The effect of a genetically reduced plasma membrane protonmotive force on vegetative growth of Arabidopsis

Abstract

The plasma membrane proton gradient is an essential feature of plant cells. In Arabidopsis (Arabidopsis thaliana), this gradient is generated by the plasma membrane proton pump encoded by a family of 11 genes (abbreviated as AHA, for Arabidopsis H(+)-ATPase), of which AHA1 and AHA2 are the two most predominantly expressed in seedlings and adult plants. Although double knockdown mutant plants containing T-DNA insertions in both genes are embryonic lethal, under ideal laboratory growth conditions, single knockdown mutant plants with a 50% reduction in proton pump concentration complete their life cycle without any observable growth alteration. However, when grown under conditions that induce stress on the plasma membrane protonmotive force (PMF), such as high external potassium to reduce the electrical gradient or high external pH to reduce the proton chemical gradient, aha2 mutant plants show a growth retardation compared with wild-type plants. In this report, we describe the results of studies that examine in greater detail AHA2's specific role in maintaining the PMF during seedling growth. By comparing the wild type and aha2 mutants, we have measured the effects of a reduced PMF on root and hypocotyl growth, ATP-induced skewed root growth, and rapid cytoplasmic calcium spiking. In addition, genome-wide gene expression profiling revealed the up-regulation of potassium transporters in aha2 mutants, indicating, as predicted, a close link between the PMF and potassium uptake at the plasma membrane. Overall, this characterization of aha2 mutants provides an experimental and theoretical framework for investigating growth and signaling processes that are mediated by PMF-coupled energetics at the cell membrane.

Figure 1.

Role of the plasma membrane H⁺-ATPase in maintaining the PMF. A, Plasma membrane PMF composed of a membrane potential (Δψ) and a proton chemical concentration gradient (ΔpH). The proton gradient (outside high), generated by the H⁺-ATPase, contributes to both a charge gradient (positive outside) and a pH gradient (acidic outside). High concentrations of potassium entering the cytoplasm result in a reduction of the charge gradient and membrane depolarization. B, Relative expression of K⁺ transporter genes, AtHAK5 and AtCHX17, in wild-type or aha2 mutant seedlings grown in the presence or absence of 100 mm KCl. The endogenous reference genes, ACT2 and UBQ10, were used to normalize and calculate the relative expression of the K⁺ transporter genes. The left panels show the quantitative analysis of gene expression, and the right panel shows images of agarose gels presenting PCR products of AtHAK5, AtCHX17, ACT2, and UBQ10. −, Negative control; W, wild type; a2, aha2-4 mutant. C, Root growth of the wild type (WT) and aha2-4 at various pH values. The asterisk indicates a statistical significance between the wild type and the aha2 mutant (P = 0.0036). D, Hypersensitivity of aha2 mutant root growth to reduced PMF. High external pH (reduced ΔpH) and high external potassium (reduced membrane potential) have an additive effect on the impaired growth of aha2 mutants. Plants were grown under various pH values in the presence or absence of 100 mm KCl. Data are shown as means of 14 seedlings ± se.

Figure 2.

Root growth responses of wild-type (WT) and aha2 mutant plants to high concentrations of basic amino acids. A, Root growth of wild-type and aha2-4 mutant plants in the presence of basic amino acids. Amino acids are supplied to medium at 100 μm Lys or 10 mm Arg. B, Images of root growth on control medium or medium supplied with amino acids. C, Dose-response effect of Arg on the root growth of wild-type and aha2 mutant plants. D, Effects of altered PMF on the root growth of wild-type and aha2 mutant plants on Arg-supplemented medium. Seedlings were grown under various pH values in the presence or absence of 50 mm KCl: the control medium (one-half-strength MS, 1% Suc, pH 5.7), medium supplied with 5 mm Arg, medium adjusted to pH 3.7 and supplied with Arg, medium adjusted to pH 7.5 and supplied with Arg, or medium supplied with Arg and 50 mm KCl. E, Effects of altered PMF on the root growth of wild-type and aha2 mutant plants on hygromycin-supplemented medium. Plants were grown under various pH values in the presence or absence of 50 mm KCl: the control medium (one-half-strength MS, 1% Suc, pH 5.7), medium supplied with 5 μg mL⁻¹ hygromycin, medium adjusted to pH 3.7 and supplied with hygromycin, medium adjusted to pH 7.5 and supplied with hygromycin, or medium supplied with hygromycin and 50 mm KCl. [See online article for color version of this figure.]

