Separation of the molecular species of intact phosphatidylethanolamines and their N-monomethyl and N,N-dimethyl derivatives by high-performance liquid chromatography on a C8 column.

We have developed a gradient reversed-phase C8 high-performance liquid chromatography method for the separation of molecular species of phosphatidylethanolamines (PEs) and their N-monomethyl and N,N-dimethyl derivatives. This method uses a 40-min linear gradient of 88-100% methanol, containing ammonium hydroxide as silanol suppressing agent, and is suitable for metabolic studies using both UV detection at 205 nm and radioactivity flow detection. The elution order of a given PE is inversely related to the polarity of its fatty acid constituents. Lipid classes studied here containing the same fatty acyl chains elute in the order: PE-N,N-dimethyl

Pathways for fatty acid elongation and desaturation in Neurospora crassa.

Neurospora crassa incorporated exogenous deuterated palmitate (16:0) and 14C-labeled oleate (18:1 delta 9) into cell lipids. Of the exogenous 18:1 delta 9 incorporated, 59% was desaturated to 18:2 delta 9,12 and 18:3 delta 9,12,15. Of the exogenous 16:0 incorporated, 20% was elongated to 18:0, while 37% was elongated and desaturated into 18:1 delta 9, 18:2 delta 9,12, and 18:3 delta 9,12,15. The mass of unsaturated fatty acids in phospholipid and triacylglycerol is 12 times greater than the mass of 18:0. Deuterium label incorporation in unsaturated fatty acids is only twofold greater than in 18:0, indicating a sixfold preferential use of 16:0 for saturated fatty acid synthesis. These results indicate that the release of 16:0 from fatty acid synthase is a key control point that influences fatty acid composition in Neurospora.

Conversion of palmitate to unsaturated fatty acids differs in a Neurospora crassa mutant with impaired fatty acid synthase activity.

The Neurospora crassa cel (fatty acid chain elongation) mutant has impaired fatty acid synthase activity. The cel mutant requires exogenous 16:0 for growth and converts 16:0 to other fatty acids. In contrast to wild-type N. crassa, which converted only 42% of the exogenous [7,7,8,8-(2)H4]16:0 that was incorporated into cell lipids to unsaturated fatty acids, cel converted 72%. In addition, cel contains higher levels of 18:3(delta 9,12,15) than wild-type, and synthesizes two fatty acids, 20:2(delta 11,14 and 20:3(delta 11,14,17, found at only trace levels in wild-type. Thus, the delta 15-desaturase activity and elongation activity on 18-carbon polyunsaturated fatty acids are higher for cel than wild-type. This altered metabolism of exogenous 16:0 may be directly due to impaired flux through the endogenous fatty acid biosynthetic pathway, or may result from altered regulation of the synthesis of unsaturated fatty acids in the mutant.

Characterization of oleoyl-12-hydroxylase in castor microsomes using the putative substrate, 1-acyl-2-oleoyl-sn-glycero-3-phosphocholine.

We have characterized the oleoyl-12-hydroxylase in the microsomal fraction of immature castor bean using the putative substrate, 1-acyl-2-oleoyl-sn-glycero-3-phosphocholine (2-oleoyl-PC). Previous characterizations of this enzyme used oleoyl-CoA as substrate and relied on the enzyme transferring oleate from oleoyl-CoA to lysophosphatidylcholine to form 2-oleoyl-PC (acyl-CoA:lysophosphatidylcholine acyltransferase) in addition to oleoyl-12-hydroxylase. The present assay system and characterization use 2-oleoyl-PC as substrate (oleoyl-12-hydroxylase alone). Use of the actual substrate for assay purposes is important for the eventual purification of the oleoyl-12-hydroxylase. Ricinoleate (product of oleoyl-12-hydroxylase) and linoleate (product of oleoyl-12-desaturase) were identified as metabolites of oleate of 2-oleoyl-PC by high-performance liquid chromatography and gas chromatography/mass spectrometry. The activity of oleoyl-12-hydroxylase in the microsomal fraction reached a peak about 44 d after anthesis of castor, while the activity of oleoyl-12-desaturase reached a peak about 23 d after anthesis. The optimal temperature for the oleoyl-12-hydroxylase was about 22.5 degrees C, and the optimal pH was 6.3. Catalase stimulated oleoyl-12-hydroxylase while bovine serum albumin and CoA did not activate oleoyl-12-hydroxylase. The phosphatidylcholine analogue, oleoyloxyethyl phosphocholine, inhibited the activity of oleoyl-12-hydroxylase. These results further support the hypothesis that the actual substrate of oleoyl-12-hydroxylase is 2-oleoyl-PC.

