JXB Advance Access originally published online on March 17, 2006
Journal of Experimental Botany 2006 57(6):1353-1362; doi:10.1093/jxb/erj111
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RESEARCH PAPER |
Root plasma membrane H+-ATPase is involved in the adaptation of soybean to phosphorus starvation
1Laboratory of Plant Nutritional Genetics and Root Biology Center, College of Resources and Environment, South China Agricultural University, Guangzhou 510642, PR China
2Research Institute for Bioresources, Okayama University, Chuo 2-20-1, Kurashiki 710-0046, Japan
* To whom correspondence should be addressed. E-mail: xlyan{at}scau.edu.cn
Received 25 June 2005; Accepted 16 December 2005
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Abstract |
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The plasma membrane H+-ATPase plays an important role in the plant response to nutrient and environmental stresses. However, the involvement of plant root plasma membrane H+-ATPase in adaptation to phosphate (P) starvation is not yet fully elucidated. In this study, experiments were performed with soybean roots in low-P nutrient solution (10 µM). Treatment with fusicoccin, an activator of the plasma membrane H+-ATPase, increased P uptake by 35%, while vanadate, an inhibitor of plasma membrane H+-ATPase, severely suppressed it. These results suggested that P uptake might be regulated via the modulation of the activity of plasma membrane H+-ATPase under P starvation. The relationship between P uptake and the activity of plasma membrane H+-ATPase was examined further by using plasma membrane H+-ATPase transgenic Arabidopsis thaliana under low-P conditions. Transgenic plants absorbed more P compared with wild-type Arabidopsis. Results from real-time RT-PCR, western-blotting and immunolocalization analysis indicated that the increase in activity of the plasma membrane H+-ATPase by P starvation was caused by its transcriptional and translational regulation. A higher expression was observed at the translational level than at the transcriptional level. P starvation could induce a transient increase of endogenous indole-3-acetic acid (IAA) in soybean roots. The exogenous application of IAA stimulated the activity of plasma membrane H+-ATPase and P uptake, while naphthylphthalamic acid (NPA), an IAA transport inhibitor, blocked IAA effects. Taken together, these results suggested an involvement of root plasma membrane H+-ATPase in the adaptation of soybean to P starvation. IAA might be involved in signal transduction of P starvation by activating the plasma membrane H+-ATPase in soybean roots.
Key words: IAA, P uptake, P starvation, plasma membrane H+-ATPase, soybean roots
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Introduction |
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Low phosphorus availability is a primary constraint to plant growth on acid soils. Plants exhibit an array of physiological, biochemical, and morphological changes in order to adapt to the altered availability of phosphates (Lynch, 1997
; Raghothama, 1999). Movement of inorganic phosphate (Pi) across the plasma membrane of root cells is a crucial step in the transport of nutrients into the plant. Although the physiological processes of Pi transport across the plasma membrane have been relatively well understood, only in recent years have the specific transport proteins, protein kinases or phosphatases involved in this transport been characterized and identified. Pi uptake into the root symplasm involves transport from the apoplast where Pi concentration is less than 2 µM, across the plasma membrane, and to the cytosol where Pi concentration ranges from 5–17 mM (Mimura et al., 1998; Mimura, 2001). The negative membrane potential and the large difference between external and internal Pi concentrations necessitate that a large electrochemical gradient must be overcome for Pi transport into root cells, thus demanding a high-affinity, energy-consuming transport mechanism driven by plasma membrane H+-ATPase (Ullrich et al., 1981, 1984).
