Integration of proteomic and transcriptomic profiles reveals multiple levels of genetic regulation of salt tolerance in cotton.
ABSTRACT: BACKGROUND:Salinity is a major abiotic stress that limits upland cotton growth and reduces fibre production worldwide. To reveal genetic regulation via transcript and protein levels after salt stress, we comprehensively analysed the global changes in mRNA, miRNA, and protein profiles in response to salt stress in two contrasting salt-tolerant cotton genotypes. RESULTS:In the current study, proteomic and mRNA-seq data were combined to reveal that some genes are differentially expressed at both the proteomic and mRNA levels. However, we observed no significant change in mRNA corresponding to most of the strongly differentially abundant proteins. This finding may have resulted from global changes in alternative splicing events and miRNA levels under salt stress conditions. Evidence was provided indicating that several salt stress-responsive proteins can alter miRNAs and modulate alternative splicing events in upland cotton. The results of the stringent screening of the mRNA-seq and proteomic data between the salt-tolerant and salt-sensitive genotypes identified 63 and 85 candidate genes/proteins related to salt tolerance after 4 and 24 h of salt stress, respectively, between the tolerant and sensitive genotype. Finally, we predicted an interaction network comprising 158 genes/proteins and then discovered that two main clusters in the network were composed of ATP synthase (CotAD_74681) and cytochrome oxidase (CotAD_46197) in mitochondria. The results revealed that mitochondria, as important organelles involved in energy metabolism, play an essential role in the synthesis of resistance proteins during the process of salt exposure. CONCLUSION:We provided a plausible schematic for the systematic salt tolerance model; this schematic reveals multiple levels of gene regulation in response to salt stress in cotton and provides a list of salt tolerance-related genes/proteins. The information here will facilitate candidate gene discovery and molecular marker development for salt tolerance breeding in cotton.
Project description:Purpose:Identification of genes and miRNAs responsible for salt tolerance in upland cotton (Gossypium hirsutum L.) would help reveal the molecular mechanisms of salt tolerance. We performed physiological experiments and transcriptome sequencing (mRNA-seq and small RNA-seq) of cotton leaves under salt stress using Illumina sequencing technology. And quantitative reverse transcription polymerase chain reaction (qRT–PCR) methods and to evaluate protocols for optimal high-throughput data analysis Methods:We investigated two distinct salt stress phases—dehydration (4 h) and ionic stress (osmotic restoration; 24 h)—that were identified by physiological changes of 14-day-old seedlings of two cotton genotypes, one salt tolerant and the other salt sensitive, during a 72-h NaCl exposure. A comparative transcriptomics approach was used to monitor gene and miRNA differential expression at two time points (4 and 24 h) in leaves of the two cotton genotypes under salinity conditions. Results:During a 24-h salt exposure, 819 transcription factor unigenes were differentially expressed in both genotypes, with 129 unigenes specifically expressed in the salt-tolerant genotype. Under salt stress, 108 conserved miRNAs from known families were differentially expressed at two time points in the salt-tolerant genotype. Conclusions:Our comprehensive transcriptome analysis has provided new insights into salt-stress response of upland cotton. The results should contribute to the development of genetically modified cotton with salt tolerance. Overall design: Leaf mRNA and miRNA profiles of 14-day-old seedlings of two cotton genotypes, one salt tolerant and the other salt sensitive during a 72-h NaCl exposure were generated by deep sequencing, using Illumina HiSeq 2000 system.
