Complex molecular mechanisms underlying seedling salt tolerance in rice revealed by comparative transcriptome and metabolomic profiling.
ABSTRACT: To understand the physiological and molecular mechanisms underlying seedling salt tolerance in rice (Oryza sativa L.), the phenotypic, metabolic, and transcriptome responses of two related rice genotypes, IR64 and PL177, with contrasting salt tolerance were characterized under salt stress and salt+abscisic acid (ABA) conditions. PL177 showed significantly less salt damage, lower Na(+)/K(+) ratios in shoots, and Na(+) translocation from roots to shoots, attributed largely to better salt exclusion from its roots and salt compartmentation of its shoots. Exogenous ABA was able to enhance the salt tolerance of IR64 by selectively decreasing accumulation of Na(+) in its roots and increasing K(+) in its shoots. Salt stress induced general and organ-specific increases of many primary metabolites in both rice genotypes, with strong accumulation of several sugars plus proline in shoots and allantoin in roots. This was due primarily to ABA-mediated repression of genes for degradation of these metabolites under salt. In PL177, salt specifically up-regulated genes involved in several pathways underlying salt tolerance, including ABA-mediated cellular lipid and fatty acid metabolic processes and cytoplasmic transport, sequestration by vacuoles, detoxification and cell-wall remodeling in shoots, and oxidation-reduction reactions in roots. Combined genetic and transcriptomic evidence shortlisted relatively few candidate genes for improved salt tolerance in PL177.
Project description:BACKGROUND:To delineate the adaptive mechanisms operative under salinity stress, it is essential to study plant responses at the very early stages of stress which are very crucial for governing plant survival and adaptation. We believe that it is the initial perception and response phase which sets the foundation for stress adaptation in rice seedlings where plants can be considered to be in a state of osmotic shock and ion buildup. RESULTS:An isobaric Tags for Relative and Absolute Quantitation (iTRAQ) approach was used to analyze the pre-existing differences as well as the very early salt shock responsive changes in the proteome of seedlings of contrasting rice genotypes, viz salt-sensitive IR64 and salt-tolerant Pokkali. In response to a quick salt shock, shoots of IR64 exhibited hyperaccumulation of Na+, whereas in Pokkali, these ions accumulated more in roots. Interestingly, we could find 86 proteins to be differentially expressed in shoots of Pokkali seedlings under non-stress conditions whereas under stress, 63 proteins were differentially expressed in Pokkali shoots in comparison to IR64. However, only, 40 proteins under non-stress and eight proteins under stress were differentially expressed in Pokkali roots. A higher abundance of proteins involved in photosynthesis (such as, oxygen evolving enhancer proteins OEE1 & OEE3, PsbP) and stress tolerance (such as, ascorbate peroxidase, superoxide dismutase, peptidyl-prolyl cis-trans isomerases and glyoxalase II), was observed in shoots of Pokkali in comparison to IR64. In response to salinity, selected proteins such as, ribulose bisphosphate carboxylase/oxygenase activase, remained elevated in Pokkali shoots. Glutamate dehydrogenase - an enzyme which serves as an important link between Krebs cycle and metabolism of amino acids was found to be highly induced in Pokkali in response to stress. Similarly, other enzymes such as peroxidases and triose phosphate isomerase (TPI) were also altered in roots in response to stress. CONCLUSION:We conclude that Pokkali rice seedlings are primed to face stress conditions where the proteins otherwise induced under stress in IR64, are naturally expressed in high abundance. Through specific alterations in its proteome, this proactive stress machinery contributes towards the observed salinity tolerance in this wild rice germplasm.
