Ammonium Accumulation Caused by Reduced Tonoplast V-ATPase Activity in Arabidopsis thaliana

Plant vacuoles are unique compartments that play a critical role in plant growth and development. The vacuolar H+-ATPase (V-ATPase), together with the vacuolar H+-pyrophosphatase (V-PPase), generates the proton motive force that regulates multiple cell functions and impacts all aspects of plant life. We investigated the effect of V-ATPase activity in the vacuole on plant growth and development. We used an Arabidopsis thaliana (L.) Heynh. double mutant, vha-a2 vha-a3, which lacks two tonoplast-localized isoforms of the membrane-integral V-ATPase subunit VHA-a. The mutant is viable but exhibits impaired growth and leaf chlorosis. Nitrate assimilation led to excessive ammonium accumulation in the shoot and lower nitrogen uptake, which exacerbated growth retardation of vha-a2 vha-a3. Ion homeostasis was disturbed in plants with missing VHA-a2 and VHA-a3 genes, which might be related to limited growth. The reduced growth and excessive ammonium accumulation of the double mutant was alleviated by potassium supplementation. Our results demonstrate that plants lacking the two tonoplast-localized subunits of V-ATPase can be viable, although with defective growth caused by multiple factors, which can be alleviated by adding potassium. This study provided a new insight into the relationship between V-ATPase, growth, and ammonium accumulation, and revealed the role of potassium in mitigating ammonium toxicity.


Introduction
The evolutionary success of higher plants is largely contributed to their unique cellular architecture [1]. Vacuoles, as the largest organelles in mature plant cells, accounting for about 90% of the cell volume, contain a substantial amount of water and solutes necessary for plant growth and development [2][3][4]. The transport of molecules across the tonoplast is driven by the proton gradient and membrane potential established by the combined activity of two proton pumps, the vacuolar H + -ATPase (V-ATPase) and vacuolar H + -pyrophosphatase (V-PPase), both of which are essential for plant growth and development [1,5].
V-ATPase and V-PPase use Mg-ATP and Mg-inorganic pyrophosphate (PPi) complexes, respectively, as substrates [6]. Through the combined activity of the two pumps, protons enter the vacuole from the cytoplasm and an electrochemical gradient is established between the cytoplasm and vacuole, which drives the transport of compounds across the tonoplast against their concentration gradient [1]. Both proton pumps are among the most abundant tonoplast proteins, thereby indicating that the amount of energy spent in vacuolar transport is enormous [7,8]. V-ATPase is structurally conserved and consists of two sub-complexes. The peripheral V 1 complex is composed of eight subunits (VHA-A to -H), which are exposed to the cytoplasmic side and are responsible for ATP hydrolysis. The membrane-integral V 0 complex consists of six subunits (VHA-a, -c, -c', c", -d, and -e), which are integrated into the membrane and act as a channel for translocation of protons

Loss of Tonoplast V-ATPase Reduces Nitrogen Absorption
To investigate whether the absence of tonoplast V-ATPase activity would re nitrogen absorption and inhibit plant growth, the total nitrogen accumulation and nitrogen content of the vha-a2 vha-a3 double mutant were determined (Figure 2a,b). nitrogen content was significantly reduced by 9.7% compared with that of the wild Additionally, the relative expression of nitrate transporter 1.1 (NRT1.1) gene was dow ulated by 25% in the tonoplast of the V-ATPase-deficient double mutant (Figure 2c). data suggest that the severe growth inhibition of the vha-a2 vha-a3 mutant may be c by the reduced total N uptake.

Loss of Tonoplast V-ATPase Reduces Nitrogen Absorption
To investigate whether the absence of tonoplast V-ATPase activity would restrain nitrogen absorption and inhibit plant growth, the total nitrogen accumulation and total nitrogen content of the vha-a2 vha-a3 double mutant were determined (Figure 2a,b). Total nitrogen content was significantly reduced by 9.7% compared with that of the wild type. Additionally, the relative expression of nitrate transporter 1.1 (NRT1.1) gene was downregulated by 25% in the tonoplast of the V-ATPase-deficient double mutant (Figure 2c). These data suggest that the severe growth inhibition of the vha-a2 vha-a3 mutant may be caused by the reduced total N uptake. (c) Relative expression of nitrate transporter 1.1 (NRT1.1) in Col-0 and vha-a2 vha-a3 was measured and Col-0 was set to 100%. Data in are mean ± S.E. (n = 3). Asterisks (*) and (**) indicate significant differences at p < 0.05 and p < 0.01, respectively. DW, Dry weight.

