R.B. van Huystee

Peroxidase-regulated elongation of segments from peanut hypocotyls

Alterations of isoperoxidases were studied in 1.0 cm segments from 3-day-peanut-seedling (Arachis hypogaea L.) hypocotyls as they were cultured on Linsmayer and Skoog (LS) medium with or without the addition of 5 mM meta-fluoro-tyrosine (MFT). The alterations in peroxidase activity and relative protein levels were correlated with the growth inhibition by MFT, particularly for the cationic isozyme. A further direct link between growth and the peroxidase has been established by the addition of either antibodies or purified isozymes to the culture medium. The enhancement of growth by the antibodies and inhibition by the peroxidase isozymes strongly suggest that peroxidase is directly involved in growth.

Progress and prospects in the use of peroxidase to study cell development

Abstract The need for peroxidase purification is stressed as a requirement for comparative studies on isoenzyme structure as well as for detailed investigations on biosynthesis. A single cationic protein possessing the major peroxidase activity was isolated from the medium in which peanut cells had grown. The antibodies raised against this pure protein were employed as a probe to study the site of synthesis of peroxidase in the cell as well as the proportion of total synthesized protein which was peroxidase. Structural studies on the purified isoenzymes suggest the presence of three gene loci for peroxidase in cultured peanut cells. The results are discussed together with potential assays for induction of this enzyme and the relationship to cell development.

Anionic peroxidase catalysed ascorbic acid and IAA oxidation in the presence of hydrogen peroxide: A defence system against peroxidative stress in peanut plant

Abstract In vitro experiments show that anionic peroxidase has a higher affinity for both ascorbic acid and IAA and a greater Vmax than cationic peroxidase in the oxidation reaction with the presence of H2O2. The peanut hypocotyls cultured in a medium with double amount of ascorbic acid (0.4 mg 1−1) and IAA (4.0 mg 1−1) show an increase of 104.3% and 128.4%, respectively, in anionic isozyme compared to the control, and only an increase of 22.6% and 12.8% in cationic isozyme. Treatment of entire plants with H2O2 (7.5%, v/v) demonstrated that a higher per cent of anionic isozyme over cationic is induced. These results suggest that cytoplasm-located anionic peroxidase probably plays a key role in the defence system against peroxidative stress through the detoxification of H2O2 during the processes of peroxidation of ascorbic acid and IAA.

An immunological study of peroxidase release by cultured peanut cells

Abstract The major increase of peroxidase activity in the medium of a suspension culture of peanut ( Arachis hypogaea , L) cells occurs between day 4 and 9 of a biweekly subculture period. This assumed release of peroxidase into the medium parallels the period of most active peroxidase synthesis in these cells. The biosynthesis and subsequent release of peroxidase were monitored more precisely by the incorporation of [ 35 S]methionine and immunoprecipitation with antibodies raised against the most abundant peroxidase isozyme. Pulse-chase experiments were used to illustrate the transport system. The drastic effect of antimycin A on the release of peroxidase indicates the need of energy for transport to proceed, probably via the Golgi apparatus.

Purification and yield of a cationic peroxidase from a peanut suspension cell culture

Abstract Variability in the purity of peanut ( Arachis hypogaea ) peroxidase has necessitated the introduction of further purification steps beyond those used formerly. This appeared to be necessary when the purity of the peroxidase obtained by the established approach was found to be deficient as determined by electrophoresis and by absorption spectra. The addition of lectin affinity and gel filtration steps yielded a homogeneous preparation as indicated by polyacrylamide gel electrophoresis (PAGE) of 20 μg of protein on a mini gel. The RZ (405/280 nm) value was as good as the best former values. A calculation of the capacity of the cultured peanut cells to produce the highly purified peroxidase is presented.

Appraisal of studies on induction of peroxidase and associated porphyrin metabolism.

