Arabidopsis plasma membrane H+-ATPase genes AHA2 and AHA7 have distinct and overlapping roles in the modulation of root tip H+ efflux in response to low-phosphorus stress

Highlight Two plasma membrane H+-ATPases, encoded by AHA2 and AHA7, enhance primary root elongation and root hair development by mediating H+ efflux in the elongation and root hair zones under low-phosphorus stress.


Introduction
Phosphorus is one of the most important nutrients for plant growth, development, and reproduction. Even when the phosphorus content of soil is high, phosphorus deficiency can arise due to mineralization and fixation processes.
Thus, low-phosphorus stress (LP) is one of the major limiting factors for plant growth in many ecosystems and a major constraint for agricultural productivity (Devaiah et al., 2007;Schachtman and Shin, 2007). In order to survive, plants have developed flexible strategies to cope with low-phosphorus soil by improving phosphorus mobilization and uptake from the soil (Vance et al., 2003;Wu et al., 2003;Hoffland et al., 2006;Li et al., 2009). These strategies include the production of enzymes, such as acid phosphatases and nucleases; increased synthesis and secretion of organic acids or protons (H + ) in the roots; enhanced expression of high-affinity phosphorus transporters; development of a larger root system capable of exploring a greater soil volume to ensure a sufficient uptake and use of phosphorus; and establishment of differential photosynthate distribution between shoots and roots, resulting in prolific root growth and increased root/shoot ratios. Among these strategies, plant plasma membrane H + -ATPase (PM H + -ATPase) plays the key role in the response to LP. Phosphorus acquisition and utilization are a highly regulated and complex set of processes relying not only on PM H + -ATPase-dependent H + release, but also on PM H + -ATPase regulatory pathways controlling the abundance and activity of transporters and enzymes involved in phosphorus deficiency responses (Yan et al., 2002).
In plants, PM H + -ATPases constitute a family of proton pumps driven by ATP hydrolysis. Their role is to provide an energy source for the transport of nutrients into the plant cell by generating an electrochemical gradient (Haruta and Sussman, 2012). Most of the membrane-bound transport proteins in plants are driven indirectly by the action of PM H + -ATPase (Palmgren, 2001). PM H + -ATPases form a large family of proteins in plants. For example, in Arabidopsis, there are 11 functional PM H + -ATPase family members, denoted AHA1 to AHA11 (for Arabidopsis H + -ATPase). Of the 11 genes encoding functional PM H + -ATPase proteins in plants, the first to be cloned were AHA1 and AHA3. Using genetic analysis, roles for AHA1 in steroid signaling, AHA2 in iron transport, AHA3 in pollen development, AHA4 in salt stress, and AHA10 in vacuole biosynthesis have been reported (Robertson et al., 2004;Baxter et al., 2005;Gévaudant et al., 2007;Santi and Schmidt, 2009;Caesar et al., 2011). Using RT-PCR, Ueno et al. (2005) determined that eight AHA genes are expressed in green leaves (AHA1,2,3,5,7,8,10,and 11), eight are expressed in roots (AHA1,2,3,4,7,8,10,and 11), and three are expressed in all tissues (AHA1, 2 and 11).
