Growth Retardation Caused by Reduced Tonoplast V-ATPase Activity in Arabidopsis thaliana

Background: Vacuoles are unique compartments in plant cells. Their multiple functions require massive fluxes of molecules across their limiting membrane, the tonoplast. Transport across the tonoplast is energized by its membrane potential and the proton gradient established by two proton pumps, the vacuolar H + -ATPase (V-ATPase), and the vacuolar H + -pyrophosphatase (V-PPase), which play important roles in the growth and development of plants. However, the mechanisms by which they affect plant growth and development, in the absence of tonoplast proton pumps, remain unclear. Results: In this study, we show that the Arabidopsis thaliana double mutant, vha-a2vha-a3 , which lacks two tonoplast-localized isoforms of the membrane-integral V-ATPase subunit VHA-a, is viable, but shows a phenotype of inhibited growth and leaf chlorosis. Reduced total nitrogen absorption and increased nitrate assimilation lead to the accumulation of ammonium in the shoot, which results in the growth retardation of vha-a2vha-a3 . Concurrently, the distribution and concentration of cations are abnormal in plants when VHA-a2 and VHA-a3 genes are absent, which is another major growth-limiting factor. Interestingly, the growth inhibition of the vha-a2vha-a3 double mutant was alleviated and senescence delayed, while the concentration of total nitrogen increased and that of ammonium decreased, when adding additional potassium. Conclusions: Our results show that plants can be viable without VHA-a2 and VHA-a3 but are affected by multiple factors that result in growth retardation, which can be alleviated by adding additional potassium, which provides a new insight on the relationship between vacuolar H + -ATPase and potassium.

In Arabidopsis, three isoforms of the integral membrane subunit VHA-a have different sub-cellular locations: VHA-a1 is located in the trans-Golgi network/early endosome (TGN/EE), while VHA-a2 and VHA-a3 are located in the tonoplast [12]. The A. thaliana double mutant, vha-a2vha-a3, which lacks the two tonoplast-localized isoforms of the membrane-integral V-ATPase subunit VHA-a, is viable but exhibits severe growth inhibition and premature aging [4].
There are multiple types of ion transporters and channels in the tonoplast that transport NO 3 − , Na + , protons, and many other ions. The activity of proton pumps in the tonoplast provides a driving force for the active transport of various solutes.
Nitrate, as an important plant nutrient, is allocated to the metabolic pool (cytoplasm) and the storage pool (vacuole) once it is absorbed by plant roots. The change in relative size of the two nitrate reservoirs is mediated by the NO 3 − /H + exchange channel CLCa in the tonoplast [4,13], and is dependent on the activity of V-ATPase and V-PPase. These vacuolar proton pumps establish an electrochemical H + gradient across the tonoplast that energizes the transport of NO 3 − against its concentration gradient. Ion balance is necessary for normal growth and development of plants, and the activity of proton pumps in vacuole membranes is crucial to the regulation of ion homeostasis in cells [14,15]. Membrane-bound transport systems regulating cytosolic ion homeostasis (K + , Na + , Ca 2+ , and Fe 2+ ) and ion accumulation in the vacuole are of crucial importance for adaptation to stress conditions [16].
Potassium (K + ) is an essential macronutrient for plant growth and development from the Krebs' laboratory and have been described previously [4]. In this study, the vha-a2vha-a3 double mutant displayed severe growth retardation and significant symptoms of premature aging, compared with wild-type (Fig. 1A), with the outcome of 74% reduction in biomass. The relative expression of the senescence-associated gene 29 ( SAG29) was up-regulated in the mutant when grown hydroponically for four weeks ( Fig. 1B and C). Furthermore, compared with WT, leaf area, total length, surface area, root volume, and number of root tips and forks were reduced by 58%, 61%, 82%, 90%, 37%, and 71%, respectively, when the tonoplast V-ATPase activity decreased (Fig. S1). Accordingly, plants had poor growth when the VHA-a2 and VHA-a3 were absent.

