Epigenetic engineering is rapidly emerging as a transformative frontier for unlocking the untapped yield potential of cottonseed oil. This review highlights the role of epigenetic mechanisms, including DNA methylation, histone modifications, and small RNAs, in regulating pathways for oil accumulation in cotton. We discuss epigenomic findings that reveal tissue and developmental stage-specific patterns affecting oil biosynthesis, including distinct regulatory signatures in the embryo versus mitochondrial/nuclear compartments. The application of advanced tools, including CRISPR-based systems and RNA-directed DNA methylation, for precise epigenetic engineering is examined, alongside challenges such as off-target effects and regulatory considerations. Future directions should extend beyond promoter editing to incorporate the regulation of distal enhancer elements. Critical steps include generating high-resolution chromatin interaction maps from developing embryos/endosperms to identify candidate enhancers for key oil genes, which can then be manipulated using CRISPR-dCas9-based activators. Integrating this enhancer biology with non-transgenic approaches, such as pollen priming, bacteriophages and nanoparticle delivery, will be essential for practical applications. We propose a framework that leverages these strategies to address oil-protein trade-offs and develop a holistic epigenetic breeding approach, underscoring the transformative potential of epigenetics for sustainable cottonseed oil yield improvement.

Cotton (Gossypium hirsutum) is globally cultivated for its high-quality fiber; yet, its seed, rich in oil and protein, offers untapped potential for various applications, including food, feed, and industry. With cottonseed oil gaining renewed attention as a valuable co-product, efforts to enhance oil content must contend with long-standing breeding priorities focused on lint yield and fiber quality. A central challenge lies in the complex and often antagonistic genetic relationships between oil accumulation and key agronomic traits. Notably, negative correlations between seed oil content and fiber yield, as well as the pleiotropic nature of several regulatory genes and Quantitative Trait Loci (QTLs), present significant barriers to dual-trait improvement. This review synthesizes current knowledge on the genetic and molecular interplay between cottonseed oil content and other agronomic traits. We examine the architecture of oil-related QTLs and pleiotropic loci, co-expression patterns of shared transcriptional regulators, and metabolic trade-offs influencing carbon allocation between seed and fiber. Recent advances in genomics, transcriptomics, and systems biology are explored as tools to disentangle these trait interactions. We highlight strategies such as multi-trait genomic selection, CRISPR-based uncoupling of antagonistic loci, and the use of wild and exotic germplasm to overcome linkage drag. By providing an integrative overview of the constraints and opportunities at the intersection of oil and agronomic trait improvement, this review lays the groundwork for the development of dual-purpose cotton ideotypes. We propose a conceptual framework for breeding programs to simultaneously enhance fiber yield and oil productivity in a sustainable and climate-resilient manner.

The membrane-bound proteins belonging to DUF677 (domain of unknown function 677) are found mainly in green plants. The function of the DUF677 gene (AT14A) has been investigated in Arabidopsis and tomato in relation to drought stress tolerance. Overexpression of AT14A improves drought tolerance in tomato, promotes growth in Arabidopsis during drought stress, and confers tolerance against oxidative damage caused by drought stress in suspension-cultured A. thaliana. However, the role of the DUF677 gene family has not yet been reported in cotton. We identified 148 DUF677 genes from 15 selected plant species using domain-based and homology-supported bioinformatics approaches and classified them into two major groups (I and II) based on phylogenetic analysis. Group I is further divided into two sub-groups (IA and IB). Structural analysis revealed the presence of a few introns in the DUF677 genes. The evolution and expansion of the DUF677 protein family were primarily driven by segmental duplication. Seventy-one miRNAs were predicted to target 29 GhDUF677 genes, including Ghi-MIR397, Ghi-MIR8722, and Ghi-MIRN1429. Several cis-elements, such as MBS, ABRE, TCA elements, and W-Box, which were known to play a role in abiotic stress response, were observed in the promoter region of GhDUF677 genes. RNA-seq data were analyzed for tissue-specific expression, and qRT‒PCR was performed on six selected genes. The outcomes revealed high levels of GhDUF677 gene expression across different tissues under abiotic stress conditions. This study provides a genome-wide bioinformatics and expression-based characterization of the DUF677 gene family in cotton, identifying candidate genes potentially associated with drought and salt stress responses. While the findings are based on evolutionary, regulatory, and transcriptomic evidence, they do not constitute direct functional validation. Instead, this study establishes a theoretical and genomic foundation for future functional studies aimed at elucidating the precise roles of DUF677 genes in cotton stress tolerance.

