Comparative transcriptome profiling of chilling stress responsiveness in two contrasting rice genotypes.
ABSTRACT: Rice is sensitive to chilling stress, especially at the seedling stage. To elucidate the molecular genetic mechanisms of chilling tolerance in rice, comprehensive gene expressions of two rice genotypes (chilling-tolerant LTH and chilling-sensitive IR29) with contrasting responses to chilling stress were comparatively analyzed. Results revealed a differential constitutive gene expression prior to stress and distinct global transcription reprogramming between the two rice genotypes under time-series chilling stress and subsequent recovery conditions. A set of genes with higher basal expression were identified in chilling-tolerant LTH compared with chilling-sensitive IR29, indicating their possible role in intrinsic tolerance to chilling stress. Under chilling stress, the major effect on gene expression was up-regulation in the chilling- tolerant genotype and strong repression in chilling-sensitive genotype. Early responses to chilling stress in both genotypes featured commonly up-regulated genes related to transcription regulation and signal transduction, while functional categories for late phase chilling regulated genes were diverse with a wide range of functional adaptations to continuous stress. Following the cessation of chilling treatments, there was quick and efficient reversion of gene expression in the chilling-tolerant genotype, while the chilling-sensitive genotype displayed considerably slower recovering capacity at the transcriptional level. In addition, the detection of differentially-regulated TF genes and enriched cis-elements demonstrated that multiple regulatory pathways, including CBF and MYBS3 regulons, were involved in chilling stress tolerance. A number of the chilling-regulated genes identified in this study were co-localized onto previously fine-mapped cold-tolerance-related QTLs, providing candidates for gene cloning and elucidation of molecular mechanisms responsible for chilling tolerance in rice.
Project description:<h4>Background</h4>Chilling stress is a major factor limiting rice production. Rice genotypes differ greatly in their seedling chilling tolerance (CT), which is known to involve differential expression of large numbers of genes and proteins. To further understand the metabolomic responses of rice to chilling stress, profiles of the 106 primary metabolites of a CT japonica variety, Lijiangxintuanhegu (LTH) and a chilling sensitive indica line, IR29, were investigated under a time-series of chilling stress and non-stress control conditions at the seedling stage.<h4>Results</h4>We identified 106 primary metabolites that were temporally and genotype-dependently regulated in LTH and IR29 under the time-series chilling stress and subsequent recovery. Three major groups of primary metabolites, amino acids (AAs), organic acids (OAs) and sugars, showed distinct change patterns in both genotypes in response to the chilling stress: a more general accumulation of most AAs, more dramatic decreased levels of most OAs, and greatly reduced levels for most sugars at early time points of stress but increased levels of specific sugars at the later time points of stress. Compared to IR29, LTH had more metabolites showing chilling induced changes, greater levels of these metabolomic changes and a greater ability to recover after stress, implying that LTH used a positive energy-saving strategy against chilling stress. During subsequent recovery, more metabolites were significantly and exclusively up-regulated in LTH, indicating their positive role in chilling tolerance. A comparative analysis of these metabolites data and differentially expressed genes data allowed identification of 7 AAs and related genes that were both chilling responsive and contributed greatly to the CT of LTH.<h4>Conclusions</h4>The metabolomic responses of rice to chilling stress at the seedling stage were dynamic and involved large numbers of the metabolites. The chilling induced changes of three major groups of metabolites, AAs, OAs and sugars, in rice were well coordinated. The high level seedling CT of LTH was apparently attributed to its increased levels of most AAs and reduced energy consumption that resulted in increased glycolysis and strong resilience on recovery. The results of this study extend our understanding of molecular mechanisms of chilling stress tolerance in rice.
Project description:Rice is sensitive to chilling stress, especially at the seedling stage. To elucidate the molecular genetic mechanisms of chilling tolerance in rice, comprehensive gene expressions of two rice genotypes (chilling-tolerant LTH and chilling-sensitive IR29) with contrasting responses to chilling stress were comparatively analyzed. Results revealed distinct global transcription reprogramming between the two rice genotypes under time-series chilling stress and subsequent recovery conditions. A set of genes with higher basal expression were identified in LTH, indicating their possible role in intrinsic tolerance to chilling stress. Under chilling stress, the major effect on gene expression was up-regulation in LTH and strong repression in IR29. Early responses to chilling stress in both genotypes featured commonly up-regulated genes related to transcription regulation and signal transduction, while functional categories for late phase chilling regulated genes were diverse with a wide range of functional adaptations to continuous stress. Following the cessation of chilling treatments, there was quick and efficient reversion of gene expression in LTH, while IR29 displayed considerably slower recovering capacity at the transcriptional level. In addition, the detection of differentially-regulated TF genes and enriched cis-elements demonstrated that multiple regulatory pathways, including CBF and MYBS3 regulons, were involved in chilling stress tolerance. In present study, comprehensive gene expression using an Affymetrix rice genome array revealed a diverse global transcription reprogramming between two rice genotypes under chilling stress and subsequent recovery conditions. The dominant change in gene expression at low temperature was up-regulation in the chilling-tolerant genotype and down-regulation in the chilling-sensitive genotype. Early responses to chilling stress common to both genotypes featured up-regulated genes related to transcription regulation and signal transduction, while functional categories of LR-chilling regulated genes were clearly diverse with a wide range of functional adaptations. At the end of the chilling treatments, there was quick and efficient reversion of gene expression in LTH, while IR29 displayed considerably slower recovery capacity at the transcriptional level. Finally, analysis of differentially-regulated TF genes and enriched cis-elements demonstrated that multiple regulatory pathways, including CBF and MYBS3 regulons, are involved in chilling stress tolerance. In this study, parallel transcriptomic analysis in two rice genotypes with contrasting chilling-tolerant phenotypes was performed to identify and characterize novel genes involved in chilling stress tolerance in rice.
