V-ATPase dysfunction under excess zinc inhibits Arabidopsis cell expansion

Abstract

Although zinc is an essential micronutrient for all living organisms, zinc is harmful to cells at high levels. In the presence of excess zinc, plants exhibit several major symptoms, including root growth inhibition, abnormal root hair morphology and chlorosis. To dissect the molecular mechanisms underlying the effects of excess zinc on plant cells, we used an iTRAQ-based quantitative proteomics approach to analyze the microsomal protein profiles of Arabidopsis roots from wild-type (WT) plants and de-etiolated 3-1 (det3-1), a vacuolar H⁺-ATPase (V-ATPase) subunit C-defective mutant. A comparative analysis of the iTRAQ data from WT and det3-1 plants exposed to excess zinc suggests that the reduction in V-ATPase subunit levels and its activity are the cause of the symptoms of zinc toxicity, including the inhibition of cell expansion. Provided that reduced V-ATPase activity in the trans-Golgi network (TGN) alone can inhibit cell expansion, it is possible that the det3-1 mutant phenotype is caused mainly by a defect in TGN acidification, leading to reduced cell wall component trafficking and cell expansion in the presence of excess zinc. To evaluate the contribution of V-ATPase activity to vacuolar acidification under excess zinc, the vacuolar pH was measured. Our results indicate clear alkalinization of Deep cell vacuoles treated with 300 µM ZnSO₄.

The plant V-ATPase, together with H⁺-pyrophosphatase (V-PPase), plays a central role in vacuole acidification, energizing the active transport of ions across the tonoplast and coordinately regulating turgor pressure and cell elongation.^1,2 On the other hand, as a strategy to avoid the toxicity of several ions, plant cells take advantage of proton gradients generated across the tonoplast to sequester ions inside vacuoles.³ While V-PPase consists of a single polypeptide, V-ATPase is a multimeric complex composed of 28 subunits.⁴ Moreover, while V-PPase is thought to be exclusively localized in the tonoplast, V-ATPase is also found in the endosome.⁵ Finally, data from mutants of several V-ATPase subunits suggest that some of them may contribute differently to the acidification of vacuoles or endosomes.^6,7

In normal cell expansion, the V-ATPase plays an important role in the TGN, which functions in the trafficking of proteins involved in the biosynthesis of cell wall components, such as cellulose.⁸ The partial dysfunction of the V-ATPase in det3-1, which is defective in V-ATPase subunit C, causes defects in cell elongation in the hypocotyl, petioles and inflorescence stems, resulting in severe plant dwarfism (Fig. 1).⁶ In Arabidopsis thaliana, V-ATPase subunit C is encoded by a single-copy gene that is essential for plant survival, and which is fundamentally important for V-ATPase function at both the tonoplast and endosome.⁴ Curiously, the extent of cell expansion inhibition in several organs was proportional to the reduction in V-ATPase subunit C mRNA and protein expression, and to the level of V-ATPase activity in the det3-1 mutant (Fig. 1). For example, the first leaf area (40.1 cotyledon area and cotyledon cell size were all reduced by ∼50% in the det3-1 mutant compared with the WT (Fig. 1C).

Figure 1.

Gross morphology and cellular phenotype of the det3-1 mutant. (A) WT and det3-1 mutant plants grown for 46 d after sowing (DAS). Bar = 4 cm. (B, left parts) Mature first leaves from WT and det3-1 mutant plants (25 DAS). Bar = 5 mm. (B, right parts) Images of paradermal palisade tissue cells from the mature first leaves of WT and det3-1 mutant plants (25 DAS). Bar = 100 µm. (C) Cotyledon area, cell number and cell size in WT and det3-1 mutant plants (25 DAS). The data shown are the means and standard deviation (n = 8). Asterisk, p < 0.0001 (two-tailed Student's t-test).