As a major abiotic constraint, drought stress adversely affects cotton growth and fiber yield. In our study, GhTrxh2 was characterized and demonstrated a positive role in the drought stress response. Treatment with ABA and PEG resulted in a marked induction of GhTrxh2 transcript level. The heterologous expression of GhTrxh2 in yeast conferred an enhanced survival rate under drought conditions. Silencing of GhTrxh2 in cotton decreased the activities of antioxidant enzymes and increased contents of MDA and H2O2, which regulated the accumulation of reactive oxygen species. The transcript levels of GhP5CS, GhDREB2A, GhCOR47, GhRAD29 decreased in silenced cotton. Conversely, overexpression of GhTrxh2 enhanced Arabidopsis tolerance to drought treatment. Transgenic Arabidopsis showed higher seed germination rate and root length, while accumulated less relative oxygen species during drought stress. Based on yeast one-hybrid and dual-luciferase assay, GhVIP1 directly bound to the promoter of GhTrxh2 and promoted GhTrxh2 expression. Yeast two-hybrid and luciferase complementation imaging (LCI) experiment confirmed a physical interaction between GhTrxh2 and GhPLA1. Collectively, these findings suggested that GhTrxh2 appeared to play an important role in plant drought tolerance by preventing ROS accumulation and regulating the expression of drought-related genes, which supplied important insights to drought tolerance mechanisms in cotton.

Vertcillium wilt (VW), a soil-borne disease causing cotton yield loss and fiber quality reduction, is a major stumbling block in cotton production. It is a challenging job to detect key genes associated with major quantitative trait loci (QTL) that can be used to develop VW-resistant varieties. In this study, several genome regions associated with major QTL controlling VW resistance were fine mapped by constructing the high-density genetic map of major QTL regions using Kompetitive Allele-Specific PCR (KASP) markers. Candidate genes were subsequently identified by combining gene annotation, genome resequencing, transcriptome sequencing and qRT-PCR, and functions of several genes associated with major QTL were demonstrated based on gene VIGS silencing and overexpressing. The results showed that six QTL were identified, among which five were regarded as major QTL detected in at least two environments, and four QTL, qVW-D05-1, qVW-D05-2, qVW-D05-3 and qVW-A01-1, were fine mapped to 15.2–241.3 kb physical regions located on chromosome D05 and A01, respectively. Polymerizing of major QTL on different chromosomes showed that the more the resistant QTL were polymerized in a line, the stronger the VW resistance of the line became, and those lines polymerizing 4 resistant QTL showed much higher and more stable VW resistance in each environments, implying the potential of multi-locus QTL polymerization in improving VW resistance. Fifteen candidate genes showing both gDNA sequence variation and expression difference between resistant and susceptible lines were identified to be associated with VW resistance in cotton. Four candidate genes associated with qVW-D05-1 were cloned and conducted function analysis, it was showed that the sequence variation of functional genes, especially the variation resulting in amino acid differences of the domains, might be the molecular mechanisms of VW resistance in cotton.

Verticillium wilt (VW) severely impacts cotton production, and it is essential to detect genes related to VW resistance and develop resistant varieties with genetic engineering in cotton. Jasmonic acid (JA) induces broad-spectrum disease resistance, while its overaccumulation hinders plant growth. However, JA can be degraded by Jasmonate-induced oxygenases (JOXs) which hydroxylating it into 12-OH-JA, balancing defense and growth. In previous research, a jasmonate oxidase gene, GhJOX2, ‌was identified to be involved in Verticillium wilt resistance in cotton. Loss-of-function of this gene in cotton enhanced the disease resistance. In this study, we further demonstrated that GhJOX2-silenced plants showed significantly elevated JA content upon pathogen challenge, and overexpression of GhJOX2 enhanced the susceptibility of Arabidopsis thaliana to Verticillium dahliae infection, downregulating the expression of JA pathway-related genes. GhJOX2 was found to interact with GhSAMS1 encoding S-adenosylmethionine synthase associated with ethylene biosynthesis, and silencing GhSAMS1 enhanced cotton resistance against Verticillium dahliae and activated multiple defense responses including reactive oxygen species (ROS) accumulation and ‌defense gene upregulation. It is speculated that GhJOX2 negatively regulates cotton's resistance to Verticillium wilt through hydroxylating jasmonic acid and interacting with the GhSAMS1.

