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Myo-Inositol phosphate synthase (MIPS) enzyme catalyses rate limiting step of myo-inositol (MI) biosynthesis. MIPS by-products are involved in many plant processes. In this study, TaMIPS2 identified from a heat subtractive cDNA library from wheat has been functionally characterized. To investigate the importance of TaMIPS2 during heat stress tolerance, TaMIPS2 overexpression transgenics were raised in Arabidopsis which were analysed physiologically and morphologically under different abiotic stress conditions. MIPS enzyme assay revealed the level of inositol in transgenics and of other soluble sugars as quantified by HPLC. Analysis of overexpression transgenics under different stress condition revealed that TaMIPS2 transgenic have reduced sensitivity to heat stress. This is the first report indicating a role of myo-Inositol during heat stress. The analysis of transgenic lines of TaMIPS2 suggests its additional role under other environmental stresses. more...

Drought and heat are the two major abiotic stresses that are detrimental to the yield and quality of crop plants such as wheat. In the present study, we cloned and characterize a membrane protein gene from wheat, previously identified through cDNA subtractive hybridization. BLAST analysis revealed that the newly identified gene belongs to Arabidopsis and rice RCI (Rare Cold Inducible) genes and hence named as TaRCI. In the present investigation, Arabidopsis transgenics were raised expressing TaRCI for functional analysis. The subcellular localization by translational fusion of TaRCI with GFP revealed the localization of GFP:TaRCI into the chloroplast. Arabidopsis transgenics expressing TaRCI performed better than the wild-type under simulated heat, drought and salinity stress conditions. Under heat stress conditions, TaRCI expressing transgenic seedlings showed faster recovery post heat stress and were healthy and greener than wild-type plants. Further, the TaRCI expressing plants accumulated more biomass in terms of size, rosette diameter and root length under simulated drought stress conditions also. Increased leaf size, and rosette diameter were also observed in the presence of ABA and SA. Transgenic plants also showed increase in physiological parameters such as maximum photosynthetic efficiency (Fv/Fm), proline and chlorophyll contents. Our study thus provides insight into a new wheat gene that could be an important regulator involved in multiple abiotic stresses and could be a potential candidate gene manipulation for improving stress tolerance in crop plants in general and wheat in particular. more...

Molecular dissection and a deeper analysis of the heat stress response mechanism in wheat have been poorly understood so far. This study delves into the molecular basis of action of TaMIPS, a heat stress-inducible enzyme that was identified through PCR-select subtraction technology, which is named here as TaMIPS2. MIPS (L-Myo-inositol-phosphate synthase) is important for the normal growth and development in plants. Expression profiling showed that TaMIPS2 is expressed during different developing seed stages upon heat stress. Also, the transcript levels increase in unfertilized ovaries and significant amounts are present during the recovery period providing evidence that MIPS is crucial for its role in heat stress recovery and flower development. Alternatively spliced forms from rice and Arabidopsis were also identified and their expression analysis revealed that apart from heat stress, some of the spliced variants were also inducible by drought, NaCl, Cold, ABA, BR, SA and mannitol. In silico promoter analysis revealed various cis-elements that could contribute for the differential regulation of MIPS in different plant systems. Phylogenetic analysis indicated that MIPS are highly conserved among monocots and dicots and TaMIPS2 grouped specifically with monocots. Comparative analyses was undertaken by different experimental approaches, i.e., semi-quantitative RT-PCR, quantitative RT-PCR, Genevestigator as a reference expression tool and motif analysis to predict the possible function of TaMIPS2 in regulating the different aspects of plant development under abiotic stress in wheat. more...

The nuclear-encoded chloroplast small heat shock proteins (sHSPs) are present in all plant species from algae to angiosperms. Expression analysis shows that the wheat chloroplastic sHSP (HSP26) is highly inducible by heat stress in almost all the vegetative and generative tissues and is also expressed constitutively in certain developmental growth stages. We characterize wheat chloroplastic sHSP 26 through transgenic approach using Arabidopsis and report cloning of the promoter and its characterization. Transgenic Arabidopsis plants were substantially tolerant under continuous high temperature regimen than wild-type plants, as measured by photosystem II (PSII) activity, accumulation of more photosynthetic pigments, higher biomass and seed yield. Transgenic plants produced bold seeds under high temperature, having higher germination potential than the wild-type plants. Further, antisense Arabidopsis plants showed negligible tolerance even for non-lethal heat shock, impaired in basal thermo-tolerance, and accumulated less biomass and seed yield under normal growth conditions. Promoter analysis revealed the presence of several heat and other abiotic stress responsive cis-acting elements along with developmental stage and tissue-specific elements. Analysis of promoter through GUS reporter system in both transgenic rice and Arabidopsis further confirms the role of chloroplastic sHsp26 in heat and other abiotic stresses as well as during seed maturation and germination. Genome-wide expression analysis of overexpression Arabidopsis plants revealed that the transcriptome remained unchanged in the transgenic plants and the tolerance was due to the overexpression of chloroplastic heat shock protein (HSP) only. more...

