Overexpression of a Plasma Membrane H⁺-ATPase Gene OSA1 Stimulates the Uptake of Primary Macronutrients in Rice Roots

Abstract

Plasma membrane (PM) H⁺-ATPase is a master enzyme involved in various plant physiological processes, such as stomatal movements in leaves and nutrient uptake and transport in roots. Overexpression of Oryza sativa PM H⁺-ATPase 1 (OSA1) has been known to increase NH₄⁺ uptake in rice roots. Although electrophysiological and pharmacological experiments have shown that the transport of many substances is dependent on the proton motive force provided by PM H⁺-ATPase, the exact role of PM H⁺-ATPase on the uptake of nutrients in plant roots, especially for the primary macronutrients N, P, and K, is still largely unknown. Here, we used OSA1 overexpression lines (OSA1-oxs) and gene-knockout osa1 mutants to investigate the effect of modulation of PM H⁺-ATPase on the absorption of N, P, and K nutrients through the use of a nutrient-exhaustive method and noninvasive microtest technology (NMT) in rice roots. Our results showed that under different concentrations of P and K, the uptake rates of P and K were enhanced in OSA1-oxs; by contrast, the uptake rates of P and K were significantly reduced in roots of osa1 mutants when compared with wild-type. In addition, the net influx rates of NH₄⁺ and K⁺, as well as the efflux rate of H⁺, were enhanced in OSA1-oxs and suppressed in osa1 mutants under low concentration conditions. In summary, this study indicated that overexpression of OSA1 stimulated the uptake rate of N, P, and K and promoted flux rates of cations (i.e., H⁺, NH₄⁺, and K⁺) in rice roots. These results may provide a novel insight into improving the coordinated utilization of macronutrients in crop plants.

1. Introduction

Plasma membrane (PM) H⁺-ATPase, belonging to the P-type ATPase superfamily, is highly conserved in plant species [1]. In rice, there are ten PM H⁺-ATPase genes (OSA1–OSA10). PM H⁺-ATPases generate membrane potential and provide proton motive force for active transport of nutrients, sugars, and other substances. PM H⁺-ATPase activities are regulated at multiple levels, including transcriptional, translational, and post-translational regulations [2]. Environmental stresses, including salinity, alkalinity, nutrient deficiency, and low pH, have been known to activate PM H⁺-ATPases [3,4,5]. The phosphorylation of PM H⁺-ATPase at the penultimate threonine (Thr) residue activates the pump by creating a binding site for 14-3-3 proteins [6,7,8]. Phosphorylation at the penultimate Thr of PM H⁺-ATPases is involved in regulating a variety of physiological processes, such as stomatal opening, hypocotyl elongation, and root growth [8,9,10,11]. While the phosphorylation of PM H⁺-ATPase plays important roles in regulating the physiological functions of plants, the transcriptional regulation of PM H⁺-ATPase genes remains the fundamental basis for controlling PM H⁺-ATPase activity, which is essential for the transmembrane transport of mineral nutrients.

