Inhibition of phosphate uptake in corn roots by aluminum-fluoride complexes

F forms stable complexes with Al at conditions found in the soil. Fluoroaluminate complexes (AlF(x)) have been widely described as effective analogs of inorganic phosphate (Pi) in Pi-binding sites of several proteins. In this work, we explored the possibility that the phytotoxicity of AlF(x) reflects their activity as Pi analogs. For this purpose, (32)P-labeled phosphate uptake by excised roots and plasma membrane H(+)-ATPase activity were investigated in an Al-tolerant variety of maize (Zea mays L. var. dwarf hybrid), either treated or not with AlF(x). In vitro, AlF(x) competitively inhibited the rate of root phosphate uptake as well as the H(+)-ATPase activity. Conversely, pretreatment of seedlings with AlF(x) in vivo promoted no effect on the H(+)-ATPase activity, whereas a biphasic effect on Pi uptake by roots was observed. Although the initial rate of phosphate uptake by roots was inhibited by AlF(x) pretreatment, this situation changed over the following minutes as the rate of uptake increased and a pronounced stimulation in subsequent (32)Pi uptake was observed. This kinetic behavior suggests a reversible and competitive inhibition of the phosphate transporter by fluoroaluminates. The stimulation of root (32)Pi uptake induced by AlF(x) pretreatment was tentatively interpreted as a phosphate starvation response. This report places AlF(3) and AlF(4)(-) among Al-phytotoxic species and suggests a mechanism of action where the accumulation of Pi-mimicking fluoroaluminates in the soil may affect the phosphate absorption by plants. The biochemical, physiological, and environmental significance of these findings is discussed.     

Salt stress in Thellungiella halophila activates Na+ transport mechanisms required for salinity tolerance

Salinity is considered one of the major limiting factors for plant growth and agricultural productivity. We are using salt cress (Thellungiella halophila) to identify biochemical mechanisms that enable plants to grow in saline conditions. Under salt stress, the major site of Na+ accumulation occurred in old leaves, followed by young leaves and taproots, with the least accumulation occurring in lateral roots. Salt treatment increased both the H+ transport and hydrolytic activity of salt cress tonoplast (TP) and plasma membrane (PM) H(+)-ATPases from leaves and roots. TP Na(+)/H+ exchange was greatly stimulated by growth of the plants in NaCl, both in leaves and roots. Expression of the PM H(+)-ATPase isoform AHA3, the Na+ transporter HKT1, and the Na(+)/H+ exchanger SOS1 were examined in PMs isolated from control and salt-treated salt cress roots and leaves. An increased expression of SOS1, but no changes in levels of AHA3 and HKT1, was observed. NHX1 was only detected in PM fractions of roots, and a salt-induced increase in protein expression was observed. Analysis of the levels of expression of vacuolar H(+)-translocating ATPase subunits showed no major changes in protein expression of subunits VHA-A or VHA-B with salt treatment; however, VHA-E showed an increased expression in leaf tissue, but not in roots, when the plants were treated with NaCl. Salt cress plants were able to distribute and store Na+ by a very strict control of ion movement across both the TP and PM.     

Aluminum inhibits the H(+)-ATPase activity by permanently altering the plasma membrane surface potentials in squash roots

Although aluminum (AL) toxicity has been widely studied in monocotyledonous crop plants, the mechanism of Al impact on economically important dicotyledonous plants is poorly understood. Here, we report the spatial pattern of Al-induced root growth inhibition, which is closely associated with inhibition of H(+)-ATPase activity coupled with decreased surface negativity of plasma membrane (PM) vesicles isolated from apical 5-mm root segments of squash (Cucurbita pepo L. cv Tetsukabuto) plants. High-sensitivity growth measurements indicated that the central elongation zone, located 2 to 4 mm from the tip, was preferentially inhibited where high Al accumulation was found. The highest positive shifts (depolarization) in zeta potential of the isolated PM vesicles from 0- to 5-mm regions of Al-treated roots were corresponded to pronounced inhibition of H(+)-ATPase activity. The depolarization of PM vesicles isolated from Al-treated roots in response to added Al in vitro was less than that of control roots, suggesting, particularly in the first 5-mm root apex, a tight Al binding to PM target sites or irreversible alteration of PM properties upon Al treatment to intact plants. In line with these data, immunolocalization of H(+)-ATPase revealed decreases in tissue-specific H(+)-ATPase in the epidermal and cortex cells (2--3 mm from tip) following Al treatments. Our report provides the first circumstantial evidence for a zone-specific depolarization of PM surface potential coupled with inhibition of H(+)-ATPase activity. These effects may indicate a direct Al interaction with H(+)-ATPase from the cytoplasmic side of the PM.     