Project description:BACKGROUND: Cotton (Gossypium spp.) is one of the major fibre crops of the world. Although it is classified as salt tolerant crop, cotton growth and productivity are adversely affected by high salinity, especially at germination and seedling stages. Identification of genes and miRNAs responsible for salt tolerance in upland cotton (Gossypium hirsutum L.) would help reveal the molecular mechanisms of salt tolerance. We performed physiological experiments and transcriptome sequencing (mRNA-seq and small RNA-seq) of cotton leaves under salt stress using Illumina sequencing technology. RESULTS: We investigated two distinct salt stress phases--dehydration (4 h) and ionic stress (osmotic restoration; 24 h)--that were identified by physiological changes of 14-day-old seedlings of two cotton genotypes, one salt tolerant and the other salt sensitive, during a 72-h NaCl exposure. A comparative transcriptomics was used to monitor gene and miRNA differential expression at two time points (4 and 24 h) in leaves of the two cotton genotypes under salinity conditions. The expression patterns of differentially co-expressed unigenes were divided into six groups using short time-servies expression miner software. During a 24-h salt exposure, 819 transcription factor unigenes were differentially expressed in both genotypes, with 129 unigenes specifically expressed in the salt-tolerant genotype. Under salt stress, 108 conserved miRNAs from known families were differentially expressed at two time points in the salt-tolerant genotype. We further analyzed the predicted target genes of these miRNAs along with the transcriptome for each time point. Important expressed genes encoding membrane receptors, transporters, and pathways involved in biosynthesis and signal transduction of calcium-dependent protein kinase, mitogen-activated protein kinase, and hormones (abscisic acid and ethylene) were up-regulated. We also analyzed the salt stress response of some key miRNAs and their target genes and found that the expressions of five of nine target genes exhibited significant inverse correlations with their corresponding miRNAs. On the basis of these results, we constructed molecular regulatory pathways and a potential regulatory network for these salt-responsive miRNAs. CONCLUSIONS: Our comprehensive transcriptome analysis has provided new insights into salt-stress response of upland cotton. The results should contribute to the development of genetically modified cotton with salt tolerance.
Project description:Soil salinity is a major abiotic stress that limits plant growth and agricultural productivity. Upland cotton (Gossypium hirsutum L.) is highly tolerant to salinity; however, large-scale proteomic data of cotton in response to salt stress are still scant. Here, an isobaric tag for relative and absolute quantitation (iTRAQ)-based proteomic technique was employed to identify the early differentially expressed proteins (DEPs) from salt-treated cotton roots. One hundred and twenty-eight DEPs were identified, 76 of which displayed increased abundance and 52 decreased under salt stress conditions. The majority of the proteins have functions related to carbohydrate and energy metabolism, transcription, protein metabolism, cell wall and cytoskeleton metabolism, membrane and transport, signal transduction, in addition to stress and defense. It is worth emphasizing that some novel salt-responsive proteins were identified, which are involved in cell cytoskeleton metabolism (actin-related protein2, ARP2, and fasciclin-like arabinogalactan proteins, FLAs), membrane transport (tonoplast intrinsic proteins, TIPs, and plasma membrane intrinsic proteins, PIPs), signal transduction (leucine-rich repeat receptor-like kinase encoding genes, LRR-RLKs) and stress responses (thaumatin-like protein, TLP, universal stress protein, USP, dirigent-like protein, DIR, desiccation-related protein PCC13-62). High positive correlation between the abundance of some altered proteins (superoxide dismutase, SOD, peroxidase, POD, glutathione S-transferase, GST, monodehydroascorbate reductase, MDAR, and malate dehydrogenase, MDH) and their enzyme activity was evaluated. The results demonstrate that the iTRAQ-based proteomic technique is reliable for identifying and quantifying a large number of cotton root proteins. qRT-PCR was used to study the gene expression levels of the five above-mentioned proteins; four patterns are consistent with those of induced protein. These results showed that the proteome of cotton roots under NaCl stress is complex. The comparative protein profiles of roots under salinity vs control improves the understanding of the molecular mechanisms involved in the tolerance of plants to salt stress. This work provides a good basis for further functional elucidation of these DEPs using genetic and/or other approaches, and, consequently, candidate genes for genetic engineering to improve crop salt tolerance.
