The enzymic conversion of hydroxycinnamic acids to p-coumarylquinic and chlorogenic acids in tomato fruits

Two enzymes thought to be involved in the biosynthesis of chlorogenic acid have been separated and purified by ion exchange chromatography and their properties studied. These two enzymes, p-coumarate CoA ligase and hydroxycinnamyl CoA: quinate hydroxycinnamyl transferase, acting together catalyse the conversion of p-coumaric acid to 5′-p-coumarylquinic acid and of caffeic acid to chlorogenic acid. The ligase has a higher affinity for p-coumaric than for caffeic acid and will in addition activate a number of other cinnamic acids such as ferulic, isoferulic and m-coumaric acids but not cinnamic acid. The transferase shows higher activity and affinity with p-coumaryl CoA than caffeyl CoA. It also acts with ferulyl CoA but only very slowly. The enzyme shows high specificity for quinic acid; shikimic acid is esterified at only 2% of the rate with quinic acid and glucose is not a substrate. The transferase activity is reversible and both chlorogenic acid and 5′-p-coumarylquinic acids are cleaved in the presence of CoA to form quinic acid and the corresponding hydroxycinnamyl CoA thioester.     

Inhibition of Chlorella growth by degradation and related products of linoleic and linolenic acids and the possible significance of polyunsaturated fatty acids in phytoplankton ecology

The activity of the degradation products of linoleic and linolenic acids in inhibiting the growth of the green alga Chlorella pyrenoidosa was determined using the paper disk-agar plate method. Although hydroperoxides derived from these acids and aldehyde biodegradation products and other related aldehydes and alcohols showed inhibitory activity, it is concluded from the weakness of the activities that the inhibitory effects of polyunsaturated fatty acids(PUFAs) on Chlorella growth is due mainly to the acids themselves and not their degradation products. The evidence for the possible ecological role of PUFAs as toxic agents against phytoplankton is presented. This revised version was published online in August 2006 with corrections to the Cover Date.     

Neoleucinodes elegantalis (Lepidoptera: Crambidae): an organism invisible to the defences of tomato fruits

Insect–plant interactions involving species of the genus Solanum have been intensively studied, resulting in several articles on insect–plant interactions. However, the interactions between herbivores and the fruits of Solanum lycopersicum (tomato) are not well known. Neoleucinodes elegantalis is a borer that causes great yield losses in S. lycopersicum crops because of the direct damage that it causes to the fruits and the difficulty of controlling it. In the field, the outside of a tomato fruit infested with the larvae of N. elegantalis is visually similar to uninfested fruits. Even a minor injury by herbivores can elicit a defensive response. Due to the lack of studies on interactions between fruit borers and S. lycopersicum, our aim in this study was to determine the locations of S. lycopersicum fruit in which the N. elegantalis larvae prefer to feed. An evaluation of nutritional sources was done through histochemical and biochemical tests and the defensive response of the S. lycopersicum fruit to attack by N. elegantalis larvae was evaluated through the detection of protease inhibitors (PIs) and lipoxygenase (LOX) activity. Our results show that the columella region is preferred by the N. elegantalis larvae and that this region has a nutritional source. Furthermore, attack by N. elegantalis larvae in the columella does not induce a significant increase in lipoxygenase activity and PIs. Thus, our results provide a better understanding of the interaction between the larvae of N. elegantalis and S. lycopersicum fruits and a better understanding of the evolution of plant–herbivore interactions, with an emphasis on the choice of feeding location as a strategy to avoid plant defences.     

Hydroperoxide isomers and ketohydroxy product from oxidation of linoleic acid by eggplant lipoxygenase

Hydroperoxides produced by oxidation of linoleic acid with purified eggplant lipoxygenase were separated by TLC and analysed by IR spectroscopy. The methyl hydroxystearates from the enzymatically produced hydroperoxides were analysed by MS and GLC. Both analyses indicated that the eggplant enzyme converted linoleic acid almost exclusively (96%) into the 13-hydroperoxy isomer whereas the 9-hydroperoxy isomer was only a minor product (4%). HPLC of the methyl ester of the isolated hydroperoxides showed three components. Each component was collected, reduced to methyl hydroxystearate and characterized by GLC, MS and IR analysis. The components were identified as 13-hydroperoxy cis-trans isomer (92.8%), 13-hydroperoxy trans-trans isomer (2.6%) and 9-hydroperoxy cis-trans isomer (4.6%). A polar by-product present in the reaction mixture was identified by IR, ¹H NMR, and MS (of the toluene-p-sulphonyl derivative) as 13-hydroxy-12-oxo-octadec-cis-9-enoic acid.     