The activity of plasma membrane H+-ATPase is a crucial factor in the survival of plants when they are under a variety of environmental stresses, such as salt stress (Vitart et al., 2001), Al stress (Ahn et al., 2001) etc. The uptake of potassium ions through specific transport proteins was found to be related to the activity of the plasma membrane H+-ATPase (Hoth et al., 1997). P uptake is postulated to involve proton co-transport (Ullrich et al., 1984; Sakano et al., 1992). A decrease in cytoplasmic pH and membrane depolarization stimulate a plasma membrane H+-ATPase that pumps protons out of the cell to maintain the intracellular pH and thus provides the proton driving force for Pi uptake (Ullrich et al., 1984). Therefore, the activation of the plasma membrane H+-ATPase may improve plant nutrition by enhancing the electrochemical proton gradient that drives ion transport across the cell membrane via secondary transport systems. It is reasonable to hypothesize that the root plasma membrane H+-ATPase could be involved in the adaptation of plant roots to P starvation. However, the evidence is still lacking for the possible involvement of this enzyme in plant roots under P starvation.
The plant hormone auxin is regarded to be the most important plant growth regulator in relation to cell division, differentiation, root initiation, and nutrient stress responses (Rayle and Cleland, 1992). Auxin was considered to be involved in mediating the P-starved effect because auxin antagonists were found to inhibit low Pi-induced root hair elongation (Bates and Lynch, 1996) and P starvation could induce auxin redistribution in Arabidopsis roots (Nacry et al., 2005). The application of auxin to Pi-sufficient white lupin (Lupinus albus) resulted in the formation of proteoid roots, a response commonly observed under Pi deficiency (Gilbert et al., 2000; Neumann et al., 2000). Furthermore, the application of auxin to P-replete plants stimulates lateral root development similar to that observed in P-deficient plants, suggesting a role of auxin in changing root morphology (López-Bucio et al., 2002; Al-Ghazi et al., 2003). Although these findings revealed the involvement of auxin in Pi deficiency responses, not much is known about its role in the Pi starvation-induced signalling pathways or in gene expression. In the present study, the activity, transcriptional and translational expression of root plasma membrane H+-ATPase and endogenous IAA have been investigated to elucidate the possible involvement of this enzyme in P uptake and signal transduction in the adaptation of soybean roots to P starvation.
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Materials and methods |
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Plant materials and growth conditions
Soybean seeds (Glycine max L., cv. Suzunari Japan) with high P efficiency were used in this study. After selection, surface-sterilized seeds were gently ground with sea sand (20–30 mesh) for 10 s to facilitate germination. Pretreated seeds were soaked in a solution containing 0.5 mM CaCl2 for 1 h and then germinated in peat moss mixed with sand quartz for 3 d at 25 °C. After germination, the seedlings were transferred to nutrient solution in 2.0 l plastic pots containing (µM): KNO3 (750), Ca(NO3)2 (250), MgSO4 (325), KH2PO4 (10 or 500), Fe-EDTA (20), H3BO3 (8), CuSO4 (0.2), ZnSO4 (0.2), MnCl2 (0.2), and (NH4)6Mo7O24 (0.2) for 7 d. The solution was adjusted to pH 6.0 with 1 mM HCl and renewed every 3 d. The seedlings were grown in a growth cabinet at 25/20 °C and 14/10 h day/night cycles, 40 µmol m–2 s–1 light intensity and 70% relative humidity. After growth for 7d in nutrient solution, soybean seedlings were selected for the following experiments. To examine the modulator effects on P uptake, fusicoccin, vanadate, IAA, or NPA was applied to low-P (10 µM) nutrient solution for 5 d. Fusicoccin, IAA, and NPA were first dissolved in methanol, and then applied to water solution. It was necessary to use a vacuum to guarantee the contact of each chemical and the roots. To increase the contact of modulators and the root surface, the first 30 min was under vacuum to guarantee that the chemicals permeated the root cells. Each chemical was dissolved in methanol or milliQ water in 1 mM as the stock solution. Methanol in each treatment was kept to less than 0.1% to minimize its effect