Further studies on the subcellular localization of lipid-degrading enzymes

Further work on the subcellular localization of two lipid-degrading enzymes, lipolytic acyl hydrolase (LAH) and lipoxygenase (LOX) has been carried out on brassica florets, potato shoots and pea roots. In all cases, the LAH profile on sucrose and Ficoll density gradients was coincident with ‘lysosomal’ acid phosphatase activity. However, the localization of LOX activity was different for each tissue. In pea roots the activity of LOX was localized in the ‘lysosomal’ fraction, whereas with brassica florets (cauliflower and calabrese) it was present in a heavy body with a similar density to plastids and in potato shoots LOX gave only low particulate recoveries.     

Subcellular localization of lipoxygenase and lipolytic acyl hydrolase enzymes in plants

The subeellular localization of two lipid-degrading enzymes, lipolytic acyl hydrolase (LAH) and lipoxygenase (LOX) was studied. In potato tubers the ac     

Purification and physiological properties of two lipolytic enzymes of Solanum tuberosum

Two lipolytic enzymes have been separated and partially purified from potato tubers. One enzyme of higher isoelectric value, possessed acyl hydrolase activity toward a number of p-nitrophenyl fatty acyl derivatives, the relative activity depending on the fatty acyl chain length. There was also some activity towards phosphatidyl choline. The other enzyme possessed phospholipase and galactolipase activity, but showed a low acyl hydrolase activity towards p-nitrophenyl fatty acyl derivatives. When applied to plant tissues, the enzyme with the greater acyl hydrolase activity caused rapid ion efflux from discs of potato tuber and beetroot, foflowed by reabsorption of ions by the tissues. The purified phospholipase did not produce this effect but induced acid phosphatase leakage from lysosome-enriched fractions of potato sprout tissue. No maceration of tissue or protoplast disruption was observed when either of the two enzymes were incubated with a variety of plant preparations.     

Phospholipase,galactolipase and acyl transferase activities of a lipolytic enzyme from potato

Characterization of reaction products showed that an enzyme (lipolytic acyl hydrolase) isolated from potato tubers could act on endogenous substrates as a galactolipase (E.C. 3.1.1.26), lysophospholipase (E.C. 3.1.1.5) or a ‘phospholipase B’ but not as a lipase (E.C. 3.1.1.3). The affinity of the enzyme for methanol as acyl acceptor (acyl transferase activity) was higher than its affinity for water (acyl hydrolase activity). The nomenclature of acyl hydrolases in plants is discussed.     

Autolysis of membrane lipids in potato leaf homogenates: Effects of calmodulin and calmodulin antagonists

Potato leaves contain high levels of lipolytic acyl hydrolase activity which degrades phospholipids and galactolipids during homogenization and organelle isolation. Four calmodulin antagonists (dibucaine, tetracaine, trifluoperazine a and chlorpromazine) were found to inhibit the rate of hydrolysis of endogenous membrane lipids in homogenates of potato leaves. In contrast, the addition of calcium and purified calmodulin stimulated the rate of hydrolysis. These results indicate that a lipolytic acyl hydrolase activity in potato leaves appears to be mediated either directly or indirectly by calcium and calmodulin.     

Wax ester formation catalysed by isoenzymes of lipolytic acyl hydrolase

Wax ester formation by esterification of a long chain fatty acid (palmitic acid) with a long chain fatty alcohol (octadecanol) was enzymically catalysed by acetone dried powder preparations of potato tubers. The enzyme responsible for wax ester formation had multiple isoenzymic forms and was identical with lipolytic acyl hydrolase, a lipid deacylating enzyme. Tubers from different varietiees of potato (Solanum tuberosum) demonstrated markedly different levels of activity and electrophoretic patterns for both wax ester formation and lipid deacylation.     

Novel study on the elicitation of hypersensitive response by polyunsaturated fatty acids in potato tuber.

