GhVTC1, the Key Gene for Ascorbate Biosynthesis in Gossypium hirsutum, Involves in Cell Elongation Under Control of Ethylene.

L-Ascorbate (Asc) plays important roles in cell growth and plant development, and its de novo biosynthesis was catalyzed by the first rate-limiting enzyme VTC1. However, the function and regulatory mechanism of VTC1 involved in cell development is obscure in Gossypium hirsutum. Herein, the Asc content and AsA/DHA ratio were accumulated and closely linked with fiber development. The GhVTC1 encoded a typical VTC1 protein with functional conserved domains and expressed preferentially during fiber fast elongation stages. Functional complementary analysis of GhVTC1 in the loss-of-function Arabidopsis vtc1-1 mutants indicated that GhVTC1 is genetically functional to rescue the defects of mutants to normal or wild type (WT). The significant shortened primary root in vtc1-1 mutants was promoted to the regular length of WT by the ectopic expression of GhVTC1 in the mutants. Additionally, GhVTC1 expression was induced by ethylene precursor 1-aminocyclopropane-1-carboxylic acid (ACC), and the GhVTC1 promoter showed high activity and included two ethylene-responsive elements (ERE). Moreover, the 5'-truncted promoters containing the ERE exhibited increased activity by ACC treatment. Our results firstly report the cotton GhVTC1 function in promoting cell elongation at the cellular level, and serve as a foundation for further understanding the regulatory mechanism of Asc-mediated cell growth via the ethylene signaling pathway.

A Cotton (Gossypium hirsutum) Myo-Inositol-1-Phosphate Synthase (GhMIPS1D) Gene Promotes Root Cell Elongation in Arabidopsis.

Myo-inositol-1-phosphate synthase (MIPS, EC 5.5.1.4) plays important roles in plant growth and development, stress responses, and cellular signal transduction. MIPS genes were found preferably expressed during fiber cell initiation and early fast elongation in upland cotton (Gossypium hirsutum), however, current understanding of the function and regulatory mechanism of MIPS genes to involve in cotton fiber cell growth is limited. Here, by genome-wide analysis, we identified four GhMIPS genes anchoring onto four chromosomes in G. hirsutum and analyzed their phylogenetic relationship, evolutionary dynamics, gene structure and motif distribution, which indicates that MIPS genes are highly conserved from prokaryotes to green plants, with further exon-intron structure analysis showing more diverse in Brassicales plants. Of the four GhMIPS members, based on the significant accumulated expression of GhMIPS1D at the early stage of fiber fast elongating development, thereby, the GhMIPS1D was selected to investigate the function of participating in plant development and cell growth, with ectopic expression in the loss-of-function Arabidopsis mips1 mutants. The results showed that GhMIPS1D is a functional gene to fully compensate the abnormal phenotypes of the deformed cotyledon, dwarfed plants, increased inflorescence branches, and reduced primary root lengths in Arabidopsis mips1 mutants. Furthermore, shortened root cells were recovered and normal root cells were significantly promoted by ectopic expression of GhMIPS1D in Arabidopsis mips1 mutant and wild-type plants respectively. These results serve as a foundation for understanding the MIPS family genes in cotton, and suggest that GhMIPS1D may function as a positive regulator for plant cell elongation.

A Gossypium hirsutum GDSL lipase/hydrolase gene (GhGLIP) appears to be involved in promoting seed growth in Arabidopsis.

GDSL lipase (GLIP) plays a pivotal role in plant cell growth as a multifunctional hydrolytic enzyme. Herein, a cotton (Gossypium hirsutum L. cv Xuzhou 142) GDSL lipase gene (GhGLIP) was obtained from developing ovules and fibers. The GhGLIP cDNA contained an open reading frame (ORF) of 1,143 base pairs (bp) and encodes a putative polypeptide of 380 amino acid residues. Sequence alignment indicated that GhGLIP includes four enzyme catalytic amino acid residue sites of Ser (S), Gly (G), Asn (N) and His (H), located in four conserved blocks. Phylogenetic tree analysis showed that GhGLIP belongs to the typical class IV lipase family with potential functions in plant secondary metabolism. Subcellular distribution analysis demonstrated that GhGLIP localized to the nucleus, cytoplasm and plasma membrane. GhGLIP was expressed predominantly at 5-15 day post anthesis (dpa) in developing ovules and elongating fibers, measured as mRNA levels and enzyme activity. Ectopic overexpression of GhGLIP in Arabidopsis plants resulted in enhanced seed development, including length and fresh weight. Meanwhile, there was increased soluble sugar and protein storage in transgenic Arabidopsis plants, coupled with the promotion of lipase activity. Moreover, the expression of cotton GhGLIP is induced by ethylene (ETH) treatment in vitro. A 1,954-bp GhGLIP promoter was isolated and expressed high activity in driving green fluorescence protein (GFP) expression in tobacco leaves. Cis-acting element analysis of the GhGLIP promoter (pGhGLIP) indicated the presence of an ethylene-responsive element (ERE), and transgenic tobacco leaves with ectopic expression of pGhGLIP::GFP-GUS showed increased GUS activity after ETH treatment. In summary, these results suggest that GhGLIP is a functional enzyme involved in ovule and fiber development and performs significant roles in seed development.