Induction of UDP-Glucose:Salicylic Acid Glucosyltransferase in Oat Roots.

A UDP-glucose:salicylic acid 3-O-glucosyltransferase (EC 2.4.1.35) (GTase) from oat (Avena sativa L. cv Dal) root extracts was assayed in vitro using [(14)C]salicylic acid (SA) and an ion exchange column to separate SA from beta-glucosylsalicylic acid. The GTase, present at a very low constitutive level, was inducible to 23 times the constitutive level. When excised roots were exposed to SA at pH 6.5, the specific activity of the enzyme increased within 1.5 h, peaked after 8 to 10 h, and then declined. The increase in specific activity depended on the concentration of SA in the induction medium. Among 16 phenolics and phenolic derivatives tested, GTase induction showed high specificity toward SA and acetylsalicylic acid. Specific activity of the enzyme was induced to higher levels in roots from 7-d-old seedlings than roots from younger plants. GTase activity was less inducible in basal compared with median or apical root sections. Induction of GTase activity was a result of de novo RNA and protein synthesis. Candidate peptides for the GTase were identified by comparison of two-dimensional electrophoresis gels of proteins labeled with [(35)S]methionine during incubation of roots in the presence or the absence of SA and a gel of a partially purified GTase preparation.

Partial purification and properties of an inducible uridine 5'-diphosphate-glucose-salicylic Acid glucosyltransferase from oat roots.

A salicylic acid (SA)-inducible uridine 5'-diphosphate (UDP)-glucose:SA 3-O-glucosyltransferase was extracted from oat (Avena sativa L. cv Dal) roots. Reverse phase high-performance liquid chromatography or anion exchange chromatography was used to separate SA from the product, beta-O-d-glucosylsalicylic acid. The soluble enzyme was purified 176-fold with 5% recovery using a combination of pH fractionation, anion exchange, gel filtration, and chromatofocusing chromatography. The partially purified protein had a native molecular weight of about 50,000, an apparent isoelectric point at pH 5.0, and maximum activity at pH 5.5. The enzyme had a K(m) of 0.28 mm for UDP-glucose and was highly specific for this sugar donor. More than 20 hydroxybenzoic and hydroxycinnamic acid derivatives were assayed as potential glucose acceptors. UDP-glucose:SA 3-O-glucosyltransferase activity was highly specific toward SA (K(m) = 0.16 mm). The enzyme was inhibited by UDP and uridine 5'-triphosphate but not by up to 7.5 mm uridine 5'-monophosphate.

Herbicide clomazone does not inhibit in vitro geranylgeranyl synthesis from mevalonate.

Clomazone reduced the chlorophyll and carotenoid contents of spinach (Spinacia oleracea L.), barley (Hordeum vulgare L.), velvetleaf (Abutilon theophrasti Medik.), and soybean (Glycine max L. Merr.) seedlings. The order of species sensitivity was velvetleaf > spinach > barley > soybean. Clomazone (100 micromolar) did not affect the in vitro activities of spinach isopentenyl pyrophosphate isomerase or prenyl transferase. Clomazone also did not affect the synthesis of isopentenyl pyrophosphate from mevalonic acid. Thus, clomazone had no direct in vitro effect on the synthesis of geranylgeranyl pyrophosphate from mevalonic acid. Greening seedlings of both soybean and velvetleaf metabolized clomazone. No qualitative differences in the metabolites were detected between soybean and velvetleaf. Thus, differential metabolism of clomazone to a toxic chemical that inhibits terpenoid synthesis is unlikely. Clomazone has either a mode of action not yet identified or a metabolite that is selective in that it is much more active in sensitive than tolerant species.

Uptake and accumulation of the herbicide bentazon by cultured plant cells.

Cellular absorption of the herbicide bentazon, a weak acid with pK(a) 3.45, was investigated using suspension-cultured cells of velvetleaf (Abutilon theophrasti Medic.). Bentazon accumulated rapidly to concentrations approximately four times that of the external medium. Bentazon accumulation against a concentration gradient was not due to its conversion to metabolites, partitioning into lipids, or binding onto cellular constituents. Bentazon uptake was related linearly to the external bentazon concentration, implying that movement of the herbicide into cells was not carrier-mediated. Bentazon was able to diffuse freely and extensively out of the cells, indicating that bentazon can readily diffuse across cell membranes. Potassium cyanide and carbonyl cyanide m-chlorophenyl hydrazone inhibited bentazon accumulation as did nitrogen gas when bubbled through the uptake medium. Absorption was pH-dependent with the greatest amount of bentazon accumulating at acidic external pH. Calculations indicated that conversion of uncharged bentazon to bentazon anion in the cytoplasm accounts for cellular accumulation of bentazon. These results provide evidence that bentazon is absorbed across membranes via simple diffusion and that bentazon accumulates in plant cells via an energy-dependent, ion-trapping mechanism which results in bentazon accumulation in the cytoplasm.