A GC-MS procedure was carried out for the simultaneous and unequivocal quantitation of both potato phytoalexin (rishitin and lubimin) accumulation and the rate of disappearance of polyunsaturated fatty acids (PUFA) and some of their esters tested as possible elicitors. Potato 5-lipoxygenase and lipolytic acyl hydrolase play a key role in hypersensitive response (HR) induction. As expected, arachidonic acid, its hydrolysable esters, and eicosapentaenoic acid elicited much higher HR than the other PUFA tested, although the latter were equally affected by potato 5-lipoxygenase. Hydroxyl radicals appear to be actively involved in the browning process. The polyaminoacid poly-L-lysine did not show any eliciting activity.     

Lipid-degrading enzymes from potato tubers

Three varieties of potato were used to investigate the activity of lipolytic acyl hydrolase, (LAH) and lipoxygenase, (LOX), for a short period after harvest. Both enzymes displayed very low levels of activity during the first few days, followed by an increase in later storage, with the hydrolase activity of Désirée tubers remaining low. An inverse relationship was found between the total LOX activity and the percentage of activity obtained in a particulate form. Only when the total LOX content was below 0.7 units (μmol/g/min fr. wt), was it possible to obtain a highly active particulate fraction. LAH particulate activity was dependent upon both enzymes remaining low. Protoplasts were isolated by the use of cell-degrading enzymes. When the total LOX activity in the tubers was low, 50% of this activity could be obtained in intact protoplasts. Once the LOX concentration in the tubers had risen, fewer intact protoplasts were isolated. No particulate activity of either enzyme was found when these protoplasts were lysed. The two lipid-degrading enzymes were not located in the amyloplasts.     

A novel patatin-like gene stimulated by drought stress encodes a galactolipid acyl hydrolase

A cDNA (Vupat1) encoding a predicted 43 kDa protein was isolated from drought-stressed cowpea (Vigna unguiculata) leaves. It has homology with patatin, a potato tuber storage protein with lipolytic acyl hydrolase activity. The recombinant protein VUPAT1 expressed in the baculovirus system displays preferentially galactolipid acyl hydrolase activity. Phospholipids are very slowly hydrolyzed and apparently triacylglycerols are not deacylated. Vupat1 promoter contains putative drought-inducible sequences. Northern blots showed that gene expression is stimulated by drought stress and is more pronounced in a drought-sensitive cultivar than in a drought-tolerant one. An involvement in drought-induced galactolipid degradation is proposed for VUPAT1.     

Regulation of Photosystem I electron flow activity by phosphatidylglycerol in thylakoid membranes as revealed by phospholipase treatment

Thylakoid membranes were treated by potato lipolytic acyl hydrolase, phospholipases A₂ from pancreas and snake venom, and by phospholipase C from Bacillus cereus under various conditions. The changes in the uncoupled rates of electron transport through Photosystem I (PS I) and in lipid composition were followed during these treatments. Pancreatic phospholipase A₂ which destroyed all phospholipids in thylakoid membranes stimulated the NADP⁺ reduction supported by reduced 2,6-dichlorophenolindophenol. This stimulation concerned only the dark but not the light reactions of this pathway. The main site of action of pancreatic phospholipase A₂ may be located on the donor side of PS I; the hydrolysis of phospholipids at this site caused an increased ability of reduced 2,6-dichlorophenolindophenol and ascorbate alone to feed electrons into PS I. A second site may be located on the acceptor side of PS I, probably between the primary acceptor and the ferredoxin system. When thylakoid membranes were first preincubated with or without lipolytic acyl hydrolase at 30°C (pH 8), the NADP⁺ photoreduction was inhibited whilst the methyl viologen-mediated O₂ uptake was stimulated. A subsequent addition of pancreatic phospholipase A₂ (which had the same hydrolysis rates for phosphatidylglycerol but not for phosphatidylcholine) further stimulated the O₂ uptake and restored NADP⁺ photoreduction. The extent of this stimulation, which depended on the presence of lipolytic acyl hydrolase, was ascribed partly to the hydrolysis of the phospholipids and partly to the generation of their lyso derivatives but not to the release of free fatty acids. On the contrary, phospholipase C which destroyed only phosphatidylcholine failed to restore this activity. It is suggested that phosphatidylglycerol is the only phospholipid associated with thylakoid membrane structures supporting PS I activities and that this lipid may play a physiological role in the regulation of these activities.     

