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The mechanism responsible for phosphorus inhibition of vesicular-arbuscular mycorrhiza formation in sudangrass (Sorghum vulgare Pers.) was investigated in a phosphorus-deficient sandy soil (0.5 micrograms phosphorus per gram soil) amended with increasing levels of phosphorus as superphosphate (0, 28, 56, 228 micrograms per gram soil). The root phosphorus content of 4-week-old plants was correlated with the amount of phosphorus added to the soil. Root exudation of amino acids and reducing sugars was greater for plants grown in phosphorus-deficient soil than for those grown in the phosphorus-treated soils. The increase in exudation corresponded with changes in membrane permeability of phosphorus-deflcient roots, as measured by K' (86Rb) efflux, rather than with changes in root content of reducing sugars and amino acids. The roots of phosphorus-deficient plants inoculated at 4 weeks with Glomusfascicuaww were 88% infected after 9 weeks as compared to less than 25% infection in phosphorus-sufficient roots; these differences were correlated with root exudation at the time of inoculation. For plants grown in phosphorusdeficient soil, infection by vesicular-arbuscular mycorrhizae increased root phosphorus which resulted in a decrease in root membrane permeability and exudation compared to nonmycorrhizal plants. It is proposed that, under low phosphorus nutrition, increased root membrane permeability leads to net loss of metaboiltes at sufficient levels to sustain the germination and growth of the mycorrhizal fungus during pre-and postinfection. Subsequently, mycorrhizal infection leads to improvement of root phosphorus nutrition and a reduction in membrane-mediated loss of root metabolites.During the past 20 years, there has been a growing appreciation of the importance of VAM' in the improvement of plant growth through increased uptake of phosphorus (P) and other mineral nutrients, especially in soils of low fertility (5, 9). As evidence has mounted for the role of VAM in the enhancement of P uptake from P-deficient soils, it has also been recognized that high soil P levels severely limit VAM infection (9, 10).Early work failed to distinguish whether P was inhibiting the activity of the mycorrhizal fungus in the soil or during the hostfungus interaction (10,16 of sudangrass inoculated with Glomusfasciculatus (Thaxt.) Gerd. and Trappe only became heavily infected if the host was not receiving adequate P from the other half of the root system. It is now clear that the P content of the host plant is the critical factor controlling the mycorrhizal symbiosis.Ratnayake et al. (12) proposed that the mechanism of P control of VAM formation was associated with a membrane-mediated decrease in root exudation. They were able to correlate low P content of sudangrass and citrus roots with a decrease in phospholipid levels and a large increase in permeability of root membranes, which results in a greater net leakage of amino acids and sugars from the root. They suggested that under P-sufficient conditions, metabolites requi...

The effects of vanadate, molybdate, and azide on ATP phosphohydrolase (ATPase) and acid phosphatase activities of plasma membrane, mitochondrial, and soluble supernatant fractions from corn (Zea mays L. WF9 x M017) roots were investigated. Azide (0.1-10 miflimolar) was a selective inhibitor of pH 9.0-ATPase activity of the mitochondrial fraction, while molybdate (0.01-1.0 milUimolar) was a relatively selective inhibitor of acid phosphatase activity in the supernatant fraction. The pH 6.4-ATPase activity of the plasma membrane fraction was inhibited by vanadate (10-500 micromolar), but vanadate, at similar concentrations, also inhibited acid phosphatase activity. This result was confirmed for oat (Avena sativa L.) root and coleoptile tissues. While vanadate does not appear to be a selective inhibitor, it can be used in combination with molybdate and azide to distinguish the plasma membrane ATPase from mitochondrial ATPase or supernatant acid phosphatase.Vanadate appeared to be a noncompetitive inhibitor of the plasma membrane ATPase, and its effectiveness was increased by KV. K -stimulated ATPase activity was inhibited by 50% at about 21 micromolar vanadate. The rate of K+ transport in excised corn root segments was inhibited by 66% by 500 micromolar vanadate.fungal (3, 4) cells. At higher concentrations (usually >50 .tM), vanadate has been shown to inhibit ATPase activities (8,13,26,35) and ion transport (8, 9, 16) in various plant tissues. However, at these concentrations, vanadate also inhibits plant and animal phosphatases (22,32,36). Mitochondrial ATPase activity is not affected by up to 500 ,UM vanadate, but higher concentrations (1 mM) have been reported to inhibit respiration and oxidative phosphorylation in rat liver mitochondria (11). Vanadate is clearly not a specific inhibitor of plasma membrane-ATPase, but it may be useful if used in combination with other inhibitors.Mitochondrial (and/or chloroplast) ATPase, acid phosphatase from the vacuole, and other phosphatases which readily utilize ATP as a substrate have caused considerable problems in studies on plasma membrane ATPase (14). It seems that molybdate is an inhibitor of phosphatase (34,36), although the specificity of molybdate for phosphatase over ATPase has not been reported. Azide is among several potent inhibitors of mitochondrial type, F1-ATPases (4).In this paper, we report the results of an investigation designed to compare the effects of vanadate, molybdate, and azide on ATPase and phosphatase activities of plasma membrane, mitochondrial, and soluble supernatant fractions from corn roots. It seems that plasma membrane ATPase activity can be distinguished by its sensitivity to vanadate and insensitivity to molybdate or azide.Progress in elucidating the role of plasma membrane-associated ATP phosphohydrolase (ATPase) in ion transport has been limited by the lack of a specific inhibitor of the enzyme. The availability of such an inhibitor is important for studies seeking to correlate effects on ATPase activity with changes in ion tra...