Project description:BACKGROUND: Rice is sensitive to salt stress, especially at the seedling stage, with rice varieties differing remarkably in salt tolerance (ST). To understand the physiological mechanisms of ST, we investigated salt stress responses at the metabolite level. METHODS: Gas chromatography-mass spectrometry was used to profile metabolite changes in the salt-tolerant line FL478 and the sensitive variety IR64 under a salt-stress time series. Additionally, several physiological traits related to ST were investigated. RESULTS: We characterized 92 primary metabolites in the leaves and roots of the two genotypes under stress and control conditions. The metabolites were temporally, tissue-specifically and genotype-dependently regulated under salt stress. Sugars and amino acids (AAs) increased significantly in the leaves and roots of both genotypes, while organic acids (OAs) increased in roots and decreased in leaves. Compared with IR64, FL478 experienced greater increases in sugars and AAs and more pronounced decreases in OAs in both tissues; additionally, the maximum change in sugars and AAs occurred later, while OAs changed earlier. Moreover, less Na+ and higher relative water content were observed in FL478. Eleven metabolites, including AAs and sugars, were specifically increased in FL478 over the course of the treatment. CONCLUSIONS: Metabolic responses of rice to salt stress are dynamic and involve many metabolites. The greater ST of FL478 is due to different adaptive reactions at different stress times. At early salt-stress stages, FL478 adapts to stress by decreasing OA levels or by quickly depressing growth; during later stages, more metabolites are accumulated, thereby serving as compatible solutes against osmotic challenge induced by salt stress.
Project description:The sucrose non-fermenting-1-related protein kinase 2 family (SnRK2s) unifies different abiotic stress signals in plants. To date, the functions of two rice SnRK2s, osmotic stress/ABA-activated protein kinase 1 (SAPK1) and SAPK2, have been unknown. We investigated their roles in response to salt stress by generating loss-of-function lines using the CRISPR/Cas9 system and by overexpressing these proteins in transgenic rice plants.Expression profiling revealed that SAPK1 and SAPK2 expression were strongly induced by drought, NaCl, and PEG treatment, but not by ABA. SAPK2 expression was highest in the leaves, followed by the roots, whereas SAPK1 was highest expressed in roots followed by leaves. Both proteins were localized to the nucleus and the cytoplasm. Under salt stress, sapk1, sapk2 and, in particular, sapk1/2 mutants, exhibited reduced germination rates, more severe growth inhibition, more distinct chlorosis, reduced chlorophyll contents, and reduced survival rates in comparison with the wild-type plants. In contrast, SAPK1- and SAPK2-overexpression lines had increased germination rates and reduced sensitivities to salt; including mild reductions in growth inhibition, reduced chlorosis, increased chlorophyll contents and improved survival rates in comparison with the wild-type plants. These results suggest that SAPK1 and SAPK2 may function collaboratively as positive regulators of salt stress tolerance at the germination and seedling stages. We also found that SAPK1 and SAPK2 affected the osmotic potential following salt stress by promoting the generation of osmotically active metabolites such as proline. SAPK1 and SAPK2 also improved reactive oxygen species (ROS) detoxification following salt stress by promoting the generation of ROS scavengers such as ascorbic acid, and by increasing the expression levels of proteins such as superoxide dismutase (SOD) and catalase (CAT). SAPK1 and SAPK2 may function collaboratively in reducing Na+ toxicity by affecting the Na+ distribution between roots and shoots, Na+ exclusion from the cytoplasm, and Na+ sequestration into the vacuoles. These effects may be facilitated through the expression of Na+-and K+-homeostasis-related genes.SAPK1 and SAPK2 may function collaboratively as positive regulators of salt stress tolerance at the germination and seedling stages in rice. SAPK1 and SAPK2 may be useful to improve salt tolerance in crop plants.
