Lysophosphatidate acyltransferase activities in the microsomes from palm endosperm, maize scutellum, and rapeseed cotyledon of maturing seeds

Lysophosphatidate (LPA) acyltransferase (EC 2.3.1.51) in the microsomes from palm endosperm (Syagrus cocoides Martius), maize scutellum (Zea mays L.), and rapeseed cotyledon (Brassica napus L.) of maturing seeds were studied for their specificities toward the acyl moiety of the substrates lysophosphatidate and acyl coenzyme A (CoA). The LPA acceptor greatly influenced the acyl CoA specificity of the enzyme and vice versa. With 1-oleoyl-lysophosphatidate (LPA-18:1), the palm enzyme was equally active on oleoyl CoA and lauroyl CoA, whereas the maize and rapeseed enzymes were more active on oleoyl CoA than on lauroyl CoA. With 1-lauroyl-lysophosphatidate (LPA-12), which generated less activity than LPA-18:1, the palm enzyme was three times more active on lauroyl CoA than on oleoyl CoA. LPA-12 was an inactive substrate for the maize and rapeseed enzymes. The selectivity of the enzymes was also studied using a mixture of LPA-18:1 and LPA-12, as well as lauroyl CoA and oleoyl CoA. Under this selectivity condition and compared to the specificity condition, the enzymes from all the three seeds exerted stronger preference for oleoyl moiety in either the LPA or acyl CoA, and again, only the palm enzyme could act on LPA-12. Similar studies, although in lesser detail, showed that the enzymes from soybean and castor bean were similar to the maize and rapeseed enzymes in having little activity on substrates containing lauroyl moiety. The results demonstrate the importance of the acyl group in the sn-1 position of LPA in determining the acyl preference in the sn-2 position in phosphatidate synthesis. The palm enzyme appears to be the only one capable of synthesizing phosphatidates containing high amounts of lauric moieties.     

Lysophosphatidate Acyltransferase in the Microsomes from Maturing Seeds of Meadowfoam (Limnanthes alba)

Lysophosphatidate (LPA) acyltransferase (EC 2.3. 1.51) in the microsomes from the maturing seeds of meadowfoam (Limnanthes alba), nasturtium (Tropaeolum majus), palm (Syagrus cocoides), castor bean (Ricinus communis), soybean (Glycine max), maize (Zea mays), and rapeseed (Brassica napus) were tested for their specificities toward 1-oleoyl-LPA or 1-erucoyl-LPA, and oleoyl coenzyme A (CoA) or erucoyl CoA. All the enzymes could use either of the two acyl acceptors and oleoyl CoA, but only the meadowfoam enzyme could use erucoyl CoA as the acyl donor to produce dierucoyl phosphatidic acid (PA). The meadowfoam enzyme was studied further. It had an optimal activity at pH 7 to 8, and its activity was inhibited by 1 millimolar MnCl₂, ZnCl₂, or p-chloromercuribenzoate. In a test of substrate specificity using increasing concentrations of either 1-oleoyl-LPA or 1-erucoyl-LPA, and either oleoyl CoA or erucoyl CoA, the enzyme activity in producing PA was highest for dioleoyl-PA, followed successively by 1-oleoyl-2-erucoyl-PA, dierucoyl-PA, and 1-erucoyl-2-oleoyl-PA. In a test of substrate selectivity using a fixed combined concentration, but varying proportions, of 1-oleoyl-LPA and 1-erucoyl-LPA, and of oleoyl CoA and erucoyl CoA, the enzyme showed a pattern of acyl preference similar to that observed in the test of substrate specificity, but the preference toward oleoyl moiety in the substrates was slightly stronger. The meadowfoam microsomes could convert [¹⁴C]glycerol-3-phosphate to diacylglycerols and triacylglycerols in the presence of erucoyl CoA. The meadowfoam LPA acyltransferase is unique in its ability to produce dierucoyl-PA, and should be a prime candidate for use in the production of trierucin oils in rapeseed via genetic engineering.     

