Genome-wide characterization of DNA methyltransferase family genes implies GhDMT6 improving tolerance of salt and drought on cotton.

BACKGROUND: DNA methylation is an important epigenetic mode of genomic DNA modification and plays a vital role in maintaining epigenetic content and regulating gene expression. Cytosine-5 DNA methyltransferase (C5-MTase) are the key enzymes in the process of DNA methylation. However, there is no systematic analysis of the C5-MTase in cotton so far, and the function of DNMT2 genes has not been studied. METHODS: In this study, the whole genome of cotton C5-MTase coding genes was identified and analyzed using a bioinformatics method based on information from the cotton genome, and the function of GhDMT6 was further validated by VIGS experiments and subcellular localization analysis. RESULTS: 33 C5-MTases were identified from three cotton genomes, and were divided into four subfamilies by systematic evolutionary analysis. After the protein domain alignment of C5-MTases in cotton, 6 highly conserved motifs were found in the C-terminus of 33 proteins involved in methylation modification, which indicated that C5-MTases had a basic catalytic methylation function. These proteins were divided into four classes based on the N-terminal difference, of which DNMT2 lacks the N-terminal regulatory domain. The expression of C5-MTases in different parts of cotton was different under different stress treatments, which indicated the functional diversity of cotton C5-MTase gene family. Among the C5-MTases, the GhDMT6 had a obvious up-regulated expression. After silencing GhDMT6 with VIGS, the phenotype of cotton seedlings under different stress treatments showed a significant difference. Compared with cotton seedlings that did not silence GhDMT6, cotton seedlings silencing GhDMT6 showed significant stress resistance. CONCLUSION: The results show that C5-MTases plays an important role in cotton stress response, which is beneficial to further explore the function of DNMT2 subfamily genes.

Systematic analysis and expression of Gossypium ATG8 family reveals the roles of GhATG8f responding to salt stress in cotton.

KEY MESSAGE: Comprehensive analysis of Gossypium ATG8 family indicates that GhATG8f could improve salt tolerance of cotton by increasing SOD, POD and CAT activity and proline accumulation. In plants, autophagy is regulated by several genes that play important roles in initiating and controlling the process. ATG8, functioning as a protein similar to ubiquitin, is involved in crucial tasks throughout the autophagosome formation process. In this research, we conducted an extensive and all-encompassing investigation of 64 ATG8 genes across four varieties of cotton. According to the subcellular localization prediction results, 49 genes were found in the cytoplasm, 6 genes in the chloroplast, 1 gene in the peroxisome, 5 genes in the nucleus, and 3 genes in the extracellular region. Phylogenetic analysis categorized a total of 5 subfamilies containing sixty-four ATG8 genes. The expression of the majority of GhATG8 genes was induced by salt, drought, cold, and heat stresses, as revealed by RNA-seq and real-time PCR. Analysis of cis-elements in the promoters of GhATG8 genes revealed the predominant presence of responsive elements for plant hormones and abiotic stress, suggesting that GhATG8 genes might have significant functions in abiotic stress response. Furthermore, we additionally performed a gene interaction network analysis for the GhATG8 proteins. The salt stress resistance of cotton was reduced due to the downregulation of GhATG8f expression, resulting in decreased activity of CAT, SOD, and POD enzymes, as well as decreased fresh weight and proline accumulation. In summary, our research is the initial exploration of ATG8 gene components in cotton, providing a basis for future investigations into the regulatory mechanisms of ATG8 genes in autophagy and their response to abiotic stress.

GhLCYε-3 characterized as a lycopene cyclase gene responding to drought stress in cotton.