Project description:The NAC transcription factors play critical roles in regulating stress responses in plants. However, the functions for many of the NAC family members in rice are yet to be identified. In the present study, a novel stress-responsive rice NAC gene, ONAC022, was identified. Expression of ONAC022 was induced by drought, high salinity, and abscisic acid (ABA). The ONAC022 protein was found to bind specifically to a canonical NAC recognition cis-element sequence and showed transactivation activity at its C-terminus in yeast. The ONAC022 protein was localized to nucleus when transiently expressed in Nicotiana benthamiana. Three independent transgenic rice lines with overexpression of ONAC022 were generated and used to explore the function of ONAC022 in drought and salt stress tolerance. Under drought stress condition in greenhouse, soil-grown ONAC022-overexpressing (N22oe) transgenic rice plants showed an increased drought tolerance, leading to higher survival ratios and better growth than wild-type (WT) plants. When grown hydroponically in Hogland solution supplemented with 150 mM NaCl, the N22oe plants displayed an enhanced salt tolerance and accumulated less Na(+) in roots and shoots as compared to WT plants. Under drought stress condition, the N22oe plants exhibited decreased rates of water loss and transpiration, reduced percentage of open stomata and increased contents of proline and soluble sugars. However, the N22oe lines showed increased sensitivity to exogenous ABA at seed germination and seedling growth stages but contained higher level of endogenous ABA. Expression of some ABA biosynthetic genes (OsNCEDs and OsPSY), signaling and regulatory genes (OsPP2C02, OsPP2C49, OsPP2C68, OsbZIP23, OsAP37, OsDREB2a, and OsMYB2), and late stress-responsive genes (OsRAB21, OsLEA3, and OsP5CS1) was upregulated in N22oe plants. Our data demonstrate that ONAC022 functions as a stress-responsive NAC with transcriptional activator activity and plays a positive role in drought and salt stress tolerance through modulating an ABA-mediated pathway.
Project description:HKT Na+ transporters correspond to major salt tolerance QTLs in different plant species and are targets of great interest for breeders. In rice, the HKT family is composed of seven or eight functional genes depending on cultivars. Three rice HKT genes, OsHKT1;1, OsHKT1;4 and OsHKT1;5, are known to contribute to salt tolerance by reducing Na+ accumulation in shoots upon salt stress. Here, we further investigate the mechanisms by which OsHKT1;4 contributes to this process and extend this analysis to the role of this transporter in plants in presence of low Na+ concentrations. By analyzing transgenic rice plants expressing a GUS reporter gene construct, we observed that OsHKT1;4 is mainly expressed in xylem parenchyma in both roots and leaves. Using mutant lines expressing artificial microRNA that selectively reduced OsHKT1;4 expression, the involvement of OsHKT1;4 in retrieving Na+ from the xylem sap in the roots upon salt stress was evidenced. Since OsHKT1;4 was found to be also well expressed in the roots in absence of salt stress, we extended the analysis of its role when plants were subjected to non-toxic Na+ conditions (0.5 and 5 mM). Our finding that the transporter, expressed in Xenopus oocytes, displayed a relatively high affinity for Na+, just above 1 mM, provided first support to the hypothesis that OsHKT1;4 could have a physiological role at low Na+ concentrations. We observed that progressive desalinization of the xylem sap along its ascent to the leaf blades still occurred in plants grown at submillimolar Na+ concentration, and that OsHKT1;4 was involved in reducing xylem sap Na+ concentration in roots in these conditions too. Its contribution to tissue desalinization from roots to young mature leaf blades appeared to be rather similar in the whole range of explored external Na+ concentrations, from submillimolar to salt stress conditions. Our data therefore indicate that HKT transporters can be involved in controlling Na+ translocation from roots to shoots in a much wider range of Na+ concentrations than previously thought. This asks questions about the roles of such a transporter-mediated maintaining of tissue Na+ content gradients in non-toxic conditions.