Project description:DNA methylation has been referred as an important player in plant genomic responses to environmental stresses but correlations between the methylome plasticity and specific traits of interest are still far from being understood. In this study, we inspected global DNA methylation levels in salt tolerant and sensitive rice varieties upon salt stress imposition. Global DNA methylation was quantified using the 5-methylcytosine (5mC) antibody and an ELISA-based technique, which is an affordable and quite pioneer assay in plants, and in situ imaging of methylation sites in interphase nuclei of tissue sections. Variations of global DNA methylation levels in response to salt stress were tissue- and genotype-dependent. We show a connection between a higher ability of DNA methylation adjustment levels and salt stress tolerance. The salt-tolerant rice variety Pokkali was remarkable in its ability to quickly relax DNA methylation in response to salt stress. In spite of the same tendency for reduction of global methylation under salinity, in the salt-sensitive rice variety IR29 such reduction was not statistically supported. In 'Pokkali', the salt stress-induced demethylation may be linked to active demethylation due to increased expression of DNA demethylases under salt stress. In 'IR29', the induction of both DNA demethylases and methyltransferases may explain the lower plasticity of DNA methylation. We further show that mutations for epigenetic regulators affected specific phenotypic parameters related to salinity tolerance, such as the root length and biomass. This work emphasizes the role of differential methylome flexibility between salt tolerant and salt sensitive rice varieties as an important player in salt stress tolerance, reinforcing the need to better understand the connection between epigenetic networks and plant responses to environmental stresses.
Project description:BACKGROUND:Salinity expansion in arable land is a threat to crop plants. Rice is the staple food crop across several countries worldwide; however, its salt sensitive nature severely affects its growth under excessive salinity. FL478 is a salt tolerant indica recombinant inbred line, which can be a good source of salt tolerance at the seedling stage in rice. To learn about the genetic basis of its tolerance to salinity, we compared transcriptome profiles of FL478 and its sensitive parent (IR29) using RNA-seq technique. RESULTS:A total of 1714 and 2670 genes were found differentially expressed (DEGs) under salt stress compared to normal conditions in FL478 and IR29, respectively. Gene ontology analysis revealed the enrichment of transcripts involved in salinity response, regulation of gene expression, and transport in both genotypes. Comparative transcriptome analysis revealed that 1063 DEGs were co-expressed, while 338/252 and 572/908 DEGs were exclusively up/down-regulated in FL478 and IR29, respectively. Further, some biological processes (e.g. iron ion transport, response to abiotic stimulus, and oxidative stress) and molecular function terms (e.g. zinc ion binding and cation transmembrane transporter activity) were specifically enriched in FL478 up-regulated transcripts. Based on the metabolic pathways analysis, genes encoding transport and major intrinsic proteins transporter superfamily comprising aquaporin subfamilies and genes involved in MAPK signaling and signaling receptor kinases were specifically enriched in FL478. A total of 1135 and 1894 alternative splicing events were identified in transcripts of FL478 and IR29, respectively. Transcripts encoding two potassium transporters and two major facilitator family transporters were specifically up-regulated in FL478 under salt stress but not in the salt sensitive genotype. Remarkably, 11 DEGs were conversely regulated in the studied genotypes; for example, OsZIFL, OsNAAT, OsGDSL, and OsELIP genes were up-regulated in FL478, while they were down-regulated in IR29. CONCLUSIONS:The achieved results suggest that FL478 employs more efficient mechanisms (especially in signal transduction of salt stress, influx and transport of k+, ionic and osmotic homeostasis, as well as ROS inhibition) to respond to the salt stress compared to its susceptible parent.
