L.S.C. Wooltorton

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

Abstract 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.

Changes in the activity of enzymes of phenylpropanoid metabolism in tomatoes stored at low temperatures

Abstract A large increase in the activity of an enzyme involved in chlorogenic acid metabolism, hydroxycinnamyltransferase occurs in tomatoes stored at low temperatures. In contrast, the activity of the enzyme remains constant or falls slightly during normal ripening at 20°. The rise in activity occurs at temperatures below 10° and fails to occur at 15° or 20°. This increase in activity during low temperature storage occurs with fruit at all stages of ripening from mature green to fully ripe. The hydroxycinnamyltransferase of chilled tomatoes falls rapidly on transfer to 20° with a lag of about 4–8 hr and within 48 hr returns to that of unchilled fruit. The effects of such warming treatments are reversible since when a chilling period is resumed following warming to 20°, the rise in hydroxycinnamyltransferase activity is also resumed. Of the 5 other enzymes of phenylpropanoid metabolism studied, only PAL shows a similar increase in activity during low temperature storage although the activity of the other enzymes was maintained at higher levels in fruit at 2° than at 20°. The possible relationship between the behaviour of hydroxycinnamyltransferase activity at various temperatures and the known susceptibility of tomatoes to chilling injury is discussed.

Stimulation of phenolic acid and lignin biosynthesis in swede root tissue by ethylene

Abstract Experiments using 14 C-phenylalanine have shown that ethylene treatment of swede root tissue promotes the utilization of phenylalanine as a precurso

Formation of coA esters of cinnamic acid derivatives by extracts of Brassica napo-brassica root tissue

Abstract An enzyme fraction from aged swede root disks catalyses the formation of CoA thioesters of cinnamic acids in the presence of CoA, ATP and Mg2+. The enzyme shows activity only to those cinnamic acid derivatives bearing a phenolic OH group, p-coumaric and ferulic acids being the most active substrates. The requirement for Mg2+ can be replaced by Mn2+, Co2+ or Ni2+. The requirement for ATP could not be replaced by GTP, CTP, UTP, ADP or AMP. ADP and AMP, but not pyrophosphate, inhibited the ATP dependent activation of p-coumarate. The activity was inhibited by N-ethylmaleimide and p-chloro-mercuribenzoate which suggests a requirement for -SH groups for activation. The activity of the enzyme is low in freshly prepared disks but rises during ageing, particularly if the ageing is carried out in the presence of low concentrations of ethylene.

The respiration climacteric in apple fruits.: The action of hydrolytic enzymes in peel tissue during the climacteric period in fruit detached from the tree

Abstract The respiration rate and the activity of four hydrolytic enzymes, chlorophyllase, lipase, ribonuclease, and acid phosphatase, were determined at intervals during the development of the respiration climacteric in detached apple fruits stored at 12°. The activities of lipase, ribonuclease and acid phosphatase rose to a peak at about the time the peak in respiration was reached and then decreased as the respiration fell. Acid phosphatase and ribonuclease are found in the soluble protein fraction in our preparations. This suggests that they are not bound within particles similar to the lysosomes which play an important role in the senescence of animal cells. Chlorophyllase activity rose before the commencement of the rise in respiration and continued beyond the peak. As the activity of chlorophyllase increased, the chlorophyll content of the tissue decreased. The possible relationship between chlorophyllase, lipase, the degradation of chlorophyll and the disorganization of the chloroplast lamellae which occurs during the climacteric is discussed.

