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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.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 NaHCOIn 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.

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.

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.

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.

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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.

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.

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.

Cotton is the most important crop for the production of natural fibres used in the textile industry. High salinity, drought, cold and high temperature represent serious abiotic stresses, which seriously threaten cotton production. Phospholipase AS has an irreplaceable role in lipid signal transmission, growth and development and stress events. Phospholipase A can be divided into three families: PLA1, PLA2 and pPLA. Among them, the PLA1 family is rarely studied in plants. In order to study the potential functions of the PLA1 family in cotton, the bioinformatics analysis of the PLA1 family was correlated with cotton adversity, and tissue-specific analysis was performed. Explore the structure-function relationship of PLA1 members. It is found that the expression of GbPLA1-32 gene is affected by a variety of environmental stimuli, indicating that it plays a very important role in stress and hormone response, and closely associates the cotton adversity with this family. Through further functional verification, we found that virus-induced GbPLA1-32 gene silencing (VIGS) caused Gossypium barbadense to be sensitive to salt stress. This research provides an important basis for further research on the molecular mechanism of cotton resistance to abiotic stress.

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 Cd

Cotton production is severely constrained by drought, especially if encountered during the seedling stage or the fiber initiation and elongation stage, but the regulatory mechanisms underlying the effects of drought remain unclear. Therefore, characterization and functional analysis of microRNA-mediated stress regulatory networks are critical to deciphering plant drought response. In this study, 357, 379 and 377 miRNAs with annotations were obtained using the drought-resistant cotton variety ZhongH177 under three treatments, CK, drought and re-watering, and divided into 73 miRNA families with varying copy numbers from 1 to 24. 136 differential expressed genes (DEGs) with significant expression changes were found, of which only 33 DEGs were upregulated, while 103 DEGs were downregulated under drought stress. However, most DEGs recovered their initial expression states when the plants were re-watered. In total, 2657 targets were identified and found to be mainly enriched in the pathways plant-pathogen interaction, plant hormone signal transduction and biosynthesis of amino acids. Drought tolerance was significantly enhanced in 2 transgenic Arabidopsis lines, showing that miRNAs were involved in cotton drought response. Analysis of the expression patterns of 2 miRNA precursors and methylation alterations of 2 targets suggested that these miRNAs or miRNA precursors may be involved in the regulation of target methylation states. Our study provides evidence of transcriptional responses to drought stress, which will be helpful for the research of drought-resistance mechanisms in the future.

Summary Although efforts have been taken to exploit diversity for yield and quality improvements, limited progress on using beneficial alleles in domesticated and undomesticated cotton varieties is limited. Given the complexity and limited amount of genomic information since the completion of four cotton genomes, characterizing significant variations and haplotype block inheritance under artificial selection has been challenging. Here we sequenced Gossypium hirsutum L. cv CRI‐12 (the cotton variety with the largest acreage in China), its parental cultivars, and progeny cultivars, which were bred by the different institutes in China. In total, 3.3 million SNPs were identified and 118, 126 and 176 genes were remarkably correlated with Verticillium wilt, salinity and drought tolerance in CRI‐12, respectively. Transcriptome‐wide analyses of gene expression, and functional annotations, have provided support for the identification of genes tied to these tolerances. We totally discovered 58 116 haplotype blocks, among which 23 752 may be inherited and 1029 may be recombined under artificial selection. This survey of genetic diversity identified loci that may have been subject to artificial selection and documented the haplotype block inheritance and recombination, shedding light on the genetic mechanism of artificial selection and guiding breeding efforts for the genetic improvement of cotton.

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.

