Ca(2+) is an essential inorganic macronutrient, involved in regulating major physiological processes in plants. It has been well established as a second messenger and is predominantly stored in the cell wall, endoplasmic reticulum, mitochondria and vacuoles. In the cytosol, the concentration of this ion is maintained at nano-molar range. Upon requirement, Ca(2+) is released from intra-cellular as well as extracellular compartments such as organelles and cell wall. In this study, we report for the first time, a whole genome transcriptome response to short (5 D) and long (14 D) term Ca(2+) starvation and restoration in rice. Our results manifest that short and long term Ca(2+) starvation involves a very different response in gene expression with respect to both the number and function of genes involved. A larger number of genes were up- or down-regulated after 14 D (5588 genes) than after 5 D (798 genes) of Ca(2+) starvation. The functional classification of these genes indicated their connection with various metabolic pathways, ion transport, signal transduction, transcriptional regulation, and other processes related to growth and development. The results obtained here will enable to understand how changes in Ca(2+) concentration or availability are interpreted into adaptive responses in plants.
In the present agricultural scenario, the major thrust is to increase crop productivity so as to ensure sustainability. In an earlier study, foliar application of thiourea (TU; a non physiological thiol based ROS scavenger) has been demonstrated to enhance the stress tolerance and yield of different crops under field condition. Towards this endeavor, present work deals with the effect of TU on photosynthetic efficiency and source-to-sink relationship of Indian mustard (Brassica juncea) for understanding its mode of action. The application of TU increased the efficiency of both PSI and PSII photosystems and vegetative growth of plant. The comparative analysis of sucrose to starch ratio and expression level of sugar transporters confirmed the higher source and sink strength in response to TU treatment. The biochemical evidence in support of this was derived from higher activities of sucrose phosphate synthase and fructose-1,6-bis-phosphatase at source; and sucrose synthase and different classes of invertases at both source and sink. This indicated an overall increase in photoassimilate level at sink. An additional contribution through pod photosynthesis was confirmed through the analysis of phosphoenol pyruvate carboxylase enzyme activity and level of organic acids. The increased photoassimilate level was also co-ordinated with acetyl coA carboxylase mediated oil biosynthesis. All these changes were ultimately reflected in the form of 10 and 20% increase in total yield and oil content, respectively under TU treatment as compared to control. Additionally, no change was observed in oil composition of seeds derived from TU treated plants. The study thus signifies the co-ordinated regulation of key steps of photosynthesis and source-to-sink relationship through the external application of TU resulting in increased crop yield and oil content.
Plant nutrition is one of the important areas for improving the yield and quality in crops as well as non-crop plants. Potassium is an essential plant nutrient and is required in abundance for their proper growth and development. Potassium deficiency directly affects the plant growth and hence crop yield and production. Recently, potassium-dependent transcriptomic analysis has been performed in the model plant Arabidopsis, however in cereals and crop plants; such a transcriptome analysis has not been undertaken till date. In rice, the molecular mechanism for the regulation of potassium starvation responses has not been investigated in detail. Here, we present a combined physiological and whole genome transcriptomic study of rice seedlings exposed to a brief period of potassium deficiency then replenished with potassium. Our results reveal that the expressions of a diverse set of genes annotated with many distinct functions were altered under potassium deprivation. Our findings highlight altered expression patterns of potassium-responsive genes majorly involved in metabolic processes, stress responses, signaling pathways, transcriptional regulation, and transport of multiple molecules including K(+). Interestingly, several genes responsive to low-potassium conditions show a reversal in expression upon resupply of potassium. The results of this study indicate that potassium deprivation leads to activation of multiple genes and gene networks, which may be acting in concert to sense the external potassium and mediate uptake, distribution and ultimately adaptation to low potassium conditions. The interplay of both upregulated and downregulated genes globally in response to potassium deprivation determines how plants cope with the stress of nutrient deficiency at different physiological as well as developmental stages of plants.
