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.

A prolific and robust whole-genome genotyping method using PCR amplification via primer-template mismatched annealing.

Whole-genome genotyping methods are important for breeding. However, it has been challenging to develop a robust method for simultaneous foreground and background genotyping that can easily be adapted to different genes and species. In our study, we accidently discovered that in adapter ligation-mediated PCR, the amplification by primer-template mismatched annealing (PTMA) along the genome could generate thousands of stable PCR products. Based on this observation, we consequently developed a novel method for simultaneous foreground and background integrated genotyping by sequencing (FBI-seq) using one specific primer, in which foreground genotyping is performed by primer-template perfect annealing (PTPA), while background genotyping employs PTMA. Unlike DNA arrays, multiple PCR, or genome target enrichments, FBI-seq requires little preliminary work for primer design and synthesis, and it is easily adaptable to different foreground genes and species. FBI-seq therefore provides a prolific, robust, and accurate method for simultaneous foreground and background genotyping to facilitate breeding in the post-genomics era.

Systematic analysis of Histidine photosphoto transfer gene family in cotton and functional characterization in response to salt and around tolerance.

BACKGROUND: Phosphorylation regulated by the two-component system (TCS) is a very important approach signal transduction in most of living organisms. Histidine phosphotransfer (HP) is one of the important members of the TCS system. Members of the HP gene family have implications in plant stresses tolerance and have been deeply studied in several crops. However, upland cotton is still lacking with complete systematic examination of the HP gene family. RESULTS: A total of 103 HP gene family members were identified. Multiple sequence alignment and phylogeny of HPs distributed them into 7 clades that contain the highly conserved amino acid residue "XHQXKGSSXS", similar to the Arabidopsis HP protein. Gene duplication relationship showed the expansion of HP gene family being subjected with whole-genome duplication (WGD) in cotton. Varying expression profiles of HPs illustrates their multiple roles under altering environments particularly the abiotic stresses. Analysis is of transcriptome data signifies the important roles played by HP genes against abiotic stresses. Moreover, protein regulatory network analysis and VIGS mediated functional approaches of two HP genes (GhHP23 and GhHP27) supports their predictor roles in salt and drought stress tolerance. CONCLUSIONS: This study provides new bases for systematic examination of HP genes in upland cotton, which formulated the genetic makeup for their future survey and examination of their potential use in cotton production.

Comprehensive genomic characterization of cotton cationic amino acid transporter genes reveals that GhCAT10D regulates salt tolerance.

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.

Genome-wide expression analysis of carboxylesterase (CXE) gene family implies GBCXE49 functional responding to alkaline stress in cotton.

BACKGROUND: Carboxylesterase (CXE) is a type of hydrolase with α/ß 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.

Structure and character analysis of cotton response regulator genes family reveals that GhRR7 responses to draught stress.

BACKGROUND: Cytokinin signal transduction is mediated by a two-component system (TCS). Two-component systems are utilized in plant responses to hormones as well as to biotic and abiotic environmental stimuli. In plants, response regulatory genes (RRs) are one of the main members of the two-component system (TCS). METHOD: From the aspects of gene structure, evolution mode, expression type, regulatory network and gene function, the evolution process and role of RR genes in the evolution of the cotton genome were analyzed. RESULT: A total of 284 RR genes in four cotton species were identified. Including 1049 orthologous/paralogous gene pairs were identified, most of which were whole genome duplication (WGD). The RR genes promoter elements contain phytohormone responses and abiotic or biotic stress-related cis-elements. Expression analysis showed that RR genes family may be negatively regulate and involved in salt stress and drought stress in plants. Protein regulatory network analysis showed that RR family proteins are involved in regulating the DNA-binding transcription factor activity (COG5641) pathway and HP kinase pathways. VIGS analysis showed that the GhRR7 gene may be in the same regulatory pathway as GhAHP5 and GhPHYB, ultimately negatively regulating cotton drought stress by regulating POD, SOD, CAT, H2O2 and other reactive oxygen removal systems. CONCLUSION: This study is the first to gain insight into RR gene members in cotton. Our research lays the foundation for discovering the genes related to drought and salt tolerance and creating new cotton germplasm materials for drought and salt tolerance.

Cotton transcriptome analysis reveals novel biological pathways that eliminate reactive oxygen species (ROS) under sodium bicarbonate (NaHCO3) alkaline stress.

