[show abstract][hide abstract] ABSTRACT: Protoplasts and vacuoles were isolated and purified in large numbers from the CAM plants Ananas comosus (pineapple) and Sedum telephium for protein characterization. Vacuoles were further fractionated to yield a tonoplast vesicle preparation. Polypeptides of protoplasts, vacuoles, and tonoplast vesicles were compared to whole leaf polypeptides from both plants by one-dimensional sodium dodecylsulfate-polyacrylamide gel electrophoresis. Approximately 100 vacuole polypeptides could be resolved of which 25 to 30% were enriched in the tonoplast vesicles. The proteins of protoplasts, vacuoles, and tonoplast vesicles from A. comosus were analyzed further by two-dimensional gel electrophoresis. When one-dimensional electrophoretograms of A. comosus polypeptides were stained with a glycoprotein-specific periodic acid Schiff stain, very few polypeptides appeared to be glycosylated, whereas a large number of glycosylated polypeptides were detected with a silver-based glycoprotein stain particularly in tonoplast vesicles. Analysis of the enzymic content of vacuoles from both plants indicated the presence of a variety of hydrolases, including bromelain as a major constituent of A. comosus. No substrate-specific ATPase, however, could be detected in vacuoles or tonoplast vesicles from either plant.
[show abstract][hide abstract] ABSTRACT: 1-Aminocyclopropane-1-carboxylate (ACC) synthase (EC 220.127.116.11), extracted from tomato pericarp tissue, was purified 6500-fold by conventional and high-performance liquid chromatography. Two-dimensional gel electrophoresis of this preparation indicated that ACC synthase activity was associated with a protein band at 50 kDa, a value consistent with size determinations by gel filtration. Monoclonal antibodies against ACC synthase were obtained from murine hybridoma cell lines. These antibodies recognized the native enzyme, as shown with an immunoprecipitation assay. A monoclonal IgG immunoaffinity gel was used to isolate, from a relatively crude enzyme preparation, a single protein, which migrated at 50 kDa in a NaDodSO(4)/polyacrylamide gel. In vivo labeling of wounded tomato pericarp tissue with [(35)S]methionine followed by immunoaffinity purification of ACC synthase yielded a radioactive protein of 50 kDa. We conclude that the 50-kDa protein represents ACC synthase in extracts of wounded tomato pericarp tissue.
Proceedings of the National Academy of Sciences 11/1986; 83(20):7755-9. · 9.74 Impact Factor
[show abstract][hide abstract] ABSTRACT: The possible role of photosynthesis in the mechanism of action of the herbicide acifluorfen (2-chloro-4-(trifluoromethyl)phenoxy-2-nitrobenzoate; AF) was examined. The sensitivity to AF of cotyledons of cucumber (Cucumis sativus L.) which had been grown under far red light (FR) and white light were compared. FR grown tissues which were photosynthetically imcompetent were hypersensitive to AF under white light and had approximately the same relative response to AF under blue and red light as green, white-light-grown tissues. Ultrastructural damage was apparent in FR-grown, AF-treated tissues within an hour after exposure to white light, with cytoplasmic and plastidic disorganization occurring simultaneously. In cucumber cotyledon tissue which had been greening for various time periods, there was no correlation between photosynthetic capacity and herbicidal efficacy of AF. PSII inhibitors (atrazine and DCMU) and the photophosphorylation inhibitor, tentoxin, had no effect on AF activity. Atrazine did not reduce AF activity at any concentration or light intensity tested, indicating that there is no second, photosynthetic-dependent mechanism of action operating at low AF concentrations or low fluence rates. Carbon dioxide-dependent O(2) evolution of intact chloroplasts of spinach (Spinacia oleracea L.) had an AF I(50) of 125 micromolar compared to 1000 micromolar for cucumber, whereas AF was much more herbicidally active in tissues of cucumber than of spinach. Differences in activity could not be accounted for by differences in uptake of AF. Our results indicate that there is no photosynthetic involvement in the mechanism of action of AF in cucumber.