Lipases in the storage tissues of peanut and other oil seeds during germination

The castor-bean endosperm-the best-studied material of reserve lipid hydrolysis in seed germination-was previously shown to have an acid lipase and an alkaline lipase having reciprocal patterns of development during germination. We studied oil seeds from 7 species, namely castor bean (Ricinus communis L.), peanut (Arachis hypogaea L.), sunflower (Helianthus annus L.), cucumber (Cucumis sativus L.), cotton (Gossypisum hirsutum L.), corn (Zea mays. L.) and tomato (Lycopersicon esculentum Mill.). The storage tissues of all these oil seeds except castor bean contained only alkaline lipase activity which increased drastically during germination. The pattern of acid and alkaline lipases in castor bean does not seem to be common in other oil seeds. The alkaline lipase of peanut cotyledons was chosen for further study. On sucrose gradient centrifugation of cotyledon homogenate from 3-d-old seedlings, about 60% of the activity of the enzyme was found to be associated with the glyoxysomes, 15% with the mitochondria, and 25% with a membrane fraction at a density of 1.12 g cm^-3. The glyoxysomal lipase was associated with the organelle membrane, and hydrolyzed only monoglyceride whereas the mitochondrial and membrane-fraction enzymes degraded mono-, di- and triglycerides equally well. Thus, although the lipase in the glyoxysomes had the highest activity, it had to cooperate with lipases in other cellular compartments for the complete hydrolysis of reserve triglycerides.     

A Vacuolar Processing Enzyme in Maturing and Germinating Seeds: Its Distribution and Associated Changes during Development

Proprotein precursors of vacuolar components are transportedfrom endoplasmic reticulum to the dense vesicles, and then targetedto the vacuoles, where they are processed proteolytically totheir mature forms by a vacuolar processing enzyme. Immunoelectronmicroscopy of the maturing endosperm of castor bean (Ricinnscommunis) revealed that the vacuolar processing enzyme is selectivelylocalized in the dense vesicles as well as in the vacuolar matrix.This indicates that the vacuolar processing enzyme is transportedto vacuoles via dense vesicles as does IIS globulin, a majorseed protein. During seed maturation of castor bean, an increasein the activity of the vacuolar processing enzyme in the endospermpreceded increases in amounts of total protein. The enzymaticactivity reached a maximum at the late stage of seed maturationand then decreased during seed germination concomitantly withthe degradation of seed storage proteins. We examined the distributionof the enzyme in different tissues of various plants. The processingenzyme was found in cotyledons of castor bean, pumpkin and soybean,as well as in endosperm, and low-level processing activity wasalso detected in roots, hypocotyls and leaves of castor bean,pumpkin, soybean, mung bean and spinach. These results suggestthat the proprotein-processing machinery is widely distributedin vacuoles of various plant tissues. (Received July 11, 1993; Accepted August 17, 1993)     

Nonspecific lipid transfer protein in castor bean cotyledon cells: subcellular localization and a possible role in lipid metabolism.

The subcellular localization and several biochemical activities of nonspecific lipid transfer protein (nsLTP) were investigated. A section of a castor bean cotyledon cell was labeled with anti-nsLTP serum followed by protein A-gold. Gold particles were more abundant in the glyoxysome matrix and the vessel cell wall than in other areas. Cell fractionation analysis of 6-day-old castor bean cotyledons by sucrose density gradient centrifugation demonstrated that 13% of nsLTP was distributed in the glyoxysomal fraction, identified on the basis of catalase as a marker, and 87% in the soluble fraction near the top of the gradient. The location of castor bean nsLTP in glyoxysomes was further confirmed by in vitro import experiments. The synthesized precursor of nsLTP (pro-nsLTP-C) was incorporated into intact castor bean glyoxysomes and processed to the mature form after import into the glyoxysomes, but it was not imported into canine pancreatic microsomes. Castor bean nsLTP-A was found to possess the ability to bind oleic acid and oleoyl-CoA by means of a method involving Lipidex 1000. The dissociation constants (Kd) for oleic acid and oleoyl-CoA binding to nsLTP-A were 4.8 and 5.0 microM, respectively. The saturated binding capacities (Bmax) for oleic acid and oleoyl-CoA per mol of nsLTP-A were 1.1 and 1.2 mol, respectively. When acyl-CoA oxidase activity was assayed in the glyoxysomal fraction, marked enhancement of the activity was observed in the presence of nsLTP. These results suggest the possibility that nsLTP regulates fatty acid beta-oxidation through the enhancement of acyl-CoA oxidase activity in glyoxysomes. The occurrence of castor bean nsLTP in the vessel wall was discussed.     

