Functional characterization of chloroplastic and cytoplasmic β-carbonic anhydrase isoforms from Leucaena leucocephala

Abstract

Carbonic anhydrases (CAs) catalyze rapid interconversion of CO2 and water to bicarbonate and protons. Carbonic anhydrases (CAs) have been extensively studied and are known to be involved in various physiological processes such as respiration, cell growth and acid/base regulation in animals, and carbon fixation and photosynthesis in plants, algae and cyanobacteria. Although the beta-type carbonic anhydrases (β-CAs) are crucial for carbon fixation in C4 plants, algae, and cyanobacteria, the role of β-CA in C3 plants is not well-studied. Therefore, the specific objective of this research is to isolate and characterize the genes encoding chloroplastic and cytoplasmic β-CAs from the C3 plant, Leucaena leucocephala (leucaena) and investigate their possible role in the plant by overexpressing the leucaena β-CA in Nicotiana tabacum and also to study the expression pattern of leucaena β-CAs under various physiological stress conditions that affect photosynthesis potential in plants. In this study, two cDNA clones, cacp and cacyt, encoding chloroplastic and cytoplasmic forms of β-CA, respectively, were isolated from a leucaena cDNA library. These clones were further extended to full-length by 5'-and 3'-rapid amplification of cDNA ends (RACE). Deduced amino acid sequences of cacp and cacyt exhibited 79 % homology with each other. To determine the possible role of these genes in leucaena under physiological stresses, quantitative PCR (qPCR) was performed from different tissues of leucaena grown under normal, drought, saline, light, bright light, and dark conditions. The expression of cacp and cacyt was found to be up regulated under drought, salt, and light conditions and down regulated under dark conditions. Bright light conditions, compared with normal light condition, did not exhibit much change in the transcript levels of cacp and cacyt. Additionally, tobacco plants were transformed with leucaena's cacp and cacyt separately and also together resulting in three types of transgenic tobacco plants (i) with cacp only (ii) with cacyt only and (iii) with both cacp and cacyt. These transgenic tobaccos were also studied under stress conditions. This study will give us a deeper insight to understand the role of chloroplastic and cytoplasmic β-CAs in leucaena and also in C3 plants in general.