Arabidopsis V-ATPase d2 Subunit Plays a Role in Plant Responses to Oxidative Stress

Vacuolar-type H+-ATPase (V-ATPase), a multisubunit proton pump located on the endomembrane, plays an important role in plant growth. The Arabidopsis thaliana V-ATPase d subunit (VHA-d) consists of two isoforms; AtVHA-d1 and AtVHA-d2. In this study, the function of AtVHA-d2 was investigated. Histochemical analysis revealed that the expression of AtVHA-d1 and AtVHA-d2 was generally highly overlapping in multiple tissues at different developmental stages of Arabidopsis. Subcellular localization revealed that AtVHA-d2 was mainly localized to the vacuole. AtVHA-d2 expression was significantly induced by oxidative stress. Analysis of phenotypic and H2O2 content showed that the atvha-d2 mutant was sensitive to oxidative stress. The noninvasive microtest monitoring demonstrated that the net H+ influx in the atvha-d2 roots was weaker than that in the wild-type under normal conditions. However, oxidative stress resulted in the H+ efflux in atvha-d2 roots, which was significantly different from that in the wild-type. RNA-seq combined with qPCR analysis showed that the expression of several members of the plasma membrane H+-ATPase gene (AtAHA) family in atvha-d2 was significantly different from that in the wild-type. Overall, our results indicate that AtVHA-d2 plays a role in Arabidopsis in response to oxidative stress by affecting H+ flux and AtAHA gene expression.


Introduction
The pH homeostasis in the endomembrane system is important for secondary active transport, cargo sorting, and protein trafficking [1][2][3][4]. Plants employ three classes of proton pumps to regulate cellular pH homeostasis: (i) plasma membrane H + -ATPase (P-ATPase); (ii) vacuolar H +pyrophosphatase (V-PPase); and (iii) vacuolar-type H + -ATPase (V-ATPase) [1]. V-ATPase uses the energy released by ATP hydrolysis to transport protons (H + ) across the membrane to regulate pH in the plant endomembrane system. V-ATPase is a highly conserved, multisubunit complex that consists of two domains, the integral membrane V 0 domain, and the cytosolic V 1 domain. The V 0 domain, which consists of three to five different subunits of five subunits (VHA-a, VHA-c, VHA-c", VHA-d, VHA-e) on different organelles, is responsible for H + translocation across membranes. The V 1 domain,

Vector Construction and Arabidopsis Transformation
To construct proAtVHA-d1:GUS and proAtVHA-d2:GUS, 1644 and 1657 bp of the AtVHA-d1 and AtVHA-d2 promoter regions, respectively, were amplified using Col-0 genomic DNA and cloned into the HindIII/BamHI or KpnI/XhoI sites of pBI121-GUS or pBI121-GUS (modified, more enzyme digestion sites were added) vectors. To construct the AtVHA-d2-GFP fusion genes, the open reading frame of AtVHA-d2, without the stop codon, was amplified using PCR and cloned into the XbaI/KpnI sites of the pBI121-PutVHA-c-GFP vector, based on a previously reported method [14]. The constructs were stably transformed into Arabidopsis via the Agrobacterium tumefaciens-mediated floral dip method [25]. The T3 transgenic plants were identified by reverse transcription PCR. The specific primers used in this study are listed in Supplementary Table S1.