Project description:Traditional varieties and landraces belonging to the aus-type group of rice (Oryza sativa L.) are known to be highly tolerant to environmental stresses, such as drought and heat, and are therefore recognized as a valuable genetic resource for crop improvement. Using two aus-type (Dular, N22) and two drought intolerant irrigated varieties (IR64, IR74) an untargeted metabolomics analysis was conducted to identify drought-responsive metabolites associated with tolerance.The superior drought tolerance of Dular and N22 compared with the irrigated varieties was confirmed by phenotyping plants grown to maturity after imposing severe drought stress in a dry-down treatment. Dular and N22 did not show a significant reduction in grain yield compared to well-watered control plants, whereas the intolerant varieties showed a significant reduction in both, total spikelet number and grain yield. The metabolomics analysis was conducted with shoot and root samples of plants at the tillering stage at the end of the dry-down treatment. The data revealed an overall higher accumulation of N-rich metabolites (amino acids and nucleotide-related metabolites allantoin and uridine) in shoots of the tolerant varieties. In roots, the aus-type varieties were characterised by a higher reduction of metabolites representative of glycolysis and the TCA cycle, such as malate, glyceric acid and glyceric acid-3-phosphate. On the other hand, the oligosaccharide raffinose showed a higher fold increase in both, shoots and roots of the sensitive genotypes. The data further showed that, for certain drought-responsive metabolites, differences between the contrasting rice varieties were already evident under well-watered control conditions.The drought tolerance-related metabolites identified in the aus-type varieties provide a valuable set of protective compounds and an entry point for assessing genetic diversity in the underlying pathways for developing drought tolerant rice and other crops.
Project description:Cold stress affects rice growth, quality and yield. The investigation of genome-wide gene expression is important for understanding cold stress tolerance in rice. We performed comparative transcriptome analysis of the shoots and roots of 2 rice seedlings (TNG67, cold-tolerant; and TCN1, cold-sensitive) in response to low temperatures and restoration of normal temperatures following cold exposure. TNG67 tolerated cold stress via rapid alterations in gene expression and the re-establishment of homeostasis, whereas the opposite was observed in TCN1, especially after subsequent recovery. Gene ontology and pathway analyses revealed that cold stress substantially regulated the expression of genes involved in protein metabolism, modification, translation, stress responses, and cell death. TNG67 takes advantage of energy-saving and recycling resources to more efficiently synthesize metabolites compared with TCN1 during adjustment to cold stress. During recovery, expression of OsRR4 type-A response regulators was upregulated in TNG67 shoots, whereas that of genes involved in oxidative stress, chemical stimuli and carbohydrate metabolic processes was downregulated in TCN1. Expression of genes related to protein metabolism, modification, folding and defense responses was upregulated in TNG67 but not in TCN1 roots. In addition, abscisic acid (ABA)-, polyamine-, auxin- and jasmonic acid (JA)-related genes were preferentially regulated in TNG67 shoots and roots and were closely associated with cold stress tolerance. The TFs AP2/ERF were predominantly expressed in the shoots and roots of both TNG67 and TCN1. The TNG67-preferred TFs which express in shoot or root, such as OsIAA23, SNAC2, OsWRKY1v2, 24, 53, 71, HMGB, OsbHLH and OsMyb, may be good candidates for cold stress tolerance-related genes in rice. Our findings highlight important alterations in the expression of cold-tolerant genes, metabolic pathways, and hormone-related and TF-encoding genes in TNG67 rice during cold stress and recovery. The cross-talk of hormones may play an essential role in the ability of rice plants to cope with cold stress.
