Biochemical and molecular characterization of stress-induced β-carbonic anhydrase from a C₄ plant, Pennisetum glaucum

Abstract

Genes encoding for many β-carbonic anhydrases and their functions in various developmental processes are well established in lower plants, however, similar studies are limited in higher plants. We report the cloning and characterization of cDNA encoding for a β-carbonic anhydrase (PgCA) from Pennisetum glaucum, a C₄ crop plant. cDNA encoding 249 amino acids and its deduced amino acid sequence analysis revealed that is related to other plant β-CA family members with an over all conserved architecture of a typical β-CA protein. Phylogenetic analysis revealed that PgCA is evolutionarily very close to chloroplast β-CA isoform. Signal sequence predicting programs identify a N-terminus putative chloroplast targeting sequence. Heterologous Escherichia coli expression system was utilized to overexpress recombinant PgCA, which showed high thermostability, an alkaline pH optima and dual activity with both reversible CO₂ hydration and esterase activities. The β-CAs studied so far possessed only CO₂ hydration activity with no detectable esterase activity. Recombinant PgCA esterase activity is inhibited by standard CA inhibitors acetazolamide, methazolamide and azide. Subcellular immunostaining studies revealed a chloroplastic localization of PgCA protein. Expression of PgCA transcript is differentially up regulated in response to various abiotic stresses wherein its accumulation in Pennisetum leaves positively correlated with the intensity and duration of stress. Biochemical and transcript analyses suggest that PgCA may play a significant role in plant's adaptation to different abiotic stresses in addition to the previously recognized role of replenishing the CO₂ supply within plant cells.