Epigenetic regulation of salinity stress responses in cereals.

Cereals are important crops and are exposed to various types of environmental stresses that affect the overall growth and yield. Among the various abiotic stresses, salt stress is a major environmental factor that influences the genetic, physiological, and biochemical responses of cereal crops. Epigenetic regulation which includes DNA methylation, histone modification, and chromatin remodelling plays an important role in salt stress tolerance. Recent studies in rice genomics have highlighted that the epigenetic changes are heritable and therefore can be considered as molecular signatures. An epigenetic mechanism under salinity induces phenotypic responses involving modulations in gene expression. Association between histone modification and altered DNA methylation patterns and differential gene expression has been evidenced for salt sensitivity in rice and other cereal crops. In addition, epigenetics also creates stress memory that helps the plant to better combat future stress exposure. In the present review, we have discussed epigenetic influences in stress tolerance, adaptation, and evolution processes. Understanding the epigenetic regulation of salinity could help for designing salt-tolerant varieties leading to improved crop productivity.

Evaluation of Spinacia oleracea (L.) for phytodesalination and augmented production of bioactive metabolite, 20-hydroxyecdysone.

In this study, adaptive features of Spinacia oleracea to different levels of salinity, its use in desalination and production of 20-Hydroxyecdysone were studied. Plants showed survival up to EC 12 dS/m with reduced growth as compared with control. Net photosynthesis rate, transpiration, stomatal conductance, and water use efficiency of salt treated plants declines with increasing salinity stress. Higher antioxidant enzyme activities and compatible solutes accumulation were observed in salt treated plants as function of osmotic adjustment. Significant Na+ sequestration and Na/K ratio were noted with increase in salt stress in comparison to the control. Since the plant accumulates a bioactive, secondary metabolite 20-Hydroxyecdysone (20E), we observed significant 20E content in plants grown at EC 4-12 dS/m in comparison to control. Furthermore, a preliminary field experiment, showed significant reduction in the soil electrical conductivity by 1.8 ds/m after 90 days of plant growth with Na+ sequestration in plant biomass. Subsequent to this growth period, the phytodesalinized soil supported the significant growth of a glycophyte (rice). Our results suggest that S. oleracea can adapt to saline conditions with antioxidant defense and osmotic adjustment. The plant can be used as a potential candidate for desalination and also for enhanced production of 20-Hydroxyecdysone.

Looking at Halophytic Adaptation to High Salinity Through Genomics Landscape.

Soil salinity is an important stress factor that limits plant growth and productivity. For a given plant species, it is critical to sense and respond to salt stimuli followed by activation of multitude of mechanisms for plants to survive. Halophytes, the wonders of saline soils, have demonstrated ability to withstand and reproduce in at least 200 mM NaCl concentration, which makes them an ideal system to study mechanism of salt adaptation for imparting salt tolerance in glycophytes. Halophytes and salt sensitive glycophytes adapt different defense strategies towards salinity stress. These responses in halophytes are modulated by a well orchestrated network of signaling pathways, including calcium signaling, reactive oxygen species and phytohormones. Moreover, constitutive expression of salt stress response related genes, which is only salt inducible in glycophytes, maintains salt tolerance traits in halophytes. The focus of this review is on the adaptive considerations of halophytes through the genomics approaches from the point of view of sensing and signaling components involved in mediating plant responses to salinity.

Brassica RNA binding protein ERD4 is involved in conferring salt, drought tolerance and enhancing plant growth in Arabidopsis.

'Early responsive to dehydration' (ERD) genes are a group of plant genes having functional roles in plant stress tolerance and development. In this study, we have isolated and characterized a Brassica juncea 'ERD' gene (BjERD4) which encodes a novel RNA binding protein. The expression pattern of ERD4 analyzed under different stress conditions showed that transcript levels were increased with dehydration, sodium chloride, low temperature, heat, abscisic acid and salicylic acid treatments. The BjERD4 was found to be localized in the chloroplasts as revealed by Confocal microscopy studies. To study the function, transgenic Arabidopsis plants were generated and analyzed for various morphological and physiological parameters. The overexpressing transgenic lines showed significant increase in number of leaves with more leaf area and larger siliques as compared to wild type plants, whereas RNAi:ERD4 transgenic lines showed reduced leaf number, leaf area, dwarf phenotype and delayed seed germination. Transgenic Arabidopsis plants overexpressing BjERD4 gene also exhibited enhanced tolerance to dehydration and salt stresses, while the knockdown lines were susceptible as compared to wild type plants under similar stress conditions. It was observed that BjERD4 protein could bind RNA as evidenced by the gel-shift assay. The overall results of transcript analysis, RNA gel-shift assay, and transgenic expression, for the first time, show that the BjERD4 is involved in abiotic stress tolerance besides offering new clues about the possible roles of BjERD4 in plant growth and development.