Sesuvium portulacastrum is a common halophyte growing well in adverse surroundings and is exploited mainly for the environmental protection including phytoremediation, desalination and stabilization of contaminated soil. In the present investigation, attempts have been made on the decolorization of a toxic textile dye Green HE4B (GHE4B) using in vitro grown Sesuvium plantlets. The plantlets exhibited significant (70%) decolorization of GHE4B (50 mg l(-1)) that sustain 200 mM sodium chloride (NaCl) within 5 days of incubation. The enzymatic analysis performed on the root and shoot tissues of the in vitro plantlets subjected to GHE4B decolorization in the presence of 200 mM NaCl showed a noteworthy induction of tyrosinase, lignin peroxidase and NADH-DCIP reductase activities, indicating the involvement of these enzymes in the metabolism of the dye GHE4B. The UV-visible spectrophotometer, HPLC and Fourier Transform Infrared Spectroscopy (FTIR) analyses of the samples before and after decolorization of the dye confirmed the efficient phytotransformation of GHE4B in the presence of 200 mM NaCl. Gas Chromatography-Mass Spectroscopy (GC-MS) analysis of the products revealed the formation of three metabolites such as p -amino benzene, p -amino toluene and 1, 2, 7-amino naphthalene after phytotransformation of GHE4B. Based on the FTIR and GC-MS results, the possible pathway for the biodegradation of GHE4B in the presence of 200 mM NaCl has been proposed. The phytotoxicity experiments confirmed the non-toxicity of the degraded products. The present study demonstrates for the first time the potential of Sesuvium for the efficient degradation of textile dyes and its efficacy on saline soils contaminated with toxic compounds.
Semi-quantitative RT-PCR based transcript expression of stress responsive genes was studied in leaves of sugarcane plants exposed to short-term (up to 24 h) salt (NaCl, 200 mM) or polyethylene glycol-PEG 8000 (20% w/v) stress. Transient increase in expression of NHX (sodium proton antiporter), SUT1 (sucrose transporter1), PDH (proline dhydrogenase) and CAT2 (catalase2) was observed in response to 2-4 h PEG stress. However, salt stress imposed repression of NHX, PDH and CAT2 at these time points. The transcript level of the delta (1)-pyrolline-5-carboxylate synthetase (P5CS) increased slightly in salt treatment while in response to the PEG stress, the gene expression increased at 4 h treatment but then decreased considerably by 80% at 24 h. The results thus indicated differential regulation of these stress responsive genes in response to salt or PEG stress in sugarcane. Further, the transcript expression data was compared with that available for the Arabidopsis homologs at Arabidopsis eFP Browser and Genevestigator V3 tools. Understanding transcript gene expression patterns of the stress responsive genes may provide insights into complex regulatory network of stress tolerance.
In the present study, the effect of arsenate (AsV) exposure either alone or in combination with calcium (Ca) was investigated in callus cultures of Brassica juncea (L.) Czern. cv. Pusa Bold grown for a period up to 24 h. The AsV?(250 ?M) + Ca (10 mM) treatment resulted in a significantly higher level of As (464 ?g g(-1) dry weight (DW)) than AsV without Ca (167 ?g g(-1) DW) treatment at 24 h. Furthermore, AsV + Ca-treated calli had a higher percent of AsIII (24-47%) than calli subjected to AsV treatment (12-14%). Despite this, AsV + Ca-treated calli did not show any signs of hydrogen peroxide (H(2)O(2)) accumulation or cell death upon in vivo staining, while AsV-exposed calli had increased H(2)O(2), shrinkage of cytoplasmic contents, and cell death. Thus, AsV treatment induced oxidative stress, which in turn elicited a response of antioxidant enzymes and metabolites as compared with control and AsV + Ca treatment. The positive effects of Ca supplementation were also correlated to an increase in thiolic constituents, viz., cysteine, reduced glutathione, and glutathione reductase in AsV + Ca than in AsV treatment. An analysis of selected signaling related genes, e.g., mitogen-activated protein kinases (MAPK3 and MAPK6) and jasmonate ZIM-domain (JAZ3) suggested that AsV and AsV + Ca followed variable pathways to sense and signal the As stress. In AsV-alone treatment, jasmonate signaling was seemingly activated, while MAPK3 was not involved. In contrast, AsV + Ca treatment appeared to specifically inhibit jasmonate signaling and activate MAPK3. In conclusion, Ca supplementation may hold promise for achieving increased As accumulation in plants without compromising their tolerance.
