The plasma membrane H+-ATPase gene family in Arabidopsis: genomic sequence of AHA10 which is expressed primarily in developing seeds

Abstract

The plasma membrane H+-ATPases in Arabidopsis thaliana represent the largest family of cation translocating P-type ATPases identified in plants or animals. We report here seven new isoforms, which were identified by polymerase chain reaction (PCR) amplification of genomic DNA. Amplifications were performed with degenerate primers corresponding to two short conserved sequence motifs (“CSDK” and “GDGV”) found in most P-type ATPases. A comparison was made of three CSDK-side primers, which were used either as totally degenerate mixtures or rendered less degenerate by substitution with deoxyinosine or fluorodeoxyuridine. Amplified genomic fragments were cloned, partially sequenced and shown to correspond to Arabidopsis genes by Southern blot analysis with gene-specific probes. One newly identified isoform, AHA10, was isolated as a cosmid clone and sequenced. The 5′ and 3′ ends of the gene were determined by comparison with the AHA10 cDNA sequence. AHA10 is the most divergent isoform characterized in the Arabidopsis family. AHA10 appears to be expressed primarily in developing seeds, as indicated by Northern blot analysis of AHA10 mRNA and by the analysis of transgenic plants expressing a β-glucuronidase (GUS) reporter gene fused to an AHA10 promoter. Our results indicate that one function of this unusually large H⁺-ATPase gene family is to allow for expression of different isoforms in different cell types.