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Abstract Vitamin C, also referred to as ascorbic acid (AsA), is recognized for its capacity to cure and avert scurvy, and it is crucial for regular human growth and development. In various crops, AsA participates in stress response mechanisms mediated by abscisic acid and has been discovered to have a crucial function in the morphogenesis, growth, development, and production of male gametes in plants. GDP-D-mannose 3′,5′-epimerase (GME) is essential in the synthesis of vitamin C. Our research identified 91, 83, 51, and 46 genes, respectively, found in G. barbadense (GbGMEs), G. hirsutum (GhGMEs), G. arboretum (GaGMEs), and G. raimondii (GrGMEs). Plants resulting from VIGS infection with GhGME31D clearly showed yellowing, water loss and wilting of leaves and black spots on stems. Measurement of MDA and AsA levels indicated that the plants were more damaged. This indicates that AsA has a substantial impact on plant growth and development.

Abstract Background Cotton is an important industrial crop and a pioneer crop for saline-alkali land restoration. However, the molecular mechanism underlying the cotton response to salt is not completely understood. Methods Here, we used metabolome data and transcriptome data to analyze the salt tolerance regulatory network of cotton and metabolic biomarkers. Results In this study, cotton was stressed at 400 m M NaCl for 0 h, 3 h, 24 h and 48 h. NaCl interfered with cotton gene expression, altered metabolite contents and affected plant growth. Metabolome analysis showed that NaCl stress increased the contents of amino acids, sugars and ABA, decreased the amount of vitamin and terpenoids. K-means cluster analysis of differentially expressed genes showed that the continuously up-regulated genes were mainly enriched in metabolic pathways such as flavonoid biosynthesis and amino acid biosynthesis. Conclusion The four metabolites of cysteine (Cys), ABA(Abscisic acid), turanose, and isopentenyladenine-7-N-glucoside (IP7G) were consistently up-regulated under salt stress, which may indicate that they are potential candidates for cotton under salt stress biomarkers. Combined transcriptome and metabolome analysis revealed accumulation of cysteine, ABA, isopentenyladenine-7-N-glucoside and turanose were important for salt tolerance in cotton mechanism. These results will provide some metabolic insights and key metabolite biomarkers for salt stress tolerance, which may help to understanding of the metabolite response to salt stress in cotton and develop a foundation for cotton to grow better in saline soil.

Abstract Background Flavonols play important roles in antioxidation and anticancer activities, longevity, and cardiovascular protection. Flavonol synthase (FLS) is a key enzyme for flavonol synthesis. Result Phenotypic, transcriptional and metabolic data were analyzed, which showed that there was a close relationship between salt stress and flavonoids, and flavonols were significantly upregulated under salt stress. Nine, seven, four, and four FLS genes were identified in Gossypium hirsutum, Gossypium barbadense, Gossypium arboreum, and Gossypium raimondii, respectively. The results of subcellular localization showed that FLS existed in the nucleus and cytoplasmic. Through phylogenetic analysis, 24 FLS genes were divided into three subfamilies. The results of the RNA sequencing showed that the expression of GhFLS genes was mainly induced by salt, drought, low temperature, and heat stress. GhFLS promoter mainly comprised plant hormone response elements and abiotic stress elements, indicating that the GhFLS gene may play a key role in abiotic stress response. The proline contents of pYL156:GhFLS1 was reduced significantly compared to pYL156 under salt stress, thereby reducing the resistance of cotton to salt stress. Conclusion This study will lay a foundation for further study on the antioxidant regulation mechanism of the FLS gene under abiotic stress.