[show abstract][hide abstract] ABSTRACT: The herbicidal action of dimethazone [FMC 57020; 2-(2-chlorophenyl) methyl-4,4-dimethyl-3-isoxalidinone] on cowpea (Vigna unguiculata L.) primary leaves was studied. Seeds were imbibed in 0.5 mM herbicide for 1 day and then seedlings were grown in darkness. In 6-day-old, etiolated seedlings, there was no effect of the herbicide on protochlorophyllide accumulation or on phototransformation of protochlorophyllide to chlorophyllide, however, the Shibata shift was greatly slowed. Accompanying this was a delay in phytylation of chlorophyllide. Protochlorophyllide resynthesis in a dark period after phototransformation of existing protochlorophyllide in etiolated tissue was also slowed by dimethazone. In the light, carotenoid accumulation and chlorophyll accumulation were slowed by the herbicide, resulting a pale green appearance of the leaves. The capacity for CO2-dependent oxygen evolution or FeCN-dependent oxygen evolution did not develop in dimethazone-treated tissue during 24 hr of light exposure. In situ measurement of variable fluorescence and cytochrome f photooxidation/dark reduction indicated that some cyclic electron transport developed very slowly in dimethazone-treated plants. No effect of the herbicide was found on either FeCN-dependent oxygen evolution or variable fluorescence in fully greened tissues. Dimethazone was concluded to have an effect on chloroplast development rather than a direct effect on photosynthesis.
[show abstract][hide abstract] ABSTRACT: The herbicide acifluorfen (2-chloro-4-(trifluoromethyl)phenoxy-2-nitrobenzoate) causes strong photooxidative destruction of pigments and lipids in sensitive plant species. Antioxidants and oxygen radical scavengers slow the bleaching action of the herbicide. The effect of acifluorfen on glutathione and ascorbate levels in cucumber (Cucumis sativus L.) cotyledon discs was investigated to assess the relationship between herbicide activity and endogenous antioxidants. Acifluorfen decreased the levels of glutathione and ascorbate over 50% in discs exposed to less than 1.5 hours of white light (450 microeinsteins per square meter per second). Coincident increases in dehydroascorbate and glutathione disulfide were not observed. Acifluorfen also caused the rapid depletion of ascorbate in far-red light grown plants which were photosynthetically incompetent.Glutathione reductase, dehydroascorbate reductase, superoxide dismutase, ascorbate oxidase, ascorbate free radical reductase, peroxidase, and catalase activities rapidly decreased in acifluorfen-treated tissue exposed to white light. None of the enzymes were inhibited in vitro by the herbicide. Acifluorfen causes irreversible photooxidative destruction of plant tissue, in part, by depleting endogenous antioxidants and inhibiting the activities of protective enzymes.
[show abstract][hide abstract] ABSTRACT: The reciprocal relationship between diurnal changes in organic acid and storage carbohydrate was examined in the leaves of three Crassulacean acid metabolism plants. It was found that depletion of leaf hexoses at night was sufficient to account quantitatively for increase in malate in Ananas comosus but not in Sedum telephium or Kalanchoë daigremontiana. Fructose and to a lesser extent glucose underwent the largest changes. Glucose levels in S. telephium leaves oscillated diurnally but were not reciprocally related to malate fluctuations.Analysis of isolated protoplasts and vacuoles from leaves of A. comosus and S. telephium revealed that vacuoles contain a large percentage (>50%) of the protoplast glucose, fructose and malate, citrate, isocitrate, ascorbate and succinate. Sucrose, a major constituent of intact leaves, was not detectable or was at extremely low levels in protoplasts and vacuoles from both plants.In isolated vacuoles from both A. comosus and S. telephium, hexose levels decreased at night at the same time malate increased. Only in A. comosus, however, could hexose metabolism account for a significant amount of the nocturnal increase in malate. We conclude that, in A. comosus, soluble sugars are part of the daily maintenance carbon cycle and that the vacuole plays a dynamic role in the diurnal carbon assimilation cycle of this Crassulacean acid metabolism plant.