Induction of nitrate uptake in maize roots: expression of a putative high-affinity nitrate transporter and plasma membrane H+-ATPase isoforms

An investigation was carried out to assess the effect of nitrate supply on the root plasma membrane (PM) H+-ATPase of etiolated maize (Zea mays L.) seedlings grown in hydroponics. The treatment induced higher uptake rates of the anion and the expression of a putative high-affinity nitrate transporter gene (ZmNRT2.1), the first to be identified in maize. Root PM H+-ATPase activity displayed a similar time-course pattern as that of net nitrate uptake and investigations were carried out to determine which of the two isoforms reported to date in maize, MHA1 and 2, responded to the treatment. MHA1 was not expressed under the conditions analysed. Genome analysis revealed that MHA2, described as the most abundant form in all maize tissues, was not present in the maize hybrid investigated, but a similar form was found instead and named MHA3. A second gene (named MHA4) was also identified and partially sequenced. Both genes, classified as members of the PM H+-ATPase subfamily II, responded to nitrate supply, although to different degrees: MHA4, in particular, proved more sensitive than MHA3, with a greater up- and down-regulation in response to the treatment. Increased expression of subfamily II genes resulted in higher steady-state levels of the enzyme in the root tissues and enhanced ATP-hydrolysing activity. The results support the idea that greater proton-pumping activity is required when nitrate inflow increases and suggest that nitrate may be the signal triggering the expression of the two members of PM H+-ATPase subfamily II.     

Seasonal changes of plasma membrane H(+)-ATPase and endogenous ion current during cambial growth in poplar plants

The plasma membrane H(+)-ATPase (PM H(+)-ATPase), potassium ions, and endogenous ion currents might play a fundamental role in the physiology of cambial growth. Seasonal changes of these parameters were studied in twigs of Populus nigra and Populus trichocarpa. Monoclonal and polyclonal antibodies against the PM H(+)-ATPase, x-ray analysis for K(+) localization and a vibrating electrode for measurement of endogenous ion currents were used as probes. In dormant plants during autumn and winter, only a slight immunoreactivity against the PM H(+)-ATPase was found in cross sections and tissue homogenates, K(+) was distributed evenly, and the density of endogenous current was low. In spring during cambial growth, strong immunoreactivity against a PM H(+)-ATPase was observed in cambial cells and expanding xylem cells using the monoclonal antibody 46 E5 B11 F6 for fluorescence microscopy and transmission electron microscopy. At the same time, K(+) accumulated in cells of the cambial region, and strong endogenous current was measured in the cambial and immature xylem zone. Addition of auxin to dormant twigs induced the formation of this PM H(+)-ATPase in the dormant cambial region within a few days and an increase in density of endogenous current in shoot cuttings within a few hours. The increase in PM H(+)-ATPase abundance and in current density by auxin indicates that auxin mediates a rise in number and activity of an H(+)-ATPase in the plasma membrane of cambial cells and their derivatives. This PM H(+)-ATPase generates the necessary H(+)-gradient (proton-motive force) for the uptake of K(+) and nutrients into cambial and expanding xylem cells.     

Phosphorylation of H+/K(+)-ATPase by inorganic phosphate. The role of K+ and SCH 28080.