Background Cotton (Gossypium hirsutum) is considered a fairly salt tolerant crop however, salinity can still cause significant economic losses by affecting the yield and deteriorating the fiber quality. We studied a salt-tolerant upland cotton cultivar under temporal salt stress to unfold the salt tolerance molecular mechanisms. Biochemical response to salt stress (400 mM) was measured at 0 h, 3 h, 12 h, 24 h and 48 h post stress intervals and single-molecule long-read sequencing technology from Pacific Biosciences (PacBio) combined with the unique molecular identifiers approach was used to identify differentially expressed genes (DEG). Results Antioxidant enzymes including, catalase (CAT), peroxidase (POD), superoxide dismutase (SOD) were found significantly induced under temporal salt stress, suggesting that reactive oxygen species scavenging antioxidant machinery is an essential component of salt tolerance mechanism in cotton. We identified a wealth of novel transcripts based on the PacBio long reads sequencing approach. Prolonged salt stress duration induces high number of DEGs. Significant numbers of DEGs were found under key terms related to stress pathways such as “response to oxidative stress”, “response to salt stress”, “response to water deprivation”, “cation transport”, “metal ion transport”, “superoxide dismutase”, and “reductase”. Key DEGs related to hormone (abscisic acid, ethylene and jasmonic acid) biosynthesis, ion homeostasis (CBL-interacting serine/threonine-protein kinase genes, calcium-binding proteins, potassium transporter genes, potassium channel genes, sodium/hydrogen exchanger or antiporter genes), antioxidant activity (POD, SOD, CAT, glutathione reductase), transcription factors (myeloblastosis, WRKY, Apetala 2) and cell wall modification were found highly active in response to salt stress in cotton. Expression fold change of these DEGs showed both positive and negative responses, highlighting the complex nature of salt stress tolerance mechanisms in cotton. Conclusion Collectively, this study provides a good insight into the regulatory mechanism under salt stress in cotton and lays the foundation for further improvement of salt stress tolerance.

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 (NaHCO

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.

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.

As an important source of fiber and edible oil, cotton has great economic value. In comparison to their individual studies, association and differentiation between salt and alkaline tolerance has not been focused yet by scientists. We have used next-generation RNA-Seq technique to analyze transcriptional changes under salt and alkaline stresses in cotton. Overall, 25,929 and 6,564 differentially expressed genes (DEGs) were identified in roots and leaves, respectively. Gene functional annotation showed that genes involving ionic homeostasis were significantly up-regulated under NaCl stress and Na2CO3 stress, and genes enriched in starch and sucrose metabolism were up-regulated under NaOH stress and Na2CO3 stress. Furthermore, a synergistic enhancing effect between NaCl and NaOH stress was also observed in this study. Likewise, our studies indicate further that genes related with starch and sucrose metabolism were regulated to respond to the high pH under Na2CO3 stress, inducing plant hormone signal transduction and key enzyme reactive oxygen species (ROS) activity to respond to ionic toxicity and intracellular ionic homeostasis. By analyzing the expression profiles of diverse tissues under different salt and alkaline stresses, this study provides valuable ideas for genetic improvements of cotton tolerance to salt-alkaline stress.

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.

Background The U-box gene encodes a ubiquitin ligase that contain U-box domain. The plant U-box gene (PUB) plays an important role in the response to stresses, but few reports about PUBs in cotton were available. Therefore research on PUBs is of great importance and a necessity when studying the mechanisms of stress- tolerance in cotton. Results In this study, we identified 93, 96, 185 and 208 PUBs from four sequenced cotton species G. raimondii (D5), G. arboreum (A2), G. hirsutum (AD1) and G. barbadense (AD2), respectively. Prediction analysis of subcellular localization showed that the PUBs in cotton were widely localized in cells, but primarily in the nucleus. The PUBs in cotton were classified into six subfamilies (A-F) on the basis of phylogenetic analysis, which was testified by the analysis of conserved motifs and exon-intron structures. Chromosomal localization analysis showed that cotton PUBs were unevenly anchored on all chromosomes, varying from 1 to 14 per chromosome. Through multiple sequence alignment analysis, 3 tandem duplications and 28 segmental duplications in cotton genome D5, 2 tandem duplications and 25 segmental duplications in A2, and 143 homologous gene pairs in A2 and D5 were found; however no tandem duplications in A2 or D5 were found. Additionally, 105, 14 and 17 homologous gene pairs were found in the intra-subgenome of At and Dt, At sub-genome and Dt sub-genome of G. hirsutum, respectively. Functional analysis of GhPUB85A and GhPUB45D showed that these genes positively responded to abiotic stresses, but the expression patterns were different. In addition, although the expression levels of these two homologous genes were similar, their contributions were different when responding to stresses, specifically showing different responses to abiotic stresses and functional differences between the two subgenomes of G. hirsutum. Conclusions This study reported the genome-wide identification, structure, evolution and expression analysis of PUBs in cotton, and the results showed that the PUBs were highly conserved throughout the evolutionary history of cotton. All PUB genes were involved in the response to abiotic stresses (including salt, drought, hot and cold) to varying degrees.