Disruption of Nitrogen Metabolism Causes Ammonium Accumulation in Shoots of Muta Plants
Altered activity of the proton pump significantly influences nitrate accumulat within the vacuole [12], affecting the nitrogen metabolism. With respect to the wild ty the vha-a2 vha-a3 mutants contained 61% less nitrate ( Figure 3a) and nitrate reductase (N activity in shoots increased four-fold ( Figure 3b). Moreover, the content of ammonium the double mutant increased by 45% compared with that in the wild type (Col-0) (Fig  3c), and the levels of glutamine synthetase (GS) increased ( Figure 3d). Chlorophyll cont as an indicator of ammonium accumulation, was significantly higher in the double mut than in Col-0 ( Figure S2A), indicating that the vha-a2 vha-a3 double mutant was possi exposed to mild ammonium stress [41]. . Nitrogen metabolism was measured using wild type and vha-a2 vha-a3 mutant four-week-old seedlings in hydroponics. Nitrate concentration (a), nitrate reductase (NR) activity (b), ammonium concentration (c), and glutamine synthetase (GS) activity (d) of Col-0 and vha-a2 vha-a3 mutant were determined in the shoots and roots. Error bars represent S.D. of three biological replicates. Asterisks (*) and (**) indicate significant differences at p < 0.05 and p < 0.01, respectively. FW, Fresh weight. in Col-0 and vha-a2 vha-a3 was measured and Col-0 was set to 100%. Data in are mean ± S.E. (n = 3). Asterisks (*) and (**) indicate significant differences at p < 0.05 and p < 0.01, respectively. DW, Dry weight.

Disruption of Nitrogen Metabolism Causes Ammonium Accumulation in Shoots of Mutant Plants
Altered activity of the proton pump significantly influences nitrate accumulation within the vacuole [12], affecting the nitrogen metabolism. With respect to the wild type, the vha-a2 vha-a3 mutants contained 61% less nitrate ( Figure 3a) and nitrate reductase (NR) activity in shoots increased four-fold ( Figure 3b). Moreover, the content of ammonium in the double mutant increased by 45% compared with that in the wild type (Col-0) (Figure 3c), and the levels of glutamine synthetase (GS) increased ( Figure 3d). Chlorophyll content, as an indicator of ammonium accumulation, was significantly higher in the double mutant than in Col-0 ( Figure S2A), indicating that the vha-a2 vha-a3 double mutant was possibly exposed to mild ammonium stress [41].

Disruption of Nitrogen Metabolism Causes Ammonium Accumulation in Shoots of Mutant Plants
Altered activity of the proton pump significantly influences nitrate accumulatio within the vacuole [12], affecting the nitrogen metabolism. With respect to the wild type the vha-a2 vha-a3 mutants contained 61% less nitrate ( Figure 3a) and nitrate reductase (NR activity in shoots increased four-fold ( Figure 3b). Moreover, the content of ammonium i the double mutant increased by 45% compared with that in the wild type (Col-0) (Figur 3c), and the levels of glutamine synthetase (GS) increased ( Figure 3d). Chlorophyll conten as an indicator of ammonium accumulation, was significantly higher in the double mutan than in Col-0 ( Figure S2A), indicating that the vha-a2 vha-a3 double mutant was possibl exposed to mild ammonium stress [41].

Absence of Tonoplast V-ATPase Affects the Long-Distance Transport of Nitrogen between Shoots and Roots
A proportion of the NO 3 absorbed by plant roots is stored within vacuoles or assimilated into organic nitrogen through a series of reactions catalyzed by NR, nitrite reductase, GS, and glutamate synthase [42]. However, most of absorbed NO 3 − is primarily transported upward through the xylem and downward through the phloem [43]. The longdistance transport and distribution of NO 3 − between the roots and shoots in Arabidopsis thaliana are mainly co-regulated by NRT1.5 and NRT1.8, two members of the peptide transporter (PTR) family, the expression of which is strongly induced by NO 3 − [44][45][46][47][48]. In a wide range of plants, the transport of ammonium across membranes is mediated by proteins of the ammonium transporter/methylammonium permease/rhesus protein (AMT/MEP/Rh) family [49,50]. In Arabidopsis thaliana, four homologs from the AMT family (AMT1;1, AMT1;2, AMT1;3, and AMT1;5) and one homolog from the MEP subfamily (AMT2;1) are expressed in roots and AMT2;1 localizes at the plasma membrane [51,52]; these function mainly in the root-to-shoot translocation of ammonium [53]. More ammonium was accumulated in the shoots of the vha-a2 vha-a3 double mutant than in those of the wild type; therefore, we measured the metabolism and long-distance transport of nitrogen between the shoots and roots. In roots, the relative expression of NRT1.5 was downregulated and that of NRT1.8 was upregulated (Figure 4a,b), which increased the transport of NO 3 − from the shoots to the roots in the mutant ( Figure 3a). Moreover, NR activity was notably induced ( Figure 3b). However, there was a significant decrease in ammonium content in the roots of the vha-a2 vha-a3 mutant (Figure 3c), and there was no significant difference in GS activity between the two lines ( Figure 3d). The relative expression of AMT2;1 in roots was upregulated two-fold, and xylem sap NH 4 + content was 66% higher than that of Col-0 ( Figure 4c,d), leading to more NH 4 + to be transported from the roots to the shoots. Similarly, the root/shoot ratio in NH 4 + decreased significantly in the vha-a2 vha-a3 mutant compared to that of the wild type ( Figure S2B), indicating a greater proportion of ammonium being allocated to the leaves in the double mutant than in the wild type. Both the reduced ammonium metabolism in the shoots and the higher transport of ammonium from roots to shoots affected the accumulation of ammonium in the shoots, thereby aggravating NH 4 + poisoning in vha-a2 vha-a3 double mutant plants.

