The Rapid Isolation of Vacuoles from Leaves of Crassulacean Acid Metabolism Plants

Autolytic lipid changes in corn (Zea mays L.) root crude homogenates and isolated membranes were examined by the use of high performance thin-layer chromatography. In the absence of added CaCI2, losses in phosphatidylcholine and other phospholipids corresponds to increase in fatty acids without the accumulation of either phosphatidic acid or lyso-phosphatidylcholine. However, in the presence of 1 millimolar CaCI2, phosphatidylcholine concentrations declined more rapidly with an immediate increase in phoshatidic acid, and slower rate of fatty acid accumulation. Autolytic phospholipid degradation yielded primarily free fatty acids in the absence of Ca and phosphatidic acid in the presence of 1 millimolar CaCI2, suggesting the presence of an acyl hydrolase and phospholipase D activities. Differential centrifugation studies indicate that 50 to 80% of the crude homogenate's phospholipase D activity is membrane-bound. Density centrifugation experiments suggest that the membranebound phospholipase D activity is localized primarily on mitochondrial membranes.Our interest in phospholipid degradation originates from a desire to minimize the loss of lipid constituents from membranes during isolation from plant tissues. Inactivation of membrane-bound CCR' from wheat aleurone and the vanadate-sensitive proton pump from maize roots during isolation was associated with degradation of membrane lipids (2, 28). lipids by a reconstitution restored transport activity to initial levels (2). Such results indicated that net proton transport was inhibited because of increased leakage of proton resdulting from phospholipid degradation. Losses in membrane phospholipids from corn root microsomes did not correspond to an accumulation of either lysophospholipids or PA (2), suggesting that neither PLase A2 nor D was responsible for the degradation. However, the exact nature of the lipolytic enzymes responsible for the phospholipid degradation in maize root homogenates has not been identified and is the focus of this report. Results from this study indicate the presence of at least two lipolytic enzyme activities, a Ca-stimulated PLase D and an acyl hydrolase. MATERIALS AND METHODS Preparation of Crude Homogenates and Membrane FractionsRoots (40-50 g fresh weight) from 3 d old maize seedlings (Zea mays L. cv WF9 x Mol7) were grown on filter paper moistened with 0.1 mm CaCl2 and harvested as described previously (19). Roots were homogenized by mortar and pestle for 3 min in the presence of 50 mm Mes titrated to pH 6.0 with BTP, 0.25 M sucrose, and 5 mM DTT using 3 mL of buffer per g of roots at 0 to 4°C. The brei was filtered through four layers of cheesecloth, and the resulting filtrate was used as the crude homogenate. Homogenates and membranes prepared as above contained less than 1 ,AM Ca2' as determined by Ca-selective electrode (data not shown). Protein concentration was determined after precipitation by TCA in the presence of deoxycholate by a modification of the Lowry method (1).When membranes in the pH 6.0 crude homogenate wer...

Section: Materials and Methods Preparation Of Crude Homogenates And Mmentioning

confidence: 99%

Evidence for and Subcellular Localization of a Ca-Stimulated Phospholipase D from Maize Roots

Bräuer¹,

Nungesser²,

Maxwell³

et al. 1990

“…Vacuoles were prepared isoosmotically from protoplasts by short-time addition of DEAE-dextran (Pharmacia), according to the method of Durr et al (8) as modified by Schmidt and Poole (29), and purified by centrifugation (100,OOOg, 30 min; SW 27, Beckman L-5/50) on a three-step discontinuous Ficoll-400 gradient (Pharmacia) (25) …”

Section: Methodsmentioning

confidence: 99%

Adenylate Levels, Energy Charge, and Phosphorylation Potential during Dark-Light and Light-Dark Transition in Chloroplasts, Mitochondria, and Cytosol of Mesophyll Protoplasts from Avena sativa L.

