Identification of the Physiological Role of Carbonic Anhydrase Using the Antisense Technique, And, Investigation of Its Transcriptional Regulation in Arabidopsis Thaliana (L.) Heynh.

Abstract

Carbonic anhydrase (CA, EC 4.2.1.1), catalyzes the interconversion of CO$\sb2$ and $\rm HCO\sb3\sp-$ in many organisms. The majority of CA activity in C3 leaves is in the chloroplast and expression of the chloroplast CA is regulated by light. To investigate the physiological role of the chloroplast CA, its expression was suppressed by insertion of a CA transgene in the antisense orientation in Arabidopsis thaliana (L.) Heynhold. Activity assays and immunoblots show that in five independent lines of antisense transgenic plants CA activity was strongly suppressed. All five independent lines died or grew poorly in the absence of sucrose whereas wild type plants grew well in the same media. Antisense plants exhibited a phenotype comparable to wild type plants on sucrose-free media only when grown in an atmosphere with elevated levels of CO$\sb2.$ The results support the notion that CA facilitates CO$\sb2$ diffusion from the atmosphere to the chloroplast and clearly show that CA plays a pivotal role in carbon assimilation in C3 plants. In order to investigate the regulation of expression of the chloroplastic CA, a CA gene from Arabidopsis thaliana was cloned, sequenced, and characterized. Sequence analysis of the 5$\sp\prime$ end of the gene encoding the chloroplastic CA revealed the presence of several cis-acting elements previously shown to mediate expression of genes encoding chloroplast proteins. Serial deletions from the 5$\sp\prime$ end of the CA promoter were fused with a reporter gene encoding $\beta$-glucuronidase and introduced into Arabidopsis thaliana. Deletion of an AT-rich palindromic sequence, designated AT-1P, resulted in the largest decrease in GUS activity in transgenic plants. However, the presence of only a second AT-rich palindromic sequence, designated TA-1P, upstream of the CAAT box conferred leaf-specific expression. An expression library from Arabidopsis thaliana was screened with a concatemer of AT-1P and yielded a partial clone. A cDNA containing the entire coding sequence was obtained by 5$\sp\prime$ RACE. Conceptual translation of this cDNA reveals a unique protein containing two putative $\rm C\sb2/C\sb2$ zinc fingers near the N-terminus followed by four putative CCHC zinc fingers.