Absence of Tonoplast V-ATPase Affects the Long-Distance Transport of Nitrogen between Shoots and Roots
A proportion of the NO3 -absorbed by plant roots is stored within vacuoles or assimilated into organic nitrogen through a series of reactions catalyzed by NR, nitrite reductase, GS, and glutamate synthase [42]. However, most of absorbed NO3 − is primarily transported upward through the xylem and downward through the phloem [43]. The longdistance transport and distribution of NO3 − between the roots and shoots in Arabidopsis thaliana are mainly co-regulated by NRT1.5 and NRT1.8, two members of the peptide transporter (PTR) family, the expression of which is strongly induced by NO3 − [44][45][46][47][48]. In a wide range of plants, the transport of ammonium across membranes is mediated by proteins of the ammonium transporter/methylammonium permease/rhesus protein (AMT/MEP/Rh) family [49,50]. In Arabidopsis thaliana, four homologs from the AMT family (AMT1;1, AMT1;2, AMT1;3, and AMT1;5) and one homolog from the MEP subfamily (AMT2;1) are expressed in roots and AMT2;1 localizes at the plasma membrane [51,52]; these function mainly in the root-to-shoot translocation of ammonium [53]. More ammonium was accumulated in the shoots of the vha-a2 vha-a3 double mutant than in those of the wild type; therefore, we measured the metabolism and long-distance transport of nitrogen between the shoots and roots. In roots, the relative expression of NRT1.5 was downregulated and that of NRT1.8 was upregulated (Figure 4a,b), which increased the transport of NO3 − from the shoots to the roots in the mutant ( Figure 3a). Moreover, NR activity was notably induced (Figure 3b). However, there was a significant decrease in ammonium content in the roots of the vha-a2 vha-a3 mutant (Figure 3c), and there was no significant difference in GS activity between the two lines ( Figure 3d). The relative expression of AMT2;1 in roots was upregulated two-fold, and xylem sap NH4 + content was 66% higher than that of Col-0 ( Figure 4c,d), leading to more NH4 + to be transported from the roots to the shoots. Similarly, the root/shoot ratio in NH4 + decreased significantly in the vha-a2 vha-a3 mutant compared to that of the wild type ( Figure S2B), indicating a greater proportion of ammonium being allocated to the leaves in the double mutant than in the wild type. Both the reduced ammonium metabolism in the shoots and the higher transport of ammonium from roots to shoots affected the accumulation of ammonium in the shoots, thereby aggravating NH4 + poisoning in vha-a2 vha-a3 double mutant plants.

Impaired Tonoplast V-ATPase Activity Influences Ion Homeostasis
We assessed whether ion content was disrupted in plants in the absence of tonoplast V-ATPase activity. Cations in rosette leaves, namely potassium (K + ), calcium (Ca 2+ ), magnesium (Mg 2+ ), sodium (Na + ), iron (Fe 2+ ), manganese (Mn 2+ ), copper (Cu 2+ ), and zinc (Zn 2+ ), were extracted from four-week-old plants and analyzed by inductively coupled plasma-mass spectrometry (ICP-MS) ( Figure 5). Compared to the wild type, the content of Ca 2+ , Mg 2+ , and Na + decreased, and the content of Fe 2+ , Mn 2+ , and Cu 2+ increased in the mutant, indicating that ion homeostasis was disturbed in plants in the absence of tonoplast proton pumps, which was correlated to decreased plant growth and premature senescence.