Hampp¹,

Goller²,

Ziegler³

1982

165

The compartmentation of celular energy relations during dark-light and light-dark transitions was studied by means of a newly developed technique to fractionate oat (Avena sativa L., var. Arnold) mesophyli protoplasts. Using an improved microgradient system with hydrophobic and hydrophilic layers of increasing density, a pure plastid pellet (up to 90% of total chloroplasts) could be separated from an interphase of only slightly contaminated mitochondria (70 to 80% of total mitochondria), and a cytoplasmic supernatant could be obtained within Energy production in heterotrophic cells of higher organisms takes place mainly in mitochondria. The ATP built up in these organelles is exported to ATP consuming sites of the cell and a back flow of ADP maintains phosphorylation (18,20).In cells of eucaryotic green plants, a similar situation probably exists in the dark. Under illumination, however, the production of ATP by chloroplasts was shown to exceed considerably that by mitochondria, both organelles yielding about 90%o of the ATP which is generated by the cell (30). Because many energy-requiring reactions proceed outside of these organelles, e.g. synthesis of protein, fatty acids, and sucrose, transfer and regulation of phosphorylation between organelles and cytosol is a prerequisite.Investigations on adenine nucleotide transfer demonstrated the existence of specific adenylate carriers on the transport-limiting membranes of both mitochondria and chloroplasts (15,18,19).While such a carrier is also highly active in plant mitochondria (similar to animal mitochondria), the exchange of phosphorylation power across the chloroplast envelope is primarily of indirect nature via a dihydroxyacetone-P/glycerate-3-P shuttle (15,18).Interactions between mitochondria, chloroplasts, and cytosol have not been studied in detail up to now. Of particular interest in this respect is the manner in which photosynthetic events interact with mitochondrial respiration. On the basis of physiological evidence, e.g. specific radioactivity of "CO2 evolved in the light or 02 effect on CO2 compensation point, many investigators favor the view that dark respiration is inhibited in the light either partially or totally.The most likely mediators of the interaction between photosynthesis, respiration, and cytosolic metabolism are the adenylates, Pi, and nicotinamide adenine dinucleotides. These cofactors are known to control many respiratory enzymes (10).Recently, we have developed a microgradient method that meets the requirements for a rapid separation of mitochondria and plastids from protoplast homogenates with acceptably low levels of cross contamination, as shown by the use of specific markers (11). Meanwhile, we have further improved this technique and used it to determine pool sizes of adenylates and inorganic phosphate.The results given in this paper on events during dark -* light and light -. dark transition for the first time clearly demonstrate the close interaction of all compartments in keeping the cytosolic energy charge const...

“…The changes in intensity of peak 5 ( Fig. 1) (8,11,12,22,26,28). Thus, it is possible that sugars may be stored temporarily in the vacuole during the day.…”

Section: Diurnal Changes In Sugars and Organic Acids Of Pineapplementioning

confidence: 99%

“…Sedum protoplasts and vacuoles were prepared by a previously published method (8). Pineapple protoplasts and vacuoles were prepared by a modification of this procedure as described elsewhere (5).…”

mentioning

confidence: 99%

Maintenance Carbon Cycle in Crassulacean Acid Metabolism Plant Leaves

Kenyon

Severson²,

Black³

1985

Self Cite

The reciprocal relationship between diurnal changes in organic acid and storage carbohydrate was examined in the leaves of three Crassulacean acid metabolism plants. between pools of malate at night and carbohydrate in the day (2,15,16).In all CAM species studied, malate is the predominant organic acid that accumulates in CAM leaf tissue at night and is depleted during the day. Nocturnal '4C02 assimilation profiles indicate that various metabolites are labeled, especially aspartate, alanine, and intermediates ofthe citric acid cycle (17, 23). The percentage of the label that accumulates in these components relative to malate, however, is low. In some species, citrate fluctuates diurnally but the amplitude is three orders of magnitude less than that exhibited by malate (10,15,20,25