Functional Genomics

Cotton fibres are specialised epidermal cells that grow from the surface of the cotton seed. We have identified three regulatory genes that are important for instigating the development of these fibre cells - two R2-R3 MYBs (GhMYB25 and GhMYB25-like) and a homeodomain-leucine zipper protein (GhHD-1) GhMYB25-like and GhMYB25 belong to a unique MIXTA clade of MYB transcription factors involved in the regulation of cell morphogenesis in the petal epidermis. Another eight of these MIXTA MYBs have been found in the cotton genome sequence. They are all expressed during early fibre development. GhHD-1 is a member of the class IV HD-ZIP transcription factor family, known to be involved in the determination of epidermal cell type. RNAi-mediated suppression of GhHD-1 reduced trichome formation and delayed fibre initiation while constitutive over-expression of this gene increased the number of fibres initiating on the seed, but did not affect leaf trichomes. GhMYB25-silenced cotton exhibits delayed fibre initiation, shorter fibres and reduced trichomes on the plant, while over-expression of this gene increased fibre initiation and leaf trichome number. GhMYB25-like silenced plants produce fibreless seeds, but normal trichomes elsewhere. Constitutive over-expression of GhMYB25-like increased the number of fibre initials. Yeast two-hybrid and one-hybrid analysis and sequencing of RNA isolated from the outer integument of -4dpa, -2dpa and 0dpa wildtype ovules and 0dpa ovules from the silenced lines have identified additional proteins involved in the regulation of early fibre development. Based on our data we have developed a model of the genes and factors involved in the early stages of cotton fibre development.

KORRIGAN1 (KOR1) is a membrane-bound endoglucanase that has been implicated in cell wall formation, largely based on studies in vegetative tissues. Its potential role in reproductive development, however, remains unknown. Here, we demonstrated that GhKOR1 played significant roles in cotton (Gossypium hirsutum) endosperm and embryo development and the establishment of early seedling vigor. Down-regulation of GhKOR1 transcript in RNAi or co-suppressed transgenic cotton resulted in smaller filial tissue and reduced seed weight. Further histological studies revealed that silencing KOR1 disrupted endosperm cellularization and delayed embryo development, leading to weak growth of seedling following germination. The transgenic plants exhibited fewer and smaller endosperm cell with irregular brittle cell walls at 10 days post anthesis (DPA) when the wild type endosperm has become highly cellularized. In contrast to the progression to heart stage of the wild type embryo, the transgenic embryo was still in the globular stage at 10 DPA. The transgenic seed also displayed reduced fiber growth. These findings demonstrate that GhKOR1 is required for cytokinesis of endosperm cell and the development of embryo and fiber. Also noteworthy is that down-regulation of GhKOR1 transcript dramatically reduced callose deposition in the seed coat transfer cells. This, however, does not appear to affect the morphology of the transfer cells. Together, the study provides several novel findings which significantly advances our understanding on the functions of KOR1 gene in seed development.