Project description:Rice yield is most sensitive to salinity stress imposed during the panicle initiation (PI) stage. In this study, we have focused on physiological and transcriptional responses of four rice genotypes exposed to salinity stress during PI. The genotypes selected included a pair of indicas (IR63731 and IR29) and a pair of japonica (Agami and M103) rice subspecies with contrasting salt tolerance. Physiological characterization showed that tolerant genotypes maintained a much lower shoot Na+ concentration relative to sensitive genotypes under salinity stress. Global gene expression analysis revealed a strikingly large number of genes which are induced by salinity stress in sensitive genotypes, IR29 and M103 relative to tolerant lines. We found 19 probe sets to be commonly induced in all four genotypes. We found several salinity modulated, ion homeostasis related genes from our analysis. We also studied the expression of SKC1, a cation transporter reported by others as a major source of variation in salt tolerance in rice. The transcript abundance of SKC1 did not change in response to salinity stress at PI stage in the shoot tissue of all four genotypes. However, we found the transcript abundance of SKC1 to be significantly higher in tolerant japonica Agami relative to sensitive japonica M103 under control and stressed conditions during PI stage.
Project description:Global increase in salinity levels has made it imperative to identify novel sources of genetic variation for tolerance traits, especially in rice. The rice landrace Horkuch, endemic to the saline coastal area of Bangladesh, was used in this study as the source of tolerance in reciprocal crosses with the sensitive but high-yielding IR29 variety for discovering transcriptional variation associated with salt tolerance in the resulting populations. The cytoplasmic effect of the Horkuch background in leaves under stress showed functional enrichment for signal transduction, DNA-dependent regulation and transport activities. In roots the enrichment was for cell wall organization and macromolecule biosynthesis. In contrast, the cytoplasmic effect of IR29 showed upregulation of apoptosis and downregulation of phosphorylation across tissues relative to Horkuch. Differential gene expression in leaves of the sensitive population showed downregulation of GO processes like photosynthesis, ATP biosynthesis and ion transport. Roots of the tolerant plants conversely showed upregulation of GO terms like G-protein coupled receptor pathway, membrane potential and cation transport. Furthermore, genes involved in regulating membrane potentials were constitutively expressed only in the roots of tolerant individuals. Overall our work has developed genetic resources and elucidated the likely mechanisms associated with the tolerance response of the Horkuch genotype.
Project description:BACKGROUND:Soil salinity is one of the primary causes of yield decline in rice. Pokkali (Pok) is a highly salt-tolerant landrace, whereas IR29 is a salt-sensitive but widely cultivated genotype. Comparative analysis of these genotypes may offer a better understanding of the salinity tolerance mechanisms in rice. Although most stress-responsive genes are regulated at the transcriptional level, in many cases, changes at the transcriptional level are not always accompanied with the changes in protein abundance, which suggests that the transcriptome needs to be studied in conjunction with the proteome to link the phenotype of stress tolerance or sensitivity. Published reports have largely underscored the importance of transcriptional regulation during salt stress in these genotypes, but the regulation at the translational level has been rarely studied. Using RNA-Seq, we simultaneously analyzed the transcriptome and translatome from control and salt-exposed Pok and IR29 seedlings to unravel molecular insights into gene regulatory mechanisms that differ between these genotypes. RESULTS:Clear differences were evident at both transcriptional and translational levels between the two genotypes even under the control condition. In response to salt stress, 57 differentially expressed genes (DEGs) were commonly upregulated at both transcriptional and translational levels in both genotypes; the overall number of up/downregulated DEGs in IR29 was comparable at both transcriptional and translational levels, whereas in Pok, the number of upregulated DEGs was considerably higher at the translational level (544 DEGs) than at the transcriptional level (219 DEGs); in contrast, the number of downregulated DEGs (58) was significantly less at the translational level than at the transcriptional level (397 DEGs). These results imply that Pok stabilizes mRNAs and also efficiently loads mRNAs onto polysomes for translation during salt stress. CONCLUSION:Under salt stress, Pok is more efficient in maintaining cell wall integrity, detoxifying reactive oxygen species (ROS), translocating molecules and maintaining photosynthesis. The present study confirmed the known salt stress-associated genes and also identified a number of putative new salt-responsive genes. Most importantly, the study revealed that the translational regulation under salinity plays an important role in salt-tolerant Pok, but such regulation was less evident in the salt-sensitive IR29.