Acyl coenzyme a preference of the glycerol phosphate pathway in the microsomes from the maturing seeds of palm, maize, and rapeseed

The acyl coenzyme A (CoA) preference of the glycerol phosphate pathway in the microsomes from the maturing seeds of palm (Butia capitata Becc.), maize (Zea mays L.), and rapeseed (Brassica napus L.) was tested. Each microsomal preparation was incubated with [¹⁴C-U]-glycerol-3-phosphate and either lauroyl CoA, oleoyl CoA, or erucoyl CoA, and the ¹⁴C-lipid products were separated and quantitated. In the presence of oleoyl CoA, the microsomes from each of the three species produced lysophosphatidic acid, phosphatidic acid, diacylglycerol, and triacylglycerol with kinetics consistent with the operation of the glycerol phosphate pathway. In the presence of erucoyl CoA, the microsomes from all the three species did not produce di- or tri-acyl lipids. In the presence of lauroyl CoA, only the microsomes from palm, but not those from maize or rapeseed, synthesized di- and tri-acyl lipids. This lack of reactivity of lauroyl CoA was also observed in the microsomes from maturing castor bean, peanut, and soybean. In maize seed and rapeseed, but not palm seed, the kinetics of labeling suggest that lauroyl and erucoyl moieties of the acyl CoAs were incorporated into lysophosphatidic acid but failed to enter into phosphatidic acid and thus the subsequent lipid products. We propose that the high degree of acyl specificity of lysophosphatidyl acyltransferase is the blocking step in the synthesis of triacylglycerols using lauroyl CoA or erucoyl CoA. The significance of the findings in seed oil biotechnology is discussed.     

Acyl coenzyme a preference of diacylglycerol acyltransferase from the maturing seeds of cuphea, maize, rapeseed, and canola

In their seed triacylglycerols, Cuphea carthagenensis contains 62% lauric acid; maize possesses 50% linoleic acid and 30% oleic acid; rapeseed (Brassica napus L. var Dwarf Essex) has 40% erucic acid; and Canola (Brassica napus L. var Tower) holds 60% oleic acid and 23% linoleic acid. Diacylglycerol acyltransferase (EC 2.3.1.20) in the microsomal preparations from maturing seeds of the above species were tested for their preference in using different forms of acyl coenzyme A (CoA). Lauroyl CoA, oleoyl CoA, and erucoyl CoA individually or in equimolar mixtures at increasing concentrations were added to the assay mixture containing diolein, and the formation of triacylglycerols from the acyl groups at 24, 32, and 40°C was analyzed. The Cuphea enzyme preferred lauroyl CoA to oleoyl CoA, and was inactive on erucoyl CoA. The maize enzyme had about equal activities on oleoyl CoA and lauroyl CoA, and was inactive on erucoyl CoA. Enzymes from both rapeseed and Canola had the same pattern of acyl CoA preference, with highest activities on lauroyl CoA. The two enzymes were more active on oleoyl CoA than on erucoyl CoA at high acyl CoA concentrations (10 and 20 micromolar) at 24°C, but were more active on erucoyl CoA than on oleoyl CoA at low acyl CoA concentrations (1.36 micromolar or less) at 32 and 40°C. These findings are discussed in terms of the contribution of the enzyme to the acyl specificity in storage triacylglycerols and the implication in seed oil biotechnology.     

