Functional and regulatory analysis of the Arabidopsis thaliana CAX2 cation transporter

Abstract

The vacuolar sequestration of metals is an important metal tolerance mechanism in plants. The Arabidopsis thaliana vacuolar transporters CAX1 and CAX2 were originally identified in a Saccharomyces cerevisiae suppression screen as Ca2+/H+ antiporters. CAX2 has a low affinity for Ca2+ but can transport other metals including Mn2+ and Cd2+. Here we demonstrate that unlike cax1 mutants, CAX2 insertional mutants caused no discernable morphological phenotypes or alterations in Ca2+/H+ antiport activity. However, cax2 lines exhibited a reduction in vacuolar Mn2+/H+ antiport and, like cax1 mutants, reduced V-type H+ -ATPase (V-ATPase) activity. Analysis of a CAX2 promoter beta-glucoronidase (GUS) reporter gene fusion confirmed that CAX2 was expressed throughout the plant and strongly expressed in flower tissue, vascular tissue and in the apical meristem of young plants. Heterologous expression in yeast identified an N-terminal regulatory region in CAX2, suggesting that Arabidopsis contains multiple cation/H+ antiporters with shared regulatory features. Furthermore, despite significant variations in morphological and biochemical phenotypes, cax1 and cax2 lines both significantly alter V-ATPase activity, hinting at coordinate regulation among transporters driven by H+ gradients and the V-ATPase.

A secretory pathway-localized cation diffusion facilitator confers plant manganese tolerance

“…The latter include secondary, proton-coupled systems such as AtCAX2 and ShMTP1. AtCAX2 has been proposed to function in the vacuolar compartmentation in Arabidopsis of transition metals such as Mn and Cd (12,18,19), whereas ShMTP1 contributes to Mn tolerance in S. hamata (13).…”

“…In accord with a role of the vacuole in Mn sequestration, two Mn transporters have been localized to the vacuolar membrane: AtCAX2 and ShMTP1. In Arabidopsis, AtCAX2, a member of a Ca 2ϩ transporter family, is a H ϩ -driven cation antiporter that is weakly selective among Mn 2ϩ , Ca 2ϩ , and Cd 2ϩ (12,18,19). Although a modest increase in Mn tolerance was conferred by ectopic expression of AtCAX2 in tobacco, overexpressing lines (12) or knockout mutants (18) for this gene did not exhibit an altered Mn tolerance.…”

AtCCX3 Is an Arabidopsis Endomembrane H+-Dependent K+ Transporter

“…Yeast cells expressing all of the AtCCX3 and AtCCX4 variants were unable to suppress the Ca 2+ sensitivity of yeast strains deficient in vacuolar Ca 2+ transport (data not shown). Yeast cells expressing AtCAXs all show increased Ca 2+ transport when truncated in their N-terminal domain (Hirschi, 1999;Shigaki et al, 2001Shigaki et al, , 2003Pittman et al, 2004b). A lack of N-terminal regulation, the ability to suppress Na + -sensitive phenotypes, and the inability to suppress Ca 2+ -sensitive phenotypes in yeast suggests functions for AtCCX3 and AtCCX4 that differ from those of CAXs.…”

Export of Vacuolar Manganese by AtNRAMP3 and AtNRAMP4 Is Required for Optimal Photosynthesis and Growth under Manganese Deficiency

“…In Arabidopsis, several pathways for Mn import into the vacuole have been identified. AtCAX2 and AtCAX4 Ca 2+ /H + antiporters are able to transport Mn into the vacuole (Hirschi et al, 2000;Pittman et al, 2004;Koren'kov et al, 2006). In addition, AtVIT1 increases the Mn content of the vacuoles when expressed in yeast (Kim et al, 2006).…”