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. 2010 Feb 16;107(7):3251-6.
doi: 10.1073/pnas.0913035107. Epub 2010 Jan 26.

Arabidopsis V-ATPase activity at the tonoplast is required for efficient nutrient storage but not for sodium accumulation

Melanie Krebs  1 , Diana Beyhl, Esther Görlich, Khaled A S Al-Rasheid, Irene Marten, York-Dieter Stierhof, Rainer Hedrich, Karin Schumacher
Affiliations

Arabidopsis V-ATPase activity at the tonoplast is required for efficient nutrient storage but not for sodium accumulation

Melanie Krebs et al. Proc Natl Acad Sci U S A. .

Abstract

The productivity of higher plants as a major source of food and energy is linked to their ability to buffer changes in the concentrations of essential and toxic ions. Transport across the tonoplast is energized by two proton pumps, the vacuolar H(+)-ATPase (V-ATPase) and the vacuolar H(+)-pyrophosphatase (V-PPase); however, their functional relation and relative contributions to ion storage and detoxification are unclear. We have identified an Arabidopsis mutant in which energization of vacuolar transport solely relies on the activity of the V-PPase. The vha-a2 vha-a3 double mutant, which lacks the two tonoplast-localized isoforms of the membrane-integral V-ATPase subunit VHA-a, is viable but shows day-length-dependent growth retardation. Nitrate content is reduced whereas nitrate assimilation is increased in the vha-a2 vha-a3 mutant, indicating that vacuolar nitrate storage represents a major growth-limiting factor. Zinc is an essential micronutrient that is toxic at excess concentrations and is detoxified via a vacuolar Zn(2+)/H(+)-antiport system. Accordingly, the double mutant shows reduced zinc tolerance. In the same way the vacuolar Na(+)/H(+)-antiport system is assumed to be an important component of the system that removes sodium from the cytosol. Unexpectedly, salt tolerance and accumulation are not affected in the vha-a2 vha-a3 double mutant. In contrast, reduction of V-ATPase activity in the trans-Golgi network/early endosome (TGN/EE) leads to increased salt sensitivity. Taken together, our results show that during gametophyte and embryo development V-PPase activity at the tonoplast is sufficient whereas tonoplast V-ATPase activity is limiting for nutrient storage but not for sodium tolerance during vegetative and reproductive growth.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
Loss of tonoplast V-ATPase causes alkalinization of vacuolar pH. (A) ATP-induced membrane polarization of wild-type (n = 5) and vha-a2 vha-a3 (n = 3) vacuoles. Error bars show SD. (B) PPi-induced H+ current densities from isolated vacuoles show similar values in wild type (n = 3) and vha-a2-vha-a3 (n = 5). Error bars represent SD. (C) Concanamycin A-inhibited V-ATPase activity and K+-stimulated PPase activity in microsomal membranes of 4-day-old etiolated seedlings. Wild-type activity was set to 100%. Error bars show SE of three independent experiments. (D) The images show emission intensities of epidermal root cell vacuoles loaded with BCECF AM. The ratio image of vha-a2 vha-a3 indicates an increased vacuolar pH. (Scale bar, 50 μm.) (E) Loss of the tonoplast V-ATPase increases the vacuolar pH in root epidermal cells by 0.5 pH units. Error bars represent SE of 40 measurements from 20 seedlings.
Fig. 2.
Fig. 2.
Limited storage capacity for nitrate causes day-length-dependent growth reduction. (A) The vha-a2 vha-a3 phenotype is severe if plants are grown under short-day conditions (8 h). Upon extended illumination periods (16 and 24 h) the growth phenotype is attenuated. Pictures show 7-week-old (8 h light) and 3-week-old (16 and 24 h light) plants. (B) Impaired vacuolar nitrate storage causes a reduction of the nitrate content in vha-a2 vha-a3. (C) Maximum nitrate reductase activity and (D) increased glutamine content in vha-a2 vha-a3 are shown. (B–D) HPLC analysis and NR assay were performed on rosette leaves of 3-week-old plants grown in soil under 16-h light/8-h dark cycles. The plant material was collected 5 h after illumination started. Error bars represent SD of four biological replicates.
Fig. 3.
Fig. 3.
Deficiency and toxicity symptoms in vha-a2 vha-a3. (A and B) vha-a2 vha-a3 mutants display leaf tip necrosis and reduced calcium content similar to cax1 cax3. (B) HPLC analysis was performed on rosette leaves of 3-week-old plants grown in soil under a 16-h light/8-h dark cycle. Error bars represent SD of fouor biological replicates. (C) vha-a2 vha-a3 mutants are hypersensitive against zinc. Seedlings were grown for 13 days under a 16-h light/8-h dark cycle on vertical plates containing different concentrations of ZnCl2. (D) Root length. Error bar indicates SD of four replicate experiments. The control condition is set to 100%.
Fig. 4.
Fig. 4.
The endosomal, but not the tonoplast V-ATPase is limiting under salt stress. (A) Inhibition of root growth through NaCl. The control condition was set to 100%. Error bars show SE with n = 16. Data are representative for one of three independent experiments. (B–D and F) HPLC analysis of sodium, chloride, potassium, and nitrate contents in 13-day-old seedlings grown on plates. Error bars represent SD of four biological replicates. (E) K+-stimulated PPase activity was determined from microsomal membranes of 4-day-old etiolated Arabidopsis seedlings. Control activity was set to 100%. Error bars represent SE of three independent experiments. (G) A RNAi against VHA-a1 was induced with 0.2% EtOH. Seedlings were grown for 15 days on vertical agar plates containing different concentrations of NaCl. The root length of the seedlings was measured (Fig. S7). (H) Inhibition of growth through salt upon EtOH induction. Differences between control samples (− EtOH) and treatment samples (+ EtOH) are expressed in percent.
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