Membrane-bound ATPase of intact vacuoles and tonoplasts isolated from mature plant tissue

Wheat irrigated with nutrient solutions containing 0, 0.2, 0.5, 1, 2, or 6 millimolar K' had maximum photosynthetic rates at 1 to 2 millimolar K+ concentrations. Rates in the 6 millimolar Ktgrown plants were not higher than the 2 millimolar K -grown wheat, and rates were inhibited below 0.5 millimolar KV. Photosynthesis was measured by both attached whole leaf CO2 uptake and by '4C02 fixation of leaf slices in solution.Exposure of leaf slices from 0.2, 2, and 6 millimolar K-grown wheat to various assay media water potentials showed that photosynthesis of the 0.2 millimolar K+-grown wheat decreased from control (high water potential) rates by 35%, that of the 2 millimolar K-grown wheat by 20.4%, and that of the 6 millimolar K+-grown wheat by only 83% at -3.11 megapascals. Also, photosynthesis of the 6 millimolar K+-grown wheat was enhanced by 28% over that of the 2 millimolar K+ wheat at the most severe water stress (-3.11 megapascals), indicating that the excess leaf K+ in the 6 millimolar K+-grown wheat partially reversed dehydration effects on photosynthesis. Oligomycin eliminated the protective effects of high K+ on photosynthesis in dehydrated leaf slices. These results suggest that the protective effect of high K+ under water stress may involve the exchange of K+ in the cytoplasm for stroma H+, thus altering stromal pH and restoring photosynthesis. The protective effect of high K+ was also observed in attached whole leaf photosynthesis of in situ water-stressed wheat grown on 0.2, 2, and 6 millimolar KV. Under water stress, rates of the 6 millimolar K+-grown wheat were enhanced by 66.2% and 113.9% over that of 2 millimolar K+-grown wheat in two separate experiments. Internal CO2 concentration of the 6 millimolar K'-grown wheat was lower than that of the 0.2 and 2 millimolar K+-grown wheat. These results suggest that the high K+ effects on chloroplast photosynthesis seen in leaf slices also occur at the whole plant level.Inhibition of photosynthesis in isolated chloroplasts, protoplasts, and leaf slices by dehydration in hypertonic solutions (4,16) diating water stress inhibition of the photosynthetic process.One line of evidence suggests that the stromal pH is too low to support optimal photosynthetic rates in dehydrated plastids, and that cellular processes which facilitate stromal alkalization may modulate stress effects on chloroplasts. Inhibition of photosynthesis of isolated chloroplasts in hypertonic medium was partially reversed by addition of K+ and NH4Cl (2, 3), which raise stromal pH (13,20

mentioning

confidence: 72%

Modulation of Water Stress Effects on Photosynthesis by Altered Leaf K⁺

Pier¹,

Berkowitz²

1987

“…Vanadate-inhibited ATPase has been used as a plasma membrane marker when molybdate is present as an inhibitor of phosphatase (2,14). In these experiments, 17.4% of the plasma membranes were present in the vacuolar fraction. Latent UDPase, a Golgi marker, and NADPH-Cyt c reductase, an ER marker, showed 7% and 16.5% contamination of the vacuolar fraction, respectively.…”

Section: Resultsmentioning

confidence: 99%

Proton Transport in Isolated Vacuoles from Corn Coleoptiles

Mandala

Taiz²

1985

Vacuoles were isolated from corn coleoptile protoplasts and ATPdependent proton transport was measured by quinacrine fluorescence quenching or by the uptake of l'4Clmethylamine. Intact vacuoles were judged to be free of a surrounding plasma membrane based on fluorescent staining with fluoroscein-diacetate. Essentially all of the detectable ATPstimulated methylamine uptake and a-mannosidase activities present in intact protoplasts were recovered in isolated vacuoles. In contrast, the activities of marker enzymes for plasma membranes, Golgi, endoplasmic reticulum, and mitochondria were reduced to 5 to 17% in vacuolar preparations. The characteristics of proton pumping by isolated vacuoles were compared to those of light microsomal membranes possibly derived from the tonoplast. ATP-dependent proton pumping by both isolated vacuoles and light microsomal vesicles was stimulated by Cl1, and inhibited by NO3-, carbonyl cyanide-m-chlorophenylhydrazone, N,N'-dicyclohexylcarbodiimide, N-ethylmaleimide, 4,4'-diisothiocyano-2,2'-stilbene disulfonic acid, diethylstilbestrol, and 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole, but not by vanadate. Both activities also showed substrate specificity for Mg-ATP. Finally, proton transport activities of vacuolar and microsomal fractions exhibited similar profiles after flotation in linear dextran gradients. We conclude that the microsomal proton pump previously charcterized in com coleoptiles Plant Physiol 70: 1738-1742) is derived from the tonoplast.The importance of electrogenic proton pumping ATPases in the transport of solutes across the plasma membrane and tonoplast of plant cells is being increasingly recognized. A cationstimulated Mg2+-ATPase on the plasma membrane is thought to be an electrogenic proton pump (25), and recent attempts to purify and reconstitute plasma membrane proteins have supported this idea (28). The presence of a proton-pumping ATPase on the tonoplast has been more controversial, although recent studies with isolated, intact vacuoles have led to the identification of a tonoplast ATPase in several systems (2-4, 7, 17, 19, 29-31). Investigations of the transport properties of sealed microsomal membranes have indicated that vesicles capable of in vitro ATPdependent proton transport can be isolated from a number of different plant tissues (26 and references therein). The bulk of this activity appears to be due to a novel ATPase which is distinguishable from other known ATPases (plasma membrane, mitochondrial, and chloroplast), but is similar to the recently characterized tonoplast ATPase.We have previously shown that microsomal membranes from corn coleoptiles have ATP-dependent proton pumping activity which is stimulated by Cl-, has a slightly alkaline pH optimum,