Confocal Laser Scanning Microscopy
Roots of Arabidopsis seedlings stably expressing AtVHA-d2-GFP were washed twice with liquid 1/2 MS medium immediately before visualizing via confocal laser scanning microscopy (Nikon, A1, Tokyo, Japan). Using the 40× oil-immersion objective lens (Plan Apochromat; numerical aperture 1.3), the observation area was 1024 × 1024 pixels, and the pixel dwell time was 0.497 µs. GFP signals were detected using a 500-530 nm emission wavelength (FITC) after excitation with a 488 nm laser.
Total RNA was extracted using the RNAprep pure plant kit (Tiangen, Beijing, China), and cDNA was synthesized from 1 µg of total RNA using the M-MLV RTase cDNA synthesis kit (TaKaRa, Shiga, Japan), according to the manufacturer's instructions. qPCR was performed on an Mx3000P QPCR system (Agilent Technologies, Palo Alto, CA, USA). The reaction components per 20 µL were as follows: 10 µL SYBR Green Mix (Agilent), 1 µL 10 µM of each primer and 1 µL cDNA, and 7 µL H 2 O. The thermal cycling program was as follows: initial denaturation at 95 • C for 120 s, and 40 cycles at 95 • C for 10 s, 60 • C for 30 s, and 72 • C for 30 s. The Arabidopsis AtActin2 gene was used as an internal control [14]. The relative quantification of gene expression was evaluated using the delta-delta-Ct method. The transcript level in untreated seedlings (control) was set as 1.0. The primers used in this study are shown in Supplementary Table S1.

Stress Tolerant Phenotype
For the stress tolerance assay, 30 seeds of Col-0 and the atvha-d2 mutant were treated at 4 • C for 3 days and then grown vertically on 1/2 MS medium (control) or 1/2 MS medium supplemented with different concentrations of NaCl (75 and 100 mM), H 2 O 2 (1 and 2 mM), or mannitol (175 and 200 mM). After 14 days, seedling phenotypes were photographed, and the root length, relative root length, and fresh weight of the seedlings were measured. The experiment was repeated three times.

Hydrogen Peroxide (H 2 O 2 ) Content Measurement
Ten-day-old seedlings of Col-0 and the atvha-d2 mutant were grown on 1/2 MS medium and transferred to 1/2 MS medium supplemented with H 2 O 2 (1, 2, and 3 mM) for 48 h. The 0.1 g seedlings were used to measure H 2 O 2 content. The H 2 O 2 content was measured using the H 2 O 2 -1-Y assay kits (Comin, Suzhou, China), according to the manufacturer's instructions.

Net H + Flux Measurement
Net H + flux was measured using the Noninvasive Microtest Technology (NMT100 Series, YoungerUSA LLC, Amherst, MA, USA) as described previously [13,26,27]. Seven-day-old seedlings of Col-0 and the atvha-d2 mutant were grown on 1/2 MS medium, and were exposed to mannitol (200 mM), NaCl (100 mM), and H 2 O 2 (2 mM) for 24 h. Root segments were immobilized in the measuring solution (0.1 mM KCl, 0.1 mM CaCl 2 , 0.1 mM MgCl 2 , 0.5 mM NaCl, and 0.3 mM MES, pH 5.8) to measure the H + flux. The roots were fixed to the bottom of the plate using resin blocks and filter paper strips. Each sample was measured continuously for 10 min. To take flux measurements, the ion-selective electrodes were calibrated using pH 5.5, 6.0, and 6.5 solutions, respectively. The H + flux rate, based on the voltages monitored between two points (0 and 20 µm), was calculated using iFluxes/imFluxes 1.0 software (Younger USA LLC, Amherst, MA, USA). The calibration slope for H + was 54.42 mV/decade. Six biological repeats were performed for each analysis.
Ion flux was calculated by Fick's law of diffusion: where J represents the ion flux in the x direction, dc/dx is the ion concentration gradient, and D is the ion diffusion constant in a particular medium.

RNA-seq and DEGs Analysis
Seedlings of Col-0 and the atvha-d2 mutant were treated in 1/2 MS medium supplemented with H 2 O 2 (2 mM) for 0, 12, and 24 h. Total RNA from the seedlings was isolated using TRIzol reagent (Invitrogen, Carlsbad, CA, USA). Subsequently, the RNA samples were sent to the Beijing Genomic Institute (Shenzhen, China) for RNA-seq.
RNA-seq data processing and DEGs analysis as previously described [28,29]. DEGs were screened with a false discovery rate threshold of 0.01 and an absolute log2 ratio of 1. All DEGs were mapped to each term of the KEGG module from a Kyoto Encyclopedia of Genes and Genomes (KEGG) databases, and significant pathways were defined based on a corrected p ≤ 0.05.

