Molecular characterization of plasma membrane H⁺-ATPase in a carrot mutant cell line with enhanced citrate excretion

The citrate efflux of a carrot mutant cell line (IPG; insoluble phosphate grower) with enhanced citrate excretion is concomitant with an electrically equivalent H⁺ release. Furthermore, plasma membrane H ⁺-ATPase activity (EC 3.6.1.35) in the IPG cell line is known to be greater than in the wild-type cell line. In the present study, molecular characterization of plasma membrane H⁺-ATPase in the IPG cell line was conducted to examine the involvement of this enzyme in enhanced citrate excretion. Four type-II (DcPA 1, 2, 4 and 5) and two type-I (DcPA 3 and 6) full-sized cDNAs encoding Daucus carota plasma membrane H⁺-ATPase were cloned. The transcript level of the most abundant isoform, DcPA 1, was three times higher in the IPG cell line than in the wild-type cells. The transcript levels of the other isoforms showed no differences between cell types. The plasma membrane H⁺-ATPase protein level in the IPG cell line was 1.6-fold that of the wild-type cell line when analysed by western blot analysis. These results indicate that the higher activity of plasma membrane H⁺-ATPase in the IPG cell line is due to up-regulation of DcPA 1. Antisense inhibition of the DcPA 1 gene also led to decreased citrate excretion from the transgenic IPG cell line, suggesting that the up-regulation of plasma membrane H⁺-ATPase is involved in enhanced citrate excretion.