Brown cotton fiber is the most widely used in the modern naturally-colored cotton industry. It was reported that brown pigments in cotton fiber belong to proanthocyanidins (PAs) and PA derives. Dark brown fiber gene (Lc1), mapped on Chr. 7, is a semi-dominant gene conferring dark brown color to fiber in upland cotton. To further understand the regulatory mechanism of brown pigmentation, we aimed to clone the Lc1 gene and to clarify its role in regulating PA biosynthesis in cotton fiber. R2R3 MYB protein TT2 plays a key role in controlling PA biosynthesis in Arabidopsis seed coat. To reveal the possible relatedness of cotton TT2 homologs to the regulation of PA biosynthesis in fibers, we identified 5 cotton TT2 homologous genes in D genome (GrTT2-1~5), and cloned 10 corresponding genes from G. hirsutum (GrTT2-1A, 1D~5A and 5D). Among these genes, only GhTT2-3A showed a significant variation in expression levels between dark brown (Lc1Lc1) and white fibers, suggesting that GhTT2-3A was related to the PA biosynthesis in dark brown fibers. Furthermore, GhTT2-3A was constructed downstream to a constitutive promoter (CaMv35S) and transformed into cotton. The expression levels of PA structural genes and PA content in GhTT2-3A overexpressing calli were significantly increased. This indicated that GhTT2-3A could activate the whole PA pathway and promote PA biosynthesis and accumulation in cotton. To determine the positional relationship between GhTT2-3A and Lc1, we perform fine-mapping in GhTT2-3A region using a RIL population, derived from a cross of a dark brown-fiber line T586 (Lc1Lc1) and a white-fiber cultivar Yumian No.1, and a secondary F2 population of 1698 individuals. Based on a BAC containing GhTT2-3A and comparative cloning of corresponding sequences in T586 and Yumian No.1, 9 polymorphic SSR/SNP makers were identified and further genetically linked to the Lc1 gene. Lc1 was finally mapped in a region between markers TT2-4a and TT2-3a3y. According to the sequenced D genome, this region harbored only two coding genes, GrTT2-3 and GrTT2-5. In conclusion, the data on expression analyses, transgenic cotton and genetic mapping collectively demonstrated that cotton TT2 homologous gene GhTT2-3A controlled PA biosynthesis in brown cotton fibers, and may be the dark brown fiber gene Lc1.

Genetic transformation method using Agrobacterium tumefaciens was developed for cotton plant species. Following the efficient regeneration of three cotton varieties, the effect of inclusion of acetosyringone in co-cultivation medium was measured. Subsequently, transformation was obtained by co- cultivation of 3 weeks old cotton apical shoot and A. tumefaciens strain LBA 4404 containing a plasmid harboring neomycin phosphotransferase and b-glucuronidase encoding genes. PCR analyses performed to identify the presence of marker gene (npt II) in the transgenic plants using primers used for amplification of a 700 bp fragment of the npt II gene showed that 87% of the transgenic plants selected for kanamycin resistance were transformed with the gene encoding b-glucuronidase.  Routine transformation efficiency of cotton was established at 1.3%. The mean number of GUS positive apices was 67% higher when acetosyringone was included in the medium.

In process of cotton (Gossypium hirsutum L.) somatic embryo development (SED), quite few embryos are able to normally mature and germinate, and successfully regenerate plantlets. Many efforts have been implemented for increasing regeneration rate, however, the maturation rate of somatic embryo is still very low. Here, a global analysis of proteome dynamics between globular and cotyledonary embryos was performed using isobaric tag for relative and absolute quantification (iTRAQ) method to better understand mechanisms underlying somatic embryo development for increasing cotton regeneration rate. A total of 5718 proteins were identified by mass spectrometry (MALDI-TOF/TOF) analysis , 209 proteins of which exhibited differential expression, including 102 upregulating proteins and 107 downregulating proteins. Based on Gene Ontology annotation, the indentified proteins could be classified into 22 biological processes and respectively localized to chloroplast, cytoplasm, thylakoid and so forth. Most of these proteins were implicated in cellular component organization or biogenesis, cellular processes and metabolic processes. Proteomic analysis of SED indentified several important proteins major involving in signal transduction, energy production and conversion, and carbohydrate metabolism. The expression level of the corresponding genes was further characterized by employing quantitative Real-time PCR. Interestedly,Pathway analysis demonstrated that abscisic acid（ABA）, giberellic acid (GA), jasmonic acid（JA） are involved in various physiological activities and has a distinct influence on SED. When exogenous hormones added in medium, the results showed that ABA promoted somatic embryo maturation, GA was negative to SED, and JA accelerated SED. The protome dynamics analysis provides a better understanding of the possible regulation mechanisms of plant hormone and some important protein function in cotton SED.