On Growth-inhibition against Aspergillus oryzae No 40 by the Compound III-3 in Rice Grain

Partially purified lipoxygenase (LOX) extracts were obtained from Fusarium proliferatum, Fusarium oxysporum, Chlorella pyrenoidosa, and Saccharomyces cerevisiae; the enzymatic extract of F. proliferatum showed the highest LOX activity while those of F. oxysporum and S. cerevisiae demonstrated only 27.8 and 16.5% of the activity at pH 8.0, and 61.2 and 9.7% of the enzyme activity at pH 10.0, respectively. The lowest LOX activity was that in the C. pyrenoidosa extract. The microbial enzymatic preparations were assayed with linoleic acid as substrate, which was bioconverted into 9- and 13-hydroperoxides (HPODEs) by all four extracts; in additon, the LOX activity in the F. oxysporum extract produced the 10- and 12-HPODEs from linoleic acid while that of the C. pyrenoidosa extract produced only the 10- HPODE. When assayed with the 9- and 13-HPODEs as substrates, the selected microbial extracts had secondary enzyme activities, one of which produced hexanal. The highest hexanal-producing activity was 1.51 and 1.39 nmol hexanol/mg protein/min in the F. proliferatum and C. pyrenoidosa extracts, respectively, while those of F. oxysporum and S. cerevisiae had approximately 15% of the HPODE-cleaving enzyme activity. The C. pyrenoidosa extract had the highest proportion of pentanone, which was produced at only one-fourth the concentration by the HPODE-cleaving enzyme activity in the three other microbial enzymatic extracts.     

Enzymic formation of carbonyls from linoleic acid in leaves of Phaseolus vulgaris

Homogenization of Phaseolus vulgaris leaves at acid pH results in the evolution of hexanal, cis-3- and trans-2-hexenal. With cell-free extracts of leaves, linoleic and linolenic acids are enzymically converted to their hydroperoxides (predominantly the 13-hydroperoxide isomers) and to hexanal or hexenal respectively. Activity was highest in young, dark-green leaves and was stimulated by Triton X-100. Oleic acid is not a substrate for these reactions. Both 9- and 13-hydroperoxides were cleaved to carbonyl fragments and are proposed as intermediates in the formation of volatile aldehydes and non-volatile ω-oxoacids in P. vulgaris leaves. Properties of the enzyme systems are described.     

Effects of flaxseed,raw soybeans and calcium salts of fatty acids on apparent total tract digestibility,energy balance and milk fatty acid profile of transition cows

Oilseeds offer some protection to the access of ruminal microorganisms and may be an alternative to calcium salts of fatty acids (FA), which are not fully inert in the ruminal environment. This study aimed to evaluate the effects of different sources of FA supplementation on apparent total tract nutrient digestibility, milk yield and composition, and energy balance (EB) of cows during the transition period and early lactation. We compared diets rich in C18:2 and C18:3 FA. Multiparous Holstein cows were randomly assigned to receive one of the four diets: control (n=11); whole flaxseed (WF, n=10), 60 and 80 g/kg (diet dry matter (DM) basis) of WF during the prepartum and postpartum periods, respectively; whole raw soybeans (WS, n=10), 120 and 160 g/kg (diet DM basis) of WS during the prepartum and postpartum periods, respectively; and calcium salts of unsaturated fatty acids (CSFA, n=11), 24 and 32 g/kg (diet DM basis) of CSFA during the prepartum and postpartum periods, respectively. Dry cows fed WF had higher DM and net energy of lactation (NE_L) intake than those fed WS or CSFA. The FA supplementation did not alter DM and NDF apparent total tract digestibility, dry cows fed WF exhibited greater NDF total tract digestion than cows fed WS or CSFA. Feeding WS instead of CSFA did not alter NE_L intake and total tract digestion of nutrients, but increased milk fat yield and concentration. Calculated efficiency of milk yield was not altered by diets. FA supplementation increased EB during the postpartum period. Experimental diets increased long-chain FA (saturated and unsaturated FA) in milk. In addition, cows fed WS and CSFA had higher C18:1 trans-11 FA and C18:2 cis, and lower C18:3 FA in milk than those fed WF. Furthermore, cows fed CSFA had higher C18:1 trans-11 and cis-9, trans-11 FA than cows fed WS. Although supplemental C18:2 and C18:3 FA did not influence the milk yield of cows, they positively affected EB and increased unsaturated long-chain FA in milk fat.     

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.     