Developmental change in c(6)-aldehyde formation by soybean leaves

Damage to plant leaves by wounding or freezing induces the production of large amounts of C₆-compounds. However, the control of formation of these compounds in leaves is not yet clear. In the current study, C₆-aldehyde formation by freeze-injured soybean leaves of different ages (based on the leaf positions on the plant) at stage R1 of plant development was investigated. The results demonstrate that C₆-aldehyde formation by the soybean (Glycine max L.) leaves changes as leaves develop. Younger leaves produce high levels of C₆-aldehydes, mainly composed of hexanal. Subsequently, as the leaves develop, the level of C₆-aldehyde formation decreases markedly, followed by an increase with a large shift from hexanal to hexenals. Lipoxygenase and lipolytic acyl hydrolase activity was reduced, and, in contrast, hydroperoxide lyase activity increased. There was little difference in lipoxygenase substrate specificity for linoleic acid and linolenic acid, but hydroperoxide lyase preferentially utilized 13-hydroperoxy-9,11,15-octadecatrienoic acid. In the in vivo lipoxygenase substrate pool, the linoleic acid level declined and the relative level of linolenic acid increased. The change in ratios of linolenic acid to linoleic acid showed a similar trend during soybean leaf development to that of hexenals to hexanal.     

Translucent Tissue Defects in Solanum tuberosum L. : II. Alterations in Lipolytic Acyl Hydrolase, Lipoxygenase, and Morphology of Mitochondria and Amyloplasts

We analyzed a physiological defect that involved translucent-like tissue which occurred randomly in potato tubers (Solanum tuberosum L., cv Kennebec) after 8 months of storage. The translucent areas had reduced lipoxygenase (0.73-fold) and lipolytic acyl hydrolase (0.27-fold) activities. The effect(s) of these reduced enzyme activities in vivo is uncertain, but they may have influenced composition, turnover and permeability of membranes because potato lipid is primarily membranous in nature. Electron micrographs of the translucent tissue revealed a discernible decrease in the number of starch granules compared to normal/healthy tissue. A few remaining amyloplasts, which still contained starch granules, possessed large electrondense areas (stroma) within the organelle. Mitochondria in translucent tissue appeared to be present in increased numbers, were aggregated, had fewer but swollen cristae, and, morphologically, were of irregular size and shape suggestive of division. The result of this tuber defect appeared, in part, to be an exaggerated or accelerated form of senescence.     

Postinfectional Changes of Lipid Metabolism in Potato Tuber Resistant and Susceptible to Phytophthora infestans

The effect of Phytophthora infestans on the lipid content and composition as well as lipid acyl hydrolase activity in the potato tuber of two cultivars, Nysa – horizontal resistant and Bintje –susceptible were investigated. In the susceptible cultivar the infection resulted in: a qualitative changes in aminolipids, an increase in steroid content, an inconsiderable decrease in amount of glycolipids and a slight drop in FFA, no significant changes in composition of total, FA and slight loss in acyl hydrolase activity. The following changes were observed in the resistant cultivar: a considerable enhancement of steroid content (reached in about 400 %), a slight increase in amount of glycolipid, a rapid drop of acyl hydrolase activity and FFA content and a considerable increase in oleic acid level in total FA. In the response to both infection and aging in both cultivars no significant quantitative changes in lipid-phosphorus and sulfolipids were noted.     

Delay of Membrane Lipid Degradation by Calcium Treatment during Cabbage Leaf Senescence

Cabbage leaf discs (Brassica oleracea L., Capitata group) were floated adaxial side up in 0, 0.05, or 0.25 m CaCl₂ solutions at 15°C for 14 d in the dark. To assess whether the delay of senescence by calcium treatment involved protection of membrane lipids, chlorophyll and protein content and the lipid composition of the membranes were determined during incubation. Chlorophyll and protein content decreased with time, in correlation with a reduction in the amount of phospholipids. The degree of unsaturation of phospholipids and free fatty acids decreased, whereas the ratio of sterol to phospholipid increased. The proportions of phospholipid classes did not change during senescence. The catabolism of phospholipids was delayed by 0.05 m calcium, but accelerated by 0.25 m, as compared to the untreated control. Based on the levels of the lipid intermediates, phospholipase D, phosphatidic acid phosphatase, lipolytic acyl hydrolase, and lipoxygenase appeared to be involved in the breakdown of phospholipids during senescence. Phospholipase D and phosphatidic acid phosphatase may be directly influenced by calcium. The calcium treatment apparently did not affect the activity of acyl hydrolase. Lipoxygenase, responsible for the peroxidation of the polyunsaturated fatty acids, was probably indirectly influenced by calcium. We conclude that the delay of senescence of cabbage leaf discs by calcium treatment involved protection of membrane lipids from degradation.     