Loss of tonoplast V-ATPase reduced nitrogen absorption
To investigate whether the absence of tonoplast V-ATPase activity would restrain the nitrogen absorption and inhibit growth, the total nitrogen (TN) accumulation and TN content of the vha-a2vha-a3 double mutant were determined ( Fig. 2A and B). TN content was significantly reduced by 9.7% compared to that of the wild-type. In addition, the relative expression of nitrate transporter 1.1 gene (NRT1.1) was downregulated by 25% in the tonoplast of the V-ATPase-deficient double mutant ( Fig. 2C). Therefore, these data suggest that the severe inhibition of growth of the vha-a2vha-a3 mutant may be due to reduced total nitrogen uptake.
Disruption of nitrogen metabolism causes ammonium accumulation in shoots of the vha-a2vha- Previous results have shown that nitrogen uptake in the vha-a2vha-a3 mutant was reduced, which is one possible reason why the mutant displayed growth inhibition [4]. Nitrate, as an important nitrogen source, is allocated to the cytoplasm and vacuole once absorbed by plant roots [30]. Nitrate accumulation within the vacuole is primarily mediated by the NO 3 − /H + exchanger AtCLCa and is dependent on the H + gradient [31]. In accordance, we investigated if the reduced tonoplast V-ATPase activity led to a shift in the relative size of the two nitrate pools. To investigate this possibility, the nitrogen metabolites of A. thaliana shoots and roots were determined after four weeks' growth under hydroponic conditions. The results revealed that vha-a2vha-a3 mutants contained 61% less nitrate (Fig. 3A); at the same time, nitrate reductase (NR) activity increased to four times that of wild-type (WT) NR in shoots (Fig. 3B). Thus, the double mutant accumulated ammonium 45% more than Col-0 ( Fig. 3C) and induced higher glutamine synthetase (GS) levels in shoots (Fig. 3D). These conditions might be inhibitory to the growth of the vha-a2vha-a3 double mutant. In this study, the chlorophyll content of the double mutant was significantly higher than that in Col-0 ( Fig. S2A).
Absence of tonoplast V-ATPase affects the long-distance transport of nitrogen between shoots and roots Since the vha-a2vha-a3 double mutant accumulated more ammonium in shoots, the metabolism and long-distance transport of nitrogen between shoots and roots were investigated. The relative expression of NRT1.5 was down-regulated and that of NRT1.8 was up-regulated ( Fig. 4A and B), which caused more nitrate to transport from shoots to roots in the vha-a2vha-a3 double mutant (Fig. 3A); moreover, NR activity was notably induced (Fig. 3B). However, we were surprised to find a significant decrease in ammonium content in roots of the vha-a2vha-a3 mutant ( Fig. 3C), and there was no significant difference in GS activity between the two strains ( Fig. 3D). To understand this in more detail, the relative expression of AMT2.1 and xylem sap NH 4 + content were measured in the double mutant. The relative expression of AMT2.1 was up-regulated two-fold, and xylem sap NH 4 + content was 66% higher than that of Col-0 ( Fig. 4C and D). Similarly, the root/shoot ratio of NH 4 + decreased significantly in the vha-a2vha-a3 mutant, which caused more NH 4 + to be transported from roots to shoots, where it was accumulated, thereby aggravating poisoning by NH 4 + in the vha-a2vha-a3 double mutant plants ( Fig. S2B, Fig. 3C).

Impaired tonoplast V-ATPase activity influenced ion homeostasis
Ion balance in cells is the basis for normal growth and development of plants, and the proton pumps located on the tonoplast play an important role in regulating ion homeostasis in cells [14,15] were supplied to the vha-a2vha-a3 double mutant during hydroponic growth for four weeks. It was observed that the yellowing phenotype of the mutant was not restored after addition of any of the three concentrations of Ca 2+ . Furthermore, plants were markedly smaller than wild-type (Fig. S3A).
Potassium, as the most abundant cation in higher plants, plays a critical role in plant nutrition, growth, ion homeostasis, enzyme activities, and osmoregulation [23]. Therefore, different doses of K + (2 mM, 4 mM, 6  indicating that exogenous potassium could alleviate the growth inhibition in these double mutant plants and delay senescence to some extent. Nitrogen absorption was promoted, and ammonium accumulation reduced with additional K + supply to the mutant It has been long noticed that K + /NO 3 − absorption and transport in vascular plants are somehow coordinated, playing a critical role in promoting nitrogen absorption [24][25][26][27]. Thus, TN accumulation and TN concentration were measured and found to have increased by 73% and 5%, respectively, compared with 2 mM K + , which aid to improve the growth of the mutant plants ( Fig. 7A and B). In contrast, the biomass and nitrogen absorption of Col-0 showed no significant difference between 2 mM and 10 mM supplied K + (Fig. 6B, 7A and B).
Previous studies have concluded that an increased K + supply or a restoration of K + transport can rapidly relieve NH 4 + toxicity [28,29], but whether it could reduce endogenous ammonium toxicity remains unclear. Surprisingly, it was found that the ammonium and chlorophyll content were also reduced by 32% and 13%, respectively, after adding 10 mM potassium in the nutrient solution ( Fig. 7C and D, Fig. S4B and C). Thus, the increase of nitrogen accumulation and the decrease of ammonium accumulation in shoots were beneficial to restore the growth performance and delay senescence when additional K + was supplied, but the specific reasons need to be further studied.