Metabolism of 1-acyl-2-oleoyl-sn-glycero-3-phosphoethanolamine in castor oil biosynthesis.

We have examined the role of 2-oleoyl-PE (phosphatidylethanolamine) in the biosynthesis of triacylglycerols (TAG) by castor microsomes. In castor microsomal incubation, the label from 14C-oleate of 1-palmitoyl-2-[1-(14)C]oleoyl-sn-glycero-3-phosphoethanolamine is incorporated into TAG containing ricinoleate. The enzyme characteristics, such as optimal pH, and the effect of incubation components of the oleoyl-12-hydroxylase using 2-oleoyl-PE as incubation substrate are similar to those for 2-oleoyl-PC (phosphatidylcholine). However, compared to 2-oleoyl-PC, 2-oleoyl-PE is a less efficient incubation substrate of oleoyl-12-hydroxylase in castor microsomes. Unlike 2-oleoyl-PC, 2-oleoyl-PE is not hydroxylated to 2-ricinoleoyl-PE by oleoyl-12-hydroxylase and is not desaturated to 2-linoleoyl-PE by oleoyl-12-desaturase. We have demonstrated the conversion of 2-oleoyl-PE to 2-oleoyl-PC and vice versa. The incorporation of label from 2-[14C]oleoyl-PE into TAG occurs after its conversion to 2-oleoyl-PC, which can then be hydroxylated or desaturated. We detected neither PE-N-monomethyl nor PE-N,N-dimethyl, the intermediates from PE to PC by N-methylation. The conversion of 2-oleoyl-PE to 2-oleoyl-PC likely occurs via hydrolysis to 1,2-diacyl-sn-glycerol by phospholipase C and then by cholinephosphotransferase. This conversion does not appear to play a key role in driving ricinoleate into TAG.

Biosynthesis of triacylglycerols containing ricinoleate in castor microsomes using 1-acyl-2-oleoyl-sn-glycero-3-phosphocholine as the substrate of oleoyl-12-hydroxylase.

We have examined the biosynthetic pathway of triacylglycerols containing ricinoleate to determine the steps in the pathway that lead to the high levels of ricinoleate incorporation in castor oil. The biosynthetic pathway was studied by analysis of products resulting from castor microsomal incubation of 1-palmitoyl-2-[14C]oleoyl-sn-glycero-3-phosphocholine, the substrate of oleoyl-12-hydroxylase, using high-performance liquid chromatography, gas chromatography, mass spectrometry, and/or thin-layer chromatography. In addition to formation of the immediate and major metabolite, 1-palmitoyl-2-[14C]ricinoleoyl-sn-glycero-3-phosphocholine, 14C-labeled 2-linoleoyl-phosphatidylcholine (PC), and 14C-labeled phosphatidylethanolamine were also identified as the metabolites. In addition, the four triacylglycerols that constitute castor oil, triricinolein, 1,2-diricinoleoyl-3-oleoyl-sn-glycerol, 1,2-diricinoleoyl-3-linoleoyl-sn-glycerol, 1,2-diricinoleoyl-3-linolenoyl-sn-glycerol, were also identified as labeled metabolites in the incubation along with labeled fatty acids: ricinoleate, oleate, and linoleate. The conversion of PC to free fatty acids by phospholipase A2 strongly favored ricinoleate among the fatty acids on the sn-2 position of PC. A major metabolite, 1-palmitoyl-2-oleoyl-sn-glycerol, was identified as the phospholipase C hydrolyte of the substrate; however, its conversion to triacylglycerols was blocked. In the separate incubations of 2-[14C]ricinoleoyl-PC and [14C]ricinoleate plus CoA, the metabolites were free ricinoleate and the same triacylglycerols that result from incubation with 2-oleoyl-PC. Our results demonstrate the proposed pathway: 2-oleoyl-PC-->2-ricinoleoyl-PC-->ricinoleate-->triacylglycerols. The first two steps as well as the step of diacylglycerol acyltransferase show preference for producing ricinoleate and incorporating it in triacylglycerols over oleate and linoleate. Thus, the productions of these triacylglycerols in this relatively short incubation (30 min), as well as the availability of 2-oleoyl-PC in vivo, reflect the in vivo drive to produce triricinolein in castor bean.

