Regulation of cotton fiber elongation by xyloglucan endotransglycosylase/hydrolase genes

Ligon lintless mutant (li1li1) with super-short fibers (5-8 mm in length) and its wild type (Li1Li1) with normal fibers (30 mm in length) were used to study the function of xyloglucan endotransglycosylase/hydrolase (XTH) genes during fiber elongation in cotton. Wild-type cotton attained the fiber elongation stage earlier (5 days post-anthesis, DPA), than the Ligon lintless mutant (12 DPA) with a higher fiber elongation velocity of about 1.76 mm/day. Xyloglucan contents in Ligon lintless mutant fibers were 5-fold higher than the wild type during 9-15 DPA. It was also observed that the activity of XTH in wild-type cotton fibers was about 2-fold higher than that of the Ligon lintless mutant with a peak at 12 DPA. DNA blot analysis indicated that the XTH gene in the Ligon lintless mutant and its wild type belonged to a multiple allelic series. However, RNA blot analysis and quantitative real-time PCR exhibited an earlier expression (10 DPA) of XTH in wild type as compared to delayed (15 DPA) expression in the Ligon lintless mutant. The study also revealed that 9-15 DPA might be a key phase for upregulation of fiber elongation via increasing XTH activity. Higher XTH activity can cleave down the xyloglucan-cellulose chains thus loosening fiber cell wall and promoting fiber cell elongation in wild type as compared to its mutant.     

Molecular characterization and expression analysis of nine cotton GhEF1A genes encoding translation elongation factor 1A

The translation elongation factor 1A, eEF1A, plays an important role in protein synthesis, catalyzing the binding of aminoacyl-tRNA to the A-site of the ribosome by a GTP-dependent mechanism. To investigate the role of eEF1A for protein synthesis in cotton fiber development, nine different cDNA clones encoding eukaryotic translation elongation factor 1A were isolated from cotton (Gossypium hirsutum) fiber cDNA libraries. The isolated genes (cDNAs) were designated cotton elongation factor 1A gene GhEF1A1, GhEF1A2, GhEF1A3, GhEF1A4, GhEF1A5, GhEF1A6, GhEF1A7, GhEF1A8, GhEF1A9, respectively. They share high sequence homology at nucleotide level (71-99% identity) in the coding region and at amino acid level (96-99% identity) among each other. Phylogenetic analysis demonstrated that the nine GhEF1A genes can be divided into 5-6 subfamilies, indicating the divergence occurred in structures of the genes as well as the deduced proteins during evolution. Real-time quantitative RT-PCR analysis revealed that GhEF1A genes are differentially expressed in different tissues/organs. Of the nine GhEF1A genes, five are expressed at relatively high levels in young fibers. Further analysis indicated that expressions of the GhEF1As in fiber are highly developmental-regulated, suggesting that protein biosynthesis is very active at the early fiber elongation.     

Role of GhHDA5 in H3K9 deacetylation and fiber initiation in Gossypium hirsutum

Cotton fibers are single‐celled trichomes that initiate from the epidermal cells of the ovules at or before anthesis. Here, we identified that the histone deacetylase (HDAC ) activity is essential for proper cotton fiber initiation. We further identified 15 HDAC s homoeologs in each of the A‐ and D‐subgenomes of Gossypium hirsutum . Few of these HDAC homoeologs expressed preferentially during the early stages of fiber development [?1, 0 and 6 days post‐anthesis (DPA )]. Among them, GhHDA 5 expressed significantly at the time of fiber initiation (?1 and 0 DPA). The in vitro assay for HDAC activity indicated that GhHDA 5 primarily deacetylates H3K9 acetylation marks. Moreover, the reduced expression of GhHDA 5 also suppresses fiber initiation and lint yield in the RNA interference (RNA i) lines. The 0 DPA ovules of GhHDA 5 RNA i lines also showed alterations in reactive oxygen species homeostasis and elevated autophagic cell death in the developing fibers. The differentially expressed genes (DEG s) identified through RNA ‐seq of RNA i line (DEP 12) and their pathway analysis showed that GhHDA 5 modulates expression of many stress and development‐related genes involved in fiber development. The reduced expression of GhHDA 5 in the RNA i lines also resulted in H3K9 hyper‐acetylation on the promoter region of few DEG s assessed by chromatin immunoprecipitation assay. The positively co‐expressed genes with GhHDA 5 showed cumulative higher expression during fiber initiation, and gene ontology annotation suggests their involvement in fiber development. Furthermore, the predicted protein interaction network in the positively co‐expressed genes indicates HDA 5 modulates fiber initiation‐specific gene expression through a complex involving reported repressors.     

The cotton ACTIN1 gene is functionally expressed in fibers and participates in fiber elongation

Single-celled cotton fiber (Gossypium hirsutum) provides a unique experimental system to study cell elongation. To investigate the role of the actin cytoskeleton during fiber development, 15 G. hirsutum ACTIN (GhACT) cDNA clones were characterized. RNA gel blot and real-time RT-PCR analysis revealed that GhACT genes are differentially expressed in different tissues and can be classified into four groups. One group, represented by GhACT1, is expressed predominantly in fiber cells and was studied in detail. A 0.8-kb GhACT1 promoter sufficient to confirm its fiber-specific expression was identified. RNA interference of GhACT1 caused significant reduction of its mRNA and protein levels and disrupted the actin cytoskeleton network in fibers. No defined actin network was observed in these fibers and, consequently, fiber elongation was inhibited. Our results suggested that GhACT1 plays an important role in fiber elongation but not fiber initiation.     

Isolation and analyses of genes preferentially expressed during early cotton fiber development by subtractive PCR and cDNA array

Cotton fibers are differentiated epidermal cells originating from the outer integuments of the ovule. To identify genes involved in cotton fiber elongation, we performed subtractive PCR using cDNA prepared from 10 days post anthesis (d.p.a.) wild-type cotton fiber as tester and cDNA from a fuzzless-lintless (fl) mutant as driver. We recovered 280 independent cDNA fragments including most of the previously published cotton fiber-related genes. cDNA macroarrays showed that 172 genes were significantly up-regulated in elongating cotton fibers as confirmed by in situ hybridization in representative cases. Twenty-nine cDNAs, including a putative vacuolar (H⁺)-ATPase catalytic subunit, a kinesin-like calmodulin binding protein, several arabinogalactan proteins and key enzymes involved in long chain fatty acid biosynthesis, accumulated to greater than 50-fold in 10 d.p.a. fiber cells when compared to that in 0 d.p.a. ovules. Various upstream pathways, such as auxin signal transduction, the MAPK pathway and profilin- and expansin-induced cell wall loosening, were also activated during the fast fiber elongation period. This report constitutes the first systematic analysis of genes involved in cotton fiber development. Our results suggest that a concerted mechanism involving multiple cellular pathways is responsible for cotton fiber elongation.     

利用mRNA荧光差异显示技术规模化筛选棉纤维特异表达基因

以陆地棉（Gossypium hirsutum L.）品种TM-1开花后9d、21d、27d三个不同发育时期的棉花纤维为材料,利用mRNA荧光差异显示 (FDD) 技术,筛选到109个差异显示的cDNA片段。在此基础上,结合两轮反Northern杂交筛选和Northern杂交分析,获得了多个仅在棉花纤维细胞中特异表达或在纤维中优先表达基因的cDNA片段,序列测定和数据库搜索分析表明,这些cDNA片段中的多数还未有报道。本工作为克隆上述基因的全长cDNA,并进一步研究它们在棉纤维发育中的功能奠定了基础。