Our recent iTRAQ analysis revealed that the levels of V-ATPase subunits A, C and E1 were significantly decreased in the presence of excess zinc in WT plants.⁹ Consistent with this, the protein expression of six V-ATPase subunits, including A, C and E1, was also significantly decreased in the det3-1 mutant.⁹ Alternatively, given that the morphologies of the Golgi stacks and Golgi-derived vesicles in vha-A and vha-E1 were abnormal, decreased levels of VHA-A and VHA-E1 expression under conditions of excess zinc might be explained by growth inhibition due to deficient acidification of the TGN.^10,11 In support of this hypothesis, Arabidopsis VHA-E1 and VHA-E3, but not VHA-E2, were found to functionally complement a vma4 zinc-sensitive mutant with a mutation in the single yeast subunit E isoform.¹² If we assume that reduced V-ATPase activity in the TGN alone can inhibit cell expansion, it is likely that the severe phenotype observed in the det3-1 mutant in the presence of excess zinc is the result of impaired TGN acidification leading to cell wall defects.

Is vacuolar V-ATPase activity also critical for zinc tolerance? To answer this question, we measured the vacuolar pH using suspension-cultured cells of Arabidopsis (strain Deep) and the ratiometric pH indicator 2′,7′-bis-(2-caroxyethyl)-5-(and-6)-carboxyfluorescein, diacetoxymethylester (BCECF-AM).¹³ Deep cells were treated with water as a control solution or MS medium containing 300 µM ZnSO₄ for 16 h and then analyzed by confocal laser-scanning microscopy. Interestingly, while the vacuolar pH was 5.25 in the untreated Deep cells, it was 5.66 in the zinc-treated cells (Fig. 2). This suggests that the pH was increased due to the inhibition of V-ATPase activity by excess zinc in the vacuole. Actually, the root cell vacuoles were alkalinized in the vha-a2 vha-a3 double mutant of Arabidopsis, which lacks both tonoplast-localized isoforms of the membrane-integral V-ATPase subunit VHA-a.⁷ Furthermore, based on our iTRAQ data, the tonoplast Zn²⁺/H⁺ antiporter MTP3, which appears to transport excess zinc from the cytosol to the vacuole, was increased 4.7-fold, probably to alleviate zinc toxicity, although the level of transport activity under conditions of excess zinc has not been reported in reference ⁹. It would be interesting to analyze the response to excess zinc in other mutants with defects in other V-ATPase subunits, such as those reported recently by Krebs et al. (2010), in a way that addresses their roles in the TGN and vacuole.⁷

Figure 2.

ZnSO₄ treatment causes alkalinization of the vacuolar pH in Deep cells. Confocal laser-scanning microscopic (CLSM, Leica TCS SP5) images of Deep cells stained with BCECF-AM. (A) Deep cell vacuoles loaded with BCECF-AM, (B) Nomarski images and (C) merged images. Bar = 10 µm. (D) Six-day-old cultured Deep cells were treated with water as a control or MS medium containing 300 µM ZnSO₄ for 16 h, after which the vacuolar pH was measured. ZnSO₄ treatment increased the vacuolar pH by 0.4 pH units. The standard deviation values provide an estimate of the variation in mean pH for 100 cells under each set of conditions.

In a recent report, we focused on a limited number of highly responsive proteins that are either directly related to zinc transport or which have a closely related function. However, the contribution of a substantial number of the zinc-responsive proteins identified from our iTRAQ data remains elusive. Given that under our experimental conditions seedlings were grown for 10 d in the presence of excess zinc, our data may include proteins that are not directly related to zinc tolerance and/or homeostasis. Moreover, we found that plants suffer an iron deficiency following long-term exposure to excess zinc.⁹ Therefore, it is important to explore zinc-responsive proteins following short-term treatment.

Very recently, Lan et al. (2011) performed an iTRAQ analysis to investigate the mechanisms of iron homeostasis under iron deficiency.¹⁴ They successfully identified 4,454 proteins and quantified 2,882. If this kind of pioneering technique is successfully applied to plant membrane proteomics, it may help dissect the intriguing response to excess zinc. The iTRAQ method is now being applied to plants to understand changes in protein profiles during normal growth/development or under a particular stress condition. Our next challenge is to identify a large enough number of proteins, including membrane proteins, to allow a more detailed comparison with the transcriptomic data available. This will boost our understanding of fundamentally important biological events and provide clues as to the integral components of the molecular network responsible for the phenotype of this interesting mutant.