Comparative analysis of Histone modifications and DNA methylation at <Emphasis Type="Italic">OsBZ8</Emphasis> locus under salinity stress in IR64 and Nonabokra rice varieties

Rice being an important cereal crop is highly sensitive to salinity stress causing growth retardation and loss in productivity. However, certain rice genotypes like Nonabokra and Pokkali show a high level of tolerance towards salinity stress compared to IR64 variety. This differential response of tolerant varieties towards salinity stress may be a cumulative effect of genetic and epigenetic factors. In this study, we have compared the salinity-induced changes in chromatin modifications at the OsBZ8 locus in salt-tolerant Nonabokra and salt-sensitive IR64 rice varieties. Expression analysis indicates that the OsBZ8 gene is highly induced in Nonabokra plants even in the absence of salt stress, whereas in IR64, the expression significantly increases only during salt stress. Sequence analysis and nucleosomal arrangement within the region ?2000 to +1000 of OsBZ8 gene show no difference between the two rice varieties. However, there was a considerable difference in histone modifications and DNA methylation at the locus between these varieties. In Nonabokra, the upstream region was hyperacetylated at H3K9 and H3K27, and this acetylation did not change during salt stress. However, in IR64, histone acetylation was observed only during salt stress. Moreover, the upstream region of OsBZ8 gene has highly dynamic nucleosome arrangement in Nonabokra, compared to IR64. Furthermore, loss of DNA methylation was observed at OsBZ8 locus in Nonabokra control plants along with low H3K27me3 and high H3K4me3. Control IR64 plants show high DNA methylation and enriched H3K27me3. Collectively these results indicate a significant difference in chromatin modifications between the rice varieties that regulates differential expression of OsBZ8 gene during salt stress.     

Spermidine and abscisic acid-mediated phosphorylation of a cytoplasmic protein from rice root in response to salinity stress

Importance of higher polyamines, spermidine, and spermine, in relation to the mechanism and adaptation to combat abiotic stress has been well established in cereals. Owing to their polycationic nature at physiological pH, polyamines bind strongly to negative charges in cellular components such as nucleic acids, various proteins, and phospholipids. To study the physiological role of polyamine during salinity stress, phosphorylation study was carried out in cytosolic soluble protein fraction isolated from the roots of salt tolerant (Nonabokra) and salt sensitive (M-1-48) rice cultivars treated with none (control), NaCl (150 mM, 16 h), spermidine (1 mM, 16 h) or with abscisic acid (100 μM, 16 h). A calcium independent auto regulatory 42 kDa protein kinase was found to phosphorylate myelin basic protein and casein but not histone. Interestingly, this was the only protein to be phosphorylated in root cytosolic fraction during NaCl/abscisic acid/spermidine treatment indicating its importance in salinity mediated signal transduction. This is the first report of polyamine as well as abscisic acid induced protein kinase activity in rice root in response to salinity stress.     

Comparative lipid peroxidation,antioxidant defense systems and proline content in roots of two rice cultivars differing in salt tolerance

The changes in the activity of antioxidant enzymes such as superoxide dismutase (SOD: EC 1.15.1.1), catalase (CAT: EC 1.11.1.6), peroxidase (POX: EC 1.11.1.7), ascorbate peroxidase (APOX: EC 1.11.1.11) and glutathione reductase (GR: EC 1.6.4.2), free proline content, and the rate of lipid peroxidation level in terms of malondialdehyde (MDA) in roots of two rice cultivars (cvs.) differing in salt tolerance were investigated. Plants were subjected to three salt treatments, 0, 60, and 120 mol m⁻³ NaCl for 7 days. The results showed that activated oxygen species may play a role in cellular toxicity of NaCl and indicated differences in activation of antioxidant defense systems between the two cvs. The roots of both cultivars showed a decrease in GR activity with increase in salinity. CAT and APOX activities increased with increasing salt stress in roots of salt-tolerant cultivar Pokkali but decreased and showed no change, respectively, in roots of IR-28 cultivar. POX activity decreased with increasing NaCl concentrations in salt-tolerant Pokkali but increased in IR-28. SOD activity showed no change in roots of both cultivars under increasing salinity. MDA level in the roots increased under salt stress in sensitive IR-28 but showed no change in Pokkali. IR-28 produced higher amount of proline under salt stress than in Pokkali. Increasing NaCl concentration caused a reduction in root fresh weight of Pokkali and root dry weight of IR-28. The results indicate that improved tolerance to salt stress in root tissues of rice plants may be accomplished by increased capacity of antioxidative system.     

