Carbonic anhydrase in arabidopsis thaliana: role and regulation of expression

Carbonic anhydrase (EC 4.2.1.1) catalyses the reversible hydration of CO2 and is a major soluble protein component of most photosynthetic organisms. There have only been two isoforms of Î² carbonic anhydrase characterized in leaves of C3 plants, a chloroplast-localized (CA1) and a cytosolic form (CA2). Proposed roles for the CA1 have included facilitation of CO2 movement across the chloroplast envelope and the stroma by rapidly equilibrating Ci speciation and the provision of CO 2 for Rubisco. In addition to a potential photosynthetic role for CA2â€”the facilitation of inorganic carbon diffusion through the cytosol, CA2 may be required for HCO3âˆ’ synthesis, the substrate for PEP carboxylase. In an attempt to further examine the roles of CA1 and CA2 in carbon metabolism, RNA anti-sense studies were carried out in the model organism Arabidopsis thaliana. Both CA1 anti-sense and co-suppression resulted in CA activity levels almost at the limit of detection and well below 1% of wild type. Plants over-expressing CA1 displayed activity levels that were as much as 600% of wild type rates. Following germination, the majority of the CA1 anti-sense and co-suppressed plants failed to develop further when grown on minimal media. The plating of CA1 anti-sense and co-suppression seeds on Â½-strength MS plates containing 1% sucrose or at 2800 Î¼lÂ· Lâˆ’1 CO 2 resulted in a significant increase in survivorship. Growth at less than ambient levels of CO2 (200 Î¼lÂ· Lâˆ’1) did result in a CA1 anti-sense phenotype. Mature plants were much smaller and exhibited a reduction in the numbers of branches and siliques, had higher conductance and less soluble hexoses compared to wild type and plants over expressing CA1. At high CO2 (1500 Î¼lÂ· Lâˆ’1), CA1 anti-sense plants accumulated significantly less anthocyanin. These data are consistent with the role of CA1 enhancing the photosynthetic capacity of the plant by reducing CO2 diffusion resistance across the chloroplast. Reducing the cytosol-localized CA2 in plants via anti-sense resulted in an increase in total CA activity, ranging from 118% to 180% of wild type levels. Conversely, the over expression of CA2 resulted in a reduction in total foliar CA activity compared to wild type as a result of a greater than 50% reduction in CA1 activity. At 200 Î¼lÂ· Lâˆ’1 CO2, CA2 anti-sense plants had less foliar hexoses and starch levels and exhibited higher dehydration rates (increased stomatal conductance) as compared to wild type and plants over expressing CA2. The above data implicate CA2 in the photosynthetic process, perhaps by facilitating carbon diffusion through the cytoplasm or an involvement in a CO2 sensing mechanism. To elucidate the cis-elements regulating CA1 expression, the CA1 promoter was ligated to the GUS reporter gene uidA. CA1 expression occurs early in the cotyledon in a light independent manner and is limited to the cotyledons, the rosette and cauline leaves and the sepals in light grown adult plants. Changes in atmospheric CO levels affected CA1 transcript in a reciprocal manner, as CO2 levels increase, CA1 levels decrease. Furthermore, it appears that CA1 expression is linked in part to the carbohydrate status of the plant in that CA1 expression is maintained in a light-independent manner if foliar carbohydrate reserves are low.