Figure 3.

Growth responses of wild-type (WT) and aha2 mutant plants to lithium and cesium. A, Root growth response to LiCl. Seedlings were germinated for 3 d on control medium (one-half-strength MS, 1% Suc, pH 5.7) and further grown on control medium or medium supplemented with LiCl at the concentrations indicated on the x axis. The asterisk indicates statistical significance between the wild type and aha2 mutants (P < 0.0001). B, Images of seedling growth on the medium supplied with LiCl. Seedlings were germinated for 3 d on control medium (one-half-strength MS, 1% Suc, pH 5.7) and further grown on medium supplemented with 20 mm LiCl. C, Root growth responses to CsCl. The asterisk indicates statistical significance between the wild type and aha2 mutants (P < 0.0001). D, Images of seedling growth on medium supplied with CsCl. E, Effect of potassium on lithium-induced root growth inhibition. F, Effect of potassium on cesium-induced root growth inhibition.

Figure 4.

Hypocotyl growth responses of wild-type (WT) and aha2 mutant plants to reduced PMF. A, Total chlorophyll content in seedlings grown under cesium stress. Seedlings were grown as described in Figure 3. Total chlorophyll was extracted from seedlings and quantified. Data shown are from one representative experiment of three experiments. B, Hypocotyl elongation of seedlings grown under low blue light on control medium (top panels) or medium supplemented with 100 mm KCl (bottom panels). C, Hypocotyl length of wild-type and aha2 plants grown under low blue light on control medium or medium supplied with 100 mm NaCl, 100 mm KCl, or 150 mm sorbitol. D, Hypocotyl length of wild-type and aha2 mutant plants grown under low blue light. Plants were grown on control medium (pH 5.7), medium supplied with 100 mm KCl, or medium adjusted to pH 7.5 or 9.5. The asterisk indicates statistical significance between wild-type and aha2 mutant plants (P < 0.001). E, Hypocotyl length of wild-type and aha2 mutant plants grown in the dark. Plants were grown as described in D. The asterisk indicates statistical significance between wild-type and aha2 mutant plants (P < 0.001). [See online article for color version of this figure.]

Figure 5.

Root tropism of wild-type and aha2 mutant plants in the presence of ATP. A, Root tropism of wild-type and aha2 mutant roots in the presence of ATP or ADP. Seedlings were vertically germinated on control medium (one-half-strength MS, 1% Suc, pH 5.7) for 3 d and transferred to control medium, medium with 200 μg mL⁻¹ ATP, or medium with 200 μg mL⁻¹ ADP. After vertical incubation for an additional 4 d, images were captured and the degree of vertical root growth of wild-type and aha2-4 mutant plants was quantified as shown in B. B, Vertical growth index of wild-type (WT) and aha2-4 mutant plants in response to ATP or ADP. Vertical growth index, described by Vicente-Agullo et al. (2004), is defined as the ratio between a vertical projection of the base-to-tip chord and the root length (Supplemental Fig. S6). C, Effect of high external pH and potassium on ATP-induced root curving. Root tropism in response to ATP was examined with plants grown on medium (one-half-strength MS, 1% Suc) adjusted to pH 7.5 or supplied with 50 mm KCl. D, ATP-induced cytoplasmic calcium increase in wild-type and aha2-4 mutant seedlings. ATP at 1 mm concentration was added to elicit cytoplasmic calcium elevation. Data are shown as averages of 10 seedlings per genotype. [See online article for color version of this figure.]