Functional association of Arabidopsis CAX1 and CAX3 is required for normal growth and ion homeostasis

Abstract

Cation levels within the cytosol are coordinated by a network of transporters. Here, we examine the functional roles of calcium exchanger 1 (CAX1), a vacuolar H+/Ca2+ transporter, and the closely related transporter CAX3. We demonstrate that like CAX1, CAX3 is also localized to the tonoplast. We show that CAX1 is predominately expressed in leaves, while CAX3 is highly expressed in roots. Previously, using a yeast assay, we demonstrated that an N-terminal truncation of CAX1 functions as an H+/Ca2+ transporter. Here, we use the same yeast assay to show that full-length CAX1 and full-length CAX3 can partially, but not fully, suppress the Ca2+ hypersensitive yeast phenotype and coexpression of full-length CAX1 and CAX3 conferred phenotypes not produced when either transporter was expressed individually. In planta, CAX3 null alleles were modestly sensitive to exogenous Ca2+ and also displayed a 22% reduction in vacuolar H+-ATPase activity. cax1/cax3 double mutants displayed a severe reduction in growth, including leaf tip and flower necrosis and pronounced sensitivity to exogenous Ca2+ and other ions. These growth defects were partially suppressed by addition of exogenous Mg2+. The double mutant displayed a 42% decrease in vacuolar H+/Ca2+ transport, and a 47% decrease in H+-ATPase activity. While the ionome of cax1 and cax3 lines were modestly perturbed, the cax1/cax3 lines displayed increased PO4(3-), Mn2+, and Zn2+ and decreased Ca2+ and Mg2+ in shoot tissue. These findings suggest synergistic function of CAX1 and CAX3 in plant growth and nutrient acquisition.

Figure 1.

Subcellular localization of CAX3. Immunoblot analysis of CAX3 in transgenic tobacco expressing HA-CAX3. Equal amounts of protein (10 μg) isolated from HA-CAX3-expressing tobacco leaf tissues were separated by SDS-PAGE, blotted, and subjected to western-blot analysis using antibodies against HA (HA-CAX3) and plant membrane markers: the plant ER luminal protein (BiP; StressGen Biotechnologies, Victoria, British Columbia), oat vacuolar ATPase (V-ATPase; Ward et al., 1992), and a PM H⁺-ATPase (PMA1; Villalba et al., 1992). HA-CAX3-expressing tobacco leaf tissue membranes were prepared and fractionated in the absence (A) or presence (B) of 1.5 mm MgCl₂, as indicated. Numbers above the blots indicate the Suc concentration of each fraction.

Figure 2.

CAX1 and CAX3 promoter::GUS expression in transgenic Arabidopsis plants. A, GUS staining in a CAX1::GUS 10-d-old seedling. Note the preferential GUS expression in the leaf tissues. B, No visible GUS staining in the leaf tissues of the CAX3::GUS seedling. C, Strong CAX1::GUS expression in young cauline leaves and lateral buds. D, Weak CAX3::GUS expression in a cauline leaf and young lateral buds. E to G, No visible GUS staining in primary and lateral roots of CAX1::GUS plants. Note the weak signals seen in vascular tissues. H to J, Strong GUS staining in primary and lateral root tips and the root elongation region of CAX3::GUS plants. K and L, CAX1::GUS expression in flower tissues and young developing embryos. M and N, CAX3::GUS expression in flower tissues and young developing embryos.

Figure 3.

Coexpression of CAX1 with CAX3 in the yeast mutant K667 confers tolerance to high Ca²⁺. Suppression of Ca²⁺ sensitivity of the pmc1 cnb vcx1 yeast mutant (K667) by various CAX constructs and their combinations. Saturated liquid cultures of K667 containing various plasmids were diluted to the cell densities, as indicated, and then spotted onto selection medium (−His −Ura) or yeast extract peptone dextrose (YPD) medium and the YPD medium containing 50 mm CaCl₂ (A) or 150 mm CaCl₂ (B). For the experiment shown in B, CAX1 in a His-selectable plasmid was coexpressed with an empty Ura-selectable plasmid (line 2), CAX3 in a Ura-selectable plasmid was coexpressed with an empty His-selectable plasmid (line 3), and CAX1 and CAX3 were coexpressed in His- and Ura-selectable plasmids, respectively (line 4). For the empty-vector-alone control (line 1), both empty His- and Ura-selectable plasmids were coexpressed.

Figure 4.