Trihelix factors constitute a family of plant-specific transcription factors, play important roles in light-regulated responses and other developmental process and in responses to environmental stresses. In this study, a novel trihelix gene, named GhSIP1, was isolated from Gossypium hirsutum, and further characterized its roles in abiotic stress tolerance. GhSIP1 can be induced by various abiotic stresses, and the encoded proteins were localized in nuclear region. In Arabidopsis protoplast assay, no transcriptional activation activity detected for GhSIP1. DNA binding analysis using Electrophoretic Mobility Shift Assay（EMSA）showed that GhSIP1 could bind to Box, Site1, GT-1box, GT2-Box, GT3-Box, GT-3b, MBS1 and MRE4. Overexpression of the GhSIP1 improved plant tolerance to salt stress in transgenic Arabidopsis plants. Moreover, GhSIP1-transgenic plant seedlings had longer root length compared to Col-0 under ABA treatment. Many stress-responsive genes were altered in GhSIP1-transgenic plants. The results indicate that GhSIP1 confer high salt tolerance through regulation of a common set of genes and specific sets of genes. Key words: GhSIP1;tihelix factor;salt tolerance;Gossypium hirsutum;transgenic Arabidopsis

Abiotic stress is a major environmental factor adversely affects cotton growth and yield. But upland cotton still has widely geographic range from most tropical to subtropical regions of the world. In this study, we sought to identify genes involved in diverse stresses including cold, drought, salinity and heat by transcriptome sequencing analysis. Our results showed that there were thousands of transcripts induced or depressed by cold, drought, heat and salinity. Of these, only less 7% transcription factors (TFs) were included which are much less than the functional genes. In addition, the D-subgenome had more genes highly expressed and being selected but less being silence and lost. These indicate the D-subgenome may contribute greater to abiotic stress tolerance of upland cotton and TFs may play critical roles to adapt to various environments.

Cotton (Gossypium spp.) is one of the most economically important crops of the world which has unique qualities. It is used in the production of textile fiber and also serves as food and feed stock. There are more than 50 species of cotton, of which four are cultivated for commercial purposes; G. hirsutum (more than 90% of world production), G. barbadense (3-4%), G. herbaceumand G. arboreum (collectively 2%). The study described here reports the optimization and use of an Agrobacterium-mediated, virus-induced gene silencing (VIGS) system in three cultivated cotton species (G. hirsutum, G. arboreum and G. herbaceum) using a Tobacco rattle virus (TRV) vector for transient silencing of an endogenous gene. GrCLA1 is a homolog of the Arabidopsis thaliana chloroplast alterados 1 (AtCLA1) gene, and is involved in chloroplast development.The GrCLA1 gene serves as a marker in this study; silencing of this gene produced a foliar albino or white phenotype within 10 to 15 day after inoculation. Three independent biological replicates were used in case of G. hirsutum (cultivarsIR3701, NIBGE2, 777). Each replicate consisted of 20 plants inoculated with control (TRV without insert) and 20 plants with TRV-GrCLA1. Plants were kept at (28+ 2 oC). All plants inoculated with TRV-GrCLA1 showed typical the albino phenotype indicating 100% efficiency of silencing. Two independent replicate were done for G. herbeceum and G. arboreum with same results and efficiencies. RT-PCR was conducted using specific primers for the GrCLA1 gene while the 18S RNA gene was used as an internal control. RT-PCR analyses showed presence of GrCLA1 transcripts in control and healthy cotton plants. No amplification was achieved from plants expressing the albino phenotype, indicating that the GrCLA1 gene had been silenced by TRV-based VIGS. The study optimized the Agrobacterium-mediated VIGS in three cultivated species of cotton with different ploidy levels; tetraploid (G. hirsutum) and diploid (G. arboreum and G. herbaceum). With the availability of the genome sequence of cotton several species, this rapid and efficient Agrobacterium-mediated VIGS assay has the potential to provide a powerful tool for robust large-scale analysis of gene function in cotton.