“…Concentrations of organic acids and ions were estimated from extracts of Cd-treated cultured cell assuming that 75% of packed cell volume was vacuolar sap and that major ion and organic acid species occur in the vacuole in vivo (9). The last assumption (for acids and nutrient ions) is based on earlier 10 and 30% ofCd present was as Cd-peptide, respectively. Here, we calculated the molar concentration of Cd-peptide using these percentages and the experimentally determined concentration of Cd.…”

Section: Methodsmentioning

confidence: 99%

“…1, if concentrations of other ligands in the vacuole system are constant, the amount of Cd complexed by peptides will depend on the amount of peptide present. According to the GEOCHEM-PC analysis using data from Table I and including the presence of Cd-peptide to complex 10 and 30% of the total Cd, vacuolar concentration of peptide ligand must reach 10-5 and 10-5 l M ( Fig. 1) for cultured tobacco cells exposed to 45 AM Cd (contain 0.027 mm Cd after 4 h).…”

Section: Methodsmentioning

confidence: 99%

See 1 more Smart Citation

Computer-Simulated Evaluation of Possible Mechanisms for Quenching Heavy Metal Ion Activity in Plant Vacuoles

Wang¹,

Evangelou²,

Nielsen³

et al. 1991

Various mechanisms have been suggested for the quenching of Cd ion activity in plant vacuoles. These include solution complexation with organic acids and sulfhydryl-containing peptides and precipitation as sulfides. Because direct experimental support for these mechanisms is lacking and difficult to obtain, we have used a computer model to evaluate the quenching role of possible organic and inorganic ligands of tobacco cultured cells exposed to Cd. Results of this thermodynamic evaluation, which assumes that a chemical equilibrium state is met in the vacuole, support the conclusion that sulfhydryl-containing peptides and certain organic acids may form soluble Cd complexes. Although complexation of malate and oxalate with Cd is predicted to be less significant, citrate in the concentration range encountered in the tobacco cultured cell vacuoles has high potential for forming soluble complexes with Cd over the entire possible vacuolar pH range, especially 4.3 to 7.0, even in the presence of low levels of Cd-binding peptides. In addition, results show that inorganic chloride, sulfide (if present), and phosphate may also act to sequester Cd ion activity in the vacuole by forming soluble Cd-Cl and insoluble CdS and Cd-phosphate.Understanding the fate ofCd in plants is of interest because of concerns of Cd transfer from plants to animals and man. Recent studies suggest that heavy metals accumulated in the higher plant are mainly compartmentalized in the vacuole (5, 7, 9, 27). Various mechanisms have been proposed to account for the accumulation of these potentially toxic heavy metal ions in the plant vacuole (18,30). In general, these mechanisms include formation of soluble metal-organic acid complexes (4, 9, 14) or metal-phytate (25, 26), formation ofmetalpeptide or metal-peptide-sulfide complexes (9, 18, 27), or precipitation of metal-sulfides (2,21,25).Support for a particular mechanism of accumulation/sequestration of an ion is gained if the compartment of accumulation/sequestration is verified and if speciation of the ion in that compartment is determined. Both direct and indirect approaches (10) used to determine compartmentation ofions, including heavy metals, can only provide qualitative and quantitative estimates of vacuole contents (28). They cannot identify the species of ion complexes occurring in vacuoles. Therefore, mechanisms proposed on the basis of compartmentation analysis alone are not sufficient to argue the validity of a mechanism of accumulation/sequestration. However, it is possible with computer assistance to simulate the ion species distribution in the plant vacuole and thus to evaluate a proposed mechanism.Computer calculations have been used to model the chemistry of xylem sap of soybean and tomato (29). Here, we use data obtained previously regarding sap composition of vacuoles from Cd-treated tobacco (Nicotiana tabacum) cultured cells and the GEOCHEM-PC computer model (16,23) to predict ion species of these vacuoles in vivo. The prediction of ion speciation in vacuoles of Zn-treate...