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

Abstract

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°C for 0.5, 1.0 and 24 h) 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 24 h of heat stress. In gel activity of PgAPX1 in Pennisetum plants also showed an increase in response to heat stress (42°C) being maximum at 24 h 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.