WRKY transcription factors had multiple functions in plant secondary metabolism, leaf senescence, fruit ripening, adaptation to biotic and abiotic stress, and plant growth and development. However, knowledge of the group III WRKY subfamily in fiber development in upland cotton (Gossypium hirsutum L.) is largely absent. Previous studies have shown that there were 21 putative group III WRKY members in G. hirsutum L. These putative amino acid sequences from the III WRKY group were phylogenetically clustered into three clades. Multiple alignment, conservative motif analysis, and gene structure analysis showed that the members clustered together in the phylogenetic tree had similar motifs and gene structures. Expression pattern analysis revealed that variation in the expression levels of these genes in different tissues and fiber development stages. To better understand the functions of putative group III WRKY genes in G. hirsutum L., we selected the cotton fiber initiation-related gene GhWRKY53 for cloning and functional identification. The subcellular localization experiment of GhWRKY53 in Nicotiana tabacum leaves showed that it was located in the nucleus. The heterologous expression of GhWRKY53 in Arabidopsis thaliana could significantly increase the density of trichomes. Twelve proteins that interacted with GhWRKY53 were screened from the cotton fiber cDNA library by yeast two-hybrid experiment. This study findings lay a foundation for further research on the role of the GhWRKY53 during cotton fiber development and provide a new insight for further studying putative group III WRKY genes in G. hirsutum L. Our research results also provide vital information for the genetic mechanism of high-quality cotton fiber formation and essential genetic resources for cotton fiber quality improvement.

Amino acid transporters (AATs) are a kind of membrane proteins that mediate the transport of amino acids across cell membranes in higher plants. The AAT proteins are involved in regulating plant cell growth and various developmental processes. However, the biological function of this gene family in cotton fiber development is not clear. In this study, 190, 190, 101, and 94 full-length AAT genes were identified from Gossypiumhirsutum, G. barbadense, G. arboreum, and G. raimondii. A total of 575 AAT genes from the four cotton species were divided into two subfamilies and 12 clades based on phylogenetic analysis. The AAT genes in the four cotton species were distributed on all the chromosomes. All GhAAT genes contain multiple exons, and each GhAAT protein has multiple conserved motifs. Transcriptional profiling and RT qPCR analysis showed that four GhATT genes tend to express specifically at the fiber initiation stage. Eight genes tend to express specifically at the fiber elongation and maturity stage, and four genes tend to express specifically at the fiber initiation and elongation stages. Our results provide a solid basis for further elucidating the biological function of AAT genes related to cotton fiber development and offer valuable genetic resources for crop improvement in the future.

The glycosyltransferase 4 (GT4) family plays essential roles in plant cell wall biosynthesis, glycolipid metabolism, and secondary metabolite glycosylation, yet its characterization in cotton remains scarce. Here, we performed a genome-wide identification of GT4 genes in four cotton species using comprehensive bioinformatics approaches, and systematically analyzed their conserved motifs, gene structures, chromosomal distributions, phylogeny, and selection pressures. A total of 49 GT4 members were identified, including seven genes in G. arboreum (A2), nine genes in G. raimondii (D5), 16 genes in G. hirsutum (AD1), and 17 genes in G. barbadense (AD2). Phylogenetic analysis classified them into four subfamilies. Chromosomal mapping revealed uneven distribution and various duplication events. Promoter cis-element analysis indicated enrichment of light-, phytohormon-, and stress-responsive regulatory elements. Selection pressure analysis indicated predominant purifying selection among homologous pairs. Expression profiling across multiple tissues, fiber developmental stages, and four abiotic stresses (cold, drought, salt, and heat) identified GhGT4_10 and GhGT4_2 as stress-responsive candidate genes; GhGT4_10 showed the most pronounced induction across the tested stresses, and both were drought-responsive by qRT-PCR. WGCNA under drought stress showed that the MEgreen module was most positively correlated with the 12 h post-treatment time point, and this module was significantly enriched in genes annotated to chloroplast components, photoresponse pathways, ribosomal machinery, and glyoxylate/dicarboxylate metabolism. Subcellular localization showed that GhGT4_2 localizes to chloroplasts, consistent with its predicted localization; this observation supports a possible chloroplast-associated role but does not establish its function or involvement in a specific pathway. In fuzz development analysis, the MEturquoise module was highly correlated with fuzz phenotype, and a co-expression network centered on GhGT4_15 identified 503 network-connected genes enriched in nucleocytoplasmic transport, phagosome, and spliceosome pathways. These enrichment results suggest a hypothesis that GhGT4_15 may be associated with these pathways. qRT-PCR showed that GhGT4_2 and GhGT4_10 were drought-responsive, whereas GhGT4_7 and GhGT4_15 were associated with fuzz development and represent candidate regulators, including possible negative regulation, pending functional validation. Collectively, this study characterizes the structural and expression features of the cotton GT4 family and provides candidate genes for molecular breeding targeting abiotic stress resistance and fiber trait improvement; functional validation is needed to establish causal roles.