Variazioni di attività della clorogenico ossidasi e di altri enzimi interessati all'ossidoriduzione del TPN durante l'attivazione di fettine di tubero di patata

Abstract

CHANGES IN THE ACTIVITY OF CHLOROGENIC ACID OXIDASE AND OTHER ENZYMES INVOLVED IN OXIDATION AND REDUCTION OF TPN IN AGEING POTATO TUBER SLICES. — The activation of respiration, and in particular of the pentose phosphate pathway, during incubation of potato tuber slices could depend on the increase of activity of oxidative enzymes mediating electron transfer from Gl. 6-P to oxygen.

The present report deals with the activity changes, in the first period of incubation, of the following enzymes: Gl. 6-P-dehydrogenase, TPNH-glutathione reductase, gluta-thione-dehydroascorbate reductase, chlorogenic acid oxidase and a TPNH diaphorase utilizing tetrazolium salts as electron acceptors.

The activity of all of these enzymes, with the exception of TPNH diaphorase, was found to bs, at all stages of incubation, in large excess respect that required to account for the estimated contribution of the pentose phosphate pathway to respiration.

Gl. 6-P dehydrogenase, glutathione reductase and chlorogenic oxidase activities markedly incresed during incubation; but their increase appeared to be clearly delayed (of some hours) respect that of oxygen uptake. This seems to indicate that the increase in activity of these anzymes is rather a consequence than a cause of the respiratory activation.

TPNH diaphorase showed a very low activity in the fresh slices, and it increased quite significantly already in the very first period (5 hours) of incubation. This behaviour suggests the possibility that this enzyme could limit TPNH oxidation, and thus the pentose phosphate pathway activity, and that its activation could be correlated with that of oxidative metabolism in the ageing slices. Further investigation of this hypothesis requires the identification of the natural electron acceptor of this enzyme.     

Localizzazione intracellulare della clorogenico ossidasi in tessuti di tubero di patata in fase di attivazione

Abstract

INTRACELLULAR LOCALIZATION OF CHLOROGENIC ACID OXIDASE IN AGED POTATO TUBER DISCS. — The localization in the cells of chlorogenic acid oxidase has been investigated in potato tubers and discs of potato tuber. It has been ascertained that the rise of activity per gram of tissue, after preparation of discs, is not due to bacterial or fungal growth. The activity is widely distributed among cell fractions. Some activity is found in mitochondria, while most of the activity is distributed among soluble fraction and a « microsomal » fraction sedimented by centrifugation in the range 15.000–50.000 x g. This fraction appears to contain mitochondrial fragments, fragments of the endoplasmic reticulum and ribosomes. When tuber discs are aged, the rise of chlorogenic acid oxidase activity is much larger in the soluble than in particulate fraction.     

Ricerche sul meccanismo di azione della cocliobolina. A: effetti della tossina su fettine di tubero di patata in riposo

Abstract

STUDIES ON MECHANISM OF COCHLIOBOLIN ACTIVITY. A: EFFECTS OF THE TOXIN ON FRESH POTATO TUBER SLICES. — Cochliobolin, a toxic compound produced by Helminthosporium oryzae, causes inorganic phosphorus leakage from fresh potato tuber slices, higher amounts being found in incubation media with higher toxin concentrations and for longer incubation periods. Organic compounds, also, are released by potato tuber slices, like phenolic products, large amounts of sugars, and some aminoacids, alanine, lysine, tyrosine, valine and γ-aminobutirric acid, which have never been observed in the incubation meida of untreated slices. Cochliobolin uptake by fresh potato tuber slices does not take place actively or, anyhow, at a metabolic level. Indeed, neither 2,4-dinitro-phenol (at concentration, pH, and incubations periods apt to reduce active uptake), nor low temperatures (0–2°C) prevent toxin uptake. Potato tuber slices are not affected by cochliobolin, unless kept at 25°C, and this fact suggests that while toxin uptake does not interest metabolic pathways, it might affect some phase, or structure, essential to cell metabolism. The locus of toxin activity seems therefore to be identified with its influence on cell absortion and retention activities, of products essential for metabolic processes. Thus the impairing of cell permeability, possibly affecting the processes or the structures which regulate the active uptake, seems to be, at the moment, the only evident interpretation of the phenomena observed.     

