An Arabidopsis thaliana plasma membrane proton pump is essential for pollen development

The plasma membrane proton pump (H+-ATPase) found in plants and fungi is a P-type ATPase with a polypeptide sequence, structure, and in vivo function similar to the mammalian sodium pump (Na+, K+-ATPase). Despite its hypothetical importance for generating and maintaining the proton motive force that energizes the carriers and channels that underlie plant nutrition, genetic evidence for such a central function has not yet been reported. Using a reverse genetic approach for investigating each of the 11 isoforms in the Arabidopsis H+-ATPase (AHA) gene family, we found that one member, AHA3, is essential for pollen formation. A causative role for AHA3 in male gametogenesis was proven by complementation with a normal transgenic gene and rescue of the mutant phenotype back to wild type. We also investigated the requirement for phosphorylation of the penultimate threonine, which is found in most members of the ARA, family and is thought to be involved in regulating catalytic activity. We demonstrated that a T948D mutant form of the AHA3 gene rescues the mutant phenotype in knockout AHA3 plants, but T948A does not, providing the first in planta evidence in support of the model in which phosphorylation of this amino acid is essential.