Abstract Background 2-oxoglutarate-dependent dioxygenase (2ODD) is the second largest family of oxidases involved in various oxygenation/hydroxylation reactions in plants. Many members in the family regulate gene transcription, nucleic acid modification/repair and secondary metabolic synthesis. The 2ODD family genes also function in the formation of abundant flavonoids during anthocyanin synthesis, thereby modulating plant development and response to diverse stresses. Results Totally, 379, 336, 205, and 204 2ODD genes were identified in G. barbadense (Gb), G. hirsutum (Gh), G. arboreum (Ga), and G. raimondii (Gb), respectively. The 336 2ODDs in G. hirsutum were divided into 15 subfamilies according to their putative functions. The structural features and functions of the 2ODD members in the same subfamily were similar and evolutionarily conserved. Tandem duplications and segmental duplications served essential roles in the large-scale expansion of the cotton 2ODD family. Ka/Ks values for most of the gene pairs were less than 1, indicating that 2ODD genes undergo strong purifying selection during evolution. Gh2ODDs might act in cotton responses to different abiotic stresses. GhLDOX3 and GhLDOX7, two members of the GhLDOX subfamily from Gh2ODDs, were significantly down-regulated in transcription under alkaline stress. Moreover, the expression of GhLDOX3 in leaves was significantly higher than that in other tissues. These results will provide valuable information for further understanding the evolution mechanisms and functions of the cotton 2ODD genes in the future. Conclusions Genome-wide identification, structure, and evolution and expression analysis of 2ODD genes in Gossypium were carried out. The 2ODDs were highly conserved during evolutionary. Most Gh2ODDs were involved in the regulation of cotton responses to multiple abiotic stresses including salt, drought, hot, cold and alkali.

Abstract Background Abscisic acid (ABA) is an important stress hormone, the changes of abscisic acid content can alter plant tolerance to stress, abscisic acid is crucial for studying plant responses to abiotic stress. The abscisic acid aldehyde oxidase (AAO) plays a vital role in the final step in the synthesis of abscisic acid, therefore, understanding the function of AAO gene family is of great significance for plants to response to abiotic stresses. Result In this study, 6, 8, 4 and 4 AAO genes were identified in four cotton species. According to the structural characteristics of genes and the traits of phylogenetic tree, we divided the AAO gene family into 4 clades. Gene structure analysis showed that the AAO gene family was relatively conservative. The analysis of cis-elements showed that most AAO genes contained cis-elements related to light response and plant hormones. Tissue specificity analysis under NaHCO3 stress showed that GhAAO2 gene was differentially expressed in both roots and leaves. After GhAAO2 gene silencing, the degree of wilting of seedlings was lighter than that of the control group, indicating that GhAAO2 could respond to NaHCO3 stress. Conclusions In this study, the AAO gene family was analyzed by bioinformatics, the response of GhAAO gene to various abiotic stresses was preliminarily verified, and the function of the specifically expressed gene GhAAO2 was further verified. These findings provide valuable information for the study of potential candidate genes related to plant growth and stress.

Abstract Background Under abiotic stress conditions, cotton growth is inhibited and yield losses are severe. Identification of calnexin family members and function analysis under abiotic stress laid the foundation for the screening of stress-related candidate genes. Results A total of 60 CNX family members have been identified in Gossypium hirsutum, G. barbadense, G. arboreum, and G. raimondii, and they were divided into two categories: CNX and CRT genes. Through the construction of a phylogenetic tree, they were subdivided into three classes. Further analysis of chromosome localization, conserved promoters, gene structure and selection under pressure showed that the family members were highly conserved in the evolution process. Analysis of cis-acting elements in the promoter regions showed that CNX family genes contain regulatory elements for growth and development, anaerobic, drought, defense and stress response, and plant hormones. Using RNA-seq data to study the expression pattern of GhCNX genes under cold, hot, salt stress and Polyethylene glycol, it was observed that the gene expression levels changed by different degrees under different stress conditions, indicating that GhCNX members were involved in the regulation of multiple biological stresses. Conclusion This study provides an insight into the members of cotton CNX genes. The results of this study suggested that CNX family members play a role in defense against adversity and provide a foundation for the discovery of stress-related genes.