Sucrose Phosphate Synthetase from Various Plant Origins

Some properties of sucrose-P synthetases obtained from various plant tissues, including sweet potato roots, potato tubers and leaves of barley, rape and ladino clover were studied. The specific enzyme activity of the sucrose-P synthetase from sweet potato roots was much lower than that of the sucrose synthetase of the other tissues. The enzyme activity decreased gradually as the roots developed. The optimum pH did not differ between enzyme preparations from sweet potato roots and barley leaves. Manganese chloride exhibited a marked stimulative effect on the sucrose-P synthetase from sweet potato roots and potato tubers, whereas it was inhibited the barley leaf enzyme.

Kinetic studies of sucrose-P synthetase showed that the behavior of the enzyme to the substrates did not differ in the enzyme sources examined. The substrate saturation curve of the enzyme with respect to fructose-6-P was sigmodal in shape, giving a straight line with a slope of 1.35~1.5 (n value) in a plot of the data using the empirical Hill equation. On the other hand, enzymes from all the various tissues exhibited a hyperbolic substrate saturation curve for UDP-glucose, obeying the ordinary Michaelis-Menten type reaction. Manganese chloride had no effect on the Km for UDP-glucose, the S_0.5 for fructose-6-P and the n value of the enzyme from potato tuber tissues.     

Caratteristiche della fosfo-gluco-isomerasi di una pianta superiore

Abstract

PHOSPHOGLUCOISOMERASE FROM PEA COTYLEDONS. — 6-P-glucose iso-merase has been purified from pea cotyledons. A 70-fold purification has been obtained by means of acetone fractionation and two absorption-elution steps on calcium phosphate gel. The partially purified enzyme is free of interfering activities.

KM values of 2.5×10^?4 and 10^?4 been measured for glucose-6-P and fructose-6-P respectively. reaction, measured at pH 7.8 and 30° C., is 3.7 (Gl-6-PIFr-6-P).

The enzyme is not inhibited by p-chloro-mercurybenzoate up to 10^?3 M. Besides the substances already known to inhibit competitively the isomerase from animal tissues, the pea enzyme has been found to be competitively inhibited by ribose-5-P and by triosespho-sphates, the K₁, being respectively 7×10^?4 and 2.5×10^?4.

The properties of the pea enzyme are compared to those of animal tissues isomerase. The possible physiological significance of these properties is discussed.     

Action de quelques phytohormones sur la respiration et ľabsorption du phosphate par des disques de tubercules de pomme de terre en survie

Action of some phytohormones on the respiration and on the absorption of phosphate by aging potato tuber discs. Discs of potato tuber incubated in aerated medium show an increase of the rates of respiration and phosphate absorption with aging time; the rates increase by two and nine respectively during the time period between 5 and 24 h of aging. Adenine or some N-6 substituted adenines [benzylaminopurine (BAP), furfurylaminopurine (FAP), methylaminopurine (MAP)], which present variable degrees of cytokinin activity, partially inhibit the increase of the rate of phosphate absorption and, to a lesser extent, the increase of the rate of respiration. Also abscisic acid (ABA), indole 3-acetic acid (IAA), and gibberellic acid (GA₃) produce inhibition of the increase of the rate of phosphate absorption with varied effects on the respiration. With regard to phosphate uptake, the effects of ABA, 1AA and GA₃ were additive to those of BAP. The effects on respiration were different from the effects on phosphate uptake, so that there is no direct relationship between inhibition of respiration and inhibition of phosphate uptake.     

Aspetti Enzimatici Della Maturazione e Germinazione dei Semi di Ricino

Abstract

Enzyme levels during ripening and germination of castor bean seeds. — During the development of the endosperm of castor bean seeds two distinct phases can be recognized: pre-maturation and germination. The former is characterized, metabolically, by the rapid conversion of carbohydrates into lipids, and storage proteins. The latter is characterized by the reconversion of these storage materials into sugars. Both these processes are dependent upon the activity of the glycolytic pathway; for this reason the behaviour of some enzymes of this pathway and, in general, of the carbohydrate metabolism have been studied during the two phases. The changes (during the evolution of the seeds) of the following enzymes have been studied:

Gl-6-P-dehydrogenase, 6-P-gluconate dehydrogenase, P-glucomutase, Hexokinase Hexoseisomerase, Aldolase, alcaline and acid Phosphatase, Pyrophosphatase and ATP-ase.

