Genes In Cotton Research Articles

Retraction Note: Genome-wide identification and characterization of TALE superfamily genes in cotton reveals their functions in regulating secondary cell wall biosynthesis

Retraction Note: Genome-wide identification and characterization of TALE superfamily genes in cotton reveals their functions in regulating secondary cell wall biosynthesis

AtZAT10/STZ1 improves drought tolerance and increases fiber yield in cotton.

Drought poses a significant challenge to global crop productivity, necessitating innovative approaches to bolster plant resilience. Leveraging transgenic technology to bolster drought tolerance in crops emerges as a promising strategy for addressing the demands of a rapidly growing global populace. AtZAT10/STZ1, a C2H2-type zinc finger protein transcription factor has shown to significantly improve Arabidopsis' tolerance to various abiotic stresses. In this study, we reports that AtSTZ1 confers notable drought resistance in upland cotton (Gossypium hirsutum), amplifying cotton fiber yield under varying conditions, including irrigated and water-limited environments, in field trials. Notably, AtSTZ1-overexpressing transgenic cotton showcases enhanced drought resilience across critical growth stages, including seed germination, seedling establishment, and reproductive phases. Morphological analysis reveals an expanded root system characterized by an elongated taproot system, increased lateral roots, augmented root biomass, and enlarged cell dimensions from transgenic cotton plants. Additionally, higher contents of proline, chlorophyll, soluble sugars, and enhanced ROS-scavenging enzyme activities are observed in leaves of transgenic plants subjected to drought, underscoring improved physiological adaptations. Furthermore, transgenic lines exhibit heightened photosynthetic rate, increased water use efficiency, and larger stomatal and epidermal cell sizes, coupled with a decline in leaf stomatal conductance and density, as well as diminished transpiration rates compared to the wild type counterparts. Transcriptome profiling unveils 106 differentially expressed genes in transgenic cotton leaves post-drought treatment, including protein kinases, transcription factors, aquaporins, and heat shock proteins, indicative of an orchestrated stress response. Collectively, these findings underscore the capacity of AtSTZ1 to augment the expression of abiotic stress-related genes in cotton following drought conditions, thus presenting a compelling candidate for genetic manipulation aimed at enhancing crop resilience.

Genetic manipulation of the genes for clonal seeds results in sterility in cotton

BackgroundHeterosis is a common phenomenon in plants and has been extensively applied in crop breeding. However, the superior traits in the hybrids can only be maintained in the first generation but segregate in the following generations. Maintaining heterosis in generations has been challenging but highly desirable in crop breeding. Recent study showed that maternally produced diploid seeds could be achieved in rice by knocking out three meiosis related genes, namely REC8, PAIR1, OSD1 to create MiMe in combination with egg cell specific expression of BBM transcription factor, a technology called clonal seeds. Interestingly, there has been very limited reports indicating the feasibility of this approach in other crops.ResultsIn this study, we aimed to test whether clonal seeds could be created in cotton. We identified the homologs of the three meiosis related genes in cotton and used the multiplex CRISPR/Cas9 gene editing system to simultaneously knock out these three genes in both A and D sub-genomes. More than 50 transgenic cotton plants were generated, and fragment analysis indicated that multiple gene knockouts occurred in the transgenic plants. However, all the transgenic plants were sterile apparently due to the lack of pollen. Pollination of the flowers of the transgenic plants using the wild type pollens could not generate seeds, an indication of defects in the formation of female sexual cells in the transgenic plants. In addition, we generated transgenic cotton plants expressing the cotton BBM gene driven by the Arabidopsis egg cell specific promoter pDD45. Two transgenic plants were obtained, and both showed severely reduced fertility.ConclusionsOverall, our results indicate that knockout of the clonal seeds related genes in cotton causes sterility and how to manipulate genes to create clonal seeds in cotton requires further research.

Predicting Cold-Stress Responsive Genes in Cotton with Machine Learning Models

Machine Learning (ML) serves as a potent tool for data mining and predictive analytics in genomic research. However, its application in identifying stress-responsive genes remains underexplored. This study identified distinct variations in the expression patterns of one-to-one homologous genes responding to cold stress in three cotton species: Gossypium hirsutum, Gossypium barbadense, and Gossypium arboreum. To better understand cold-responsive genes, we developed ML predictive models (LightGBM, XGBoost, and Random Forest) utilizing 121 biochemical features. The incorporating of these features significantly enhanced model accuracy. Moreover, incorporating evolutionary information further refined the models, achieving an impressive 80.80% accuracy in predicting cold-stress responsive genes. Notably, models trained on sequence features from G. hirsutum showed transferability to the closely related species of G. barbadense, with accuracies ranging from 78.65% to 83.04%. This research presents a promising workflow for identifying candidate genes for experimental exploration of cold stress responses and establishes a systematic framework for predicting cold-stress related genes using ML methodologies.