Drought stress significantly affects crop productivity. Carotenoids are essential photosynthetic pigment for plants, bacteria, and algae, with signaling and antioxidant functions. Lutein is a crucial branch product in the carotenoid synthesis pathway, which effectively improves the stress tolerance of higher plants. lycopene cyclase, a central enzyme for lutein synthesis, holds great significance in regulating lutein production. This research establishes a correlation between lutein content and stress resistance by measuring the drought resistance and lutein content of various cotton materials. To identify which crucial genes are associated with lutein, the lycopene cyclase family (LCYs) was analyzed. The research found that LCYs form a highly conserved family divided into two subfamilies, LCY-ε (lycopene ε-cyclase) and LCY-ß (lycopene ß-cyclase). Most members of the LCY family contain photoresponsive elements and abscisic acid elements. qRT-PCR demonstrates showed that most genes responded positively to drought stress, and GhLCYε-3 was expressed significantly differently under drought stress. Virus-induced gene silencing (VIGS) assay showed that the content of GhLCYε-3 was significantly increased with MDA and PRO, and the contents of chlorophyll and lutein were significantly decreased in pYL156 plants. The decrease in GhLCYε-3 expression is speculated to lead to reduced lutein content in vivo, resulting in the accumulation of reactive oxygen species (ROS) and decreased drought tolerance. This research enriched the understanding of LCY gene family and lutein function, and provided a new reference for cotton planting in arid areas. Synopsis: Lycopene cyclase plays an important role in enhancing the ability of scavenging ROS and drought resistance of plants.

Structural and functional characterization of exopolysaccharide from Leuconostoc citreum BH10 discovered in birch sap.

In this study, Leuconostoc citreum BH10, an endophytic strain, was isolated from aseptically collected xylem sap of birch for the first time, and its exopolysaccharide (LCEPS) production was up to 46.31 g/L in glucan producing medium. The produced LCEPS was purified to obtain two water-soluble fractions, named as LCEPS-1 and LCEPS-2, respectively. The major fraction LCEPS-1 was characterized to be comprised of glucose with average molecular weight of 6.34 × 106 Da. The structure of LCEPS-1 was investigated by spectroscopy analysis, which revealed that LCEPS-1 was identified with containing 90.45 % α-(1,6) linkages in the main chains and 9.55 % α-(1,3) branch linkages. The scanning electron microscope results demonstrated that the dried LCEPS-1 appeared porous surface overlaid with an irregular glittering. The water solubility index (WSI) and water holding capacity (WHC) of LCEPS-1 were 88.02 ± 1.69 % and 241.43 ± 6.38 %, respectively. Besides, it exhibited high thermal stability as well as fine antioxidant activities. Taken together, the results indicated that LCEPS-1 could have good potentiality to be applied in fields of foods, cosmetics, nutraceuticals and pharmaceutical industries as the natural agent.

Enhancing stimulation of cyaniding, GhLDOX3 activates reactive oxygen species to regulate tolerance of alkalinity negatively in cotton.

Anthocyanins belong to flavonoid secondary metabolites that act as plant pigments to give flowers and fruits different colors and as "scavengers" of reactive oxygen species (ROS) to protect plants from abiotic and biotic stresses. Few studies linked anthocyanins to alkaline resistance so far. In this study, anthocyanin synthesis-related gene leucoanthocyanidin dioxygenase (LDOX) was screened as a candidate gene to explore its relationship with alkali stress. The results found that pYL156: GhLDOX3 lines treated with 50 mM Na2CO3 (pH 11.11) for 24 h showed a significant increase in peroxidase (POD) activity, a decrease in total anthocyanin content and an increase in cyanidin content and a decrease in ROS accumulation compared to pYL156. The overexpressed (OE) lines, ldox mutant and wild-type (WT) lines in Arabidopsis were treated with 50 mM Na2CO3, 100 mM Na2CO3 and 150 mM Na2CO3 for 8 d, respectively. The wilted degree of the OE lines was more severe than WT lines, and less severe in the mutant lines in the 150 mM Na2CO3 treatment. After treatment, the expression levels of AtCAT and AtGSH genes related to antioxidant system in OE lines were significantly lower than in WT, and the expression levels of AtCAT and AtGSH in mutant lines were significantly higher than in WT. In conclusion, the above results suggest GhLDOX3 played a negative regulatory role in the mechanism of resisting Na2CO3 stress. Therefore, it can be considered in cotton breeding to improve the alkali tolerance of cotton by regulating the expression of related genes.