Project description:Salinity-induced osmotic, ionic and oxidative stress responses were investigated on Asakaze/Manas wheat/barley addition lines 7H, 7HL and 7HS, together with their barley (salt-tolerant) and wheat (relatively salt-sensitive) parents. Growth, photosynthetic activity, chlorophyll degradation, proline, glycine betaine accumulation, sugar metabolism, Na+ and K+ uptake and transport processes and the role of polyamines and antioxidants were studied in young plants grown in hydroponic culture with or without salt treatment. Changes in plant growth and photosynthetic activity of plants demonstrated that the salt tolerance of the addition lines 7H and 7HL was similar to that of barley parent cv. Manas, while the sensitivity of the addition line 7HS was similar to that of the wheat parent cv. Asakaze. The Na accumulation in the roots and shoots did not differ between the addition lines and wheat parent. The activation of various genes related to Na uptake and transport was not correlated with the salt tolerance of the genotypes. These results indicated that the direct regulation of Na transport processes is not the main reason for the salt tolerance of these genotypes. Salt treatment induced a complex metabolic rearrangement in both the roots and shoots of all the genotypes. Elevated proline accumulation in the roots and enhanced sugar metabolism in the shoots were found to be important for salt tolerance in the 7H and 7HL addition lines and in barley cv. Manas. In wheat cv. Asakaze and the 7HS addition line the polyamine metabolism was activated. It seems that osmotic adjustment is a more important process in the improvement of salt tolerance in 7H addition lines than the direct regulation of Na transport processes or antioxidant defence.
Project description:BACKGROUND:Introgression as a means of generating phenotypic novelty, including altered stress tolerance, is increasingly being recognized as common. The underlying basis for de novo genesis of phenotypic variation in the introgression lines remains largely unexplored. In this investigation, we used a rice line (RZ35) derived from introgressive hybridization between rice (Oryza sativa L.) and wild rice (Zizania latifolia Griseb.), along with its rice parental line (cv. Matsumae) as the experimental materials. We compared effects of salt stress on growth, ion homeostasis, and relevant gene expression between RZ35 and Matsumae, to explore possible mechanisms of heritable alteration in stress tolerance induced by the introgression. RESULTS:Contrary to our expectation, the results showed that the inhibitory effect of salt stress on growth of RZ35 was significantly greater than that of Matsumae. We further found that a major underlying cause for this outcome is that the introgression process weakened the capacity in Na+ exclusion under the salt stress condition, and hence, escalated the injuries of Na+ and Cl- in shoots of RZ35. Accordingly, based on q-RT-PCR analysis, four genes known to be involved in the Na+ exclusion, i.e., OsHKT1;5, OsSOS1, OsCIPK24 and OsCBL4, were found to be significantly down-regulated in roots of RZ35 relative to its rice parental line under the salt stress condition, thus implicating a gene expression regulation-based molecular mechanism underlying the difference in salt stress-tolerance between the introgression line and its rice parental line. CONCLUSIONS:We show that introgression represents a potent means for rapidly generating de novo heritable variations in physiological traits like stress tolerance in plants, although the direction of the alteration appears unpredictable.
Project description:High salt stress caused by ionic and osmotic stressors eventually results in the suppression of plant growth and a reduction in crop productivity. In our previous reports, we isolated the endophytic bacterium Bacillus oryzicola YC7007 from the rhizosphere of rice (Oryza sativa L.), which promoted plant growth and development and suppressed bacterial disease in rice by inducing systemic resistance and antibiotic production. In this study, Arabidopsis thaliana seedlings under salt stress that were bacterized with YC7007 displayed an increase in the number of lateral roots and greater fresh weight relative to that of the control seedlings. The chlorophyll content of the bacterized seedlings was increased when compared with that of untreated seedlings. The accumulation of salt-induced malondialdehyde and Na+ in seedlings was inhibited by their co-cultivation with YC7007. The expression of stress-related genes in the shoots and roots of seedlings was induced by YC7007 inoculation under salt stress conditions. Interestingly, YC7007-mediated salt tolerance requires SOS1, a plasma membrane-localized Na+/H+ antiporter, given that plant growth in sos2-1 and sos3-1 mutants was promoted under salt-stress conditions, whereas that of sos1-1 mutants was not. In addition, inoculation with YC7007 in upland-crops, such as radish and cabbage, increased the number of lateral roots and the fresh weight of seedlings under salt-stress conditions. Our results suggest that B. oryzicola YC7007 enhanced plant tolerance to salt stress via the SOS1-dependent salt signaling pathway, resulting in the normal growth of salt-stressed plants.