Diclofop-methyl increases the proton permeability of isolated oat-root tonoplast.

Diclofop-methyl (methyl ester of 2-[4-(2',4'-dichlorophenoxy)phenoxy]propionate; 100 micromolar) and diclofop (100 micromolar) inhibited both ATP- and PPi-dependent formation of H(+) gradients by tonoplast vesicles isolated from oat (Avena sativa L., cv Dal) roots. Diclofop-methyl (1 micromolar) significantly reduced the steady-state H(+) gradient generated in the presence of ATP. The ester (diclofop-methyl) was more inhibitory than the free acid (diclofop) at pH 7.4, but this relative activity was reversed at pH 5.7. Neither compound affected the rate of ATP or PPi hydrolysis by the proton-pumping enzymes. Diclofop-methyl (50, 100 micromolar), but not diclofop (100 micromolar), accelerated the decay of nonmetabolic H(+) gradients established across vesicle membranes. Diclofop-methyl (100 micromolar) did not collapse K(+) gradients across vesicle membranes. Both the (+)- and (-)-enantiomers of diclofop-methyl dissipated nonmetabolic H(+) gradients established across vesicle membranes. Diclofop-methyl, but not diclofop (each 100 micromolar), accelerated the decay of H(+) gradients imposed across liposomal membranes. These results show that diclofop-methyl causes a specific increase in the H(+) permeability of tonoplast.

Characterization of the inhibition of k absorption in oat roots by salicylic Acid.

The phenolic compounds salicylic acid (o-hydroxybenzoic acid) and ferulic acid (4-hydroxy-3-methoxycinnamic acid) inhibited K(+) ((86)Rb(+)) absorption in excised oat (Avena sativa L. cv. Goodfield) root tissue. Salicylic acid was the most inhibitory. The degree of inhibition was both concentration- and pH-dependent. With decreasing pH, the inhibitory effect of the phenolic increased. During the early stages of incubation, the time required to inhibit K(+) absorption was also pH- and concentration-dependent. At pH 4.0, 5x10(-4) molar salicylic acid inhibited K(+) absorption about 60% within 1 minute; whereas, at pH 6.5, this concentration affected absorption only after 10 to 15 minutes. However, at 5 x 10(-3) molar and pH 6.5, salicylic acid was inhibitory within 1 minute. The capacity of the tissue to recover following a 1-hour treatment in 5 x 10(-4) molar salicylic acid ranged from no recovery at pH 4.5 to complete recovery at pH 7.5. The absorption of salicylic acid was pH-dependent, also. As pH decreased, more of the phenolic compound was absorbed by the tissue. The increased absorption of the compound at low pH most likely contributed to apparent tissue damage at pH 4.5 and might have accounted for the lack of recovery of K(+) absorption as pH decreased.Under the proper conditions of pH and concentration, phenolic acids such as salicylic acid could significantly affect mineral absorption by plants in the field.

Inhibition of adenosine triphosphatase activity of the plasma membrane fraction of oat roots by diethylstilbestrol.

Diethylstibestrol (DES) inhibited noncompetitively the ATPase in the plasma membrane fraction from Avena sativa L. cv. Goodfield roots when assayed in the presence of MgSO(4) or MgSO(4) plus KCl. In the presence of MgSO(4), 7.1x10(-5) molar DES inhibited the enzyme 50%; whereas in the presence of MgSO(4) and KCl, 1.3x10(-4) molar DES was required for the same inhibition. Dixon plots indicated that in the presence of MgSO(4), one molecule of DES bound to one molecule of ATPase; however, in the presence of MgSO(4) and KCl, two or more molecules bound to one ATPase molecule. These results suggested that KCl causes a conformational change in the enzyme which exposes additional binding sites for DES, but that these sites are not as inhibitory as the first binding site.In addition to KCl, other factors also affected the DES inhibition of the ATPase. Plasma membrane vesicles warmed to 38 C were inhibited more than vesicles kept on ice prior to assay. DES inhibited the Triton X-100-treated ATPase less than the ATPase which was not detergent-treated. Finally, studies with DES analogs showed that the hydroxyl groups of DES were essential for inhibition and that steric configurations of the molecule were important.DES inhibition of the ATPase suggests that DES inhibits K(+) absorption in oat roots by inhibiting the ATPase. Inhibition of K(+) absorption was greater than inhibition of the ATPase, and thus DES may also inhibit other aspects of metabolism that are involved with ion absorption.

Effect of diethylstilbestrol on ion fluxes in oat roots.

