Summary: The transition from vegetative to reproductive growth is very important for early maturity in cotton. However, the genetic control of this highly dynamic and complex developmental process remains unclear. A high‐resolution tissue‐ and stage‐specific transcriptome profile was generated from six developmental stages using 72 samples of two early‐maturing and two late‐maturing cotton varieties. The results of histological analysis of paraffin sections showed that flower bud differentiation occurred at the third true leaf stage (3TLS) in early‐maturing varieties, but at the fifth true leaf stage (5TLS) in late‐maturing varieties. Using pairwise comparison and weighted gene co‐expression network analysis, 5312 differentially expressed genes were obtained, which were divided into 10 gene co‐expression modules. In the MElightcyan module, 46 candidate genes regulating cotton flower bud differentiation were identified and expressed at the flower bud differentiation stage. A novel key regulatory gene related to flower bud differentiation, GhCAL, was identified in the MElightcyan module. Anti‐GhCAL transgenic cotton plants exhibited late flower bud differentiation and flowering time. GhCAL formed heterodimers with GhAP1‐A04/GhAGL6‐D09 and regulated the expression of GhAP1‐A04 and GhAGL6‐D09. GhAP1‐A04‐ and GhAGL6‐D09‐silenced plants also showed significant late flowering. Finally, we propose a new flowering regulatory pathway mediated by GhCAL. This study elucidated the molecular mechanism of cotton flowering regulation and provides good genetic resources for cotton early‐maturing breeding.

Background: Cotton fiber length and strength are both key traits of fiber quality, and fiber strength (FS) is tightly correlated with secondary cell wall (SCW) biosynthesis. The three-amino-acid-loop-extension (TALE) superclass homeoproteins are involved in regulating diverse biological processes in plants, and some TALE members has been identified to play a key role in regulating SCW formation. However, little is known about the functions of TALE members in cotton (Gossypium spp.). Results: In the present study, based on gene homology, 46, 47, 88 and 94 TALE superfamily genes were identified in G. arboreum, G. raimondii, G. barbadense and G. hirsutum, respectively. Phylogenetic and evolutionary analysis showed the evolutionary conservation of two cotton TALE families (including BEL1-like and KNOX families). Gene structure analysis also indicated the conservation of GhTALE members under selection. The analysis of promoter cis-elements and expression patterns suggested potential transcriptional regulation functions in fiber SCW biosynthesis and responses to some phytohormones for GhTALE proteins. Genome-wide analysis of colocalization of TALE transcription factors with SCW-related QTLs revealed that some BEL1-like genes and KNAT7 homologs may participate in the regulation of cotton fiber strength formation. Overexpression of GhKNAT7-A03 and GhBLH6-A13 significantly inhibited the synthesis of lignocellulose in interfascicular fibers of Arabidopsis. Yeast two-hybrid (Y2H) experiments showed extensive heteromeric interactions between GhKNAT7 homologs and some GhBEL1-like proteins. Yeast one-hybrid (Y1H) experiments identified the upstream GhMYB46 binding sites in the promoter region of GhTALE members and defined the downstream genes that can be directly bound and regulated by GhTALE heterodimers. Conclusion: We comprehensively identified TALE superfamily genes in cotton. Some GhTALE members are predominantly expressed during the cotton fiber SCW thicking stage, and may genetically correlated with the formation of FS. Class II KNOX member GhKNAT7 can interact with some GhBEL1-like members to form the heterodimers to regulate the downstream targets, and this regulatory relationship is partially conserved with Arabidopsis. In summary, this study provides important clues for further elucidating the functions of TALE genes in regulating cotton growth and development, especially in the fiber SCW biosynthesis network, and it also contributes genetic resources to the improvement of cotton fiber quality.