on the activity of the plasma membrane H+-ATPase and P uptake. Chemicals were purchased from Sigma Chemical Co. (St Louis). For experiments with transgenic plants, Arabidopsis seedlings were cultured as follows: sterilized seeds were incubated on a nylon mesh square cup (mesh size, 300 µm; cup size: 4 cm width x 4 cm length x 1 cm height) under irradiation at 50 µE m–2 s–1, 16/8 h light/dark at 22 °C. The square cup was kept floating with a sponge supporter on 140 ml of 1/3 Murashige and Skoog solution containing 3% (w/v) sucrose. After 7 d incubation, the seedlings were transferred to the 1/3 Murashige and Skoog solution with a P level of 10 µM for 5 d. After treatment, P uptake and the activity of plasma membrane H+-ATPase in Arabidopsis were analysed. Each experiment was performed at least twice independently. Transgenic lines of Arabidopsis (Ecotype: Landsberg erecta) was obtained by cloning plasma membrane H+-ATPase into a binary vector and expressing under the control of the CaMV 35S promoter in a sense or an antisense orientation according to Harper et al. (1989)
. Binary vector constructions were transformed in planta by Agrobacterium tumefaciens GV3101/Pmp90 by electroporation. The transformed plants were allowed to grow and set seeds. Positive transformants were identified as normal seedlings grown on Murashige and Skoog agar plates supplemented with 50 µg ml–1 kanamycin. Single-locus insertions were harvested by observing a 3:1 ratio (kanamycin-resistant versus kanamycin-sensitive) in the T2 progenies that resulted from the self-pollination of T1 parents. Homologous T3 progenies (nine lines) were used for subsequent assays.
Isolation of root plasma membranes
Root plasma membrane vesicles were prepared according to the method of Faraday and Spanswick (1992) with minor modifications. Briefly, the collected root samples were ground in ice-cold homogenization buffer contained 250 mM sucrose, 2 mM EGTA, 10% (v/v) glycerol, 0.5% (w/v) BSA, 2 mM DTT, 1 mM PMSF, 5 mM 2-mercaptoethanol, and 50 mM BTP, adjusted to pH 7.8. The homogenate was filtered through two layers of Miracloth and centrifuged in a swinging bucket rotor at 10 000 g for 10 min, 4 °C. The supernatants were centrifuged at 100 000 g for 55 min. The microsomal pellets were resuspended in phase buffer (250 mM sucrose, 3 mM KCl, and 5 mM KH2PO4, pH 7.8). The microsomal membrane preparation was fractionated by two-phase partitioning in aqueous dextran T-500 (Sigma) and PEG-3350 (Sigma) according to the method of Ahn et al. (2001). After centrifugation, the residue was dissolved in microsome suspension buffer, and centrifuged again at 100 000 g for 30 min. The pellet was dissolved in the microsome suspension solution, and then pooled into 12 g with microsome suspension solution. The phase component solution was prepared by mixing different phase components with a weight of 24 g. After phase partitioning, the upper phase containing the plasma membrane was retained, mixed with microsome suspension buffer, and then centrifuged. The plasma membrane was dissolved in suspension buffer, and stored at –80 °C. The protein concentration was determined with BSA as the standard according to Bradford (1976).
Assay of plasma membrane H+-ATPase activity
In a reaction volume of 0.5 ml that contained 30 mM BTP/MES, pH 6.5, 5 mM MgSO4, 50 mM KCl, and 4 mM TRIS-ATP, plasma membrane H+-ATPase activity was measured. Brij 58 (0.02% w/v) was applied to obtain membrane vesicles of uniform sideness (Johansson et al., 1995). Reactions were initiated by adding 3–5 µg of membrane protein. Reactions proceeded for 30 min at 30 °C and were stopped with 1 ml of stopping solution (2% [v/v]) concentrated H2SO4, 5% (w/v) sodium dodecyl sulphate, 0.7% (w/v) sodium molybdate, followed by 50 µl of 10% (w/v) ascorbic acid. Colour development of the phosphomolybdate complex proceeded for 30 min. Absorbance at 700 nm was measured with a spectrophotometer. ATPase activity was calculated as the Pi liberated in the excess of boiled-membrane controls. To assess the purification of the H+-ATPase, its activity was expressed as the difference in activity in the presence and absence of 0.1 mM vanadate. Since the oxidation state of vanadate varies greatly, solid vanadate is directly added to a reaction after calculation. A standard curve of Pi in the reaction mixture was included in each assay.