BackgroundThe YUCCA (YUC) genes play a pivotal regulatory role in plant growth and development; however, those in cotton have not been systematically characterized.ResultsIn this study, 104 YUC members were identified across four cotton species using bioinformatics analysis. Phylogenetic analysis revealed that the YUC genes clustered into four distinct clades, and both gene structures and motif compositions were highly conserved within each subgroup. Additionally, cis-acting elements associated with growth and development, hormone response, and abiotic stress were found in the promoter regions of GhYUCs. GhYUC19 is a homologous gene of AtYUC1/4 in Arabidopsis thaliana. The quantitative reverse transcription polymerase chain reaction (qRT-PCR) analysis demonstrated that GhYUC19 was highly expressed in the main shoot apex, young leaves, and flower buds of cotton plants. Overexpression of GhYUC19 in Arabidopsis resulted in increased plant height, downward-curling leaves, reduced silique size, and lower fertility. Conversely, downregulation of GhYUC19 via virus-induced gene silencing (VIGS) in cotton resulted in abnormal floral organ development, including missing petals and fewer stamens.ConclusionsThis study conducted a systematic analysis of the YUC gene family members and validated the regulatory role of GhYUC19 in plant height, branching, and flower and fruit development. These findings provide a foundation for further investigation into the biological functions of GhYUCs in cotton.

Semigamy is a rare fertilization anomaly in plants that enables haploid induction (HI), a valuable strategy for accelerating crop breeding; however, its molecular basis remains largely unexplored. We investigated transcriptional and epigenetic mechanisms underlying semigamy mutant VSg in island cotton (Gossypium barbadense), which exhibits a high haploid induction rate during double fertilization. We combined cytological observations with time-resolved transcriptome profiling and whole-genome bisulfite sequencing across key fertilization stages. This integrative approach captured dynamic molecular changes associated with gamete nuclear fusion and early zygotic development. Compared with the wild-type, which displayed rapid polar nuclei fusion and normal free nuclear endosperm formation, the semigamy mutant showed delayed polar nuclei fusion and impaired sperm-egg nuclear fusion. Transcriptomic analyses identified differentially expressed genes enriched in membrane fusion processes, while epigenomic profiling revealed dynamic DNA methylation changes in genes encoding transmembrane proteins, cyclins, and kinesins, suggesting disrupted regulation of membrane dynamics and cell cycle progression. These results indicate that coordinated transcriptional and epigenetic regulation of nuclear fusion and cell cycle pathways underlie semigamy-induced developmental arrest and haploid induction. The study provides mechanistic insights into fertilization biology and highlights semigamy as a promising system for improving haploid breeding strategies in crops.

Blue fescue (Festuca glauca) is a widely used ornamental grass worldwide. Drought is an important limiting factor for the growth and development of blue fescue; therefore, cultivating new strains of blue fescue with a strong drought tolerance is of great significance for its production practice. To investigate the drought tolerance mechanism of ds-1, this study subjected both ds-1 and “Festina” to a natural drought treatment and measured their physiological and biochemical indicators. A transcriptomic analysis was also conducted to explore the underlying molecular mechanisms. The results showed that, after the drought treatment, the relative water content (RWC), water use efficiency (WUE), and photosynthetic rate (Pn) of ds-1 leaves were significantly higher than those of “Festina”; in addition, the contents of H2O2 and O2−, the relative electrical conductivity (REC), the malondialdehyde (MDA) content, the gas conductance (Gs), and the transpiration rate (Tr) were significantly lower than those of “Festina”. The peroxidase (POD) activity of ds-1 was significantly higher than that of “Festina”, while the superoxide dismutase (SOD) activity of ds-1 was significantly lower than that of “Festina”. The transcriptome data analysis showed that there were a total of 9475 differentially expressed genes (DEGs) between ds-1 and “Festina”. A Venn plot analysis showed 692 DEGs between ds-1—8d vs. “Festina”—8d and ds-1—16d vs. “Festina”—16d. A KEGG enrichment analysis showed that these 692 genes were mainly enriched in 86 pathways, including those related to the photosynthesis antenna protein, plant hormone signal transduction, MAPK signaling, starch and sucrose metabolism, and arginine and proline metabolism. Further screening identified genes that may be associated with drought stress, including PYL, PP2C, SnRK2, ABF, BRI1, JAZ, MYC2, Lhc, and MPK6. The qRT-PCR results indicated that the expression trends of the DEGs were consistent with the transcriptome sequencing results. Our research results can provide a basis for exploring candidate genes for drought tolerance in blue fescue. In addition, our research results provide valuable genetic resources for the development of drought-resistant ornamental grass varieties, which can help reduce water consumption in cities and decrease labor and capital investment.

