Cyclophilins (CYPs) are a member of the immunophilin superfamily (in addition to FKBPs and parvulins) and play a significant role in peptidyl-prolyl cis-trans isomerase (PPIase) activity. Previous studies have shown that CYPs have important functions in plants, but no genome-wide analysis of the cotton CYP gene family has been reported, and the specific biological function of this gene is still elusive. Based on the release of the cotton genome sequence, we identified 75, 78, 40 and 38 CYP gene sequences from G. barbadense, G. hirsutum, G. arboreum, and G. raimondii, respectively; 221 CYP genes were unequally located on chromosomes. Phylogenetic analysis showed that 231 CYP genes clustered into three major groups and eight subgroups. Collinearity analysis showed that segmental duplications played a significant role in the expansion of CYP members in cotton. There were light-responsiveness, abiotic-stress and hormone-response elements upstream of most of the CYPs. In addition, the motif composition analysis revealed that 49 cyclophilin proteins had extra domains, including TPR (tetratricopeptide repeat), coiled coil, U-box, RRM (RNA recognition motif), WD40 (RNA recognition motif) and zinc finger domains, along with the cyclophilin-like domain (CLD). The expression patterns based on qRT-PCR showed that six CYP expression levels showed greater differences between Xinhai21 (long fibres, G. barbadense) and Ashmon (short fibres, G. barbadense) at 10 and 20 days postanthesis (DPA). These results signified that CYP genes are involved in the elongation stage of cotton fibre development. This study provides a valuable resource for further investigations of CYP gene functions and molecular mechanisms in cotton.

Chalcone isomerase (CHI) is a key component of phenylalanine metabolism that can produce a variety of flavonoids. However, little information and no systematic analysis of CHI genes is available for cotton. Here, we identified 33 CHI genes in the complete genome sequences of four cotton species (Gossypium arboretum L., Gossypium raimondii L., Gossypium hirsutum L., and Gossypium barbadense L.). Cotton CHI proteins were classified into two main groups, and whole-genome/segmental and dispersed duplication events were important in CHI gene family expansion. qRT-PCR and semiquantitative RT-PCR results suggest that CHI genes exhibit temporal and spatial variation and respond to infection with Fusarium wilt race 7. A preliminary model of CHI gene involvement in cotton evolution was established. Pairwise comparison revealed that seven CHI genes showed higher expression in cultivar 06-146 than in cultivar Xinhai 14. Overall, this whole-genome identification unlocks a new approach to the comprehensive functional analysis of the CHI gene family, which may be involved in adaptation to plant pathogen stress.

Background: Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is the central enzyme of glycolysis and plays important regulatory roles in plant growth and development and responses to adverse stress conditions. However, studies on the characteristics and functions of cotton GAPDH family genes are still lacking. Methods: In this study, genome-wide identification of the cotton GAPDH gene family was performed, and the phylogeny, gene structures, promoter progenitors and expression profiles of upland cotton GAPDH gene family members were explored by bioinformatics analysis to highlight potential functions. The functions of GhGAPDH9 in response to drought stress were initially validated based on RNA-seq, qRT‒PCR, VIGS techniques and overexpression laying a foundation for further studies on the functions of GAPDH genes. Results: This study is the first systematic analysis of the cotton GAPDH gene family, which contains a total of 84 GAPDH genes, among which upland cotton contains 27 members. Quantitative, phylogenetic and covariance analyses of the genes revealed that the GAPDH gene family has been conserved during the evolution of cotton. Promoter analysis revealed that most cis-acting elements were related to MeJA and ABA. Based on the identified promoter cis-acting elements and RNA-seq data, it was hypothesized that Gh_GAPDH9, Gh_GAPDH11, Gh_GAPDH19 and Gh_GAPDH21 are involved in the response of cotton to abiotic stress. The expression levels of the Gh_GAPDH9 gene in two drought-resistant and two drought-sensitive materials were analyzed by qRT‒PCR and found to be high early in the treatment period in the drought-resistant material. The silencing of Gh_GAPDH9 based on virus-induced gene silencing (VIGS) technology resulted in significant leaf wilting or whole-plant dieback in silenced plants after drought stress compared to the control. The content of—malondialdehyde (MDA) in cotton leaves was significantly increased, and the content of proline (Pro) and chlorophyll (Chl) was reduced. In addition, the leaf wilting and dryness of transgenic lines under drought stress were lower than those of wild-type Arabidopsis, indicating that Gh_GAPDH9 is a positive regulator of drought resistance. In conclusion, our results demonstrate that GAPDH genes play an important role in the response of cotton to abiotic stresses and provide preliminary validation of the function of the Gh_GAPDH9 gene under drought stress. These findings provide an important theoretical basis for further studies on the function of the Gh_GAPDH9 gene and the molecular mechanism of the drought response in cotton.

Background/Objectives: The terrestrialization of plants has been a significant driver of plant evolution over hundreds of millions of years, and epidermal development plays a crucial role in adapting to terrestrial environments. It is therefore of great interest that the HD-Zip IV subfamily serves as a key regulator of epidermal and cuticle development in plants. However, research on their expansion trajectory, adaptive evolution, and functional divergence remains scarce. Methods: We conducted a functional divergence and adaptive evolution analysis in the plant HD-Zip IV subfamily and confirmed the functional differentiation caused by positive selection site mutations through binding affinity prediction analysis and EMSA experiments. Results: Our findings revealed that the HD-Zip IV subfamily has diversified into five distinct branches, with progressive expansion throughout plant evolution. These variations in the HD (homeodomain) and START drive the functional differentiation among the evolutionary branch, in particular, a distinct leucine-rich motif in HD and lipid-binding pockets. Furthermore, we identified several amino acid sites within the START domain that have been under selective pressure during plant evolution, as well as convergent evolutionary sites shared between early land plants and seed plants. Conclusions: Our findings show that the HD-Zip IV subfamily experienced a significant expansion in gene number and diversification of evolutionary branch from streptophyte algae to seed plants. During plant evolution, genomic duplication events and variations in the HD and START have contributed to its expansion in gene number and diversification, respectively. We suggest that the balanced coexistence of functional robustness and relaxed constraints in the HD-Zip IV subfamily may have underpinned its successful response to the challenges of land environment.

Gossypium barbadense possesses a superior fiber quality because of its fiber length and strength. An in-depth analysis of the underlying genetic mechanism could aid in filling the gap in research regarding fiber strength and could provide helpful information for Gossypium barbadense breeding. Three quantitative trait loci related to fiber strength were identified from a Gossypium barbadense recombinant inbred line (PimaS-7 × 5917) for further analysis. RNA sequencing was performed in the fiber tissues of PimaS-7 × 5917 0–35 days postanthesis. Four specific modules closely related to the secondary wall-thickening stage were obtained using the weighted gene coexpression network analysis. In total, 55 genes were identified as differentially expressed from 4 specific modules. Gene Ontology and the Kyoto Encyclopedia of Genes and Genomes were used for enrichment analysis, and Gbar_D11G032910, Gbar_D08G020540, Gbar_D08G013370, Gbar_D11G033670, and Gbar_D11G029020 were found to regulate fiber strength by playing a role in the composition of structural constituents of cytoskeleton and microtubules during fiber development. Quantitative real-time PCR results confirmed the accuracy of the transcriptome data. This study provides a quick strategy for exploring candidate genes and provides new insights for improving fiber strength in cotton.