Project description:Salinity is one of the major constraints in rice production. To date, development of salt-tolerant rice cultivar is primarily focused on salt-exclusion strategies, which incur greater energy cost. The present study aimed to evaluate a balancing strategy of ionic discrimination vis-à-vis tissue tolerance, which could potentially minimize the energy cost of salt tolerance in rice. Four rice genotypes, viz., FL478, IR29, Kamini, and AC847, were grown hydroponically and subjected to salt stress equivalent to 12 dS m-1 at early vegetative stage. Different physiological observations (leaf chlorophyll content, chlorophyll fluorescence traits, and tissue Na+ and K+ content) and visual scoring suggested a superior Na+-partitioning strategy operating in FL478. A very low tissue Na+/K+ ratio in the leaves of FL478 after 7 days of stress hinted the existence of selective ion transport mechanism in this genotype. On the contrary, Kamini, an equally salt-tolerant genotype, was found to possess a higher leaf Na+/K+ ratio than does FL478 under similar stress condition. Salt-induced expression of different Na+ and K+ transporters indicated significant upregulation of SOS, HKT, NHX, and HAK groups of transporters in both leaves and roots of FL478, followed by Kamini. The expression of plasma membrane and vacuolar H+ pumps (OsAHA1, OsAHA7, and OsV-ATPase) were also upregulated in these two genotypes. On the other hand, IR29 and AC847 showed greater salt susceptibility owing to excess upward transport of Na+ and eventually died within a few days of stress imposition. But in the "leaf clip" assay, it was found that both IR29 and Kamini had high tissue-tolerance and chlorophyll-retention abilities. On the contrary, FL478, although having higher ionic-discrimination ability, showed the least degree of tissue tolerance as evident from the LC50 score (amount of Na+ required to reduce the initial chlorophyll content to half) of 336 mmol g-1 as against 459 and 424 mmol g-1 for IR29 and Kamini, respectively. Overall, the present study indicated that two components (ionic selectivity and tissue tolerance) of salt tolerance mechanism are distinct in rice. Unique genotypes like Kamini could effectively balance both of these strategies to achieve considerable salt tolerance, perhaps with lesser energy cost.
Project description:Identification and cloning of cold-tolerant genes that can stably express under different cold environments are crucial for molecular rice breeding for cold tolerance. In the previous study, we identified a cold-tolerant QTL at the seedling stage, qCTS-9 which could be detected under different cold environments using a recombinant inbred line (RIL) population derived from a cold-tolerant variety Lijiangxintuanheigu (LTH) and a cold-sensitive variety Shanhuangzhan 2 (SHZ-2). In this study, eight candidate genes within the qCTS-9 interval were identified through integrated analysis of QTL mapping with genomewide differential expression profiling of LTH. The qRT-PCR assay showed that only Os09g0410300 exhibited different expression patterns between LTH and SHZ-2 during cold stress, and significantly positive correlation was found between cold induction of Os09g0410300 and seedling cold tolerance in the RI lines. Five SNPs and one InDel in the promoters of Os09g0410300 were detected between LTH and SHZ-2, and the InDel marker ID410300 designed based on the insertion-deletion polymorphism in the promoter was significantly associated with seedling cold tolerance in RIL population. Further, Os09g0410300 over-expression plants exhibited enhanced cold tolerance at the seedling stage compared with the wild-type plants. Thus, our results suggest that Os09g0410300 is the functional gene underlying qCTS-9. To our knowledge, it is a novel gene contributed to enhance cold tolerance at the seedling stage in rice. Identification of the functional gene underlying qCTS-9 and development of the gene-specific marker will facilitate molecular breeding for cold tolerance at the seedling stage in rice through transgenic approach and marker-assisted selection (MAS).
Project description:Salt stress causes significant reductions in rice production worldwide; thus, improving salt tolerance is a promising approach to meet the increasing food demand. Wild rice germplasm is considered a valuable genetic resource for improving rice cultivars. However, information regarding the improvement of salt tolerance in cultivated rice using wild rice genes is limited. In this study, we identified a salt-tolerant line Dongxiang/Ningjing 15 (DJ15) under salt-stress field conditions from the population of a salt tolerant Dongxiang wild rice × a cultivated rice variety Ningjing16 (NJ16). Genomic resequencing analysis of NJ16, DJ15 and Dongxiang wild rice revealed that the introgressed genomic fragments were unevenly distributed over the 12 chromosomes (Chr.) and mainly identified on Chr. 6, 7, 10, and 11. Using quantitative trait locus (QTL) mapping, we found 9 QTL for salt tolerance (qST) at the seedling stage located on Chr. 1, 3, 4, 5, 6, 8, and 10. In addition, sequence variant analysis within the QTL regions demonstrated that SKC1/HKT8/HKT1;5 and HAK6 transporters along with numerous transcriptional factors were the candidate genes for the salt tolerant QTL. The DJ15/Koshihikari recombinant inbred lines that contained both qST1.2 and qST6, two QTL with the highest effect for salt tolerance, were more tolerant than the parental lines under salt-stress field conditions. Furthermore, the qST6 near-isogenic lines with IR29 background were more tolerant than IR29, indicating that qST1.2 and qST6 could improve salt tolerance in rice. Overall, our study indicates that wild rice genes could markedly improve the salt tolerance of cultivated rice.