Real-time RT-PCR analysis of gene expression of plasma membrane H+-ATPase
After treatment, the root apices of soybean were excised and used for RNA isolation using an extraction reagent (TRIZOL Reagent, Invitrogen Life Technologies). The total RNA was purified by RNase-free DNase. First-strand cDNA was synthesized in a 20 µl reaction solution containing 2 µg of total RNA using the SuperScript II RT-amplification system for first-strand cDNA synthesis and oligo (dT)12-18 as a primer. Using the synthesized first-strand cDNA as a template and two gene-specific primers derived from the plasma membrane H+-ATPase gene (5'-CTTGGGATAATC-TTTTGGAGAAC-3') as a sense primer and (5'-CTCGGCACGTCTCTTA-G-3') as an antisense primer according to the gene number AF091303
[GenBank]
. PCR with 25 cycles was performed. The 
-tubulin gene was used to normalize the amount of first-strand cDNA. -Tubulin sense primer (5'-CTCAGGTGATTTCATCTTTG-3') and antisense primer (5'-GAATTCAG TCACATCCAC-3') were derived according to the gene number CA936138
[GenBank]
. Transcriptional levels were quantified using LightCycler Data Analysis software.
Western blotting analysis and immunolocalization of plasma membrane H+-ATPase
Plasma membrane proteins were separated by SDS-PAGE using the system of Laemmli (1970). Membrane vesicles were solubilized in SDS-loading buffer containing 0.125 mM TRIS–HCl, pH 7.4, 10% (w/v) SDS, 10% (v/v) glycerol, 0.2 M DTT, 0.002% (w/v) bromocresol blue, and 5 mM PMSF. The samples were loaded on a discontinuous SDS-polyacrylamide gel [5% (w/v) acrylamide stacking gel and 8% (w/v) acrylamide separating gel]. For the western blot analysis, after separation by SDS-PAGE, samples were transferred to PVDF membrane (0.2 µm). To identify and quantify the plasma membrane H+-ATPase, the blots were incubated with polyclonal antibodies against maize plasma membrane H+-ATPase. The antiserum was diluted to 1:1000 in TBS-T buffer [1 mM TRIS-HCl pH 8.0 mM NaCl, and 0.1% (v/v) Tween 20] and the incubation was kept for 1 h at room temperature. After rinsing in TBS-T, PVDF membrane was incubated at room temperature for 1 h with a 1:10 000 (v/v) diluted secondary antibody (ECL Anti-rabbit IgG, peroxidase-linked species-specific whole antibody from donkey). After washing in TBS-T, the membrane was incubated for 5 min in ECL-based signal detection solution. Intensities of signals on the film were quantified by a densitometer (Bio-Rad Laboratories). Immunolocalization of plasma membrane H+-ATPase was examined as described by Ahn et al. (2001) as follows. After treatment, the root apices (5 mm) were excised, transferred to a stabilizing buffer (SB: 50 mM PIPES, 5 mM EGTA, and 5 mM MgSO4, pH 6.9) containing 5% (v/v) dimethyl sulphoxide for 30 min at room temperature, and then fixed with 4% (w/v) paraformaldehyde for 60 min with an initial 10 min under vacuum. After three 10-min rinses in PBS (pH 7.4), they were digested with an enzymatic cocktail [(1% (w/v) hemicellulase (from Aspergillus niger, Sigma-Aldrich, Tokyo), 1% (w/v) pectolyase (Seishin Corporation, Tokyo), 0.5 M EGTA, 0.4 M mannitol, 1% (v/v) Triton X-100, 0.3 mM phenylmethylsulphonyl fluoride, all dissolved in SB] for 60 min. The digestion reaction was stopped by transferring the roots to SB for 15 min followed by 1% (v/v) Triton X-100 in SB for 10 min. After a brief rinse in SB, the samples were extracted in methanol at –20 °C, rehydrated in PBS (2 h), and incubated with rabbit polyclonal antibody raised against maize H+-ATPase diluted 1:200 in PBS for 12 h in the dark at room temperature. The roots were then incubated with TRITC conjugated anti-rabbit IgG raised in goat (Sigma-Aldrich, Tokyo) diluted 1:100 in PBS for 12 h at room temperature. Parallel sets of roots processed without primary antibodies served as negative controls. The procedure was completed by transferring the labelled roots to 0.01% (w/v) toluidine blue in PBS to diminish the autofluorescence of the tissue and mounted in mowiol (Calbiochem, La Jolla, CA). The images of plasma membrane H+-ATPases from roots were captured using the 543 nm excitation line of He–Ne laser fitted in a Zeiss (Oberkochen, Germany) confocal microscope with Ph3-Plan-Neofluar x100 oil immersion objective. The root surface images were the overlay of 7–11 optical sections (0.75 µm thick), and scan configurations were kept constant among treatments using the recycle option of the LSM 510 software to assess the intensity differences. A model F-4500 fluorescence spectrophotometer (Hitachi Instruments, San Jose, CA) was used to measure the fluorescence intensity, and the relative induction was calculated.
