Functional Genomics

Cotton (Gossypium hirsutum L.) is the most important textile fiber crop in the world. Cotton fiber, a highly elongated and thickened single cell derived from the ovule epidermis, provides an excellent system for study on cell elongation and secondary cell wall biosynthesis. In this study, a gene, GhKNL1 (KNOTTED1-LIKE), encoding a classical Class II KNOX protein was identified in cotton. GhKNL1 was preferentially expressed in developing fibers at the stage of secondary cell wall (SCW) biosynthesis. GhKNL1 protein is localized in cell nucleus, and could interact with GhOFP4, as well as AtOFP1, AtOFP4 and AtMYB75. However, GhKNL1 protein lacks the activity of transcriptional activation. Dominant repression of GhKNL1 gene affected fiber development of cotton. The expression levels of the genes related to fiber elongation and SCW biosynthesis were altered in the transgenic fibers of cotton. As a result, the transgenic cotton plants produced the aberrant, shrunken and collapsed fiber cells. Length and cell wall thickness of fibers of the transgenic cotton plants were significantly reduced, compared with those of wild type. Furthermore, overexpression and dominant repression of GhKNL1 in Arabidopsis resulted in a reduction in interfascicular fiber cell wall thickening of basal stems of transgenic plants. Complementation test revealed that GhKNL1 rescued the defective phenotype of Arabidopsis knat7 mutant in some extent. Given the data together, it is suggested that GhKNL1 participates in regulation of fiber development via modulating expression of the genes related to cell elongation and secondary wall biosynthesis of cotton fibers. Additionally, our study also provides the candidate intrinsic gene for improving fiber quality by genetic manipulation.

The Gossypium species are characterized by the presence of small and darkly pigmented lysigenous glands filled with gossypol. Duplicate recessive genes gl2gl3 and/or another dominant gene Gl2e, a multi-allele of Gl2 can eliminate all glands on the aerial plant parts and seeds and result in low gossypol content. However, the gene conferring glandless phenotype was previously unknown. Based on our precious study, the Gl2e locus was mapped on Chr12 between molecular marker CLR362 and NAU2251b，NAU3860b，STV033，with a genetic distance 9.27 and 0.96 cM . Further, we narrow the target region to 47-kb interval. In the interval, we detected seven candidate genes, and only one has altered expression level between Hai1 and TM-1. Therefore, it is likely the gene at Gl2e locus. Functional validation of the candidate gene through transcriptome profiling and virus induced gene silencing is in progress.

Hydroxyproline-rich glycoproteins (HRGPs) 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 to wild-type plants. In contrast, knock-down of GhPRP5 expression by RNA interference in cotton enhanced fiber development. The fiber length of transgenic cotton lines was longer than that of wild type. 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. In addition, a fasciclin-like arabinogalactan protein gene (GhFLA1) was identified in cotton. 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. Moreover, 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. Together, our results suggested that PRPs and AGPs function in fiber initiation and elongation.

Glycerophospholipids (GPLs) severed as the major components of plasma membranes and signaling molecules are classified according to their head groups, backbones and fatty acyl chains. Previous transcriptome studies showed that lipid biosynthesis pathway was preferentially expressed during the rapid and vigorous cotton fiber elongation. To examine GPL composition changes during fiber elongation, here, by application of ultra-performance liquid chromatography coupled to electrospray ionization and tandem mass spectrometry, we find that lipid biogenesis is activated in the same growth stage. Membrane lipids targeted on phosphatidylcholine, phosphatidylethanolamine, phosphatidylinositol (PtdIns), phosphatidic acid (PA), phosphatidylserine and phosphatidylglycerol were extracted and separated from 10 day post-anthesis, fast-elongating wild-type cotton (G. hirsutum) fibers, wild-type ovules with fibers removed and ovules from the fuzzless-lintless (fl) mutant harvested at the same age. A total of 1566 GPL species were identified and further quantified. Fiber cells contained significantly higher amount of PtdIns and PA than that extracted from ovule samples of both genotypes, with 34:3 (16:0, 18:3) being the most predominant molecular species of PtdIns. Exogenously applied soybean L-α-PtdIns or C18:3 per se rather than other phospholipids in ovule culture medium significantly promoted fiber cell elongation. Genes encoding fatty acid desaturases, PtdIns synthase and phosphatidylinositol-specific phospholipase C were activated during this growth stage with substantial increases in endogenous levels of phosphatidylinositol monophosphate, 1,4,5-triphosphate and inositol hexaphosphate. Our data thus lay a basis for future investigation of how PtdIns and its phosphorylated derivates are involved in regulation of cotton fiber cell growth.

