Transcriptome analysis of grapevine under salinity and identification of key genes responsible for salt tolerance.

The negative effects of soil salinity towards grape yield depend upon salt concentration, cultivar type, developmental stage, and rootstock. Thompson Seedless variety of grape plant is considered moderately sensitive to salinity when grown upon its own root stock. In recent epoch, identification of key genes responsive to salinity offers hope to generate salinity-tolerant crop plants by their overexpression through genetic manipulation. In the present report, salt responsive transcriptome analysis of Thompson Seedless grape variety was done to identify vital genes involved in salinity tolerance which could be used further to generate salt liberal grape plant or other crop plants. Transcriptome libraries for control and 150-mM-NaCl-treated grape leaves were sequenced on Illumina platform where 714 genes were found to be differentially expressed. Gene ontology analysis indicated that under salinity conditions, the genes involved in metabolic process were highly enriched. Keto Encyclopedia of Genes and Genomes analysis revealed that, among the top 22 enriched pathways for the salt stress upregulated genes, the carbohydrate metabolism, signal transduction, energy metabolism, amino acid metabolism, biosynthesis of secondary metabolite, and lipid metabolism pathways possessed the largest number of transcripts. Key salinity-induced genes were selected and validated through qRT-PCR analysis which was comparable to RNA-seq results. Real-time PCR analysis also revealed that after 24 days of salinity, the expression of most of the selected key genes was highest. These salinity-induced genes will be characterized further in a model plant and also in Vitis vinifera through transgenic approach to disclose their role towards salt tolerance.

Abiotic stress regulates expression of galactinol synthase genes post-transcriptionally through intron retention in rice.

MAIN CONCLUSION: Expression of the Galactinol synthase genes in rice is regulated through post-transcriptional intron retention in response to abiotic stress and may be linked to Raffinose Family Oligosaccharide synthesis in osmotic perturbation. Galactinol synthase (GolS) is the first committed enzyme in raffinose family oligosaccharide (RFO) synthesis pathway and synthesizes galactinol from UDP-galactose and inositol. Expression of GolS genes has long been implicated in abiotic stress, especially drought and salinity. A non-canonical regulation mechanism controlling the splicing and maturation of rice GolS genes was identified in rice photosynthetic tissue. We found that the two isoforms of Oryza sativa GolS (OsGolS) gene, located in chromosomes 3(OsGolS1) and 7(OsGolS2) are interspersed by conserved introns harboring characteristic premature termination codons (PTC). During abiotic stress, the premature and mature transcripts of both isoforms were found to accumulate in a rhythmic manner for very small time-windows interrupted by phases of complete absence. Reporter gene assay using GolS promoters under abiotic stress does not reflect this accumulation profile, suggesting that this regulation occurs post-transcriptionally. We suggest that this may be due to a surveillance mechanism triggering the degradation of the premature transcript preventing its accumulation in the cell. The suggested mechanism fits the paradigm of PTC-induced Nonsense-Mediated Decay (NMD). In support of our hypothesis, when we pharmacologically blocked NMD, the full-length pre-mRNAs were increasingly accumulated in cell. To this end, our work suggests that a combined transcriptional and post transcriptional control exists in rice to regulate GolS expression under stress. Concurrent detection and processing of prematurely terminating transcripts coupled to repressed splicing can be described as a form of Regulated Unproductive Splicing and Translation (RUST) and may be linked to the stress adaptation of the plant, which is an interesting future research possibility.

Functional characterization of two myo-inositol-1-phosphate synthase (MIPS) gene promoters from the halophytic wild rice (Porteresia coarctata).