Diacylglycerol acyltransferase in maturing sunflower seeds

Developing sunflower seeds exhibit a high diacylglycerol acyltransferase (DAGAT, EC 2.3.1.20) activity. The distribution of the enzyme has been studied in subcellular fractions prepared by differential centrifugation of seed homogenate. Its activity was characterized using [1-(14)C]oleoyl-CoA and diolein dispersed in Tween 20. Some properties of the microsomal fraction of DAGAT were investigated. Hyperbolic kinetics were observed, the apparent K(m) was 60 microM and the specific activity of the reaction 15 pmol/min/mg of protein. Addition of BSA (0.1%) stimulated oleate incorporation, which was not dependent on the presence of exogenous diacylglycerol. Detergents which might solubilize DAGAT, Triton X-100 and CHAPS, were tested for enzyme inhibition, and CHAPS was found to be the least denaturing.     

Properties of acid phosphatase from scutella of germinating maize seeds

One acid phosphatase (optimum pH at 5.4) was purified from maize scutellum after 96 hr of germination. The purified enzyme was homogeneous on polyacrylamide gel electrophoresis (PAGE) with or without sodium dodecyl sulfate (SDS). The enzyme has a MW of 65 000 ± 4000 as determined by Sephadex G-200 gel filtration and SDS-PAGE. The enzyme contained 16% neutral sugars, and cations are not required for activity. The purified enzyme was not inactivated by DTNB at pH 8. The hydrolysis of glucose-6-phosphate in the presence of 4 mM fluoride and 4 mm EDTA, at pH 6.7 (optimum pH), seems to be catalysed by this acid phosphatase.     

Life tables of Neoseiulus cucumeris exclusively fed with seven different pollens

The juvenile development and survival, and demographic parameters of the predatory mite Neoseiulus cucumeris (Oudemans) (Acari: Phytoseiidae) fed on pollen of castor bean, tulip, apple, Christmas cactus, horse-chestnut, maize, and birch were assessed under laboratory conditions. Deprivation of food and pollen of castor bean plants resulted in 100 % juvenile mite mortality. Feeding mites with tulip and horse-chestnut pollen resulted in the shortest development and the highest total fecundity. Adult mites fed on birch, tulip, maize, and apple pollen lived significantly longer compared with those fed on pollen of horse-chestnut and Christmas cactus. The intrinsic rate of natural increase ranged between 0.1013 ♀♀/♀/day for maize and 0.1806 ♀♀/♀/day for horse-chestnut pollen as food. Net reproductive rate was the lowest when fed with maize pollen and highest when fed with horse-chestnut pollen. Population doubling time was highest on maize pollen and shortest on horse-chestnut pollen. Our study revealed that birch, tulip, horse-chestnut, apple, and maize pollen can be used by N. cucumeris from early spring to late summer as a suitable alternative food in periods when prey in the field are scarce or absent.     

Changes in stability of isocitrate dehydrogenase (NADP+) during germination of castor bean seeds

In crude extract of castor bean endosperm, isocitrate dehydrogenase (NADP⁺) (EC 1.1.1.42) was stable at 57°C at the beginning of seed germination as well as in maturing and dry seeds. The enzyme gradually became less thermostable as germination proceeded and became unstable after 4 days. Extract from 5-day-old endosperm reduced the thermostability of the thermostable enzyme. The destabilizing factor accumulated in the endosperm as germination progressed and was identified as ricinoleate. Ricinoleate destabilized the purified enzyme which was stabilized by isocitrate and Mg²⁺, but ricinoleate did not affect the activity of NADP⁺-isocitrate dehydrogenase itself. Stearate, oleate, palmitate and myristate were similar to ricinoleate in their effect on the thermostability of the enzyme. The thermolabile enzyme in the crude extract of 5-day-old endosperm was readily inactivated by trypsin and in low concentrations of buffer. The thermostable enzyme in the crude extract of 2-day-old endosperm was not affected by these treatments. The thermostable enzyme treated with ricinoleate showed the same instabilities as the thermolabile enzyme. The role of ricinoleate in ther germinating castor bean endosperm is discussed.     