Statistical Analysis
All experiments were conducted at least in three independent biological and three technical replicates. The data were analyzed using a one-way analysis of variance in SPSS (SPSS, Inc., Chicago, IL, USA), and statistically significant differences were calculated using the Student's t-test, with p < 0.05 (*) and p < 0.01 (**) as the threshold for significance.

Tissue Specificity of AtVHA-d Genes Expression
AtVHA-d1 and AtVHA-d2 were two highly similar genes, and they shared 99.4% identity in the amino acid sequence ( Figure 1A). Transmembrane prediction using a hidden Markov model prediction showed that both AtVHA-d1 and AtVHA-d2 proteins had no transmembrane domain ( Figure 1B). To verify whether functions of AtVHA-d1 and AtVHA-d2 are redundant, their expression patterns were investigated by promoter-driven GUS reporter transgene. During early seedling development, the expression of proAtVHA-d1:GUS and proAtVHA-d2:GUS was detected in all tissues of the seedlings, including roots, stems, leaves, and stipule primordia (Figure 2A,B). In mature plants, both proAtVHA-d1:GUS and proAtVHA-d2:GUS were expressed in flowers and siliques, including sepal, anther, and embryo sac ( Figure 2C,D). In conclusion, AtVHA-d1 and AtVHA-d2 genes are broadly expressed in plant tissues and exhibit overlapping expression patterns.

Subcellular Localization of AtVHA-d2
Only two amino acids of AtVHA-d1 and AtVHA-d2 were different; thus the subcellular localization of one member (AtVHA-d2) in Arabidopsis was investigated using GFP as a fusion protein marker. The confocal images showed that the GFP signals were mainly localized to the vacuoles in root cells of wild-type Arabidopsis seedlings stably expressing AtVHA-d2-GFP ( Figure 3).

Subcellular Localization of AtVHA-d2
Only two amino acids of AtVHA-d1 and AtVHA-d2 were different; thus the subcellular localization of one member (AtVHA-d2) in Arabidopsis was investigated using GFP as a fusion protein marker. The confocal images showed that the GFP signals were mainly localized to the vacuoles in root cells of wild-type Arabidopsis seedlings stably expressing AtVHA-d2-GFP ( Figure  3).

Sensitivity of the Atvha-d2 Mutant to Multiple stresses
qPCR analysis showed that the expression of AtVHA-d1 was not affected by salt, osmotic, oxidative, and ABA stress, but AtVHA-d2 was significantly induced by these stressors. Under cold and heat treatment, the expression of both AtVHA-d1 and AtVHA-d2 was not obviously affected ( Figure 4A,B). The result suggests that AtVHA-d2 may play a role in the Arabidopsis response to multiple stresses. was investigated by qPCR. The AtActin2 gene was used as an internal control, and the transcript level in untreated seedlings was set as 1.0. Asterisks indicate a significant difference between untreated and stress-treated seedlings (* p < 0.05; ** p < 0.01; Student′s t-test). Error bars represent the SD (n = 3).

Sensitivity of the Atvha-d2 Mutant to Multiple Stresses
qPCR analysis showed that the expression of AtVHA-d1 was not affected by salt, osmotic, oxidative, and ABA stress, but AtVHA-d2 was significantly induced by these stressors. Under cold and heat treatment, the expression of both AtVHA-d1 and AtVHA-d2 was not obviously affected ( Figure 4A,B). The result suggests that AtVHA-d2 may play a role in the Arabidopsis response to multiple stresses.

Subcellular Localization of AtVHA-d2
Only two amino acids of AtVHA-d1 and AtVHA-d2 were different; thus the subcellular localization of one member (AtVHA-d2) in Arabidopsis was investigated using GFP as a fusion protein marker. The confocal images showed that the GFP signals were mainly localized to the vacuoles in root cells of wild-type Arabidopsis seedlings stably expressing AtVHA-d2-GFP ( Figure  3).