MAIN CONCLUSION: The promoter deletion mutants from second isoform of INO1 (gene-encoding MIPS) from Porteresia coarctata of 932 bp (pPcINO1.2.932) and 793 bp (pPcINO1.2.793) prove to be very efficient as salt/drought stress-inducible promoters, while pPcINO1.2.932 is found to be responsive to cold stress as well. The promoters of the two identified myo-inositol-1-phosphate synthase (INO1) isoforms from salt-tolerant wild rice, Porteresia coarctata (PcINO1.1 and PcINO1.2) have been compared bioinformatically with their counterparts present in the salt-sensitive rice, Oryza sativa. PcINO1.2 promoter was found to be enriched with many abiotic stress-responsive elements, like abscisic acid-responsive elements, MYC-responsive elements, MYB-binding sites, low-temperature stress-responsive elements, and heat-shock elements similar to the ones found in the conserved motifs of the promoters of salt/drought stress-inducible INO1 promoters across Kingdom Planta. To have detailed analysis on the arrangement of cis-acting regulatory elements present in PcINO1 promoters, 5' deletion mutational studies were performed in dicot model plants. Both transient as well as stable transformation methods were used to check the influence of PcINO1 promoter deletion mutants under salt and physiologically drought conditions using ß-glucuronidase as the reporter gene. The deletion mutant from the promoter of PcINO1.2 of length 932 bp (pPcINO1.2.932) was found to be significantly upregulated under drought stress and also in cold stress, while another deletion mutant, pPcINO1.2.793 (of 793 bp), was significantly upregulated under salt stress. P. coarctata being a halophytic species, the high inducibility of pPcINO1.2.932 upon exposure to low-temperature stress was an unexpected result.

Different dehydrins perform separate functions in Physcomitrella patens.

MAIN CONCLUSION: Dehydrins, PpDHNA and PpDHNB from Physcomitrella patens provide drought and cold tolerance while PpDHNC shows antimicrobial property suggesting different dehydrins perform separate functions in P. patens. The moss Physcomitrella patens can withstand extremes of environmental condition including abiotic stress such as dehydration, salinity, low temperature and biotic stress such as pathogen attack. Osmotic stress is inflicted under both cold and drought stress conditions where dehydrins have been found to play a significant protective role. In this study, a comparative analysis was drawn for the three dehydrins PpDHNA, PpDHNB and PpDHNC from P. patens. Our data shows that PpDHNA and PpDHNB play a major role in cellular protection during osmotic stress. PpDHNB showed several fold upregulation of the gene when P. patens was subjected to cold and osmotic stress in combination. PpDHNA and PpDHNB provide protection to enzyme lactate dehydrogenase under osmotic as well as freezing conditions. PpDHNC possesses antibacterial activity and thus may have a role in biotic stress response. Overexpression of PpDHNA, PpDHNB and PpDHNC in transgenic tobacco showed a better performance for PpDHNB with respect to cold and osmotic stress. These results suggest that specific dehydrins contribute to tolerance of mosses under different stress conditions.

Identification and organization of chloroplastic and cytosolic L-myo-inositol 1-phosphate synthase coding gene(s) in Oryza sativa: comparison with the wild halophytic rice, Porteresia coarctata.

The gene coding for rice chloroplastic L-myo-inositol-1-phosphate synthase (MIPS; EC 5.5.1.4) has been identified by matrix-assisted laser desorption time-of-flight mass spectrometry analysis of the purified and immunologically cross-reactive approximately 60 kDa chloroplastic protein following two-dimensional polyacrylamide gel electrophoresis, which exhibited sequence identity with the cytosolic MIPS coded by OsINO1-1 gene. A possible chloroplastic transit peptide sequence was identified upstream of the OsINO1-1 gene upon analysis of rice genome. RT-PCR and confocal microscope studies confirmed transcription, effective translation and its functioning as a chloroplast transit peptide. Bioinformatic analysis mapped the chloroplastic MIPS (OsINO1-1) gene on chromosome 3, and a second MIPS gene (OsINO1-2) on chromosome 10 which lacks conventional chloroplast transit peptide sequence as in OsINO1-1. Two new PcINO1 genes, with characteristic promoter activity and upstream cis-elements were identified and cloned, but whether these proteins can be translocated to the chloroplast or not is yet to be ascertained. Electrophoretic mobility shift assay carried out with nuclear extract of Porteresia coarctata leaves grown under both control and stressed condition shows binding of nuclear proteins with the upstream elements. Nucleotide divergence among the different Oryza and Porteresia INO1 genes were calculated and compared.