The GhEB1C gene mediates resistance of cotton to Verticillium wilt.

The GhEB1C gene of the EB1 protein family functions as microtubule end-binding protein and may be involved in the regulation of microtubule-related pathways to enhance resistance to Verticillium wilt. The expression of GhEB1C is induced by SA, also contributing to Verticillium wilt resistance. Cotton, as a crucial cash and oil crop, faces a significant threat from Verticillium wilt, a soil-borne disease induced by Verticillium dahliae, severely impacting cotton growth and development. Investigating genes associated with resistance to Verticillium wilt is paramount. We identified and performed a phylogenetic analysis on members of the EB1 family associated with Verticillium wilt in this work. GhEB1C was discovered by transcriptome screening and was studied for its function in cotton defense against V. dahliae. The RT-qPCR analysis revealed significant expression of the GhEB1C gene in cotton leaves. Subsequent localization analysis using transient expression demonstrated cytoplasmic localization of GhEB1C. VIGS experiments indicated that silencing of the GhEB1C gene significantly increased susceptibility of cotton to V. dahliae. Comparative RNA-seq analysis showed that GhEB1C silenced plants exhibited altered microtubule-associated protein pathways and flavonogen-associated pathways, suggesting a role for GhEB1C in defense mechanisms. Overexpression of tobacco resulted in enhanced resistance to V. dahliae as compared to wild-type plants. Furthermore, our investigation into the relationship between the GhEB1C gene and plant disease resistance hormones salicylic axid (SA) and jasmonic acid (JA) revealed the involvement of GhEB1C in the regulation of the SA pathway. In conclusion, our findings demonstrate that GhEB1C plays a crucial role in conferring immunity to cotton against Verticillium wilt, providing valuable insights for further research on plant adaptability to pathogen invasion.

Pan-genome analysis of GT64 gene family and expression response to Verticillium wilt in cotton

BackgroundThe GT64 subfamily, belonging to the glycosyltransferase family, plays a critical function in plant adaptation to stress conditions and the modulation of plant growth, development, and organogenesis processes. However, a comprehensive identification and systematic analysis of GT64 in cotton are still lacking.ResultsThis study used bioinformatics techniques to conduct a detailed investigation on the GT64 gene family members of eight cotton species for the first time. A total of 39 GT64 genes were detected, which could be classified into five subfamilies according to the phylogenetic tree. Among them, six genes were found in upland cotton. Furthermore, investigated the precise chromosomal positions of these genes and visually represented their gene structure details. Moreover, forecasted cis-regulatory elements in GhGT64s and ascertained the duplication type of the GT64 in the eight cotton species. Evaluation of the Ka/Ks ratio for similar gene pairs among the eight cotton species provided insights into the selective pressures acting on these homologous genes. Additionally, analyzed the expression profiles of the GT64 gene family. Overexpressing GhGT64_4 in tobacco improved its disease resistance. Subsequently, VIGS experiments conducted in cotton demonstrated reduced disease resistance upon silencing of the GhGT64_4, may indicate its involvement in affecting lignin and jasmonic acid biosynthesis pathways, thus impacting cotton resistance. Weighted Gene Co-expression Network Analysis (WGCNA) revealed an early immune response against Verticillium dahliae in G. barbadense compared to G. hirsutum. Quantitative Reverse Transcription Polymerase Chain Reaction (qRT-PCR) analysis indicated that some GT64 genes might play a role under various biotic and abiotic stress conditions.ConclusionsThese discoveries enhance our knowledge of GT64 family members and lay the groundwork for future investigations into the disease resistance mechanisms of this gene in cotton.