Thiourea (TU) has been found to enhance the stress tolerance of plants in our earlier field trials. In the present study, the TU mediated effect on the redox and antioxidant responses were studied in response to salinity (NaCl) stress in Indian mustard (Brassica juncea (L.) Czern.) seedlings. Biochemical analyses of reactive oxygen species (ROS) and lipid peroxidation revealed that TU supplementation to NaCl brought down their levels to near control values as compared to that of NaCl stress. These positive effects could be correlated to the significant increases in the 1,1-diphenyl-2-picrylhydrazyl (DPPH)-radical scavenging activity, in the levels of reduced glutathione (GSH) and GSH/GSSG (reduced/oxidized glutathione) ratio and in the activities of superoxide dismutase (SOD; EC 188.8.131.52.1) and glutathione reductase (GR; EC 184.108.40.206) in NaCl+TU treatment as compared to that of NaCl treatment. Further, TU supplementation allowed plants to avoid an over-accumulation of pyridine nucleotides, to stimulate alternative pathways (through higher glycolate oxidase activity; EC 220.127.116.11) for channeling reducing equivalents and thus, to maintain the redox state to near control levels. These positive responses were also linked to an increased energy utilization (analyzed in terms of ATP/ADP ratio) and presumably to an early signaling of the stress through stimulated activity of ascorbate oxidase (EC 18.104.22.168), an important component of stress signaling. A significant reduction observed in the level of sodium ion (Na(+)) accumulation indicated that TU mediated tolerance is attributable to salt avoidance. Thus, the present study suggested that TU treatment regulated redox and antioxidant machinery to reduce the NaCl-induced oxidative stress.
Arsenic (As) is a potential hazard to plants health, however the mechanisms of its toxicity are yet to be properly understood. To determine the impact of redox state and energetic in stress imposition, plants of Hydrilla verticillata (L.f.) Royle, which are known to be potential accumulator of As, were exposed to 100 and 500 ?M arsenate (AsV) for 4 to 96 h. Plants demonstrated significant As accumulation with the maximum being at 500 ?M after 96 h (568 ?g g(-1) dry weight, dw). The accumulation of As led to a significant increase in the level of reactive oxygen species, nitric oxide, carbonyl, malondialdehyde, and percentage of DNA degradation. In addition, the activity of pro-oxidant enzymes like NADPH oxidase and ascorbate oxidase also showed significant increases. These parameters collectively indicated oxidative stress, which in turn caused an increase in percentage of cell death. These negative effects were seemingly linked to an altered energetic and redox equilibrium [analyzed in terms of ATP/ADP, NADH/NAD, NADPH/NADP, reduced glutathione/oxidized glutathione, and ascorbate/dehydroascobate ratios]. Although there was significant increase in the levels of phytochelatins, the As chelating ligands, a large amount of As was presumably present as free ion particularly at 500 ?M AsV, which supposedly produced toxic responses. In conclusion, the study demonstrated that the magnitude of disturbance to redox and energetic equilibrium of plants upon AsV exposure determines the extent of toxicity to plants.
An efficient plant regeneration protocol using axillary shoots of the salt accumulator halophyte, Sesuvium portulacastrum (L.) L. was established and in vitro responses of six Sesuvium clones were studied. The shoot and root induction responses to cytokinins and auxins in clone MH (Maharashtra) were concentration specific. Significantly the highest number of shoots, average shoot elongation and percent shoot regeneration per explant were observed on MS medium supplemented with 40 ?M 2-isopentenyl adenine (2iP) followed by 20 ?M benzyladenine (BA). Higher cytokinin (60 ?M), however, inhibited shoot induction and shoot length. The lower concentrations (5 or 10 ?M) of ?-napthaleneacetic acid (NAA) proved more effective for root induction, number of roots and average root length. Well-developed plantlets were successfully hardened and established in the field with more than 85 % survival rate. In vitro response of six Sesuvium clones cultured on MS + 20 ?M BA revealed higher multiplication rate in clone MH and KA (Karnataka, India) compared to other clones. The results offer the prospect of selecting clones of this species with characteristics desirable for utilization and/or restoration in specific ecological zones.
