High V‐PPase activity is beneficial under high salt loads, but detrimental without salinity

Summary The membrane‐bound proton‐pumping pyrophosphatase (V‐PPase), together with the V‐type H+‐ATPase, generates the proton motive force that drives vacuolar membrane solute transport. Transgenic plants constitutively overexpressing V‐PPases were shown to have improved salinity tolerance, but the relative impact of increasing PPi hydrolysis and proton‐pumping functions has yet to be dissected. For a better understanding of the molecular processes underlying V‐PPase‐dependent salt tolerance, we transiently overexpressed the pyrophosphate‐driven proton pump (NbVHP) in Nicotiana benthamiana leaves and studied its functional properties in relation to salt treatment by primarily using patch‐clamp, impalement electrodes and pH imaging. NbVHP overexpression led to higher vacuolar proton currents and vacuolar acidification. After 3 d in salt‐untreated conditions, V‐PPase‐overexpressing leaves showed a drop in photosynthetic capacity, plasma membrane depolarization and eventual leaf necrosis. Salt, however, rescued NbVHP‐hyperactive cells from cell death. Furthermore, a salt‐induced rise in V‐PPase but not of V‐ATPase pump currents was detected in nontransformed plants. The results indicate that under normal growth conditions, plants need to regulate the V‐PPase pump activity to avoid hyperactivity and its negative feedback on cell viability. Nonetheless, V‐PPase proton pump function becomes increasingly important under salt stress for generating the pH gradient necessary for vacuolar proton‐coupled Na+ sequestration.


Fig. S1
Vectors generated for transient overexpression of pyrophosphatases together with free GFP in N. benthamiana using the agroinfiltration method.         Images from leaves overexpressing free GFP alone (control) or together with AtVHP1 three days after agroinfiltration. (f) Maximal pyrophosphate-induced changes in current density of vacuoles released from AtVHP1/GFP-overexpressing mesophyll cells, plotted against the pyrophosphate concentration. Current responses were normalized to those recorded from the same vacuole during application of 150 µM pyrophosphate. Experiments were performed at luminal pH 7.5 (triangle, n = 3-17) or pH 5.5 (circles, n = 3 -9). Data points (means ± SE) were globally fitted with a

Michaelis-Menten equation (solid line).
Asterisks in (c, d) indicate significant differences (*P < 0.05 and ***P < 0.001, Student's t-test) between the given values.   The hydrolytic pyrophosphate activity of the soluble fraction of N. benthamiana leaves overexpressing free GFP alone (control) or together with IPP1 or NbVHP1. Data points (means ± SE, **P < 0.01, Student's t-test) represent three independent experiments. leaves were directly injected with NaCl (triangle markers) or NaCl was applied in solution to the soil ('poured', circle and square markers). The NaCl content of the 3 rd and 4 th leaves from the base of the plant were measured separately. Note, that when pots with soil-grown plants were soaked with either water or NaCl, the Na + content in the third and fourth leaf increased over 48 h to the same Na + level as observed in Na + infiltrated leaves, and likewise, then remained stable.  Maximal pyrophosphate-induced current responses of vacuoles released from mesophyll cells overexpressing free GFP alone (control, n = 10) or free GFP together with either AtVHP1 (n = 22), NbVHP1 (n = 14) or NbVHP2 (n = 4) after agroinfiltration. Pyrophosphate was applied at a concentration of 150 µM. Data points represent means ± SE (***P < 0.001, Student's t-test). Method S1 Generation of mGFP-NbVHP1 construct According to Segami et al. (2014), NbVHP1 was labelled with mGFP (monomeric Green Fluorescence Protein) which was inserted into loop 1 exactly between the amino acids Gly53 and Ala54 (Fig. S1b) via USER cloning (Nour-Eldin et al., 2006). Additionally, the following linker For staining the plasma membrane, isolated protoplasts were briefly incubated with FM TM 4-64 (10 µM) (Thermo Fisher Scientific). The FM4-64 dye was excited with 514 nm and the emission detected at 625-690 nm.