IAA analysis
After treatment, soybean root apices were excised, and immediately placed in liquid nitrogen then stored at –80 °C until extraction. Samples were ground with a mortar and pestle using IAA extraction buffer (65% isopropanol and 35% 0.2 M imidazole buffer, pH 7.0). The extracts were passed through a C18-reversed phase prepacked column immediately under dim light conditions at 4 °C. Extracts were centrifuged at 10 000 g for 5 min. The supernatant was diluted by 0.5 ml of TBS-buffer (150 mM NaCl, 1 mM MgCl2, and 50 mM TRIS, pH 7.5) and treated with diazomethane to convert the acid form to its methyl ester form for an immunological IAA assay (ELISA) as described by Hager et al. (1991).
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Results |
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Activity of plasma membrane H+-ATPase and P uptake
To evaluate the purity of plasma membrane, the activity of various inhibitor-sensitive ATPases in both the microsome and membrane fractions was measured (Fig. 1). In the microsome fraction, molybdate-sensitive ATPase was the major enzyme, which suggested the presence of unspecific acid phosphatases (Yan et al., 2002
). However, vanadate-sensitive ATPase occupied 90% of the total activity in plasma membrane fraction, and other inhibitor-sensitive enzyme activity was negligible. The results indicated that the isolation techniques used here for the plasma membrane were correct and practicable.
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To investigate whether plasma membrane H+-ATPase in soybean roots responds to P starvation, soybean roots were transferred into low-P (10 µM) nutrient solution for 5 d. Results indicated that, under the normal P level, P uptake into roots did not vary greatly in response to both vanadate and fusicoccin treatment, although the activity of the plasma membrane H+-ATPase changed (Fig. 2). P starvation enhanced the activity of plasma membrane H+-ATPase in soybean roots. Under P starvation, the addition of vanadate, an inhibitor of plasma membrane H+-ATPase, severely inhibited P uptake and the activity of the plasma membrane H+-ATPase. While fusicoccin, an activator of plasma membrane H+-ATPase, increased P uptake by 35% and the activity of plasma membrane H+-ATPase by 42%, respectively (Fig. 2). In Neurospora crassa, Bowman (1983)
found that vanadate was absorbed via the phosphate transport system. In this experiment, pretreatment with vanadate also inhibited P uptake (data not shown), suggesting that the vanadate-induced inhibition of P uptake in soybean seedlings was not caused by the competitive uptake of vanadate. To investigate the correlation between P uptake and the activity of plasma membrane H+-ATPase, transgenic Arabidopsis was generated by overexpressing plasma membrane H+-ATPase using the CaMV 35S promoter. When plants were grown on 1/3 Murashige and Skoog medium for 7 d, then exposed to 1/3 Murashige and Skoog solution with low P (10 µM) for 5 d, transgenic plants absorbed more P compared with the wild-type Arabidopsis. Interestingly, under normal P supply, no differences in P uptake were observed between wild-type and transgenic Arabidopsis (Fig. 3). One of the classical features of Pi starvation in soybean seedlings is the development of dark-green shoots due to the accumulation of anthocyanins, a class of red/purple coloured flavonoids. During this accumulation, a plasma membrane H+-ATPase is required for the formation of proanthocyanidins (Baxter et al., 2005). These results suggested that plasma membrane H+-ATPase in soybean roots was involved in the response to P starvation.
