Z. Zmrhal

The role of peroxidase in the metabolism of indole-3-acetic acid and phenols in wheat

Abstract A crude peroxidase preparation from leaves of young wheat plants oxidizes IAA in the presence of Mn 2+ and a phenolic cofactor in the absence of erogenous H 2 O 2 . When erogenous H 2 O 2 is supplied the enzyme oxidizes ferulic and p -coumaric acids. Ferulic acid causes a lag period in the oxidation of IAA and is oxidized itself during the lag.

Isolation and characterization of wheat peroxidase isoenzyme B1

Abstract Two pure peroxidase isoenzymes B1 and D4 were isolated from the upper parts of 10-day-old wheat seedlings by means of gel and ion-exchange chromatography. Their MWs were 85000 and 24000 respectively. B1 was unstable and under various conditions it was converted to another isoenzyme, electrophoretically identical with D4. B1 contains about 40% of neutral sugars: 17.2% arabinose, 15.3% galactose, 5% glucose and traces of mannose. D4 is free of neutral sugars. None of the isoenzymes contained amino sugars. B1 oxidizes ferulic and p -coumaric acids. This oxidation has two pH optima of 4.4 and 5.4–5.6 and is inhibited by high concentrations of substrates, cyanide and azide. B1 oxidizes IAA in the presence of phenolic cofactor and Mn 2+ ions. IAA oxidation has two pH optima of 4.5 and 5.6 and is inhibited by high substrate concentration, cyanide and azide, and by a number of indole derivatives. The main products of IAA oxidation are 3-methyleneoxindole and indole-3-methanol. o - and p - diphenols induce a lag period prior to IAA oxidation. Ferulic acid is oxidized during this lag period, probably to a dimer. B1 is able to produce H 2 O 2 from oxygen. Mn 2+ ions, a phenolic cofactor and an electron donor (IAA or NADH) are needed. B1 oxidizes α-keto-γ- methylmercaptobutyric acid to ethylene. D4 has a low peroxidatic activity and is inactive as an IAA oxidase. Thus B1 is probably an active cell wall-bound peroxidase isoenzyme, whereas D4 is its decomposition product.

Characterization of phosphate absorption in maize root cortex segments

Uptake of phosphate ions by 1 mm segments of isolated maize root cortex layers was studied. Cortex segments (from roots of 8 days old maize plants) absorb phosphate ions from 1 mM KH2PO4 in 0.2 mM CaSCO4 at the average rate of 34.3 ±3.2 μg Pi g−1 (fr. m.) h−1,i.e. 0.35± 0.02 μmol Pi g−1 (fr. m.) h−1. Phosphate uptake considerably increases after a certain period of “augmentation”,i.e. washing in aerated 0.2 mM CaSO4. This increase is completely blocked by the presence of 10 μg ml−1 cycloheximide.The relation of uptake rate to phosphate concentration in the medium was shown to have 3 phases in the concentration range of 0.02 - 40 mM. Transition points were found between 0.8–1 mM and 10–20 mM. Following Km and Vmax values were found: Km[mM] : 0.37 - 3.82 - 27.67 Vmax[μg Pi g−1 (fr. m.) h−1] : 3.33 - 39.40 - 66.67We have found no sharp pH optimum for phosphate uptake. It proceeds at almost constant rate till pH 6.0 and then the uptake rate drops with increasing pH. At low phosphate concentrations (1 mM) the lowest uptake rate was found at 5 and 13 °C, while the uptake is higher at 5 °C than at 13 °C at phosphate concentrations higher than 1 mM. At these concentrations uptake rate at 35 °C is lower than at 25 °C.Phosphate uptake considerably decreased in anaerobic conditions. DNP and iodoacetate (0.1 mM) completely blocked phosphate uptake from 1 mM KH2PO4, while uptake from 5 and 10 mM KH2PO4 was left unaffected by these substances. The inhibitors of active - SH groups NEM and PCMB inhibited phosphate uptake: 10−3 M NEM by 81.6%, 104 M NEM by 42% and 10−4 M PCMB by 42%.