The effects of K+ on the phosphorylation of H+/K(+)-ATPase with inorganic phosphate were studied using H+/K(+)-ATPase purified from porcine gastric mucosa. The phosphoenzyme formed by phosphorylation with Pi was identical with the phosphoenzyme formed with ATP. The maximal phosphorylation level obtained with Pi was equal to that obtained with ATP. The Pi phosphorylation reaction of H+/K(+)-ATPase was, like that of Na+/K(+)-ATPase, a relatively slow reaction. The rates of phosphorylation and dephosphorylation were both increased by low concentrations of K+, which resulted in hardly any effect on the phosphorylation level. A decrease of the steady-state phosphorylation level was caused by higher concentrations of K+ in a noncompetitive manner, whereas no further increase in the dephosphorylation rate was observed. The decreasing effect was caused by a slow binding of K+ to the enzyme. All above-mentioned K+ effects were abolished by the specific H+/K(+)-ATPase inhibitor SCH 28080 (2-methyl-8-[phenyl-methoxy]imidazo-[1-2-a]pyrine-3-acetonitrile). Additionally, SCH 28080 caused a 2-fold increase in the affinity of H+/K(+)-ATPase for Pi. A model for the reaction cycle of H+/K(+)-ATPase fitting the data is postulated.     

在耐热性诱导中豌豆叶片过氧化氢和水杨酸含量与质膜ATP酶活性的变化及相互关系

在高温锻炼(37℃,2h)过程中,豌豆(Pisum sativum L.)叶片过氧化氢(H_2O_2)和游离态水杨酸(SA)含量与质膜ATP酶(H~ -ATPase)活性都有一个高峰,H_2O_2的迸发早于游离态SA的积累,而质膜H~ -ATPase活性高峰的出现则迟于SA高峰;活性氧清除剂、抗氧化剂、质膜NADPH氧化酶抑制剂和H_2O_2的淬灭剂预处理均可有效地阻止高温下H_2O_2和SA的积累以及质膜H~ -ATPase活性的增加。根据以上结果推测,H_2O_2、质膜H~ -ATPase和SA均参与耐热性诱导相关的信号传递,前者作用于SA的上游,而后者在SA下游起作用。     

磷饥饿时番茄幼苗根系质膜H+-ATP酶活性

磷饥饿提高了番茄幼苗质膜H+-ATP酶活性并促进了番茄幼苗根部的H+分泌。动力学分析表明,磷饥饿使番茄幼苗根部质膜H+-ATP酶的Km值明显降低,亦即提高了该酶对其底物的亲和力,但对该酶的Vmax影响不大。另外,磷饥饿并不改变ATP酶的最适pH值(最适pH值为6.5)。钒酸盐显著抑制番茄幼苗根部质膜ATP酶的活性以及H+分泌,也显著抑制番茄幼苗的Pi吸收。与对照相比,上述抑制作用在饥饿处理的植物中表现得更强。以上结果表明,磷饥饿时高亲和性Pi传递系统的诱导很可能包含质膜H+-ATP酶的参与。     

Dual regulation of proton- and sodium-coupled phosphate transport systems in the Yarrowia lipolytica yeast by extracellular phosphate and pH

In this study we used a newly isolated osmo-, salt-, and alkali-tolerant Yarrowia lipolytica yeast strain, with a unique capacity to grow over a wide pH range (3.5-10.5). A procedure was elaborated to follow phosphate accumulation by Y. lipolytica cells grown at different pH values. In this paper we demonstrate that Pi-starved Y. lipolytica cells are endowed by derepressible high-affinity, high-capacity H(+)- and Na(+)-driven Pi uptake systems and that activities of these transport systems are under the dual control by the prevailing extracellular Pi concentrations and pH values.     

Requirements for activation of the signal-transduction network that leads to regulatory phosphorylation of leaf guard-cell phosphoenolpyruvate carboxylase during fusicoccin-stimulated stomatal opening