Sensitivity of the Atvha-d2 Mutant to Multiple stresses
qPCR analysis showed that the expression of AtVHA-d1 was not affected by salt, osmotic, oxidative, and ABA stress, but AtVHA-d2 was significantly induced by these stressors. Under cold and heat treatment, the expression of both AtVHA-d1 and AtVHA-d2 was not obviously affected ( Figure 4A,B). The result suggests that AtVHA-d2 may play a role in the Arabidopsis response to multiple stresses.  -d2 (B) was investigated by qPCR. The AtActin2 gene was used as an internal control, and the transcript level in untreated seedlings was set as 1.0. Asterisks indicate a significant difference between untreated and stress-treated seedlings (* p < 0.05; ** p < 0.01; Student′s t-test). Error bars represent the SD (n = 3). To better understand the role of AtVHA-d2 in response to multiple stresses, the T-DNA insertion mutant of AtVHA-d2 was identified. The genomic PCR and sequencing analysis showed that T-DNA was inserted into the fifth exon of the AtVHA-d2 gene ( Figure 5A-C; Supplementary Figure S1). qPCR analysis revealed that the AtVHA-d2 mRNA level in atvha-d2 was approximately 10% of that in Col-0. However, the AtVHA-d1 mRNA level in atvha-d2 was similar to that in Col-0 ( Figure 5D).
To better understand the role of AtVHA-d2 in response to multiple stresses, the T-DNA insertion mutant of AtVHA-d2 was identified. The genomic PCR and sequencing analysis showed that T-DNA was inserted into the fifth exon of the AtVHA-d2 gene (Figure 5A-C; Supplementary Figure S1). qPCR analysis revealed that the AtVHA-d2 mRNA level in atvha-d2 was approximately 10% of that in Col-0. However, the AtVHA-d1 mRNA level in atvha-d2 was similar to that in Col-0 ( Figure 5D). The phenotypes of the atvha-d2 mutant and Col-0 were compared under normal and multiple stress conditions. On 1/2 MS medium or 1/2 MS medium supplemented with NaCl (75 and 100 mM), H2O2 (1 and 2 mM), and mannitol (175 and 200 mM), the primary root length of the atvha-d2 mutant was generally lower than that of Col-0 ( Figure 6A, B). Under H2O2 and mannitol stress, the fresh weight of the atvha-d2 seedlings was significantly lower than that of Col-0, and there were no significant differences in the normal conditions (1/2 MS medium; Figure 6C). The relative root length analysis showed that the inhibition ratio of H2O2 on the primary root growth of the atvha-d2 mutant was significantly higher than that of Col-0 ( Figure 6D). Moreover, the H2O2 accumulation level in the atvha-d2 mutant was significantly higher than that in Col-0 under both normal and H2O2 stress ( Figure 7). These results suggest that the atvha-d2 mutant is sensitive to oxidative stress. The phenotypes of the atvha-d2 mutant and Col-0 were compared under normal and multiple stress conditions. On 1/2 MS medium or 1/2 MS medium supplemented with NaCl (75 and 100 mM), H 2 O 2 (1 and 2 mM), and mannitol (175 and 200 mM), the primary root length of the atvha-d2 mutant was generally lower than that of Col-0 ( Figure 6A,B). Under H 2 O 2 and mannitol stress, the fresh weight of the atvha-d2 seedlings was significantly lower than that of Col-0, and there were no significant differences in the normal conditions (1/2 MS medium; Figure 6C). The relative root length analysis showed that the inhibition ratio of H 2 O 2 on the primary root growth of the atvha-d2 mutant was significantly higher than that of Col-0 ( Figure 6D). Moreover, the H 2 O 2 accumulation level in the atvha-d2 mutant was significantly higher than that in Col-0 under both normal and H 2 O 2 stress (Figure 7). These results suggest that the atvha-d2 mutant is sensitive to oxidative stress.

