Background: Members of the AT-HOOK MOTIF CONTAINING NUCLEAR LOCALIZED (AHL) family are involved in various plant biological processes via protein-DNA and protein-protein interaction. However, no the systematic identification and analysis of AHL gene family have been reported in cotton. Results: To investigate the potential functions of AHLs in cotton, genome-wide identification, expressions and structure analysis of the AHL gene family were performed in this study. 48, 51 and 99 AHL genes were identified from the G.raimondii, G.arboreum and G.hirsutum genome, respectively. Phylogenetic analysis revealed that the AHLs in cotton evolved into 2 clades, Clade-A with 4–5 introns and Clade-B with intronless (excluding AHL20–2). Based on the composition of the AT-hook motif(s) and PPC/DUF 296 domain, AHL proteins were classified into three types (Type-I/−II/−III), with Type-I AHLs forming Clade-B, and the other two types together diversifying in Clade-A. The detection of synteny and collinearity showed that the AHLs expanded with the specific WGD in cotton, and the sequence structure of AHL20–2 showed the tendency of increasing intron in three different Gossypium spp. The ratios of non-synonymous (Ka) and synonymous (Ks) substitution rates of orthologous gene pairs revealed that the AHL genes of G.hirsutum had undergone through various selection pressures, purifying selection mainly in A-subgenome and positive selection mainly in D-subgenome. Examination of their expression patterns showed most of AHLs of Clade-B expressed predominantly in stem, while those of Clade-A in ovules, suggesting that the AHLs within each clade shared similar expression patterns with each other. qRT-PCR analysis further confirmed that some GhAHLs higher expression in stems and ovules. Conclusion: In this study, 48, 51 and 99 AHL genes were identified from three cotton genomes respectively. AHLs in cotton were classified into two clades by phylogenetic relationship and three types based on the composition of motif and domain. The AHLs expanded with segmental duplication, not tandem duplication. The expression profiles of GhAHLs revealed abundant differences in expression levels in various tissues and at different stages of ovules development. Our study provided significant insights into the potential functions of AHLs in regulating the growth and development in cotton.

The SWEET (Sugars will eventually be exported transporters) gene family plays multiple roles in plant physiological activities and development process. It participates in reproductive development and in the process of sugar transport and absorption, plant senescence and stress responses and plant-pathogen interaction. However, thecomprehensive analysis of SWEET genes has not been reported in cotton. In this study, we identified 22, 31, 55 and 60 SWEET genes from the sequenced genomes of Gossypium arboreum, G. raimondii, G. hirsutum and G. barbadense, respectively. Phylogenetic tree analysis showed that the SWEET genes could be divided into four groups, which were further classified into 14 sub-clades. Further analysis of chromosomal location, synteny analysis and gene duplication suggested that the orthologs showed a good collinearity and segmental duplication events played a crucial role in the expansion of the family in cotton. Specific MtN3_slv domains were highly conserved between Arabidopsis and cotton by exon-intron organization and motif analysis. In addition, the expression pattern in different tissues indicated that the duplicated genes in cotton might have acquired new functions as a result of sub-functionalization or neo-functionalization. The expression pattern of SWEET genes showed that the different genes were induced by diverse stresses. The identification and functional analysis of SWEET genes in cotton may provide more candidate genes for genetic modification. SWEET genes were classified into four clades in cotton. The expression patterns suggested that the duplicated genes might have experienced a functional divergence. This work provides insights into the evolution of SWEET genes and more candidates for specific genetic modification, which will be useful in future research.

Drought-induced 19 (Di19) protein is a Cys2/His2 (C2H2) type zinc-finger protein, which plays a crucial role in plant development and in response to abiotic stress. This study systematically investigated the characteristics of the GhDi19 gene family, including the member number, gene structure, chromosomal distribution, promoter cis-elements, and expression profiles. Transcriptomic analysis indicated that some GhDi19s were up-regulated under heat and salt stress. Particularly, two nuclear localized proteins, GhDi19-3 and GhDi19-4, were identified as being in potential salt stress responsive roles. GhDi19-3 and GhDi19-4 decreased sensitivity under salt stress through virus-induced gene silencing (VIGS), and showed significantly lower levels of H₂O₂, malondialdehyde (MDA), and peroxidase (POD) as well as significantly increased superoxide dismutase (SOD) activity. This suggested that their abilities were improved to effectively reduce the reactive oxygen species (ROS) damage. Furthermore, certain calcium signaling and abscisic acid (ABA)-responsive gene expression levels showed up- and down-regulation changes in target gene-silenced plants, suggesting that GhDi19-3 and GhDi19-4 were involved in calcium signaling and ABA signaling pathways in response to salt stress. In conclusion, GhDi19-3 and GhDi19-4, two negative transcription factors, were found to be responsive to salt stress through calcium signaling and ABA signaling pathways.

Summary: Branch length is an important plant architecture trait in cotton (Gossypium) breeding. Development of cultivars with short branch has been proposed as a main object to enhance cotton yield potential, because they are suitable for high planting density. Here, we report the molecular cloning and characterization of a semi‐dominant quantitative trait locus, Short Branch Internode 1(GhSBI1), which encodes a NAC transcription factor homologous to CUP‐SHAPED COTYLEDON 2 (CUC2) and is regulated by microRNA ghr‐miR164. We demonstrate that a point mutation found in sbi1 mutants perturbs ghr‐miR164‐directed regulation of GhSBI1, resulting in an increased expression level of GhSBI1. The sbi1 mutant was sensitive to exogenous gibberellic acid (GA) treatments. Overexpression of GhSBI1 inhibited branch internode elongation and led to the decreased levels of bioactive GAs. In addition, gene knockout analysis showed that GhSBI1 is required for the maintenance of the boundaries of multiple tissues in cotton. Transcriptome analysis revealed that overexpression of GhSBI1 affects the expression of plant hormone signalling‐, axillary meristems initiation‐, and abiotic stress response‐related genes. GhSBI1 interacted with GAIs, the DELLA repressors of GA signalling. GhSBI1 represses expression of GA signalling‐ and cell elongation‐related genes by directly targeting their promoters. Our work thus provides new insights into the molecular mechanisms for branch length and paves the way for the development of elite cultivars with suitable plant architecture in cotton.