Plants respond to heat stress by enhancing the expression of genes encoding heat shock protein (HSPs) genes through activation of heat shock factors (HSFs) which interact with heat shock elements present in the promoter of HSP genes. Plant HSFs have been divided into three conserved classes viz A, B and C. In the present study, a detailed analysis has been done of all rice HSFs, along with their spliced variants. Their chromosomal localization reveals that six HSFs are segmentally duplicated and four pairs of these segmentally duplicated HSF encoding genes show pseudo-functionalization. Expression profiling through microarray and quantitative real-time PCR showed that eight OsHsfs express at a higher level during seed development, while six HSFs are up-regulated in all the abiotic stresses studied. The expression of OsHsfA2a gene in particular was greatly stimulated by heat stress in both root and shoot tissues and also during panicle and seed development. OsHsfA3 was found more responsive to cold and drought stress, while OsHsfA7 and OsHsfA9 showed developing seed-specific expression. This study also revealed that spliced variants generally accumulated at a higher level in all the tissues examined. Different hormones/elicitors like ABA, brassinosteroids and salicylic acid also alter OsHsf gene expression. Calcium in combination with heat stress elevated further the level of HSF transcripts. Expression analysis by both microarray and real-time RT-PCR revealed a unique stable constitutive expression of OsHsfA1 across all the tissues and stresses. A detailed in silico analysis involving identification of unidentified domains has been done by MEME-motif tool in their full-length proteins as well as in DNA-binding domains. Analysis of 1 kb putative promoter region revealed presence of tissue-specific, abiotic stress and hormone-related cis-acting elements, correlating with expression under stress conditions. more...

To elucidate the effect of high temperature, wheat plants (Triticum aestivum cv. CPAN 1676) were given heat shock at 37 and 42°C for 2 h, and responsive genes were identified through PCR-Select Subtraction technology. Four subtractive cDNA libraries, including three forward and one reverse subtraction, were constructed from three different developmental stages. A total of 5,500 ESTs were generated and 3,516 high quality ESTs submitted to Genbank. More than one-third of the ESTs generated fall in unknown/no hit category upon homology search through BLAST analysis. Differential expression was confirmed by cDNA macroarray and by northern/RT-PCR analysis. Expression analysis of wheat plants subjected to high temperature stress, after 1 and 4 days of recovery, showed fast recovery in seedling tissue. However, even after 4 days, recovery was negligible in the developing seed tissue after 2 h of heat stress. Ten selected genes were analyzed in further detail including one unknown protein and a new heat shock factor, by quantitative real-time PCR in an array of 35 different wheat tissues representing major developmental stages as well as different abiotic stresses. Tissue specificity was examined along with cross talk with other abiotic stresses and putative signalling molecules. more...

To elucidate the effects of high temperatures, wheat plants ( Triticum aestivum cv. CPAN 1676) were given heat shocks at 37°C and 42°C for two hours, and responsive genes were identified through PCR-Select Subtraction technology. Four subtractive cDNA libraries, including three forward and one reverse subtraction, were constructed from three different developmental stages. A total of 5500 ESTs were generated and 3516 high quality ESTs were submitted to Genbank. More than one third of the ESTs generated fall in unknown/no hit categories upon a homology search through BLAST analysis. A large number of high temperature responsive genes have been identified and characterized. Reverse subtraction analysis in developing grains showed extensive transcriptional changes upon heat stress as revealed by comparative analysis with forward subtraction. Differ - ential expression was confirmed by cDNA macroarray and by northern/RT-PCR analysis. Expression analysis of wheat plants subjected to high temperature stress, after one and four days of recovery, showed fast recovery in seedling tissues. However, recovery was small in the developing seed tissue after two hours of heat stress. Ten selected genes were analysed in further detail by quantitative real-time PCR in an array of 35 different wheat tissues representing major developmental stages as well as different abiotic stresses. Tissue specificity was ex more...