Alkaline stress is one of the abiotic stresses limiting cotton production. Though RNA-Seq analyses, have been conducted to investigate genome-wide gene expression in response to alkaline stress in plants, the response of sodium bicarbonate (NaHCO3) stress-related genes in cotton has not been reported. To explore the mechanisms of cotton response to this alkaline stress, we used next-generation sequencing (NGS) technology to study transcriptional changes of cotton under NaHCO3 alkaline stress. A total of 18,230 and 11,177 differentially expressed genes (DEGs) were identified in cotton roots and leaves, respectively. Gene ontology (GO) analysis indicated the enrichment of DEGs involved in various stimuli or stress responses. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis showed that DEGs associated with plant hormone signal transduction, amino acid biosynthesis, and biosynthesis of secondary metabolites were regulated in response to the NaHCO3 stress. We further analyzed genes enriched in secondary metabolic pathways and found that secondary metabolites were regulated to eliminate the reactive oxygen species (ROS) and improve the cotton tolerance to the NaHCO3 stress. In this study, we learned that the toxic effect of NaHCO3 was more profound than that of NaOH at the same pH. Thus, Na+, HCO3- and pH had a great impact on the growth of cotton plant. The novel biological pathways and candidate genes for the cotton tolerance to NaHCO3 stress identified from the study would be useful in the genetic improvement of the alkaline tolerance in cotton.

Genome-wide identification and expression analysis of Raffinose synthetase family in cotton.

BACKGROUND: The Raffinose synthetase (RAFS) genes superfamily is critical for the synthesis of raffinose, which accumulates in plant leaves under abiotic stress. However, it remains unclear whether RAFS contributes to resistance to abiotic stress in plants, specifically in the Gossypium species. RESULTS: In this study, we identified 74 RAFS genes from G. hirsutum, G. barbadense, G. arboreum and G. raimondii by using a series of bioinformatic methods. Phylogenetic analysis showed that the RAFS gene family in the four Gossypium species could be divided into four major clades; the relatively uniform distribution of the gene number in each species ranged from 12 to 25 based on species ploidy, most likely resulting from an ancient whole-genome polyploidization. Gene motif analysis showed that the RAFS gene structure was relatively conservative. Promoter analysis for cis-regulatory elements showed that some RAFS genes might be regulated by gibberellins and abscisic acid, which might influence their expression levels. Moreover, we further examined the functions of RAFS under cold, heat, salt and drought stress conditions, based on the expression profile and co-expression network of RAFS genes in Gossypium species. Transcriptome analysis suggested that RAFS genes in clade III are highly expressed in organs such as seed, root, cotyledon, ovule and fiber, and under abiotic stress in particular, indicating the involvement of genes belonging to clade III in resistance to abiotic stress. Gene co-expressed network analysis showed that GhRFS2A-GhRFS6A, GhRFS6D, GhRFS7D and GhRFS8A-GhRFS11A were key genes, with high expression levels under salt, drought, cold and heat stress. CONCLUSION: The findings may provide insights into the evolutionary relationships and expression patterns of RAFS genes in Gossypium species and a theoretical basis for the identification of stress resistance materials in cotton.

Genome-wide identification and characteristic analysis of the downstream melatonin metabolism gene GhM2H in Gossypium hirsutum L.

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.

Transcriptome Analysis Reveals Cotton (Gossypium hirsutum) Genes That Are Differentially Expressed in Cadmium Stress Tolerance.

High concentrations of heavy metals in the soil should be removed for environmental safety. Cadmium (Cd) is a heavy metal that pollutes the soil when its concentration exceeds 3.4 mg/kg. Although the potential use of cotton to remediate heavy Cd-polluted soils is known, little is understood about the molecular mechanisms of Cd tolerance. In this study, transcriptome analysis was used to identify Cd tolerance genes and their potential mechanisms in cotton. We exposed cotton plants to excess Cd and identified 4627 differentially expressed genes (DEGs) in the root, 3022 DEGs in the stem and 3854 DEGs in the leaves through RNA-Seq analysis. Among these genes were heavy metal transporter coding genes (ABC, CDF, HMA, etc.), annexin genes and heat shock genes (HSP), amongst others. Gene ontology (GO) analysis showed that the DEGs were mainly involved in the oxidation⁻reduction process and metal ion binding. The DEGs were mainly enriched in two pathways, the influenza A and pyruvate pathway. GhHMAD5, a protein containing a heavy-metal binding domain, was identified in the pathway to transport or to detoxify heavy metal ions. We constructed a GhHMAD5 overexpression system in Arabidopsis thaliana that showed longer roots compared to control plants. GhHMAD5-silenced cotton plants showed more sensitivity to Cd stress. The results indicate that GhHMAD5 is involved in Cd tolerance, which gives a preliminary understanding of the Cd tolerance mechanism in upland cotton. Overall, this study provides valuable information for the use of cotton to remediate soils polluted with Cd and potentially other heavy metals.