The cotton fiber, as a single-celled trichome, is a biological model system for studying cell differentiation and elongation. However, the complexity of gene expression and regulation in the fiber complicates genetic research. In this study, we investigated the genome-wide transcriptome profiling in Texas Marker-1 (TM-1) and five naked seed or fuzzless mutants (three dominant and two recessive) during the fuzz initial development stage. More than three million clean tags were generated from each sample representing the expression data for 27,325 genes, which account for 72.8% of the annotated Gossypium raimondii primary transcript genes. Thousands of differentially expressed genes (DEGs) were identified between TM-1 and the mutants. Based on functional enrichment analysis, the DEGs downregulated in the mutants were enriched in protein synthesis-related genes and transcription factors, while DEGs upregulated in the mutants were enriched in DNA/chromatin structure-related genes and transcription factors. Pathway analysis showed that ATP synthesis, and sugar and lipid metabolism-related pathways play important roles in fuzz initial development. Also, we identified a large number of transcription factors from families such as MYB, bHLH, HB, WRKY, AP2/EREBP, bZIP and C2H2 zinc finger that were differently expressed between TM-1 and the mutants, and which were also related to trichome development in Arabidopsis.

Male reproduction in flowering plants is highly sensitive to high temperature (HT). To investigate molecular mechanisms of the response of cotton anthers to HT, a relatively complete comparative transcriptome analysis was performed during anther development of Gossypium hirsutum ‘84021’ and ‘H05’ under normal temperature and HT. Totally, 4599 differentially expressed genes (DEGs) were screened; the DEGs were mainly related to epigenetic modifications, carbohydrate metabolism, and plant hormone signaling. Detailed studies showed that deficiency in S-ADENOSYL-L-HOMOCYSTEINE HYDROLASE1 (SAHH1) and the inhibition of methyltransferases contributed to genome-wide hypomethylation in ‘H05’, and the increased expression of histone constitution genes contributed to DNA stability in ‘84021’. Furthermore, HT induced expression of CASEIN KINASE I (GhCKI) in ‘H05’, coupled with the suppression of starch synthase activity, decreases in the glucose level during anther development, and increases in the indole-3-acetic acid (IAA) level in late-stage anthers. The same changes also were observed in Arabidopsis GhCKI overexpression lines, and GhCKI activates the accumulation of abscisic acid (ABA) in Arabidopsis buds, thereby disturbing the balance of reactive oxygen species (ROS) and eventually disrupting tapetal PCD, leading to anther abortion or indehiscence. These results suggest that GhCKI, sugar, and auxin may be key regulators of the anther response to HT stress. Moreover, PHYTOCHROME-INTERACTING FACTOR GENES (PIFs), which are involved in linking sugar and auxin and are regulated by sugar, might positively regulate IAA biosynthesis in the cotton anther response to HT. Additionally, exogenous IAA application revealed that high background IAA may be a disadvantage for late-stage cotton anthers during HT stress. Overall, the linking of HT, sugar, PIFs, and IAA, together with our reported data on GhCKI, may provide dynamic coordination of plant anther responses to HT stress.

Cotton fiber, a highly elongated, thickened single cell of the seed epidermis, is a powerful cell wall research model. Fiber length and strength are the key trait that determines fiber quality in cotton. To further reveal the mechanism of fiber length and strength formation, we used Digital Gene Expression Tag Profiling to compare transcriptome data from longer and/or stronger fiber chromosome introgressed lines (CSILs) containing segments of various Gossypium barbadense chromosomes with data from its recurrent parent TM-1 (from 5 DPA to 25 DPA). During the fiber elongation stage, a large number of differentially expressed genes (DEGs) involved in carbohydrate, fatty acid and secondary metabolism, particularly cell wall biosynthesis, were highly up-regulated, as determined by functional enrichment and pathway analysis. Furthermore, DEGs related to hormone responses and transcription factors showed up-regulated expression levels in the CSILs. During secondary cell wall stage, functional classification and enrichment analysis revealed that these DEGs were mainly enriched for secondary cell wall biogenesis and carbohydrate metabolism. In both stages, metabolic and regulatory network analysis indicated that the same pathways were differentially altered, and distinct pathways exhibited altered gene expression, in the CSILs. Our results provide important information about the molecular mechanisms controlling fiber length and strength, which are mainly tied to carbohydrate metabolism, cell wall biosynthesis, fatty acid metabolism, secondary metabolism, hormone responses and Transcription factors. Moreover, our results provide new insights into the critical factors associated with cell elongation and secondary cell wall biosynthesis, and facilitate further research aimed at understanding the mechanisms underlying cotton fiber length and strength.