All these activities have been measured in the 20.000 × g supernatant fraction of cell homogenates.

The results show that all the enzymes activities measured increase rapidly during the period of accumulation of storage materials. In the following period all of these activities decrease until the stage of ripeness of the seed. During the first few days of germination the activities increase again rapidly. A particular behaviour is the one of Fr-1-6-P-phosphatase (the enzyme cleaving the phosphate bond in C ¹ position). This enzyme reaches during germination a level much higher than the maximal observed during the ripening process. This might be an important fact correlated with the inversion of the glycolytic reactions during germination.     

类芽孢杆菌QHZ11对马铃薯黑痣病的生防效果

[背景] 马铃薯黑痣病是由立枯丝核菌（Rhizoctonia solani）引起的一种典型土传病害,目前该病害生物防治的菌种资源比较有限,相应菌株生防机制的研究更是缺乏。[目的] 明确马铃薯黑痣病病原菌立枯丝核菌（R. solani） JT18的拮抗菌QHZ11对马铃薯黑痣病的生防效果,揭示QHZ11对黑痣病的部分防治机理。[方法] 在灭菌土壤中分别接种R. solani JT18（CK）,R. solani JT18和普通有机肥（Organic Fertilized,OF）,R.solaniJT18和氨基酸有机肥（AA+OF）及R. solani JT18和QHZ11生物有机肥（BOF11）,结合实时荧光定量PCR （Real-Time Fluorescence Quantitative PCR,RT-qPCR）等方法,研究马铃薯全生育期不同处理R.solaniJT18在马铃薯根际和植株不同部位的数量变化及拮抗菌QHZ11与R.solaniJT18的数量消长规律,同时比较不同处理黑痣病的病情指数及相应的防效。[结果] RT-qPCR结果表明,随马铃薯生育进程的推进,马铃薯根际、根系和匍匐茎R.solaniJT18的数量在各处理中均呈现先升高至块茎膨大期到达峰值后下降的趋势,而且各部位R.solaniJT18的数量为CK>OF>AA+OF>BOF11且根际>根系>匍匐茎;拮抗菌QHZ11的数量变化趋势与R.solaniJT18相同,但峰值在块茎形成期,并且同时期同一部位QHZ11的定殖数量均显著高于R.solaniJT18,甚至高出2个数量级,说明QHZ11占用了一定的营养资源和生态位点,严重抑制了R.solaniJT18的生长和繁殖。病情结果表明：CK病情指数最高,OF、AA+OF和BOF11处理均显著低于CK,其中BOF11处理发病最轻;生防结果则相反,为BOF11>AA+OF>OF处理,说明普通有机肥、氨基酸有机肥及生物有机肥均可不同程度地防治马铃薯黑痣病,其中以生物有机肥效果最显著。[结论] QHZ11以有机肥为载体施入土壤后,可以通过在马铃薯根际及植株不同部位竞争营养和生态位点,从而有效抑制黑痣病病原菌R.solaniJT18的生存和繁殖,起到显著的生防效果,这对QHZ11生物有机肥的应用和推广具有重要意义,并为进一步研究QHZ11的生防机制奠定了基础。     

Photocontrol of chlorogenic acid biosynthesis in potato tuber discs

The appearance of phenylalanine ammonia-lyase activity and the accumulation of chlorogenic acid in potato tuber discs are stimulated by illumination with white light, whereas the appearance of cinnamic acid 4-hydroxylase activity is unaffected by illumination. The photosensitive step in chlorogenic acid biosynthesis may be by-passed by treatment of discs with exogenous supplies of cinnamic acid, whereas treatment of discs with phenylalanine does not isolate the photosensitive step. Therefore, the site of photocontrol of chlorogenic acid biosynthesis in potato tuber discs is the reaction catalysed by phenylalanine ammonia-lyase. Cinnamic acid 4-hydroxylase activity in vitro is unaffected by p-coumaric acid, caffeic acid or chlorogenic acid. Phenylalanine ammonia-lyase activity in vitro is sensitive to inhibition by cinnamic acid. The in vitro properties of the two enzymes are also consistent with the hypothesis that phenylalanine ammonia-lyase rather than cinnamic acid 4-hydroxylase is important in the regulation of chlorogenic acid biosynthesis in potato tuber discs.     