Plants require nitrogen (N), phosphorus (P), and potassium (K) to grow and maintain a variety of metabolic processes [12,13,14]. PM H⁺-ATPase is required for low P adaptation in plants such as soybeans [15]. As a result of P deficiency in white lupin, PM H⁺-ATPase activity is induced in cluster roots, which is associated with citrate release and rhizosphere acidification [16]. Overexpression of a tomato PM H⁺-ATPase gene, SlHA8, promotes N and P uptakes in tomato roots [17]. Furthermore, it has been demonstrated that K⁺ transport is chemically linked to ATP hydrolysis [18]. PM H⁺-ATPase provides a driving force for cellular K⁺ uptake mediated by K⁺ channels or H⁺/K⁺ symporters. PM H⁺-ATPase energizes multiple ion channels and various H⁺-coupled transporters, such as HAK1 (a high affinity K⁺ transporter), PHTs (H⁺-coupled phosphate transporters), and NRTs (H⁺/NO₃⁻ symporters) [19], by generating H⁺ gradient across the PM and membrane potential to drive the transport of these nutrient ions [20]. Several isoforms of PM H⁺-ATPase in rice have been demonstrated to mediate N, P, and K uptakes. For example, NO₃⁻ was found to induce expression of OSA2, OSA5, OSA7, and OSA8 in rice roots [21]. Rice osa8 knockout mutants showed reduced P concentration in the shoots [3]. In some cases, overexpression of OsHAK5, a potassium transporter gene, improved PM H⁺-ATPase activity [22]. In our previous studies, we found that overexpression of the OSA1 gene could improve the NH₄⁺ uptake rate, increase plant growth, and enhance the total nutrient amount per plant, such as P and K [23]. However, we did not determine the P and K uptake rates in previous studies, and it is still ambiguous whether OSA1 gene overexpression could stimulate P and K uptakes. In this study, we first estimated the N, P, and K uptake rates of OSA1 overexpression plants and knockout mutant plants by cultivating them with hydroponic culture under different concentrations of P and K by using nutrient-exhaustion methods. We also used noninvasive microtest technology (NMT) methods to analyze the flux rates of NH₄⁺, K⁺, and H⁺ with OSA1 overexpression plants and knockout mutant plants under the high-affinity transport conditions. We found that the overexpression of OSA1 significantly improved P and K uptake rates in roots. This study could help to demonstrate the crucial role of PM H⁺-ATPases in coordinating N, P, and K absorptions.

2. Results

2.1. OSA1-Overexpression Promotes Rice Growth under Different Concentrations of P and K

Hydroponically grown OSA1 overexpression lines (OSA1-oxs), osa1 mutants, and WT rice plants were investigated for phenotypic differences under different concentrations of P and K (normal concentrations of P and K (PK treatment), 0.5-fold P and 0.5-fold K (0.5PK treatment), and 2-fold P and 2-fold K (2PK treatment)). OSA1-oxs exhibited improved root and shoot growth when compared with WT (Figure 1A). Under PK treatment, root biomass of OSA1-oxs was 30% higher than that of WT. However, when supplied with 2PK, the root biomass reached to 130% higher than that of WT (Figure 1B). In addition, osa1 mutants showed reduced root biomass by approximately 37–44% under 0.5PK treatment when compared with WT (Figure 1B). It is interesting that the root dry weight of WT under 2PK treatment decreased by 41% compared with that of control (PK treatment), whereas OSA1-oxs did not exhibit any growth defects (Figure 1B). These results suggest that high concentrations of P and K inhibit root growth, but higher activities of PM H⁺-ATPases could alleviate this inhibition. The shoot biomass also significantly increased in OSA1-oxs under all treatments but decreased in osa1 mutants under 2PK treatment when compared with WT (Figure 1C). Furthermore, we determined the root surface area of OSA1-oxs and osa1 mutants using root scanner; the root surface area is positively correlated with root dry weight. Under PK and 2PK treatments, the root surface area of OSA1-oxs was significantly higher than that of WT, and the root surface area of osa1 mutants was reduced under 0.5PK and PK treatments (Figure 1D). These results demonstrate that overexpression of OSA1 can improve plant growth under moderate, low, and high concentrations of P and K.

2.2. PM H⁺-ATPase Activity in Rice Roots of OSA1-Modified Plants under Different Concentrations of P and K

We next performed qRT-PCR assay to determine OSA1 gene expression levels in WT, OSA1-oxs, and osa1 mutants under different concentrations of P and K. Results showed that the relative expression levels of OSA1 were significantly enhanced in the roots of OSA1-oxs but were decreased in osa1 mutants as compared with WT under different concentrations of P and K (Figure 2A). Further investigation of rice root PM H⁺-ATPase activity was conducted under different concentrations of P and K. Results showed that PM H⁺-ATPase activity of WT increased by 18% under the 0.5PK treatment and was suppressed by approximately 33% under the 2PK treatment compared to that of the PK treatment (Figure 2B), which indicates that high concentrations of P and K inhibit PM H⁺-ATPase activity. In addition, OSA1-oxs showed significantly higher PM H⁺-ATPase hydrolysis activity under all treatments when compared with WT. By contrast, osa1 mutants showed significantly reduced PM H⁺-ATPase activity compared to WT under the 0.5 Pk and PK treatments (Figure 2B).