H + Flux in Root of the Atvha-d2 Mutant under Multiple Stresses
The net H + flux in the roots of the atvha-d2 and Col-0 seedlings was monitored using NMT. The H + influx in the roots of atvha-d2 and Col-0 seedlings grown on 1/2 MS medium was observed, but the rate of H + influx in atvha-d2 was significantly lower than that in Col-0 ( Figure 8A,B). The result indicated that H + flux in the atvha-d2 roots is impaired. Furthermore, the H + flux in the roots of atvha-d2 and Col-0 was compared under multiple stresses. Similar to that in untreated roots (1/2 MS medium), the mannitol treatment had no obvious effect on H + flux in the atvha-d2 and Col-0 roots. The NaCl treatment resulted in H + efflux in both atvha-d2 and Col-0 roots, but there was no significant difference between them. Under the H 2 O 2 treatment, a H + influx in the Col-0 roots was observed; however, in the atvha-d2 roots, a H + efflux was observed ( Figure 8A,B). The result indicates that H 2 O 2 treatment significantly affects H + flux in the atvha-d2 roots.

H + flux in Root of the Atvha-d2 Mutant under Multiple Stresses
The net H + flux in the roots of the atvha-d2 and Col-0 seedlings was monitored using NMT. The H + influx in the roots of atvha-d2 and Col-0 seedlings grown on 1/2 MS medium was observed, but the rate of H + influx in atvha-d2 was significantly lower than that in Col-0 ( Figure 8A,B). The result indicated that H + flux in the atvha-d2 roots is impaired. Furthermore, the H + flux in the roots of atvha-d2 and Col-0 was compared under multiple stresses. Similar to that in untreated roots (1/2 MS medium), the mannitol treatment had no obvious effect on H + flux in the atvha-d2 and Col-0 roots. The NaCl treatment resulted in H + efflux in both atvha-d2 and Col-0 roots, but there was no significant difference between them. Under the H2O2 treatment, a H + influx in the Col-0 roots was observed; however, in the atvha-d2 roots, a H + efflux was observed ( Figure 8A, B). The result indicates that H2O2 treatment significantly affects H + flux in the atvha-d2 roots.

Identification of DEGs between Atvha-d2 and Wild-Type under Normal and Oxidative Stress Conditions
To identify DEGs between the atvha-d2 mutant and Col-0, RNA-seq was performed. Under normal and oxidative stress conditions, 278 DEGs (Supplementary Table S2) were identified ( Figure  9A, B). These DEGs were most significantly enriched in the V-type ATPase, eukaryotes module from the KEGG database ( Figure 9C). This module contains 28 genes (Supplementary Table S3) that, with the exception of AtVHA-d2, were upregulated in the atvha-d2 mutant ( Figure 9D). The result showed that the inhibition of AtVHA-d2 expression results in an upregulation of the expression of other V-ATPase assembly subunits under normal and oxidative stress conditions. Moreover, the

Identification of DEGs between Atvha-d2 and Wild-Type under Normal and Oxidative Stress Conditions
To identify DEGs between the atvha-d2 mutant and Col-0, RNA-seq was performed. Under normal and oxidative stress conditions, 278 DEGs (Supplementary Table S2) were identified ( Figure 9A,B). These DEGs were most significantly enriched in the V-type ATPase, eukaryotes module from the KEGG database ( Figure 9C). This module contains 28 genes (Supplementary Table S3) that, with the exception of AtVHA-d2, were upregulated in the atvha-d2 mutant ( Figure 9D). The result showed that the inhibition of AtVHA-d2 expression results in an upregulation of the expression of other V-ATPase assembly subunits under normal and oxidative stress conditions. Moreover, the expression of genes encoding P-ATPase (AtAHA) and V-PPase (AtAVP) in Col-0 and atvha-d2 were different under normal and oxidative stress conditions ( Figure 9E,F).