Impaired Tonoplast V-ATPase Activity Influences Ion Homeostasis
We assessed whether ion content was disrupted in plants in the absence of tonoplast V-ATPase activity. Cations in rosette leaves, namely potassium (K + ), calcium (Ca 2+ ), magnesium (Mg 2+ ), sodium (Na + ), iron (Fe 2+ ), manganese (Mn 2+ ), copper (Cu 2+ ), and zinc (Zn 2+ ), were extracted from four-week-old plants and analyzed by inductively coupled plasmamass spectrometry (ICP-MS) ( Figure 5). Compared to the wild type, the content of Ca 2+ , Mg 2+ , and Na + decreased, and the content of Fe 2+ , Mn 2+ , and Cu 2+ increased in the mutant, indicating that ion homeostasis was disturbed in plants in the absence of tonoplast proton pumps, which was correlated to decreased plant growth and premature senescence.

Growth Retardation is Alleviated and Senescence is Delayed by Supplementing K + to the Mutant
Reduced leaf growth of the vha-a2 vha-a3 double mutant under control conditions was accompanied by leaf etiolation; these symptoms were similar to those caused by calcium deficiency in the cax1 cax3 double mutant that lacked two vacuolar Ca 2+ /H + antiporters [54,55]. To address the lower calcium content in the double mutant, additional doses of Ca 2+ (0.5 mM, 2.5 mM, 5 mM) were supplied to the vha-a2 vha-a3 double mutant during the four-week hydroponic cultivation. 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 those of the wild type ( Figure S3A).
The effect of K + on plants was examined by supplying different doses of K + (2 mM, 4 mM, 6 mM, 8 mM, 10 mM, 15 mM, and 20 mM) to the plants. The difference in plant size between the vha-a2 vha-a3 double mutant and the wild type gradually diminished, and the symptoms of leaf chlorosis were alleviated with increasing external K + concentrations from 2 mM to 10 mM ( Figure S3B). However, these effects were reduced when the concentration of K + in the culture solution was 15 mM and 20 mM. Compared with those of the 2 mM K + supplementation, the changes in the wild type were not significant after the addition of different K + doses. The content of NO3 -, NH4 + , and chlorophyll decreased within the potassium concentration range of 2 mM to 10 mM, and slightly increased at 15 mM and 20 mM ( Figure S4A-C).
The vha-a2 vha-a3 double mutant demonstrated increased shoot and root biomass after supplying 10 mM external K + when compared to that in the treatment with 2 mM external K + (Figure 6a). The biomass of mutant plants was increased by 74% and relative

Growth Retardation is Alleviated and Senescence is Delayed by Supplementing K + to the Mutant
Reduced leaf growth of the vha-a2 vha-a3 double mutant under control conditions was accompanied by leaf etiolation; these symptoms were similar to those caused by calcium deficiency in the cax1 cax3 double mutant that lacked two vacuolar Ca 2+ /H + antiporters [54,55]. To address the lower calcium content in the double mutant, additional doses of Ca 2+ (0.5 mM, 2.5 mM, 5 mM) were supplied to the vha-a2 vha-a3 double mutant during the four-week hydroponic cultivation. 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 those of the wild type ( Figure S3A).
The effect of K + on plants was examined by supplying different doses of K + (2 mM, 4 mM, 6 mM, 8 mM, 10 mM, 15 mM, and 20 mM) to the plants. The difference in plant size between the vha-a2 vha-a3 double mutant and the wild type gradually diminished, and the symptoms of leaf chlorosis were alleviated with increasing external K + concentrations from 2 mM to 10 mM ( Figure S3B). However, these effects were reduced when the concentration of K + in the culture solution was 15 mM and 20 mM. Compared with those of the 2 mM K + supplementation, the changes in the wild type were not significant after the addition of different K + doses. The content of NO 3 -, NH 4 + , and chlorophyll decreased within the potassium concentration range of 2 mM to 10 mM, and slightly increased at 15 mM and 20 mM (Figure S4A-C).
The vha-a2 vha-a3 double mutant demonstrated increased shoot and root biomass after supplying 10 mM external K + when compared to that in the treatment with 2 mM external K + (Figure 6a). The biomass of mutant plants was increased by 74% and relative expression levels of SAG29 were decreased by 87% at potassium concentrations from 2 mM to 10 mM (Figure 6b,c). Concomitantly, the leaf area, total root length, root surface area, root volume, and number of root tips and forks were enhanced in wild type and mutant plants when grown in 10 mM K + ( Figure S5); a much greater growth difference was observed in the vha-a2 vha-a3 double mutant, indicating that exogenous potassium could alleviate the growth inhibition and delay senescence to some extent in double mutant plants.
expression levels of SAG29 were decreased by 87% at potassium concentrations from mM to 10 mM (Figure 6b,c). Concomitantly, the leaf area, total root length, root surfa area, root volume, and number of root tips and forks were enhanced in wild type a mutant plants when grown in 10 mM K + ( Figure S5); a much greater growth differen was observed in the vha-a2 vha-a3 double mutant, indicating that exogenous potassiu could alleviate the growth inhibition and delay senescence to some extent in double m tant plants.