2.3. Overexpression of OSA1 Stimulates P, K, and N Uptake Rates under Different Concentrations of P and K

Using the nutrient-exhaustion method, we examined the uptake rates of N, P, and K in rice roots after incubation in a nutrient solution with different concentrations of P and K for 1, 2, and 4 h. As a result of incubation for one hour, the N uptake rate averagely increased by approximately 20.7%, 22.8%, and 24% in OSA1-oxs roots, and averagely decreased by approximately 16.1%, 14.7%, and 13.2% in osa1 mutant roots under the 0.5PK, PK, and 2PK treatments, respectively (Figure 3A). OSA1-oxs roots absorbed K with an average rate of about 14.2%, 24.5%, and 34.9% higher than that of WT under the 0.5PK, PK, and 2PK treatments, respectively, but the uptake rate showed no significant difference in osa1 mutants (Figure 3B). The P uptake rate increased averagely by 11.5% in OSA1-oxs under the 0.5PK treatment and there was no significant difference between the osa1 mutants and WT under all treatments (Figure 3C). After incubation for 2 h, the N uptake rate of OSA1-oxs roots averagely increased by approximately 31.3%, 38.4%, and 43.7% under the 0.5PK, PK, and 2PK treatments, respectively, and it exhibited no significant difference in osa1 mutant roots (Figure 3D). The average K absorption rate of OSA1-oxs roots was 27.4%, 20.5%, and 24.6% higher under 0.5PK, PK, and 2PK treatments, respectively, whereas there was no difference between osa1 mutants and WT (Figure 3E). The average P uptake rate increased by 38%, 15.8, and 8.4% in OSA1-oxs under 0.5PK, PK, and 2PK treatments, respectively, and no significant difference was found in osa1 mutants (Figure 3F). After incubation for 4 h, N uptake rate by OSA1-oxs roots averagely increased by approximately 36.7%, 60.4%, and 53.5% under 0.5PK, PK, and 2PK treatments, respectively, and no significant difference was found in osa1 mutant roots (Figure 3G). In addition, the average K absorption rate of OSA1-oxs roots was 23.3%, 27%, and 17.2% higher than WT, and was reduced by 26.6%, 21.3%, and 19.5% in osa1 mutant roots under 0.5PK, PK, and 2PK treatments, respectively (Figure 3H). As shown in Figure 3I, the average P uptake rate increased by 54%, 25.2%, and 19.9% in OSA1-oxs under 0.5PK, PK, and 2PK treatment, respectively. These results indicate that overexpression of OSA1 could improve macronutrient uptake rates under moderate, low, and high P and K concentrations.

After 1, 2, and 4 h of incubation at different concentrations of P and K, we determined the total amount of N, P, and K in the whole plants. Under 0.5PK, PK, and 2PK treatments, the NH₄⁺ amount averagely increased by 114%, 140%, and 165%, respectively, in OSA1-oxs and decreased by 42.4%, 28.5%, and 29.4%, respectively, in osa1 mutants compared to WT (Figure S1A). P content averagely increased by 78%, 103%, and 124% in OSA1-oxs, and averagely decreased by 33.8%, 34.7%, and 31.7% in osa1 mutants under 0.5PK, PK, and 2PK treatments, respectively (Figure S1B). In addition, the K content in OSA1-oxs averagely increased by 119%, 115%, and 112%, and averagely decreased by 24.8%, 40.3%, and 41% in osa1 mutants when compared with WT under 0.5PK, PK, and 2PK treatments, respectively (Figure S1C).