Plant PM H + -ATPases are regulated by a number of factors, including blue light, fusicoccin, protein kinase, 14-3-3 proteins, and auxin (Palmgren, 2001;Hager, 2003;Rodrigues et al., 2014). The C-terminus of plant PM H + -ATPases serves as an autoinhibitory domain to regulate enzymatic activity. The regulatory C-terminal domain of PM H + -ATPases is phosphorylated in vivo. In vitro, the domain becomes phosphorylated within seconds of exposure to blue light and fusicoccin. Phosphorylation of PM H + -ATPase Thr-947 by a protein kinase generates a binding site for 14-3-3 proteins. Activated PM H + -ATPases consist of six phosphorylated PM H + -ATPase molecules, assembled in a hexameric structure, together with six 14-3-3 molecules. Auxin also regulates PM H + -ATPase activity by phosphorylation; auxin-enhanced PM H + -ATPase activity lowers the pH of the cell wall, activates pH-sensitive enzymes and proteins within the cell wall, and initiates cell-wall loosening and extension growth. Auxin can also increase the quantity of certain PM ATPases by influencing protein expression. This results not only in enhanced H + extrusion, but also eventually in the restoration of the number of PM H + -ATPases in a given membrane area.
Root growth, including primary root elongation and root hair formation, is important for the uptake and use of phosphorus (Shen et al., 2006). Optimal primary root elongation or root hair formation requires fine regulation of root tip H + secretion by PM H + -ATPase (Palmgren, 2001;Staal et al., 2011;Haruta & Sussmann, 2012). Acidification of the aqueous fraction of the cell wall apoplast by H + excretion via PM H + -ATPase is a critical growth-promoting event and a key factor in triggering elongation of the primary root (Moloney, 1981). Root hair development in Arabidopsis is associated with alkalinization of the cytoplasm and acidification of the cell wall, and these processes are regulated by PM H + -ATPase-independent H + influx and efflux (Bibikova et al., 1998). However, little is known regarding the role of PM H + -ATPases in mediating root H + flux under LP. Thus, in this study, we investigated the involvement and function of different Arabidopsis PM H + -ATPase genes in root H + flux in response to LP.
Before planting, Arabidopsis seeds were surface-sterilized with 70% ethanol for 1 min, then with 1% sodium hypochlorite solution plus sodium dodecyl sulfate for 9 min, and subsequently rinsed with sterile deionized water six times. Seeds were then kept in the dark for 3 days at 4 °C for stratification and grown hydroponically using a sugar-free agar medium-based culture system, as described by Xu and Shi (2008). Sterilized seeds were sown on a sucrose-free medium containing full-strength Murashige and Skoog nutrients and 6 g L -1 agar. This sugar-free agar medium was dispensed into bottomremoved Eppendorf tubes, which were held in a plastic platform. Two to three seeds were sown in each tube. Seedlings were thinned to one plant per tube after 7 days of growth. The nutrient solution consisted of: 5 mM KNO 3 , 1 mM KH 2 PO 4 , 2 mM MgSO 4 , 2 mM Ca(NO 3 ) 2 , 0.5 mM Fe-Na-EDTA, 70 μM H 3 BO 3 , 14 μM MnCl 2 , 0.5 μM CuSO 4 , 1 μM ZnSO 4 , 0.2 μM Na 2 MoO 4 , 10 μM NaCl, and 0.01 μM CoCl 2 . The pH of the solution was adjusted to 6.0 every day and the solution renewed every 2 days. Low-phosphorous (1 μM) conditions were achieved in the nutrient solution by replacing 1 mM KH 2 PO 4 with 0.999 mM KCl and 0.001 mM KH 2 PO 4 . Arabidopsis plants were grown at 22 ± 1 °C, with a light intensity of 120 μmol photons m -2 s -1 , a 16 h light/8 h dark photoperiod, and a relative humidity of 70% in the growth chamber (Sanyo Electric Co., Ltd, Kyoto, Japan). Twenty-two-day-old Arabidopsis plants were treated with 1 mM phosphorus (control) or 1 μM phosphorus (LP: lowphosphorus stress) for 8 days within the hydroponic system. Control plants and LP-treated plants were harvested at 0-, 1-, 2-, 4-, 6-, and 8-day time points. After harvesting, Arabidopsis plants were frozen immediately in liquid nitrogen and stored at -80 °C until analysis.