Abstract Background The cationic amino acid transporters (CAT) play indispensable roles in maintaining metabolic functions, such as synthesis of proteins and nitric oxide (NO), biosynthesis of polyamine, and flow of amino acids, by mediating the bidirectional transport of cationic amino acids in plant cells. Results In this study, we performed a genome-wide and comprehensive study of 79 CAT genes in four species of cotton. Localization of genes revealed that CAT genes reside on the plasma membrane. Seventy-nine CAT genes were grouped into 7 subfamilies by phylogenetic analysis. Structure analysis of genes showed that CAT genes from the same subgroup have similar genetic structure and exon number. RNA-seq and real-time PCR indicated that the expression of most GhCAT genes were induced by salt, drought, cold and heat stresses. Cis-elements analysis of GhCAT promoters showed that the GhCAT genes promoters mainly contained plant hormones responsive elements and abiotic stress elements, which indicated that GhCAT genes may play key roles in response to abiotic stress. Moreover, we also conducted gene interaction network of the GhCAT proteins. Silencing GhCAT10D expression decreased the resistance of cotton to salt stress because of a decrease in the accumulation of NO and proline. Conclusion Our results indicated that CAT genes might be related with salt tolerance in cotton and lay a foundation for further study on the regulation mechanism of CAT genes in cationic amino acids transporting and distribution responsing to abiotic stress.

Abstract Background Carboxylesterase (CXE) is a type of hydrolase with α/β sheet hydrolase activity widely found in animals, plants and microorganisms, which plays an important role in plant growth, development and resistance to stress. Results A total of 72, 74, 39, 38 CXE genes were identified in Gossypium barbadense, Gossypium hirsutum, Gossypium raimondii and Gossypium arboreum, respectively. The gene structure and expression pattern were analyzed. The GBCXE genes were divided into 6 subgroups, and the chromosome distribution of members of the family were mapped. Analysis of promoter cis-acting elements showed that most GBCXE genes contain cis-elements related to plant hormones (GA, IAA) or abiotic stress. These 6 genes we screened out were expressed in the root, stem and leaf tissues. Combined with the heat map, GBCXE49 gene was selected for subcellular locate and confirmed that the protein was expressed in the cytoplasm. Conclusions The collinearity analysis of the CXE genes of the four cotton species in this family indicated that tandem replication played an indispensable role in the evolution of the CXE gene family. The expression patterns of GBCXE gene under different stress treatments indicated that GBCXE gene may significantly participate in the response to salt and alkaline stress through different mechanisms. Through the virus-induced gene silencing technology (VIGS), it was speculated that GBCXE49 gene was involved in the response to alkaline stress in G. barbadense.

Identification, evolution, and expression patterns of BSK (BR signaling kinase) family genes revealed that BSKs participated in the response of cotton to abiotic stress and maintained the growth of cotton in extreme environment. The steroidal hormone brassinosteroids (BR) play important roles in different plant biological processes. This study focused on BSK which were downstream regulatory element of BR, in order to help to decipher the functions of BSKs genes from cotton on growth development and responses to abiotic stresses and lean the evolutionary relationship of cotton BSKs. BSKs are a class of plant-specific receptor-like cytoplasmic kinases involved in BR signal transduction. In this study, bioinformatics methods were used to identify the cotton BSKs gene family at the cotton genome level, and the gene structure, promoter elements, protein structure and properties, gene expression patterns and candidate interacting proteins were analyzed. In the present study, a total of 152 BSKs were identified by a genome-wide search in four cotton species and other 11 plant species, and phylogenetic analysis revealed three evolutionary clades. It was identified that BSKs contain typical PKc and TPR domains, the N-terminus is composed of extended chains and helical structures. Cotton BSKs genes show different expression patterns in different tissues and organs. The gene promoter contains numerous cis-acting elements induced by hormones and abiotic stress, the hormone ABA and Cold-inducing related elements have the highest count, indicating that cotton BSK genes may be regulated by various hormones at different growth stages and involved in the response regulation of cotton to various stresses. The expression analysis of BSKs in cotton showed that the expression levels of GhBSK06, GhBSK10, GhBSK21 and GhBSK24 were significantly increased with salt-inducing. This study is helpful to analyze the function of cotton BSKs genes in growth and development and in response to stress.