Method S2 Protein extraction and enzyme activity measurements
Two days after agroinfiltration, tobacco leaves were harvested, immediately frozen in liquid nitrogen and stored at -80 °C until microsomal membranes and soluble protein fractions were prepared as described previously (Krebs et al., 2010), with minor modifications. Tissue was homogenized with 2 ml homogenization buffer per g fresh weight (350 mM sucrose, 70 mM Tris-HCl pH 8.0, 10 % (v/v) glycerol, 3 mM Na2EDTA, 0.15 % (w/v) BSA, 1.5 % (w/v) PVP-40, 4 mM DTT, and 1 x complete protease inhibitor mixture (Roche)). The homogenate was filtered through two layers of Miracloth (Calbiochem) and centrifuged at 15,000 g for 15 min at 4 °C. The supernatant was filtered again through Miracloth, then centrifuged at 100,000 g for 1 h at 4 °C. To determine enzyme activity of the soluble protein fraction, 200 µl of the supernatant was retained and the remaining supernatant was carefully removed and discarded. The pellet (microsomal membranes) was re-suspended in 350 mM sucrose, 10 mM Tris-Mes pH 7.0, 2 mM DTT and 1 × complete protease inhibitor mixture.
PPase activity was calculated as the difference measured in the presence and absence of 50 mM KCl.

Method S3 Quantification of leaf sodium content
N. benthamiana leaves infiltrated only with 200 mM NaCl were harvested at different times after salt treatment, dried at 60 °C to constant weight, pulverized and homogenized. After the exact amount of the samples (usually 10 -20 mg) were precisely weighed in a quartz digestion vessel, the leaf tissue was treated with 1 ml of nitric acid (65%, suprapur, Merck KGaA, Darmstadt) and digested at 180 °C for 10 hours inside a Teflon pressure vessel. After cooling, samples were diluted 1:20 with nanopure water. The sodium content was determined using a flame atomic absorption spectrometer (AAnalyst 400, PerkinElmer) three times and averaged.
For tobacco leaves infiltrated with a NaCl-free or 200 mM NaCl-containing agrobacterium suspension, leaves were collected two days after infiltration, immediately frozen in liquid nitrogen and stored at -80 °C until use. After re-suspension in double-distilled water the plant leaf material was subjected to complete hydrolysis using a temperature step gradient with a maximum temperature of 210 °C. Hydrolysis was carried in a MLS-Ethos microwave oven (http://www.mlsmikrowellen.de/) with 5 ml HNO3 (60% v/v), 2 ml H2O2 (30% v/v), and the preparation was diluted to a final volume of 12 ml with double-distilled water. Quantification was performed by inductively coupled plasma/optical emission spectrometry, on an iCAP 6300 DUO apparatus (Thermo-Fischer). Sodium was detected and quantified at 589.5 nm (Krebs et al., 2010;Müller et al., 2014).

Method S4 Apoplast washing
Agromix solution with or without 200 mM NaCl was infiltrated into the apoplast of wild type N. Plant material was immediately frozen in liquid nitrogen, crushed with a pestle and mortar and stored at -80°C until use. Total RNA of the leaf was isolated using Plant RNA Kit R6827-02 (OMEGA, www.omegabiotek.com). Each of the collected RNAs was treated with recombinant DNase (Thermo Scientific) to remove any genomic DNA contamination. Generation of cDNA and Quantitative real-time PCR (pPCR) were done like described in the main text. Transcription data were normalized to the coexpressed GFP molecules using standard curves calculated for the individual PCR products. GFPfwd (5´-CCT GAA GTT CAT CTG CAC CA-3´) and GFPrev (5´-TGC TCA GGT AGT GGT TGT CG-3´) (Gaddam et al., 2013).

Method S6 pH calibration with BCECF
A BCECF calibration curve was generated ex vivo with exactly the same imaging parameters and setup used for the in vivo measurements described in the main text by dissolving the free acid form of the dye (2.5 µM) in a medium containing 1 mM CaCl2, 1 mM MES adjusted to the designated pH with TRIS (Fig. S8). The generated calibration curve (n = 3) was used to convert the ratio values into pH-values.