Effects of diethylstilbestrol (DES) on ion fluxes in oat roots (Avena sativa L.) were investigated by measuring K(+) and Cl(-) absorption and K(+) efflux. DES rapidly decreased the absorption of K(+) ((86)Rb) and (36)Cl(-) by excised roots; 10(-4) molar DES inhibited Cl(-) absorption in 1 minute and K(+) absorption in 1 to 2 minutes. With a 10-minute incubation period, K(+) and Cl(-) absorption were inhibited 50% by 1.1x10(-5) molar and 8.4x10(-6) molar DES, respectively. Treatment for 3 minutes with 10(-4) molar DES caused irreversible inhibition of K(+) absorption. Increasing concentrations of KCl in the absorption media decreased the DES inhibition. Experiments with the DES analogs, DES dipropionate, dienestrol and hexestrol, showed that the steric configuration and the hydroxyl group of the DES molecule are important in determining the inhibitory capacity of the compound.DES increased the efflux of (86)Rb from excised roots only after a 10-minute lag period. In 10(-4) molar DES, roots lost 82% of their radionuclide content in 1 hour. Comparison of efflux curves for roots loaded for 20 hours and those loaded for 15 minutes suggested that DES increased the permeability of the plasma membrane after about 10 minutes and the permeability of the tonoplast after 10 to 20 minutes. Oligomycin and dinitrophenol also increased the loss of (86)Rb, but the lag period was about 4 hours.The rapid effect of DES on ion absorption and the slower effect on ion efflux suggest that DES initially inhibits ion uptake by affecting the transport mechanism at the plasma membrane in some manner other than alteration of membrane permeability.

Comparison of Reductions in Adenosine Triphosphate Content, Plasma Membrane-associated Adenosine Triphosphatase Activity, and Potassium Absorption in Oat Roots by Diethylstilbestrol.

The possibility was investigated that diethylstilbestrol (DES) inhibits potassium absorption in oat (Avena sativa L. cv. Goodfield) roots by inhibiting mitochondrial functions in addition to inhibiting the plasma membrane ATPase. DES at 10(-6) molar stimulated the mitochondrial ATPase slightly, but higher concentrations had no effect. Oxidative phosphorylation by isolated mitochondria was inhibited 50% by 2.6 x 10(-5) molar DES; concentrations of 10(-4) molar or greater were completely inhibitory. After a lag of about 2 minutes, 10(-4) molar DES produced a linear decrease in ATP content of excised roots. After 20 minutes, the ATP content of the tissue was about 50% of the control and remained at that level after 30 minutes in DES.Comparison of changes in ATP content, plasma membrane ATPase activity, and K(+) absorption rate with time in the presence of DES showed that the rapid decrease in K(+) absorption rate corresponded more closely with the decrease in ATPase activity than the decrease in ATP content. Total inhibition of the ATPase was calculated by multiplying together the percentage decreases in ATPase activity and ATP content. At times greater than 10 minutes this "net" ATPase activity corresponded very closely with the K(+) absorption rate.These results show that DES can inhibit potassium absorption by reducing mitochondrial ATP production in addition to inhibiting the plasma membrane ATPase. However, the rapid (less than 5 minutes) inhibition of absorption is caused by direct inhibition of the ATPase rather than a reduced ATP supply because the ATP content is lowered only slightly whereas the ATPase is inhibited dramatically in that time. The relationship between plasma membrane ATPase activity and K(+) absorption rate as inhibited by DES supports the hypothesis that the ATPase is involved in cation absorption by plant roots.

Plasma membrane adenosine triphosphatase of oat roots: activation and inhibition by mg and ATP.

ATPase activity of plasma membrane vesicles isolated from oat (Avena sativa L. cv. Goodfield) roots was examined in the presence of various concentrations of MgCl(2) and ATP. A Mg(2+): ATP ratio of about 1 was required for maximal activity regardless of the concentrations used; the optimum concentration for both Mg(2+) and ATP was 9 mm. Based on the ATPase activity at different concentrations of complexed Mg.ATP and free ATP, it is concluded that Mg.ATP is the true substrate of this enzyme.Under certain experimental conditions, high concentrations of MgCl(2) and ATP inhibited the plasma membrane ATPase. On the basis of the relative amounts of free and complexed ATP and Mg(2+), it was found that the different moieties caused different amounts of inhibition. Free ATP inhibited the ATPase at concentrations in excess of 2 mm. Mg.ATP concentrations above 11 mm inhibited the enzyme. Free Mg(2+) caused only a slight inhibition of the ATPase.The Km for Mg.ATP was found to vary from 0.64 to 1.24 mm depending on the experimental conditions. This variation is thought to be due to variable amounts of Mg.ATP, which serves as an inhibitor as well as the substrate, and free ATP, which also inhibits the enzyme.