Biosynthesis of ricinoleate in castor oil.

Castor oil is 90% ricinoleate (12-hydroxyoleate) and has numerous industrial uses. Components of castor bean (Ricinus communis L.) pose serious problems to processors. Other researchers have cloned the gene for the oleoyl hydroxylase, but transgenic plants produce only about 20% hydroxy fatty acid. To improve such transgenic substitutes for castor, we are using HPLC analysis of castor bean microsomal suspensions to follow the hydroxylase reaction and the movement of 14C-ricinoleate through phospholipid into triacylglycerol. Most labeled ricinoleate is rapidly removed from the phospholipid fraction as free fatty acid and incorporated into triacylglycerol, with triricinolein predominating. Elucidation of the basis for high incorporation of ricinoleate and exclusion of oleate from triacylglycerols will identify genes that can be used to engineer high ricinoleate production in transgenic plants.

A comparison of the conversion of 1-amino-2-ethylcyclopropane-1-carboxylic acid stereoisomers to 1-butene by pea epicotyls and by a cell-free system.

The characteristics of the conversion of 1-aminocyclopropane-1-carboxylic acid (ACC) to ethylene by pea (Pisum sativum L.) epicotyls and by pea epicotyl enzyme are compared. Of the four stereoisomers of 1-amino-2-ethylcyclopropane-1-carboxylic acid (AEC), only (1R,2S)-AEC is preferentially converted to 1-butene in pea epicotyls. This conversion is inhibited by ACC, indicating that butene production from (1R,2S)-AEC and ethylene production from ACC are catalyzed by the same enzyme. Furthermore, pea epicotyls efficiently convert ACC to ethylene with a low K m (66 µM) for ACC and do not convert 4-methylthio-2-oxo-butanoic acid (KMB) to ethylene, thus demonstrating high specificity for its substrate. In contrast, the reported pea epicotyl enzyme which catalyzes the conversion of ACC to ethylene had a high K m (389 mM) for ACC and readily converted KMB to ethylene. We show, moreover, that the pea enzyme catalyzes the conversion of AEC isomers to butene without stereodiscrimination. Because of its lack of stereospecificity, its low affinity for ACC and its utilization of KMB as a substrate, we conclude that the reported pea enzyme system is not related to the in-vivo ethylene-forming enzyme.

Single-strand-preferring nuclease activity in wheat leaves is increased in senescence and is negatively photoregulated.

Single-strand-preferring nucleases (EC 3.1.30.1) selectively cleave internucleotide bonds in single-stranded regions of predominantly duplex DNA and DNA.RNA hybrids and extensively degrade denatured DNA and RNA. The functions of single-strand-preferring nuclease in plants are unknown. We have monitored this nuclease activity in flag leaves of wheat (Triticum aestivum L. cv. Chinese Spring) undergoing natural senescence and in primary leaves of wheat seedlings undergoing dark-induced senescence. In falg leaves, nuclease activity remained at basal levels during the first 2 weeks after anthesis, while chlorophyll content increased to a maximum. Nuclease activity then rose in concert with a decline in chlorophyll, reaching a 16-fold elevation at 5 weeks post-anthesis, when 53% of the chlorophyll had been lost. When 8-day-old wheat seedlings were induced to senesce by placing them in darkness, nuclease activity rose without apparent lag, reaching a 13-fold elevation in 7 days, when 61% of the chlorophyll had been lost. The increase in nuclease activity was reversible upon reexposure of seedlings to light, a decline beginning without apparent lag. Reversibility was complete for plants that had been held in darkness for 5 days, with activity returning to the control level in 2 days. These senescence-related changes in nuclease activity, measured in conventional assays, were consistent with concomitant analysis by activity staining of sodium dodecyl sulfate/polyacrylamide gels. We conclude that an increase in single-strand-preferring nuclease activity is closely associated with wheat leaf senescence and that nuclease activity is subject to negative photoregulation.

Purification and characterization of the stearoyl-acyl carrier protein desaturase and the acyl-acyl carrier protein thioesterase from maturing seeds of safflower.