Premature senescence can reduce the yield and quality of crops. WRKY transcription factors (TFs) play important roles during leaf senescence, but little is known about their ageing mechanisms in cotton. In this study, a group III WRKY TF, GhWRKY27, was isolated and characterized. The expression of GhWRKY27 was induced by leaf senescence and was higher in an early-ageing cotton variety than in a non-early-ageing cotton variety. Overexpression of GhWRKY27 in Arabidopsis promoted leaf senescence, as determined by reduced chlorophyll content and elevated expression of senescence-associated genes (SAGs). Yeast two-hybrid (Y2H) and bimolecular fluorescence complementation (BiFC) assays showed that GhWRKY27 interacted with an MYB TF, GhTT2. Putative target genes of GhWRKY27 were identified via chromatin immunoprecipitation followed by sequencing (ChIP-seq). Yeast one-hybrid (Y1H) assay and electrophoretic mobility shift assay (EMSA) revealed that GhWRKY27 binds directly to the promoters of cytochrome P450 94C1 (GhCYP94C1) and ripening-related protein 2 (GhRipen2–2). In addition, the expression patterns of GhTT2, GhCYP94C1 and GhRipen2–2 were identified during leaf senescence. Transient dual-luciferase reporter assay indicated that GhWRKY27 could activate the expression of GhCYP94C1 and GhRipen2–2. Our work lays the foundation for further study of the functional roles of WRKY genes during leaf senescence in cotton. In addition, our data provide new insights into the senescence-associated mechanisms of WRKY genes in cotton.

Gossypium hirsutum L., or upland cotton, is an important renewable resource for textile fiber. To enhance understanding of the genetic basis of cotton earliness, we constructed an intra-specific recombinant inbred line population (RIL) containing 137 lines, and performed linkage map construction and quantitative trait locus (QTL) mapping. Using restriction-site associated DNA sequencing, a genetic map composed of 6,434 loci, including 6,295 single nucleotide polymorphisms and 139 simple sequence repeat loci, was developed from RIL population. This map spanned 4,071.98 cM, with an average distance of 0.63 cM between adjacent markers. A total of 247 QTLs for six earliness-related traits were detected in 6 consecutive years. In addition, 55 QTL coincidence regions representing more than 60 % of total QTLs were found on 22 chromosomes, which indicated that several earliness-related traits might be simultaneously improved. Fine-mapping of a 2-Mb region on chromosome D3 associated with five stable QTLs between Marker25958 and Marker25963 revealed that lines containing alleles derived from CCRI36 in this region exhibited smaller phenotypes and earlier maturity. One candidate gene (EMF2) was predicted and validated by quantitative real-time PCR in early-, medium- and late-maturing cultivars from 3- to 6-leaf stages, with highest expression level in early-maturing cultivar, CCRI74, lowest expression level in late-maturing cultivar, Bomian1. We developed an SNP-based genetic map, and this map is the first high-density genetic map for short-season cotton and has the potential to provide deeper insights into earliness. Cotton earliness-related QTLs and QTL coincidence regions will provide useful materials for QTL fine mapping, gene positional cloning and MAS. And the gene, EMF2, is promising for further study.

WRKY transcription factors (TFs) play essential roles in the plant response to leaf senescence and abiotic stress. However, the WRKY TFs involved in leaf senescence and stress tolerance in cotton (Gossypium hirsutum L.) are still largely unknown. In this study, a WRKY gene, GhWRKY91, was isolated and thoroughly characterized. Transcriptional activity assays showed that GhWRKY91 could activate transcription in yeast. The expression pattern of GhWRKY91 during leaf senescence, and in response to abscisic acid (ABA) and drought stress was evaluated. β-Glucuronidase (GUS) activity driven by the GhWRKY91 promoter in transgenic Arabidopsis was reduced upon exposure to ABA and drought treatments. Constitutive expression of GhWRKY91 in Arabidopsis delayed natural leaf senescence. GhWRKY91 transgenic plants exhibited increased drought tolerance and presented delayed drought-induced leaf senescence, as accompanied by reinforced expression of stress-related genes and attenuated expression of senescence-associated genes (SAGs). Yeast one-hybrid (Y1H) assays and electrophoretic mobility shift assays (EMSAs) revealed that GhWRKY91 directly targets GhWRKY17, a gene associated with ABA signals and reactive oxygen species (ROS) production. A transient dual-luciferase reporter assay demonstrated that GhWRKY91 activated the expression of GhWRKY17. Our results suggest that GhWRKY91 might negatively regulate natural and stress-induced leaf senescence and provide a foundation for further functional studies on leaf senescence and the stress response in cotton.