Nitrogen Absorption is Promoted and Ammonium Accumulation is Reduced with Additional K + Provision to the Mutant
It has been long noticed that K + /NO3 -absorption and transport in vascular plants a somehow coordinated, playing a critical role in promoting NO3 -absorption [31][32][33][34]. To nitrogen accumulation and total nitrogen content of the double mutant increased by 73 and 5%, respectively, in 10 mM K + treatment compared with those in 2 mM K + treatmen this, in turn, improved the growth of mutant plants (Figure 7a,b). In contrast, the bioma and nitrogen absorption of the wild type showed no significant difference between tre ments with 2 mM and 10 mM K + (Figures 6b and 7a,b).
Surprisingly, ammonium and chlorophyll content in the shoots of mutant plants w reduced by 32% and 13%, respectively, after adding 10 mM potassium in the nutrient s lution (Figure 7c,d and Figure S4B,C). Increased nitrogen accumulation and decreas ammonium content in shoots resulting from additional K + supplementation helped to store the growth performance and delayed the senescence, but the specific mechanism involved in these processes remain uncertain and warrant further studies.

Nitrogen Absorption is Promoted and Ammonium Accumulation is Reduced with Additional K + Provision 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 NO 3 absorption [31][32][33][34]. Total nitrogen accumulation and total nitrogen content of the double mutant increased by 73% and 5%, respectively, in 10 mM K + treatment compared with those in 2 mM K + treatments; this, in turn, improved the growth of mutant plants (Figure 7a,b). In contrast, the biomass and nitrogen absorption of the wild type showed no significant difference between treatments with 2 mM and 10 mM K + (Figures 6b and 7a,b).

Discussion
The two proton pumps in plant tonoplast, vacuolar H + -ATPase and vacuol PPase, are highly abundant tonoplast proteins that play important roles in plant g and development [1,5]. Both proton pumps energize the massive fluxes of ions an tabolites that are required for vacuolar function throughout the plant life cycle [4]. T cidate the role of V-ATPase in the growth of plants, we selected an Arabidopsis thalia with loss of V-ATPase in the tonoplast, vha-a2 vha-a3, which lacks the two tonoplast ized isoforms of the membrane-integral V-ATPase subunit VHA-a.
The mutant plants are viable but present severe growth inhibition and prem aging, including reduced biomass, leaf area, total root length, root surface area, ro ume, and the number of root tips and forks. Nitrate, as the main source of nitro partitioned between the cytoplasm and the vacuole after its uptake by the root [21 uolar NO3 − storage and its subsequent remobilization are a central feature of the nitrogen economy [4]. The reduced vacuolar uptake capacity can lead to increased solic NO3 − levels, which in turn stimulate nitrate assimilation, a process that req substantial electron flow via either ferredoxin or NAD(P)H [56].
Compared to that of the wild type, the content of ammonium was increased b and the chlorophyll content was higher in mutant line, a condition which is pote toxic to plants. Chlorophyll content is an indicator of ammonium stress and its c increases under mild ammonium stress in Arabidopsis thaliana [41]. Our data reveale the NO3 − metabolism was blocked when V-ATPase activity was absent, likely due creased vacuolar pH. Consequently, the vacuoles cannot retain optimal acidity and cellular environment conducive to normal metabolism of nitrate in cells. Surprisingly, ammonium and chlorophyll content in the shoots of mutant plants was reduced by 32% and 13%, respectively, after adding 10 mM potassium in the nutrient solution (Figure 7c,d and Figure S4B,C). Increased nitrogen accumulation and decreased ammonium content in shoots resulting from additional K + supplementation helped to restore the growth performance and delayed the senescence, but the specific mechanisms involved in these processes remain uncertain and warrant further studies.