Project description:BACKGROUND:Salinity is a major abiotic stress seriously hindering crop yield. Development and utilization of tolerant varieties is the most economical way to address soil salinity. Upland cotton is a major fiber crop and pioneer plant on saline soil and thus its genetic architecture underlying salt tolerance should be extensively explored. RESULTS:In this study, genome-wide association analysis and RNA sequencing were employed to detect salt-tolerant qualitative-trait loci (QTLs) and candidate genes in 196 upland cotton genotypes at the germination stage. Using comprehensive evaluation values of salt tolerance in four environments, we identified 33 significant single-nucleotide polymorphisms (SNPs), including 17 and 7 SNPs under at least two and four environments, respectively. The 17 stable SNPs were located within or near 98 candidate genes in 13 QTLs, including 35 genes that were functionally annotated to be involved in salt stress responses. RNA-seq analysis indicated that among the 98 candidate genes, 13 were stably differentially expressed. Furthermore, 12 of the 13 candidate genes were verified by qRT-PCR. RNA-seq analysis detected 6640, 3878, and 6462 differentially expressed genes at three sampling time points, of which 869 were shared. CONCLUSIONS:These results, including the elite cotton accessions with accurate salt tolerance evaluation, the significant SNP markers, the candidate genes, and the salt-tolerant pathways, could improve our understanding of the molecular regulatory mechanisms under salt stress tolerance and genetic manipulation for cotton improvement.
Project description:BACKGROUND:Salt stress is one of the most damaging abiotic stresses in production of Upland cotton (Gossypium hirsutum). Upland cotton is defined as a medium salt-tolerant crop. Salinity hinders root development, shoots growth, and reduces the fiber quality. RESULTS:Our previous study verified a GhCIPK6a gene response to salt stress in G. hirsutum. The homologs of GhCIPK6a were analyzed in A2 (G. arboreum), D5 (G. raimondii), and AD1 (G. hirsutum) genomes. GhCIPK6a localized to the vacuole and cell membrane. The GhCBL1-GhCIPK6a and GhCBL8-GhCIPK6a complexes localized to the nucleus and cytomembrane. Overexpression of GhCIPK6a enhanced expression levels of co-expressed genes induced by salt stress, which scavenged ROS and involved in MAPK signaling pathways verified by RNA-seq analysis. Water absorption capacity and cell membrane stability of seeds from GhCIPK6a overexpressed lines was higher than that of wild-type seeds during imbibed germination stage. The seed germination rates and seedling field emergence percentages of GhCIPK6a overexpressed lines were higher than that of control line under salt stress. Moreover, overexpressing of GhCIPK6a in cotton increased lint percentage, and fiber length uniformity under salt stress. CONCLUSIONS:We verified the function of GhCIPK6a by transformation and RNA-seq analysis. GhCIPK6a overexpressed lines exhibited higher tolerance to abiotic stresses, which functioned by involving in ROS scavenging and MAPK pathways. Therefore, GhCIPK6a has the potential for cotton breeding to improve stress-tolerance.
Project description:Soil salinization is one of the major problems in global agricultural production. Cotton is a pioneer crop with regard to salt stress tolerance, and can be used for saline-alkali land improvement. The large-scale detection of salt tolerance traits in cotton accessions, and the identification of elite quantitative trait loci (QTLs)/genes for salt-tolerance have been very important in salt tolerance breeding. Here, 43 advanced salt-tolerant and 31 highly salt-sensitive cultivars were detected by analyzing ten salt tolerance related traits in 304 upland cotton cultivars. Among them, 11 advanced salt-tolerance and eight highly salt-sensitive cultivars were consistent with previously reported results. Association analysis of ten salt-tolerance related traits and 145 SSRs was performed, and a total of 95 significant associations were detected; 17, 41, and 37 of which were associated with germinative index, seedling stage physiological index, and four seedling stage biochemical indexes, respectively. Of these associations, 20 SSR loci were simultaneously associated with two or more traits. Furthermore, we detected 117 elite alleles associated with salt-tolerance traits, 4 of which were reported previously. Among these loci, 44 (37.60%) were rare alleles with a frequency of less than 5%, 6 only existed in advanced salt-tolerant cultivars, and 2 only in highly salt-sensitive cultivars. As a result, 13 advanced salt-tolerant cultivars were selected to assemble the optimal cross combinations by computer simulation for the development of salt-tolerant accessions. This study lays solid foundations for further improvements in cotton salt-tolerance by referencing elite germplasms, alleles associated with salt-tolerance traits, and optimal crosses.