Analysis of the histidine kinase gene family and the role of GhHK8 in response to drought tolerance in cotton.

As an important member of the two-component system (TCS), histidine kinases (HKs) play important roles in various plant developmental processes and signal transduction in response to a wide range of biotic and abiotic stresses. So far, the HK gene family has not been investigated in Gossypium. In this study, a total of 177 HK gene family members were identified in cotton. They were further divided into seven groups, and the protein characteristics, genetic relationship, gene structure, chromosome location, collinearity, and cis-elements identification were comprehensively analyzed. Whole genome duplication (WGD) / segmental duplication may be the reason why the number of HK genes doubled in tetraploid Gossypium species. Expression analysis revealed that most cotton HK genes were mainly expressed in the reproductive organs and the fiber at initial stage. Gene expression analysis revealed that HK family genes are involved in cotton abiotic stress, especially drought stress and salt stress. In addition, gene interaction networks showed that HKs were involved in the regulation of cotton abiotic stress, especially drought stress. VIGS experiments have shown that GhHK8 is a negative regulatory factor in response to drought stress. Our systematic analysis provided insights into the characteristics of the HK genes in cotton and laid a foundation for further exploring their potential in drought stress resistance in cotton.

Fabrication of flexible accelerated-wound-healing chitosan/dopamine-based bilayer hydrogels for strain sensors.

Flexible conductive hydrogels have great potential for healthcare and human motion sensing. However, it is difficult to simultaneously achieve conductive hydrogel epidermal sensors with reliable adhesion capabilities and excellent sensing properties, as well as accelerated wound healing performance in wearable hydrogels. Here, an epidermal sensor with excellent adhesion (0.6 kPa) and tensile strain (218.0 %) properties was assembled from an easy-to-prepare bilayer antimicrobial hydrogel, which effectively accelerates wound healing, as well as for human motion sensing. The upper hydrogel layer was composed of PVA, which could effectively enhance the mechanical properties of the bilayer hydrogel. The lower hydrogel layer consisted of polyacrylamide (PAm) and chitosan-dopamine (CC-DA). PAm with good adhesion properties adhered effectively to the skin surface. CC-DA not only had adhesion properties, but also has good antibacterial effects. It inhibited the growth of bacteria, which assisted in wound healing and infection prevention. Therefore, the design of the bilayer hydrogel combined the mechanical enhancement of PVA with the adhesion properties and antimicrobial effect of PAm and CC-DA to provide better wound repair. In addition, the double-layer hydrogel with good electrical conductivity (1.65 S·m-1) could sensitively monitor the tiny electrophysiological signals emitted by the human body during exercise rehabilitation training.

GhIMP10D, an inositol monophosphates family gene, enhances ascorbic acid and antioxidant enzyme activities to confer alkaline tolerance in Gossypium hirsutum L.