Genomewide discovery of DNA polymorphisms in rice cultivars with contrasting drought and salinity stress response and their functional relevance

Next‐generation sequencing technologies provide opportunities to understand the genetic basis of phenotypic differences, such as abiotic stress response, even in the closely related cultivars via identification of large number of DNA polymorphisms. We performed whole‐genome resequencing of three rice cultivars with contrasting responses to drought and salinity stress (sensitive IR64, drought‐tolerant Nagina 22 and salinity‐tolerant Pokkali). More than 356 million 90‐bp paired‐end reads were generated, which provided about 85% coverage of the rice genome. Applying stringent parameters, we identified a total of 1 784 583 nonredundant single‐nucleotide polymorphisms (SNPs) and 154 275 InDels between reference (Nipponbare) and the three resequenced cultivars. We detected 401 683 and 662 509 SNPs between IR64 and Pokkali, and IR64 and N22 cultivars, respectively. The distribution of DNA polymorphisms was found to be uneven across and within the rice chromosomes. One‐fourth of the SNPs and InDels were detected in genic regions, and about 3.5% of the total SNPs resulted in nonsynonymous changes. Large‐effect SNPs and InDels, which affect the integrity of the encoded protein, were also identified. Further, we identified DNA polymorphisms present in the differentially expressed genes within the known quantitative trait loci. Among these, a total of 548 SNPs in 232 genes, located in the conserved functional domains, were identified. The data presented in this study provide functional markers and promising target genes for salinity and drought tolerance and present a valuable resource for high‐throughput genotyping and molecular breeding for abiotic stress traits in rice.     

Oxidative Stress Tolerance Mechanism in Rice under Salinity

The research was conducted to investigate comparative oxidative damage including probable protective roles of antioxidant and glyoxalase systems in rice (Oryza sativa L.) seedlings under salinity stress. Seedlings of two rice genotypes: Pokkali (tolerant) and BRRI dhan28 (sensitive) were subjected to 8 dSm⁻¹ salinity stress for seven days in a hydroponic system. We observed significant variation between Pokkali and BRRI dhan28 in phenotypic, biochemical and molecular level under salinity stress. Carotenoid content, ion homeostasis, antioxidant enzymes, ascorbate and glutathione redox system and proline accumulation may help Pokkali to develop defense system during salinity stress. However, the activity antioxidant enzymes particularly superoxide dismutase (SOD), catalase (CAT) and non-chloroplastic peroxidase (POD) were observed significantly higher in Pokkali compared to salt-sensitive BRRI dhan28. Higher glyoxalase (Gly-I) and glyoxalase (Gly-II) activity might have also accompanied Pokkali genotype to reduce potential cytotoxic MG through non-toxic hydroxy acids conversion. However, the efficient antioxidants and glyoxalase system together increased adaptability in Pokkali during salinity stress.     

Protective role of exogenous polyamines on salinity-stressed rice (Oryza sativa) plants

Salt-tolerant Pokkali rice plants accumulate higher polyamines (PAs) such as spermidine (Spd) and spermine (Spm) in response to salinity stress, while the sensitive cultivar M -1–48 is unable to maintain high titres of these PAs under similar conditions. The effects of the triamine Spd and the tetramine Spm on physiological and biochemical changes in 12-day-old rice seedlings were investigated during salinity stress to determine whether they could protect the sensitive plants from stress effects. At physiological concentrations Spd and Spm significantly prevented the leakage of electrolytes and amino acids from roots and shoots induced by salinity stress. To different degrees they also prevented chlorophyll loss, inhibition of photochemical reactions of photosynthesis as well as downregulation of chloroplast-encoded genes like psbA , psbB , psbE and rbcL , indicating a positive correlation between salt tolerance and accumulation of higher PAs in rice. The inhibitory effect of salinity stress and its reversal by exogenous PAs were more pronounced in the salt-sensitive M -1–48 plants than in the tolerant Pokkali plants.     

