Knockout of multiple Arabidopsis cation/H(+) exchangers suggests isoform-specific roles in metal stress response, germination and seed mineral nutrition

Abstract

Cation/H(+) exchangers encoded by CAX genes play an important role in the vacuolar accumulation of metals including Ca(2+) and Mn(2+). Arabidopsis thaliana CAX1 and CAX3 have been previously shown to differ phylogenetically from CAX2 but the physiological roles of these different transporters are still unclear. To examine the functions and the potential of redundancy between these three cation transporters, cax1/cax2 and cax2/cax3 double knockout mutants were generated and compared with wild type and cax single knockouts. These double mutants had equivalent metal stress responses to single cax mutants. Both cax1 and cax1/cax2 had increased tolerance to Mg stress, while cax2 and cax2/cax3 both had increased sensitivity to Mn stress. The cax1/cax2 and cax2/cax3 mutants did not exhibit the deleterious developmental phenotypes previously seen with the cax1/cax3 mutant. However, these new double mutants did show alterations in seed germination, specifically a delay in germination time. These alterations correlated with changes in nutrient content within the seeds of the mutants, particularly the cax1/cax2 mutant which had significantly higher seed content of Ca and Mn. This study indicates that the presence of these Arabidopsis CAX transporters is important for normal germination and infers a role for CAX proteins in metal homeostasis within the seed.

Figure 1. CAX1, CAX2 and CAX3 expression.

Changes in CAX expression in cax1, cax2 and cax3 single and double knockout lines relative to Col-0 (wild type) were determined by real-time PCR using actin and ubiquitin as constitutive control primers. RNA was extracted from whole seedling tissue grown on 0.5×MS plates without additional metal supplementation for 21 d. Expression relative to actin is shown and expression relative to ubiquitin was equivalent. Relative fold changes in gene expression were calculated using the 2^−ΔΔCt method. Bars indicate the mean log expression±SE from three samples each replicated twice. ‘n’ denotes no increase in expression detected.

Figure 2. Mg and Mn sensitivity of CAX mutant plants.

Fresh weight of Col-0 (wild type) and cax knockout mutant plants following germination and growth on solid 0.5×MS media (adjusted to pH 5.6) in the absence (A) or presence of 25 mM MgCl₂ (B) or 1.5 mM MnCl₂ (C). Bars indicate the mean±SE (n = 12–15) fresh weight measured in 21 d-old plants. ** (P<0.01) and * (P<0.05) denotes significant difference between CAX mutant lines and Col-0 control as determined by one-way ANOVA.

Figure 3. Na sensitivity of CAX mutant plants.

Total chlorophyll content was measured in leaf and shoot tissue of 21 d-old Col-0 (wild type) and cax knockout mutant plants following germination and growth on solid 0.5×MS media (adjusted to pH 5.6) in the absence or presence of 50 mM NaCl. The mean±SE (n = 4–6) chlorophyll content is shown. ** (P<0.01) denotes significant difference between control and NaCl treatments as determined by one-way ANOVA.

Figure 4

Germination profile of CAX mutant seedlings over three time periods. Approximately 100 seeds from Col-0 (wild type) and cax knockout mutant plants were sterilized and sown on 1% agar plates. After a 2-d incubation period at 4°C plates were moved to an environmentally-controlled growth chamber at 22°C. Radicle emergence from the testa was taken as the indicator of germination. Stacked bars indicate the percentage of seeds germinating 0–9 hours, 9–21 hours and 21–37 hours after transfer to 22°C. All values are corrected for seed non-viability, determined by quantifying the absence of seed germination after 10 d.

Figure 5

Germination of CAX mutant seedlings in response to abscisic acid (ABA). The germination of seeds from Col-0 (wild type) and cax knockout mutant plants was quantified after 24 h on 0.1 µM ABA. Approximately 100 seeds of each line were sterilized and sown on 1% agar plates containing ABA. After a 2-d incubation period at 4°C plates were moved to an environmentally-controlled growth chamber at 22°C. Radicle emergence from the testa was taken as the indicator of germination. Bars indicate the mean±SE percentage of germination. ** (P<0.01) denotes significant difference between CAX mutant lines and Col-0 control as determined by one-way ANOVA.

Figure 6. Difference in elemental concentration in dry seeds from CAX mutant plants.

The concentrations of known or putative substrates of CAX transporters: Ca, Mn, Na and Zn (A) and of metals not thought to be transported by CAX: Fe, K, Mg and P (B) are shown as % difference from Col-0 (wild type) seeds. Dry seeds (approximately 15 mg per sample) were obtained from plants grown on soil without additional metal supplementation. Elemental analysis was performed by ICP-AES measurement. Bars indicate the mean±SE of three replicates. * (P<0.05) denotes significant difference from Col-0 as determined by one-way ANOVA.

Figure 7. Vacuolar H⁺-ATPase, Ca²⁺/H⁺ and Mn²⁺/H⁺ exchanger activity in CAX mutant plants.

(A) Initial rates of V-ATPase H⁺ transport activity in purified vacuolar-enriched membrane vesicles from Col-0 (wild type) and cax knockout mutant plants determined from the rates of acridine orange fluorescence quenching during the first 60 sec following the addition of Mg-ATP, as shown in Figure S6. (B) and (C) Initial rates of ΔpH-dependent cation/H⁺ exchange activity in purified vacuolar-enriched membrane vesicles from Col-0 and cax knockout mutant plants determined from the rates of acridine orange fluorescence recovery during the first 60 sec following the establishment of a steady-state pH gradient and the addition of 200 µM CaCl₂ (A) or MnCl₂ (B), as shown in Figure S6. F indicates relative fluorescence intensity. Membrane vesicles were prepared from 2-week-old plants grown on solid 0.5×MS media and pre-treated with 50 mM CaCl₂ and 1.5 mM MnCl₂ 14 h before harvest. The data represent means±SE from three experiments. ** (P<0.01) and * (P<0.05) denotes significant difference between CAX mutant lines and Col-0 control as determined by one-way ANOVA.