2.4. Overexpression of OSA1 Increases the Expression of Nutrient Transporter Genes in Roots under Different Concentrations of P and K

Next, we determined the relative expression levels of the corresponding genes related to N, P, and K uptakes under different concentrations of P and K in OSA1 overexpression and knockout mutant plants by qRT-PCR assay. OsAMT1;1 and OsAMT1;2 are two critical NH₄⁺ transporter genes that are highly induced by N deficiency [24]. Here, we found that the relative expression level of OsAMT1;1 was about 2–3 times higher in OSA1-oxs roots and decreased significantly in osa1 roots when compared with that of WT under all treatments of different concentrations of P and K (Figure 4A). In addition, the relative expression level of OsAMT1;2 in OSA1-oxs roots was also significantly higher than that of WT under all these conditions and decreased significantly in osa1 mutants under the PK condition (Figure 4B). OsPHT1;1 and OsPHT1;2, belonging to OsPHT gene family, are involved in phosphate uptake in rice roots [25]. Here, we found that OsPHT1;1 expression was significantly enhanced in OSA1-oxs roots but showed no significant difference in osa1 mutants when compared with WT (Figure 4C). However, the expression of OsPHT1;2 showed no significant difference among OSA1-oxs, osa1 mutants, and WT (Figure 4D). Then, we detected expression levels of K transporter genes, HAK1 and HAK5 [26]. OSA1-oxs plants exhibited at least a two times higher expression level of HAK1 in the roots under all these treatments, and osa1 mutants showed a reduced expression level of HAK1 under PK treatment (Figure 4E), but no significant difference was found for HAK5 (Figure 5F). All these results suggest that the overexpression of OSA1 could activate the expression of N, P, and K transporter genes under moderate, low, and high P and K concentrations.

2.5. Correlation between N/P/K Uptakes and Root Morphology

A correlation analysis was conducted between N, P, and K uptakes and root morphology. These nutrient uptakes were all positively correlated with root dry weight and root surface area (Figure 5 and Figure S2). Interestingly, the increment trend of nutrient uptake with root dry weight or root surface area was in the order of N > K > P under all treatments (Figure 5 and Figure S2). The R² values and regression equations for their correlations were shown in Tables S1 and S2. The correlation coefficients were all significant at p value < 0.001. According to these results, improved NH₄⁺, PO₄^3-, and K⁺ uptake could not only attribute by the enhanced PM H⁺-ATPase activity, but also by the increased root growth conferred by OSA1 overexpression.

2.6. OSA1 Overexpression Promotes Cation Fluxes in Rice Roots under High-Affinity Transport Concentrations

To measure the net flux rates of NH₄⁺, K⁺, and H⁺ in roots of WT and OSA1 transgenic plants under high-affinity transport concentrations of NH₄⁺ and K⁺ (0.1 mM NH₄⁺ and 0.1 mM K⁺), we used a noninvasive microtest system (NMT) to perform the microelectrode ion flux measurement (MIFE) with intact roots of 5-day-old seedlings. The nutrient flux rate was measured along the root tip, around 150 μm from the root cap (Figure 6A). As a result, the NH₄⁺ influx rate, K⁺ influx rate, and H⁺ efflux rate increased in three OSA1-oxs by 64.2%, 28.3%, and 96.1%, respectively (Figure 6B–D), and decreased significantly in the roots of three osa1 mutants by approximately 35.7%, 27.3%, and 21%, respectively, when compared with that of WT (Figure 7A–C). These results suggest that OSA1 plays a positive role in controlling NH₄⁺ and K⁺ uptake rates, as well as the H⁺ excretion rate in rice roots under a high-affinity transport system.

3. Discussion

Numerous studies have been conducted on the effects of N, P, and K on crop growth and development [27,28]. Our previous study demonstrated that the overexpression of OSA1 significantly enhanced NH₄⁺ uptake and improved grain yield in rice [21]. The present result showed that the overexpression of OSA1 in rice significantly increased the PM H⁺-ATPase activity (Figure 2B), and activated the expression of several nutrient transporter genes, such as OsAMT1;1, OsAMT1;2, OsPHT1;1, and OsHAK1 (Figure 4), and improved the uptake rates of macronutrients such as N, P, and K by roots under different concentrations of P and K (Figure 3).