[show abstract][hide abstract] ABSTRACT: The time course for development of herbicidal effects of acifluorfen on the ultrastructure and physiology of cotyledon discs from 6- to 8-day-old, light-grown cucumber (Cucumis sativus L.) seedlings was examined. After 20 hr of incubation in darkness in 30 μM acifluorfen no herbicidal damage was detected, but within 1.5 hr after subsequent exposure to 420 μEin m−2 sec−1 PAR, chloroplast envelope, tonoplast, and plasma membrane disruption was observed. Choloroplast thylakoid disruption occurred after envelope disruption. CO2-Dependent O2 evolution was reduced ca. 30% within 1 hr of the onset of light exposure, whereas dark O2 uptake was not affected during a 5-hr time course. Reduction of in vivo variable chlorophyll fluorescence and cytochrome f oxidation/reduction was observable within 1 hr and was reduced by ca. 90% within 5 hr. Neither carotenoid nor chlorophyll content declined significantly during 5 hr of light, whereas acifluorfen caused increases in ethane, ethylene, and malondialdehyde within 2 hr. Increased leakage of electrolytes was detectable within 1 hr. These data indicate that loss of membrane integrity, perhaps beginning with the plastid envelope, precedes most of the other symptoms of acifluorfen-caused cellular damage.
[show abstract][hide abstract] ABSTRACT: Diurnal changes in levels of selected metabolites associated with glycolysis, the C(3) cycle, C(4)-organic acids, and storage carbohydrates were analyzed in active Kalanchoë daigremontiana Crassulacean acid metabolism leaves. Three metabolic transition periods occurred each day. During the first two hours of light, nearly all of the metabolite pools underwent transient changes. Beginning at daylight, stomata opened transiently and closed again within 30 minutes; malate synthesis continued for about 1 hour into the light; C(3) photosynthesis began within 30 minutes; and net quantities of starch and glucan began to accumulate after 2 hours, continuing linearly throughout the rest of the day.THE SECOND TRANSITION OCCURRED IN MIDAFTERNOON: stomata reopened; malate decarboxylation nearly terminated; and the assimilation of ambient CO(2) occurred primarily via the C(3) cycle. The third transition occurred at dark: stomata transiently closed before opening again; the C(3) cycle stopped; malate synthesis started in about 1 hour; starch and glucan degradation began within 1 hour; and the bulk of carbon flow was through glycolysis leading to the synthesis and accumulation of malate throughout the night. At night, the levels of metabolites involved in acidification and glycolysis (except for phosphoenolpyruvate) generally accumulated. Phosphoenolpyruvate levels peaked near midday and were minimal at night. The ribulose 1,5-bisphosphate pool was depleted at night, while sedoheptulose 1,7-bisphosphate, fructose 1,6-bisphosphate, glucose 6-phosphate, and fructose 6-phosphate accumulated.
[show abstract][hide abstract] ABSTRACT: A technique is presented for the isolation of vacuoles from Sedum telephium L. leaves. Leaf material is digested enzymically to produce protoplasts rapidly which are partially lysed by gentle osmotic shock and the inclusion of 5 millimolar ethyleneglycol-bis (beta-aminoethyl ether)N,N'-tetraacetic acid in the wash medium. Vacuoles are isolated from the partially lysed protoplasts by brief centrifugation on a three-step Ficoll-400 gradient consisting of 5, 10, and 15% (w/v) Ficoll-400. A majority of the vacuoles accumulate at the 5 to 10% Ficoll interface, whereas a smaller proportion sediments at the 10 to 15% Ficoll-400 interface. The total time required for vacuole isolation is 2 to 2.5 hours, beginning from leaf harvest.The yield of vacuoles is approximately 44%. The major vacuole layer is < 7% contaminated by marker enzymes from the cytoplasm and other organelles but shows no contamination by chloroplasts. Isolated vacuoles were stable for >15 hours when left in Ficoll; however, dispersion into media of various osmotic concentrations resulted in decreased stability. Addition of mercaptobenzothiazole, CaCl(2), MgCl(2), bovine serum albumin, ethylenediaminetetraacetic acid, polyethylene glycol 600, and KH(2)PO(4) to the vacuole isolation media did not increase the stability of the isolated vacuoles.THIS TECHNIQUE WITH ONLY SLIGHT MODIFICATIONS HAS BEEN USED TO ISOLATE LEAF CELL VACUOLES FROM THE FOLLOWING CRASSULACEAN ACID METABOLISM PLANTS: pineapple, Kalanchoë fedtschenkoi, and Echeveria elegans. Spinach leaves also were used successfully.