Comparison of the effect of some phenolic compounds on wheat coleoptile section growth with their effect on iaa-oxidase activity

P-coumaric acid (HCA), 2,4-dichlorophenol (DCP) and resorcionol acted as cofactors for IAA-oxidase isolated from young wheat plants. Ferulic acid (FA) and 3,4-dihydroxybenzoic acid (DHBA) induced a lag phase prior to IAA oxidation. HCA, FA (0.2-1 mg ml-1) and DCP (0.03-1 mg ml-1) strongly inhibited wheat coleoptile section growth. DHBA (0.01-1 mg ml-1) slightly stimulated it and resorcinol was without effect. HCA inhibited IAA-induced growth of coleoptile sections and FA stimulated it at low IAA levels and inhibited it at higher ones. DHBA, DCP and resorcinol did not affect IAA-induced growth of coleoptile sections.AbstractKyselina p-kumarová (HCA), 2,4-dichlorfenol (DCP) a resorcin působilyin vitro jako kofaktory IAA-oxidasy isolované z mladých rostlin pšenice. Kyseliny ferulová (FA) a 3,4-dihydroxybenzoová (DHBA) indukovaly před oxidací IAA lag fázi. HCA a FA (0,2-1 mg ml-1) a DCP (0,03-1 mg ml-1) silně inhibovaly růst segmentů pšeničných koleoptilí. DHBA (0,01-1 mg ml-1 jej mírně stimulovala a resoroin jej téměř neovlivnil. HCA brzdila IAA-indukovaný růst segmentů koleoptilí. FA jej při nízkýoh koncentracích IAA stimulovala, ale při vyšších koncentracích IAA brzdila. DHBA, DCP a resorcin neměly na IAA-indukovaný růst segmentů koleoptilí žádný vliv.

The effect of new male sterilants: Exo-3,4-methano-L-proline and cis-α-(carboxycyclopropyl)glycine on the IAA- and ACC-induced ethylene formation ia wheat coleoptile segments

The male sterilants exo-3,4-methano-L-proline (MP) and cis-α-(carboxycyclopropyl)glycine (CCPG) were not converted to ethylene, eitherin vitro or in wheat coleoptile segments. Neither of these substances was able to affect IAA- and ACC-induced ethylene biosynthesis in wheat coleoptile segments to such an extent that it could explain their effect ay male sterilants.

The effect of phenylacetic acid on ethylene formation in wheat seedlings

Phenylacetic acid (PAA) was found to induce ethylene formation in wheat coleoptile segments. In its most effective concentration (0.5 mM) PAA was by approximately 60 % less active than 0.1 mM indole-3-acetic acid (IAA). PAA-induced ethylene formation was stimulated with 0.1 mM L-methionine by 24 % and totally inhibited by 2.5 and 5 μ gml-1 aminoethoxyvinylglycin (AVG) and 10 μg ml-1 cycloheximide. Cyoloheximide in lower concentration (5 μg ml-1) and actinomycin D (10 μg ml-1) inhibited PAA-induced ethylene formation by 50 % and 40 %, respectively.After the simultaneous addition of PAA and IAA ethylene formation was by 35 % lower than in the presence of IAA itself. Further, the coleoptile segments preincubated in IAA and then incubated in PAA solution produced by 35 % less ethylene than those incubated in plain buffer after preincubation in IAA. Quite the opposite effect was found when the segments were preincubated in PAA and then transferred into IAA solution. This treatment resulted in 70 % stimulation of ethylene formation over segments preincubated in PAA and incubated in buffer.

