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.

Cotton is one of the major industrial crops that supply natural fibers and oil for industries. This study was conducted to understand the mechanism of delayed gland morphogenesis in seeds of Gossypium bickii. In this study, we compared glandless seeds of G. bickii with glanded seeds of Gossypium arboreum. High-throughput sequencing technology was used to explore and classify the expression patterns of gland-related genes in seeds and seedlings of cotton plants. Approximately 131.33 Gigabases of raw data from 12 RNA sequencing samples with three biological replicates were generated. A total of 7196 differentially-expressed genes (DEGs) were identified in all transcriptome data. Among them, 3396 genes were found up-regulated and 3480 genes were down-regulated. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) annotations were performed to identify different functions between genes unique to glandless imbibed seeds and glanded seedlings. Co-expression network analysis revealed four modules that were identified as highly associated with the development of glandless seeds. Here the hub genes in each module were identified by weighted gene co-expression network analysis (WGCNA). In total, we have selected 13 genes involved in transcription factors, protein and MYB-related functions, that were differentially expressed in transcriptomic data and validated by quantitative reverse-transcription polymerase chain reaction (qRT-PCR). These selected genes may play an important role for delayed gland morphogenesis. Our study provides comprehensive insight into the key genes related to glandless traits of seeds and plants, and can be further exploited by functional and molecular studies.

Background: Cotton, a key global economic crop, suffers yield and quality losses due to salt stress. This study aims to analyze the cotton STARD gene family and its role in salt stress responses. Methods: We conducted a genome-wide analysis of the STARD gene family in four cotton species, using phylogenetic trees, chromosomal mapping, and collinearity analyses to explore their evolutionary relationships and expansion mechanisms. We also examined gene structures, conserved motifs, and promoter cis-elements. Results: STARD genes are evenly distributed across the four cotton species. Segmental duplication was found to be the main driver of gene expansion, with most pairs undergoing purifying selection. Distinct structural features and potential roles in plant growth and stress responses were identified. Notably, 11 GhSTARD genes showed significant expression changes under salt stress, especially GhSTARD45 in root tissues. Conclusions: This study provides new insights into the function and salt stress response mechanisms of the cotton STARD gene family, suggesting GhSTARD45 plays a key role in root-mediated salt tolerance and highlighting the potential of STARD genes in enhancing cotton’s salt tolerance.

The red plant phenotype of R1 cotton is a genetic marker produced by light-induced anthocyanin accumulation. GhPAP1D controls this trait. There are two 228 bp tandem repeats upstream of GhPAP1D in R1 cotton. In this study, GUS staining assays in transgenic Arabidopsis thaliana (L.) Heynh. demonstrated that tandem repeats in the GhPAP1D promoter-enhanced transcriptional activity. GhPAP1D is a homolog of A. thaliana AtPAP1. AtPAP1’s expression is regulated by photomorphogenesis-related transcription factors such as AtHY5 and AtBBXs. We identified the homologs of A. thaliana AtHY5, AtBBX21, and AtBBX24 in R1 cotton, designated as GhHY5, GhBBX21, and GhBBX24, respectively. Y1H assays confirmed that GhHY5, GhBBX21, and GhBBX24 each bound to the GhPAP1D promoter. Dual-luciferase reporter assays revealed that GhHY5 weakly activated the promoter activity of GhPAP1D. Heterologous expression assays in A. thaliana indicated that GhBBX21 promoted anthocyanin accumulation, whereas GhBBX24 had the opposite effect. Dual-luciferase assays showed GhBBX21 activated GhPAP1D transcription, while GhBBX24 repressed it. Further study indicated that GhHY5 did not enhance GhBBX21-mediated transcriptional activation of GhPAP1D but alleviates GhBBX24-induced repression. Together, our results demonstrate that GhBBX21 and GhBBX24 antagonistically regulate anthocyanin accumulation in R1 cotton under GhHY5 mediation, providing insights into light-responsive anthocyanin biosynthesis in cotton.

Upland cotton ( Gossypium hirsutum L.) is one of the most economically important crops worldwide due to the significant source of natural fiber, feed, oil and biofuel products. Cottonseed can also serve as an excellent source of edible protein and oil. However,the presence of gossypol in pigment gland has limited it utilization In the past few decades, some progress has been made in the understanding molecular mechanism of the formation of the pigment gland. However, little is known about the specific mechanism of pigment gland formation in cotton. In this study, the cDNA sequence of a ethylene transcription factor gene, designated GhERF105a, was cloned from upland cotton CCRI12. Sequence alignment revealed that GhERF105a gene contained a typical AP2/ERF domain of 61 amino acids, and belonged to the ERF subgroup of the ERF supfamily. It was highly expressed in the leaves and stems of glanded plants but had substantially lower expression of the glandless plants. GhERF105a, localized to the nucleus, could bind to GCC-box and DRE. Some development, phytohormone and stress related cis-elements were enriched in the promoters of GhERF105a/d . Split ubiquitin assays in yeast and BiFC experiments showed extensive interactions between GhERF105a and Gh_A07G1044. In addition, GhERF105a was highly similar with GhERF105d in the gene length, molecular weight, protein molecule, gene structure and expression pattern. The overall results suggested that GhERF105a might participate in the pigment gland formation and stree-response processes.