BACKGROUND: Inositol monophosphates (IMP) are key enzymes in the ascorbic acid (AsA) synthesis pathways, which play vital roles in regulating plant growth and development and stresses tolerance. To date, no comprehensive analysis of the expression profile of IMP genes and their functions under abiotic stress in cotton has been reported. RESULTS: In this study, the genetic characteristics, phylogenetic evolution, cis-acting elements and expression patterns of IMP gene family in cotton were systematically analyzed. A total of 28, 27, 13 and 13 IMP genes were identified in Gossypium hirsutum (G. hirsutum), Gossypium barbadense (G. barbadense), Gossypium arboreum (G. arboreum), and Gossypium raimondii (G. raimondii), respectively. Phylogenetic analysis showed that IMP family genes could cluster into 3 clades. Structure analysis of genes showed that GhIMP genes from the same subgroup had similar genetic structure and exon number. And most GhIMP family members contained hormone-related elements (abscisic acid response element, MeJA response element, gibberellin response element) and stress-related elements (low temperature response element, defense and stress response element, wound response element). After exogenous application of abscisic acid (ABA), some GhIMP genes containing ABA response elements positively responded to alkaline stress, indicating that ABA response elements played an important role in response to alkaline stress. qRT-PCR showed that most of GhIMP genes responded positively to alkaline stress, and GhIMP10D significantly upregulated under alkaline stress, with the highest up-regulated expression level. Virus-induced gene silencing (VIGS) experiment showed that compared with 156 plants, MDA content of pYL156:GhIMP10D plants increased significantly, while POD, SOD, chlorophyII and AsA content decreased significantly. CONCLUSIONS: This study provides a thorough overview of the IMP gene family and presents a new perspective on the evolution of this gene family. In particular, some IMP genes may be involved in alkaline stress tolerance regulation, and GhIMP10D showed high expression levels in leaves, stems and roots under alkaline stress, and preliminary functional verification of GhIMP10D gene suggested that it may regulate tolerance to alkaline stress by regulating the activity of antioxidant enzymes and the content of AsA. This study contributes to the subsequent broader discussion of the structure and alkaline resistance of IMP genes in cotton.

Stem cell-based therapeutic strategies for rotator cuff tendinopathy.

Rotator cuff tendinopathy is a common musculoskeletal disorder that imposes significant health and economic burden. Stem cell therapy has brought hope for tendon healing in patients with final stage rotator cuff tendinopathy. Some clinical trials have confirmed the effectiveness of stem cell therapy for rotator cuff tendinopathy, but its application has not been promoted and approved. There are still many issues that should be solved prior to using stem cell therapy in clinical applications. The optimal source and dose of stem cells for rotator cuff tendinopathy should be determined. We also proposed novel prospective approaches that can overcome cell population heterogeneity and standardize patient types for stem cell applications. The translational potential of this article: This review explores the optimal sources of stem cells for rotator cuff tendinopathy and the principles for selecting stem cell dosages. Key strategies are provided for stem cell population standardization and recipient selection.

Melatonin receptor, GhCAND2-D5 motivated responding to NaCl signaling in cotton.

As a receptor for plant melatonin, CAND2/PMTR plays an important role in melatonin signaling. Most of the CANDs are membrane proteins and play indispensable roles in signal transduction. In this study, the CANDs from four cotton species were characterized, and the phylogenetic relationships, expression patterns, stress responses of cotton CANDs were analyzed by bioinformatics. Through the analysis of phylogenetic and protein structure, it was found that the CANDs in clade Ⅱ might function as cotton melatonin receptors, and most of the GhCANDs in clade Ⅱ were induced by melatonin. A putative cotton melatonin receptor, GhCAND2-D5, was functionally probed by gene silencing. The plants with silenced expression of this gene exhibited decreased salt tolerance. Protein interaction prediction identified that GhCAND2-D5 interacted with several membrane proteins and played an important role in melatonin signaling. This study provided a theoretical reference for further investigation of melatonin signaling in cotton.

GhCYS2 governs the tolerance against cadmium stress by regulating cell viability and photosynthesis in cotton.

Cysteine, an early sulfur-containing compound in plants, is of significant importance in sulfur metabolism. CYS encodes cysteine synthetase that further catalyzes cysteine synthesis. In this investigation, CYS genes, identified from genome-wide analysis of Gossypium hirsutum bioinformatically, led to the discovery of GhCYS2 as the pivotal gene responsible for Cd2+ response. The silencing of GhCYS2 through virus-induced gene silencing (VIGS) rendered plants highly susceptible to Cd2+ stress. Silencing GhCYS2 in plants resulted in diminished levels of cysteine and glutathione while leading to the accumulation of MDA and ROS within cells, thereby impeding the regular process of photosynthesis. Consequently, the stomatal aperture of leaves decreased, epidermal cells underwent distortion and deformation, intercellular connections are dramatically disrupted, and fissures manifested between cells. Ultimately, these detrimental effected culminating in plant wilting and a substantial reduction in biomass. The association established between Cd2+ and cysteine in this investigation offered a valuable reference point for further inquiry into the functional and regulatory mechanisms of cysteine synthesis genes.