Lipolytic and peroxidative enzyme expressions in cultured potato tuber cells

Potato cells (cv. Norchip) were cultured from tuber parenchymal tissue and subcultured to dissociate and habituate the despecialized cells. After several subculturings on a minimal nutrient media, this line of cells demonstrated repeatable physical growth profiles for dry weight (DW), fresh weight (FW) and protein. Two enzymes of plant lipid metabolism were investigated, lipolytic acyl hydrolase (LAH) and lipoxygenase (LOX), which respectively liberate and peroxidize fatty acids from lipid in cellular membranes. LAH, measured as p-nitrophenyl palmitate hydrolase, was present in this line of cells in easily detectable amounts (317 units g^-1 DW) albeit much lower than that found in mother tuber (9878 units g^-1 DW). The presence of LAH in this line is significant because LAH isozymes are often described as storage proteins, yet activity per gram fresh weight in these unorganized cells is reasonably constant until culture growth exits the linear phase. However, LOX, the most active free fatty acid metabolizing enzyme in potato tubers (89,800 units g^-1 DW), was not detectable in this line of callus or suspension cultured cells. The absence of LOX activity in this line of cells was verified by a number of assay approaches and was confirmed by activity staining of extracted enzymes separated in polyacrylamide gels. The absence of LOX in these cultured cells is especially important in determining the functions of this lipid peroxidation system and how it may be genetically regulated.Mention of company or trade name does not imply endorsement by the United States Department of Agriculture over others not named.A laboratory cooperatively operated by the Midwest Area, Agricultural Research Service, U.S. Department of Agriculture, The Minnesota Agricultural Experiment Station, the North Dakota Agrcultural Experiment Station, and the Red River Valley Potato Grower's Association.     

Lipoxygenase- mediated cleavage of fatty acids in plant mitochondria

Incubation of cauliflower bud mitochondria in the presence of 5 mM CaCl2 results in a rapid hydrolysis of the main membrane phospholipsds. Under the action of phospholipase D, phosphatidic acid is produced and forms, within the membranes, a very labile complex with Ca2+ and HPO42-ions present in the incubation medium. With time, one observes a first step characterized by the formation of phosphatidic acid, followed by a second step linked to the breakdown of this phospholipid. The enzyme responsible for the disappearance of phosphalidic acid has been identified as lipoxygenase. In the presence of molecular oxygen, this enzyme acts on the polyun-saturated fatty acids of phosphatidic add (mainly C18:2 and C18:3) yielding small water-soluble molecules, one of them being identified as malondialdehyde (1, 3-propanedial). Experiments involving inhibitory conditions of the breakdown of phosphatidic acid indicate that lipoxygenase acts directly on membrane-bound phosphatidic acid without previous, involvement of a lipolytic acyl hydrolase activity. In addition, the lipoxygenase activity is fully sensitive to hydroxamate derivatives. It is proposed that the lipoxygenase activity may account for a part of the mitochondrial alternative electron pathway that is insensitive to cyanide.     

Isoenzymes of lipolytic acyl hydrolase and esterase in potato tuber

Five varieties of potato (Solanum tuberosum) were shown by gel- and free-flow-electrophoresis to exhibit multiple forms of lipolytic acyl hydrolase (LAH) and esterase enzymes. The electrophoretic patterns of LAH and esterase activities and protein differed with the variety and were characteristic for a given variety. In the variety (Golden Wonder) with the highest LAH activity (p-nitrophenylpalmitate as substrate), this was 200-fold greater than the esterase activity (p-nitrophenylacetate as substrate) and isoenzyme patterns for both enzymes were the most complex. In the variety with a very low LAH activity (Désirée), the LAH and esterase activities were similar and more simple isoenzyme patterns for these enzymes were observed.     

