Comprehensive analysis of differentially expressed genes and transcriptional regulation induced by salt stress in two contrasting cotton genotypes.
ABSTRACT: 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: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: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: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: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:Long non-coding RNAs (lncRNAs) represent a class of riboregulators that either directly act in long form or are processed into shorter microRNAs (miRNAs) and small interfering RNAs. Long noncoding RNAs (lncRNAs) are arbitrarily defined as RNA genes larger than 200 nt in length that have no apparent coding potential. lncRNAs have emerged as playing important roles in various biological regulatory processes and are expressed in a more tissue-specific manner than mRNA. Emerging evidence shows that lncRNAs participate in stress-responsive regulation.In this study, in order to develop a comprehensive catalogue of lncRNAs in upland cotton under salt stress, we performed whole-transcriptome strand-specific RNA sequencing for three-leaf stage cotton seedlings treated with salt stress (S_NaCl) and controls (S_CK). In total we identified 1117 unique lncRNAs in this study and 44 differentially expressed RNAs were identified as potential non-coding RNAs. For the differentially expressed lncRNAs that were identified as intergenic lncRNAs (lincRNA), we analysed the gene ontology enrichment of cis targets and found that cis target protein-coding genes were mainly enriched in stress-related categories. Real-time quantitative PCR confirmed that all selected lincRNAs responsive to salt stress. We found lnc_388 was likely as regulator of Gh_A09G1182. And lnc_883 may participate in regulating tolerance to salt stress by modulating the expression of Gh_D03G0339 MS_channel. We then predicted the target mimics for miRNA in Gossypium. six miRNAs were identified, and the result of RT-qPCR with lncRNA and miRNA suggested that lnc_973 and lnc_253 may regulate the expression of ghr-miR399 and ghr-156e as a target mimic under salt stress.We identified 44 lincRNAs that were differentially expressed under salt stress. These lincRNAs may target protein-coding genes via cis-acting regulation. We also discovered that specifically-expressed lincRNAs under salt stress may act as endogenous target mimics for conserved miRNAs. These findings extend the current view on lincRNAs as ubiquitous regulators under stress stress.
Project description:BACKGROUND:Previous transcriptome profiling studies have investigated the molecular mechanisms of pollen and anther development, and identified many genes involved in these processes. However, only 51 anther ESTs of Upland cotton (Gossypium hirsutum) were found in NCBI and there have been no reports of transcriptome profiling analyzing anther development in Upland cotton, a major fiber crop in the word. METHODOLOGY/PRINCIPAL FINDING:Ninety-eight hundred and ninety-six high quality ESTs were sequenced from their 3'-ends and assembled into 6,643 unigenes from a normalized, full-length anther cDNA library of Upland cotton. Combined with previous sequenced anther-related ESTs, 12,244 unigenes were generated as the reference genes for digital gene expression (DGE) analysis. The DGE was conducted on anthers that were isolated at tetrad pollen (TTP), uninucleate pollen (UNP), binucleate pollen (BNP) and mature pollen (MTP) periods along with four other tissues, i.e., roots (RO), stems (ST), leaves (LV) and embryos (EB). Through transcriptome profiling analysis, we identified 1,165 genes that were enriched at certain anther development periods, and many of them were involved in starch and sucrose metabolism, pentose and glucuronate interconversion, flavonoid biosynthesis, and ascorbate and aldarate metabolism. CONCLUSIONS/SIGNIFICANCE:We first generated a normalized, full-length cDNA library from anthers and performed transcriptome profiling analysis of anther development in Upland cotton. From these results, 10,178 anther expressed genes were identified, among which 1,165 genes were stage-enriched in anthers. And many of these stage-enriched genes were involved in some important processes regulating anther development.
Project description:Gossypium arboreum possesses many favorable traits including robust defense against biotic and abiotic stress although it has been withdrawn from the market because of lower yield and fiber quality compared to G. hirsutum (upland cotton). It is therefore important to explore and utilize the beneficial genes of G. arboretum for G. hirsutum cultivar breeding. Here, the function of G. arboreum JAZ1 in tolerance to salt stress was determined through loss-of-function analysis. GaJAZ1can interact with GaMYC2 to repress expression of downstream genes whose promoters contain a G-box cis element, affecting plant tolerance to salinity stress. The experimental data from NaCl treatments and a 2 year continuous field trial with natural saline-alkaline soil showed that the ectopically overexpressed GaJAZ1 significantly increased salt tolerance in upland cotton compared to the wild type, showing higher growth vigor with taller plants, increased fresh weight, and more bolls, which is due to reprogrammed expression of tolerance-related genes and promotion of root development. High-throughput RNA sequencing of GaJAZ1 transgenic and wild-type plants showed many differentially expressed genes involved in JA signaling and biosynthesis, salt stress-related genes, and hormone-related genes, suggesting that overexpressing GaJAZ1 can reprogram the expression of defense-related genes in G. hirsutum plants to increase tolerance to salt stress. The research provides a foundation to explore and utilize favorable genes from Gossypium species for upland cotton cultivar breeding.
Project description:BACKGROUND:Late embryogenesis abundant (LEA) proteins are large groups of hydrophilic proteins with major role in drought and other abiotic stresses tolerance in plants. In-depth study and characterization of LEA protein families have been carried out in other plants, but not in upland cotton. The main aim of this research work was to characterize the late embryogenesis abundant (LEA) protein families and to carry out gene expression analysis to determine their potential role in drought stress tolerance in upland cotton. Increased cotton production in the face of declining precipitation and availability of fresh water for agriculture use is the focus for breeders, cotton being the backbone of textile industries and a cash crop for many countries globally. RESULTS:In this work, a total of 242, 136 and 142 LEA genes were identified in G. hirsutum, G. arboreum and G. raimondii respectively. The identified genes were classified into eight groups based on their conserved domain and phylogenetic tree analysis. LEA 2 were the most abundant, this could be attributed to their hydrophobic character. Upland cotton LEA genes have fewer introns and are distributed in all chromosomes. Majority of the duplicated LEA genes were segmental. Syntenic analysis showed that greater percentages of LEA genes are conserved. Segmental gene duplication played a key role in the expansion of LEA genes. Sixty three miRNAs were found to target 89 genes, such as miR164, ghr-miR394 among others. Gene ontology analysis revealed that LEA genes are involved in desiccation and defense responses. Almost all the LEA genes in their promoters contained ABRE, MBS, W-Box and TAC-elements, functionally known to be involved in drought stress and other stress responses. Majority of the LEA genes were involved in secretory pathways. Expression profile analysis indicated that most of the LEA genes were highly expressed in drought tolerant cultivars Gossypium tomentosum as opposed to drought susceptible, G. hirsutum. The tolerant genotypes have a greater ability to modulate genes under drought stress than the more susceptible upland cotton cultivars. CONCLUSION:The finding provides comprehensive information on LEA genes in upland cotton, G. hirsutum and possible function in plants under drought stress.
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: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.