Vacuolar Processing Enzyme of Soybean That Converts Proproteins to the Corresponding Mature Forms

A vacuolar processing enzyme was detected in soybean proteinbodies. A 39-kDa immunore-active polypeptide obtained by chromatographyon a hydroxyapatite column processed both a decapeptide substrateand proproteins. A cDNA was isolated for a 55-kDa protein with71% identity to the castor bean vacuolar processing enzyme. (Received March 18, 1994; Accepted April 18, 1994)     

Use of a low-cost methodology for biodetoxification of castor bean waste and lipase production

The castor bean (Ricinus communis) represents a potential candidate for biodiesel production. The Petrobras Research Center is developing a biodiesel production process from castor bean seeds, in which an unwanted byproduct named castor bean waste is produced. This extremely alkaline waste is toxic and allergenic and, as such, poses a significant environmental problem. Solid-state fermentation (SSF) of castor bean waste was carried out to achieve ricin detoxification, reduce allergenic potential and stimulate lipase production. The fungus, Penicillium simplicissimum, an excellent lipase producer, was able to grow and produce lipase enzyme. After an optimization process, the maximum lipase activity achieved was 44.8 U/g. Moreover, the fungus P. simplicissimum was able to reduce the ricin content to non-detectable levels in addition to diminishing castor bean waste allergenic potential by approximately 16%. In this way, SSF of castor bean waste by P. simplicissimum may increase the utility of the waste by promoting enzyme production and eliminating the principal toxic element, ricin.     

Ascorbate free-radical reduction by glyoxysomal membranes

Glyoxysomal membranes from germinating castor bean (Ricinus communis L. cv Hale) endosperm contain an NADH dehydrogenase. This enzyme can utilize extraorganellar ascorbate free-radical as a substrate and can oxidize NADH at a rate which can support intraglyoxysomal demand for NAD⁺. NADH:ascorbate free-radical reductase was found to be membrane-associated, and the activity remained in the membrane fraction after lysis of glyoxysomes by osmotic shock, followed by pelleting of the membranes. In whole glyoxysomes, NADH:ascorbate free-radical reductase, like NADH:ferricyanide reductase and unlike NADH:cytochrome c reductase, was insensitive to trypsin and was not inactivated by Triton X-100 detergent. These results suggest that ascorbate free-radical is reduced by the same component which reduces ferricyanide in the glyoxysomal membrane redox system. NADH:ascorbate free-radical reductase comigrated with NADH:ferricyanide and cytochrome c reductases when glyoxy-somal membranes were solubilized with detergent and subjected to rate-zonal centrifugation. The results suggest that ascorbate free-radical, when reduced to ascorbate by membrane redox system, could serve as a link between glyoxysomal metabolism and other cellular activities.     

Phosphatidylethanolamine synthesis by castor bean endosperm. Intracellular distribution and characteristics of CTP:ethanolaminephosphate cytidylyltransferase.

The intracellular distribution and catalytic properties of CTP: ethanolaminephosphate cytidylyltransferase from endosperm of castor bean (Ricinus communis L. var. Hale) have been studied. This enzyme was confined to membranes, with about 80% of the activity occurring in mitochondria and the rest in endoplasmic reticulum (ER) following sucrose density gradient centrifugation. The mitochondrial location of this enzyme was supported by further purifying mitochondria on Percoll density gradients. The mitochondrial cytidylyltransferase was detected largely in outer membrane fractions, and lost its activity after trypsin treatment, indicating that the active sites are exposed to the cytoplasm. Both mitochondrial and ER cytidylyltransferase required cations for activity; Mg2+ was preferred over Mn2+ and Ca2+. The pH optima both were 6.5. The apparent Km values for ethanolamine phosphate were 143 and 83 microM and those for CTP were 125 and 1010 microM, respectively, for the mitochondrial and ER activities. The mitochondrial cytidylyltransferase reached a maximal velocity of 3.0 nmol/min/mg protein, whereas ER cytidylyltransferase was 0.424 nmol/min/mg protein. These findings reveal that the majority of the cytidylyltransferase activity in castor bean endosperm is not closely associated with ethanolaminephosphotransferase (predominantly in ER) which catalyzes the subsequent reaction in the synthesis of phosphatidyl-ethanolamine by a nucleotide pathway. The possible roles of these enzymes in phosphatidylethanolamine synthesis in plants are discussed.     