Abstract Background The internal NAD(P)H dehydrogenase (NDA) gene family was a member of the NAD(P)H dehydrogenase (ND) gene family, mainly involved in the non-phosphorylated respiratory pathways in mitochondria and played crucial roles in response to abiotic stress. Methods The whole genome identification, structure analysis and expression pattern of NDA gene family were conducted to analyze the NDA gene family. Results There were 51, 52, 26, and 24 NDA genes identified in G. hirsutum, G. barbadense, G. arboreum and G. raimondii, respectively. According to the structural characteristics of genes and traits of phylogenetic tree, we divided the NDA gene family into 8 clades. Gene structure analysis showed that the NDA gene family was relatively conservative. The four Gossypium species had good collinearity, and segmental duplication played an important role in the evolution of the NDA gene family. Analysis of cis-elements showed that most GhNDA genes contained cis-elements related to light response and plant hormones (ABA, MeJA and GA). The analysis of the expression patterns of GhNDA genes under different alkaline stress showed that GhNDA genes were actively involved in the response to alkaline stress, possibly through different molecular mechanisms. By analyzing the existing RNA-Seq data after alkaline stress, it was found that an NDA family gene GhNDA32 was expressed, and then theGhNDA32 was silenced by virus-induced gene silencing (VIGS). By observing the phenotype, we found that the wilting degree of silenced plants was much higher than that of the control plant after alkaline treatment, suggesting that GhNDA32 gene was involved in the response to alkaline stress. Conclusions In this study, GhNDAs participated in response to alkaline stress, especially NaHCO3 stress. It was of great significance for the future research on the molecular mechanism of NDA gene family in responding to abiotic stresses.

Abstract Background Melatonin 2-hydroxylase (M2H) is the first enzyme in the catabolism pathway of melatonin, which catalyzes the production of 2-hydroxymelatonin (2-OHM) from melatonin. The content of 2-hydroxymelatonin in plants is much higher than that of melatonin. So M2H may be a key enzyme in the metabolic pathway of melatonin. Method We conducted a systematic analysis of the M2H gene family in Gossypium hirsutum based on the whole genome sequence by integrating the structural characteristics, phylogenetic relationships, expression profile, and biological stress of the members of the Gossypium hirsutum M2H gene family. Result We identified 265 M2H genes in the whole genome of Gossypium hirsutum, which were divided into 7 clades (clades I-VII) according to phylogenetic analysis. Most M2H members in each group had similar motif composition and gene structure characteristics. More than half of GhM2H members contain ABA-responsive elements and MeJA-responsive elements. Under different stress conditions, the expression levels of the gene changed, indicating that GhM2H members were involved in the regulation of abiotic stress. Some genes in the GhM2H family were involved in regulating melatonin levels in cotton under salt stress, and some genes were regulated by exogenous melatonin. Conclusion This study is helpful to explore the function of GhM2H, the downstream metabolism gene of melatonin in cotton, and lay the foundation for better exploring the molecular mechanism of melatonin improving cotton's response to abiotic stress.

Drought stress is one of the abiotic stresses that limits crop production and greatly affects crop yield. Enhancement of plant stress resistance by NaCl pretreatment has been reported, but the mechanism by which NaCl pretreatment activates cotton stress resistance remains unclear. In this study, upland cotton (Gossypium hirsutum cv H177) was used as the material to conducted the treatments with three replications: 0 Mm NaCl + 0% PEG6000 (Polyethylene glycol), 0 mM NaCl + 15% PEG6000, 50 mM NaCl + 15% PEG6000 to explore the molecular mechanism by which NaCl improves the drought tolerance of cotton. The results showed that pretreatment with 50 mM NaCl could alleviate the adverse effects of PEG on cotton seeds while promoting the elongation of root length. RNA-seq showed that NaCl specifically induced the expression of carotenoid-related genes. By silencing the upstream gene GHLUT2 of lutein synthesis, it was found that the chlorophyll of silenced plants decreased, and leaf wilting was more sensitive to drought. We found that NaCl enhanced the drought resistance of cotton by regulating genes related to the carotenoid and abscisic acid downstream synthesis pathways. This study provides a new reference for the study of drought resistance in cotton and a theoretical basis for the molecular breeding of cotton.