Notably, the 0.5PK treatment activated PM H⁺-ATPase in WT roots compared with that of normal conditions (PK treatment) (Figure 2B), which is consistent with previous findings that low K⁺ or low P concentrations in apoplast could significantly improve PM H⁺-ATPase activity [3,29]. In addition, we found higher concentrations of P and K improved NH₄⁺ uptake rates in WT, and this effect was even enhanced in OSA1-oxs plants (Figure 3A,D,G). Previous studies showed that a high K concentration reduced the NH₄⁺ uptake rate in rice due to the competitive absorption between K⁺ and NH₄⁺ [30]. It is possible that the increased uptake of phosphate could help to maintain the charge balance in the cytoplast and further facilitate the uptake of K⁺. Theoretically, absorption of N, P, and K will increase H⁺ concentrations in root cytoplasm, which need to be pumped out of cells by PM H⁺-ATPase to maintain the membrane potential and cytosolic pH. Interestingly, overexpression of OSA1 activated the expression of several transporter genes related to N, P, and K uptakes in rice under low-affinity P and K conditions (Figure 4). However, the underlying molecular mechanism of the linkage between PM H⁺-ATPase activity and the expression of nutrient transporter genes deserves to be investigated in the future.

PM H⁺-ATPase has been known to be involved in auxin-induced root growth in the model plant Arabidopsis [9,10,31,32]. In rice, it has been demonstrated that overexpression of OSA1 enhanced root biomass under different concentrations of NH₄⁺ [21]. In this study, we found that biomass also increased significantly under different concentrations of P and K in OSA1-oxs and decreased in osa1 mutants (Figure 1). Interestingly, we found that 2PK treatment significantly inhibited root growth in WT and osa1 mutants, but the growth inhibition was alleviated in OSA1-oxs (Figure 1). Increases in the uptakes of P and K may lead to cytosolic H⁺ accumulation and even H⁺ toxicity in the cytoplast of rice roots, which could negatively affect root growth [33,34]. However, improved PM H⁺-ATPase activity by overexpression of OSA1 could alleviate H⁺ accumulation in root cells and then contribute to the tolerance to high concentrations of P and K. In addition, N, P, and K contents in OSA1-oxs showed linear relationships with root biomass and root surface area (Figure 5 and Figure S2), which is consistent with previous studies which found that nutrient uptake amounts are correlated with root biomass [35,36]. However, the underlying mechanism of how PM H⁺-ATPase activity is linked to the root growth of rice under different nutrient concentrations needs to be investigated in the future, which may possibly be associated with auxin signaling.

In the high-affinity transport system, it has been well documented that PM H⁺-ATPase is involved in nutrient uptakes by supplying proton motive force to facilitate ion transports across the PM [37]. However, the detailed information is unclear about the effect of PM H⁺-ATPase overexpression on nutrient transportation in rice roots. Here, we performed MIFE experiment with NMT system to measure the net ion fluxes around the root tips of OSA1-modified plants and WT. In the root tip region, comprising the meristem and elongation zone (0–600 µm from the tip), a strong ion flux can be detected [38]. In this study, we detected one at the position of 150 µm far from the root tip, which belongs to the elongation region close to the meristem region (Figure 6A). The results showed that the net NH₄⁺ and K⁺ influx rates were significantly increased in OSA1-oxs and were significantly reduced in osa1 mutants under the high-affinity transport system (0.1 mM NH₄⁺ and 0.1 mM K⁺) when compared with that of WT (Figure 6 and Figure 7). NMT uses specialized flux sensors that are derived from microelectrodes to measure the dynamic ion/molecule activity (i.e., diffusion flux). Diffusion flux is the amount of substance per unit area per unit time around an intact sample [39]. Therefore, the overexpression of OSA1 improved nutrient uptake rate directly, possibly by generating more polarized membrane potential, which is independent of the root biomass. However, to our knowledge, NMT methods are not permitted for determining phosphate influx rates in rice roots, which is expected to be explored in the future.