Porteresia coarctata (Roxb.) Tateoka, a wild rice: a potential model for studying salt-stress biology in rice.

Porteresia coarctata (Syn = Oryza coarctata) is a tetraploid wild rice growing abundantly in the coastal region of India and some other Asian countries. The salt tolerance property of this mangrove associate has been dealt with by a number of workers earlier. The distinct morphology and leaf architecture enabling the plant to exclude salt is a characteristic feature of Porteresia in comparison with Oryza sp. A number of genes have been isolated and characterized from Porteresia that are related to the salt-tolerance property of the plant. Evidence have accumulated that some pathways critical to salt tolerance are in operation in Porteresia of which the inositol metabolic pathway has been recently elaborated. Some of the enzymes of Porteresia have been shown to function as salt-tolerant under in vitro studies giving a clue that this wild halophytic rice may have evolved genes and proteins capable of functioning under a salt environment. Bioprospecting of such genes and proteins coupled with genomic and proteomic approaches remain an exciting area of research in evaluating this plant as a model for salt tolerance for the rice plant.

Insight into the salt tolerance factors of a wild halophytic rice, Porteresia coarctata: 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.

Selective manipulation of the inositol metabolic pathway for induction of salt-tolerance in indica rice variety.

Halophytes are rich sources of salt stress tolerance genes which have often been utilized for introduction of salt-tolerance character in salt-sensitive plants. In the present study, we overexpressed PcINO1 and PcIMT1 gene(s), earlier characterized in this laboratory from wild halophytic rice Porteresia coarctata, into IR64 indica rice either singly or in combination and assessed their role in conferring salt-tolerance. Homozygous T3/T4 transgenic plants revealed that PcINO1 transformed transgenic rice lines exhibit significantly higher tolerance upto 200 mM or higher salt concentration with negligible compromise in their growth or other physiological parameters compared to the untransformed system grown without stress. The PcIMT1-lines or the double transgenic lines (DC1) having PcINO1 and PcIMT1 introgressed together, were less efficient in such respect. Comparison of inositol and/or pinitol pool in three types of transgenic plants suggests that plants whose inositol production remains uninterrupted under stress by the functional PcINO1 protein, showed normal growth as in the wild-type plants without stress. It is conceivable that inositol itself acts as a stress-ameliorator and/or as a switch for a number of other pathways important for imparting salt-tolerance. Such selective manipulation of the inositol metabolic pathway may be one of the ways to combat salt stress in plants.

Inositol methyl tranferase from a halophytic wild rice, Porteresia coarctata Roxb. (Tateoka): regulation of pinitol synthesis under abiotic stress.

Methylated inositol D-pinitol (3-O-methyl-D-chiro-inositol) accumulates in a number of plants naturally or in response to stress. Here, we present evidence for accumulation and salt-enhanced synthesis of pinitol in Porteresia coarctata, a halophytic wild rice, in contrast to its absence in domesticated rice. A cDNA for Porteresia coarctata inositol methyl transferase 1 (PcIMT1), coding for the inositol methyl transferase implicated in the synthesis of pinitol has been cloned from P. coarctata, bacterially overexpressed and shown to be functional in vitro. In silico analysis confirms the absence of an IMT1 homolog in Oryza genome, and PcIMT1 is identified as phylogenetically remotely related to the methyl transferase gene family in rice. Both transcript and proteomic analysis show the up-regulation of PcIMT1 expression following exposure to salinity. Coordinated expression of L-myo-inositol 1-phosphate synthase (PcINO1) gene along with PcIMT1 indicates that in P. coarctata, accumulation of pinitol via inositol is a stress-regulated pathway. The presence of pinitol synthesizing protein/gene in a wild halophytic rice is remarkable, although its exact role in salt tolerance of P. coarctata cannot be currently ascertained. The enhanced synthesis of pinitol in Porteresia under stress may be one of the adaptive features employed by the plant in addition to its known salt-exclusion mechanism.