Metabolism of polyunsaturated fatty acids by larvae of the waxmoth,Galleria mellonella

The metabolic fates of linoleic (18:2n6) and linolenic (18:3n3) acids injected into the hemocoel of fifth instar larvae of the waxmoth, Galleria mellonella, were examined by radio-high-pressure liquid chromatography and radio-gas-liquid chromatography. In addition to undergoing β-oxidation and incorporation into neutral and phospholipid fractions, a portion of both of these C18 fatty acids was elongated and desaturated to longer chain and more unsaturated polyenoics. Radioactivity from linoleic acid was recovered in components that coeluted with 18:3, 18:4, 20:3, and 20:4. Radioactivity from linolenic acid was recovered in an unidentified component and in components that coeluted with 18:4, 20:3, and 20:5. Labeled arachidonic and eicosapentaenoic acids injected into waxmoth larvae were converted to prostaglandins, suggesting that one aspect of the biological significance of the elongation/desaturation reactions is to generate precursors for prostaglandin biosynthesis.     

Distribution of acid invertase in the tomato plant

Acid invertase activity in Lycopersicon esculentum was highest in the locular wall of ripe fruit and lowest in roots. Activity was greater in leaf laminae than in petiole tissue and increased with leaf age, whereas there was more invertase in the upper part of the stem compared with the older portion. Activity in whole fruit increased with increasing ripeness and was greatest in overripe fruit. Of various tissues from a number of wild tomato species examined, the fruit of L. pimpinellifolium were particularly rich in the enzyme, in contrast to the fruit of L. hirsutum, L. hirsutum, var. glabratum and L. peruvianum which had low activity.     

Omega-6 and omega-3 fatty acids metabolism pathways in the body of pigs fed diets with different sources of fatty acids

This study was carried out on 24 gilts (♀ Polish Large White × ♂ Danish Landrace) grown with body weight (BW) of 60 to 105 kg. The pigs were fed diets designed on the basis of a standard diet (appropriate for age and BW of pigs) where a part of the energy content was replaced by different fat supplements: linseed oil in Diet L, rapeseed oil in Diet R and fish oil in Diet F (6 gilts per dietary treatment). The fat supplements were sources of specific fatty acids (FA): in Diet L α-linolenic acid (C18:3 n?3, ALA); in Diet R linoleic acid (C18:2 n?6, LA) and in Diet F eicosapentaenoic acid (C20:5 n?3, EPA), docosapentaenoic acid (C22:5 n?3, DPA) and docosahexaenoic acid (C22:6 n?3, DHA). The protein, fat and total FA contents in the body did not differ among groups of pigs. The enhanced total intake of LA and ALA by pigs caused an increased deposition of these FA in the body (p < 0.01) and an increased potential body pool of these acids for further metabolism/conversions. The conversion efficiency of LA and ALA from the feed to the pig’s body differed among groups (p < 0.01) and ranged from 64.4% to 67.2% and from 69.4% to 81.7%, respectively. In Groups L and R, the level of de novo synthesis of long-chain polyunsaturated FA was higher than in Group F. From the results, it can be concluded that the efficiency of deposition is greater for omega-3 FA than for omega-6 FA and depends on their dietary amount. The level of LA and ALA intake influences not only their deposition in the body but also the end products of the omega-3 and omega-6 pathways.     

Endopolygalacturonase from tomato fruit

A polygalacturonase was extracted from ripening tomato fruit. A four step procedure was developed producing a 44-fold increase in specific activity with 9% recovery. The enzyme was found to rapidly degrade pectic acid but not pectin. No transeliminase activity was detected. Viscosity and per cent hydrolysis studies formed a basis for suggesting that this enzyme cleaves its substrate in a random manner and is likely to be an endopolygalacturonase.     

Effects of growth regulators on fatty acids of soybean suspension cultures

Soybean suspension cultures were grown for 24 weeks in the absence of plant growth regulators and in the presence of 1 ppm levels each of an auxin (indole-3-butyric acid), a cytokinin (kinetin) and a gibberellin (gibberellic acid), individually and in all possible combinations. Cells grown in the presence of the auxin with and without gibberellin contained relatively greater amounts of palmitic and smaller amounts of polyunsaturated acids than did cells grown under other regimens. The combination of cytokinin and gibberellin caused a higher proportion of linoleic and a lower proportion of linolenic acids than in cells of the other groups. Neither of these regulators by itself produced the effect, and addition of auxin to the other two diminished the effect.     

Production of hexanal from hydrolyzed sunflower oil by lipoxygenase and hydroperoxid lyase enzymes

Hexanal was produced from hydrolyzed sunflower oil in two steps: 1) 13-hydroperoxy-9-(Z),11(E)-octadecadienoic acid (13-HPOD) was formed from linoleic acid (100 mM) by soybean lipoxygenase-1 isoenzyme (Lox-1) with O₂, the reaction resulted 68.7 mM 13-HPOD with a yield of 72%. 2) 13-HPOD (15 mM) was cleaved by spinach leaf hydroperoxide lyase resulting 8.2 mM hexanal (54% yield). Hexanal was isolated from the reaction mixture by repeated steam distillation.     