Setaria italica seed coat represents an underutilized source of bioactive polyphenols. This study employed an integrated approach combining spatial metabolomics, computational screening, and in vitro assays to characterize its polyphenol composition and antioxidant properties. UPLC-MS/MS analysis revealed distinct metabolite profiles with numerous polyphenols significantly enriched in seed coats relative to kernels. Molecular docking identified Homoplantaginin as a high-affinity ligand for catalase, with molecular dynamics simulations confirming complex stability. In vitro antioxidant assays demonstrated concentration-dependent scavenging activity of Homoplantaginin against ABTS•+, O₂•-, and •OH radicals. These findings highlight the potential of Setaria italica seed coat as a sustainable source of natural antioxidants for functional food applications, providing scientific basis for the valorization of this agricultural byproduct.

Miscanthus is extensively cultivated as a pioneer plant, demonstrates excellent performance in coping with heavy metal stress. However, existing research on Miscanthus and its mechanisms for heavy metal transport is limited, particularly regarding the genetic mechanisms underlying of its tolerance to heavy metals. This study aimed to fill the knowledge gap concerning the role of NRAMP (natural resistance associated macrophage protein) genes in Miscanthus, as NRAMP has been widely reported to facilitate the transport of heavy metal ions in other plants. A total of seventeen MsNRAMP genes were identifed in Miscanthus sinensis, which can be classified into two major groups and are characterized by a high degree of conservation in their secondary structure, the presence of ion-binding sites characteristic of the Leu-T domain, and an average of 9-12 transmembrane domains. Our findings significantly enhance the understanding of metal ion balance and stress response mechanisms in Miscanthus sinensis. Specifically, we discovered that only MsNRAMP3 and MsNRAMP7 are expressed under normal conditions, while the remaining MsNRAMP genes exhibit low or no expression. Additionally, MsNRAMP14 and MsNRAMP8 were found to have numerous ABA and MeJA response elements in their promoter regions, suggesting a potential role in stress response.

Vesicle-associated membrane protein (VAMP)-associated proteins (VAPs) serve as crucial molecular bridges, connecting the endoplasmic reticulum (ER) and plasma membrane (PM), VAPs play important roles in cell elongation, material transport and metabolism, and maintain the normal structure and morphology of cells. However, the mechanism of VAP regulates cotton fiber development is not understood. In this study, a candidate protein, GhVAP1, was screened via a previous yeast two-hybrid assay for interaction with GhAlaRP, an alanine-rich protein that regulates cotton fiber cell development, further inferred that GhVAP1 have an important function in cotton fiber development. The expression pattern of GhVAP1 exhibited that it is predominantly expressed in the elongating fibers. GhVAP1 is localized on the ER. Further knockout of the GhVAP1 through genome editing technology inhibited fiber elongation and reduced fiber strength. Moreover, transcriptome data analysis of cotton fibers from vap1 plants identified 904 differentially expressed genes (DEGs), mainly involved in SNARE interactions in the vesicle transport pathway, etc. Furthermore, protein interaction analysis revealed that GhVAP1 also interacts with GhSyntaxins, which are vesicle transport-associated proteins that are predominantly expressed in fibers. These findings revealed that the possible pathway by which GhVAP1 interacts with GhAlaRP and GhSyntaxins to form a regulatory network involved in cotton fiber development. Collectively, our findings elucidate that GhVAP1 is pivotal in the regulation of fiber cell elongation development.

BACKGROUND:BRVIS RADIX (BRX) family is a small gene family with the highly conserved plant-specific BRX domains, which plays important roles in plant development and response to abiotic stress. Although BRX protein has been studied in other plants, the biological function of cotton BRX-like (BRXL) gene family is still elusive.RESULT:In this study, a total of 36 BRXL genes were identified in four cotton species. Whole genome or segmental duplications played the main role in the expansion of GhBRXL gene family during evolutionary process in cotton. These BRXL genes were clustered into 2 groups, α and β, in which structural and functional conservation within same groups but divergence among different groups were found. Promoter analysis indicated that cis-elements were associated with the phytohormone regulatory networks and the response to abiotic stress. Transcriptomic analysis indicated that GhBRXL2A/2D and GhBRXL5A/5D were up/down-regulated in response to the different stress. Silencing of GhBRXL5A gene via virus-induced gene silencing (VIGS) improved salt tolerance in cotton plants. Furthermore, yeast two hybrid analysis suggested homotypic and heterotypic interactions between GhBRXL1A and GhBRXL5D.CONCLUSIONS:Overall, these results provide useful and valuable information for understanding the evolution of cotton GhBRXL genes and their functions in salt stress.

