Molecular cloning and characterization of gene encoding for cytoplasmic Hsc70 from Pennisetum glaucum may play a protective role against abiotic stresses.

Molecular chaperones (Hsps) have been shown to facilitate protein folding or assembly under various developmental and adverse environmental conditions. The aim of this study was to unravel a possible role of heat-shock proteins in conferring abiotic stress tolerance to plants. We isolated a cDNA encoding a cytoplasmic Hsp70 (PgHsc70) from Pennisetum glaucum by screening heat-stress cDNA library. PgHsc70 cDNA encoding 649 amino acids represents all conserved signature motifs characteristic of Hsp70s. The predicted molecular model of PgHsc70 protein suggests that the N-terminus ATP-binding region is evolutionarily conserved, in comparison to C-terminus peptide-binding domains. A single intron in ATPase domain coding region of PgHsc70 exhibited a high degree of conservation with respect to its position and phasing among other plant Hsp70 genes. Recombinant PgHsc70 protein purified from E. coli possessed in vitro chaperone activity and protected PgHsc70 expressing bacteria from damage caused by heat and salinity stress. Nucleotide sequence analysis of 5' flanking promoter region of PgHsc70 gene revealed a potential heat-shock element (HSE) and other putative stress-responsive transcription factor binding sites. Positive correlation existed between differentially up-regulated PgHsc70 transcript levels and the duration and intensity of different environmental stresses. Molecular and biochemical analyses revealed that PgHsc70 gene was a member of the Hsp70 family and suggested that its origin was from duplication of a common ancestral gene. Transcript induction data, presence of several putative stress-responsive transcription factor-binding sites in the promoter region of PgHsc70 and the presence of a protective in vitro chaperone activity of this protein against damage caused by heat and salinity, when expressed in E. coli, suggest its probable role in conferring abiotic stress tolerance to this plant.

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

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.

Molecular cloning and characterization of genes encoding Pennisetum glaucum ascorbate peroxidase and heat-shock factor: interlinking oxidative and heat-stress responses.

The recent genetic and biochemical studies reveal a considerable overlap among cellular processes in response to heat and oxidative stress stimuli in plants suggesting an intimate relationship between the heat-shock response and oxidative stress responses. Pennisetum glaucum (Pg) seedlings were exposed to heat stress (42 degrees C for 0.5, 1.0 and 24h) and a mixture of RNA from all the heat stressed seedlings was used to prepare cDNA. Full-length cDNA clones encoding for cytoplasmic ascorbate peroxidase 1 (PgAPX1) and heat-shock factor (PgHSF) were isolated by screening heat stress-specific cDNA library using corresponding EST sequences as radioactive probes. These full-length cDNAs were expressed in E. coli and their recombinant proteins were purified to near homogeneity. The recombinant PgAPX1 preferred ascorbate but did not accept guaiacol as a reducing substrate. Over-expression of PgAPX1 protects E. coli cells against methyl viologen-induced oxidative stress. Sequence analysis of PgAPX1 promoter identified a number of putative stress regulatory cis-elements including a heat-shock element (HSE). Heat-shock transcription factors (HSFs) play a central role in mediating these overlapping cellular processes. Gel shift analysis and competition with specific and non-specific unlabeled DNA probes showed a specific interaction between HSE of PgAPX1 and the PgHSF protein. Expression analysis of PgHSF in Pennisetum showed maximum increase in transcript level in response to heat stress within 30 min of exposure and slowed down at subsequent time points of heat stress, indicating a typical characteristic of HSF in terms of early responsiveness. Expression of PgAPX1 significantly increased under heat-stress condition; however, the maximum expression observed at 24h of heat stress. In gel activity of PgAPX1 in Pennisetum plants also showed an increase in response to heat stress (42 degrees C) being maximum at 24h and these trends are in conformity with the expression pattern of PgAPX1. Expression patterns and interactive specificity of HSF with HSE (PgAPX1) suggest a probable vital interlink in heat and oxidative stress signaling pathways that plays a significant role in comprehending the underlying mechanisms in plant abiotic stress tolerance.

Isolation and characterization of expressed sequence tags (ESTs) from subtracted cDNA libraries of Pennisetum glaucum seedlings.

Pearl millet (Pennisetum glaucum), used as forage and grain crop is a stress tolerant species. Here we identify differentially regulated transcripts in response to abiotic (salinity, drought and cold) stresses from subtracted cDNA libraries by single-pass sequencing of cDNA clones. A total of 2,494 EST sequences were clustered and assembled into a collection of 1,850 unique sequences with 224 contigs and 1,626 singleton sequences. By sequence comparisons the putative functions of many ESTs could be assigned. Genes with stress related functions include those involved in cellular defense against abiotic stresses and transcripts for proteins involved in stress response signaling and transcription in addition to ESTs encoding unknown functions. These provide new candidate genes for investigation to elucidate their role in abiotic stress. The relative mRNA abundance of 38 selected genes, quantified using real time quantitative RT-PCR, demonstrated the existence of a complex gene regulatory network that differentially modulates gene expression in a kinetics-specific manner in response to different abiotic stresses. Notably, housekeeping and non-target genes were effectively reduced in these subtracted cDNA libraries constructed. These EST sequences are a rich source of stress-related genes and reveal a major part of the stress-response transcriptome that will provide the foundation for further studies into understanding Pennisetum's adaptability to harsh environmental conditions.

The severe acute respiratory syndrome coronavirus nucleocapsid protein is phosphorylated and localizes in the cytoplasm by 14-3-3-mediated translocation.

The severe acute respiratory syndrome coronavirus(SARS-CoV) nucleocapsid (N) protein is one of the four structural proteins of the virus and is predicted to be a 46-kDa phosphoprotein. Our in silico analysis predicted N to be heavily phosphorylated at multiple residues. Experimentally, we have shown in this report that the N protein of the SARS-CoV gets serine-phosphorylated by multiple kinases, in both the cytoplasm and the nucleus. The phosphoprotein is stable and localizes in the cytoplasm and coprecipitates with the membrane fraction. Also, using specific inhibitors of phosphorylation and an in vitro phosphorylation assay, we show that the nucleocapsid protein is a substrate of cyclin-dependent kinase (CDK), glycogen synthase kinase, mitogen-activated protein kinase, and casein kinase II. Further, we show that the phosphorylated protein is translocated to the cytoplasm by binding to 14-3-3 (tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein). 14-3-3 proteins are a family of highly conserved, ubiquitously expressed eukaryotic proteins that function primarily as adapters that modulate interactions between components of various cellular signaling and cell cycle regulatory pathways through phosphorylation-dependent protein-protein interactions. Coincidentally, the N protein was also found to downregulate the expression of the theta isoform of 14-3-3 (14-3-3theta), leading to the accumulation of phosphorylated N protein in the nucleus, in the absence of growth factors. Using short interfering RNA specific to 14-3-3theta we have inhibited its expression to show accumulation of phosphorylated N protein in the nucleus. Thus, the data presented here provide a possible mechanism for phosphorylation-dependent nucleocytoplasmic shuttling of the N protein. This 14-3-3-mediated transport of the phosphorylated N protein and its possible implications in interfering with the cellular machinery are discussed.