Quantitative real-time expression profiling of aquaporins-isoforms and growth response of Brassica juncea under arsenite stress.

The present study analyzed the expression level of aquaporins of plasma membrane intrinsic protein (PIP) class in response to arsenite (AsIII) exposure of 100 µM from 0.5 h to 8 days in Brassica juncea. The expression levels of most of the PIPs were down-regulated during the course of AsIII exposure. This led to decrease in total water content of plants, which in turn hampered seedling growth. The level of reactive oxygen species (superoxide radicals and hydrogen peroxide), lipid peroxidation and root oxidizability increased significantly upon exposure to AsIII as compared to that of control leading to an increase in cell death. The study proposes that the down-regulation of PIPs happened presumably to regulate AsIII levels, which, however, occurred at the cost of reduced growth, disturbed water balance and induced oxidative stress.

Genome-wide analysis of thiourea-modulated salinity stress-responsive transcripts in seeds of Brassica juncea: identification of signalling and effector components of stress tolerance.

BACKGROUND AND AIMS: Abiotic stresses including salinity are the major constraints to crop production. In this regard, the use of thiourea (TU) in imparting salinity-stress tolerance to Indian mustard (Brassica juncea) has been demonstrated earlier. To gain an insight into the mechanism of TU action, various molecular and biochemical studies were conducted. METHODS: Microarray analysis was performed in seeds subjected to distilled water (control), 1 m NaCl, 1 m NaCl + 6·5 mm TU and 6·5 mm TU alone for 1 h. Real-time PCR validation of selected genes and biochemical studies were conducted under similar treatments at 1 h and 6 h. KEY RESULTS: The microarray analysis revealed a differential expression profile of 33 genes in NaCl- and NaCl + TU-treated seeds, most of which are established markers of stress tolerance. The temporal regulation of eight selected genes by real-time PCR indicated their early and co-ordinated induction at 1 h in NaCl + TU only. Besides, NaCl + TU-treated seeds also maintained a higher level of abscisic acid, reduced to oxidized glutathione (GSH : GSSG) ratio and activities of catalase, phenylalanine ammonia lyase and glutathione-S-transferases, as compared with that of NaCl treatment. The addition of LaCl(3) (a specific calcium-channel blocker) restricted the responses of TU both at molecular and biochemical level suggesting the possible involvement of a cytosolic calcium burst in the TU-mediated response. The TU-alone treatment was comparable to that of the control; however, it reduced the expression of some transcription factors and heat-shock proteins presumably due to the stabilization of the corresponding proteins. CONCLUSIONS: The TU treatment co-ordinately regulates different signalling and effector mechanisms at an early stage to alleviate stress even under a high degree of salinity. This also indicates the potential of TU to be used as an effective bioregulator to impart salinity tolerance under field conditions.

A potential seedling-stage evaluation method for heat tolerance in Indian mustard (Brassica juncea L. Czern and Coss).

Heat stress is one of the major limitations to crop productivity. In the present study, an efficient method of screening was adopted for identification of heat tolerant Indian Mustard genotypes by applying 4-day cycle of heat stress to seedlings. Thirty-four genotypes were screened based upon lipid peroxidation and survival percentage and classified them into five different classes according to membership function value (MFV) for response against high temperature. The maximum and minimum value of mean MFV were 0.89 (highly heat tolerant, TPM1) and 0.12 (highly heat sensitive, JM2), respectively. The coefficient of determination (R 2) between the mean MFV and the heat tolerance index (HTI) of MDA content, survival percentage was 0.914 and 0.808 suggesting that these parameters are reliable traits to evaluate the heat tolerance of Brassica juncea genotypes. The evaluation method was further validated using identified contrasting genotypes and assessment of heat stress associated biochemical parameters. Results showed efficient recovery of tolerant genotype as compared to sensitive genotype. Expression profiling of heat stress-related genes (HSP21 and HSFA7A) showed significant upregulation in the tolerant genotype (TPM1) (9.73- and 4.87-fold, respectively) as compared to the sensitive genotype (JM2) (4.18- and 1.73-fold, respectively) under heat stress condition. The results imply development of an efficient screening method which is useful for evaluation and breeding of thermo-tolerant B. juncea.

