Glyceraldehyde-3-phosphate dehydrogenase Gh_GAPDH9 is associated with drought resistance in Gossypium hirsutum.

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.

Weighted Gene Co-Expression Network Analysis Reveals Hub Genes for Fuzz Development in Gossypium hirsutum.

Fuzzless Gossypium hirsutum mutants are ideal materials for investigating cotton fiber initiation and development. In this study, we used the fuzzless G. hirsutum mutant Xinluzao 50 FLM as the research material and combined it with other fuzzless materials for verification by RNA sequencing to explore the gene expression patterns and differences between genes in upland cotton during the fuzz period. A gene ontology (GO) enrichment analysis showed that differentially expressed genes (DEGs) were mainly enriched in the metabolic process, microtubule binding, and other pathways. A weighted gene co-expression network analysis (WGCNA) showed that two modules of Xinluzao 50 and Xinluzao 50 FLM and four modules of CSS386 and Sicala V-2 were highly correlated with fuzz. We selected the hub gene with the highest KME value among the six modules and constructed an interaction network. In addition, we selected some genes with high KME values from the six modules that were highly associated with fuzz in the four materials and found 19 common differential genes produced by the four materials. These 19 genes are likely involved in the formation of fuzz in upland cotton. Several hub genes belong to the arabinogalactan protein and GDSL lipase, which play important roles in fiber development. According to the differences in expression level, 4 genes were selected from the 19 genes and tested for their expression level in some fuzzless materials. The modules, hub genes, and common genes identified in this study can provide new insights into the formation of fiber and fuzz, and provide a reference for molecular design breeding for the genetic improvement of cotton fiber.

Identification and functional analysis of PIN family genes in Gossypium barbadense.

Background: PIN proteins are an important class of auxin polar transport proteins that play an important regulatory role in plant growth and development. However, their characteristics and functions have not been identified in Gossypium barbadense. Methods: PIN family genes were identified in the cotton species G. barbadense, Gossypium hirsutum, Gossypium raimondii, and Gossypium arboreum, and detailed bioinformatics analyses were conducted to explore the roles of these genes in G. barbadense using transcriptome data and quantitative reverse-transcription polymerase chain reaction (qRT-PCR) technology. Functional verification of the genes was performed using virus-induced gene silencing (VIGS) technology. Results: A total of 138 PIN family genes were identified in the four cotton species; the genes were divided into seven subgroups. GbPIN gene family members were widely distributed on 20 different chromosomes, and most had repeated duplication events. Transcriptome analysis showed that some genes had differential expression patterns in different stages of fiber development. According to 'PimaS-7' and '5917' transcript component association analysis, the transcription of five genes was directly related to endogenous auxin content in cotton fibers. qRT-PCR analysis showed that the GbPIN7 gene was routinely expressed during fiber development, and there were significant differences among materials. Transient silencing of the GbPIN7 gene by VIGS led to significantly higher cotton plant growth rates and significantly lower endogenous auxin content in leaves and stems. This study provides comprehensive analyses of the roles of PIN family genes in G. barbadense and their expression during cotton fiber development. Our results will form a basis for further PIN auxin transporter research.

Analysis of transcriptome data and quantitative trait loci enables the identification of candidate genes responsible for fiber strength in Gossypium barbadense.

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.

Genome-wide identification of the DUF668 gene family in cotton and expression profiling analysis of GhDUF668 in Gossypium hirsutum under adverse stress.

BACKGROUND: Domain of unknown function 668 (DUF668) may play a crucial role in the plant growth and developmental response to adverse stress. However, our knowledge of the function of the DUF668 gene family is limited. RESULTS: Our study was conducted based on the DUF668 gene family identified from cotton genome sequencing. Phylogenetic analysis showed that the DUF668 family genes can be classified into four subgroups in cotton. We identified 32 DUF668 genes, which are distributed on 17 chromosomes and most of them located in the nucleus of Gossypium hirsutum. Gene structure and motif analyses revealed that the members of the DUF668 gene family can be clustered in G. hirsutum into two broad groups, which are relatively evolutionarily conserved. Transcriptome data analysis showed that the GhDUF668 genes are differentially expressed in different tissues under various stresses (cold, heat, drought, salt, and Verticillium dahliae), and expression is generally increased in roots and stems. Promoter and expression analyses indicated that Gh_DUF668-05, Gh_DUF668-08, Gh_DUF668-11, Gh_DUF668-23 and Gh_DUF668-28 in G. hirsutum might have evolved resistance to adverse stress. Additionally, qRT-PCR revealed that these 5 genes in four cotton lines, KK1543 (drought resistant), Xinluzao 26 (drought sensitive), Zhongzhimian 2 (disease resistant) and Simian 3 (susceptible), under drought and Verticillium wilt stress were all significantly induced. Roots had the highest expression of these 5 genes before and after the treatment. Among them, the expression levels of Gh_DUF668-08 and Gh_DUF668-23 increased sharply at 6 h and reached a maximum at 12 h under biotic and abiotic stress, which showed that they might be involved in the process of adverse stress resistance in cotton. CONCLUSION: The significant changes in GhDUF668 expression in the roots after adverse stress indicate that GhDUF668 is likely to increase plant resistance to stress. This study provides an important theoretical basis for further research on the function of the DUF668 gene family and the molecular mechanism of adverse stress resistance in cotton.