Analysis of cax3 alleles. A, Diagram of the CAX3 gene depicting the sites of T-DNA insertions. The approximately 4.0 kb of genomic CAX3 DNA is represented by nine introns (lines) and 10 exons (boxes). Triangles indicate the sites of the T-DNA locations. The lines harboring these insertions are termed cax3-1 and cax3-2. B, RNA gel-blot analysis of CAX3 expression in wild-type and cax3 alleles. Total RNA was extracted from 3-week-old wild-type and cax3 seedlings pretreated with 50 mm CaCl₂ overnight. Ten micrograms of total RNA from each sample was loaded, blotted, and hybridized with a ³²P-labeled cax3-specific probe. Ethidium bromide (EtBr) staining of the agarose gel is shown as a loading control. C, Reduction of growth mass in cax3 alleles. Five-day-old seedlings of wild-type (Col-0) and cax3 alleles were transferred and grown on one-half-strength MS medium in the presence or absence of 25 mm CaCl₂ for 10 d. Fresh growth mass of individual seedling for each line was measured. All results are means ± se (n ≥ 50). Data are representative of three independent experiments.

Figure 5.

Vacuolar H⁺/Ca²⁺ antiport and H⁺-ATPase activity in cax1 and cax3 mutants. A, Time courses of Mg²⁺-ATP-energized ΔpH-dependent 10 μm ⁴⁵Ca²⁺ uptake into vacuolar membrane vesicles isolated from wild-type, cax1, and cax3 Arabidopsis plants and determined in the presence of 0.5 mm orthovanadate (a Ca²⁺-ATPase inhibitor). Eighteen hours prior to harvest and membrane isolation, plants were pretreated with 100 mm CaCl₂. H⁺/Ca²⁺ antiport activity was determined as the difference between Ca²⁺ uptake in the absence and presence of 5 μm carbonyl cyanide p-(trifluoromethoxy) phenylhydrazone (FCCP; protonophore) and net H⁺/Ca²⁺ antiport activity is shown after the subtraction of the FCCP background values. The Ca²⁺ ionophore A23187 (5 μm) was added at the 12-min time point and significantly dissipated Ca²⁺ accumulation mediated by H⁺/Ca²⁺ antiport when measured at the 22-min time point. B, V-ATPase activity in vacuolar membrane vesicles isolated from wild-type, cax1, and cax3 plants. Activity was determined in the absence or presence of the V-ATPase inhibitor bafilomycin and net activity following subtraction of the bafilomycin background values are shown. Percentages (%) indicate percent of wild-type activity.

Figure 6.

Morphological phenotypes and ion sensitivity of the cax1/cax3 double-mutant lines. A, Four-week-old wild type (Col-0), cax1, cax3, and cax1/cax3 plants were grown on soil under continuous light. The cax1/cax3 plants grew smaller and showed leaf tip necrosis. B, The mature cax1/cax3 plants have more branches and appear bushy in comparison with wild-type plants. The cax1/cax3 double-mutant lines rarely produce normal siliques and seeds. C, The cax1/cax3 plants have curly dark green leaves showing tip necrosis. D, The cax/cax3 plant displayed shoot apex necrosis. E, The cax1/cax3 mature plants have smaller siliques with few seeds. F to H, Five-day-old seedlings of wild type (Col-0), cax1, cax3, and cax1/cax3 grown on one-half-strength MS medium were transferred onto one-half-strength MS medium (F), one-half-strength MS medium supplemented with 25 mm CaCl₂ (G), and one-half-strength MS medium supplemented with 15 mm MgCl₂ and grown for an additional 10 d (H).

Figure 7.

Vacuolar H⁺/Ca²⁺ antiport and H⁺-ATPase activity in cax1/cax3 mutants. A, Time courses of Mg²⁺-ATP-energized ΔpH-dependent 10 μm ⁴⁵Ca²⁺ uptake into vacuolar membrane vesicles isolated from wild-type and cax1/cax3 Arabidopsis plants pretreated with 100 mm CaCl₂. H⁺/Ca²⁺ antiport activity was determined as described in the legend to Figure 5. B, V-ATPase activity in vacuolar membrane vesicles isolated from wild-type, cax1, cax3, and cax1/cax3 plants. Values for cax1 and cax3 activity are from the same experiment as shown in Figure 5B. Activity was determined in the absence or presence of the V-ATPase inhibitor bafilomycin, and net activity following subtraction of the bafilomycin background values is shown.

Figure 8.

Alterations in the ionome in cax1/cax3, cax1, and cax3 mutants. Shoot samples from cax1/cax3, cax1, cax3, and wild-type plants were analyzed for Li⁺, B⁺, Na⁺, Mg²⁺, PO₄³⁻, K⁺, Ca²⁺, Mn²⁺, Fe³⁺, Co²⁺, Ni²⁺, Cu²⁺, Zn²⁺, As²⁺, Se²⁺, Mo²⁺, Cd²⁺, and Pb²⁺ using ICP-MS. Ion profile data of mutants and wild type grown under the same condition were compared, and elements showing a significant difference (P ≥ 0.05; n = 13–18) are shown as black bars as percentage change from the wild type.