Abstract ZAT (Zinc Finger of Arabidopsis thaliana) proteins are composed of a plant-specific transcription factor family, which play an important role in plant growth, development, and stress resistance. To study the potential function of ZAT family in cotton, the whole genome identification, expression, and structure analysis of ZAT gene family were carried out. In this study, our analysis revealed the presence of 115, 56, 59, and 115 ZAT genes in Gossypium hirsutum, G. raimondii, G. arboreum and G. barbadense, respectively. According to the number of domains and phylogenetic characteristics, we divided ZAT genes of four Gossypium species into 4 different clades, and further divided them into 11 subfamilies. The results of collinearity analysis showed that segmental duplication was the main method to amplify the cotton ZAT genes family. Analysis of cis-elements of promoters indicated that most GhZAT genes contained cis-elements related to plant hormones and abiotic stress. According to heatmap analysis, the expression patterns of GhZAT genes under different stresses indicated that GhZAT genes were significantly involved in the response to cold, heat, salt, and PEG stress, possibly through different mechanisms. Among the highly expressed genes, we cloned a G. hirsutum gene GhZAT67. Through virus-induced gene silencing (VIGS), we found that its expression level decreased significantly after being silenced. Under alkaline treatment, the wilting degree of silenced plants was even greater than the wild type, which proved that GhZAT67 gene was involved in the response to alkaline stress.

Abstract Salinity is an extensive and adverse environmental stress to crop plants across the globe, and a major abiotic constraint responsible for limited crop production threatening the crop security. Soil salinization is a widespread problem across the globe, threatening the crop production and food security. Salinity impairs plant growth and development via reduction in osmotic potential, cytotoxicity due to excessive uptake of ions such as sodium (Na+) and chloride (Cl−), and nutritional imbalance. Cotton, being the most cultivated crop on saline-alkaline soils, it is of great importance to elucidate the mechanisms involved in Na2SO4 tolerance which is still lacking in upland cotton. Zhong 9835, a Na2SO4 resistant cultivar was screened for transcriptomic studies through various levels of Na2SO4 treatments, which results into identification of 3329 differentially expressed genes (DEGs) in roots, stems and leave at 300 mM Na2SO4 stress till 12 h in compared to control. According to gene functional annotation analysis, genes involved in reactive oxygen species (ROS) scavenging system including osmotic stress and ion toxicity were significantly up-regulated, especially GST (glutathione transferase). In addition, analysis for sulfur metabolism, results in to identification of two rate limiting enzymes [APR (Gh_D05G1637) and OASTL (Gh_A13G0863)] during synthesis of GSH from SO4 2−. Furthermore, we also observed a crosstalk of the hormones and TFs (transcription factors) enriched in hormone signal transduction pathway. Genes related to IAA exceeds the rest of hormones followed by ubiquitin related genes which are greater than TFs. The analysis of the expression profiles of diverse tissues under Na2SO4 stress and identification of relevant key hub genes in a network crosstalk will provide a strong foundation and valuable clues for genetic improvements of cotton in response to various salt stresses.