Genome-wide analysis and prediction of chloroplast and mitochondrial RNA editing sites of AGC gene family in cotton (Gossypium hirsutum L.) for abiotic stress tolerance

BackgroundCotton is one of the topmost fiber crops throughout the globe. During the last decade, abrupt changes in the climate resulted in drought, heat, and salinity. These stresses have seriously affected cotton production and significant losses all over the textile industry. The GhAGC kinase, a subfamily of AGC group and member of serine/threonine (Ser/Thr) protein kinases group and is highly conserved among eukaryotic organisms. The AGC kinases are compulsory elements of cell development, metabolic processes, and cell death in mammalian systems. The investigation of RNA editing sites within the organelle genomes of multicellular vascular plants, such as Gossypium hirsutum holds significant importance in understanding the regulation of gene expression at the post-transcriptional level.MethodsIn present work, we characterized twenty-eight GhAGC genes in cotton and constructed phylogenetic tree using nine different species from the most primitive to the most recent.ResultsIn sequence logos analyses, highly conserved amino acid residues were found in G. hirsutum, G. arboretum, G. raimondii and A. thaliana. The occurrence of cis-acting growth and stress-related elements in the promoter regions of GhAGCs highlight the significance of these factors in plant development and abiotic stress tolerance. Ka/Ks levels demonstrated that purifying selection pressure resulting from segmental events was applied to GhAGC with little functional divergence. We focused on identifying RNA editing sites in G. hirsutum organelles, specifically in the chloroplast and mitochondria, across all 28 AGC genes.ConclusionThe positive role of GhAGCs was explored by quantifying the expression in the plant tissues under abiotic stress. These findings help in understanding the role of GhAGC genes under abiotic stresses which may further be used in cotton breeding for the development of climate smart varieties in abruptly changing climate.

GhCASPL1 regulates secondary cell wall thickening in cotton fibers by stabilizing the cellulose synthase complex on the plasma membrane.

Cotton (Gossypium hirsutum) fibers are elongated single cells that rapidly accumulate cellulose during secondary cell wall (SCW) thickening, which requires cellulose synthase complex (CSC) activity. Here, we describe the CSC-interacting factor CASPARIAN STRIP MEMBRANE DOMAIN-LIKE1 (GhCASPL1), which contributes to SCW thickening by influencing CSC stability on the plasma membrane. GhCASPL1 is preferentially expressed in fiber cells during SCW biosynthesis and encodes a MARVEL domain protein. The ghcaspl1 ghcaspl2 mutant exhibited reduced plant height and produced mature fibers with fewer natural twists, lower tensile strength, and a thinner SCW compared to the wild type. Similarly, the Arabidopsis (Arabidopsis thaliana) caspl1 caspl2 double mutant showed a lower cellulose content and thinner cell walls in the stem vasculature than the wild type but normal plant morphology. Introducing the cotton gene GhCASPL1 successfully restored the reduced cellulose content of the Arabidopsis caspl1 caspl2 mutant. Detergent treatments, ultracentrifugation assays, and enzymatic assays showed that the CSC in the ghcaspl1 ghcaspl2 double mutant showed reduced membrane binding and decreased enzyme activity compared to the wild type. GhCASPL1 binds strongly to phosphatidic acid (PA), which is present in much higher amounts in thickening fiber cells compared to ovules and leaves. Mutating the PA-binding site in GhCASPL1 resulted in the loss of its colocalization with GhCesA8, and it failed to localize to the plasma membrane. PA may alter membrane structure to facilitate protein-protein interactions, suggesting that GhCASPL1 and PA collaboratively stabilize the CSC. Our findings shed light on CASPL functions and the molecular machinery behind SCW biosynthesis in cotton fibers.

Transcriptional regulation of phospholipid transport in cotton fiber elongation by GhMYB30D04-GhHD1 interaction complex.

Cotton fiber length is basically determined by well-coordinated gene expression and phosphatidylinositol phosphates (PIPs) accumulation during fiber elongation but the regulatory mechanism governing PIPs transport remains unknown. Here, we report a MYB transcription factor GhMYB30D04 in Gossypium hirsutum that promotes fiber elongation through modulating the expression of PIP transporter gene GhLTPG1. Knockout of GhMYB30D04 gene in cotton (KO) results in a reduction of GhLTPG1 transcripts with lower accumulation of PIPs, leading to shorter fibers and lower fiber yield. Conversely, GhMYB30D04 overexpression (GhMYB30D04-OE) causes richer PIPs and longer cotton fibers, mimicking the effects of exogenously applying PIPs on the ovules of GhMYB30D04-KO and wild type. Furthermore, GhMYB30D04 interacts with GhHD1, the crucial transcription factor of fiber initiation, to form an activation complex stabilized by PIPs, both of which upregulate GhLTPG1 expression. Comparative omics-analysis revealed that higher and extended expressions of LTPG1 in fiber elongation mainly correlate with the variations of the GhMYB30D04 gene between two cotton allotetraploids, contributing to longer fiber in G. babardense. Our work clarifies a mechanism by which GhHD1-GhMYB30D04 form a regulatory module of fiber elongation to tightly control PIP accumulation. Our work still has an implication that GhMYB30D04-GhHD1 associates with development transition from fiber initiation to elongation.