Changes in terpene biosynthesis and submergence tolerance in cotton.

BACKGROUND: Flooding is among the most severe abiotic stresses in plant growth and development. The mechanism of submergence tolerance of cotton in response to submergence stress is unknown. RESULTS: The transcriptome results showed that a total of 6,893 differentially expressed genes (DEGs) were discovered under submergence stress. Gene Ontology (GO) enrichment analysis showed that DEGs were involved in various stress or stimulus responses. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis indicated that DEGs related to plant hormone signal transduction, starch and sucrose metabolism, glycolysis and the biosynthesis of secondary metabolites were regulated by submergence stress. Eight DEGs related to ethylene signaling and 3 ethylene synthesis genes were identified in the hormone signal transduction. For respiratory metabolism, alcohol dehydrogenase (ADH, GH_A02G0728) and pyruvate decarboxylase (PDC, GH_D09G1778) were significantly upregulated but 6-phosphofructokinase (PFK, GH_D05G0280), phosphoglycerate kinase (PGK, GH_A01G0945 and GH_D01G0967) and sucrose synthase genes (SUS, GH_A06G0873 and GH_D06G0851) were significantly downregulated in the submergence treatment. Terpene biosynthetic pathway-related genes in the secondary metabolites were regulated in submergence stress. CONCLUSIONS: Regulation of terpene biosynthesis by respiratory metabolism may play a role in enhancing the tolerance of cotton to submergence under flooding. Our findings showed that the mevalonate pathway, which occurs in the cytoplasm of the terpenoid backbone biosynthesis pathway (ko00900), may be the main response to submergence stress.

Biomimetic Scaffolds for Tendon Tissue Regeneration.

Tendon tissue connects muscle to bone and plays crucial roles in stress transfer. Tendon injury remains a significant clinical challenge due to its complicated biological structure and poor self-healing capacity. The treatments for tendon injury have advanced significantly with the development of technology, including the use of sophisticated biomaterials, bioactive growth factors, and numerous stem cells. Among these, biomaterials that the mimic extracellular matrix (ECM) of tendon tissue would provide a resembling microenvironment to improve efficacy in tendon repair and regeneration. In this review, we will begin with a description of the constituents and structural features of tendon tissue, followed by a focus on the available biomimetic scaffolds of natural or synthetic origin for tendon tissue engineering. Finally, we will discuss novel strategies and present challenges in tendon regeneration and repair.

The Biological Roles of Puccinia striiformis f. sp. tritici Effectors during Infection of Wheat.

Puccinia striiformis f. sp. tritici (Pst) is the causative agent of wheat stripe rust, which can lead to a significant loss in annual wheat yields. Therefore, there is an urgent need for a deeper comprehension of the basic mechanisms underlying Pst infection. Effectors are known as the agents that plant pathogens deliver into host tissues to promote infection, typically by interfering with plant physiology and biochemistry. Insights into effector activity can significantly aid the development of future strategies to generate disease-resistant crops. However, the functional analysis of Pst effectors is still in its infancy, which hinders our understanding of the molecular mechanisms of the interaction between Pst and wheat. In this review, we summarize the potential roles of validated and proposed Pst effectors during wheat infection, including proteinaceous effectors, non-coding RNAs (sRNA effectors), and secondary metabolites (SMs effectors). Further, we suggest specific countermeasures against Pst pathogenesis and future research directions, which may promote our understanding of Pst effector functions during wheat immunity attempts.

Characterization and gene expression patterns analysis implies BSK family genes respond to salinity stress in cotton.

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.

Combined transcriptomic and metabolomic analyses elucidate key salt-responsive biomarkers to regulate salt tolerance in cotton.

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.

Allergenicity evaluation of five types of commercial food-derived oligopeptide products.