Real-time RT-PCR
Real-time RT-PCR was performed according to the method described by Xu and Shi (2006). Total RNA was extracted from Arabidopsis plants under control and LP conditions. Gene-specific primers for real-time RT-PCR were designed using Primer 5 software (see Supplementary Table S1). At-ACT2 is a strongly and constitutively expressed 'housekeeping' gene in Arabidopsis plants; the mRNA expression level of this gene was designated to be 100 relative expression units (REU). Expression levels of the genes of interest are described relative to At-ACT2, specifically, as the ratio of the copy number of the cDNA of interest to the copy number of At-ACT2 multiplied by 100 REU.

Measurement of root growth and phosphorus concentration
Root growth was measured according to the method of Xu et al. (2013b). Root surface area was measured using a root analysis instrument (WinRHIZO; Regent Instruments Inc., Quebec, ON, Canada). The primary root elongation rate of Arabidopsis (μm h -1 ) was calculated based on the displacement of the primary root apex relative to the primary root length over the duration of the experiment. Root hairs in the region 0-5000 μm from the root cap junction were identified in Arabidopsis plants using confocal laser scanning microscopy (Olympus FV-1000 spectral type SPD mar/G/R IX81 Fluoview laser confocal system). Average root hair densities in 100 × 100 μm 2 sections of the 0-5000 μm region were calculated. The phosphorus concentrations of Arabidopsis roots were determined colorimetrically by the phosphovanadate method (Hanson, 1950) after digestion in a mixture of HNO 3 , HClO 4 , and H 2 SO 4 (3:1:1, v/v).

Analysis of proton extrusion
The proton extrusion rate of Arabidopsis roots was analysed following the method of Jin et al. (2009). Briefly, 22-day-old Arabidopsis plants were treated with 1 mM phosphorus (control) or 1 μM phosphorus (LP) for 8 days. Arabidopsis plants were transferred to 100-mL pots filled with 1 mM phosphorus solution or 1 μM phosphorus solution. After 12 h, each solution was filtered, and the pH was adjusted to 6.0. The quantity of protons excreted by roots was determined by titrating the solution with 1 mM NaOH.

PM H + -ATPase activity and H + flux in the root tip of Arabidopsis plants
PM H + -ATPase activity was determined according to the method described by Xu et al. (2012). H + fluxes were measured non-invasively using the scanning ion-selective electrode technique (SIET; SIET system BIO-003A; Younger USA Science and Technology Corp.; Applicable Electronics Inc.; Science Wares Inc., Falmouth, MA, USA), also according to the method described by Xu et al. (2012). Under normal growth conditions (control) or low-phosphorus stress (LP), proton (H + ) flux was measured along the root tip of Arabidopsis plants in the following zones: meristem zone (0-200 μm from the root cap junction), transition zone (200-520 μm from the root cap junction), elongation zone (520-850 μm from the root cap junction), and root hair zone (850-1500 μm from the root cap junction).

Expression of Arabidopsis PM H + -ATPase genes in response to LP
To investigate whether Arabidopsis PM H + -ATPase gene family members are regulated by soil phosphorus levels, we examined the expression of PM H + -ATPase genes (AHA1-AHA11) under LP conditions using real-time RT-PCR. To avoid biased results, real-time RT-PCR data are typically expressed relative to the expression of a housekeeping gene (i.e. At-ACT2) as an internal control. Using real-time RT-PCR, expression patterns of the 11 PM H + -ATPase genes were analysed in whole Arabidopsis plants (leaf and root) under phosphorous-sufficient conditions (control) or low-phosphorus stress (LP) for 4 days (Fig. 1). Under LP, the steady state transcript levels of most PM H + -ATPase gene family members appeared relatively unchanged relative to the control condition, but significant differences in three genes, AHA1, AHA2, and AHA7, were observed. The expression of selected PM H + -ATPase genes (AHA1, AHA2, and AHA7) was further investigated in Arabidopsis roots under control or LP conditions over 0, 1, 2, 4, 6, and 8 days (Fig. 2). During LP, from day 0 to day 4, the expression of AHA1, AHA2, and AHA7 in the roots increased approximately 2-, 4-, and 5-fold, respectively, compared with the control condition. After LP for 8 days, the expression level of AHA2 and AHA7 in the roots was approximately 2.5-fold that of the phosphorus-sufficient plants, but no significant change was found in the expression of AHA1 in roots over this time period.