Disrupted oxylipin biosynthesis mitigates pathogen infections and pest infestations in cotton (Gossypium hirsutum).

Cotton (Gossypium hirsutum) is the world's most important fiber crop, critical to global textile industries and agricultural economies. However, cotton yield and harvest quality are undermined by the challenges introduced from invading pathogens and pests. Plant-synthesized oxylipins, specifically 9-hydroxy fatty acids resulting from 9-lipoxygenase activity (9-LOX), enhance the growth and development of many microbes and pests. We hypothesized that targeted disruption of 9-LOX-encoding genes in cotton could bolster crop resilience against prominent agronomic threats. Fusarium oxysporum f. sp. vasinfectum (FOV), Aphis gossypii (cotton aphid), and Tobacco rattle virus induced the expression of 9-oxylipin biosynthesis genes, suggesting that the 9-LOX gene products were susceptibility factors to these stressors. Transiently disrupting the expression of the 9-LOX-encoding genes by virus-induced gene silencing significantly reduced target transcript accumulation, and this correlated with impaired progression of FOV infections and a significant decrease in the fecundity of cotton aphids. These findings emphasize that the cotton 9-LOX-derived oxylipins are leveraged by multiple pathogens and pests to enhance their virulence in cotton, and reducing the expression of 9-LOX-encoding genes can benefit cotton crop vitality.

Application of an endogenous pGhαGloA promoter in CRISPR/Cas12a system for efficient genome editing to creat glandless cotton germplasm

The efficient genome editing tool (the CRISPR/Cas12a system) has been used in research on plant functionional genomics and improvement of agronomic traits. In this study, CRISPR/Cas12a system was optimized by using the endogenous pGhαGloA promoter in cotton. Using this system, crRNAs was driven by the Pol II pGhaGloA promoter to construct the pGhRBE3-pGhαGloA-GhPGF vector and carry out genetic transformation. The vector could work efficiently in all positive transgenic plants and the editing efficiency at the crRNA1 target site was up to 93.37%, and the editing efficiency of the crRNA2 target was up to 88.24%, which is significantly higher in editing efficiency of the pGhRBE3 system with Pol III promoter-Ubi 6.7 promoter, this result indicates that the Pol II promoter is more suitable for expressing multiple sgRNA or crRNA than the pol III promoter in cotton. The vector mainly generated the editing type of fragment deletion and the deletion size was in the range of 3-12 bp with the editing sites spanning at the 14th to 29th bases downstream of the protospacer adjacent motif (PAM). All the targeted mutation loci were stably inherited from T0 to T2 generation and three transgene-free lines with target site mutations of GhPGF gene were obtained and these glandless and gossypol-free/(low contents) cotton germplasm will play key role for healthy cottonseeds oil/cake production. Therefore, the CRISPR/Cas12a system driven by the pGhαGloA promoter can efficiently edit target genes in cotton, which provides a powerful tool for cotton functionional genomics and genetic improvement.

GhJUB1_3-At positively regulate drought and salt stress tolerance under control of GhHB7, GhRAP2-3 and GhRAV1 in Cotton.

Climate change severely affects crop production. Cotton is one of the primary fiber crops in the world and its production is susceptible to various environmental stresses, especially drought and salinity. Development of stress tolerant genotypes is the only way to escape from these environmental constraints. We identified sixteen homologs of the Arabidopsis JUB1 gene in cotton. Expression of GhJUB1_3-At was significantly induced in the temporal expression analysis of GhJUB1 genes in the roots of drought tolerant (H177) and susceptible (S9612) cotton genotypes under drought. The silencing of the GhJUB1_3-At gene alone and together with its paralogue GhJUB1_3-Dt reduced the drought tolerance in cotton plants. The transgenic lines exhibited tolerance to the drought and salt stress as compared to the wildtype (WT). The chlorophyll and relative water contents of wildtype decreased under drought as compared to the transgenic lines. The transgenic lines showed decreased H2O2 and increased proline levels under drought and salt stress, as compared to the WT, indicating that the transgenic lines have drought and salt stress tolerance. The expression analysis of the transgenic lines and WT revealed that GAI was upregulated in the transgenic lines in normal conditions as compared to the WT. Under drought and salt treatment, RAB18 and RD29A were strongly upregulated in the transgenic lines as compared to the WT. Conclusively, GhJUB1_3-At is not an auto activator and it is regulated by the crosstalk of GhHB7, GhRAP2-3 and GhRAV1. GhRAV1, a negative regulator of abiotic stress tolerance and positive regulator of leaf senescence, suppresses the expression of GhJUB1_3-At under severe circumstances leading to plant death.