Expression of AtAHA and AtAVP genes in atvha-d2 and wild-type under normal and oxidative stress conditions
The expression of 11 AtAHA and two AtAVP genes in the atvha-d2 mutant and Col-0 under normal and oxidative stress conditions was compared by qPCR. Among the 11 AtAHA genes, the expression of AtAHA1, AtAHA2, and AtAHA7 in the atvha-d2 mutant was significantly lower than that in Col-0, whereas that of AtAHA4, AtAHA5, and AtAHA8 was significantly higher than that in Col-0 under normal conditions. After 24 h of oxidative stress, the expression of AtAHA1, AtAHA2, AtAHA4, and AtAHA5 in the atvha-d2 mutant was significantly higher than that in Col-0 ( Figure  10A). Two AtAVP genes, AtAVP1 and AtAVP2, did not show significant changes in expression between Col-0 and the atvha-d2 mutant ( Figure 10B). The results showed that the inhibition of the AtVHA-d2 expression causes changes in the expression of most members of AtAHA genes under normal and oxidative stress conditions.

Expression of AtAHA and AtAVP Genes in atvha-d2 and Wild-Type under Normal and Oxidative Stress Conditions
The expression of 11 AtAHA and two AtAVP genes in the atvha-d2 mutant and Col-0 under normal and oxidative stress conditions was compared by qPCR. Among the 11 AtAHA genes, the expression of AtAHA1, AtAHA2, and AtAHA7 in the atvha-d2 mutant was significantly lower than that in Col-0, whereas that of AtAHA4, AtAHA5, and AtAHA8 was significantly higher than that in Col-0 under normal conditions. After 24 h of oxidative stress, the expression of AtAHA1, AtAHA2, AtAHA4, and AtAHA5 in the atvha-d2 mutant was significantly higher than that in Col-0 ( Figure 10A). Two AtAVP genes, AtAVP1 and AtAVP2, did not show significant changes in expression between Col-0 and the atvha-d2 mutant ( Figure 10B). The results showed that the inhibition of the AtVHA-d2 expression causes changes in the expression of most members of AtAHA genes under normal and oxidative stress conditions.

Discussion
AtVHA-d1 and AtVHA-d2 are highly similar in sequence ( Figure 1A), and it is presumed that they may have arisen from a gene replication event [24]. Moreover, their expression pattern is highly overlapping. The promoter-driven GUS expression analysis showed that AtVHA-d1 and AtVHA-d2 were generally expressed in multiple tissues at different developmental stages of Arabidopsis, including all tissues of seedlings, especially the stipule primordia, as well as pollens and embryo sacs (Figure 2), indicating that they are nonredundant. In addition, the tissue specificity of the expression of AtVHA-d genes is highly consistent with that of AtVHA-c genes reported previously [14,30]. VHA-d and VHA-c are essential subunits of the complete V-ATPase enzyme [6]. VHA-d itself does not have a transmembrane domain; it can be located on the membrane by combining with VHA-c [5]. As was expected, confocal observation showed that AtVHA-d2 was mainly localized to the vacuoles (Figure 3). Furthermore, the localization pattern of AtVHA-d2 was similar to that of AtVHA-c5 [13]. The consistency of the expression pattern and subcellular localization suggests that AtVHA-d2, together with AtVHA-c, may be involved in the assembly of the V-ATPase in the expressed tissues. However, our observations cannot completely exclude AtVHA-d2-GFP localization on other endomembranes.
Studies have shown that V-ATPase is involved in multiple stress responses [12,23,31]. The expression of AtVHA-d genes was investigated under abiotic stress conditions that significantly inhibited the growth of Arabidopsis seedlings, including 150 mM NaCl, 300 mM mannitol, 5 mM H2O2, 100 µM ABA, cold (4 °C), and heat (37 °C). qPCR analysis showed that AtVHA-d2 was significantly induced by multiple stresses, especially oxidative stress (Figure 4). The phenotype of the atvha-d2 mutant and wild-type were compared under the conditions of abiotic stress that

