Carotenoids are natural pigments that are widely distributed in the flowers, fruits, and seeds of many plant species. These compounds not only endow diverse colors but also exhibit antioxidant, immune-modulatory, anti-aging, and photoprotective properties. Although carotenoid metabolism has been studied extensively in microbial and plant science, the genetic mechanisms underlying carotenoid metabolism in cotton remain underexplored. Here, we isolated gene GbDYA that regulates a dark-yellow anther color by map-based cloning using a BC₁F₁ population derived from a cross of Gossypium barbadense acc. Hai7124 with dark-yellow anthers and G. hirsutum acc. TM-1 with light-yellow anthers backcrossed with TM-1. GbDYA encodes phytoene synthase, a key rate-limiting enzyme in the carotenoid biosynthesis pathway. A long terminal repeat retrotransposon in the first exon of GhDYA (an ortholog of GbDYA in G. hirsutum acc. TM-1) caused loss of function and led to the light-yellow anther color. GbDYA is predominantly expressed in the early stages of anther development. Transcriptome, RT-qPCR and KEGG enrichment analyses revealed that GbDYA influences the synthesis and accumulation of carotenoids in anthers by modulating expression of key genes in the carotenoid biosynthesis pathway. Integrated transcriptomic and metabolomic analyses indicated that the accumulation of lutein, violaxanthin, antherxanthin, cryptoxanthin, zeaxanthin, and β-carotene contributed to yellow coloration of anthers. Dual-luciferase and yeast one-hybrid assays confirmed that transcription factor GbMYB105 (GB_A11G3511) binds to the promoter of GbDYA and activates its expression. High-temperature stress treatment indicated that carotenoids accumulation in anthers enhances pollen antioxidant activity. This study unravels the role of GbDYA in conferring the anther coloration, and provides the potential utilization by modulating accumulation of carotenoids in anthers to enhance pollen viability in high-temperature tolerance breeding in cotton.

Mitochondrial calcium uniporter (MCU) is a conserved calcium ion (Ca²⁺) transporter in the mitochondrial inner membrane of eukaryotic cells. How MCU proteins regulate Ca²⁺ flow and modulate plant cell development remain largely unclear. Here, we identified the gene GhMCU4 encoding a MCU protein that negatively regulates plant development and fiber elongation in cotton (Gossypium hirsutum). GhMCU4 expressed constitutively in various tissues with the higher transcripts in elongating fiber cells. Knockdown of GhMCU4 in cotton significantly elevated the plant height and root length. The calcium signaling pathway was significantly activated and calcium sensor genes, including Ca²⁺ dependent modulator of interactor of constitutively active ROP (GhCMI1), calmodulin like protein (GhCML46), calcium-dependent protein kinases (GhCPKs), calcineurin B-like protein (GhCBLs), and CBL-interacting protein kinases (GhCIPKs), were dramatically upregulated in GhMCU4-silenced plants. Metabolic processes were preferentially enriched, and genes related to regulation of transcription were upregulated in GhMCU4-silenced plants. The contents of Ca²⁺ and H₂O₂ were significantly increased in roots and leaves of GhMCU4-silenced plants. Fiber length and Ca²⁺ and H₂O₂ contents in fibers were significantly increased in GhMCU4-silenced plants. This study indicated that GhMCU4 plays a negative role in regulating cell elongation in cotton, thus expanding understanding in the role of MCU proteins in plant growth and development.

Although a few cases of genetic epistasis in plants have been reported, the combined analysis of genetically phenotypic segregation and the related molecular mechanism remains rarely studied. Here, we have identified a gene (named GaPC) controlling petal coloration in Gossypium arboreum and following a heritable recessive epistatic genetic model. Petal coloration is controlled by a single dominant gene, GaPC. A loss-of-function mutation of GaPC leads to a recessive gene Gapc that masks the phenotype of other color genes and shows recessive epistatic interactions. Map-based cloning showed that GaPC encodes an R2R3-MYB transcription factor. A 4814-bp long terminal repeat retrotransposon insertion at the second exon led to GaPC loss of function and disabled petal coloration. GaPC controlled petal coloration by regulating the anthocyanin and flavone biosynthesis pathways. Expression of core genes in the phenylpropanoid and anthocyanin pathways was higher in colored than in white petals. Petal color was conferred by flavonoids and anthocyanins, with red and yellow petals rich in anthocyanin and flavonol glycosides, respectively. This study provides new insight on molecular mechanism of recessive epistasis, also has potential breeding value by engineering GaPC to develop colored petals or fibers for multi-functional utilization of cotton.

WRKY proteins are members of a family of transcription factors in higher plants that function in plant responses to various physiological processes. We identified 120 candidate WRKY genes from Gossypium raimondii with corresponding expressed sequence tags in at least one of four cotton species, Gossypium hirsutum, Gossypium barbadense, Gossypium arboreum, and G. raimondii. These WRKY members were anchored on 13 chromosomes in G. raimondii with uneven distribution. Phylogenetic analysis showed that WRKY candidate genes can be classified into three groups, with 20 members in group Ⅰ, 88 in group Ⅱ, and 12 in group Ⅲ. The 88 genes in group Ⅱ were further classified into five subgroups, groups Ⅱa–e, containing 7, 16, 37, 15, and 13 members, respectively. We characterized diversity in amino acid residues in the WRKY domain and/or other zinc finger motif regions in the WRKY proteins. The expression patterns of WRKY genes revealed their important roles in diverse functions in cotton developmental stages of vegetative and reproductive growth and stress response. Structural and expression analyses show that WRKY proteins are a class of important regulators of growth and development and play key roles in response to stresses in cotton.