Discussion
The two proton pumps in plant tonoplast, vacuolar H + -ATPase and vacuolar H + -PPase, are highly abundant tonoplast proteins that play important roles in plant growth and development [1,5]. Both proton pumps energize the massive fluxes of ions and metabolites that are required for vacuolar function throughout the plant life cycle [4]. To elucidate the role of V-ATPase in the growth of plants, we selected an Arabidopsis thaliana line with loss of V-ATPase in the tonoplast, vha-a2 vha-a3, which lacks the two tonoplast-localized isoforms of the membrane-integral V-ATPase subunit VHA-a.
The mutant plants are viable but present severe growth inhibition and premature aging, including reduced biomass, leaf area, total root length, root surface area, root volume, and the number of root tips and forks. Nitrate, as the main source of nitrogen, is partitioned between the cytoplasm and the vacuole after its uptake by the root [21]. Vacuolar NO 3 − storage and its subsequent remobilization are a central feature of the plant nitrogen economy [4]. The reduced vacuolar uptake capacity can lead to increased cytosolic NO 3 − levels, which in turn stimulate nitrate assimilation, a process that requires a substantial electron flow via either ferredoxin or NAD(P)H [56].
Compared to that of the wild type, the content of ammonium was increased by 31% and the chlorophyll content was higher in mutant line, a condition which is potentially toxic to plants. Chlorophyll content is an indicator of ammonium stress and its content increases under mild ammonium stress in Arabidopsis thaliana [41]. Our data revealed that the NO 3 − metabolism was blocked when V-ATPase activity was absent, likely due to increased vacuolar pH. Consequently, the vacuoles cannot retain optimal acidity and intracellular environment conducive to normal metabolism of nitrate in cells.
Interestingly, in Arabidopsis thaliana, the lack of tonoplast V-ATPase activity could affect the long-distance transport of nitrogen between shoots and roots. In roots, the amount of nutrients destined for long-distance transportation is determined by the activity of transporters that load the xylem vessels, while in shoots, transporters unloading into the xylem can increase the overall root-to-shoot translocation [44][45][46]57,58]. The relative expression of NRT1.5 was downregulated and that of NRT1.8 was upregulated in mutant plants, which caused more NO 3 − to accumulate and induced a notable increase in NR activity in the roots. However, surprisingly, a notable decrease in ammonium content was detected in the roots of mutant plants, but there was no difference in GS activity between the two lines. A previous study has revealed that AMT2;1, which encodes a high-affinity ammonium transporter, functions mainly in the translocation of ammonium from roots to shoots [53]. The relative expression of AMT2;1 was upregulated in the mutant, and xylem sap NH 4 + content was about twice as high as that in the wild type. As a result, more ammonium was transported from roots into the shoots, which in turn caused the accumulation of ammonium, aggravating the ammonium toxicity in the shoots of vha-a2 vha-a3 double mutant plants.
The proton pumps in the tonoplast affect the vacuolar transport capacities and are considered crucial for translocation of harmful ions from the cytoplasm into the vacuoles, reduction of excessive ion toxicity to cells, and stabilization of ion homeostasis in the cell [59], as well as for the regulation of metabolite homeostasis, plant growth, and plant adaptation to abiotic stress conditions [7,12,[17][18][19][20]. In the mutant, the content of Ca 2+ , Mg 2+ , and Na + was decreased, and that of Fe 2+ , Mn 2+ , and Cu 2+ was increased. The disturbance of ions in cells may be caused by a decrease in the electrochemical potential gradient between the tonoplast and cytoplasm, which leads to the alkalization of vacuoles, thereby affecting the activity of various ion transporters and channel proteins in the tonoplast and plant growth.
The impaired growth of the vha-a2 vha-a3 double mutant under normal growth conditions was accompanied by smaller leaf area and etiolated leaves. These are similar to the symptoms of calcium deficiency also found in the cax1 cax3 double mutant that lacks two vacuolar Ca 2+ /H + antiporters [54,55]. Our results showed a reduced content of Ca 2+ in the mutant. We aimed to investigate whether the growth inhibition of mutant plants was caused by Ca 2+ deficiency. To explore this aspect, we supplemented the hydroponic nutrient solution with different concentrations of Ca 2+ ; the growth limitation of mutants did not abate, indicating that Ca 2+ deficiency was not the major factor inhibiting plant growth. Considering the fact that NO 3 − is abundant in plants and its absorption and transport are believed to be accompanied by K + [33,34], we supplied different K + doses to the double mutant. The growth retardation of mutants was alleviated to some extent by 2 mM to 10 mM potassium supplementation-biomass and leaf area increased, and the total root length, root surface area, root volume, and the number of root tips and forks was enhanced. However, the double mutants remained smaller than the wild type. The activity of V-PPase, another main proton pump in the tonoplast, was increased six-fold by potassium supplementation [38], which can explain the partial rescue of the plant phenotype at high potassium concentrations; however, the growth phenotype indicated that V-PPase cannot completely replace and compensate for the functions of V-ATPase. The positive effect of potassium supplementation was reduced when the concentration of K + in the culture solution was 15 mM and 20 mM, which exposed the negative effect of high potassium concentration on plant growth. The increase in external K + to 10 or 20 mM KCl significantly affected plant growth, leading to a severe growth reduction [60].
Additionally, the acquisition rates of K + and NO 3 − are often positively correlated and enhance each other [33,34]; potassium supplementation increased total nitrogen accumulation and reduced the content of ammonium, delaying senescence and death of rosette leaves. Compared with those of the 2 mM K + treatment, the changes in the wild type were not significant after potassium addition likely due to homeostasis that was established in its cells, and the addition of potassium had no further effect on plant growth. Therefore, potassium supplementation alleviates the growth inhibition. One possible explanation for this is that the interactions between NO 3 − and K + may aid in the absorption of nitrogen by plants, leading to increased biomass of mutants [61]. Moreover, potassium may alleviate ammonium toxicity in mutants; this notion is supported by a previous work showing that an increased K + supply or a restoration of K + transport can rapidly alleviate NH 4 + toxicity [39,40].
In conclusion, we revealed the important role of V-ATPase activity in growth of Arabidopsis thaliana plants. An impaired growth and excessive ammonium accumulation were observed in the vha-a2 vha-a3 double mutant. Interestingly, the limited growth can be alleviated by adding additional potassium. This study provided a new insight into the relationship between vacuolar H + -ATPase, growth, and ammonium accumulation. Concomitantly, we revealed the role of potassium in mitigating ammonium stress and, via adjustment of the activity of tonoplast V-ATPase and the content of potassium, for regulation of ammonium metabolism and growth.

