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.

Background: Wall-associated kinases (WAK), one of the receptor-like kinases (RLK), function directly in the connection and communication between the plant cell wall and the cytoplasm. WAK genes are highly conserved and have been identified in plants, such as rice, but there is little research on the WAK gene family in cotton. Results: In the present study, we identified 29 GhWAK genes in Gossypium hirsutum. Phylogenetic analysis showed that cotton WAK proteins can be divided into five clades. The results of synteny and Ka/Ks analysis showed that the GhWAK genes mainly originated from whole genome duplication (WGD) and were then mainly under purifying selection. Transcriptome data and real-time PCR showed that 97% of GhWAK genes highly expressed in cotton fibers and ovules. β-glucuronidase (GUS) staining assays showed that GhWAK5 and GhWAK16 expressed in Arabidopsis leaf trichomes. Fourteen GhWAK genes were found to possess putative gibberellin (GA) response elements in the promoter regions, 13 of which were significantly induced by GA treatment. Ten GhWAK genes contained auxin (IAA) response elements and the expression level of nine GhWAKs significantly increased under auxin treatment. Conclusions: We provide a preliminary analysis of the WAK gene family in G. hirsutum, which sheds light on the potantial roles of GhWAK genes in cotton fiber cell development. Our data also provides a useful resource for future studies on the functional roles of GhWAK genes.

Gossypium hirsutum L. is a worldwide economical crop; however, premature leaf senescence reduces its production and quality which is regulated by stresses, hormones, and genes. DNA binding with the one zinc finger (Dof) transcription factors (TFs) participate widely in plant development and responses to biotic and abiotic stresses, but there have been few reports of these TFs in cotton. Here, we perform a genome-wide study of G. Hirsutum L. Dof (GhDof) genes and analyze their phylogeny, duplication, and expression. In total, 114 GhDof genes have been identified and classified into nine subgroups (A, B1, B2.2, B2.1, C1, C2.1, C2.2, D1, and D2) based on phylogenetic analysis. An MCScanX analysis showed that the GhDof genes expanded due to segmental duplications. Quantitative real-time polymerase chain reaction (qRT-PCR) analysis showed that GhDofD9.6 was not only differentially expressed between CCRI10 (with premature senescence) and Liao4086 (without premature senescence) but also responded to salinity stress; GhDofA5.7, GhDofA7.4, GhDofA8.2, GhDof11.1, GhDofD7.2, and GhDofD11.3 signfificantly responded to cold (4 °C) stress. This work lays the foundation for further analysis of the function of GhDof genes in G. hirsutum, which will be helpful for improving the production and quality of cotton.

In plants, brassinosteroids (BRs) are a class of steroidal hormones that are involved in numerous physiological responses. However, the function of BRs in cold tolerance in cotton has not been explored. In this study, cotton seedlings were treated with five concentrations (0, 0.05, 0.1, 0.2, 0.5 and 1.0 mg/L) of 24-Epibrassinolide (EBR) at 4°C. We measured the electrolyte leakage, malondialdehyde (MDA) content, proline content, and net photosynthesis rate (Pn) of the seedlings, which showed that EBR treatment increased cold tolerance in cotton in a dose-dependent manner, and that 0.2 mg/L is an optimum concentration for enhancing cold tolerance. The function of EBR in cotton cotyledons was investigated in the control 0 mg/L (Cold+water) and 0.2 mg/L (Cold+EBR) treatments using RNA-Seq. A total of 4,001 differentially expressed genes (DEGs), including 2,591 up-regulated genes and 1,409 down-regulated genes were identified. Gene Ontology (GO) and biochemical pathway enrichment analyses showed that EBR is involved in the genetic information process, secondary metabolism, and also inhibits abscisic acid (ABA) and ethylene (ETH) signal transduction. In this study, physiological experiments showed that EBR can increase cold tolerance in cotton seedlings, and the comprehensive RNA-seq data shed light on the mechanisms through which EBR increases cold tolerance in cotton seedlings.