In this research, methylation-sensitive amplification polymorphism (MSAP) method based on capillary electrophoresis was used to analyze DNA methylation level in a cotton hybrid CCRI 29, so as to discuss the molecular mechanism of salt tolerance in cotton. Totally 24 primer combinations were adopted and 69.2 and 56.7 methylated CCGG loci per primer combination were detected in CCRI 29 treated with 0.4% of salt water and control respectively, which were significantly different. Comparing to water control, 52.6% of the loci under salt stress showed increased methylation level, meaning hypermethylation happened; whereas 19.7% of the loci showed decreased methylation level, meaning hypomethylation happened; the hypermethylation rate is significantly higher than that of hypomethylation. The results showed that all kinds of variation of DNA methylation happened in CCRI 29 under salt stress, including hypermethylation, hypomethylation and other patterns; hypermethylation happened at a significantly higher rate than that of hypomethylation. DNA methylation level significantly increased under salt stress in CCRI 29, suggesting that CCRI 29 might have the potential mechanism of increasing methylation level when responding to salt stress. The increase of overall level of DNA methylation in cotton genome might play an important role in tolerance to salt stress in cotton. Preliminary cloning and analysis of methylated DNA sequences showed that some ATP synthase subunit genes maintained the same methylation level after salt stress, so their expression should not be inhibited by DNA methylation, which might be part of the reason that CCRI 29 is salt tolerant and can maintain normal growth after salt stress in a certain period.

As a main economical and oil crop, cotton was cultivated in many countries. But the domestication of cotton by human beings made this crop loss many genes which made cultivars vulnerable to biotic and abiotic stresses. Meanwhile, due to more attentions to high yield, the modern cultivars of cotton show worse fiber quality with high yield improved somewhere. To cope with these weaknesses, we have been trying to discover genes conferring fiber quality and diverse resistances. Some genes were functionally evaluated via genetic transformation. We found that a series of genes involves in fiber development, such as GbPDF1, GbTCP, GhPSK, and GhCaM7, even a metabolic pathway such as flavonoid was proved to be involved in fiber development. We are sure these results will contribute to fiber quality improvement in the coming breeding program. Verticillium wilt disease is threatening cotton production in China, even causing 30% yield loss. We found several signaling pathways contribute to disease resistance, the key genes in these pathways are under functional analysis. Besides above, some genes conferring high temperature tolerance and drought tolerance were proved to have high potential in abiotic tolerance improvement in breeding cotton.

Somatic embryogenesis (SE) is the developmental process by which somatic cells of higher plants can dedifferentiate and reorganize into new plants. It is a notable illustration of cell totipotency, representing a unique developmental pathway of characteristic events, during which cells dedifferentiate, activate their cell division, and reprogram their physiology, metabolism, and gene expression patterns. However, the precise molecular mechanisms regulating SE remain unclear. The Digital Gene Expression (DGE) system, in combination with small RNA sequencing, parallel analysis of RNA ends (PARE) and comparative transcriptome analysis using materials from different development time-point/stages of somatic embryogenesis and zygotic embryos (ZEs) from Gossypium hirsutum cv. YZ1, were conducted in cotton, a typical plant species in SE. Genome-wide profiling of gene expression allowed the identification of novel molecular markers characteristic of this developmental process. DGE profiling and functional assignments of the genes differentially expressed during cotton somatic embryogenesis indicated significant transcriptional complexity during this process. Bioinformatics analysis showed that the genes were enriched for basic processes such as metabolic pathways and biosynthesis of secondary metabolites. Transcription factor–encoding genes were found to be differentially regulated during SE. The complex pathways of auxin abundance, transport and response with differentially regulated genes revealed that the auxin-related transcripts belonged to IAA biosynthesis, indole-3-butyric acid (IBA) metabolism, IAA conjugate metabolism, auxin transport, auxin-responsive protein / indoleacetic acid-induced protein (Aux/IAA), auxin response factor (ARF), small auxin-up RNA (SAUR), Aux/IAA degradation, and other auxin-related proteins, which allow an intricate system of auxin utilization to achieve multiple purposes in SE. A total of 4242 differentially expressed genes were identified in at least one developmental stage by comparative transcriptome analysis using three parallel development stages of the two types of embryos from Gossypium hirsutum cv. YZ1. Expression pattern and functional classification analysis based on these differentially expressed genes revealed that SE development exhibited remarkable and broad stress responses. RT-PCR for selected stress-related genes with different expression levels further confirmed that the higher expression levels of stress-related genes in SEs than in ZEs. Moreover, stress treatment induced by NaCl and ABA accelerated SE development and increased the transcription of genes related to stress response, in parallel with decelerated proliferation of embryogenic calli under stress treatment. Our data reveal that SE development is the process of plant cells adapting to stress conditions, through genetic adjustment of the balance between cell proliferation and differentiation. In the small RNA profiling, A total of 36 known miRNA families were found to be differentially expressed, by comparing seedling hypocotyl and embryogenic callus (EC) of G. hirsutum YZ1, of which 19 miRNA families were represented by 29 precursors. 25 novel miRNAs were identified. 234 transcripts in EC and 322 transcripts in control (CK) were found to be the targets of 23 and 30 known miRNA families respectively, and 16 transcripts were targeted by 8 novel miRNAs. Several targets, which were enriched in auxin signaling pathway, were further validated via RNA ligase-mediated 5′ rapid amplification of cDNA ends (RLM 5′-RACE). The comprehensive transcriptome dynamics, together with biochemical and histological approaches, present a new insight into the molecular mechanisms of somatic embryogenesis and provide strategies that can be used for regulating the developmental processes of somatic embryogenesis in plants.