Two enzymes involved in oleic acid biosynthesis have been purified from immature safflower seed. The stearoyl-acyl carrier protein (ACP) desaturase which catalyzes the formation of the double bond of oleate has been purified 200-fold and is a dimer with a molecular weight of 68,000. The enzyme shows strong preference for stearoyl-ACP as substrate; by comparison of its activity with stearoyl-CoA and palmitoyl-ACP as substrates, it appears that the ACP moiety is primarily important for binding of substrate and the chain length is important for catalytic activity. The desaturase requires 56 microM oxygen for half-maximal activity, 400 microM oxygen for maximal activity, and is stimulated severalfold by catalase. The acyl-ACP thioesterase has been purified 700-fold and is also a dimer of molecular weight 74,000. It shows a 5-fold preference for oleoyl-ACP versus stearoyl-ACP and is relatively inactive with corresponding acyl-CoAs.

Calcium ion improves electrophoretic transfer of calmodulin and other small proteins.

Inclusion of 2 mM CaCl2 in standard electrotransfer buffer improves detection of transferred calmodulin 20-fold. Detection of the calcium-binding proteins calmodulin and alpha-lactalbumin displays similar improvement, some other small proteins show slightly improved detection, while other proteins, especially those greater than or equal to 30 kDa, show none. The effect of CaCl2 on transfer is a result of enhanced retention of protein by the binding matrix. Our results suggest that addition of CaCl2 more effectively removes sodium dodecyl sulfate from proteins, increasing protein-binding to polyvinylidene difluoride and nitrocellulose membranes. The enhanced membrane-binding of calmodulin is not due to specific binding of calcium ion, as other divalent cations are similarly effective. Detection of native calmodulin is also slightly enhanced after transfer with added CaCl2, suggesting an additional, minor effect of the added ion. The increased binding brought about by inclusion of calcium ion in transfer buffer offers a very useful means for improving the sensitivity of assays that involve electro-transfer.

Three RNases in Senescent and Nonsenescent Wheat Leaves : Characterization by Activity Staining in Sodium Dodecyl Sulfate-Polyacrylamide Gels.

We have described three RNases in wheat leaves (Triticum aestivum L. cv Chinese Spring) and developed assays for measuring each RNase individually in crude leaf extracts. We initially used activity staining in sodium dodecyl sulfate-polyacrylamide gels to characterize RNases in extracts of primary and flag leaves. We thus identified acid RNase (EC 3.1.27.1, here designated RNase WL(A)), and two apparently novel enzymes, designated RNases WL(B) and WL(C). RNase WL(B) activity displays a distinctive isozyme pattern, a molecular mass of 26 kilodaltons (major species), a broad pH range with an optimum near neutrality, insensitivity to EDTA, and stimulation by moderate concentrations of KCl and by MgCl(2). RNase WL(C) activity exhibits a molecular mass of 27 kilodaltons, a neutral pH optimum, insensitivity to EDTA, and inhibition by KCl, MgCl(2), and tri-(hydroxymethyl)aminomethane. Based on distinctive catalytic properties established in gels, we designed conventional solution assays for selective quantitation of each RNase activity. We used the assays to monitor the individual RNases after gel filtration chromatography and native gel electrophoresis of extracts. In accompanying work, we used the assays to monitor RNases WL(A), WL(B), and WL(C), which are present in senescent and nonsenescent leaves, during the course of leaf senescence.

Expression of Three RNase Activities during Natural and Dark-Induced Senescence of Wheat Leaves.

We have monitored the activities of RNases WL(A), WL(B), and WL(C) (A Blank, TA McKeon [1991] Plant Physiol 97: 1402-1408) during leaf senescence in wheat (Triticum aestivum L. cv Chinese Spring). When seedlings were induced to senesce in darkness, protein loss from primary leaves began immediately. RNase WL(B) activity was unchanged for 2 days and then rose linearly, reaching a sixfold elevation in 7 days. RNase WL(C) activity declined for 2 days and then rose linearly, reaching a twofold elevation in 7 days. RNase WL(A) activity declined in the first 2 days and was unchanged thereafter. Although differentially expressed, these RNase activities may respond to a common regulatory mechanism(s) which, at 2 days of darkness, signals progression into a more advanced stage of senescence. The RNase activities were also differentially expressed during light-induced recovery, returning to normal levels in dissimilar patterns. In flag leaves of greenhouse-grown wheat, the three RNase activities increased during the early postanthesis period when protein content was stable and underwent further, accelerated accumulation during senescence. RNase WL(B) activity showed the largest overall senescence-associated elevation (sixfold), followed by RNase WL(C) (fourfold) and RNase WL(A) (threefold).