Comparative biochemical and transcriptional profiling of two contrasting varieties of Brassica juncea L. in response to arsenic exposure reveals mechanisms of stress perception and tolerance.

The mechanisms of perception of arsenic (As)-induced stress and ensuing tolerance in plants remain unresolved. To obtain an insight into these mechanisms, biochemical and transcriptional profiling of two contrasting genotypes of Brassica juncea was performed. After screening 14 varieties for As tolerance, one tolerant (TPM-1) and one sensitive (TM-4) variety were selected and exposed to arsenate [As(V)] and arsenite [As(III)] for 7 d and 15 d for biochemical analyses. The tolerant variety (TPM-1) demonstrated higher accumulation of As upon exposure to both 500 microM As(V) and 250 microM As(III) [49 microg g(-1) and 37 microg g(-1) dry weight (dw) after 15 d] as well as a better response of thiol metabolism as compared with the responses observed in the sensitive variety (TM-4). Transcriptional profiling of selected genes that are known to be responsive to sulphur depletion and/or metal(loid) stress was conducted in 15-d-old seedlings after 3 h and 6 h exposure to 250 microM As(III). The results showed an up-regulation of sulphate transporters and auxin and jasmonate biosynthesis pathway genes, whereas there was a down-regulation of ethylene biosynthesis and cytokinin-responsive genes in TPM-1 within 6 h of exposure to As(III). This suggested that perception of As-induced stress was presumably mediated through an integrated modulation in hormonal functioning that led to both short- and long-term adaptations to combat the stress. Such a coordinated response of hormones was not seen in the sensitive variety. In conclusion, an early perception of As-induced stress followed by coordinated responses of various pathways was responsible for As tolerance in TPM-1.

FT-IR profiling reveals differential response of roots and leaves to salt stress in a halophyte Sesuvium portulacastrum (L.) L.

In a halophyte, Sesuvium portulacastrum (L.) L., we have applied Fourier Transform InfraRed (FT-IR) spectroscopy to detect the corresponding changes associated with salt-induced physiological changes under long- term treatment with 0, 100 and 500 mM NaCl. FT-IR profiles showed changes in chemical composition and functional groups of proteins, lipids and carbohydrates due to salt treatments, evident as differential FT-IR profiles in both roots and leaves specific to these metabolites. Further, the Principle Component Analysis (PCA) was applied to identify the main sources of variation in FT-IR data due to differential treatment. In PCA, the PC1 showed 85.55% and PC2 showed 18.18% variability in data and confirmed differential response of root and leaves to salt treatment in Sesuvium. The results suggest that FT-IR spectrometry can be used to study stress-induced metabolic changes in plants in relation to their salt tolerance.

Nitrogen supply influences arsenic accumulation and stress responses of rice (Oryza sativa L.) seedlings.

In the present study, the effects of nitrogen supply (low nitrogen: LN and high nitrogen: HN) on As stress (25 µM) responses of rice seedlings were monitored for 7 d. The mean length of primary, adventitious and lateral roots and number of adventitious and lateral roots were significantly improved in LN+As, while further reduced in HN+As, as compared to As alone treatment at 7 d. The LN+As treatment resulted in significant decline in As (848 µg g-1 DW) than that in As alone treatment (1434 µg g-1 DW) in roots but no significant effect was seen in shoot. In contrast, HN+As treatment showed significant increase in shoot As (6.86 µg g-1 DW) as compared to As alone treatment (3.43 µg g-1 DW). The level of nitrate was increased in roots but declined in shoots in As alone treatment. Surprisingly, no improvement in nitrate level was seen in HN+As as compared to that in As alone treatment in both root and shoot. The expression analysis of nitrate transporters (NRT2;1, NRT2;3a, NRT2;4) showed significant differences in expression patterns in As, LN+As and HN+As treatments. In conclusion, nitrogen supply had profound influences on responses of rice plants to As.

Temporal and spatial changes in ion homeostasis, antioxidant defense and accumulation of flavonoids and glycolipid in a halophyte Sesuvium portulacastrum (L.) L.