Discussion
The Nitrate is the main source of nitrogen, and once taken up into the root, it is partitioned between a small active cytosolic metabolite pool and a large storage pool in the vacuole [30]. Nitrate accumulation in the vacuole is primarily mediated by the NO 3 − /H + exchanger AtCLCa and depends on the pH gradient [31]. Our data suggest that reduced V-ATPase activity could lead to higher nitrate levels in the cytosol, which in turn stimulate the activity of nitrate reductase. This notion was supported by previous studies in which the tonoplast V-ATPase-deficient mutant vha-a2vha-a3 contained 80% less nitrate, but also displayed an increase by 90% of NR activity [4]. Accordingly, the vha-a2vha-a3 double mutant accumulated 31% more ammonium in shoots than the wild-type, a condition that might be toxic to plants. It is obvious from the results obtained in this study that chlorophyll content of vha-a2vha-a3 double mutants was higher than that in wild-type. Our data revealed that the nitrate metabolism was blocked when V-ATPase activity was absent in the tonoplast; one possible scenario to explain this is that the vacuolar pH was increased in the vha-a2vha-a3 double mutant. Thus, vacuoles cannot retain optimal acidity and the intracellular environment conducive to the normal metabolism of nitrate in cells.
It is interesting that in Arabidopsis, lack of tonoplast V-ATPase activity could affect the long-distance transport of nitrogen between shoots and roots. Our results showed that the relative expression of NRT1.5 was down-regulated and that of NRT1.8 up-regulated in the mutant plants, which caused more nitrate to accumulate and induced a notable increase in NR activity in the roots of mutant plants.
However, we were surprised to find a notable decrease in ammonium content in roots of mutant plants, but no difference in GS activity between WT and mutant. To explain the decrease in the accumulation of ammonium in the roots of the mutant, we considered AMT2.1, which encodes a high-affinity ammonium transporter responsible for the transport of ammonium from roots to shoots. We found that the relative expression of AMT2.1 was up-regulated in the mutant, and xylem sap NH 4 + content was about twice as high as that in WT, which caused more ammonium to be transported from roots to shoots. This in turn caused ammonium accumulation, aggravating the ammonium poisoning in shoots of vha-a2vha-a3 double mutant plants.
The activity of the proton pump in vacuole membranes can regulate the ion homeostasis in cells, which is necessary for normal growth and development of plants [14,15]. Membrane-bound transport systems regulating cytosolic ion balance (K + , Na + , Ca 2+ , and Fe 2+ ) and ion accumulation in the vacuole can be considered of crucial importance for adaptation to stress conditions [16]. To corroborate the fact that lack of tonoplast V-ATPase activity leads to ion disorder, the Ca 2+ , Mg 2+ , and Na + contents were decreased and the content of Fe 2+ , Mn 2+ , and Cu 2+ increased.
Vacuolar membrane proton pumps, as the power sources of substance exchange in the barrier between cytoplasm and vacuoles, play an important role in separating harmful ions from vacuoles, thus reducing excessive ions' toxicity to cells [34].
There are various metal ion transporters on the vacuolar membrane, which transport metal ions, depending on the energy produced by the activity of the vacuolar membrane proton pumps and H + gradient [35]. The disturbance of ions in cells may be due to the decrease of the electrochemical potential gradient between the vacuole membrane and cytoplasm, which leads to the alkalization of vacuoles, thereby affecting the activity of various ion transporters and channel proteins in vacuole membranes.
Reduced leaf growth of the vha-a2vha-a3 double mutant under normal growth conditions was accompanied by less leaf area and etiolated leaves. These are similar to symptoms of calcium deficiency also found in the cax1cax3 double mutant that lacks two vacuolar Ca 2+ /H + antiporters [32,33]. At the same time, our results showed that the calcium content in the mutants was reduced. Therefore, was the inhibition of mutant growth caused by calcium deficiency? To answer this question, we supplemented the hydroponic nutrient solution with different concentrations of calcium; the growth inhibition of mutants did not abate, indicating that calcium deficiency was not the main factor inhibiting plant growth. Taking into consideration that nitrate is one of the most abundant anions in plants, whose absorption and transport are believed to be accompanied by K + [ 26,27], we supplied different K + doses to the double mutant. The growth inhibition of mutants was alleviated to some extent by potassium supplementation; this included increased biomass and leaf area, enhanced total length, surface area, root volume, and number of root tips and forks. However, the effect was reduced when the concentration of K + in the culture solution was 15 mM and 20 mM. This indicated that the mutants are not potassium-deficient and that the V-ATPases may play a different role in the cell, such as acting as activators of certain K + -dependent enzymes, thereby promoting plant growth. In addition, the acquisition rates of K + and NO 3 − are often positively correlated and enhance each other [26,27]; potassium supplementation increased TN accumulation, and ammonium accumulation was also reduced, delaying cell senescence and death. These data demonstrate that the potassium supplementation has a positive effect on alleviating the growth inhibition phenotype of mutants. One possible explain this is that potassium and nitrogen interact in beneficial ways to aid in the absorption of nitrogen by plants, leading to increased biomass of mutants. Conversely, potassium may alleviate ammonium toxicity in mutants; this notion is supported by previous work showing that an increased K + supply or a restoration of K + transport can rapidly alleviate NH 4 + toxicity [28,29]. and nitrate was extracted from tissue samples (shoot: 1 g; root: 0.5 g). The samples were soaked in deionized water and bathed in a boiling water bath for 30 min. Then 0.1 mL of the sample solution was taken, 0.4 mL of 5% salicylic acid-H 2 SO 4 solution (5 g salicylic acid dissolved in 100 mL H 2 SO 4 ) was added, mixed well and allowed to react at room temperature for 20 min. Subsequently, 9.5 mL of 8% sodium hydroxide solution was added to the mixture and allowed to cool to room temperature. The NO 3 − concentration was measured spectrophotometrically at 410 nm following the method of Cataldo et al [40].