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Expression analysis and immunolocalization of plasma membrane H+-ATPase
Plant plasma membrane H+-ATPases form a large gene family that is either transcriptionally or/and translationally regulated (Michelet and Boutry, 1995
). In soybean, plasma membrane H+-ATPase was encoded by a gene, accession number AF091303. To clone the plasma membrane H+-ATPase, the gene-specific primers were designed based on AF091303
[GenBank]
, and thus the same gene was detected by RT-PCR in soybean. To examine the effects of P starvation and IAA on the transcriptional expression of the plasma membrane H+-ATPase gene, the mRNA accumulation of plasma membrane H+-ATPase gene was analysed by real-time RT-PCR. -Tubulin (primers were designed according to CA936138
[GenBank]
), a housekeeping gene, was selected as an internal standard. Results indicated that P starvation tended to increase the mRNA accumulation of the plasma membrane H+-ATPase gene after 5 d treatment, however, transcript levels of plasma membrane H+-ATPase declined when exposed to 15 d P starvation. Addition of IAA to the low-P solution slightly enhanced the mRNA accumulation of the plasma membrane H+-ATPase gene (Fig. 4A).
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Effects of P starvation and IAA on plasma membrane H+-ATPase protein were also examined using a polyclonal antibody against maize plasma membrane H+-ATPase. Western-blotting analysis indicated that 27% and 38% increases in plasma membrane H+-ATPase protein were observed for 1 d and 5 d P starvation, respectively, in comparison to their controls (Fig. 4B). The exogenous application of IAA stimulated the accumulation of plasma membrane H+-ATPase protein by 41% for 1 d and by 29% for 5 d P starvation. But with a further increase of P-starved time, plasma membrane H+-ATPase protein declined. The polyclonal antibody also decorated the plasma membrane of all cells along the root apices of soybean seedlings (Fig. 5). P starvation increased the fluorescence intensity of plasma membrane H+-ATPase by 31% for 1 d, 46% for 5 d, and 16% for 15 d treatments, respectively. Figure 5E was the negative control of the root apex incubated without primary antibody.
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Endogenous IAA influenced by P starvation
In maize coleoptiles, auxin could stimulate the activity of plasma membrane H+-ATPase (Hager et al., 1991
; Frias et al., 1996; Coenen et al., 2002). To investigate the regulatory mechanism that P starvation enhanced the activity of plasma membrane H+-ATPase, endogenous IAA was measured in soybean root apices using the ELISA method (Fig. 6). The results indicated that a transient increase in endogenous IAA was induced by P starvation (1 d). With a prolonged P-starved peroid, 5 d and 15 d, endogenous IAA declined rapidly. At 15 d P starvation, it was decreased to 1488 pg g–1 fresh root weight, which accounted for 38% of that of the control. Under the normal P level, endogenous IAA in soybean root apices did not vary greatly.
