Cotton (Gossypium) fiber length, a key trait determining fiber yield and quality, is highly regulated by a class of recently identified phytohormones, strigolactones (SLs). However, the underlying molecular mechanisms of SL signaling involved in fiber cell development are largely unknown. Here, we show that the SL signaling repressors MORE AXILLARY GROWTH2-LIKE7 (GhSMXL7) and GhSMXL8 negatively regulate cotton fiber elongation. Specifically, GhSMXL7 and GhSMXL8 inhibit the polyubiquitination and degradation of the gibberellin (GA)-triggered DELLA protein (GhSLR1). Biochemical analysis revealed that GhSMXL7 and GhSMXL8 physically interact with GhSLR1, which interferes with the association of GhSLR1 with the E3 ligase GA INSENSITIVE2 (GhGID2), leading to the repression of GA signal transduction. GhSMXL7 also interacts with the transcription factor GhHOX3, preventing its binding to the promoters of essential fiber elongation regulatory genes. Moreover, both GhSMXL7 and GhSMXL8 directly bind to the promoter regions of the AUXIN RESPONSE FACTOR (ARF) genes GhARF18-10A, GhARF18-10D, and GhARF19-7D to suppress their expression. Cotton plants in which GhARF18-10A, GhARF18-10D, and GhARF19-7D transcript levels had been reduced by virus-induced gene silencing (VIGS) displayed reduced fiber length compared with control plants. Collectively, our findings reveal a mechanism illustrating how SL integrates GA and auxin signaling to coordinately regulate plant cell elongation at the single-cell level.

Caffeic acid O-methyltransferases (COMTs) play an essential role in lignin synthesis procession, especially in the plant’s phenylalanine metabolic pathway. The content of COMT genes in cotton and the relationship between their expression patterns have not been studied clearly in cotton. In this study, we have identified 190 COMT genes in cotton, which were classified into three groups (I, II and III), and mapped on the cotton chromosomes. In addition, we found that 135 of the 190 COMT genes result from dispersed duplication (DSD) and whole-genome duplication (WGD), indicating that DSD and WGD were the main forces driving COMT gene expansion. The Ka/Ks analysis showed that GhCOMT43 and GhCOMT41 evolved from GaCOMT27 and GrCOMT14 through positive selection. The results of qRT-PCR showed that GhCOMT13, GhCOMT28, GhCOMT39 and GhCOMT55 were related to lignin content during the cotton fiber development. GhCOMT28, GhCOMT39, GhCOMT55, GhCOMT56 and GhCOMT57 responded to Verticillium Wilt (VW) and maybe related to VW resistance through lignin synthesis. Conclusively, this study found that GhCOMTs were highly expressed in the secondary wall thickening stage and VW. These results provide a clue for studying the functions of GhCOMTs in the development of cotton fiber and VW resistance and could lay a foundation for breeding cotton cultivates with higher quantity and high resistance to VW.

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.

Phytocyanins (PCs) are a class of plant-specific blue copper proteins that have been demonstrated to play a role in electron transport and plant development. Through analysis of the copper ligand residues, spectroscopic properties, and domain architecture of the protein, PCs have been grouped into four subfamilies: uclacyanins (UCs), stellacyanins (SCs), plantacyanins (PLCs), and early nodulin-like proteins (ENODLs). The present study aimed to identify and characterise the PCs present in three distinct cotton species (Gossypium hirsutum, Gossyium arboreum, and Gossypium raimondii) through the identification of 98, 63, and 69 genes respectively. We grouped PCs into four clades by using bioinformatics analysis and sequence alignment, which exhibit variations in gene structure and motif distribution. PCs are distributed across all chromosomes in each of the three species, with varying numbers of exons per gene and multiple conserved motifs, and with a minimum of 1 and maximum of 11 exons found on one gene. Transcriptomic data and qRT-PCR analysis revealed that two highly differentiated PC genes were expressed at the fibre initiation stage, while three highly differentiated PCs were expressed at the fibre elongation stage. These findings serve as a foundation for further investigations aimed at understanding the contribution of this gene family in cotton fibre production.

Plant NAC (NAM, ATAF1/2, and CUC2) family is involved in various development processes including Programmed Cell Death (PCD) associated development. However, the relationship between NAC family and PCD-associated cotton pigment gland development is largely unknown. In this study, we identified 150, 153 and 299 NAC genes in newly updated genome sequences of G. arboreum, G. raimondii and G. hirsutum, respectively. All NAC genes were divided into 8 groups by the phylogenetic analysis and most of them were conserved during cotton evolution. Using the vital regulator of gland formation GhMYC2-like as bait, expression correlation analysis screened out 6 NAC genes which were low-expressed in glandless cotton and high-expressed in glanded cotton. These 6 NAC genes acted downstream of GhMYC2-like and were induced by MeJA. Silencing CGF1(Cotton Gland Formation1), another MYC-coding gene, caused almost glandless phenotype and down-regulated expression of GhMYC2-like and the 6 NAC genes, indicating a MYC-NAC regulatory network in gland development. In addition, predicted regulatory mechanism showed that the 6 NAC genes were possibly regulated by light, various phytohormones and transcription factors as well as miRNAs. The interaction network and DNA binding sites of the 6 NAC transcription factors were also predicted. These results laid the foundation for further study of gland-related genes and gland development regulatory network.