Project description:Salt tolerance in cotton is highly imperative for improvement in the response to decreasing farmland and soil salinization. However, little is known about the genetic basis underlying salt tolerance in cotton, especially the seedling stage. In this study, we evaluated two salt-tolerance-related traits of a natural population comprising 713 upland cotton (Gossypium hirsutum L.) accessions worldwide at the seedling stage and performed a genome-wide association study (GWAS) to identify marker-trait associations under salt stress using the Illumina Infinium CottonSNP63K array. A total of 23 single nucleotide polymorphisms (SNPs) that represented seven genomic regions on chromosomes A01, A10, D02, D08, D09, D10, and D11 were significantly associated with the two salt-tolerance-related traits, relative survival rate (RSR) and salt tolerance level (STL). Of these, the two SNPs i46598Gh and i47388Gh on D09 were simultaneously associated with the two traits. Based on all loci, we screened 280 possible candidate genes showing different expression levels under salt stress. Most of these genes were involved in transcription factors, transporters and enzymes and were previously reported as being involved in plant salt tolerance, such as NAC, MYB, NXH, WD40, CDPK, LEA, and CIPK. We further validated six putative candidate genes by qRT-PCR and found a differential expression level between salt-tolerant and salt-sensitive varieties. Our findings provide valuable information for enhancing the understanding of complicated mechanisms of salt tolerance in G. hirsutum seedlings and cotton salt tolerance breeding by molecular marker-assisted selection.
Project description:Cotton is a pioneer of saline land crop, while salt stress still causes its growth inhibition and fiber production decrease. Phenotype identification showed better salt tolerance of a wild diploid cotton species Gossypium klotzschianum. To elucidate the salt-tolerant mechanisms in G. klotzschianum, we firstly detected the changes in hormones, H2O2 and glutathione (GSSH and GSH), then investigated the gene expression pattern of roots and leaves treated with 300 mM NaCl for 0, 3, 12, 48 h, and each time control by RNA-seq on the Illumina-Solexa platform. Physiological determination proved that the significant increase in hormone ABA at 48 h, while that in H2O2 was at 12 h, likewise, the GSH content decrease at 48 h and the GSSH content increase at 48 h, under salt stress. In total, 37,278 unigenes were identified from the transcriptome data, 8,312 and 6,732 differentially expressed genes (DEGs) were discovered to be involved in salt stress tolerance in roots and leaves, respectively. Gene function annotation and expression analysis elucidated hormone biosynthesis and signal transduction, reactive oxygen species (ROS), and salt overly sensitive (SOS) signal transduction related genes revealed the important roles of them in signal transmission, oxidation balance and ion homeostasis in response to salinity stress. This is a report which focuses on primary response to highly salty stress (upto 300 mM NaCl) in cotton using a wild diploid Gossypium species, broadening our understanding of the salt tolerance mechanism in cotton and laying a solid foundation of salt resistant for the genetic improvement of upland cotton with the resistance to salt stress.
Project description:The capacity for ion compartmentalization among different tissues and cells is the key mechanism regulating salt tolerance in plants. In this study, we investigated the ion compartmentalization capacity of two upland cotton genotypes with different salt tolerances under salt shock at the tissue, cell and molecular levels. We found that the leaf glandular trichome could secrete more salt ions in the salt-tolerant genotype than in the sensitive genotype, demonstrating the excretion of ions from tissue may be a new mechanism to respond to short-term salt shock. Furthermore, an investigation of the ion distribution demonstrated that the ion content was significantly lower in critical tissues and cells of the salt-tolerant genotype, indicating the salt-tolerant genotype had a greater capacity for ion compartmentalization in the shoot. By comparing the membrane H(+)-ATPase activity and the expression of ion transportation-related genes, we found that the H(+)-ATPase activity and Na(+)/H(+) antiporter are the key factors determining the capacity for ion compartmentalization in leaves, which might further determine the salt tolerance of cotton. The novel function of the glandular trichome and the comparison of Na(+) compartmentalization between two cotton genotypes with contrasting salt tolerances provide a new understanding of the salt tolerance mechanism in cotton.
Project description: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.