Dataset Information

Thermo and pH stable ATP-independent chaperone activity of heat-inducible Hsp70 from Pennisetum glaucum.

ABSTRACT: Heat shock proteins (Hsps) are a class of molecular chaperones that play an essential role in preserving cellular functions under stressful conditions. The over production of recombinant proteins often causes cellular stress that results in aggregation/misfolding of proteins, which sometimes leads to the formation of inclusion bodies. Here we report the cloning and characterization of heat-inducible PgHsp70 from Pennisetum glaucum, a heat and drought tolerant plant that showed stability and chaperone activity at elevated temperatures. The predicted amino acid sequence of PgHsp70 revealed a high homology with Hsp70 from other plants, and the overall 3D structure homology modeling is similar to that of the constitutively expressed bovine cytosolic Heat Shock Cognate (HSC)-70. The purified recombinant protein had an apparent molecular mass of 70 kDa and displayed optimal chaperone activity at 50 degrees C, and pH 8.0. Under these conditions, the T(1/2) of PgHsp70 increased from 10 to 15 h in the presence of glycerol. The PgHsp70 exhibited a higher chaperone activity towards glutamate dehydrogenase than alcohol dehydrogenase. The expression of recombinant carbonic anhydrase (CA) in E. coli in a catalytically active soluble form rather than in inclusion bodies was made feasible by co-expression of PgHsp70. Circular dichroism (CD) studies of the recombinant PgHsp70 did not reveal any discernible changes in the alpha-helix content, with increase in temperature from 35 to 85 degrees C, thus suggesting a critical role of alpha-helix content in maintaining the chaperone activity.

SUBMITTER: Rao JL

PROVIDER: S-EPMC2884110 | biostudies-literature | 2010 Feb

REPOSITORIES: biostudies-literature

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Thermo and pH stable ATP-independent chaperone activity of heat-inducible Hsp70 from Pennisetum glaucum.

Rao J L Uma Maheswar JL Reddy Palakolanu Sudhakar PS Mishra Rabi N RN Gupta Dinesh D Sahal Dinkar D Tuteja Narendra N Sopory Sudhir K SK Reddy Malireddy K MK

Plant signaling & behavior 20100209 2

Heat shock proteins (Hsps) are a class of molecular chaperones that play an essential role in preserving cellular functions under stressful conditions. The over production of recombinant proteins often causes cellular stress that results in aggregation/misfolding of proteins, which sometimes leads to the formation of inclusion bodies. Here we report the cloning and characterization of heat-inducible PgHsp70 from Pennisetum glaucum, a heat and drought tolerant plant that showed stability and chap ...[more]

PMID: 20023401

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Project description:Pennisetum glaucum (L.) R. Br., being widely grown in dry and hot weather, frequently encounters heat stress at various stages of growth. The crop, due to its inherent capacity, efficiently overcomes such stress during vegetative stages. However, the same is not always the case with the terminal (flowering through grain filling) stages of growth, where recovery from stress is more challenging. However, certain pearl millet genotypes such as 841-B are known to overcome heat stress even at the terminal growth stages. Therefore, we performed RNA sequencing of two contrasting genotypes of pearl millet (841-B and PPMI-69) subjected to heat stress (42°C for 6 h) at flowering stages. Over 274 million high quality reads with an average length of 150 nt were generated, which were assembled into 47,310 unigenes having an average length of 1,254 nucleotides, N50 length of 1853 nucleotides, and GC content of 53.11%. Blastx resulted in the annotation of 35,628 unigenes, and functional classification showed 15,950 unigenes designated to 51 Gene Ontology terms. A total of 13,786 unigenes were allocated to 23 Clusters of Orthologous Groups, and 4,255 unigenes were distributed to 132 functional Kyoto Encyclopedia of Genes and Genomes database pathways. A total of 12,976 simple sequence repeats and 305,759 SNPs were identified in the transcriptome data. Out of 2,301 differentially expressed genes, 10 potential candidate genes were selected based on log2 fold change and adjusted p value parameters for their differential gene expression by qRT-PCR. We were able to identify differentially expressed genes unique to either of the two genotypes, and also, some DEGs common to both the genotypes were enriched. The differential expression patterns suggested that 841-B 6 h has better ability to maintain homeostasis during heat stress as compared to PPMI-69 6 h. The sequencing data generated in this study, like the SSRs and SNPs, shall serve as an important resource for the development of genetic markers, and the differentially expressed heat responsive genes shall be used for the development of transgenic crops.

