Carbonic Anhydrase-Deficient Mutant of Chlamydomonas reinhardii Requires Elevated Carbon Dioxide Concentration for Photoautotrophic Growth

Abstract

A mendelian mutant of the unicellular green alga Chlamydomonas reinhardii has been isolated which is deficient in carbonic anhydrase (EC 4.2.1.1) activity. This mutant strain, designated ca-1-12-1C (gene locus ca-1), was selected on the basis of a high CO(2) requirement for photoautotrophic growth. Photosynthesis by the mutant at atmospheric CO(2) concentration was very much reduced compared to wild type and, unlike wild type, was strongly inhibited by O(2). In contrast to a CO(2) compensation concentration of near zero in wild type at all O(2) concentrations examined, the mutant exhibited a high, O(2)-stimulated CO(2) compensation concentration. Evidence of photorespiratory activity in the mutant but not in wild type was obtained from the analysis of photosynthetic products in the presence of (14)CO(2). At air levels of CO(2) and O(2), the mutant synthesized large amounts of glycolate, while little glycolate was synthesized by wild type under identical conditions. Both mutant and wild type strains formed only small amounts of glycolate at saturating CO(2) concentration. At ambient CO(2), wild type accumulated inorganic carbon to a concentration several-fold higher than that in the suspension medium. The mutant cells accumulated inorganic carbon internally to a concentration 6-fold greater than found in wild type, yet photosynthesis was CO(2) limited. The mutant phenotype was mimicked by wild type cells treated with ethoxyzolamide, an inhibitor of carbonic anhydrase activity. These observations indicate a requirement for carbonic anhydrase-catalyzed dehydration of bicarbonate in maintaining high internal CO(2) concentrations and high photosynthesis rates. Thus, in wild type cells, carbonic anhydrase rapidly converts the bicarbonate taken up to CO(2), creating a high internal CO(2) concentration which stimulates photosynthesis and suppresses photorespiration. In mutant cells, bicarbonate is taken up rapidly but, because of a carbonic anhydrase deficiency, is not dehydrated at a rate sufficiently rapid to maintain a high internal CO(2) concentration.