In conclusion, we demonstrated that the overexpression of a rice PM H⁺-ATPase gene, OSA1, promoted root growth and stimulated N, P, and K uptake rates by roots under different concentrations of P and K in rice. Actually, this report firstly provided evidence that the engineering of a PM H⁺-ATPase gene in rice could improve plant growth under moderate, low, and high P and K concentrations. This study could also provide a novel insight into the cultivating crop plants growing in agricultural soil, either with inadequate abundance of nutrients or with excessive abundance of nutrients. Further studies are required to explore the underlying mechanism by which OSA1 overexpression enhances root growth and nutrient uptake under moderate, low, and high concentrations of P and K.

4. Materials and Methods

4.1. Plant Cultivation

All the rice seeds used in this study were kept in our own lab [21]. Seeds of WT (Oryza sativa L. ssp. japonica cv. Nipponbare); overexpression lines of OSA1#1, OSA1#2, and OSA1#3; and mutant lines osa1-1 (TOS17 line ND3017), osa1-2 (TOS17 line ND3025), and osa1-3 (TOS17 line ND3033) were surface-sterilized in 10% H₂O₂ for 30 min and then soaked in water before germination. Rice seedlings were grown in a greenhouse under a light intensity of 400 μmol m⁻² s⁻¹, a relative humidity of approximately 60–80%, and a 14 h light (30 °C)/10 h dark (22 °C) photoperiod. Pots with PO₄³⁻ and K⁺ treatments were filled with a minor modified IRRI nutrient solution as follows: 0.5PK treatment contains 0.25 mM K₂SO₄, and 0.15 mM NaH₂PO₄; 2PK treatment contains 1 mM K₂SO₄, and 0.6 mM NaH₂PO₄, and PK treatment (control) contains 0.5 mM K₂SO₄ and 0.3 mM NaH₂PO₄. The full chemical composition of the IRRI nutrient solution (pH 5.5) is listed below: 1 mM (NH₄)₂SO₄, 0.5 mM K₂SO₄, 0.3 mM NaH₂PO₄, 1 mM CaCl₂, 1 mM MgSO₄, 9 μM MnCl₂, 0.39 μM Na₂MoO₄, 20 μM H₃BO₄, 0.77 μM ZnSO₄, 0.32 μM CuSO₄, and 20 μM EDTA-Fe [21]. Solutions were changed every 3 days. Rice seedlings were grown in 1/2 IRRI nutrient solution for 1 week, then transferred to full IRRI nutrient solution containing different concentrations of P and K (0.5PK, PK, 2PK) for 4 weeks.

4.2. Nutrient Uptake Rate Determination

Three plants exhibiting similar growth in each treatment were used for the exhaustion test according to [40,41] with some modifications. The rice seedlings were placed in a N-, P-, and K-free nutrient solution for 24 h, then plants were transferred into 250 mL IRRI nutrient solutions with different concentrations of P and K to analyze nutrient uptake rates. All experiments were performed in a growth chamber at 28 °C with a light intensity of 400 μmol m⁻² s⁻¹ and relative humidity of 60%. The culture medium was sampled at 1 h, 2 h, 4 h. After that, one mL of exhaustion fluid was used for the determination of the N, P, and K concentration and was replenished with 1 mL of deionized water at the same time to ensure that the volume of depleted fluid was constant. Control solution was used as negative control, which contained the same nutrient solution with the treatment but without root incubation. At the end of the exhaustion test, the fresh root weight was calculated after the moisture was removed with filter paper. K concentration in the exhaustion fluid and control solution was measured by flame emission photometry. P concentration was determined using the molybdate yellow method. The concentration of NH₄⁺ was determined by the continuous-flow analytical system AA3 (SEAL, Gernany) (The detection limit is 0.0165 mg·L⁻¹).