Translational Regulation of Duplicated Gene Expression Evolution in Allopolyploid Cotton.

Polyploidy, a prevalent event in plant evolution, drives phenotypic diversification and speciation. While transcriptional changes and regulation in polyploids have been extensively studied, the translational level impact remains largely unexplored. To address this gap, we conducted a comparative transcriptomic and translatomic analysis of cotton leaves from allopolyploid species G. hirsutum (AD1) and G. barbadense (AD2) relative to their model A-genome and D-genome diploid progenitors. Our data revealed that while allopolyploidization significantly affects the transcriptional landscape, its impact on translation was relatively modest, evidenced by a narrower expression range and fewer expression changes in ribosome-protected fragments than in mRNA levels. Allopolyploid-specific changes commonly identified in both AD1 and AD2 were observed in 7393 genes at either transcriptional or translational levels. Interestingly, the majority of translational changes exhibited concordant down-regulation in both ribosome-protected fragments and mRNA, particularly associated with terpenoid synthesis and metabolism (352 genes). Regarding translational efficiency (TE), at least one-fifth of cotton genes exhibit translational level regulation, with a general trend of more down-regulation (13.9-15.1%) than up-regulation (7.3-11.2%) of TE. The magnitude of translational regulation was slightly reduced in allopolyploids compared with diploids, and allopolyploidy tends to have a more profound impact on genes and functional associations with ultra-low TE. Moreover, we demonstrated a reduced extent of homeolog expression biases during translation compared with transcription. Our study provides insights into the regulatory consequences of allopolyploidy post-transcription, contributing to a comprehensive understanding of regulatory mechanisms of duplicated gene expression evolution.

Exogenous Melatonin Enhances Cold Tolerance by Regulating the Expression of Photosynthetic Performance, Antioxidant System, and Related Genes in Cotton.

In China, cotton is a significant cash crop, and cold stress negatively impacts the crop's development, production, and quality formation. Recent studies have shown that melatonin (MT) can alleviate the damage to plants under cold stress and promote good growth and development. In this study, the morphological and physiological changes induced by exogenous melatonin pretreatment on 'Xinluzao 33' cotton seedlings under cold stress were examined to investigate its defensive effects. The results showed that 100 μM MT pretreatment improved the cold resistance of cotton most significantly. It also improved the wilting state of cotton under cold stress, greatly increased the photosynthetic rate (Pn), stomatal conductance (Gs), maximum photochemical efficiency (Fv/Fm), and photosynthetic performance index (PIabs) by 116.92%, 47.16%, 32.30%, and 50.22%, respectively, and mitigated the adverse effects of low-temperature. In addition, MT supplementation substantially reduced the accumulation of superoxide anion (O2•-) and hydrogen peroxide (H2O2) by 14.5% and 45.49%, respectively, in cold-stressed cotton leaves by modulating the antioxidant system, thereby mitigating oxidative damage. Furthermore, MT pretreatment increased the endogenous melatonin content (23.80%) and flavonoid content (21.44%) and considerably induced the expression of biosynthesis enzyme-related genes. The above results indicate that exogenous melatonin improves the low-temperature resistance of cotton seedlings by regulating photosynthetic performance, antioxidant enzyme activity, antioxidant content, endogenous melatonin and flavonoid content, and the expression levels of genes related to their synthesis.

Genome-Wide Identification and Expression Analysis of ADK Gene Family Members in Cotton under Abiotic Stress.