The calmodulin-binding protein 60 (CBP60) family is a gene family unique to plants, and its members play a crucial role in plant defense responses to pathogens and growth and development. Considering that cotton is the primary source of natural cotton textile fiber, the functional study of its CBP60 gene family members is critical. In this research, we successfully identified 162 CBP60 members from the genomes of 21 species. Of these, 72 members were found in four cotton species, divided into four clades. To understand the function of GhCBP60B in cotton in depth, we conducted a detailed analysis of its sequence, structure, cis-acting elements, and expression patterns. Research results show that GhCBP60B is located in the nucleus and plays a crucial role in cotton growth and development and response to salt and drought stress. After using VIGS (virus-induced gene silencing) technology to conduct gene silencing experiments, we found that the plants silenced by GhCBP60B showed dwarf plants and shortened stem nodes, and the expression of related immune genes also changed. In further abiotic stress treatment experiments, we found that GhCBP60B-silenced plants were more sensitive to drought and salt stress, and their POD (peroxidase) activity was also significantly reduced. These results imply the vital role of GhCBP60B in cotton, especially in regulating plant responses to drought and salt stress. This study systematically analyzed CBP60 gene family members through bioinformatics methods and explored in depth the biological function of GhCBP60B in cotton. These research results lay a solid foundation for the future use of the GhCBP60B gene to improve cotton plant type and its drought and salt resistance.

Climate deterioration, water shortages, and abiotic stress are the main threats worldwide that seriously affect cotton growth, yield, and fiber quality. Therefore, research on improving cotton yield and tolerance to biotic and abiotic stresses is of great importance. The NAC proteins are crucial and plant-specific transcription factors (TFs) that are involved in cotton growth, development, and stress responses. The comprehensive utilization of cotton NAC TFs in the improvement of cotton varieties through novel biotechnological methods is feasible. Based on cotton genomic data, genome-wide identification and analyses have revealed potential functions of cotton NAC genes. Here, we comprehensively summarize the recent progress in understanding cotton NAC TFs roles in regulating responses to drought, salt, and Verticillium wilt-related stresses, as well as leaf senescence and the development of fibers, xylem, and glands. The detailed regulatory network of NAC proteins in cotton is also elucidated. Cotton NAC TFs directly bind to the promoters of genes associated with ABA biosynthesis and secondary cell-wall formation, participate in several biological processes by interacting with related proteins, and regulate the expression of downstream genes. Studies have shown that the overexpression of NAC TF genes in cotton and other model plants improve their drought or salt tolerance. This review elucidates the latest findings on the functions and regulation of cotton NAC proteins, broadens our understanding of cotton NAC TFs, and lays a fundamental foundation for further molecular breeding research in cotton.

GEX1 (gamete expressed 1) proteins are critical membrane proteins conserved among flowering plants that are involved in the nuclear fusion and embryonic development. Herein, we identified the 32 GEX1 proteins from representative land plants. In cotton, GEX1 genes expressed in various tissues across all stages of the life cycle, especially in pollen. Subcellular localization indicated the position of GhGEX1 protein was localized in the endoplasmic reticulum. Experimental research has demonstrated that GhGEX1 has the potential to improve the partial abortion phenotype in Arabidopsis. CRISPR/Cas9-mediated knockout of GhGEX1 exhibited the seed abortion. Paraffin section of the ovule revealed that the polar nuclear fusion of ghgex1 plants remains at a standstill when the wild type has developed into a normal embryo. Comparative transcriptome analysis showed that the DEGs of reproductive-related processes and membrane-related processes were repressed in the pollen of knockout lines. The predicted protein interactions showed that GhGEX1 probably functioned through interactions with proteins related to reproduction and membrane. From all these investigations, it was possible to conclude that the GEX1 proteins are evolutionarily conserved in flowering plants and elucidated the pivotal roles during fertilization and early embryonic development in cotton.