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IAA and NPA effect on P uptake and activity of plasma membrane H+-ATPase
P starvation can induce a transient increase in endogenous IAA. To explore the possible role of elevated endogenous IAA in response to P starvation, the effect of exogenous IAA and NPA (an auxin transport inhibitor) on P uptake and plasma membrane H+-ATPase was examined in soybean root apices. With exposure to a normal P level, IAA or NPA treatment did not influence P uptake and the activity of plasma membrane H+-ATPase, but under low-P conditions, IAA treatment enhanced P uptake and the activity of plasma membrane H+-ATPase, and the application of NPA to an IAA solution blocked the IAA effects (Fig. 7). These results suggest that endogenous IAA was involved in the response to P starvation.
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Discussion |
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Activity and expression analysis of plasma membrane H+-ATPase
Plasma membrane H+-ATPase is involved in multiple physiological functions. This ‘master enzyme’ creates and maintains the transmembrane electrochemical gradient for nutrient uptake (Ullrich et al., 1984
; Sakano et al., 1992). Hyperpolarization may drive the uptake of nutrients into cells via secondary active transport and cause the opening of ion channels, for instance H+, 
and 
channels (Michelet and Boutry, 1995; Hoth et al., 1997). In maize, the high activity of plasma membrane H+-ATPase induced by nitrate coincided with the increased rate of 
uptake, which was driven by an 
symport carrier (RuizCristin and Briskin, 1991). Low K+ availability led to a 25-fold increase in K+ influx although a slight increase in the expression of the plasma membrane H+-ATPase was observed (Samuels et al., 1992). In this study, P starvation enhanced the activity of the plasma membrane H+-ATPase (Figs 2, 3). Fusicoccin and vanadate, activator and inhibitor of plasma membrane H+-ATPase, had stimulatory and inhibitory effects on P uptake, respectively. Plasma membrane H+-ATPase transgenic plants also showed a higher P uptake compared with their wild-type seedlings (Fig. 3). The results suggested that variation in the activity of the plasma membrane H+-ATPase modulated P uptake under P starvation. In accordance with these results, Song et al. (2001) indicated that plasma membrane H+-ATPase in tomato roots was involved in the induction of high-affinity Pi transport system under P starvation. Grinsted et al. (1982) and Moorby et al. (1988) indicated that P starvation could induce H+ efflux from rape roots, and this pH change influenced the P concentration in the soil solution. Since proton efflux is regulated by plasma membrane H+-ATPase, these results suggested that plasma membrane H+-ATPase in soybean roots played an important role in regulating P uptake, especially under P starvation.
When P was absorbed by plant roots, the root plasma membrane would repolarize and hyperpolarize. The recovery process is considered to be a result of the activation of the electrogenic H+-ATPase (Michelet and Boutry, 1995; Mimura, 2001). To investigate the molecular mechanism how P starvation enhances the activity of plasma membrane H+-ATPase, the effect of P starvation on the transcriptional and translational expression of plasma membrane H+-ATPase was measured in soybean roots. Real-time RT-PCR results indicated that P starvation slightly influenced the mRNA accumulation of the plasma membrane H+-ATPase gene, but enhanced the accumulation of plasma membrane H+-ATPase protein in root apices of soybean seedlings significantly (Fig. 4). Yan et al. (2002) found that P starvation could induce a high expression of plasma membrane H+-ATPase protein in proteoid roots, and this response contributed to P uptake. An increase in the translational expression might elevate the activity of the plasma membrane H+-ATPase, and thus facilitate P uptake. Recently, it was found that Al could induce the phosphorylation of the plasma membrane H+-ATPase, and enhance this enzyme activity (Shen et al., 2005). It is likely that P starvation might influence the phosphorylation or dephosphorylation of plasma membrane H+-ATPase, and thus modulate its activity.