Adenosine kinase (ADK) is a key enzyme widely distributed in plants, playing an important role in maintaining cellular energy homeostasis and regulating plant growth, development, and responses to environmental stresses. However, research on ADK genes in cotton (Gossypium hirsutum), an economically significant crop, has been limited. This study identified 92 ADK genes from four cotton species (G. arboreum, G. raimondii, G. hirsutum, and G. barbadense) using HMMER and Local BLASTP methods and classified them into six groups. Chromosomal localization revealed a random distribution of ADK genes in G. hirsutum, with 13 genes located on the At subgenome and 14 genes on the Dt subgenome. Gene structure analysis showed consistency in exon-intron organization within subgroups, while conserved motif analysis identified subgroup-specific motifs, indicating functional diversity. Synteny and collinearity mapping analysis revealed that the primary expansion mechanisms of the ADK gene family in cotton are polyploidy and segmental duplication. Cis-regulatory elements in GhADK promoters were classified into light response, hormone response, developmental regulation, and stress response. We also analyzed the expression patterns of GhADK genes under a low temperature (4 °C) and drought conditions. Most GhADK genes responded to cold stress with different expression patterns, indicating their roles in rapid response and long-term cold adaptation. Under drought stress, expression patterns varied, with some genes showing sustained high expression levels. The qRT-PCR validation of transcriptomic data confirmed the stress-induced expression patterns of selected GhADK genes. Functional analysis through the VIGS silencing of GhADK25 demonstrated its importance in cold and drought stress responses, with silencing resulting in poor growth under stress, highlighting its significance in stress tolerance. This study provides a basis for further understanding the evolutionary relationships and functions of the cotton ADK gene family.

Volatiles emitted by Pseudomonas aurantiaca ST-TJ4 trigger systemic plant resistance to Verticillium dahliae

Verticillium dahliae is among the most devastating fungal pathogens, causing significant economic harm to agriculture and forestry. To address this problem, researchers have focused on eliciting systemic resistance in host plants through utilizing volatile organic compounds (VOCs) produced by biological control agents. Herein, we meticulously measured the quantity of V. dahliae pathogens in plants via RTqPCR, as well as the levels of defensive enzymes and pathogenesis-related (PR) proteins within plants. Finally, the efficacy of VOCs in controlling Verticillium wilt in cotton was evaluated. Following treatment with Pseudomonas aurantiaca ST-TJ4, the expression of specific VdEF1-α genes in cotton decreased significantly. The incidence and disease indices also decreased following VOC treatment. In cotton, the salicylic acid (SA) signal was strongly activated 24 h posttreatment; then, hydrogen peroxide (H2O2) levels increased at 48 h, and peroxidase (POD) and catalase (CAT) activities increased to varying degrees at different time points. The malondialdehyde (MDA) content and electrolyte leakage in cotton treated with VOCs were lower than those in the control group, and the expression levels of chitinase (CHI) and PR genes (PR10 and PR17), increased at various time points under the ST-TJ4 treatment. The activity of phenylalanine ammonia lyase (PAL) enzymes in cotton treated with VOCs was approximately 1.26 times greater than that in control plants at 24 h，while the contents of phenols and flavonoids increased significantly in the later stage. Additionally, 2-undecanone and 1-nonanol can induce a response in plants that enhances disease resistance. Collectively, these findings strongly suggest that VOCs from ST-TJ4 act as elicitors of plant defence and are valuable natural products for controlling Verticillium wilt.

Evolution and subfunctionalization of CIPK6 homologous genes in regulating cotton drought resistance

The occurrence of whole-genome duplication or polyploidy may promote plant adaptability to harsh environments. Here, we clarify the evolutionary relationship of eight GhCIPK6 homologous genes in upland cotton (Gossypium hirsutum). Gene expression and interaction analyses indicate that GhCIPK6 homologous genes show significant functional changes after polyploidy. Among these, GhCIPK6D1 and GhCIPK6D3 are significantly up-regulated by drought stress. Functional studies reveal that high GhCIPK6D1 expression promotes cotton drought sensitivity, while GhCIPK6D3 expression promotes drought tolerance, indicating clear functional differentiation. Genetic and biochemical analyses confirm the synergistic negative and positive regulation of cotton drought resistance through GhCBL1A1-GhCIPK6D1 and GhCBL2A1-GhCIPK6D3, respectively, to regulate stomatal movement by controlling the directional flow of K+ in guard cells. These results reveal differentiated roles of GhCIPK6 homologous genes in response to drought stress in upland cotton following polyploidy. The work provides a different perspective for exploring the functionalization and subfunctionalization of duplicated genes in response to polyploidization.