BACKGROUND:Phospholipases As (PLAs) are acyl hydrolases that catalyze the release of free fatty acids in phospholipids and play multiple functions in plant growth and development. The three families of PLAs are: PLA1, PLA2 (sPLA), and patatin-related PLA (pPLA). The diverse functions that pPLAs play in the growth and development of a broad range of plants have been demonstrated by prior studies.METHODS:Genome-wide analysis of the pPLA gene family and screening of genes for expression verification and gene silencing verification were conducted. Additionally, pollen vitality testing, analysis of the pollen expression pattern, and the detection of POD, SOD, CAT, MDA, and H2O2 were performed.RESULT:In this study, 294 pPLAs were identified from 13 plant species, including 46 GhpPLAs that were divided into three subfamilies (I-III). Expression patterns showed that the majority of GhpPLAs were preferentially expressed in the petal, pistil, anther, and ovule, among other reproductive organs. Particularly, GhpPLA23 and GhpPLA44, were found to be potentially important for the reproductive development of G. hirsutum. Functional validation was demonstrated by VIGS which showed that reduced expression levels of GhpPLA23 and GhpPLA44 in the silenced plants were associated with a decrease in pollen activity. Moreover, a substantial shift in ROS and ROS scavengers and a considerable increase in POD, CAT, SOD, and other physiological parameters was found out in these silenced plants. Our results provide plausibility to the hypothesis that GhpPLA23 and GhpPLA44 had a major developmental impact on cotton reproductive systems. These results also suggest that pPLAs are important for G. hirsutum's reproductive development and suggest that they could be employed as potential genes for haploid induction.CONCLUSIONS:The findings of the present research indicate that pPLA genes are essential for the development of floral organs and sperm cells in cotton. Consequently, this family might be important for the reproductive development of cotton and possibly for inducing the plant develop haploid progeny.

BACKGROUND:Cytokinin oxidase/dehydrogenase (CKX) plays a vital role in response to abiotic stress through modulating the antioxidant enzyme activities. Nevertheless, the biological function of the CKX gene family has yet to be reported in cotton.RESULT:In this study, a total of 27 GhCKXs were identified by the genome-wide investigation and distributed across 18 chromosomes. Phylogenetic tree analysis revealed that CKX genes were clustered into four clades, and most gene expansions originated from segmental duplications. The CKXs gene structure and motif analysis displayed remarkably well conserved among the four groups. Moreover, the cis-acting elements related to the abiotic stress, hormones, and light response were identified within the promoter regions of GhCKXs. Transcriptome data and RT-qPCR showed that GhCKX genes demonstrated higher expression levels in various tissues and were involved in cotton's abiotic stress and phytohormone response. The protein-protein interaction network indicates that the CKX family probably participated in redox regulation, including oxidoreduction or ATP levels, to mediate plant growth and development. Functionally identified via virus-induced gene silencing (VIGS) found that the GhCKX14 gene improved drought resistance by modulating the antioxidant-related activitie.CONCLUSIONS:In this study, the CKX gene family members were analyzed by bioinformatics, and validates the response of GhCKX gene to various phytohormone treatment and abiotic stresses. Our findings established the foundation of GhCKXs in responding to abiotic stress and GhCKX14 in regulating drought resistance in cotton.

Abstract Background Phospholipases As (PLAs) are acyl hydrolase that can catalyze the release of free fatty acids in phospholipids and play multiple functions in plant growth and development. PLAs can be divided into three families, PLA1, PLA2 (sPLA) and patatin-related PLA (pPLA). Previous studies have elucidated the versatile roles of pPLAs in growth and development of many plants.Methods Genome-wide analysis of the pPLA family and screening of genes for expression verification and gene silencing verification. After gene silencing, analysis of pollen expression pattern, pollen vitality test and POD, SOD, CAT, MDA and H2O2 detection.Result In this study, a total of 294 pPLAs were identified from 13 species, including 46 GhpPLAs divided into three subfamilies (I-III-γ). Expression pattern analysis revealed that most of GhpPLAs were preferentially expressed in the reproductive organs (petal, pistil, anther and ovule), especially two genes (GhpPLA23 and GhpPLA44) were deduced that may affect the reproductive development of G. hirsutum. VIGS experiment of GhpPLA23 and GhpPLA44 showed that the pollen activity decreased with their lower expression levels in the silenced plants. The contents of POD, CAT, SOD and other physiological indicators were significantly increased, while ROS and ROS scavengers were significantly changed after genes silencing, indicating that these two genes had important effects on cotton reproductive development. These findings suggest that pPLAs play a crucial role in reproductive development of G. hirsutum and show the potential to be utilized as candidate genes for haploid inducing.Conclusions In this study, pPLA genes play an important role in the reproductive organs of cotton, affecting the development of flower organs and sperm cells, so this family may play an important role in the reproductive development progress of cotton, and even in inducing cotton to produce haploid.