Author Correction: An Endophytic Bacterial Consortium modulates multiple strategies to improve Arsenic Phytoremediation Efficacy in Solanum nigrum.

A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has not been fixed in the paper.

An Endophytic Bacterial Consortium modulates multiple strategies to improve Arsenic Phytoremediation Efficacy in Solanum nigrum.

Endophytic microbes isolated from plants growing in contaminated habitats possess specialized properties that help their host detoxify the contaminant/s. The possibility of using microbe-assisted phytoremediation for the clean-up of Arsenic (As) contaminated soils of the Ganga-Brahmaputra delta of India, was explored using As-tolerant endophytic microbes from an As-tolerant plant Lantana camara collected from the contaminated site and an intermediate As-accumulator plant Solanum nigrum. Endophytes from L. camara established within S. nigrum as a surrogate host. The microbes most effectively improved plant growth besides increasing bioaccumulation and root-to-shoot transport of As when applied as a consortium. Better phosphate nutrition, photosynthetic performance, and elevated glutathione levels were observed in consortium-treated plants particularly under As-stress. The consortium maintained heightened ROS levels in the plant without any deleterious effect and concomitantly boosted distinct antioxidant defense mechanisms in the shoot and root of As-treated plants. Increased consortium-mediated As(V) to As(III) conversion appeared to be a crucial step in As-detoxification/translocation. Four aquaporins were differentially regulated by the endophytes and/or As. The most interesting finding was the strong upregulation of an MRP transporter in the root by the As + endophytes, which suggested a major alteration of As-detoxification/accumulation pattern upon endophyte treatment that improved As-phytoremediation.

Introgression of a novel salt-tolerant L-myo-inositol 1-phosphate synthase from Porteresia coarctata (Roxb.) Tateoka (PcINO1) confers salt tolerance to evolutionary diverse organisms.

We have previously demonstrated that introgression of PcINO1 gene from Porteresia coarctata (Roxb.) Tateoka, coding for a novel salt-tolerant L-myo-inositol 1-phosphate synthase (MIPS) protein, confers salt tolerance to transgenic tobacco plants (Majee, M., Maitra, S., Dastidar, K.G., Pattnaik, S., Chatterjee, A., Hait, N.C., Das, K.P. and Majumder, A.L. (2004) A novel salt-tolerant L-myo-inositol-1-phosphate synthase from Porteresia coarctata (Roxb.) Tateoka, a halophytic wild rice: molecular cloning, bacterial overexpression, characterization, and functional introgression into tobacco-conferring salt-tolerance phenotype. J. Biol. Chem. 279, 28539-28552). In this communication we have shown that functional introgression of the PcINO1 gene confers salt-tolerance to evolutionary diverse organisms from prokaryotes to eukaryotes including crop plants albeit to a variable extent. A direct correlation between unabated increased synthesis of inositol under salinity stress by the PcINO1 gene product and salt tolerance has been demonstrated for all the systems pointing towards the universality of the application across evolutionary divergent taxa.

Diversification and evolution of L-myo-inositol 1-phosphate synthase.

L-myo-Inositol 1-phosphate synthase (MIPS, EC 5.5.1.4), the key enzyme in the inositol and phosphoinositide biosynthetic pathway, is present throughout evolutionarily diverse organisms and is considered an ancient protein/gene. Analysis by multiple sequence alignment, phylogenetic tree generation and comparison of newly determined crystal structures provides new insight into the origin and evolutionary relationships among the various MIPS proteins/genes. The evolution of the MIPS protein/gene among the prokaryotes seems more diverse and complex than amongst the eukaryotes. However, conservation of a 'core catalytic structure' among the MIPS proteins implies an essential function of the enzyme in cellular metabolism throughout the biological kingdom.

