtained. Alignment studies revealed that GHSP26 comprises a single open reading frame of 230 amino acids. Expression studies of the gene in different tissues showed that the gene is expressed mainly in leaves of stressed plants. We also carried out high throughput analysis of drought tolerant genes from G. arboreum, and developed cDNA libraries from mRNA isolated from leaves and roots of drought stressed plants. Twelve thousand clones were cultured of which 1000 showed novelty in that there was no homology to genes in the NCBI data bank. Selected clones were spotted on the microarray slides and hybridized with probes from control and water stressed plants. Thirty seven ESTs were found up-regulating under drought stressed conditions. Besides, wax mutants were developed by physical and chemical mutagens and cDNA libraries were constructed from the leaf mRNA to yield clones 78% of which showed no homology with genes in the NCBI databank. Therefore, there is great potential in the search for novel genes from G. arboreum. Selected clones were spotted on the microarray slides and hybridized to yield 40 ESTs involved in wax biosynthesis. GHSP26 and GUSP genes were cloned in a plant expression vector,pcambia-1301 (CaMv 35S promoter, neomycin phosphotransferase and GUS, a reporter gene). Transformation of Gossypium hirsutum variety CIM 496 with these genes yielded transgenic plants that exhibited 6-fold and 60-fold over expression of GUSP and GHSP26 genes under eight days of drought stress conditions. Experiments are in progress for longer exposure of transgenic plants to drought stresses. The upstream region of the promoter of stress resistant gene GHSP26 was analyzed. Each deletion construct was analyzed by Agroinfection in Tobacco leaves after treatment of Abscisic acid (ABA), heavy metals and dehydration. Promoter fragment of 716bp showed two-fold or greater induction after each treatment. Similarly, the promoter of 949bp upstream from translation initiation of the GUSP gene encoding universal stress protein was isolated from genomic DNA of Gossypium arboreum. Deletion analysis revealed that dehydration of abscisic acid and salt responses was affected. A deletion between -208bp and -949bp showed 2-4 fold induction.

Proline-rich proteins (PRP) 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. Subcellular localization showed that GhPRP5 protein was mainly localized on the plasma membrane. The transgenic Arabidopsis plants with overexpressing GhPRP5 displayed reduced cell growth, resulting in smaller cell size and consequently plant dwarfs, in comparison to wild-type plants. In contrast, knock-down of GhPRP5 expression by RNA interference in cotton enhanced fiber development. The lint fiber length of transgenic cotton lines was longer than that of wild type. Interestingly, the length of fuss fibers was also longer in transgenic lines. A yeast two-hybrid screen results suggested that GhPRP5 may form homodimers in cotton fiber cells. In addition, a number of genes that are involved in the cell wall-plasma membrane (PM)-cytoskeleton continuum were up-regulated or down-regulated in the transgenic cotton plants owing to suppression of GhPRP5. Collectively, these data suggested that GhPRP5 is involved in fiber development of cotton.

A virus-induced gene silencing (VIGS) system developed from the Cotton leaf crumple geminivirus (CLCrV) is effective in vegetative cotton tissues, but it had not been tested in the most economically important part of the plant—cotton fiber. A CLCrV:GFP reporter construct revealed GFP fluorescence in the ovule vasculature but not the fiber. This raised the possibility that the CLCrV-derived silencing signal might not be expressed in cotton fiber, making fiber gene silencing dependent on translocation of siRNAs from adjacent tissues. To test this possibility, we inoculated a cotton line expressing 35S:GFP with CLCrV:GFP and showed that GFP fluorescence was reduced in the fiber from 7 to 20 days post anthesis (DPA). Unlike sectored vegetative silencing, GFP silencing within locules appeared uniform and was correlated with silencing in the adjacent boll wall. Reduced GFP transcript was verified by qRT-PCR at 12, 15, and 20 DPA. Since transgenes are typically more easily silenced, the CLCrV-VIGS system was also used to silence the endogenous cell wall loosening gene, α-expansin1 (GhEXP1), resulting in 8.8% shorter fiber compared to controls. Analysis by qRT-PCR showed reduced GhEXP1 transcript at 16 DPA but not at 12 DPA. Since cotton fibers are symplastically isolated at about 10 to 15 DPA, CLCrV-VIGS of endogenous genes may be hindered during this period. Characterizing the silencing dynamics in this economically important plant cell provides a guide for future VIGS experiments in cotton fiber. For research support we thank NSF (Grant # IOS1025947) and Cotton Inc. (Cary, NC).

The regulatory mechanism of cotton fiber development is always the key part of cotton research. However, this process is not fully understtod. In this research, we chose the high-quality fiber bearing cultivar, Gossypium barbadense, as the main material to uncover the mechanism during fiber development at early stage. By combination of the effects in gene cloning, evolution study, expression analysis, protein-DNA binding, protein-protein interaction and functional study by transgenic plants, we achieaved the results as follows; A new HD-Zip IV family member, designated as GbML1, was cloned from early tage ovules by RACE technique. GbML1 could form homodimers by its ZLZ domain and this dimmer formation is critical for its binding to DNA. GbML1 could pecifically bind to L1 box by its HD domain. The expression of GbML1 and GbMYB25 was similar during cotton ovule development and both protein could be ocalized into nucleus. Moreover, GbML1 could interact with the C terminal part of GbMYB25 by its START-SAD domain and the three dimentional structure of START domain was important for the binding. Though GbML1 itself was a weak ctivator, but the C2 domain of GbMYB25 had strong activation activity. Thus the complex formed by GbML1 and GbMYB25 both had the DNA specific binding activity and transcriptional activation activity. This complex could bind to the promoters of fiber specific genes and then activated their expression. Overexpression of GbML1 in Arabidopsis not only increased the trichome number in the leaf and stem epidermis, but also activated seed fiber formation in Arabidopsis, which supported that GbML1 was a key regulator in cotton fiber differentiation and development.