Cloning and Characterization of MxVHA-c, a Vacuolar H+-ATPase Subunit C Gene Related to Fe Efficiency from Malus xiaojinensis

The vacuolar H+-ATPase plays a crucial role in secondary transport and in plant response to environmental stress. In this study, a vacuolar H+-ATPase (MxVHA-c) gene, consisting of an ORF of 498 base pairs and 165 amino acid residues, has been cloned from the iron-efficient genotype of Malus xiaojinensis. Subsequently, this gene has been targeted to the tonoplast using transient expression analysis. Quantitative real-time (qRT) PCR results reveal that the MxVHA-c gene is expressed in both roots and leaves of Fe-deficient plants; however, it is sensitive to iron stress in roots. This suggests that MxVHA-c expression in roots may mediate iron-dependent responses. MxVHA-c expression is up-regulated following exogenous treatment with abscisic acid (ABA) and down-regulated following treatment with CaCl2. Overexpression of the MxVHA-c gene in yeast strains has revealed that MxVHA-c transiently alleviated cadmium toxicity via the Cd2+/H+ antiport protein. H+-ATPase activity is slightly increased in yeast overexpressing the MxVHA-c gene compared to that in yeast transformed with an empty vector. In addition, this transgenic yeast strain can grow in a liquid medium containing 40 mu M ferrozine. These findings may provide useful information in elucidating molecular mechanisms that mediate resistance to iron deficiency.