Glycosyltransferases mainly catalyse the glycosylation reaction in living organisms and widely exists in plants. UGTs have been identified from G. raimondii, G. arboreum and G. hirsutum. However, Genome-wide systematic analysis of UGTs superfamily have not been studied in G. barbadense. 752 UGTs were identified from four cotton species and grouped into 18 clades, of which R was newly discovered clades. Most UGTs were clustered at both ends of the chromosome and showed a heterogeneous distribution. UGT proteins were widely distributed in cells, with the highest distribution in chloroplasts. UGTs of the same clade shared similar intron/exon structural features. During evolution, the gene family has undergone strong selection for purification. UGTs were significantly enriched in “transcriptional activity (GO:0016758)” and “metabolic processes (GO:0008152)”. Genes from the same clade differed in function under various abiotic stresses. The analysis of cis-acting element and qRT–PCR may indicate that GHUGTs play important roles in plant growth, development and abiotic stress. We further found that GHUGT74-2 plays an important role under submergence. The study broadens the understanding of UGTs in terms of gene characteristics, evolutionary processes, and gene function in cotton and provides a new way to systematically and globally understand the structure–function relationship of multigene families in the evolutionary process.

Glutamate decarboxylase (GAD) mainly regulated the biosynthesis of γ-aminobutyric acid (GABA) and played an important role in plant growth and stress resistance. To explore the potential function of GAD in cotton growth, the genome-wide identification, structure, and expression analysis of GAD genes were performed in this study. There were 10, 9, 5, and 5 GAD genes identified in G. hirsutum, G. barbadense, G. arboreum, and G. raimondii, respectively. GAD was divided into four clades according to the protein motif composition, gene structure, and phylogenetic relationship. The segmental duplication was the main way of the GAD gene family evolution. Most GhGADs respond to abiotic stress. Clade Ⅲ GAD was induced by Cd2+ stress, especially GhGAD6, and silencing GhGAD6 would lead to more serious Cd2+ poisoning in cotton. The oxidative damage caused by Cd2+ stress was relieved by increasing the GABA content. It was speculated that the decreased expression of GhGAD6 reduced the content of GABA in vivo and caused the accumulation of ROS. This study will further expand our understanding of the relationship between the evolution and function of the GhGAD gene family and provide new genetic resources for cotton breeding under environmental stress and phytoremediation.

Serotonin N-acetyltransferase (SNAT) is a key enzyme in the biosynthesis of melatonin, and plays an important role in the regulation of melatonin synthesis. The study of SNAT is of great significance to understand the function of melatonin. In this study, we analyzed the structural characteristics, phylogenetic relationship, gene structure, expression pattern, evolutionary relationship and stress response of the members of the SNAT gene family in upland cotton through bioinformatics. A putative Serotonin n-acetyltransferase gene GhSNAT3D was identified, and preliminarily function of GhSNAT3D was verified by virus-induced gene silencing. We identified a total of 52 SNAT genes in the whole genome of G. hirsutum, and part of the GhSNATs were regulated by exogenous melatonin. The content of melatonin, antioxidant enzyme activity and Ca2+ content of GhSNAT3D gene silenced plants decreased, and the salt tolerance of GhSNAT3D gene silenced plants was reduced. Exogenous melatonin supplementation restored the salt tolerance of GhSNAT3D gene silenced plants. GhSNAT3D may interact with GhSNAT25D and ASMT to regulate melatonin synthesis. This study provided an important basis for further study on the regulation of melatonin in cotton against abiotic stress.

Plant 3-ketoacyl-CoA synthase (KCS) gene family catalyzed a β ketoacyl-CoA synthase, which was the rate-limiting enzyme for the synthesis of very long chain fatty acids (VLCFAs). Gossypium barbadense was well-known not only for high-quality fiber, which was perceived as a cultivated species of Gossypium. In this study, a total of 131 KCS genes were identified in four cotton species, there were 38, 44, 26, 23 KCS genes in the G. barbadense, the G. hirsutum, the G. arboreum and G. raimondii, respectively. The gene structure and expression pattern were analyzed. GBKCS genes were divided into six subgroups, the chromosome distribution of members of the family were mapped. The prediction of cis-acting elements of the GBKCS gene promoters suggested that the GBKCS genes may be involved in hormone signaling, defense and the stress response. Collinearity analysis on the KCS genes of the four cotton species were formulated. Tandem duplication played an indispensable role in the evolution of the KCS gene family. Specific expression analysis of 20 GBKCS genes indicated that GBKCS gene were widely expressed in the first 25 days of fiber development. Among them, GBKCS3, GBKCS8, GBKCS20, GBKCS34 were expressed at a high level in the initial long-term level of the G. barbadense fiber. This study established a foundation to further understanding of the evolution of KCS genes and analyze the function of GBKCS genes.