The effect of indole-3-Acetic acid on ethylene formation in wheat seedlings

Isoperoxidase B 1 isolated from winter wheat (Triticum aestivum L., cv. Jubilar) seedlings was shown to catalyze ethylene formation from α-keto, γ-methylmercaptobutyric acid (KMBA). In the presence of Mn2+, indole-3-acetic acid (IAA), andp-coumaric acid, the kinetics by isoperoxidase B 1 catalyzed conversion of KMBA into ethylene and other products was similar to that of IAA oxidation. The reaction rate was therefore controlled by IAA through its electrondonating properties.Exogenous IAA induced ethylene formation in the segments of etiolated wheat coleoptiles. IAA-induced ethylene production was enhanced by L-methionine and mitomycin C. Aminoethoxy-analogue of rhizobitoxine, ferulic acid, sodium benzoate, cycloheximide and actinomyoin D exhibited significant inhibitory effects. These data indicate that the overall reaction mechanism in coleoptile segments involves RNA and protein synthesis.The site of IAA action is not specific; 2,4-dichlorophenoxyacetic, α-naphthylacetic and indole-3-butyric acids, respectively, possessed comparable inductive effect as IAA. Indole-3-propionic acid, indole, L-tryptophan and glucobrassicin had only low inductive efficiency, and moreover indole and L-tryptophan slowed down IAA-induced ethylene formation.

The effect of some micronutrients and heavy metals on phosphate absorption by maize root cortex segments

The effect of salts (nitrates, chlorides, and sulfates) of microelements, Cd2+, Ni2+, and Co2+ and the effect of boric acid and ammonium molybdate on phosphate uptake by maize root cortex segments were tested.Higher concentration (0.1 mM) of Cu2+ salts caused enhancement of phosphate efflux to the extent that efflux was higher than influx.Inhibitory action on phosphate uptake by maize root cortex segments was exerted by following salts: 0.01 mM Cu2+ salts (20–30% inhibition), 0.5 mM ZnSO4 (9.7%), 0.5 and 0.05 mM ZnCl2 (34.3% and 20.8%), 0.1 mM salts of Cd2+, Ni2+, Co2+ (35–78%).1 mM FeSO4 had significant stimulatory effect (92%) on phosphate uptake. Much weaker stimulatory effect was exerted by 1 mM FeCl3 (14%), 0.05 mM ZnSO4 (9.6%), 0.005 mM ZnCla and ZnSO4 (8.4 and 18.5%) and 0.001 mM CdCl2 and CdSO4 (20.8 and 12.4%).All other tested salts-salts of Mn2+ (0.1 and 0.01 mM), 0.01 and 0.001 mM salts of Co2+ and Ni2+, 0.001 mM salts of Cu2+, 0.001–10 mM boric acid, and 0.001–0.1 mM ammonium molybdate left phosphate uptake unaffected.

Peroxidase activity and isoenzyme patterns in wheat during ontogenesis

Protein content, total and specific peroxidase activity and isoperoxidase patterns were determined in crude protein preparations from individual parts of field-grown wheat (Triticum aestivum L., cv. Jubilar). Protein content in roots, leaves, and stalks increased at the beginning of ontogenesis and then decreased from 6th, 9th, and 10th development phase (according to Feekes), respectively. Steady increase of the protein content in the ears was observed.Highest peroxidase activity was found in the roots; it diminished from the onset of ontogenesis till maturity of the plants. In the leaves and stalks a slight decrease of peroxidase activity till the 10th development phase and then an increase till maturity was found. The ears exhibited a gradual increase of peroxidase activity. The course of specific peroxidase activity was found to be very similar to that of total activity.Isoperoxidase patterns did not change significantly. In the leaves, a decrease of activity of C4 and C5 isoperoxidases was recorded. In the stalks, C l isoenzyme emerged at the end of ontogenesis. A gradual increase of A1 and A5 isoperoxidase intensity took place both in the leaves and stalks.

Histochemical IAA-oxidase localization in the shoot of wheat

A histochemical method for the determination of IAA-oxidase has been used in sections of various aerial parts of winter wheat plants. High IAA-oxidase activity was localized in the cell walls of sclerenchyma near the periphery of the stem, in the vascular bundle sheath of sclerenchyma and in xylem, both in the stem and in the leaf. The cell wall—bound IAA-oxidase activity therefore appeared in lignifying tissues. The staining was very weak or absent in the cell walls of parenchyma tissues and phloem. The positive reaction of the cytosol at the bulbous ends of guard cells and in the leaf primordia is presumed to be due to cytosolic IAA-oxidase. These results are discussed in relation to peroxidase localization and to our previousin vitro studies.