Background DNA methylation, with a cryptic role in genome stability, gene transcription and expression, is involved in the drought response process in plants, but the complex regulatory mechanism is still largely unknown. Results Here, we performed whole-genome bisulfite sequencing (WGBS) and identified long non-coding RNAs on cotton leaves under drought stress and re-watering treatments. We obtained 31,223 and 30,997 differentially methylated regions (representing 2.48% of the genome) after drought stress and re-watering treatments, respectively. Our data also showed that three sequence contexts, including mCpG, mCHG, mCHH, all presented a hyper-methylation pattern under drought stress and were nearly restored to normal levels after the re-watering treatment. Among all the methylation variations, asymmetric CHH methylation was the most consistent with external environments, suggesting that methylation/demethylation in a CHH context may constitute a novel epigenetic modification in response to drought stress. Combined with the targets of long non-coding RNAs, we found that long non-coding RNAs may mediate variations in methylation patterns by splicing into microRNAs. Furthermore, the many hormone-related genes with methylation variations suggested that plant hormones might be a potential mechanism in the drought response. Conclusions Future crop-improvement strategies may benefit by taking into account not only the DNA genetic variations in cotton varieties but also the epigenetic modifications of the genome. Electronic supplementary material The online version of this article (doi:10.1186/s12864-017-3681-y) contains supplementary material, which is available to authorized users.

The CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)/Cas9 system has been widely used for genome editing in various plants because of its simplicity, high efficiency and design flexibility. However, to our knowledge, there is no report on the application of CRISPR/Cas9-mediated targeted mutagenesis in cotton. Here, we report the genome editing and targeted mutagenesis in upland cotton (Gossypium hirsutum L., hereafter cotton) using the CRISPR/Cas9 system. We designed two guide RNAs to target distinct sites of the cotton Cloroplastos alterados 1 (GhCLA1) and vacuolar H+-pyrophosphatase (GhVP) genes. Mutations in these two genes were detected in cotton protoplasts. Most of the mutations were nucleotide substitutions, with one nucleotide insertion and one substitution found in GhCLA1 and one deletion found in GhVP in cotton protoplasts. Subsequently, the two vectors were transformed into cotton shoot apexes through Agrobacterium-mediated transformation, resulting in efficient target gene editing. Most of the mutations were nucleotide deletions, and the mutation efficiencies were 47.6–81.8% in transgenic cotton plants. Evaluation using restriction-enzyme-PCR assay and sequence analysis detected no off-target mutations. Our results indicated that the CRISPR/Cas9 system was an efficient and specific tool for targeted mutagenesis of the cotton genome.

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&ndash, 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.

Mitogen-activated protein kinase (MAPK) cascades are conserved signal transduction pathways in all eukaryotic organisms. MAPKKKs (MAPK kinase kinases) operate at the top levels of these cascades. Recently, this family of genes has been systematically investigated in Arabidopsis, rice and maize, but has not yet been characterized in cotton. In this study, we identified 78 putative MAPKKK genes in the genome of the diploid cotton, Gossypium raimondii. They were classified into three subfamilies, of which 12 were ZIK, 22 were MEKK and 44 were Raf. The ZIK and MEKK genes displayed a scattered genomic distribution across 11 of the 13 chromosomes, whereas Raf genes were distributed across the entire genome. Their conserved patterns observed for introns and additional domains were consistent with the evolutionary relationships inferred from the phylogenetic analysis within subfamily. Transcriptome sequencing data were used to investigate their transcript profiles in mature leaves, 0 day and 3 days post-anthesis (DPA) ovules. Sixty MAPKKK genes were expressed, of which 41 were strongly expressed in mature leaves. Twelve MAPKKK genes were more highly expressed in 3-DPA ovules than in 0-DPA ovules. Our results provide a foundation for future evolutionary and functional characterizations of MAPKKK genes in cotton and probably other Gossypium plants.