Genome wide identification and characterization of fertility associated novel CircRNAs as ceRNA reveal their regulatory roles in sheep fecundity.

Reproductive traits play a vital role in determining the production efficiency of sheep. Maximizing the production is of paramount importance for breeders worldwide due to the growing population. Circular RNAs (circRNAs) act as miRNA sponges by absorbing miRNA activity through miRNA response elements (MREs) and participate in ceRNA regulatory networks (ceRNETs) to regulate mRNA expression. Despite of extensive research on role of circRNAs as miRNA sponges in various species, their specific regulatory roles and mechanism in sheep ovarian tissue are still not well understood. In this study, we performed whole genome sequencing of circRNAs, miRNA and mRNA employing bioinformatic techniques on ovine tissues of two contrasting sheep breeds "Small tail Han (X_LC) and Dolang sheep (D_LC)", which results into identification of 9,878 circRNAs with a total length of 23,522,667 nt and an average length of 2,381.32 nt. Among them, 44 differentially expressed circRNAs (DECs) were identified. Moreover, correlation between miRNA-mRNA and lncRNA-miRNA provided us with to prediction of miRNA binding sites on nine differentially expressed circRNAs and 165 differentially expressed mRNAs using miRanda. miRNA-mRNA and lncRNA-miRNA pairs with negative correlation were selected to determine the ceRNA score along with positively correlated pairs from lncRNA and mRNA network. Integration of ceRNA score and positively correlated pairs exhibit a significant ternary relationship among circRNAs-miRNA-mRNA demonestrated by ceRNA, comprising of 50 regulatory pairs sharring common nodes and predicted potential differentially expressed circRNAs-miRNAs-mRNAs regulatory axis. Based on functional enrichment analysis shortlisted key ceRNA regulatory pairs associated with reproduction including circRNA_3257-novel579_mature-EPHA3, circRNA_8396-novel130_mature-LOC101102473, circRNA_4140- novel34_mature > novel661_mature-KCNK9, and circRNA_8312-novel339_mature-LOC101110545. Furthermore, expression profiling, functional enrichments and qRT-PCR analysis of key target genes infer their implication in reproduction and metabolism. ceRNA target mRNAs evolutionary trajectories, expression profiling, functional enrichments, subcellular localizations following genomic organizations will provide new insights underlying molecular mechanisms of reproduction, and establish a solid foundation for future research. Graphical abstract summarizing the scheme of study.

A high-quality assembled genome and its comparative analysis decode the adaptive molecular mechanism of the number one Chinese cotton variety CRI-12.

BACKGROUND: Gossypium hirsutum L. is the most widely cultivated cotton species, and a high-quality reference genome would be a huge boost for researching the molecular mechanism of agronomic traits in cotton. FINDINGS: Here, Pacific Biosciences and Hi-C sequencing technologies were used to assemble a new upland cotton genome of the No. 1 Chinese cotton variety CRI-12. We generated a high-quality assembled CRI-12 genome of 2.31 Gb with a contig N50 of 19.65 Mb, which was superior to previously reported genomes. Comparisons between CRI-12 and other reported genomes revealed 7,966 structural variations and 7,378 presence/absence variations. The distribution of the haplotypes among A-genome (Gossypium arboreum), D-genome (Gossypium raimondii), and AD-genome (G. hirsutum and Gossypium barbadense) suggested that many haplotypes were lost and recombined in the process of polyploidization. More than half of the haplotypes that correlated with different tolerances were located on chromosome D13, suggesting that this chromosome may be important for wide adaptation. Finally, it was demonstrated that DNA methylation may provide advantages in environmental adaptation through whole-genome bisulfite sequencing analysis. CONCLUSIONS: This research provides a new reference genome for molecular biology research on Gossypium hirsutum L. and helps decode the broad environmental adaptation mechanisms in the No. 1 Chinese cotton variety CRI-12.

Transcriptome analysis of upland cotton revealed novel pathways to scavenge reactive oxygen species (ROS) responding to Na2SO4 tolerance.

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 SO42-. 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.

Exploring potential of copper and silver nano particles to establish efficient callogenesis and regeneration system for wheat (Triticum aestivum L.).

