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

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 (VATPase) 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 vhaa2 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.proton-pump | pH-homeostasis | vacuole | nitrate | salt tolerance T he productivity of higher plants as a major source of food and renewable energy is linked to their ability to cope with fluctuations in essential as well as toxic ion and metabolite concentrations. In plants the large central vacuoles function as reservoirs for ions and metabolites that allow buffering of changes in nutrients as well as challenges by toxic components that plants, as sessile, photoautotrophic organisms, frequently encounter. Furthermore, vacuoles are essential for plant growth and development. Expansion of plant cells is achieved by osmotically driven water influx into the vacuole that, in combination with the cell wall, generates turgor, the driving force for hydraulic stiffness and plant growth.All vacuolar functions require massive fluxes of ions and metabolites that are channeled by a battery of vacuolar transport proteins, many of which have been well characterized physiologically, but the nature of several transporters remains to be identified (1). Among those characterized on the molecular level are, e.g., those transporting NO 3 − , Na + , and protons. Nitrate, a major plant nutrient, is accumulated and stored in the vacuole from where it can be retrieved according to metabolic deman...

Tonoplast calcium sensors CBL2 and CBL3 control plant growth and ion homeostasis through regulating V-ATPase activity in Arabidopsis

“…Previously, several mutants with reduced V-ATPase activity appeared to be hypersensitive to Na + , supporting the notion that increased V-ATPase activity is required for Na + compartmentalization under salt stress [49,50]. However, consistent with our results, the vha-a2 vha-a3 double mutant does not show hypersensitivity to external Na + , suggesting that tonoplast V-ATPase activity may not be a limiting factor for Na + sequestration and tolerance [34]. In contrast, reduction of V-ATPase activity in the early endosome results in increased salt sensitivity [34].…”

“…The most dramatic phenotypic changes in the cbl2 cbl3 double mutant include stunted growth, leaf necrosis and reduced fertility. When we correlated the developmental defects with possible functions of these two CBLs in vacuolar transport, we found that cbl2 cbl3 double mutant phenotype is largely shared by the vha-a2 vha-a3 double mutant as well, as the cax1 cax3 double mutant, both of which have defects in V-ATPase activity [34,48]. It is very likely that the reduced V-ATPase activity accounts for growth inhibition of all these mutants.…”

“…All these developmental abnormalities are reminiscent of the recently characterized vha-a2 vha-a3 double mutant lacking the tonoplast-localized membrane-integral vacuolar H + -ATPase subunit VHA-a ( Figure 5A, Supplementary information, Figure S8, and [34]). As both CBL2 and CBL3 are tonoplast calcium sensors, we thus speculated that the phenotypes of cbl2 cbl3 could be attributed to the malfunction of vacuolar H + -ATPase (V-ATPase).…”

“…As both CBL2 and CBL3 are tonoplast calcium sensors, we thus speculated that the phenotypes of cbl2 cbl3 could be attributed to the malfunction of vacuolar H + -ATPase (V-ATPase). To this end, we measured total V-ATPase activity as Concanamycin A and NO 3 − -sensitive ATP hydrolysis in microsomal membranes from rosette leaves of the wildtype, cbl2 and cbl3 single mutants, as well as cbl2 cbl3 and vha-a2 vha-a3 double mutants [34][35][36]. Compared to the wild-type control, similar levels of V-ATPase activity was found in both single mutants.…”

TNO1 Is Involved in Salt Tolerance and Vacuolar Trafficking in Arabidopsis

“…It was hypothesized that a cation transporter in the osm1/syp61 mutant may not function correctly, affecting guard cell turgor and salt sensitivity (Zhu et al, 2002). Extensive investigations of cation transporters in yeast, plants, and mammalian cells have shown that cation/H + antiporters in the Golgi, TGN, endosome, and PVC regulate the pH and cation concentration in these organelles, affecting vesicle trafficking, and knockdown of VHAa1 in Arabidopsis also leads to increased salt sensitivity (Brett et al, 2002(Brett et al, , 2005Nakamura et al, 2005;Pardo et al, 2006;Fuji et al, 2007;Krebs et al, 2010). Thus, one possibility is that the salt-sensitive phenotype of the tno1 and osm1/ syp61 mutants could be caused by defects in the Table I.…”