The activity of the plasma membrane H+-ATPase was regulated by the membrane environment (Kasamo, 2003). The activity of the plasma membrane H+-ATPase induced by P starvation might be associated with changes in the composition of the plasma membrane. Gniazdowska et al. (1999) reported that P starvation could reduce the phospholipids by 50% in the seedling roots of kidney bean, but the total lipid content per protein of plasma membrane fraction was not changed. Dörmann and Benning (2002) and Härtel et al. (2000) indicated that plant roots could adapt to Pi deprivation by functionally replacing phospholipids with non-phosphorus galactolipids and sulpholipids, thus avoiding severe disruption in plasma membrane integrity and other functions relying on plasma membranes. In oat, the galactolipid digalactosyldiacylglycerol can constitute a substantial proportion of oat plasma membrane lipids in roots, and activate plasma membrane H+-ATPase (Andersson et al., 2003). In Ustilago maydis, complex lipid-bound unsaturated fatty acids could activate plasma membrane H+-ATPase hydrolytic activity (Hernandez et al., 2002).
IAA signal transduction
The acid growth hypothesis holds that susceptible cells exposed to auxin secrete protons into the apoplast, which causes hyperpolarization of the cells, a decrease in apoplastic pH, and an increase in the growth rate (Rayle and Cleland, 1992). In the coleoptile of maize, auxin not only stimulated gene expression of the plasma membrane H+-ATPase, but also enhanced the membrane flow from the endoplasmic reticulum to the plasma membrane or the fusion of Golgi vesicles with the plasma membrane, resulting in a high steady-state H+-ATPase concentration in the plasma membrane (Hager et al., 1991). In accordance with these results, the exogenous application of IAA to P-starved seedling roots enhanced the accumulation of plasma membrane H+-ATPase protein. Interestingly, IAA did not influence the expression of this enzyme protein under normal P conditions. This phenomenon suggested that P starvation might modify the plasma membrane H+-ATPase protein, and thus result in a long life expectancy and a slow degradation of this enzyme (Hager et al., 1991; Hager, 2003). Karthikeyan et al. (2002) indicated that the phytohormones, auxin and cytokinin, were involved in the regulation of some components of the P-starvation response pathway in Arabidopsis roots. These results supported the above view that the P signal was transferred by IAA. But other pathways for P signal transduction might exist, because exogenous application of IAA to P-supplied seedling roots did not increase the activity of the plasma membrane H+-ATPase and P uptake (Fig. 7). IAA probably triggers a signal-transduction chain by binding at a plasma membrane receptor molecule (Hertel et al., 1972; Schmidt et al., 2003), or by inducing phosphatidylinositol metabolism via G-proteins, finally resulting in the formation of inositol trisphosphate (Palmgren et al., 1988). In vitro experiments indicated that ABP57 was able to bind auxin and activate plasma membrane H+-ATPase in rice (Kim et al., 2001). However, IAA signal transduction in response to P nutrition needs to be further investigated.
Recently, Tang et al. (2003) indicated that the application of exogenous ATP could alter the distribution of auxin, decrease basipetal auxin transport, and increase the retention of indole-3-acetic acid in root tips of maize. One day of P starvation might stimulate root respiration, increase ATP accumulation, and thus induce a transient increase in endogenous IAA in soybean roots. Vice versa, long-term P starvation would decrease the concentration of ATP and endogenous IAA in the root apices of soybean seedlings (Fig. 6). Worley et al. (2000) demonstrated that the degradation of Aux/IAA proteins was essential for normal auxin signalling. This study's results suggested that endogenous IAA might trigger a cascade that activates transcription factors and protein synthesis and at the same time alters the activity of particular enzymes like the plasma membrane H+-ATPase that plays an important role in regulating P uptake, especially under P starvation. IAA, as a signal molecule is probably involved in the signal transduction of P starvation by stimulating the activity and expression of plasma membrane H+-ATPase.
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Acknowledgements |
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This research was supported by the Major State Basic Research Development Program of China (2005CB120902), National Natural Science Foundation of China (No. 30100110/30230220/30471040), the International Foundation for Science (No. C/3042-1,2), Guangdong Province Natural Science Foundation (No. 000642) and the McKnight Foundation Collaborative Crop Research Program (Grant 01-1565/05-780).
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