In vitro recalcitrance of wheat to regeneration is the major bottleneck for its improvement through callus-based genetic transformation. Nanotechnology is one of the most dynamic areas of research, which can transform agriculture and biotechnology to ensure food security on sustainable basis. Present study was designed to investigate effects of CuSO4, AgNO3 and their nanoparticles on tissue culture responses of mature embryo culture of wheat genotypes (AS-2002 and Wafaq-2001). Initially, MS-based callus induction and regeneration medium were optimized for both genotypes using various concentrations of auxin (2,4-D, IAA) and cytokinins (BAP, kinetin). The genotypes differed for embryogenic callus induction and regeneration potential. Genotype AS-2002 yielded maximum embryogenic calli in response to 3.0 mg/l 2,4-D, whereas Wafaq-2001 offered the highest embryogenic calli against 3.5 mg/l 2,4-D supplemented in the induction medium. Genotype AS-2002 showed maximum regeneration (59.33%) in response to regeneration protocol comprising 0.5 mg/l IAA, 0.3 mg/l BAP and 1.0 mg/l Kin, while Wafaq-2001 performed best in response to 0.5 mg/l IAA, 0.3 mg/l BAP and 1.5 mg/l Kin with 55.33% regeneration efficiency. The same optimized basal induction and regeneration medium for both genotypes were further used to study effects of CuSO4, AgNO3 and their nano-particles employing independent experiments. The optimized induction medium fortified with various concentrations of CuSO4 or CuNPs confirmed significant effects on frequency of embryogenic callus. Addition of either 0.020 mg/l or 0.025 mg/l CuSO4, or 0.015 mg/l CNPs showed comparable results for embryogenic callus induction and were statistically at par with embryogenic callus induction of 74.00%, 75.67% and 76.83%, respectively. Significantly higher regeneration was achieved from MS-based regeneration medium supplemented with 0.015 mg/l or 0.020 mg/l CuNPs than standard 0.025 mg/l CuSO4. In another study, the basal induction and regeneration medium were fortified with AgNO3 or AgNPs ranging from 1 to 7 mg/l along with basal regeneration media devoid of AgNO3 or AgNPs (control). The maximum embryogenic calli were witnessed from medium fortified with 3.0 mg/l or 4.0 mg/l AgNPs compared with control and rest of the treatments. The standardized regeneration medium fortified with 5.0 mg/l AgNO3 or 3.0 mg/l AgNPs showed pronounced effect on regeneration of wheat genotypes and offered maximum regeneration compared with control. The individual and combined effect of Cu and Ag nanoparticles along with control (basal regeneration media of each genotype) was also tested. Surprisingly, co-application of metallic NPs showed a significant increase in embryogenic callus formation of genotypes. Induction medium supplemented with 0.015 mg/l CuNPs + 4.0 mg/l AgNPs or 0.020 mg/l CuNPs + 2.0 mg/l AgNPs showed splendid results compared to control and other combination of Cu and Ag nanoparticles. The maximum regeneration was achieved by co-application of 0.015 mg/l CuNP and 4.0 mg/l AgNPs with 21% increment of regeneration over control. It is revealed that CuNPs and AgNPs are potential candidate to augment somatic embryogenesis and regeneration of mature embryo explants of wheat.Abbreviations: 2,4-D (2,4-dichlorophenoxyacetic acid), BAP (6-benzylaminopurine), IAA (Indole-3-acetic acid), AgNPs (silver nanoparticles), CuNPs (copper nanoparticles).

Genome-wide identification of CK gene family suggests functional expression pattern against Cd2+ stress in Gossypium hirsutum L.

Choline kinase (CK) gene plays an important role in plants growth, development and resistance to stress. It mainly regulates the biosynthesis of phosphatidylcholine. This study aims to explore the structure-function relationship, and to provide a framework for functional validation and biochemical characterization of various CK genes. Our analysis showed that 87 CK genes were identified in cotton and 7 diploid plants, of which 43 genes encode CK proteins in 4 cotton species, and 13 genes were identified in Gossypium hirsutum. Most of GhCK genes are affected by the abiotic stress conditions, indicating the importance of CK proteins for plant development and response to abiotic stress. RT-qPCR analysis showed the tissue specificity of GhCK genes in response to Cd2+ and other abiotic stresses. Under Cd2+ stress, the expression level of GhCK gene family members has undergone different changes. The expression level of GhCK5 was enhanced, indicating that Cd2+ stress caused the increase of phosphatidylcholine content, which in turn reacted on the plant cell membrane, finally reached the absorption of Cd2+ into plant cells to repair Cd2+ the purpose of contaminated soil. This study will further broaden our understanding of the association between evolution and function of the GhCK gene family.