Recent researches on long noncoding RNAs (lncRNAs) have expanded our horizon of gene regulation and the cellular complexity. However, the number, characteristics and expression patterns of lncRNAs remain poorly characterized and how these lncRNAs biogenesis are regulated in response to drought stress in cotton are still largely unclear. In the study, using a reproducibility-based RNA-sequencing and bioinformatics strategy to analyze the lncRNAs of 9 samples under three different environment stresses (control, drought stress and re-watering, three replications), we totally identified 10,820 lncRNAs of high-confidence through five strict steps filtration, of which 9,989 were lincRNAs, 153 were inronic lncRNAs, 678 were anti-sense lncRNAs. Coding function analysis showed 6,470 lncRNAs may have the ability to code proteins. Small RNAs precursor analysis revealed that 196 lncRNAs may be the precursors to small RNAs, most of which (35.7%, 70) were miRNAs. Expression patterns analysis showed that most of lncRNAs were expressed at a low level and most inronic lncRNAs (75.95%) had a consistent expression pattern with their adjacent protein-coding genes. Further analysis of transcriptome data uncovered that lncRNAs XLOC_063105 and XLOC_115463 probably function in regulating two adjacent coding genes CotAD_37096 and CotAD_12502, respectively. Investigations of the content of plant hormones and proteomics analysis under drought stress also complemented the prediction. We analyzed the characteristics and the expression patterns of lncRNAs under drought stress and re-watering treatment, and found lncRNAs may be likely to involve in regulating plant hormones pathway in response to drought stress.

Salinity stress is a major abiotic factor that affects crop output, and as a pioneer crop in saline and alkaline land, salt tolerance study of cotton is particularly important. In our experiment, four salt-tolerance varieties with different salt tolerance indexes including CRI35 (65.04%), Kanghuanwei164 (56.19%), Zhong9807 (55.20%) and CRI44 (50.50%), as well as four salt-sensitive cotton varieties including Hengmian3 (48.21%), GK50 (40.20%), Xinyan96-48 (34.90%), ZhongS9612 (24.80%) were used as the materials. These materials were divided into salt-tolerant group (ST) and salt-sensitive group (SS). Illumina Cotton SNP 70K Chip was used to detect SNP in different cotton varieties. SNPv (SNP variation of the same seedling pre- and after- salt stress) in different varieties were screened; polymorphic SNP and SNPr (SNP related to salt tolerance) were obtained. Annotation and analysis of these SNPs showed that (1) the induction efficiency of salinity stress on SNPv of cotton materials with different salt tolerance index was different, in which the induction efficiency on salt-sensitive materials was significantly higher than that on salt-tolerant materials. The induction of salt stress on SNPv was obviously biased. (2) SNPv induced by salt stress may be related to the methylation changes under salt stress. (3) SNPr may influence salt tolerance of plants by affecting the expression of salt-tolerance related genes.

Upland cotton (Gossypium hirsutum L.), an important source of natural fiber, can tolerate relatively high salinity and drought stresses. In the present study, a plasma membrane Na+/H+ antiporter gene, GhSOS1, was cloned from a salt-tolerant genotype of G. hirsutum, Zhong 9807. The expression level of GhSOS1 in cotton roots was significantly upregulated in the presence of high concentrations of NaCl (200 mM), while its transcript abundance was increased when exposed to low temperature and drought stresses. Localization analysis using onion epidermal cells showed that the GhSOS1 protein was localized to the plasma membrane. The overexpression of GhSOS1 in Arabidopsis enhanced tolerance to salt stress, as indicated by a lower MDA content and decreased Na+/K+ ratio in transgenic plants. Moreover, the transcript levels of stress-related genes were significantly higher in GhSOS1 overexpression lines than in wild-type plants under salt treatment. Hence, GhSOS1 may be a potential target gene for enhancing salt tolerance in transgenic plants.