Evidence that cation transport in animals is mediated by cation-activated ATPase is now unequivocal. In addition to strong correlative evidence relating ATPase to transport in a number of animal tissues (3, 21), the (Na+ + K+)-ATPase of the plasma membrane (7, 25) and the Ca2t-ATPase of sarcoplasmic reticulum (12) have been removed from their membranes, purified, incorporated into artificial lipid membranes, and showed to catalyze ATP-dependent cation transport. This is direct proof that the ATPase is the cation pump.Based on the assumption that the mechanism of cation transport in plants is likely to be fundamentally similar to that in animals, attempts have been made to demonstrate the presence of ion-stimulated ATPase in plant membranes and to correlate this activity with ion transport (8,9,13,15 This paper is concerned with the properties of ATPase activity associated with a plasma membrane fraction from primary roots of corn (17). The results show that the plasma membrane ATPase of corn roots has characteristics similar to those for the plasma membrane ATPase of oat roots and strengthen the hypothesis that this enzyme functions in cation transport in plants.MATERIALS AND METHODS Plasma membranes were isolated from primary roots (6-8 cm in length) of 3-day-old etiolated corn seedlings (Zea mays L., WF9 x M14) as previously described (10, 17). Briefly, the rinsed roots (15-20 g fresh weight) were ground (mortar and pestle) in 0.25 M sucrose, 3 mm EDTA, and 25 mm tris-MES, pH 7.7. The filtered homogenate (pH 7.2) was successively centrifuged at 13,000g for 15 min and 80,000g for 30 min. The 13,000 to 80,000 g pellet was suspended in 0.25 M sucrose in 1 mM tris-MES, pH 7.2, and centrifuged for 2 hr at 82,500g in the gradient shown in Figure 1. Membranes banding at the interface between 34 and 45% (w/w) sucrose were about 70% plasma membranes (17).Mitochondria were obtained by centrifugation of the 13,000 g pellet in the gradient shown in Figure 1. The 34 to 45% sucrose interface contained more than 60% of the total Cyt c oxidase activity and was rich in mitochondria (17). ATPase activity was measured at 38 C in a 1-ml volume containing 3 mm ATP (tris salt) at desired pH, 30 mm tris-MES at desired pH, variable amounts of mono-and divalent ions (see tables and figure legends), and 25 to 40,g of membrane protein by determining inorganic phosphate released as previously described (10, 17). ATPase activity was linear for at least 45 min over the range of membrane protein added to the assay. Substrate blanks were subtracted to calculate all enzyme activities.

SUMMARYThe mechanism responsible for inhibition of the establishment of mycorrhizal associations in Sorghum vulgare Pers. (herbaceous monocot) and Citrus aurantium L. (woody dicot) under high levels of soil phosphorus (P) was studied. Plants were grown on low fertility loamy sand (4.5 ppm P), receiving superphosphate [Ca (H2PO4)2H2O] at 0, 6, 28, 56, 228 and 556 ppm P along with all the other necessary nutrients. The percentage P content of root tissue was correlated with the amount of P added to the soil. Root exudation, measured in terms of the net leakage of soluble amino acids and reducing sugars from the roots within a 17-h period, was significantly higher under low P levels (0, 6 and 28 ppm P) than under high P levels (56, 228 and 556 ppm P). The amount of exudation was correlated with a Pinduced decrease in phospholipid levels and associated changes in permeability properties of root membranes, rather than with changes in the root content of sugars and amino nitrogen. The hypothesis is proposed that phosphorus inhibition of mycorrhizal symbiosis is associated with a membrane-mediated decrease in root exudation.