Physiological and genomic basis of mechanical-functional trade-off in plant vasculature.

Some areas in plant abiotic stress research are not frequently addressed by genomic and molecular tools. One such area is the cross reaction of gravitational force with upward capillary pull of water and the mechanical-functional trade-off in plant vasculature. Although frost, drought and flooding stress greatly impact these physiological processes and consequently plant performance, the genomic and molecular basis of such trade-off is only sporadically addressed and so is its adaptive value. Embolism resistance is an important multiple stress- opposition trait and do offer scopes for critical insight to unravel and modify the input of living cells in the process and their biotechnological intervention may be of great importance. Vascular plants employ different physiological strategies to cope with embolism and variation is observed across the kingdom. The genomic resources in this area have started to emerge and open up possibilities of synthesis, validation and utilization of the new knowledge-base. This review article assesses the research till date on this issue and discusses new possibilities for bridging physiology and genomics of a plant, and foresees its implementation in crop science.

Galactinol synthase across evolutionary diverse taxa: functional preference for higher plants?

Galactinol synthase (GolS), a GT8 family glycosyltransferase, synthesizes galactinol and raffinose series of oligosaccharides (RFOs). Identification and analysis of conserved domains in GTs among evolutionarily diverse taxa, structure prediction by homology modeling and determination of substrate binding pocket followed by phylogenetic analysis of GolS sequences establish presence of functional GolS predominantly in higher plants, fungi having the closest possible ancestral sequences. Evolutionary preference for a functional GolS expression in higher plants might have arisen in response to the need for galactinol and RFO synthesis to combat abiotic stress, in contrast to other organisms lacking functional GolS for such functions.

Salt-induced abnormalities on root tip mitotic cells of Allium cepa: prevention by inositol pretreatment.

Salt-induced growth reduction of plants is a well-known phenomenon which poses major problem in crop productivity in places where vast majority of land plants are affected by salt. In this report, studies were carried out to reveal the effect of salt injury on the cell division pattern in roots and the role of myo-inositol in preventing the salt-induced ion disequilibrium on the chromosome and DNA degradation in roots. Present study revealed induction of various chromosomal abnormalities on the root tip mitotic cells of Allium cepa by treatment with different concentrations of NaCl (0-500 mM) for 24 h as also the amelioration of such effect by prior treatment of the roots with different concentration of myo-inositol (0-300 mM). Results showed that a narrow albeit definite range of extracellular myo-inositol (100-150 mM) is effective in preventing internucleosomal fragmentation which is the early response in roots under salt stress. Transgenic tobacco plants overexpressing Oryza (OsINO1) as well as Porteresia (PcINO1) cytosolic L: -myo-inositol-1-phosphate synthase coding genes can withstand and retain their chromosomal and DNA integrity in 100 mM NaCl solution and can subsequently prevent DNA fragmentation, caused by intracellular endonuclease activity at this salt concentration.

Enhanced salt tolerance of transgenic tobacco plants by co-expression of PcINO1 and McIMT1 is accompanied by increased level of myo-inositol and methylated inositol.

Introgression and functional expression of either the PcINO1 (L: -myo-inositol 1-phosphate synthase or MIPS coding gene from the wild halophytic rice, Porteresia coarctata) or McIMTI (inositol methyl transferase, IMTI coding gene from common ice plant Mesembryanthemum crystallinum) has earlier been shown to confer salt tolerance to transgenic tobacco plants (Sheveleva et al., Plant Physiol 115:1211-1219, 1997; Majee et al., J Biol Chem 279:28539-28552, 2004). In this communication, we show that transgenic tobacco plants co-expressing PcINO1 and McIMT1 gene either in cytosol or in chloroplasts accumulate higher amount of total inositol (free and methyl inositol) compared to non-transgenic plants. These transgenic plants were more competent in terms of growth potential and photosynthetic activity and were less prone to oxidative stress under salt stress. A positive correlation between the elevated level of total inositol and methylated inositol and the capability of the double transgenic plants to withstand a higher degree of salt stress compared to the plants expressing either PcINO1 or McIMT1 alone is inferred.