Response of PM H + -ATPase-deficient mutant roots to LP
PM H + -ATPases play an important role not only in nutrient uptake by roots, but also in root adaption to environmental stress (Desnos, 2008;Xu et al., 2013a). We therefore analysed root phosphorus content, root surface area, primary root elongation, root hair density, PM H + -ATPase activity, proton extrusion, and proton flux in the roots of Col-0 (WT Arabidopsis plant), aha1 mutant lines (aha1-1 and aha1-2), aha2 mutant lines (aha2-1 and aha2-2), and aha7 mutant lines (aha7-1 and aha7-2) under normal growth conditions (control) and LP (Table 1 and Fig. 3). As shown in Table 1A, there was no significant difference in phosphorus content between the AHA-deficient mutants and Col-0 under control conditions. Under LP, no significant change was found in the phosphorus content of aha1 mutant lines compared with Col-0; however, the phosphorus contents of aha2 and aha7 mutant lines were significantly lower than that of Col-0. As shown in Table 1B, there was no significant difference in the root surface area between AHA-deficient mutants and Col-0 under control conditions. Under LP, no significant change was found in the root surface area of aha1 mutant lines compared with Col-0; however, the root surface areas of aha2 and aha7 mutant lines were significantly lower than that of Col-0. As shown in Table 1C, there was no significant difference in primary root elongation between AHA-deficient mutants and Col-0 under control conditions. Under LP, no significant change was found in the primary root elongation of aha1 or aha7 mutant lines compared with Col-0; however, the primary root elongation of aha2 mutant lines was significantly lower than that of Col-0. As shown in Table 1D, there was no significant difference in root hair density between AHA-deficient mutants and Col-0 under control conditions. Under LP, no significant change was found in root hair density of aha1 or aha2 mutant lines compared with Col-0; however, the root hair density of aha7 mutant lines was significantly lower than that of Col-0. As shown in Table 1E, there was no significant difference in PM H + -ATPase activity between AHA-deficient mutants and Col-0 under control conditions. Under LP, no significant change was found in PM H + -ATPase activity of aha1 mutant lines compared with Col-0; however, the PM H + -ATPase activities of aha2 and aha7 mutant lines were significantly lower than that of Col-0. As shown in Table 1F, there was no significant difference in proton extrusion between AHA-deficient mutants and Col-0 under control condition. Under LP, no significant change was found in proton extrusion in aha1 mutant lines compared with Col-0; however, proton extrusion was significantly lower in aha2 and aha7 mutant lines relative to Col-0. We also investigated proton (H + ) flux in the meristem zone (MZ), transition zone (TZ), elongation zone (EZ), and root hair zone (RHZ) in the root tip of WT Arabidopsis (Col-0), aha2 mutant lines (aha2-1 and aha2-2), and aha7 mutant lines (aha7-1 and aha7-2) under normal growth conditions (control) and LP. As shown in Fig. 3, there was no significant difference in proton flux between AHA-deficient mutants and Col-0 under control conditions. Under LP, there was no significant difference in proton flux in the MZ and TZ of AHA-deficient mutants and Col-0. However, under LP, proton efflux in the EZ of aha2 mutant lines was significantly lower than in the EZ of Col-0 and aha7. Finally, under LP, proton efflux in the RHZ of aha7 mutant lines was significantly lower than in the EZ of Col-0 and aha2.