SummaryThe enzyme peroxidase has acquired popularity with plant scientists partly because its activity is inversely related to growth rate, perhaps as a consequence of the enzyme’s IAA oxidase activity, and partly because its “isozymes” vary with the developmental state of the tissue studied. The changes in both activity and “isozyme” patterns could be accounted for by de novo synthesis of the enzyme or by post-synthesis alterations of existing enzyme molecules. The paucity of information on the structural nature of peroxidase “isozymes” has hampered a distinction being made in most physiological studies between synthesis and post-synthesis mechanisms of change. This leaves the basic question of peroxidase regulation unanswered.Analogies between peroxidase and other hemoproteins like hemoglobin and leghemoglobin lead to the suggestion that heme might function in regulating apoperoxidase synthesis. Investigations with peanut cells in suspension culture indicate that the heme of peroxidase is drawn from a cellular heme pool. Whereas peroxidase activity in these cells exhibits a phytochrome-mediated light response, the heme pool responds otherwise to light. The lack of parallels in the response of peroxidase and the heme pool to light argue for a lack of heme involvement in at least the lightmediated changes in peroxidase activity although the role of heme in establishing the baseline levels of peroxidase in the dark has yet to be explored.The use of a peroxidase-specific mRNA is proposed to facilitate studies of the mechanisms of change in activity and “isozyme” pattern and to facilitate identification of the factors involved in the regulation of peroxidase synthesis.ResuméL’enzyme appelée peroxydase a acquis une popularité auprès des botanistes à cause de son activité qui est inversement proportionnelle au taux de croissance, ceci etant probablement une conséquence de l’activité enzymatique de l’oxydase AIA, d’autre part a cause de ses differents “Isoenzymes” dont le nombre varie selon l’état de développement du tissu etudié. Les changements au niveau de son activité enzymatique et de son nombre d’isoenzymes pourraient être dûs à la synthèse de novo de l’enzyme ou à des alterations des molécules enzymatiques existantes. Le manque d’information concernant la structure des “isoenzymes” de la peroxydase a gêné une distinction, faite dans la plupart des études physiologiques, entre les mécanismes de changement au cours de la synthèse et ceux après la synthèse. Ceci laisse la question fondamentale du mécanisme de régulation de la peroxydase sans réponse.Des analogies entre la peroxydase et d’autres hemoprotéines comme l’hemoglobine et la leg-hemoglobine conduisent a suggérer que l’heme pourrait intervenir dans la régulation de la synthèse de l’apoperoxydase. Des investigations faites avec des cellules d’arachide en culture liquide, indiquent que l’heme de la peroxydase provient d’un pool hemique cellulaire. Différemment a celle du pool hemique, la réponse à la lumiere de l’activité peroxydasique se fait par l’intermediaire du phytochrome. Le manque de parallele entre la réponse de la peroxydase et celle du, pool hemique a la lumière indiquerait que l’heme ne serait pas impliqué dans les changements de l’activité peroxydasique interverant sous l’effet de la lumière. Cependant le rôle de l’heme, lors de l’étude de la réponse de l’activité peroxydasique à l’obscurité rstera etre déterminé.L’utilisation d’un mRNA specifique de la peroxydase est proposée pour faciliter les études des mecanismes de changement au niveau de l’activité et au niveau du nombre d’isoenzymes et pour faciliter l’identification des facteurs intervenant dans la régulation de la synthèse de la peroxydase.ZusammenfassungPeroxidase ist bei Pflanzenwissenschaftlern deshalb beliebt geworden, weil einerseits bei diesem Enzym eine entgegengesetzte Wechselwirkung zur Wachstumsrate besteht (wahrscheinlich als Konsequenz der IAA Oxidase Aktivität des Enzyms) und andererseits, weil ihre “Isoenzyme” mit dem Wachstumsstadium des untersuchten Gewebes wechseln. Die Veränderung in Aktivität und “Isoenzym” —Muster kann durch de novo—Synthese des Enzyms oder durch nach-synthetische Veränderungen bestehender Enzym-Moleküle erklärt werden. Da die Struktur von Peroxidase “Isoenzymen” kaum beschrieben ist, ist in den meisten physiologischen Untersuchungen eine Unterscheidung erschwert zwischen der Synthese und dem nach-synthetischen Veränderungs-mechanismus. Dies lässt die Grundfrage der Peroxidase Regulation unbeantwortet.Analogien zwischen Peroxidase und anderen Hamoproteinen wie Hamoglobin und Leghamoglobin lassen erwarten, dass die Regulation von Apoperoxidase Synthese eine Häm Funktion ist. Experimente mit Erdnusszellen in Suspensionskultur zeigen, dass das Häm der Peroxidase einem zellulären Pool entzogen wird. Während Peroxidase Aktivität in diesen Zellen eine Phytochrom-vermittelte Lichtreaktion zeigen, reagiert der Häm Pool insgesamt anders zu Licht. Mangel an Parallelen in der Reaktion der Peroxidase und dem Häm Pool zu Licht argumentieren dagegen, dass das Häm in der Licht-vermittelten Veränderung in der Peroxidase Aktivität eine grosse Rolle spielt, obwohl die Rolle des Häm in der Bestimmung des Grundniveaus der Peroxidase im Dunkeln noch zu untersuchen ist.Es wird vorgeschlagen, Peroxidase-spezifisches mRNS zu benutzen, um den Mechanismus der Veränderung in Aktivität und “Isoenzym” Muster zu untersuchen, und um die Bestimmung der Faktoren zu erleichtern, die in der Regulation von Peroxidase Synthese am Werk sind.