Discussion
AtVHA-d1 and AtVHA-d2 are highly similar in sequence ( Figure 1A), and it is presumed that they may have arisen from a gene replication event [24]. Moreover, their expression pattern is highly overlapping. The promoter-driven GUS expression analysis showed that AtVHA-d1 and AtVHA-d2 were generally expressed in multiple tissues at different developmental stages of Arabidopsis, including all tissues of seedlings, especially the stipule primordia, as well as pollens and embryo sacs (Figure 2), indicating that they are nonredundant. In addition, the tissue specificity of the expression of AtVHA-d genes is highly consistent with that of AtVHA-c genes reported previously [14,30]. VHA-d and VHA-c are essential subunits of the complete V-ATPase enzyme [6]. VHA-d itself does not have a transmembrane domain; it can be located on the membrane by combining with VHA-c [5]. As was expected, confocal observation showed that AtVHA-d2 was mainly localized to the vacuoles ( Figure 3). Furthermore, the localization pattern of AtVHA-d2 was similar to that of AtVHA-c5 [13]. The consistency of the expression pattern and subcellular localization suggests that AtVHA-d2, together with AtVHA-c, may be involved in the assembly of the V-ATPase in the expressed tissues. However, our observations cannot completely exclude AtVHA-d2-GFP localization on other endomembranes.
Studies have shown that V-ATPase is involved in multiple stress responses [12,23,31]. The expression of AtVHA-d genes was investigated under abiotic stress conditions that significantly inhibited the growth of Arabidopsis seedlings, including 150 mM NaCl, 300 mM mannitol, 5 mM H 2 O 2 , 100 µM ABA, cold (4 • C), and heat (37 • C). qPCR analysis showed that AtVHA-d2 was significantly induced by multiple stresses, especially oxidative stress (Figure 4). The phenotype of the atvha-d2 mutant and wild-type were compared under the conditions of abiotic stress that moderately inhibited the growth of Arabidopsis seedlings, including 75 and 100 mM NaCl, 175 and 200 mM mannitol, and 1 and 2 mM H 2 O 2 . Phenotypic analysis showed that the relative root length and fresh weight of atvha-d2 were significantly lower than those of Col-0 under oxidative stress ( Figure 6). Furthermore, the H 2 O 2 content in atvha-d2 was higher than that in Col-0 under normal conditions and oxidative stress (Figure 7). Under normal culture conditions (1/2 MS), H + influx occurred in the atvha-d2 roots, whereas H 2 O 2 treatment caused H + efflux, which was significantly different from that in Col-0 ( Figure 8). These results suggest that atvha-d2 is sensitive to oxidative stress, which may be associated with abnormal H + flux in roots. The extrusion of H + in roots is directly regulated by P-ATPase [32]. In Arabidopsis, P-ATPase is encoded by 11 genes, AtAHA1 to AtAHA11, of which AtAHA1 and AtAHA2 are the most highly expressed isoforms [32,33]. RNA-seq combined with qPCR analysis showed that the expression of AtAHA1 and AtAHA2 in atvha-d2 was significantly higher than that in Col-0 after oxidative stress (Figures 9E and 10A). The results suggest that the H + efflux in atvha-d2 roots under oxidative stress (for 24 h) may be caused by the higher expression of AtAHA1 or AtAHA2. V-ATPase together with P-ATPase and V-PPase, co-regulates the cytosolic pH in the plant cells [1,3]. Compared to that in Col-0, the expression of genes encoding the other V-ATPase assembly subunits, P-ATPase and V-PPase in the atvha-d2 mutant were altered, as shown RNA-seq and qPCR (Figures 9 and 10). The result suggests that V-ATPase, P-ATPase, and V-PPase play synergistic roles in H + balance through gene expression regulation. For example, the expression of AtVHA-c, AtAVP1, AtAHA1, and AtAHA2 is simultaneously affected in the atsos1 mutant (SOS1, a plasma membrane Na + /H + -antiporter) [34].

Conclusions
In conclusion, our results indicate that AtVHA-d2 plays a role in Arabidopsis in response to oxidative stress by affecting H + flux, which may be related to differential expression of AtAHA genes.