Calcium- and calmodulin-regulated breakdown of phospholipid by microsomal membranes from bean cotyledons

Evidence for the involvement of Ca²⁺ and calmodulin in the regulation of phospholipid breakdown by microsomal membranes from bean cotyledons has been obtained by following the formation of radiolabeled degradation products from [U-¹⁴C]phosphatidylcholine. Three membrane-associated enzymes were found to mediate the breakdown of [U-¹⁴C] phosphatidylcholine, viz. phospholipase D (EC 3.1.4.4), phosphatidic acid phosphatase (EC 3.1.3.4), and lipolytic acyl hydrolase. Phospholipase D and phosphatidic acid phosphatase were both stimulated by physiological levels of free Ca²⁺, whereas lipolytic acyl hydrolase proved to be insensitive to Ca²⁺. Phospholipase D was unaffected by calmodulin, but the activity of phosphatidic acid phosphatase was additionally stimulated by nanomolar levels of calmodulin in the presence of 15 micromolar free Ca²⁺. Calmidazolium, a calmodulin antagonist, inhibited phosphatidic acid phosphatase activity at IC₅₀ values ranging from 10 to 15 micromolar. Thus the Ca²⁺-induced stimulation of phosphatidic acid phosphatase appears to be mediated through calmodulin, whereas the effect of Ca²⁺ on phospholipase D is independent of calmodulin. The role of Ca²⁺ as a second messenger in the initiation of membrane lipid degradation is discussed.     

Ethylene-Mediated Phospholipid Catabolic Pathway in Glucose-Starved Carrot Suspension Cells

Glucose (Glc) starvation of suspension-cultured carrot (Daucus carota L.) cells resulted in sequential activation of phospholipid catabolic enzymes. Among the assayed enzymes involved in the degradation, phospholipase D (PLD) and lipolytic acyl hydrolase were activated at the early part of starvation, and these activities were followed by β-oxidation and the glyoxylate cycle enzymes in order. The activity of PLD and lipolytic acyl hydrolase was further confirmed by in vivo-labeling experiments. It was demonstrated that Glc added to a medium containing starving cells inhibited the phospholipid catabolic activities, indicating that phospholipid catabolism is negatively regulated by Glc. There was a burst of ethylene production 6 h after starvation. Ethylene added exogeneously to a Glc-sufficient medium activated PLD, indicating that ethylene acts as an element in the signal transduction pathway leading from Glc depletion to PLD activation. Activation of lipid peroxidation, suggestive of cell death, occurred immediately after the decrease of the phospholipid degradation, suggesting that the observed phospholipid catabolic pathway is part of the metabolic strategies by which cells effectively survive under Glc starvation.     

Modification of Phospholipid Catabolism in Microsomal Membranes of [gamma]-Irradiated Cauliflower (Brassica oleracea L.)

Acceleration of membrane deterioration has been observed recently during storage of [gamma]-irradiated cauliflower (Brassica oleracea L., Botrytis group). In the present study, the activity of microsome-associated lipolytic enzymes was investigated in cauliflower florets exposed to 0 or 4 kilograys of [gamma] radiation and stored for 8 d at 13[deg]C. Radiolabeled breakdown products obtained from the metabolism of (16:0/18:2*)-phosphatidylcholine and (16:0/16:0)-phosphatidyl-[N-methyl-3H]choline by microsomal membranes indicated that phospholipase D (EC 3.1.4.4), phosphatidic acid phosphatase (EC 3.1.3.4), and lipolytic acyl hydrolase were associated with the membranes. The rate of phosphatidylcholine catabolism by the membranes increased slowly in control cauliflower during storage. [gamma] irradiation caused an immediate rise in phosphatidylcholine catabolism that remained higher than that of the controls during subsequent storage. Collectively, the data suggest that enhancement of membrane lipolytic activity results from free-radical-induced stress. Rapid increase of the membrane-associated phospholipase D activity may be a key event leading to accelerated membrane deterioration following [gamma] irradiation.