AHA2 plays a regulatory role in H + efflux in the root elongation zone in the context of primary root elongation in response to LP
One of the most pronounced adaptations of plants to low-phosphorus soil conditions is the enhancement of root elongation (Richardson, 2009). Primary root elongation, regulated by a sensory zone in the root tip, plays an important role in the plastic acclimation response to fluctuating soil environments (Baluska et al., 2010). PM H + -ATPase-regulated proton (H + ) excretion has an important function in this process. The acid growth theory implicates that apoplastic H + is the major wall-loosening factor that causes root cell extension (Staal et al., 2011). Apoplastic H + is mainly attributed to H + efflux mediated by PM H + -ATPase (Palmgren, 2001). Wang et al. (2014) proposed that AHA2 is useful for promoting plant growth by modulating light-induced stomatal opening. AHA2 is also important in maintaining the plasma membrane proton motive force (PMF) during plant seedling growth (Haruta and Sussman, 2012). We found that the expression of AHA2 is up-regulated in roots under LP (Figs 1 and 2). As shown in Table 1, when AHA2 is deficient in Arabidopsis plants, as in the aha2 mutant, root phosphorus content, root surface area, PM H + -ATPase activity, and root proton extrusion are lower than in the Col-0 (WT) and the aha1 mutant. Although there was no significant difference in root hair density between aha2 and Col-0, primary root elongation was lower in aha2 than in Col-0 and aha7. Further, under LP, although no significant difference in H + efflux of the root hair zone was found between Col-0 and aha2, H + efflux in the root elongation zone of aha2 was significantly lower than that in Col-0 (Fig. 3). Thus, our results suggest that AHA2 plays a regulatory role in H + efflux in the root elongation zone in the context of the primary root elongation response to LP.

AHA7-H + -mediated efflux in the root hair zone is important for root hair formation under LP
Plants have developed physiological and morphological adaptations to acquire phosphorus from the soil. These adaptations include not only increased root length for phosphorus uptake over a greater soil volume, but also root hair formation for more efficient acquisition of phosphorus (Raghothama, 1999;Hammond et al., 2004;Lambers et al., 2006). Studies have shown that PM H + -ATPase-regulated H + flux is important for root hair development (Palmgren, 2001). For instance, PM H + -ATPase protein is highly concentrated in root hairs, and several isoforms of plant PM H + -ATPase localize to root hairs. H + circuits have also been found in root hairs: H + enters the root hair at the root tip and leaves the root hair below this tip. AHA7 is the PM H + -ATPase responsible for iron deficiency-induced differentiation of rhizodermic cells. The action of AHA7 is not affected by the modulation of Arabidopsis 14-3-3 protein GRF11 under conditions of iron deficiency (Yang et al., 2013). We also found that the expression of AHA7 is up-regulated in roots under LP ( Fig. 1  and Fig. 2). According to Table 1, when AHA7 is deficient in Arabidopsis plants, as in the aha7 mutant, root phosphorus content, root surface area, PM H + -ATPase activity, and root proton extrusion are lower than in the Col-0 (WT) and aha1 mutant. Although there was no significant difference in primary root elongation between aha7 and Col-0, root hair density of aha7 was lower than that of Col-0 and aha2. Table 1. Phosphorus content, root surface area, elongation rate of primary root, root hair density, activity of PM H + -ATPase and proton extrusion in the roots of Col-0 (wild-type Arabidopsis plant), aha1 mutant lines (aha1-1 and aha1-2), aha2 mutant lines (aha2-1 and aha2-2) and aha7 mutant lines (aha7-1 and aha7-2) under normal growth condition (Control)

or low phosphorus stress (LP)
Twenty-two-day-old Arabidopsis plants (wild-type and mutant lines) were treated with 1 mM phosphorus (Control) and 1 μM phosphorus (LP) for 8 days under hydroponic system. Then, 30-day-old Arabidopsis plants were used for experimental analysis. The data were subjected to analysis of variance and post hoc comparisons were performed with Duncan's multiple range test at the P<0.05 level. The statistical software program used was SPSS version 13.0. The values are the means and SD of six replicates from two independent experiments. Values with the same letter (a or b or c) are not significantly different at P<0.05 level under the same treatment (control or LP).