Genome-Wide Identification of the WRKY Gene Family in Four Cotton Varieties and the Positive Role of GhWRKY31 in Response to Salt and Drought Stress.

The WRKY gene family is ubiquitously distributed in plants, serving crucial functions in stress responses. Nevertheless, the structural organization and evolutionary dynamics of WRKY genes in cotton have not been fully elucidated. In this study, a total of 112, 119, 217, and 222 WRKY genes were identified in Gossypium arboreum, Gossypium raimondii, Gossypium hirsutum, and Gossypium barbadense, respectively. These 670 WRKY genes were categorized into seven distinct subgroups and unequally distributed across chromosomes. Examination of conserved motifs, domains, cis-acting elements, and gene architecture collectively highlighted the evolutionary conservation and divergence within the WRKY gene family in cotton. Analysis of synteny and collinearity further confirmed instances of expansion, duplication, and loss events among WRKY genes during cotton evolution. Furthermore, GhWRKY31 transgenic Arabidopsis exhibited heightened germination rates and longer root lengths under drought and salt stress. Silencing GhWRKY31 in cotton led to reduced levels of ABA, proline, POD, and SOD, along with downregulated expression of stress-responsive genes. Yeast one-hybrid and molecular docking assays confirmed the binding capacity of GhWRKY31 to the W box of GhABF1, GhDREB2, and GhRD29. The findings collectively offer a systematic and comprehensive insight into the evolutionary patterns of cotton WRKYs, proposing a suitable regulatory framework for developing cotton cultivars with enhanced resilience to drought and salinity stress.

Optimization of Agrobacterium-mediated transformation conditions for efficient Bacillus thuringiensis (Bt) cotton transformation

Cotton is one of the world’s largest fiber-producing crops. It is also known as the important cash crop of Pakistan. Cotton crop products contribute to economy as cottonseed oil, animal feed and the fiber support the textile industries, thus, playing a significant role in uplifting the economy of a country. But over time, bollworm attacks had escalated, severely reducing the yield of cotton and damaging cotton production. Bt (Bacillus thuringiensis) cotton is being widely used nowadays due to its enhanced resistance against cotton bollworms which are responsible for reducing crop production leading to high economic loss. It greatly affects the economy by increasing the cost of production and protection. Using resistant strains of Bt cotton not only makes it easier to combat bollworm attacks but also reduces the substantial expense issues that farmers face. Bt has many toxic proteins among which the two crystal proteins, Cry1Ac and Cry2Ab, are used to develop resistance against bollworms in cotton. This study aimed to optimize the developing transgenic callus containing Cry1Ac and Cry2Ab genes in cotton. The dual gene construct (Cry1Ac + Cry2Ab) provides more resistance to the cotton crop as compared to the single gene construct against whom the most bollworms have developed resistance. The vector was inoculated into the hypocotyl segments and the transformation process was preceded by shifting the hypocotyls at different mediums Agrobacterium tumefaciens strain LBA4404 was used for cotton transformation. The construct-containing vector was introduced into a standard cotton line. A molecular confirmation test was carried out utilizing PCR and gene-specific primers after the formation of the transgenic callus. As a result of the research an indigenous cotton line that expresses double Bt genes for insect resistance was developed.

Genome-wide identification and mining elite allele variation of the Monoacylglycerol lipase (MAGL) gene family in upland cotton (Gossypium hirsutum L.)

BackgroundMonoacylglycerol lipase (MAGL) genes belong to the alpha/beta hydrolase superfamily, catalyze the terminal step of triglyceride (TAG) hydrolysis, converting monoacylglycerol (MAG) into free fatty acids and glycerol.ResultsIn this study, 30 MAGL genes in upland cotton have been identified, which have been classified into eight subgroups. The duplication of GhMAGL genes in upland cotton was predominantly influenced by segmental duplication events, as revealed through synteny analysis. Furthermore, all GhMAGL genes were found to contain light-responsive elements. Through comprehensive association and haplotype analyses using resequencing data from 355 cotton accessions, GhMAGL3 and GhMAGL6 were detected as key genes related to lipid hydrolysis processes, suggesting a negative regulatory effect.ConclusionsIn summary, MAGL has never been studied in upland cotton previously. This study provides the genetic mechanism foundation for the discover of new genes involved in lipid metabolism to improve cottonseed oil content, which will provide a strategic avenue for marker-assisted breeding aimed at incorporating desirable traits into cultivated cotton varieties.