Measurement of NH 4 + concentration
To determine the concentration of NH 4 + , shoots and roots were sampled as described above and extracted with deionized water for 30 min. NH 4 + concentration was measured using indophenol blue colorimetry at 630 nm [41].
Determination of the activities of nitrate reductase (NR) and glutamine synthetase (GS) The method for determining NR activity was slightly modified on the basis of previous studies [42,43]. The fresh plants were divided into shoots and roots and then frozen. A small amount of quartz sand and 5 mL phosphate buffer (0.1 M, pH = 7.5) were added to a pre-frozen mortar, and the frozen samples were ground to homogeneity using a chilled pestle. The homogenates were centrifuged at 2,000 x g  Table 1, and the expression data were normalized to Actin [46,47].

Determination of ammonium in xylem sap
Seedlings were gathered and stripped of leaves, and the stems were cut at about 1 cm from the root and shielded from light. The xylem sap was collected with capillaries within 2 h of cutting. Each plant was covered with one capillary for xylem sap collection. Three biological replicates were conducted for each kind of plant.
The xylem sap was collected from 20 plants and the total volume was approximately 50 µL for each replicate [48]. Ammonium was determined as described by Santoni et al [41].

Determination of cation content
The method for determining cationic content was slightly modified from a previous study [49]. The seedlings were hydroponically grown for four weeks and the shoots oven-dried at 105 °C for 30 min, followed by 65 °C to constant weight, and then ground into a fine powder. Following this, 5 mL HNO 3  A v a i l a b i l i t y o f d a t a a n d m a t e r i a l The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.
C o m p e t i n g i n t e r e s t s The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. Nitrogen metabolism was measured using wild-type and vha-a2vha-a3 mutant four-week-old Nitrogen transport over long distance between shoots and roots. Relative expression levels o Figure 5 Impaired tonoplast V-ATPase activity prejudiced ion homeostasis. The cationic concentration Figure 6 The growth performance of vha-a2vha-a3 mutant was improved with the additional K+ suppli