Antioxidants and stress-related metabolites in the seedlings of two indica rice varieties exposed to cadmium chloride toxicity

We present here a comprehensive study depicting the differences in biochemical responses to increasing CdCl₂ concentrations (0.1, 0.25, 0.5 and 1.5 mM) in the two indica rice varieties, IR-29 (salt-sensitive) and Nonabokra (salt-tolerant), in order to contribute to our understanding of genotypic variation of cadmium tolerance. The oxidative damages in both the varieties enhanced with the increase in CdCl₂ concentrations, the susceptibility of IR-29 being more pronounced than Nonabokra. The detrimental effects in IR-29 were reflected in greater chlorophyll loss, higher H₂O₂ and malondialdehyde content even at lower concentrations and drastically higher lipoxygenase activity, protein oxidation and putrescine accumulation, especially at higher CdCl₂ levels. The antioxidants like anthocyanin and carotenoids, antioxidative enzymes like guaiacol peroxidase (GPX) and ascorbate peroxidase (APX), osmolytes like proline, reducing sugars, spermidine and spermine, increased in both the varieties with CdCl₂ levels. While anthocyanin, reducing sugars and spermine showed greater increment in IR-29, the GPX/APX activity was more enhanced in Nonabokra; the increase in carotenoids, proline and spermidine being similar in both the varieties. However, reverse trends were noted for cysteine level and CAT activity; IR-29 showed marked decrease in cysteine content and CAT activity with increased cadmium exposure, whereas in Nonabokra, both the parameters increased, particularly at higher cadmium levels. Thus, the detoxification mechanism in the more-susceptible IR-29 was probably rendered by anthocyanin, reducing sugars and spermine in particular, as well as by GPX/APX, rather than cysteine and CAT, which showed cadmium sensitivity. Thus, the CdCl₂ stress-dependent comparative biochemical analyses displayed major differences in the two rice varieties in terms of tolerance to Cd toxicity. Our data provides evidence that Nonabokra, which is a well-known variety tolerant to sodium chloride toxicity, also shows promising tolerance to cadmium toxicity, and hints at their possible utilization in Cd remediation.     

Insight into the salt tolerance factors of a wild halophytic rice, <Emphasis Type="Italic">Porteresia coarctata</Emphasis>: a physiological and proteomic approach

Salinity poses a serious threat to yield performance of cultivated rice in South Asian countries. To understand the mechanism of salt-tolerance of the wild halophytic rice, Porteresia coarctata in contrast to the salt-sensitive domesticated rice Oryza sativa, we have compared P. coarctata with the domesticated O. sativa rice varieties under salinity stress with respect to several physiological parameters and changes in leaf protein expression. P. coarctata showed a better growth performance and biomass under salinity stress. Relative water content was conserved in Porteresia during stress and sodium ion accumulation in leaves was comparatively lesser. Scanning electron microscopy revealed presence of two types of salt hairs on two leaf surfaces, each showing a different behaviour under stress. High salt stress for prolonged period also revealed accumulation of extruded NaCl crystals on leaf surface. Changes induced in leaf proteins were studied by two-dimensional gel electrophoresis and subsequent quantitative image analysis. Out of more than 700 protein spots reproducibly detected and analyzed, 60% spots showed significant changes under salinity. Many proteins showed steady patterns of up- or downregulation in response to salinity stress. Twenty protein spots were analyzed by MALDI-TOF, leading to identification of 16 proteins involved in osmolyte synthesis, photosystem functioning, RubisCO activation, cell wall synthesis and chaperone functions. We hypothesize that some of these proteins confer a physiological advantage on Porteresia under salinity, and suggest a pattern of salt tolerance strategies operative in salt-marsh grasses. In addition, such proteins may turn out to be potential targets for recombinant cloning and introgression in salt-sensitive plants. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Effects of exogenous abscisic acid on some physiological responses in a popular aromatic indica rice compared with those from two traditional non-aromatic indica rice cultivars