Oxazolomycin Esters,Specific Inhibitors of Plant Transformation

Oxazolomycin diacetate, dipropionate, monobutyrate and dibutyrate were derived from oxazolomycin, a product of Streptomyces sp. KBFP-2025. These esters were potent inhibitors of crown gall formation on potato tuber disks upon infection with Agrobacterium tumefaciens. They showed neither antibacterial nor phytotoxic activity, whereas oxazolomycin showed both antibacterial and phytotoxic activities. Further, they had no inhibitory activity against A. tumefaciens on the potato tuber disk. The inhibitory activity of these esters against crown gall formation seems to be due to specific inhibition of the transformation of plants with A. tumefaciens.     

Respiration and Carbohydrate Metabolisms of Potato Tuber

When a potato tuber is cut into slices, the activity of dehydrogenase system linked to DPN decreases but the activities of tyrosinase system and G-6-P dehydrogenase increase in the slices. Accompanying the changes of these enzymatic activities, the metabolic pathway in the slices turns from glycolytic path to pentose phosphate path. Tyrosinase oxidizes phenols which are formed from an intermediate of the pentose phosphate path, sedo-heptulose via shikimate as in microorganisms, resulting in increase in the oxygen uptake of the slices. It seems impossible to play a role as a terminal oxidase linked to diphosphopyridine nucleotide.     

Abscisic Acid Suppresses the Hypersensitive Necrosis of Potato Tubers and Reverses the Arachidonic Acid-Induced Inhibition of Plasma Membrane-Bound ATPase

Abscisic acid (ABA) fully suppressed the development of the hypersensitive necrosis of potato tuber slices to crude elicitor from Phytophthora infestans and arachidonic acid (AA) if supplied before or up to 6 to 8 h after elicitor treatments. Later application of ABA only reduced the necrotic response. Added to highly purified plasma membrane preparation from tuber tissue, ABA eliminated the AA-induced inhibition of the plasma membrane-bound (K⁺+ Na⁺+ Mg²⁺) ATPase. It is suggested that ABA counteracts AA in the membrane.     

Glycolytic activity in embryos of Pisum sativum and of non-dormant or dormant seeds of Avena sativa L. expressed through activities of PFK and PPi-PFK

Summary A quantative cytochemical assay for PP_i-PFK activity in the presence of Fru-2,6-P₂ is described along with its application to determine levels of activity in embryos of Pisum sativum and Avena sativa. The activity of ATP-PFK has also been studied in parallel as have PFK activities during the switch from dormant to non-dormant embryos in Avena sativa. PP_i-PFK activity, has been demonstrated in all tissues of Pisum sativum embryos and of Avena sativa embryos including the scutellum and the aleurone layers. The PP_i-PFK activity was greater than that of ATP-PFK in both dormant and non-dormant seeds though with only marginally more activity in the dormant as opposed to the non-dormant state.Abbreviations AMP adenosine monophosphate - ATP adenosine triphosphate - Fru-1,6-P₂ fructose 1,6-bisphosphate - Fru-2,6-P₂ fructose 2,6-bisphosphate - Fru-6-P fructose 6-phosphate - FB Pase 2 fructose 2,6-bisphosphatase (EC 3.1.3.46) - Gl-3-PD glyceraldehyde-3-phosphate dehydrogenase - NAD nicotinamide adenine dinucleotide - NBT nitroblue tetrazolium - PEP phosphoenolpyruvate - PFK 6-phosphofructokinase (EC 2.7.1.11) - PFK₂ 6-phosphofructo-2-kinase (EC 2.7.1.105) - PP_i pyrophosphate - PP_i-PFK pyrophosphate: fructose 6-phosphate 1-phosphotransferase (EC 2.7.1.90) - PVA polyvinyl alcohol (G04/140 Wacke Chemical Company)     

Starch and fatty acid synthesis in plastids from developing embryos of oilseed rape (Brassica napus L.)