Biochemical aspects of castor oil biosynthesis.

Castor oil is 90% ricinoleate (12-hydroxy-oleate) and has numerous industrial uses. Components of castor bean (Ricinus communis L.) pose serious problems to processors. We are evaluating two complementary approaches to providing a safe source of castor oil.

The effect of plant-hormone pretreatments on ethylene production and synthesis of 1-aminocyclopropane-1-carboxylic acid in water-stressed wheat leaves.

Excised wheat (Triticum aestivum L.) leaves, when subjected to drought stress, increased ethylene production as a result of an increased synthesis of 1-aminocyclopropane-1-carboxylic acid (ACC) and an increased activity of the ethyleneforming enzyme (EFE), which catalyzes the conversion of ACC to ethylene. The rise in EFE activity was maximal within 2 h after the stress period, while rehydration to relieve water stress reduced EFE activity within 3 h to levels similar to those in nonstressed tissue. Pretreatment of the leaves with benzyladenine or indole-3-acetic acid prior to water stress caused further increase in ethylene production and in endogenous ACC level. Conversely, pretreatment of wheat leaves with abscisic acid reduced ethylene production to levels produced by nonstressed leaves; this reduction in ethylene production was accompanied by a decrease in ACC content. However, none of these hormone pretreatments significantly affected the EFE level in stressed or nonstressed leaves. These data indicate that the plant hormones participate in regulation of water-stress ethylene production primarily by modulating the level of ACC.

Purification and characterization of two ribonucleases from developing tomato fruit.

Two neutral ribonucleases have been purified from developing tomato fruit. Their activity is maximal 5 days after anthesis, declines during maturation, and then increases slightly in the mature green through breaker stages. The ribonucleases Tf1 and Tf2 have molecular weights of 59 and 29 K, respectively, based on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and are glycoproteins. The reduced and denatured Tf1 is composed of two subunits, 30 and 29 K, of which only the 30-K subunit displays ribonuclease activity after renaturation. Reduced and denatured Tf2 is a single 29-K polypeptide that is renaturable to an active ribonuclease. Only the 30-K, active subunit of Tf1 is immunologically cross-reactive with Tf2. Both ribonucleases are cyclyzing endoribonucleases with a strong preference for cleavage at pyrimidine residues, thus generating oligonucleotide products ending with pyrimidine 2',3'-cyclic phosphate. These tomato fruit ribonucleases share a number of properties in common with the S-glycoprotein ribonucleases that are involved in self-incompatibility reactions in some solanaceous plants.

Metabolism of ricinoleate by Neurospora crassa.

Neurospora crassa is a potential expression system for evaluating fatty-acid-modifying genes from plants producing uncommon fatty acids. One such gene encodes the hydroxylase that converts oleate to ricinoleate, a fatty acid with important industrial uses. To develop this expression system, it is critical to evaluate the metabolism and physiological effects of the expected novel fatty acid(s). We therefore examined effects of ricinoleate on lipid biosynthesis and growth of N. crassa. Ricinoleate inhibited growth and reduced levels of phospholipids and 2-hydroxy fatty acids in glycolipids, but led to increased lipid accumulation on a mass basis. To evaluate incorporation and metabolism of ricinoleate, we followed the fate 14 microM-3mM [1-14C]ricinoleate. The fate of the [14C]ricinoleate was concentration-dependent. At higher concentrations, ricinoleate was principally incorporated into triacylglycerols. At lower concentrations, ricinoleate was principally metabolized to other compounds. Thus, N. crassa transformants expressing the hydroxylase gene can be detected if the level of hydroxylase expression allows both growth and ricinoleate accumulation.

Fatty acid biosynthesis in novel ufa mutants of Neurospora crassa.

New mutants of Neurospora crassa having the ufa phenotype have been isolated. Two of these mutants, like previously identified ufa mutants, require an unsaturated fatty acid for growth and are almost completely blocked in the de novo synthesis of unsaturated fatty acids. The new mutations map to a different chromosomal location than previously characterized ufa mutations. This implies that at least one additional genetic locus controls the synthesis of unsaturated fatty acids in Neurospora.