The poor productivity and local confinement of indigenous aromatic rice varieties are mostly due to their susceptibility to salinity/drought/abscisic acid (ABA)-mediated abiotic stresses. It is thus essential to study the effects of several stress factors on their physiological parameters so as to improve their tolerance mechanism and enhance their global demand. Previously, we studied the effect of salinity stress on the physiological and molecular responses of the common aromatic rice Gobindobhog. The objective of this study was to understand the influence of exogenous ABA on some biochemical parameters in Gobindobhog, and comparison with those from non-aromatic M-1-48 and Nonabokra rice. The highest endogenous hydrogen peroxide content and membrane lipid peroxidation (increased malondialdehyde and lipoxygenase activity) were found in ABA-treated Gobindobhog leaves. While the catalase activity was down regulated the most in ABA-treated Gobindobhog leaves, the guaiacol peroxidase activity was induced maximally, indicating the protective role of peroxidase rather than catalase, during ABA-induced oxidative damages. The antioxidant, anthocyanin, showed the highest level in ABA-treated Nonabokra. Enhanced cysteine, following ABA exposure and the highest levels of reducing sugars, total amino acids, proline, and polyamines (putrescine and spermidine) recorded in Gobindobhog, probably served to shield from ABA-induced stress injuries, whereas the spermine levels were comparable in ABA-treated Nonabokra and Gobindobhog. The aroma content, intensified after ABA treatment, was markedly noted in Gobindobhog. Thus, the systematic examination of ABA-mediated stress revealed the most prominent oxidative damages in Gobindobhog, even higher than M-1-48, with a concomitant enhancement in peroxidase system and particularly osmolyte or polyamine levels to ensure its sustenance.     

Effects of salinity on protein and lipopolysaccharide pattern in a salt-tolerant strain of Mesorhizobium ciceri

AIMS: To characterize the physiological and metabolic responses of Mesorhizobium ciceri strain ch-191 to salt stress, investigating the changes induced by salinity in protein and lipopolysaccharide profiles, as well as determining the accumulation of amino acids, glutamate and proline. METHODS AND RESULTS: Strain ch-191 of M. ciceri was grown with different NaCl concentrations. Protein and lipopolysaccharide patterns were determined by electrophoresis. The strain ch-191 tolerated up to 200 mmol l-1 NaCl, although higher salt dosages limited its growth and induced changes in the protein profile. The most noteworthy change in the LPS-I pattern was the decrease in the slowest band and the appearance of an intermediate mobility band. The accumulation of proline in response to salt stress surpassed that of glutamate. CONCLUSION: The protein profile showed major alterations at salinity levels which inhibited growth. However, the alterations in the LPS profile and accumulation of compatible solutes were evident from the lowest levels, suggesting that these changes may constitute adaptative responses to salt, allowing normal growth. SIGNIFICANCE AND IMPACT OF THE STUDY: The selection and characterization of salt-tolerant strains, which also show efficient symbiotic performance under salinity, may constitute a strategy for improving Cicer arietinum-Mesorhizobium ciceri symbiosis in adverse environments.     

Biochemical and molecular changes in rice seedlings (Oryza sativa L.) to cope with chromium stress