The aim of this work was to investigate the capacity for synthesis of starch and fatty acids from exogenous metabolites by plastids from developing embryos of oilseed rape (Brassica napus L.). A method was developed for the rapid isolation from developing embryos of intact plastids with low contamination by cytosolic enzymes. The plastids contain a complete glycolytic pathway, NADP-glucose-6-phosphate dehydrogenase, NADP-6-phosphogluconate dehydrogenase, fructose-1,6-bisphosphatase, NADP-malic enzyme, the pyruvate dehydrogenase complex (PDC), and acetyl-CoA carboxylase. Organelle fractionation studies showed that 67% of the total cellular PDC activity was in the plastids. The isolated plastids were fed with ¹⁴C-labelled carbon precursors and the incorporation of ¹⁴C into starch and fatty acids was determined. ¹⁴C from glucose-6-phosphate (G-6-P), fructose, glucose, fructose-6-phosphate and dihydroxyacetone phosphate (DHAP) was incorporated into starch in an intactness- and ATP-dependent manner. The rate of starch synthesis was highest from G-6-P, although fructose gave rates which were 70% of those from G-6-P. Glucose-1-phosphate was not utilized by intact plastids for starch synthesis. The plastids utilized pyruvate, G-6-P, DHAP, malate and acetate as substrates for fatty acid synthesis. Of these substrates, pyruvate and G-6-P supported the highest rates of synthesis. These studies show that several cytosolic metabolites may contribute to starch and/or fatty acid synthesis in the developing embryos of oilseed rape.     

Ribonucleic Acid Changes in Germinating Potato Tubers

Abstract

The changes of the total RNA occurring during germination in potato tubers have been investigated. During germination the development of the new sprouts is accompanied by a progressive decrease of RNA. A preferential breakdown of high molecular weight RNA has been observed. Tubers deprived of the buds mantain the level of RNA of the quiescent tuber. Gibberellic acid treatment promotes the development of the sprouts, but is ineffective in inducing changes in the RNA level when supplied to isolated explants of potato tuber both germinating or deprived of buds.     

Okadaic acid-induced transcriptional downregulation of type I collagen gene expression is mediated by protein phosphatase 2A

Summary In Saccharomyces cerevisiae, a small proportion of the glucose-6-P dehydrogenase activity is firmly associated with the mitochondrial fraction and is not removed by repeated washing or density-gradient centrifugation. However, the enzyme is released by sonic disruption. Mitochondrial glucose-6-P dehydrogenase that is released by sonication and partially purified has been found to be similar to cytosol glucose-6-P dehydrogenase with respect to electrophoretic mobility, isoelectric point, pH optimum, molecular size, and apparent K _m 's for NADP⁺ and glucose-6-P. These results indicate that a single species of glucose-6-P dehydrogenase is synthesized in S. cerevisiae and that the enzyme has more than one intracellular location. Mitochondrial glucose-6-P dehydrogenase may be a source of intramitochondrial NADPH and may function with hexokinase and transhydrogenase to provide a pathway for glucose oxidation that is coupled to the synthesis of mitochondrial ATP. A constant proportion of total glucose-6-P dehydrogenase activity remains compartmented in the mitochondrial fraction throughout the growth cycle.     

Field evaluation of transgenic potato plants expressing an antisense granule-bound starch synthase gene: increase of the antisense effect during tuber growth

Transgenic plants of a tetraploid potato cultivar were obtained in which the amylose content of tuber starch was reduced via antisense RNA-mediated inhibition of the expression of the gene encoding granule-bound starch synthase (GBSS). GBSS is one of the key enzymes in the biosynthesis of starch and catalyses the formation of amylose. The antisense GBSS genes, based on the full-length GBSS cDNA driven by the 35S CaMV promoter or the potato GBSS promoter, were introduced into the potato genome by Agrobacterium tumefaciens-mediated transformation. Expression of each of these genes resulted in the complete inhibition of GBSS gene expression, and thus in the production of amylose-free tuber starch, in mature field-grown plants originating from rooted in vitro plantlets of 4 out of 66 transgenic clones. Clones in which the GBSS gene expression was incompletely inhibited showed an increase of the extent of inhibition during tuber growth. This is likely to be due to the increase of starch granule size during tuber growth and the specific distribution pattern of starch components in granules of clones with reduced GBSS activity. Expression of the antisense GBSS gene from the GBSS promoter resulted in a higher stability of inhibition in tubers of field-grown plants as compared to expression from the 35S CaMV promoter. Field analysis of the transgenic clones indicated that inhibition of GBSS gene expression could be achieved without significantly affecting the starch and sugar content of transgenic tubers, the expression level of other genes involved in starch and tuber metabolism and agronomic characteristics such as yield and dry matter content.     

