Targeted Lipidomics Studies Reveal that Linolenic Acid Promotes Cotton Fiber Elongation by Activating Phosphatidylinositol and Phosphatidylinositol Monophosphate Biosynthesis.

The membrane lipids from fast-elongating wild-type cotton (Gossypium hirsutum) fibers at 10 days post-anthesis, wild-type ovules with fiber cells removed, and ovules from the fuzzless-lintless mutant harvested at the same age, were extracted, separated, and quantified. Fiber cells contained significantly higher amounts of phosphatidylinositol (PI) than both ovule samples with PI 34:3 being the most predominant species. The genes encoding fatty acid desaturases (Δ(15)GhFAD), PI synthase (PIS) and PI kinase (PIK) were expressed in a fiber-preferential manner. Further analysis of phosphatidylinositol monophosphate (PIP) indicated that elongating fibers contained four- to five-fold higher amounts of PIP 34:3 than the ovules. Exogenously applied linolenic acid (C18:3), soybean L-α-PI, and PIPs containing PIP 34:3 promoted significant fiber growth, whereas a liver PI lacking the C18:3 moiety, linoleic acid, and PIP 36:2 were completely ineffective. The growth inhibitory effects of carbenoxolone, 5-hydroxytryptamine, and wortmannin were reverted by C18:3, PI, or PIP, respectively, suggesting that PIP signaling is essential for fiber cell growth. Furthermore, cotton plants expressing virus-induced gene-silencing constructs that specifically suppressed GhΔ(15)FAD, GhPIS, or GhPIK expression, resulted in significantly short-fibered phenotypes. Our data provide the basis for in-depth studies on the roles of PI and PIP in mediating cotton fiber growth.

Quantitative proteomics and transcriptomics reveal key metabolic processes associated with cotton fiber initiation.

An iTRAQ-based proteomics of ovules from the upland cotton species Gossypium hirsutum and its fuzzless-lintless mutant was performed, and finally 2729 proteins that preferentially accumulated at anthesis in wild-type ovules were identified. We confirmed that the gene expression levels of 2005 among these proteins also increased by performing an RNA sequencing transcriptomics. Expression of proteins involved in carboxylic acid metabolism, small-molecule metabolic processes, hormone regulation, and lipid metabolism was significantly enhanced in wild-type ovules. Quantitative real-time PCR verified the increased expression of 26 genes involved in these processes. Cotton 3-hydroxyacyl-CoA dehydratase (GhPAS2) catalyzing the third reaction of very long-chain fatty acid (VLCFA) biosynthesis, accumulated at anthesis in wild-type ovules. Heterogeneous expression of GhPAS2 restored viability to the Saccharomyces cerevisiae haploid psh1-deletion strain deficient in PAS2 activity. Application of VLCFA biosynthesis inhibitor acetochlor (2-chloro-N-[ethoxymethyl]-N-[2-ethyl-6-methyl-phenyl]-acetamide; ACE) and gibberellic acid to the unfertilized cotton ovules significantly suppressed fiber cell protrusion. In this study, the profiling of gene expression at both transcriptome and proteome levels provides new insights into cotton fiber cell initiation. BIOLOGICAL SIGNIFICANCE: Cotton fiber initiation determines the ultimate number of fibers per ovule, thereby determining fiber yield. In total, 2729 proteins were preferentially accumulated in wild-type ovules at anthesis. The most up-regulated proteins were assigned to carboxylic acid metabolism, small-molecule metabolic processes, hormone regulation, and lipid metabolism. In consistence with these findings, we characterized GhPAS2 gene coding for the enzyme that catalyzes VLCFA production. VLCFA biosynthesis inhibitor, acetochlor, was shown to significantly suppress fiber initiation. This study provides a genome-scale transcriptomic and proteomic characterization of fiber initial cells, laying a solid basis for further investigation of the molecular processes governing fiber cell development.