Chromium (Cr) is very toxic to both humans and plants. This investigation aimed to understand the physiological and molecular responses of rice seedlings to Cr stress. Cr toxicity did not significantly affect morphological features and Cr accumulation in roots and shoots in Pokkali but not in BRRI 51, although there was a reduction in chlorophyll concentration in leaves of both genotypes. These results imply that Pokkali has mechanisms to cope with Cr supplementation. We therefore performed quantitative real‐time PCR on the expression pattern of two chelator genes, OsPCS1 and OsMT1, but there were no significant changes in expression in roots and shoots of Pokkali and BRRI 51 following Cr stress. This suggests that there was no metal sequestration following heavy metal stress in roots of these genotypes. Moreover, no expression of two heavy metal transporter genes, OsHMA3 and OsNRAMP1, was induced after Cr stress in roots and shoots, suggesting that these transporter genes are not induced by Cr stress or might not be involved in Cr uptake in rice. We also performed a targeted study on the effect of Cr on Fe uptake mechanisms. Our studies showed a consistent reduction in Fe uptake, Fe reductase activity and expression of Fe‐related genes (OsFRO1 and OsIRT1) under Cr stress in both roots and leaves of Pokkali. In contrast, these parameters and genes were significantly increased in Cr‐sensitive BRRI 51 under Cr stress. The results confirm that limiting Fe uptake through the down‐regulation of Fe reductase and Fe transporter genes is the main strategy of Cr‐tolerant Pokkali to cope with Cr stress. Finally, increased CAT, POD and GR activity and elevated glutathione and proline synthesis might provide strong antioxidant defence against Cr stress in Pokkali. Taken together, our findings reveal that Cr stress tolerance in rice (Pokkali) is not related to metal sequestration but is associated with reduced Fe transport and increased antioxidant defence.     

The role of root apoplastic transport barriers in salt tolerance of rice (<Emphasis Type="Italic">Oryza sativa</Emphasis> L.)

Increasing soil salinity reduces crop yields worldwide, with rice being particularly affected. We have examined the correlation between apoplastic barrier formation in roots, Na⁺ uptake into shoots and plant survival for three rice (Oryza sativa L.) cultivars of varying salt sensitivity: the salt-tolerant Pokkali, moderately tolerant Jaya and sensitive IR20. Rice plants grown hydroponically or in soil for 1 month were subjected to both severe and moderate salinity stress. Apoplastic barriers in roots were visualized using fluorescence microscopy and their chemical composition determined by gas chromatography and mass spectrometry. Na⁺ content was estimated by flame photometry. Suberization of apoplastic barriers in roots of Pokkali was the most extensive of the three cultivars, while Na⁺ accumulation in the shoots was the least. Saline stress induced the strengthening of these barriers in both sensitive and tolerant cultivars, with increase in mRNAs encoding suberin biosynthetic enzymes being detectable within 30 min of stress. Enhanced barriers were detected after several days of moderate stress. Overall, more extensive apoplastic barriers in roots correlated with reduced Na⁺ uptake and enhanced survival when challenged with high salinity. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Low salinity causes oxidative stress and modulates specific antioxidant gene expression in the toxic dinoflagellate Alexandrium pacificum

Salinity is an important factor in the physiological regulation of algae; however, its influence on the genomic responses in toxic dinoflagellates is insufficiently understood. In the present study, we evaluated the effect of salinity stress on the physiology, photosynthesis, and molecular responses of the toxic dinoflagellate Alexandrium pacificum (group IV). When exposed cells to different salinities of 20–40 psu, we detected the lowest cell density (3.25?×?10³ cells mL^?1) and highest cell size (30.6 µm) at 20 psu. Photosynthesis efficiency considerably decreased at 20 and 40 psu compared to the control (33 psu). Quantitative real-time polymerase chain reaction revealed that psbA, psbD, and atpC expression levels were significantly downregulated under conditions of salinity stress for 72 h. In contrast, the expression levels of antioxidant genes MnSOD and GPx were greatly upregulated at 20 psu (13.2- and 15.2-fold changes at 6 h; 8.8- and 8.3-fold changes at 24 h, respectively). The expression levels of other antioxidant genes, CuZnSOD, GST, and APx, increased steadily over time under salinity stress. Such conditions increased the relative levels of reactive oxygen species by 2.2-fold in 6 h and 2.4-fold in 24 h at 20 psu. These results suggest that low salinity may cause cellular oxidative stress, leading to a decrease in photosynthesis and affecting specific antioxidant systems in toxic dinoflagellates.

     

Salinity-induced Physiological Modification in the Callus from Halophyte Nitraria tangutorum Bobr.