Salinity is an important environmental constraint limiting plant productivity. Understanding adaptive responses of halophytes to high saline environments may offer clues to manage and improve salt stress in crop plants. We have studied physiological, biochemical and metabolic changes in a perennial, fast growing halophyte, Sesuvium portulacastrum under 0 mM (control), 150 mM (low salt, LS) and 500 mM (high salt, HS) NaCl treatments. The changes in growth, relative water content, cation, osmolyte accumulation, H2O2 and antioxidant enzyme activity (SOD, CAT and APX) were observed under different treatment conditions. A positive correlation was revealed for sodium ion accumulation with malondialdehyde (r2 = 0.77), proline (r2 = 0.88) and chlorophyll content (r2 = 0.82) under salt treatment while a negative correlation was observed with relative tissue water content (r2 = -0.73). The roots and leaves showed contrasting accumulation of potassium and sodium ions under LS treatment. Temporal and spatial study of sodium and potassium ion content indicated differential accumulation pattern in roots and leaves, and, high potassium levels in root. Higher H2O2 content was recorded in roots than leaves and the antioxidant enzyme activities also showed significant induction under salt treatment conditions. Gene expression profiling of sodium transporters, Sodium proton exchanger (NHX3), Vacuolar ATPase (vATPase) and Salt overly sensitive1 (SOS1) showed up regulation under salt stress after 6-24 hr of NaCl treatment. Metabolite changes in the salt stressed leaves showed increased accumulation of flavonoids (3,5-dihydroxy-6,4'-dimethoxy-flavone-7-O-[α-L-rhamnopyranosyl-(1→6)-ß-D-glucopyranoside], and3,5-dihydroxy-6,3',4'-trimethoxy-flavone-7-O-[α-L-rhamnopyranosyl-(1→6)-ß-D-glucopyranoside] in both LS and HS treatments, while a glycolipid, 1-O-linolenyl-2-O-(palmitoyl)-3-O-galactopyranosyl glycerol, accumulated more in LS over HS treatments and control. The results suggest that differential spatial and temporal cation levels in roots and leaves, and accumulation of flavanoid and glycolipid could be responsible for salt adaptation of S. portulacastrum.

Salt responsive physiological, photosynthetic and biochemical attributes at early seedling stage for screening soybean genotypes.

Salt stress affects all the stages of plant growth however seed germination and early seedling growth phases are more sensitive and can be used for screening of crop germplasm. In this study, we aimed to find the most effective indicators of salt tolerance for screening ten genotypes of soybean (SL-295, Gujosoya-2, PS-1042, PK-1029, ADT-1, RKS-18, KDS-344, MAUS-47, Bragg and PK-416). The principal component analysis (PCA) resulted in the formation of three different clusters, salt sensitive (SL-295, Gujosoya-2, PS-1042 and ADT-1), salt tolerant (MAUS-47, Bragg and PK-416) and moderately tolerant/sensitive (RKS-18, PK-1029 and KDS-344) suggesting that there was considerable genetic variability for salt tolerance in the soybean genotypes. Subsequently, genotypes contrasting in salt tolerance were analyzed for their physiological traits, photosynthetic efficiency and mitochondrial respiration at seedling and early germination stages under different salt (NaCl) treatments. It was found that salt mediated increase in AOX-respiration, root and shoot K+/Na+ ratio, improved leaf area and water use efficiency were the key determinants of salinity tolerance, which could modulate the net photosynthesis (carbon assimilation) and growth parameters (carbon allocation). The results suggest that these biomarkers could be can be useful for screening soybean genotypes for salt tolerance.

Study of trace element correlations with drought tolerance in different sorghum genotypes using energy-dispersive X-ray fluorescence technique.

Drought-tolerant and drought-susceptible genotypes of sorghum (Sorghum bicolor L. Monech) were analyzed by the energy-dispersive X-ray fluorescence (EDXRF) technique to study the correlation of trace elements with drought-tolerance capacities. Samples prepared from mature seeds, young seedlings, and old plants were analyzed using a 109Cd radioisotope source and a Si(Li) semiconductor detector of resolution 170 eV for 5.9-keV MnKalpha X-rays. Elements such as K, Fe, Cu, Zn, Rb and Sr and Y were found to be present in varying concentrations in different samples. The trace element profile studied in the seeds of 11 genotypes and in seedlings (young and old) of 4 sorghum genotypes showed considerable variation. The genotype Arfa Gadamak (AG) showed a distinct presence of a high level of Zn in its young seedling. It was observed that in most of the genotypes (seeds), K and Fe concentrations were more in the tolerant genotype as compared to the susceptible type. The concentration of Fe decreased with maturity in the tolerant group and it increased with maturity in the susceptible group.

Identification of redox-regulated components of arsenate (As(V)) tolerance through thiourea supplementation in rice.