Activity of enzymes involved in lignin biosynthesis in swede root disks

Abstract The activity of nine enzymes involved in the biosynthesis of lignin precursors has been studied during the ageing of swede root disks in the presence and absence of ethylene. Peroxidase, aromatic alcohol dehydrogenase and phenylalanine transaminase show very little change in activity during ageing under either ageing condition. O-methyl transferase, shikimate dehydrogenase and ferulyl CoA reductase show only a 2–3 fold increase on ageing and are relatively insensitive to ethylene treatment. A third group (comprising phenylalanine ammonia lyase, cinnamic acid-4-hydroxylase and hydroxycinnamate CoA ligase) show 20–30 fold increase on ageing and are most sensitive to ethylene treatment. Phenylalanine ammonia lyase and cinnamic acid-4-hydroxylase behave very similarly in respect of their time course of ageing and in their responses to metabolic inhibitors such as cycloheximide, puromycin and actinomycin D. In addition the properties of the O-methyl transferase of swede root tissue are described.

Metabolic changes in excised fruit tissue—III. The development of ethylene biosynthesis during the ageing of disks of apple peel

Abstract The production of ethylene by disks of peel from pre-climacteric apples is induced by ageing the disks aerobically at 25°. The development of the ethylene-producing system is dependent upon protein synthesis. Ethylene evolution is stimulated by peroxidation products from linolenic acid, but not by methionine. The relationship between changes occurring during the ageing of pre-climacteric apple peel disks during the development of the climacteric in whole fruit is discussed. A rapid and sensitive method for the determination of ethylene is described.

Metabolic changes in excised fruit tissue-I. Factors affecting the development of a malate decarboxylation system during the ageing of disks of pre-climacteric apples

Abstract A system develops in peel from preclimacteric apples during ageing for periods up to 48 hr which rapidly decarboxylates added malate (the malate effect). This development is preceded by a peak of incorporation of 14C-valine into the protein of the tissue. During the period of increased 14C-valine incorporation the tissue develops the capacity to produce ethylene, and exogenous ethylene reduces the time taken to reach the full malate effect. Cycloheximide and other inhibitors of protein synthesis prevent the incorporation of 14C-valine and the development of the malate effect. Changes in uptake of the various substrates do not appear to be involved in these phenomena. It is suggested that ethylene, RNA and protein synthesis are all involved in the development of the malate effect.

The respiration climacteric in apple fruits: Some possible regulatory mechanisms

Abstract The inhibition of succinate and malate oxidation by added oxaloacetate (OAA) has been studied in mitochondria isolated from the peel of apple fruits at various stages in the development of the respiration climacteric in fruit either attached or detached from the tree. The rate of relief of inhibition increases as the climacteric peak is reached. It is suggested that this faster relief of inhibition is related to the development of a special enzyme system for metabolizing OAA rather than to an increase in the rate of turnover of the Krebs cycle. The effect of addition of glucose and hexokinase to mitochondria oxidizing succinate and malate results in an enhancement of the rate of oxidation, and again the effect is most pronounced at the climacteric peak; the effect on CO 2 -output is greater than an O 2 -uptake. The effect of glucose and hexokinase is probably to provide a continuous supply of phosphate acceptors, but this does not explain the greater effect on CO 2 -output.

Purification and properties of hydroxycinnamoyl CoA quinate hydroxycinnamoyl transferase from potatoes

Abstract A rapid method for the purification of hydroxycinnamoyl CoA quinate hydroxycinnamoyl transferase (CQT) from potato tubers which had been stored at low temperatures is described. The method involves affinity chromatography on Blue Sepharose with biospecific desorption of CQT with its substrate, CoA. Elution of the Blue Sepharose column with a gradient of CoA leads to the resolution of CQT, a protein with MW of ca 41500, into 3 peaks of activity; the largest peak elutes first. This fraction is purified × 1440 and gives a single band of protein after PAGE which suggests a high degree of purity. The properties of the 3 fractions of CQT, with respect to substrates and to a number of inhibitors, are described. The first and last eluting CQT fractions are specific for quinate and show no activity towards shikimate. The second peak, however, shows a small activity towards shikimate but this is thought to be due to an underlying peak of a shikimate specific enzyme. The major peak of CQT activity found in potatoes stored at 0° is absent from those stored at 10° throughout the period after harvest.