Differentially expressed bZIP transcription factors confer multi-tolerances in Gossypium hirsutum L.

Basic leucine zipper (bZIP) transcription factor plays an important role in various biological processes, such as response to biotic and abiotic stresses. In this study we performed a systematic investigation and analysis of bZIP gene family in Gossypium hirsutum to predict their functions in response to different abiotic stresses. A total of 207 bZIP genes were identified from Gossypium hirsutum genome and classified into 13 subfamilies through phylogenetic analysis, which was testified by the analysis of conserved motifs and exon-intron structures. Annotation of GHbZIPs was performed based on well-studied Arabidopsis bZIPs to speculate the gene function. RNA-seq analysis was conducted to identify the co-expressed and differentially expressed bZIPs under cold, heat, salt and PEG treatments. Promoter analysis and interaction network of GHbZIP proteins demonstrated that ABA-activated signaling pathway was pivotal in the regulation of GHbZIPs, and GHbZIPs involved in ER stress were supposed to function through interaction with other GHbZIPs and ABA pathway. Cis-elements in the upstream and downstream of GHbZIPs interaction network were also discussed. These findings provided us with clues about functions of bZIP in Gossypium hirsutum.

Genome-wide expression analysis suggests glutaredoxin genes response to various stresses in cotton.

Oxidative stress reflects an imbalance between the systemic manifestation of reactive oxygen species (ROS) and a biological system's ability to readily detoxify the reactive intermediates or to repair the resulting damage. Glutaredoxins (GRXs) are ubiquitous oxidoreductase enzymes involved in diverse cellular processes and play a key role in oxidative stress responsive mechanisms. This study was aimed to explore the structure-function relationship and to provide a framework for functional validation and biochemical characterization of various GRX members. In this study, our analysis revealed the presence of 127 genes encoding GRX proteins in G. hirsutum. A total of 758 genes from two typical monocot and nine dicot species were naturally divided into four classes based on phylogenetic analysis. The classification was supported with organization of conserved protein motifs and sequence logos comparison between cotton, rice and Arabidopsis. Cotton GRX gene family has underwent strong purifying selection with limited functional divergence. A good collinearity was observed in the synteny analysis of four Gossypium species. Majority of cotton GRXs were influenced by various phytohormones and abiotic stress conditions during expression analysis, suggesting an important role of GRX proteins in response to oxidative stress. Cis-regulatory elements, gene enrichments and co-expression network analysis also support their predicted role against various abiotic stresses. Whole genome and segmental duplication were determined to be the two major impetuses for the expansion of gene numbers during the evolution. The identification of GRX genes showing differential expression in specific tissues or in response to environmental stimuli provides a new avenue for in-depth characterization of selected genes of importance. This study will further broaden our insights into the evolution and functional elucidation of GRX gene family in cotton.

Structure and character analysis of cotton response regulator genes family reveals that GhRR7 responses to draught stress

BACKGROUND: Cytokinin signal transduction is mediated by a two-component system (TCS). Two-component systems are utilized in plant responses to hormones as well as to biotic and abiotic environmental stimuli. In plants, response regulatory genes (RRs) are one of the main members of the two-component system (TCS). METHOD: From the aspects of gene structure, evolution mode, expression type, regulatory network and gene function, the evolution process and role of RR genes in the evolution of the cotton genome were analyzed. RESULT: A total of 284 RR genes in four cotton species were identified. Including 1049 orthologous/paralogous gene pairs were identified, most of which were whole genome duplication (WGD). The RR genes promoter elements contain phytohormone responses and abiotic or biotic stress-related cis-elements. Expression analysis showed that RR genes family may be negatively regulate and involved in salt stress and drought stress in plants. Protein regulatory network analysis showed that RR family proteins are involved in regulating the DNA-binding transcription factor activity (COG5641) pathway and HP kinase pathways. VIGS analysis showed that the GhRR7 gene may be in the same regulatory pathway as GhAHP5 and GhPHYB, ultimately negatively regulating cotton drought stress by regulating POD, SOD, CAT, H2O2 and other reactive oxygen removal systems. CONCLUSION: This study is the first to gain insight into RR gene members in cotton. Our research lays the foundation for discovering the genes related to drought and salt tolerance and creating new cotton germplasm materials for drought and salt tolerance.

Genome-wide identification and characteristic analysis of the downstream melatonin metabolism gene GhM2H in Gossypium hirsutum L

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.