Leaves regulate gas exchange through control of stomata in the epidermis. Stomatal aperture increases when the flanking guard cells accumulate K+ or other osmolytes. K+ accumulation is stoichiometric with H+ extrusion, which is compensated for by phosphoenolpyruvate carboxylase (PEPC, EC 4.1.1.31)-mediated malate synthesis. Plant PEPCs are regulated allosterically and by phosphorylation. Aspects of the signal-transduction network that control the PEPC phosphorylation state in guard cells are reported here. Guard cells were preloaded with [32P]orthophosphate (32Pi); then stomata were incubated with fusicoccin (FC), which activates the guard-cell plasma membrane H+-ATPase. [32P]PEPC was assessed by immunoprecipitation, electrophoresis, immunoblotting, and autoradiography. In -FC controls, stomatal size, guard-cell malate, and [32P]PEPC were low; maximum values for these parameters were observed in the presence of FC after a 90-min incubation and persisted for an additional 90 min. This high steady-state phosphorylation status resulted from continuous phosphorylation and dephosphorylation, even after the malate-accumulation phase. PEPC phosphorylation was diminished by approximately 80% when K+ uptake was associated with Cl- uptake and was essentially abolished when stomatal opening was sucrose--rather than K+--dependent. Finally, alkalinization by NH4+ in the presence of K+ did not cause PEPC phosphorylation (as it does in C4 plants). As discussed, a role for cytoplasmic protons cannot be completely excluded by this result. In summary, activation of the plasma membrane H+-ATPase was essential, but not sufficient, to cause phosphorylation of guard-cell PEPC. Network components downstream of the H+-ATPase influence the phosphorylation state of this PEPC isoform.     

Functional Characterization of 14 Pht1 Family Genes in Yeast and Their Expressions in Response to Nutrient Starvation in Soybean

Background

Phosphorus (P) is essential for plant growth and development. Phosphate (Pi) transporter genes in the Pht1 family play important roles in Pi uptake and translocation in plants. Although Pht1 family genes have been well studied in model plants, little is known about their functions in soybean, an important legume crop worldwide.

Principal Findings

We identified and isolated a complete set of 14 Pi transporter genes (GmPT1-14) in the soybean genome and categorized them into two subfamilies based on phylogenetic analysis. Then, an experiment to elucidate Pi transport activity of the GmPTs was carried out using a yeast mutant defective in high-affinity Pi transport. Results showed that 12 of the 14 GmPTs were able to complement Pi uptake of the yeast mutant with Km values ranging from 25.7 to 116.3 µM, demonstrating that most of the GmPTs are high-affinity Pi transporters. Further results from qRT-PCR showed that the expressions of the 14 GmPTs differed not only in response to P availability in different tissues, but also to other nutrient stresses, including N, K and Fe deficiency, suggesting that besides functioning in Pi uptake and translocation, GmPTs might be involved in synergistic regulation of mineral nutrient homeostasis in soybean.

Conclusions

The comprehensive analysis of Pi transporter function in yeast and expression responses to nutrition starvation of Pht1 family genes in soybean revealed their involvement in other nutrient homeostasis besides P, which could help to better understand the regulation network among ion homeostasis in plants.     

The High-Affinity Phosphate Transporter GmPT5 Regulates Phosphate Transport to Nodules and Nodulation in Soybean

Legume biological nitrogen (N) fixation is the most important N source in agroecosystems, but it is also a process requiring a considerable amount of phosphorus (P). Therefore, developing legume varieties with effective N(2) fixation under P-limited conditions could have profound significance for improving agricultural sustainability. We show here that inoculation with effective rhizobial strains enhanced soybean (Glycine max) N(2) fixation and P nutrition in the field as well as in hydroponics. Furthermore, we identified and characterized a nodule high-affinity phosphate (Pi) transporter gene, GmPT5, whose expression was elevated in response to low P. Yeast heterologous expression verified that GmPT5 was indeed a high-affinity Pi transporter. Localization of GmPT5 expression based on β-glucuronidase staining in soybean composite plants with transgenic roots and nodules showed that GmPT5 expression occurred principally in the junction area between roots and young nodules and in the nodule vascular bundles for juvenile and mature nodules, implying that GmPT5 might function in transporting Pi from the root vascular system into nodules. Overexpression or knockdown of GmPT5 in transgenic composite soybean plants altered nodulation and plant growth performance, which was partially dependent on P supply. Through both in situ and in vitro (33)P uptake assays using transgenic soybean roots and nodules, we demonstrated that GmPT5 mainly functions in transporting Pi from roots to nodules, especially under P-limited conditions. We conclude that the high-affinity Pi transporter, GmPT5, controls Pi entry from roots to nodules, is critical for maintaining Pi homeostasis in nodules, and subsequently regulates soybean nodulation and growth performance.     

