MOLECULAR-CLONING AND BIOCHEMICAL-CHARACTERIZATION OF CARBONIC-ANHYDRASE FROM POPULUS-TREMULA X TREMULOIDES

A leaf cDNA library from hybrid aspen, Populus tremula x tremuloides, was constructed. From this two different cDNA clones, denoted CAla and CAlb, encoding a chloroplastic carbonic anhydrase (CA) were isolated and DNA sequenced. Analysis of the deduced amino acid sequences showed that the isolated CAs belong to the beta-CA family, and have identiti es around 70% to other dicotyledonous plant CAs. The two hybrid aspen cDNA clones display a high nucleotide sequence identity, only 12 nucle otides differ. Since only one gene copy of this soluble chloroplastic CA is present in the nuclear genome, we postulate that the two isolate d cDNA clones are alleles. Northern blot hybridization revealed a CA t ranscript of ca. 1300 bases, 140 bases shorter than in pea. Western an d northern blot hybridizations on crude protein extracts and on total RNA, respectively, isolated from stem and leaves, showed that hybrid a spen CA is expressed specifically in the leaf under the growth conditi ons used. Based on the deduced amino acid sequence, the mature hybrid aspen CA enzyme subunit has a molecular mass of 24.8 kDa. The enzyme w as over-expressed in Escherichia coli, and purified by affinity chroma tography. Biochemical characterization showed that the protein structu re and the CO2-hydration activity are similar to the pea enzyme. Molec ular characterization of a CA from a perennial plant has not previousl y been performed, and it demonstrates that both the structure and acti vity of hybrid aspen CA resembles CAs from annual plants.