Vie di ossidazione della frazione extramitocondriale dei coenzimi piridinici: I. - Sull'Efficienza In Vivo del sistema ascorbato/deidroascorbato

Abstract

OXIDATION PATHWAYS OF EXTRAMITOCHONDRIAL PIRIDINE COENZYMES. I. - ON THE « IN VIVO » EFFICIENCY OF THE ASCORBATE-DEYHDROASCORBATE SYSTEM. — An evaluation of the efficiency in vivo of the AA-DHA couple as an electron carrier system has been attempted, by measuring after short time of anaerobiosis the rate of the increase of AA and of the dicrease of DHA in etiolated pea internode segments and in potato tuber disks. The changes of reduced glutathione (GSH) contents as induced by anaerobiosis or by the addition of DHA to the incubation medium were also followed.

In the pea segments anaerobiosis induced a significant increase of AA and a corresponding decrease of DHA. These changes were almost completed after 10 minutes from starting anaerobiosis. The value (extrapolated to 0 time) of the initial rate of DHA desappearance under anaerobiosis was taken as representing the rate of DHA reduction to AA « in vivo », under aerobic conditions. As this rate — in a steady state situation — corresponds to that of the inverse process of oxidation of AA to DHA, this value should give and indication on the « in vivo » efficiency of the AA-DHA system as an electron carrier in respiration. As some AA was probably reoxidized to DHA in the very short period required to kill the tissue, the value of the AA DHA turnover thus calculated is probably somewhat lower than the real one.

According to the present work, the oxidative turnover of the AA-DHA system would results of 0,7 micromoles/g. fr. weght/h. for the pea internode tissues and of 0,9 micromoles/g. fr. weght^?h for the potato tuber (aged disks). These values would account for 5% of total oxygen uptake, in the former, and for 3% in the latter material.

The very high AA/DHA ratio usually prevailing in living cells suggests that the contents in DHA (and thus the activity of the AA oxidizing systems) is a limiting factor for the efficiency of the AA-DHA system as an electron carrier. This view is supported also by experiments in which DHA (at pH 5) was fed to pea internode segments and to potato tuber disks : as the presence of DHA into the medium induced — under anaerobiotic conditions — a rapid increase of the level of AA in both types of materials. In aerobiosis uptake and reduction of DHA to AA was evident in the potato tuber tissue, while it appeared very scarce in the pea internodes. As an interpretation of this behaviour it is suggested that, in aerobiosis, the very active and probably surface localized ascorbic acid oxidase of the pea tissue re-oxidises the AA formed from reduction of the DHA fed; an accumulation of DHA into the cells would follow, and this excess of DHA would inhibit the enzyme GSH-DHA reductase. This enzyme, in fact, appears, from « in vitro » experiments, to be strongly inhibited by DHA when the DHA/GSH ratio becomes higher than 1. On the other hand, the same hypothesis is also supported by the finding that the addition of DHA to the medium induces a significant drop in the GSH level (probably due to its oxidation to GSSG) only under those conditions in which DHA is absorbed and reduced to AA; that is, in the pea internodes, under anaerobiosis, and in the potato disks, under both anaerobiosis and aerobiosis. These results are also taken as confirming the indication from the enzymatic data that GSH is acting, in vivo as a reducing agent for DHA. The results of this investigation are thus interpred as showing that a comparatively small, but by no means negligeable fraction of respiration is mediated, in higher plant tissues such as those of the pea stem and the potato tuber, by and electron transfer system including glutathione and the ascorbate-dehydroascorbate couple. The efficiency of this system in the materials investigated appears to account for 3–5% of the total 0₂ uptake (minimum value). As enzyme systems transferring electrons from TPNH to ox. glutathione are widely distributed and generally very active in higher plant tissues, it is suggested that the sequence TPNH-GSH-AA/DHA - O₂ is probably of considerable importance in mediating the reoxidation of extramitochondrial trophosphoridine nucleotide and thus in permitting the operation of the TPN requiring pentose phosphate pathway of respiration.