Characterization of a Pi transport system in cartilage matrix vesicles. Potential role in the calcification process.

The mechanisms by which calcium (Ca2+) and inorganic phosphate (Pi) accumulate into matrix vesicles (MV) have not been elucidated. In the present study the characteristics of Pi uptake into MV isolated from mildly rachitic chicken growth plate cartilage have been investigated. The results indicate that Pi accumulates into MV mainly via a Na(+)-dependent Pi transport system. In the absence of NaCl in the extravesicular medium, Pi uptake was a nonsaturable process. In the presence of 150 mM NaCl, the initial rate of Pi uptake was 4.38 +/- 1.02-fold higher than with 150 mM choline chloride (mean +/- S.E., n = 8, p less than 0.005). Other cations showed partial activity to drive Pi into MV as compared to Na+:Li+ (64.4%) greater than K+ (39.8%) greater than choline (39.0%) greater than tetramethylammonium (30.0%) greater than N-methylglucamine (26.3%). Na(+)-dependent Pi transport activity displayed saturability towards increasing extra-vesicular concentrations of Na+ and Pi. The apparent Km for Pi was 0.68 +/- 0.16 mM. The Na+ concentration producing half-maximum Pi transport activity was 106.2 +/- 11.0 mM. Kinetic analysis suggests that Na+ interacts with the Pi carrier with a stoichiometry of more than one Na+ ion with one Pi molecule. In MV isolated from normal chicken growth plate cartilage, this Na(+)-dependent Pi transport system was barely expressed. In contrast to the effect on Pi uptake by MV, the activity of alkaline phosphatase was not changed when NaCl was substituted for choline chloride in the assay medium. In addition to this observation which suggests that this enzyme is not related to the Pi transport activity described in this study, levamisole, which inhibited alkaline phosphatase activity did not affect the Na(+)-dependent uptake of Pi. Both arsenate and phosphonoformic acid, two inhibitors of the epithelial Na(+)-dependent Pi transport systems, were active inhibitors of the Na(+)-dependent Pi uptake by MV with a higher potency for phosphonoformic acid. Associated with the expression of a facilitated Na(+)-coupled Pi transport in MV, in vitro calcification assessed by 45Ca2+ uptake also showed a marked dependence on extravesicular sodium. This relationship was markedly attenuated in MV isolated from normal chicken growth plate cartilage expressing a weak Na(+)-facilitated Pi transport activity. In conclusion, a saturable Na(+)-dependent Pi carrier has been characterized which facilitates Pi transport in MV. Its potential role for Ca-Pi accumulation into MV and subsequent development of vesicular calcification followed by mineralization of the osteogenic matrix is proposed and remains to be further investigated.     

Cadmium-induced sulfate uptake in maize roots

The effect of cadmium (Cd) on high-affinity sulfate transport of maize (Zea mays) roots was studied and related to the changes in the levels of sulfate and nonprotein thiols during Cd-induced phytochelatin (PC) biosynthesis. Ten micromolar CdCl(2) in the nutrient solution induced a 100% increase in sulfate uptake by roots. This was not observed either for potassium or phosphate uptake, suggesting a specific effect of Cd(2+) on sulfate transport. The higher sulfate uptake was not dependent on a change in the proton motive force that energizes it. In fact, in Cd-treated plants, the transmembrane electric potential difference of root cortical cells was only slightly more negative than in the controls, the external pH did not change, and the activity of the plasma membrane H(+)-ATPase did not increase. Kinetics analysis showed that in the range of the high-affinity sulfate transport systems, 10 to 250 microM, Cd exposure did not influence the K(m) value (about 20 microM), whereas it doubled the V(max) value with respect to the control. Northern-blot analysis showed that Cd-induced sulfate uptake was related to a higher level of mRNA encoding for a putative high-affinity sulfate transporter in roots. Cd-induced sulfate uptake was associated to both a decrease in the contents of sulfate and glutathione and synthesis of a large amount of PCs. These results suggest that Cd-induced sulfate uptake depends on a pretranslational regulation of the high-affinity sulfate transporter gene and that this response is necessary for sustaining the higher sulfur demand during PC biosynthesis.