Comparative studies of glyoxysomes from various Fatty seedlings

The separation of various organelles from cotton cotyledon (Gossypium hirsutum L.), cucumber cotyledon (Cucumis sativus L.), peanut cotyledon (Archis hypogaea L.), pine megagametophyte (Pinus ponderosa Laws), and watermelon cotyledon (Citrullus vulgaris Schrad.) by sucrose density gradient centrifugation was found to be similar to that described for castor bean endosperm (Ricinus communis L.). Equilibrium densities were 1.12 to 1.13 g cm³ for endoplasmic reticulum, 1.17 to 1.19 g/cm³ for mitochondria, and 1.25 g/cm³ for glyoxysomes. Isolated glyoxysomes from different fatty seedlings have striking similar specific activities of individual enzymes. The only exception is alkaline lipase activity which, when assayed with an artificial substrate, varies some 10-fold in glyoxysomes from different fatty seedlings. The properties of individual enzymes in glyoxysomes from different fatty seedlings are qualitatively similar as regard to sub-organelle localization and behavior in the presence of KCl and Triton X-100. In pine megagametophyte, the glyoxysomes and not the mitochondria are the intracellular site for the breakdown of stored lipid.     

Effect of some food sources associated with cassava in Africa on the development, fecundity and longevity of Euseius fustis (Pritchard and Baker) (Acari: Phytoseiidae)

Various foods associated with cassava were tested for their effect on the development, fecundity and longevity of Euseius fustis, the most common phytoseiid species found on cassava in Africa. Euseius fustis developed successfully to adulthood on the spider mite prey species Mononychellus tanajoa (Bondar) and Oligonychus gossypii (Zacher) and on pollen from maize, castor bean and cassava. Euseius fustis also completed development on water-diluted phloem exudate from cassava, diluted honeydew from the cassava mealybug and on various pollen and prey combinations. When reared on Tetranychus urticae Koch prey or free water only, E. fustis did not develop past the deutonymphal stage. All larvae held on clean leaf discs on water-soaked cotton died without moulting, suggesting that E. fustis must feed in order to moult to the nymphal stages. Diets of maize plus castor bean pollen and maize pollen plus M. tanajoa resulted in the highest rate of development, the highest fecundity and the greatest longevity. Castor bean pollen alone and maize pollen alone produced a higher fecundity and greater longevity than M. tanajoa tested alone. A colony of E. fustis reared continuously for seven generations on castor bean pollen produced nine times more adult females than a colony of E. fustis reared continuously on M. tanajoa. No negative effects on the development and fecundity of E. fustis were observed after seven generations were reared on pollen.     

Subcellular distribution of gluconeogenetic enzymes in germinating castor bean endosperm

The intracellular distribution of enzymes capable of catalyzing the reactions from oxaloacetate to sucrose in germinating castor bean endosperm has been studied by sucrose density gradient centrifugation. One set of glycolytic enzyme activities was detected in the plastids and another in the cytosol. The percentages of their activities in the plastids were less than 10% of total activities except for aldolase and fructose diphosphatase. The activities of several of the enzymes present in the plastids seem to be too low to account for the in vivo rate of gluconeogenesis whereas those in the cytosol are quite adequate. Furthermore, phosphoenolypyruvate carboxykinase, sucrose phosphate synthetase, and sucrose synthetase, which catalyze the first and final steps in the conversion of oxaloacetate to sucrose, were found only in the cytosol. It is deduced that in germinating castor bean endosperm the complete conversion of oxaloacetate to sucrose and CO₂ occurs in the cytosol. The plastids contain some enzymes of the pentose phosphate pathway, pyruvate dehydrogenase and fatty acid synthetase in addition to the set of glycolytic enzymes. This suggests that the role of the plastid in the endosperm of germinating castor bean is the production of fatty acids from sugar phosphates, as it is known to be in the endosperm during seed development.