Arsenic (As) is a ubiquitously present environmental carcinogen that enters into the human food chain through rice grains. In our previous research, the application of thiourea (TU; a non-physiological thiol based ROS scavenger) has been demonstrated to enhance salt and UV stress tolerance as well as the crop yield under field conditions. These effects were associated with the ability of TU to maintain plant redox homeostasis. Since As stress also induces redox imbalance, the present research was initiated to evaluate the efficiency of TU in regulating As tolerance/accumulation in rice. The supplementation of TU (75 µM) to As(V) (25 µM) improved the root growth and also reduced the As concentration by 56% in the aerial parts, which could be attributed to significant downregulation of the Lsi2 transporter responsible for the translocation of As from root to shoot. The fact that these effects were not due to direct interaction between As and TU was confirmed from complexation studies using HPLC-(ICP-MS)-(ESI-MS). Short-term kinetic studies of GSH levels and the GSH/GSSG ratio confirmed the establishment of differential redox states in As and As + TU treated seedlings. The real-time RT-PCR based comparative expression profiling under As with/without TU treatment identified Sultr1;1 and Sultr1;2 as major redox-regulated sulfate transporters. Their specific induction in shoots coupled with enhanced root-to-shoot sulfate translocation (analyzed using (35)S-sulfate as a radiotracer) was observed under TU supplementation. Furthermore, the level of thiolic metabolites (PC2 in roots and GSH and PC3 in shoots) and activities of sulfur metabolism enzymes (ATP sulfurylase and cysteine synthase in roots and 5'-adenylylsulfate reductase in shoot) were also increased with As + TU as compared to As treatment. Thus, this study utilizes the interaction between As and TU to identify the critical redox regulated components of As tolerance in rice.

Investigation of arsenic accumulation and tolerance potential of Sesuvium portulacastrum (L.) L.

Sesuvium portulacastrum (L.) L., a facultative halophyte, is considered a suitable candidate for the phytoremediation of metals. An investigation of As accumulation and tolerance was conducted in Sesuvium plants upon exposure to As(V) (100-1000 µM) for 30 d. Plants demonstrated a good growth even after prolonged exposure (30 d) to high As(V) concentrations (1000 µM) and a significant As accumulation (155 µg g⁻¹ dry weight) with a bioaccumulation factor of more than ten at each concentration. The results of shoot and root dry weight, malondialdehyde accumulation, photosynthetic pigments, and total soluble proteins demonstrated that plants did not experience significant toxicity even at 1000 µM As(V) after 30 d. However, metabolites (total non-protein thiols and cysteine) and enzymes (serine acetyltransferase, cysteine synthase and γ-glutamylcysteine synthetase) of thiol metabolism, in general, remained either unaffected or showed slight decline. Hence, plants tolerated high As(V) concentrations without an involvement of thiol metabolism as a major component. Taken together, the results indicate that plants are potential As accumulator and may find application in the re-vegetation of As contaminated sites.

Plantlet regeneration from glume calli of maize (Zea mays L.).

Totipotent callus cultures were established from anther-free glumes of 'Sweet corn', 'Seed corn,' 'DHM 103' and 'DHM 101' on MS medium supplemented with 1-2 mg/l 2,4-D. The callusing response of the glumes was tested on six different media. Glumes at the uninucleate stage of pollen development callused with a high frequency compared to other stages. Organogenesis was observed in 40% of the cultures on media devoid of hormones. A total of 76 plantlets were regenerated on medium with 0.5-1.0 mg/l of both IAA and kinetin. Cytological observations in root tips indicated a diploid chromosome number (2n=20).

Propagation of banana through encapsulated shoot tips.

Plants were regenerated from encapsulated shoot tips of banana. Shoot tips (ca 4 mm) isolated from multiple shoot cultures of banana cv. Basrai were encapsulated in 3% sodium alginate containing different gel matrices. The encapsulated shoot tips regenerated in vitro on different substrates. Use of White's medium resulted in 100% conversion of encapsulated shoot tips into plantlets. The plantlets were successfully established in soil.

In Vitro Production Of L-dopa In Tissue Cultures Of Banana.

Shoot and callus cultures of banana ( Musa sp.) were analyzed for the accumulation of L-DOPA. Treatment of cultures with L-tyrosine and L-phenylalanine yielded higher levels of DOPA compared to those in control cultures without any treatment. Among the two amino acids, phenylalanine induced higher accumulation of DOPA. The study suggests that banana may become an useful system for the production of DOPA.