Project description:The survival, biomass, and grain yield of most of the crops are negatively influenced by several environmental stresses. The present study was carried out by using transcript expression profiling for functionally clarifying the role of genes belonging to a small heat shock protein (sHSP) family in pearl millet under high-temperature stress. Transcript expression profiling of two high-temperature-responsive marker genes, Pgcp70 and PgHSF, along with physio-biochemical traits was considered to screen out the best contrasting genotypes among the eight different pearl millet inbred lines in the seedling stage. Transcript expression pattern suggested the existence of differential response among different genotypes upon heat stress in the form of accumulation of heat shock-responsive gene transcripts. Genotypes, such as WGI 126, TT-1, TT-6, and MS 841B, responded positively toward high-temperature stress for the transcript accumulation of both Pgcp70 and PgHSF and also indicated a better growth under heat stress. PPMI-69 showed the least responsiveness to transcript induction; moreover, it supports the membrane stability index (MSI) data for scoring thermotolerance, thereby suggesting the efficacy of transcript expression profiling as a molecular-based screening technique for the identification of thermotolerant genes and genotypes at particular crop growth stages. The contrasting genotypes, such as PPMI-69 (thermosusceptible) and WGI-126 and TT-1 (thermotolerant), are further utilized for the characterization of thermotolerance behavior of sHSP by cloning a PgHSP16.97 from the thermotolerant cv. WGI-126. In addition, the investigation was extended for the identification and characterization of 28 different HSP20 genes through a genome-wide search in the pearl millet genome and an understanding of their expression pattern using the RNA-sequencing (RNA-Seq) data set. The outcome of the present study indicated that transcript profiling can be a very useful technique for high-throughput screening of heat-tolerant genotypes in the seedling stage. Also, the identified PgHSP20s genes can provide further insights into the molecular regulation of pearl millet stress tolerance, thereby bridging them together to fight against the unpredicted nature of abiotic stress.

Project description:BACKGROUND: Pearl millet landraces display an important variation in their cycle duration. This diversity contributes to the stability of crop production in the Sahel despite inter-annual rainfall fluctuation. Conservation of phenological diversity is important for the future of pearl millet improvement and sustainable use. Identification of genes contributing to flowering time variation is therefore relevant. In this study we focused on three flowering candidate genes, PgHd3a, PgDwarf8 and PgPHYC. We tested for signatures of past selective events within polymorphism patterns of these three genes that could have been associated with pearl millet domestication and/or landraces differentiation. In order to implement ad hoc neutrality tests, a plausible demographic history of pearl millet domestication was inferred through Approximate Bayesian Computation by using eight neutral STS loci. RESULTS: Domesticated pearl millet exhibited 84% of the nucleotide diversity level found in the wild population. No specific polymorphisms were found either in the wild or in the domestic populations. The bayesian approach and previous studies suggest that gene flow between wild relatives and domesticated pearl millets is a main factor explaining these results. Early and late landraces did not show significant genetic differentiation at both the neutral and the candidate loci. A positive selection was evidenced in PgHd3a and PgDwarf8 genes of domestic forms but not in the wild population. CONCLUSION: Our results strongly suggest that PgHd3a and PgDwarf8 were likely targeted by selection during domestication. However, a potential role of any of the three candidate genes in the phenological differentiation between early and late landraces was not supported by our data. Reasons why these results contrast with previous results that have shown a slight but significant association between PgPHYC polymorphisms and variation in flowering time in pearl millet are discussed.

Dataset Information

Thermo and pH stable ATP-independent chaperone activity of heat-inducible Hsp70 from Pennisetum glaucum.

Publications

Thermo and pH stable ATP-independent chaperone activity of heat-inducible Hsp70 from Pennisetum glaucum.

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