In the past two to three decades, great progress has been achieved in the field of plant genetic manipulation. This progress is based on fine-tuning gene transfer methods, selection of transformed cells and regulation of transgene expression. Transgenic plant production requires selectable marker genes that enable the selection of transformed cells, tissue and plants. The most used are those that exhibit resistance to antibiotics or herbicides. Although this type of selection is routinely practiced, there are perceived risks in the deployment of transgenic plants containing these markers. A number of strategies have emerged on the development of alternate selection systems referred to as positive selection and marker-free systems. Transgenes that permit plant cells to utilize new carbon sources are being employed in transformation research. Current research on development of novel selection methods in transgenics is growing rapidly and its application is being tested in different plant species.
Identification of genes whose expression enables plants to adapt to any kind of stresses is integral to developing stress tolerance in crop plants. In this study, PCR-based cDNA suppression subtractive hybridization technique was used to construct sugarcane salt (NaCl) stress specific forward and reverse subtracted cDNA library. For this, mRNAs were pooled from the shoot and root tissues stressed with NaCl (200 mM) for various time intervals (0.5 to 18 h). Sequencing the clones from the forward subtracted cDNA library, we identified shaggy-like protein kinase (hereafter referred as sugarcane shaggy-like protein kinase, SuSK; NCBI GenBank EST database Acc: FG804674). The sequence analysis of the SuSK revealed homology to Arabidopsis thaliana shaggy-related protein kinase delta (E value, 1e(-108)), dzeta and iota. Alignment of the catalytic domain sequence of GSK-3/shaggy-like kinase with partial sequence of SuSK performed using ClustalW tool indicated kinase active-site signature sequence. Spatial and temporal transcript expression profiling of the SuSK gene based on Real-Time PCR revealed significant induction of transcript expression in response to short-term salt (NaCl 200 mM) or polyethylene glycol-8,000 (PEG; 20% w/v) induced osmotic stress in leaves and shoots of sugarcane plants. The transcript expression increased progressively under salt stress and reached to 1.5-fold of the control up to 8 h treatment. In response to PEG stress, the transcript expression increased by 1.5-fold over the control in 2-h treatment in leaf, whereas in shoots, the expression remained unchanged in response to the various treatments. Differences in growth parameters, relative water content, and membrane damage rate were statistically insignificant in the short-term salt or PEG-stressed plants as compared to the control, non-stressed plants. Expression analysis revealed the differential and temporal regulation of this gene under salt and PEG stress and that its early induction may indicate involvement in stress signaling.
Comparative antioxidant profiling of tolerant (TPM-1) and sensitive (TM-4) variety of Brassica juncea L. was performed after exposure to arsenate [As(V)] and arsenite [As(III)]. TPM-1 demonstrated higher accumulation of As upon exposure to both 500 microM As(V) and 250 microM As(III) (49 and 37 microg g(-1) dw after 15 days) as compared with that observed in TM-4. The activities of various antioxidant enzymes and the level of glutathione and proline demonstrated, in general, a comparatively better response in TPM-1 than in TM-4 that presumably allowed TPM-1 to tolerate higher As concentrations as compared with that of TM-4.
MicroRNAs (miRNAs) constitute a novel mechanism of gene regulation affecting plant development, growth, and stress response. To study the role of miRNAs in arsenic (As) stress, microarray profiling of miRNAs was performed in Brassica juncea using a custom Phalanx Plant OneArray containing 381 unique miRNA probes representing 618 miRNAs from 22 plant species. miRNA microarray hybridization of roots exposed to As for 1h and 4h revealed that a total of 69 miRNAs belonging to 18 plant miRNA families had significantly altered expression. The As-responsive miRNAs also exhibited a time- and organ-dependent change in their expression. Putative target prediction for the miRNAs suggested that they regulate various developmental processes (e.g. miR156, miR169, and miR172), sulphur uptake, transport, and assimilation (miR395, miR838, and miR854), and hormonal biosynthesis and/or function (e.g. miR319, miR167, miR164, and miR159). Notable changes were observed in the level of auxins [indole-3-acetic acid (IAA), indole-3- butyric acid, and naphthalene acetic acid], jasmonates [jasmonic acid (JA) and methyl jasmonate], and abscisic acid. The exogenous supply of JA and IAA improved growth of plants under As stress and altered expression of miR167, miR319, and miR854, suggesting interplay of hormones and miRNAs in the regulation of As response. In conclusion, the present work demonstrates the role of miRNAs and associated mechanisms in the plants response towards As stress.