Little is known about the physiological adaptation mechanisms of the desert halophyte Nitraria tangutorum Bobr. to the environment. In this study, callus from Nitraria tangutorum Bobr. was used to investigate physiological responses to salinity and the regulatory function of nitric oxide (NO) on catalase (CAT) activity. Increased dry weight and soluble proteins were observed in the callus exposed to lower salinity (50 and 100 mM NaCl), whereas 200 mM NaCl led to significant decreases of these two growth parameters, and the levels of proline and soluble carbohydrates also were enhanced under NaCl treatment. In addition, short-term stress from 50 mM NaCl and the application of lower sodium nitroprusside (SNP, a NO donor) concentration resulted in decreased levels of malondialdehyde (MDA). In contrast, higher concentrations of NaCl and SNP induced significant oxidative damage in Nitraria tangutorum Bobr. callus. Analysis based on the fluorescent probe DAF-FM DA revealed that NaCl and SNP treatment led to enhanced levels of NO in the callus cells. Moreover, the NO scavenger 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (c-PTIO) reduced endogenous NO concentrations and abolished the enhancement in dry weight and the decrease in MDA level under 50-mM-NaCl treatment. CAT activity increased under salt stress, and the 50-mM-NaCl effect was alleviated by treatment with c-PTIO or the nitric oxide synthase inhibitor N^ω-nitro-l-arginine. We suggest that Nitraria tangutorum Bobr. callus exhibited tolerance to lower-salinity stress. We also showed that increased NO generation in response to salinity might be associated with regulation of growth, protection against oxidative damage, and excitation of CAT activity in Nitraria tangutorum Bobr. callus under salt stress.     

Genetic variation in microsatellite DNA, physiology and morphology of coastal saline rice (Oryza sativa L.) landraces of Bangladesh

Genetic variation of Oryza sativa L. landraces (LRs) collected from the saline coastal belt of Bangladesh, modern varieties (MVs), as well as Pokkali, Nona Bokra and salt tolerant modern varieties (SMVs) derived from the last two were analyzed with 60 evenly distributed rice microsatellite DNA markers. A total of 196 reproducible polymorphic alleles were identified from the band loci. Heterozygosity among the 31 LRs was found to be 0.57, 0.46 in the 5 MVs and 0.40 in the 8 SMVs. Computation of genetic similarity with this data, using Jaccard's coefficient followed by UPGMA clustering, divided the landraces into 6 distinct groups. Three groups were composed of LRs only from the highly saline southwest. Two groups consisted of LRs from the mild to moderately saline mid-east and northeast coasts. The sixth group was heterogeneous, with LRs from the northeast, LRs from the southwest and Nona Bokra. Pokkali and Gunshi, a LR of the southwest, branched out individually. When all the 46 O. sativa L. cultivars were clustered together, most of the MVs and SMVs were found to be linked within the heterogeneous group. The measure of seedling Na and K concentration, Na/K ratios, affected leaf area as well as survival under salinity stress in hydroponics identified 6 LRs from the highly saline southwest as the most tolerant. These group with Pokkali when UPGMA clustering using the Pearson product-moment correlation coefficient suitable for the quantitative physiological data on seedling saline stress was computed. Morphological observations of plant type and height, days to maturity and yield components in non-saline soil indicated low variability among the different LRs. When yield performance as well as tolerance scores were considered, 7 LRs from the southwest and 1 LR from the mid-northeast show potential as donors for breeding salt tolerant rice. The microsatellite fingerprinting analysis thus revealed that some of the salt tolerant landraces of the coastal region have unique polymorphic loci, quite distinct from the popular salt tolerance donor Pokkali. The similarity matrices between the O. sativa L. cultivars chosen for the study can be used as a valuable tool for the proper choice of parents for mapping or breeding purposes. Abbreviations: BARC – Bangladesh Agricultural Research Council; BRRI – Bangladesh Rice Research Institute; CTAB – Hexadecyltrimethyl ammonium bromide; DMRT – Duncan's multiple range test; DU – University of Dhaka; Het – heterogeneous; IRIS – International Rice Information System; IRRI – International Rice Research Institute; LRs – land races; MNE – mid & north east; MVs – modern varieties; SMVs – salt tolerant modern varieties; SRDI – soil resources Development Institute; SSR – simple sequence repeat; SW – south west.