Recent studies have shown that lipid metabolism is a key factor affecting anther development and male fertility. However, how plants regulating the metabolic balance of multiple lipids to ensure proper anther development and male fertility remains unclear. Analyzing lipid molecules related to anther fertility and genes responsible for their biosynthesis is crucial for understanding the physiological significance of lipid metabolism in crop fertility. In this study, we compared the transcriptome and the composition and content of lipids in anthers of two upland cotton (Gossypium hirsutum) materials, Shida 98 (WT) and its nearly-isogenic male sterile line Shida 98A (MS). Transcriptomics analysis identified many differentially expressed genes (DEGs) between the two materials, with the genes of the alpha-linolenic acid metabolism pathway being the most significantly associated with the male sterility phenotype. Investigations on lipids revealed that the MS anthers over-accumulated free fatty acids (FFAs), phosphatidic acid (PA), mono- and di-galactosyldiacylglycerol (MGDG and DGDG), and had a decreased content of triacylglycerol (TAG), which was closely related to the abnormal metabolism of alpha-linolenic acid (C18:3); therefore, the major lipids containing C18:3-acyl chains, such as PA, MGDG, DGDG, and TAG, are proposed to play a major role in cotton anther development. We also showed that an excessive level of MGDG and DGDG caused jasmonic acid (JA) overaccumulation in MS anthers, which in turn inhibited the expression of GhFAD3 and consequently reduced the C18:3 content, presumably via a feedback regulation mechanism, ultimately affecting plant fertility. Together, our results revealed the importance of a balanced lipid metabolism in regulating the development of cotton anther and pollen and consequently male fertility.

Fiber length is one of the most important quality parameters of cotton fibers. Transcriptomic analyses of developing cotton fibers have identified genes preferentially expressing in fiber elongation stage, but few have been functionally characterized. Here, on the basis of confirmation of the preferential expression profile of GhAlaRP (Gh_A09G1166 and Gh_D09G1172), an alanine rich protein gene, in the rapid elongating fibers, we investigated the role of GhAlaRP in fiber development by generating transgenic cottons with an increased or decreased expression level of GhAlaRP. Our results showed that the fiber length was consistently significantly shorter in both the GhAlaRP-RNAi lines and the alarp mutant generated by genome editing than in the control YZ-1. GhAlaRP was localized on plasma membrane, nucleus and endoplasmic reticulum. The yeast two-hybrid assay and bimolecular fluorescence complementation assay showed that GhAlaRP co-expresses and interacts with GhAnnexin (Gh_D11G2184) and GhEXPA (Gh_A10G2323) that are involved in fiber elongation. Down-regulation of GhAlaRP co-suppressed the expression levels of GhAnnexin and GhEXPA. These results suggest a role of GhAlaRP in regulation of cotton fiber elongation, which could be achieved by regulating the functions of GhAnnexin and GhEXPA.

WRKY transcription factors (TFs) play important roles in the regulation of biotic and abiotic stresses. However, the functions of most WRKY TFs in upland cotton (Gossypium hirsutum) are still unknown. In this study, we functionally identified a group Ⅲ WRKY transcription factor, GhWRKY70, in upland cotton. Reverse transcription-quantitative PCR analysis showed that GhWRKY70 expression was induced by Verticillium dahliae, salicylic acid (SA) and methyl jasmonate. Virus-induced gene silencing of GhWRKY70 increased the resistance of cotton to V. dahliae. Specifically, jasmonic acid (JA) response-associated genes were upregulated and SA-related genes were downregulated in GhWRKY70-silenced cotton plants. Overexpression of GhWRKY70 reduced tolerance to V. dahliae in Arabidopsis thaliana. Transgenic Arabidopsis plants showed increased expression of SA-associated genes and reduced expression of JA response-associated genes. These results suggest that GhWRKY70 negatively regulates tolerance to V. dahliae in at least two ways: (ⅰ) by upregulating the expression of SA-associated genes and (ⅱ) by reducing the expression of JA-associated genes.