Plant Materials and Growth Conditions
The wild type Arabidopsis thaliana line Columbia-0 (Col-0) was used as a control for the tonoplast V-ATPase double mutant (vha-a2 vha-a3). The vha-a2 vha-a3 double mutant was kindly provided by Dr. M. Krebs (Heidelberg Institute for Plant Science, University of Heidelberg, Germany). The mutant was identified in the F 2 generation derived from a cross between homozygous vha-a2 (SALK_142642) and vha-a3 (SALK_29786) individuals [4]. The experiments were carried out in a phytotron (MGC-800HP-2; Bluepard Test Equipment Company, Shanghai, China) at the Hunan Agricultural University, Changsha, China. The phytotron was set at a 16-h light/8-h dark photoperiod, 70% relative humidity, and constant temperature of 22 • C. Seeds were sown in a matrix consisting of vermiculite and perlite at a ratio of 3:2, and allowed to germinate and grow for 10 days [62,63]. Subsequently, the seedlings at the two-leaf stage were transferred and grown hydroponically in 600 mL plastic pots covered with a black film on the outside, as per methods described by Gong et al. [64]. Nine plants were grown in each plastic pot. To eliminate edge effect, the positions of the pots were interchanged when refreshing the media. The nutrient solution (pH = 5.8) provided to plants was replaced every five days, as described in Han et al. [65].

Measurement of Leaf Rosette Areas
Whole rosette leaves were collected from individual Arabidopsis thaliana seedlings grown for four weeks. Leaf area was measured using a leaf area meter (CI-202 Laser Area Meter; CID Bio-Science, Camas, WA, USA).

Measurement of Root Configuration
The roots were sampled from individual Arabidopsis thaliana seedlings grown for four weeks. They were spread completely in 1000 cm 3 (25 cm by 20 cm by 2 cm) transparent plastic containers filled with distilled water. Root surface was washed to remove impurities, and the total root length, root surface area, root volume, and the number of root tips and forks were determined using WinRHIZO (EPSON Expression 11000XL, Japan).

Determination of Nitrogen Content
Whole seedlings of Arabidopsis thaliana grown hydroponically for four weeks were sampled and oven-dried at 105 • C for 30 min, followed by drying at 65 • C to a constant weight. The samples were digested with H 2 SO 4 -H 2 O 2 (5 mL H 2 SO 4 ) at 350 • C and analyzed using an AA3 continuous-flow auto-analyzer [65]. Nitrogen concentration was calculated using the following formula: Total N = [N] × biomass.

Assays for NO 3 − Content Estimation
Fresh plants were divided into shoots and roots as per methods described by Han et al. [65], and nitrate was extracted from tissue samples (shoot: 1 g; root: 0.5 g). The samples were soaked in deionized water and heated in a boiling water bath for 30 min. Then, 0.1 mL of the sample solution was mixed well with 0.4 mL of 5% salicylic acid-H 2 SO 4 solution (5 g salicylic acid dissolved in 100 mL H 2 SO 4 ) 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 left to cool to room temperature. The content of NO 3 − was measured spectrophotometrically at 410 nm following the method reported by Cataldo et al. [66].

Determination of NH 4 + Content
To determine the content of NH 4 + , shoots and roots were sampled as described above and extracted with deionized water for 30 min. NH 4 + content was measured using indophenol blue colorimetry at an absorbance wavelength of 630 nm [67].