Food-derived oligopeptides are low in molecular weight and have a variety of biological activities. However, many of them are derived from allergens, which may pose a threat to allergic consumers. This study aimed to assess the allergenicity of five types of oligopeptides industrially derived from allergenic foods (soybean, wheat, oyster, salmon skin, and haddock skin). Referring to the decision tree proposed by the FAO/WHO for the allergenicity evaluation of genetically modified food, we included three kinds of bioinformatic tools (AlgPred, AllerCatPro, and AllerTOP), SDS-PAGE, ELISA, cell and animal experiments in this study. The variation of effector levels (IL-4, IFN-γ, His, and mMCP-1) was determined in mouse models. The results revealed that the oligopeptides were all predicted to contain allergenic peptides, of which the soybean one had the highest number of allergenic peptides. Moreover, there were anti-enzymatic peptides present in the soybean oligopeptide. Unexpectedly, the serum IgE binding ability of the peptides was lower than the positive threshold. In addition, no statistical divergence was found between the oligopeptide groups and the control groups in the effector and ß-hexosaminidase levels. Overall, the five types of oligopeptides, though derived from allergens, had low allergenicity.

Integrating Widely Targeted Lipidomics and Transcriptomics Unravels Aberrant Lipid Metabolism and Identifies Potential Biomarkers of Food Allergies in Rats.

SCOPE: Oral food challenges (OFCs) are currently the gold standard for determining the clinical reactivity of food allergy (FA) but are time-consuming, expensive, and risky. To screen novel peripheral biomarkers of FA and characterize the aberrant lipid metabolism in serum, 24 rats are divided into four groups: peanut, milk, and shrimp allergy (PA, MA, and SA, respectively) and control groups, with six rats in each group, and used for widely targeted lipidomics and transcriptomics analysis. METHODS AND RESULTS: Widely targeted lipidomics reveal 144, 162, and 206 differentially accumulated lipids in PA, MA, and SA groups, respectively. The study integrates widely targeted lipidomics and transcriptomics and identifies abnormal lipid metabolism correlated with widespread differential accumulation of diverse lipids (including triacylglycerol, diacylglycerol, sphingolipid, and glycerophospholipid) in PA, MA, and SA. Simplified random forest classifier is constructed through five repetitions of 10-fold cross-validation to distinguish allergy from control. A subset of 15 lipids as potential biomarkers allows for more reliable and more accurate prediction of FA. Independent replication validates the reproducibility of potential biomarkers. CONCLUSION: The results reveal the major abnormalities in lipid metabolism and suggest the potential role of lipids as novel molecular signatures for FA.

Systematic analysis and expression of Gossypium 2ODD superfamily highlight the roles of GhLDOXs responding to alkali and other abiotic stress in cotton.

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.

Raffinose degradation-related gene GhAGAL3 was screened out responding to salinity stress through expression patterns of GhAGALs family genes.

A-galactosidases (AGALs), the oligosaccharide (RFO) catabolic genes of the raffinose family, play crucial roles in plant growth and development and in adversity stress. They can break down the non-reducing terminal galactose residues of glycolipids and sugar chains. In this study, the whole genome of AGALs was analyzed. Bioinformatics analysis was conducted to analyze members of the AGAL family in Gossypium hirsutum, Gossypium arboreum, Gossypium barbadense, and Gossypium raimondii. Meanwhile, RT-qPCR was carried out to analyze the expression patterns of AGAL family members in different tissues of terrestrial cotton. It was found that a series of environmental factors stimulated the expression of the GhAGAL3 gene. The function of GhAGAL3 was verified through virus-induced gene silencing (VIGS). As a result, GhAGAL3 gene silencing resulted in milder wilting of seedlings than the controls, and a significant increase in the raffinose content in cotton, indicating that GhAGAL3 responded to NaCl stress. The increase in raffinose content improved the tolerance of cotton. Findings in this study lay an important foundation for further research on the role of the GhAGAL3 gene family in the molecular mechanism of abiotic stress resistance in cotton.