Background: Cotton (Gossypium spp.) is the world’s leading natural fiber and the third largest oilseed crop. Cotton fibers are trichomes that terminally differentiate from single cells located in the epidermis of the ovule and a model system for studying cell elongation. To identify differentially expressed genes related to differences in fiber length between two near-isogenic lines that will facilitate the uncovering of the mechanism of fiber elongation, a high-throughput sequencing technology was applied. Results: Six sequencing libraries were constructed and sequenced of ovules and developing fibers at the early cotton fiber development stages including the initiation stage (0 days post anthesis (DPA), 3 DPA and the fast elongation stage (10 DPA) in two near-isogenic lines differing in fiber length. As a result, 28 G raw data were obtained and then de novo assembled into 98,464 unigenes with a mean length of 896 bp and a N50 length of 1397 bp. Approximately 71.5% of the unigenes were annotated by aligning against the public protein databases including NR, SwissProt, KEGG and COG. Differentially expressed genes (DEGs) were investigated using the FPKM method, leading to identification of 1536 up-regulated genes and 395 down-regulated genes between the two lines at 10 DPA. Based on GO enrichment and metabolic pathway enrichment analysis, a number of candidate genes involved in fiber elongation related pathways were selected and discussed. Moreover, genes related to previous linear cell growth mode were also confirmed in our study. Conlusion: we investigate the transcriptome profiling of two near-isogenic lines differing in fiber length at fast elongation period using the Illumina RNA-Seq technology. Studies of DEGs involved in fiber elongation related pathways facilitate the discovery of genes related to fiber length and the mechanism of fiber elongation. Keywords：Gossypium hirsutum, near-isogenic lines, Fiber elongation, de novo, RNA-Seq

Phytosterol, as the precursor of bioactive brassinosteroids and a kind of content of cell membrane, plays an important role in plant growth and development. Sitosterol and campesterol are two major phytosterols in plant cell. Their content and the ratio of campesterol to sitosterol influenced the membrane fluidity and permeability, the membrane-associated metabolic processes, the activity of proteins within the membrane, Vesicle trafficking, and bioactivity BRs biosynthesis. Sterols become functional only after removal of the two methyl groups at C-4 site. In Animals and fungi, two C-4 demethylation is performed in one step. In the plant, however, is performed by two reactions separated by several steps. The biological significance of the phytosterol biosynthesis pathway in plants is largely unknown. Enzymology study showed each C-4 demethylation included three consecutive reaction performed by a C-4 demethylation complex which composed of a sterol methyl oxidase (SMO), a 3 beta steroid dehydrogenase/C-4 decarboxylase (3β HSD/D), and a 3-ketone reductase (SR). In plant, SMO1 and SMO2 mediated the first and the second demethylation reaction, respectively. To understand the role of two reactions in plant cells, we have cloned SMO1 and SMO2 genes from cotton fiber. There were two GhSMO1 genes in upland cotton and each protein contained 304 amino acid residues. Both GhSMO1-1 and GhSMO1-2 were membrane protein while four transmembrane domains were in GhSMO1-1 and three transmembrane domains were in GhSMO1-1. 89.5% identical amino acid shared by two proteins. Similarly, there were two GhSMO2 genes in upland cotton. The GhSMO2-1 and GhSMO2-2 contained 271 and 269 amino acid residues, respectively. Both proteins were soluble protein and there were 90.0% identical amino acid in two GhSMO2. The homology between GhSMO1-1 and GhSMO2-1 was 40.6% while the homology between GhSMO1-2 and GhSMO2-2 was 42.4%. These results suggested that the GhSMO mediated two demethylation was very different on sequence and the subcellular localization. The GhSMO1-1 and GhSMO1-2 expressed similarly in fibers and ovules. Following fiber and ovule growth, their expression levels increased gradually and reached to peak in 10 dpa, after that gradually decreased. However, there was not significant difference between various developmental stages of fiber and ovule. The GhSMO2-1 and GhSMO2-2 expressed preferentially in fiber cell. As the fiber grows, their expression levels increased drastically and reached to peak at rapid elongation stage of fiber (8-12 DPA). Furthermore, their expression levels elevated with ovule development in Xuzhou 142 wild type while extremely low level was detected and decreases gradually with ovule growth. These results suggested that the GhSMO1 might be a house-keeping gene and the first reaction was the basis for plant growth. The GhSMO2 and the second demethylation were closely related to the fiber cell elongation.