Functional identification of sll1383 from Synechocystis sp PCC 6803 as L-myo-inositol 1-phosphate phosphatase (EC 3.1.3.25): molecular cloning, expression and characterization.

The genome sequence of the cyanobacterium Synechocystis sp. PCC6803 revealed four Open reading frame (ORF) encoding putative inositol monophosphatase or inositol monophosphatase-like proteins. One of the ORFs, sll1383, is approximately 870 base pair long and has been assigned as a probable myo-inositol 1 (or 4) monophosphatase (IMPase; EC 3.1.3.25). IMPase is the second enzyme in the inositol biosynthesis pathway and catalyses the conversion of L-myo-inositol 1-phosphate to free myo-inositol. The present work describes the functional assignment of ORF sll1383 as myo-inositol 1-phosphate phosphatase (IMPase) through molecular cloning, bacterial overexpression, purification and biochemical characterization of the gene product. Affinity (K (m)) of the recombinant protein for the substrate DL-myo-inositol 1-phosphate was found to be much higher (0.0034 +/- 0.0003 mM) compared to IMPase(s) from other sources but in comparison V (max) ( approximately 0.033 mumol Pi/min/mg protein) was low. Li(+) was found to be an inhibitor (IC(50) 6.0 mM) of this enzyme, other monovalent metal ions (e.g. Na(+), K(+) NH (4) (+) ) having no significant effect on the enzyme activity. Like other IMPase(s), the activity of this enzyme was found to be totally Mg(2+) dependent, which can be substituted partially by Mn(2+). However, unlike other IMPase(s), the enzyme is optimally active at approximately 42 degrees C. To the best of our knowledge, sll1383 encoded IMPase has the highest substrate affinity and specificity amongst the known examples from other prokaryotic sources. A possible application of this recombinant protein in the enzymatic coupled assay of L-myo-inositol 1-phosphate synthase (MIPS) is discussed.

An insight into the molecular basis of salt tolerance of L-myo-inositol 1-P synthase (PcINO1) from Porteresia coarctata (Roxb.) Tateoka, a halophytic wild rice.

The molecular basis of salt tolerance of L-myo-inositol 1-P synthase (MIPS; EC 5.5.1.4) from Porteresia coarctata (Roxb.) Tateoka (PcINO1, AF412340) earlier reported from this laboratory, has been analyzed by in vitro mutant and hybrid generation and subsequent biochemical and biophysical studies of the recombinant proteins. A 37-amino acid stretch between Trp-174 and Ser-210 has been confirmed as the salt-tolerance determinant domain in PcINO1 both by loss or gain of salt tolerance by either deletion or by addition to salt-sensitive MIPS(s) of Oryza (OsINO1) and Brassica juncea (BjINO1). This was further verified by growth analysis under salt environment of Schizosaccharomyces pombe transformed with the various gene constructs and studies on the differential behavior of mutant and wild proteins by Trp fluorescence, aggregation, and circular dichroism spectra in the presence of salt. 4,4'-Dianilino-1,1'-binaphthyl-5,5-disulfonic acid binding experiments revealed a lower hydrophobic surface on PcINO1 than OsINO1, contributed by this 37-amino acid stretch explaining the differential behavior of OsINO1 and PcINO1 both with respect to their enzymatic functions and thermodynamic stability in high salt environment. Detailed amino acid sequence comparison and modeling studies revealed the interposition of polar and charged residues and a well-connected hydrogen-bonding network formed by Ser and Thr in this stretch of PcINO1. On the contrary, hydrophobic residues clustered in two continuous stretches in the corresponding region of OsINO1 form a strong hydrophobic patch on the surface. It is conceivable that salt-tolerant MIPS proteins may be designed out of the salt-sensitive plant MIPS proteins by replacement of the corresponding amino acid stretch by the designated 37-amino acid stretch of PcINO1.