Effect on AHA2 or AHA7 regulation of the action of 14-3-3 proteins or auxin modulation in the root response to LP
In plants, 14-3-3 proteins regulate the activity of PM H + -ATPases (Chevalier et al., 2009). The binding of 14-3-3 proteins to PM H + -ATPases blocks the autoinhibitory action of the PM H + -ATPase C-terminus, thus increasing PM H + -ATPase activity. The 14-3-3 binding sites on Arabidopsis were mapped to the extreme C-terminus, where the penultimate threonine is phosphorylated. The plant 14-3-3 protein family has many members, with high levels of conservation, and most can bind PM H + -ATPase. Environmental factors, such as light, can stimulate 14-3-3 interactions with PM H + -ATPase (Kinoshita and Shimazaki, 1999). This increase in PM H + -ATPase activity causes guard cells to swell, opening the stomata. Our previous work showed that the tomato 14-3-3 protein TFT7 acts mainly in the root and is involved in the local response of plants to LP by activating root plasma membrane H + -ATPase (Xu et al., 2012). By interbreeding strains, we obtained TFT7-overexpressing Arabidopsis plants (OE-TFT7) that lacked AHA2 or AHA7. As shown in Table 2, under LP, root phosphorus content, root surface area, PM H + -ATPase activity, and root proton extrusion of OE-TFT7/aha2 and OE-TFT7/aha7 were significantly lower  Table 1.
root proton extrusion of aha2 or aha7 was significantly lower than that of Col-0 (wild-type) under LP. And also, when AHA2 or AHA7 was deficient in TFT7-overexpressing plants (OE-TFT7), root proton extrusion of OE-TFT7 under LP was decreased greatly. Thus, our results suggest that AHA2 or AHA7 is involved in the action of 14-3-3 proteins in the root response to LP.
In plants, auxin also regulates the activity of PM H + -ATPases. The acid growth theory implicates (Hager, 2003) that (i) PM H + -ATPase is responsible for the extrusion of H + into the cell wall; (ii) auxin enhances the activity of PM H + -ATPase, thus causing a lowering of the cell wall pH; (iii) the reduced pH in the cell wall activates cell wall-loosening enzymes and initiates the enlargement of the cell. Our previous results also suggest that auxin modulation and auxin cell-cell transport are key regulators of acidity-induced cell elongation in the root under alkaline stress (Xu et al., 2013a). In this study, under LP, primary root elongation and H + efflux in the root elongation zone of OE-TFT7 were significantly higher than in TFT7/aha2 (Fig. 5). In addition, the root hair density and H + efflux in the root hair zone of OE-TFT7 were significantly higher than in TFT7/aha7. Under PM H + -ATPase inhibition or auxin flux inhibition, however, no significant difference in these indexes was found among these treated plants (Fig. 5). Thus, our results suggest that the H + -mediated regulation of AHA2 and AHA7 is affected by auxin modulation in the root response to LP. In conclusion, H + secretion, regulated by PM H + -ATPases, plays an important role in the plant response to LP. On the one hand, H + extrusion in the rhizosphere is important for the release of phosphorus from the soil. On the other hand, H + efflux (cell-cell transport) in the root can loosen the cell wall to promote root cell elongation to further increase phosphorus uptake. In this process, AHA2 plays a regulatory role in primary root elongation by modulating H + efflux in the root elongation zone in response to LP. Similarly, AHA7 is important for root hair formation, because it mediates H + efflux in the root hair zone under LP. The H + -mediated regulation of AHA2 or AHA7 are affected by 14-3-3 proteins and auxin modulation in the root response to LP. We conclude that AHA2 and AHA7, which encode two Arabidopsis plasma membrane H + -ATPases, differentially modulate H + efflux in the root tip and thereby have distinct and overlapping roles in response to LP.

Supplementary data
Supplementary data are available at JXB online. Table S1. Gene specific primers used in PCR for PM ATPase gene family members (AHA1-AHA11) or Salk lines in Arabidopsis plants.