Biophysical parameters of linoleic acid hydroperoxides as assessed by surface behavior and Fourier Transform Infra-Red spectrometry: possible pertinence to senescence

Hydroperoxidation of linoleic acid (LA) by two major lipoxygenases (LOX) produced octadecanoic trans, cis ( 10, 12) 9-hydroperoxide (9 LAHP) and octadecanoic cis, trans (, 9, 11) 13-hydroperoxide (13, LAHP). The effect of hydroperoxidation on surface tension-related parameters of simulated monolayer membranes was studied. Langmuir-Blodgett isotherms demonstrated that hydroperoxidation of LA markedly reduces molecular area, and hence increases monolayer rigidity. The reduction in the surface area of 13 LAHP was more pronounced than that of 9 LAHP. Surface tension and Contact Angle measurements demonstrated a similar effect. However, in contrast to the molecular area data, both HP species behaved similarly in that as compared to the unoxidized LA, they both increased Contact angles to the same extent. Fourier-Transform Infra-Red studies indicated that 13 LAHP undergoes non-enzymatic cyclization, and hence molecular area reduction. The results described are discussed in terms of membrane senescence and possible relevance to plant prostaglandin-like compounds of the jasmonate group.Abbreviations FTIR Fourier transform Infra-Red spectrometry - HP hydroperoxides - LA linoleic acid - LAHP linoleic acid hydroperoxides - 9 LAHP octadecanoic trans, cis ( 10, 12) 9-hydroperoxide - 13 LAHP octadecanoic cis,trans ( 9,11) 13-hydroperoxide - LOX lipoxygenase - OTS octadecyltrichlorosilane - PL phospholipids - PUFA polyunsaturated fatty acids - SP-HPLC -straight phase high pressure liquid chromatography - THF tetrahydrofuran     

Behaviour of some mitochondrial enzymes in ripening tomato fruit

The activities of four mitochondrial enzymes were studied in four stages of ripening tomato fruit. The highest enzyme activity was recorded for malate dehydrogenase followed by cytochrome c oxidase. Succinate dehydrogenase and NADH oxidase levels were low and could only be determined in the green stage of the fruit. However, peaks of various enzyme activities coincided in identical mitochondrial fractions on the sucrose density gradient. Moreover, the levels of malate dehydrogenase and cytochrome c oxidase were constant during the ripening process while the other two enzymes, succinate dehydrogenase and NADH oxidase, declined. This might indicate that mitochondria retain some of their essential functions through the ripening process.     

The predominance of polyunsaturated fatty acids in the butterfly Morpho peleides before and after metamorphosis

We hypothesized that the polyunsaturated fatty acids of the butterfly were probably derived from the diet and that there might be a great loss of body fat during metamorphosis. To substantiate these hypotheses, we analyzed the fatty acid composition and content of the diet, the larva, and the butterfly Morpho peleides. Both the diet and the tissues of the larva and butterfly had a high concentration of polyunsaturated fatty acids. In the diet, linolenic acid accounted for 19% and linoleic acid for 8% of total fatty acids. In the larva, almost 60% of the total fatty acids were polyunsaturated: linolenic acid predominated at 42% of total fatty acids, and linoleic acid was at 17%. In the butterfly, linolenic acid represented 36% and linoleic acid represented 11% of total fatty acids. The larva had a much higher total fatty acid content than the butterfly (20.2 vs. 6.9 mg). Our data indicate that the transformation from larva to butterfly during metamorphosis drastically decreased the total fatty acid content. There was bioenhancement of polyunsaturated fatty acids from the diet to the larva and butterfly. This polyunsaturation of membranes may have functional importance in providing membrane fluidity useful in flight.     

The wilty tomato mutants flacca and sitiens are impaired in the oxidation of ABA-aldehyde to ABA

Abstract. Deuterium-labelled ABA-aldehyde was fed to various tomato genotypes. Normal and notabilis mutant plants incorporated substantial amounts of the label into ABA. In contrast, two ABA-deficient mutants, flacca and sitiens , reduced ABA-aldehyde to a mixture of cis- and trans -ABA alcohol rather than oxidizing it to ABA. It was concluded that ABA-aldehyde is the immediate precursor of ABA in higher plants. It appears that the flacca and sitiens lesions both act to block the last step of the ABA biosynthetic pathway. The mutant gene loci are likely to be involved in coding for different sub-units of the same dehydrogenase enzyme.