4.3. Measurements of Net Proton, K⁺, and NH₄⁺ Fluxes Rate in Roots

A BIO-IM Series NMT Physiolyzer^® system (YoungerUSA, MA, USA) was applied to determine the net H⁺, K⁺, and NH₄⁺ fluxes in the root tip based on a previous study [42]. Five-day-old rice roots were subsequently placed in 50 mL of growth solution with 2 mM NH₄⁺ for 12 h. After washing with deionized water, the roots were placed in Petri dishes containing 20 mL detection buffer containing 0.2 mM CaCl₂, 0.1 mM KCl, 0.1 mM MgCl₂, 0.5 mM NaCl, 0.3 mM MES (2-morpholinoethanesulfonic acid sodium salt), and 0.2 mM Na₂SO₄ (pH 6.0) for the determination of the H⁺ and K⁺ fluxes. For the NH₄⁺ flux assay, the detection buffer containing 0.1 mM NH₄NO₃, 0.1 mM KCl, 0.1 mM CaCl₂, and 0.3 mM MES (pH 6.0) was used. The fluxes of rice plants were measured along the root tip around 150 μm from the root cap. The microelectrodes in the NMT system were positioned 0 ± 2 μm away from the samples. At least three individual plants were analyzed for each sample.

4.4. Quantitative Reverse-Transcription PCR

qRT-PCR was performed using the Step One Real-Time PCR system (Applied Biosystems, Foster City, CA, USA), as described previously [43]. Briefly, total RNA was extracted from the roots of 4-week-old WT and transgenic plants using the RNeasy Plant Mini Kit (Qiagen). First-strand cDNAs were synthesized from total RNA using the PrimeScript II First Strand cDNA Synthesis Kit using oligo (dT) primers (TakaRa). Relative quantification was performed using an SYBR Premix Ex Taq II (Perfect Real Time) Kit (TaKaRa Biotechnology, Dalian, China) on a Step One Plus Real-Time PCR System (Applied Biosystems, Bio-Rad, CA, USA). For amplification of OSA1 (Os03g0689300), OsAMT1;1 (Os04g0509600), OsAMT1;2 (Os02g0620600), OsPHT1;1 (Os03g0150600), OsPHT1;2 (Os03g0150800), OsHAK1 (Os04g0401700), and OsHAK5 (Os01g0930400), the gene-specific primers were used (Table S3). Relative gene expression levels were normalized to that of the internal control gene, OsActin (Os03g0718100), using the comparative cycle threshold (ΔΔCt) method [44]. All analyses were repeated at least three times.

4.5. Measurement of PM H⁺-ATPase Activity

The detection of ATP hydrolytic activity of PM H⁺-ATPase was completed mainly according to [16], with a few modifications. Root tissues of 4-week-old WT and transgenic plants treated with different concentrations of P and K (0.5 PK, PK, and 2PK) were ground in ice-cold homogenization buffer to isolate the PM in a two-phase partitioning method. The protein concentration of the membrane vesicle was determined by using Bio-Rad protein assay dye reagent concentrate (BIO-RAD, Hercules, CA, USA) according to the manufacturer’s instruction. Na₃VO₄ (0.1 mM) was used as an inhibitor of H⁺-ATPase in 0.5 mL reaction solution, which contained 30 mM BTP/MES, 5 mM MgSO₄, 50 mM KCl, 50 mM KNO₃, 1 mM Na₂MoO₄, 1 mM NaN₃, 0.02% (w/v) Brij 58, and 5 mM disodium-ATP (substrate for PM H⁺-ATPase). Firstly, 30 μL of a membrane vesicle suspension containing 1–2 μg total protein was added to the reaction solution for 30 min at 30 °C; the inorganic phosphate was liberated after the hydrolysis of ATP. The reaction was stopped by adding 1 mL reagent (2% (v/v) concentrated H₂SO₄, 5% (w/v) sodium dodecyl sulfate, and 0.7% (w/v) (NH₄)₂MoO₄), followed by 50 μL 10% (w/v) ascorbic acid. After 10 min, 1.45 mL arsenide-citrate reagent (2% (w/v) sodium citrate, 2% (w/v) sodium arsenide, and 2% (w/v) glacial acetic acid) was added. The color will be stable after 30 min and measured spectrophotometrically at 720 nm. In each test, H⁺-ATPase activity was calculated as the amount of phosphate liberated within 30 min mg⁻¹ membrane protein in excess of the boiled-membrane protein control.

4.6. Statistical Analysis

Data were analyzed for statistics and significance by two-tailed student’s t test and two-way ANOVA test with a significant difference at p < 0.05 using GraphPad Prism 9 software (GraphPad Software, San Diego, CA, USA).