Determination of the Activities of Nitrate Reductase (NR) and Glutamine Synthetase (GS)
The method for determining NR activity was as previously described [68,69], with slight modification. Fresh plants were separated into shoot (0.5 g) and root (0.5 g) samples.
The samples were washed with deionized water and wiped dry, snap-frozen in liquid nitrogen at −196 • C for 30 min, and stored at −80 • C for determination of enzymatic activity. 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 2000× g for 15 min at 4 • C. The supernatant fluid was used to determine the activity of NADH-dependent NR. The reaction mixture consisted of 0.4 mL supernatant, 0.1 M KNO 3 , and 3 mM NADH. The reaction was terminated after 30 min at 25 • C by adding 1% sulfanilamide and α-naphthylamine. The amount of reaction product was measured at 540 nm using a UV-2600 spectrophotometer (Shimadzu Corp., Kyoto, Japan).
To measure GS activity, frozen plant samples (shoot: 0.5 g; root: 0.5 g) were ground to obtain homogeneous samples as presented above with 3 mL 50 mM Tris-HCl buffer (pH = 8.0), containing 2 mM dithiothreitol, 2 mM Mg 2+ , and 0.4 M sucrose. The homogenate was centrifuged at 10,000× g for 10 min at 4 • C. The supernatant was examined for GS activity according to the method described by Zhang et al. [70].

Chlorophyll Content Assays
Fresh rosette leaves (0.2 g) were sampled and extracted in 10 mL absolute ethanol: Acetone (1:1) for 48 h in the dark at 4 • C [71]. The absorbance of the extract was measured at 663 and 645 nm using a UV-VIS spectrophotometer (UV-2600, Shimadzu, Kyoto, Japan) to estimate total chlorophyll content.

RNA Extraction and qRT-PCR Assay
The seedlings were grown under normal conditions for 30 days after which shoots (0.1 g) and roots (0.1 g) were harvested for RNA analysis. Total RNA was extracted with 1 mL TRIzol reagent (Invitrogen, Carlsbad, CA, USA), precipitated with an equal volume of isopropanol, washed with 75% ethanol, and dissolved in RNAase-free water. The first-strand cDNA was synthesized from total RNA using the PrimeScript reverse transcription (RT) reagent kit (TaKaRa, Shiga, Japan) in accordance with the manufacturer's instructions. The relative gene expression was determined using the SYBR Green RT-PCR kit (TaKaRa) with a pair of gene-specific primers. The RT-PCR analysis was performed using the StepOnePlus™ Real-Time PCR Instrument (StepOnePlus™, Life Technologies Holdings Pte Ltd., Singapore) following the manufacturer's instructions. The thermal cycles were as follows: 95 • C for 3 min, followed by 40 cycles of 95 • C for 10 s and 60 • C for 30 s. A melt curve analysis was performed to ensure the primer gene specificity as follows: 95 • C for 15 s, 60 • C for 1 min, 60-95 • C for 15 s. The primers used in this analysis are presented in Supplementary Table S1, and the expression data were normalized to those of Actin2 [72,73].

Determination of Ammonium in Xylem Sap
Seedlings were gathered and stripped of leaves, and the stems were cut 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 prepared for the double mutant and wild type. The xylem sap was collected from 20 plants; the total collected volume was approximately 50 µL per replicate [74]. Ammonium content was determined as described by Santoni et al. [67].

Determination of Cation Content
The method for determining cation content was a slightly modified protocol used in a previous study [75]. Shoots of the seedlings that were hydroponically grown for four weeks were collected and oven-dried at 105 • C for 30 min and then at 65 • C to constant weight. Dried samples were ground into a fine powder and about 100 mg of sample was mixed with 5 mL HNO 3 and heated in boiling water for 2 h. The volume of the extracting solution was adjusted to 50 mL with deionized water after cooling. The cation content was measured by ICP-MS (ELAN DRC-e, Perkin Elmer, Shelton, CT, USA).

Statistical Analyses
SPSS 17.0 (Ibm Corp., Chicago, IL, USA) was used for one-way analysis of variance (ANOVA) and Tukey's honestly significant difference (HSD) multiple comparison tests. All experiments were conducted using a completely randomized design. Three samples were prepared per treatment, with two technical replicates per sample. Differences between the wild type and vha-a2 vha-a3 double mutant and/or between different treatments were considered statistically significant for p < 0.05.

Supplementary Materials:
The following are available online at https://www.mdpi.com/1422-0 067/22/1/2/s1, Figure S1: The leaf area and root configuration of wild type and the vha-a2 vha-a3 double mutant, Figure S2: Chlorophyll concentration and NH4+ root/shoot ratio (R/S) of wild type and vha-a2 vha-a3 mutant, Figure S3: The phenotype of wild type and vha-a2 vha-a3 mutant with different Ca 2+ and K + supply levels; Figure S4: Nitrogen metabolites in wild type and vha-a2 vha-a3 mutant with different potassium concentrations applied, Figure S5: The leaf area and root configuration of wild type and the vha-a2 vha-a3 mutant with different K + doses applied; Table S1: Primers of the genes targeted in the qRT-PCR assays.