Some Aspects of Peroxidase Synthesis by Cultured Peanut Cells

Summary Free and membrane-bound polysomes isolated from cultured peanut cells were used for in vitro protein synthesis. Using antibodies raised against a pure peroxidase isozyme, it was possible to show, by immunoprecipitation, that about twice as much peroxidase was synthesized on the membrane-bound polysomes as on the free polysomes. In vitro translation of poly(A) -containing mRNA purified from total polysomal RNA, followed by immunoprecipitation with peroxidase specific antibodies indicated that about 2% of the total poly(A)-containing mRNA coded for one major peroxidase isozyme in cultured peanut cells. In addition, two other peroxidase isozymes present in the cell suspension medium were purified by ion exchange chromatography. The peptide mappings derived from proteolysis of the three peroxidase fractions showed that there is a difference in the protein structure. Consequently, there may be three different loci for synthesis of peroxidase in these cultured cells.

Porphyrin Metabolism in Chill Stressed Maize (Zea mays L.)

Summary Etiolated seedlings of maize ( Zea mays L.), when illuminated at various temperatures, exhibited differences in greening kinetics.When illuminated at 28°C and 25°C, no significant differences could discerned.However, at temperatures ranging from 12°C to 22°C only negligible greening could be observed, even after 9 h of illumination.Incubation of etiolated tissue in ALA, a porphyrin precursor, revealed an enhanced synthesis of protochlorophyll(ide) at temperatures ranging from 18°C to about 28°C, at the expense of accumulations of protoporphyrin, Mg-protoporphyrin and coproporphyrin.Below 18°C all porphyrin synthesis declined.No difference in protoheme content was detected in tissues incubated at 28°C, 20°C, 12°C.It is suggested that chill induced chlorosis is a result of at least two metabolic blocks in the porphyrin path that is situated prior to the formation of coproporphyrin.

Rapid Simultaneous Estimation of Protoporphyrin and Mg-Porphyrins in Higher Plants

Summary A rapid, inexpensive method has been derived that allows the simultaneous determination of Protoporphyrin and Mg-Porphyrins in the same extract.The procedure allows for accurate (> 90%), repeatable estimates in the nanomolar range.Analyses of experimental conditions known to alter porphyrin metabolism, such as illumination of etiolated material or incubation in 5-aminolevulinic acid, give results that are consistent with those reported by others. Using this procedure, it is shown that one characteristic of chilling induced chlorosis involves a non-lethal alteration in the ability of etiolated tissue to synthesize chlorophylls.

Early differences in production of mRNAs for phenylalanine ammonia-lyase and chalcone synthase in resistant and susceptible cultivars of soybean inoculated with Phytophthora megasperma f. sp. glycinea☆

Abstract The production of mRNAs for phenylalanine ammonia-lyase and chalcone synthase was determined in the first 5 h following infection of intact etiolated soybean hypocotyls with zoospores of Phytophthora megasperma f. sp. glycinea . mRNA was extracted from tissue excised from inoculated sites and mRNAs for the two enzymes detected by dot hybridization using corresponding cDNA probes. A major increase in mRNAs for both enzymes was detected by 3 h following inoculation in an incompatible interaction but not in a compatible interaction. The results are consistent with the development of early differences in glyceollin biosynthesis in the two types of interaction.