Seed priming is a well known pre-germination strategy that improves seed performance. However, biochemical and molecular mechanisms underlying priming mediated stress tolerance are little understood. Here, we report results of the study on growth, physiological characteristics and expression of stress responsive genes in salt primed sugarcane cv. Co 86032 plants in response to salt (NaCl, 150 mM) or iso-osmotic (-0.7 MPa) polyethylene glycol-PEG 8000 (20 % w/v) stress exposure for 15 days. Variable growth, osmolyte accumulation and antioxidant capacity was revealed among the primed and non-primed plants. The primed plants showed better tolerance to the salt or PEG stress, as revealed by better growth and lower membrane damage, through better antioxidant capacity as compared to the respective non-primed controls. Further, steady state transcript expression analysis revealed up regulation of sodium proton antiporter (NHX) while, down regulation of sucrose transporter (SUT1), delta ( 1 )-pyrolline-5-carboxylate synthetase (P5CS) and proline dehydrogenase (PDH) in primed plants on exposure to the stress as compared to the non-primed plants. Transcript abundance of catalase (CAT2) decreased by about 25 % in leaves of non-primed stressed plants, however, the expression was maintained in leaves of the stressed primed plants to that of non-stressed controls. Thus, the results indicated priming mediated salt and PEG stress tolerance through altered gene expression leading to improved antioxidant capacity in sugarcane.
Arsenic (As) contamination of the environment has emerged as a serious problem. Consequently, there is an urge to understand plants responses to As. The analysis of various hypertolerant and hyperaccumulator plants and comparison of their responses with non-tolerant and nonaccumulators have provided valuable information about the mechanisms of As tolerance and detoxification. Therefore, we understand why most of the pteridophytes are able to hyperacumulate As, why it is difficult to find hyperaccumulators among angiosperms and why rice is able to translocate As to its grains more efficiently than any other cereal crop. This information can be employed to generate As hyperaccumulators in angiosperms and to develop safe cultivars of rice for human consumption through biotechnological approaches. Although measurable success, in terms of application in the field, has so far not been achieved, transgenic research has yielded promising results, which shed light on the approaches to be taken up in future endeavor. In this review, we discuss the mechanisms of As tolerance and detoxification in plants and transgenic research conducted.
Functional annotation of uncharacterized genes is the main focus of computational methods in the post genomic era. These tools search for similarity between proteins on the premise that those sharing sequence or structural motifs usually perform related functions, and are thus particularly useful for membrane proteins. Early responsive to dehydration (ERD) genes are rapidly induced in response to dehydration stress in a variety of plant species. In the present work we characterized function of Brassica juncea ERD4 gene using computational approaches. The ERD4 protein of unknown function possesses ubiquitous DUF221 domain (residues 312-634) and is conserved in all plant species. We suggest that the protein is localized in chloroplast membrane with at least nine transmembrane helices. We detected a globular domain of 165 amino acid residues (183-347) in plant ERD4 proteins and expect this to be posited inside the chloroplast. The structural-functional annotation of the globular domain was arrived at using fold recognition methods, which suggested in its sequence presence of two tandem RNA-recognition motif (RRM) domains each folded into ?????? topology. The structure based sequence alignment with the known RNA-binding proteins revealed conservation of two non-canonical ribonucleoprotein sub-motifs in both the putative RNA-recognition domains of the ERD4 protein. The function of highly conserved ERD4 protein may thus be associated with its RNA-binding ability during the stress response. This is the first functional annotation of ERD4 family of proteins that can be useful in designing experiments to unravel crucial aspects of stress tolerance mechanism.
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