Gossypium barbadense is a cultivated cotton not only known for producing superior fiber but also for its salt and alkaline resistance. Here, we used Whole Genome Bisulfite Sequencing (WGBS) technology to map the cytosine methylation of the whole genome of the G. barbadense hypocotyl at single base resolution. The methylation sequencing results showed that the mapping rates of the three samples were 75.32, 77.54, and 77.94%, respectively. In addition, the Bisulfite Sequence (BS) conversion rate was 99.78%. Approximately 71.03, 53.87, and 6.26% of the cytosine were methylated at CG, CHG, and CHH sequence contexts, respectively. A comprehensive analysis of DNA methylation and transcriptome data showed that the methylation level of the promoter region was a positive correlation in the CHH context. Saline-alkaline stress was related to the methylation changes of many genes, transcription factors (TFs) and transposable elements (TEs), respectively. We explored the regulatory mechanism of DNA methylation in response to salt and alkaline stress during cotton hypocotyl elongation. Our data shed light into the relationship of methylation regulation at the germination stage of G. barbadense hypocotyl cell elongation and salt-alkali treatment. The results of this research help understand the early growth regulation mechanism of G. barbadense in response to abiotic stress.

As one of the cash crops, cotton is facing the threat of abiotic stress during its growth and development. It has been reported that melatonin is involved in plant defense against salt stress, but whether melatonin can improve cotton salt tolerance and its molecular mechanism remain unclear. We investigated the role of melatonin in cotton salt tolerance by silencing melatonin synthesis gene and exogenous melatonin application in upland cotton. In this study, applicating of melatonin can improve salt tolerance of cotton seedlings. The content of endogenous melatonin was different in cotton varieties with different salt tolerance. The inhibition of melatonin biosynthesis related genes and endogenous melatonin content in cotton resulted in the decrease of antioxidant enzyme activity, Ca2+ content and salt tolerance of cotton. To explore the protective mechanism of exogenous melatonin against salt stress by RNA-seq analysis. Melatonin played an important role in the resistance of cotton to salt stress, improved the salt tolerance of cotton by regulating antioxidant enzymes, transcription factors, plant hormones, signal molecules and Ca2+ signal transduction. This study proposed a regulatory network for melatonin to regulate cotton’s response to salt stress, which provided a theoretical basis for improving cotton’s salt tolerance.

Glutamate decarboxylase (GAD) mainly regulated the biosynthesis of γ-aminobutyric acid (GABA) and played an important role in plant growth and stress resistance. To explore the potential function of GAD in cotton growth, the genome-wide identification, structure, and expression analysis of GAD genes were performed in this study. There were 10, 9, 5, and 5 GAD genes identified in G. hirsutum, G. barbadense, G. arboreum, and G. raimondii, respectively. GAD was divided into four clades according to the protein motif composition, gene structure, and phylogenetic relationship. The segmental duplication was the main way of the GAD gene family evolution. Most GhGADs respond to abiotic stress. Clade III GAD was induced by Cd2+ stress, especially GhGAD6, and silencing GhGAD6 would lead to more serious Cd2+ poisoning in cotton. The oxidative damage caused by Cd2+ stress was relieved by increasing the GABA content. It was speculated that the decreased expression of GhGAD6 reduced the content of GABA in vivo and caused the accumulation of ROS. This study will further expand our understanding of the relationship between the evolution and function of the GhGAD gene family and provide new genetic resources for cotton breeding under environmental stress and phytoremediation. [ABSTRACT FROM AUTHOR]