Hydrogen Peroxide-Dependent Oxidation of Flavonoids and Hydroxycinnamic Acid Derivatives in Epidermal and Guard Cells of Tradescantia virginiana L.

Luteolin, kaempferol, quercetin, caffeic acid and ferulic acidwere identified in acid-hydrolyzed epidermal strips of Tradescantiavirginiana using HPLC and spectrophotometry. The amount of flavonoidswas much smaller than that of cinnamic acid derivatives. Morethan 80% of the flavonoids were found in methanol extracts ofepidermal strips. Caffeic acid was found in both methanol extractsand the residues in nearly equal amounts, while more than 80%of the ferulic acid was found in the residues after methanolextraction. These data suggest that most of the ferulic acidand part of the caffeic acid bind to macromolecules as estersin the cell wall and that flavonoids are localized mainly inthe cytoplasm. The localization of esters of hydroxycinnamicacids in cell walls was ascertained by fluorometric analysis.These phenolic compounds were oxidized by H₂O₂ (0.025–1mM) in epidermal and guard cells and the oxidation was inhibitedby KCN and NaN₃: luteolin glycosides were less sensitive toH₂O₂ than quercetin and kaempferol glycosides in flavonoids.Ferulic acid esters were more sensitive to H₂O₂ than caffeicacid esters in hydroxycinnamic acid derivatives. On the basisof these data, the physiological significance of the oxidationof phenolic compounds by H₂O₂ is discussed. (Received October 9, 1987; Accepted February 3, 1988)     

Hydrogen Peroxide-dependent Synthesis of Flavonols in Mesophyll Cells of Vicia faba L.

Mesophyll cells of Vicia faba contain kaempferol and quercetinglycosides. When isolated mesophyll cells were treated with0.1 mM H₂O₂ for 2 h, the levels of these flavonols increasedby 10–70% of the control values (mean values, 19.6% and34.4% for kaempferol and quercetin glycosides, respectively).Such increases in levels of flavonols were also observed inisolated vacuoles of mesophyll cells. However, when mesophyllcells and vacuoles were treated with 10 mM H₂O₂₎degradationof flavonols was observed. These data suggest that H₂O₂ hastwo effects on the metabolism of flavonols: induction of theirsynthesis and stimulation of their oxidation. (Received March 6, 1989; Accepted July 10, 1989)     

A Role of Hydrogen Peroxide in the Metabolism of Phenolics in Mesophyll Cells of Viciafaba L.

3,4-Dihydroxyphenylalanine (DOPA) and flavonols were oxidizedby externally added H₂O₂and the oxidation was inhibited by KCN(5 mM) in protoplasts of mesophyll cells of Viciafaba. DOPAwas also oxidized by light in the presence of methyl viologen(MV), which can stimulate formation of O^–₂ and H₂O₂ invivo, both in the light and in the dark, in isolated mesophyllcells. The light-dependent oxidation of DOPA was partially inhibitedby removal of MV or addition of NaN₃ (10 mM), an inhibitor ofperoxidases, suggesting the participation of H₂O₂, generatedin vivo, in the oxidation. The effects of light on the levelof flavonols in isolated mesophyll cells were rather complicated.Level of flavonols increased by about 10–20% in the darkin the presence of MV. The levels in the light in the presenceof MV were lower than those in the dark. The data suggest thatflavonols can be oxidized by O^–₂ and/or H₂O₂ generatedin cells. Based on the data, the role of H₂O₂ in the metabolismof phenolics in mesophyll cells is discussed. (Received June 8, 1988; Accepted January 13, 1989)     

Effect of O2 Concentration on Dark H2 Oxidation in Whole Cells of a Marine Sulfur Photosynthetic Bacterium (Chromatium sp. Strain Miami PBS 1071)

Whole cells of photoanaerobically grown Chromatium sp. strainMiami PBS 1071, a marine purple sulfur bacterium, oxidized H₂in the dark through the oxyhydrogen reaction. Oxidation of H₂was measured by injecting either H₂ into an air-equilibratedcell suspension (microaerobic H₂ oxidation) or O₂ into an H₂/Ar-equilibratedcell suspension (microaerobic H₂ oxidation). Both types of H₂oxidation were strongly inhibited by azide (40 mM), indicatingthat the oxidation proceeds via a terminal oxidase system. 2,5-Dibromo-3-methyl-6-isopropyl-p-benzoquinone(16 µM) inhibited aerobic H₂ oxidation by 49% but it acceleratedmicroacrobic H₂ oxidation. The sensitivity of H₂ oxidation torotenone was higher under aerobic conditions. The results indicatethat H₂ oxidation proceeds via two different pathways; one containsubiquinone and NAD, and the other does not. The contributionof each pathway depends on the O₂ partial pressure. ⁴ Present address: Institute of Oceanic Research and Development,Tokai University, Shimizu, Shizuoka 424, Japan. (Received May 24, 1985; Accepted August 29, 1985)     

Hydrogen peroxide-dependent oxidation of flavonols by intact spinach chloroplasts

Externally added quercetin (100 micromolar) was oxidized by intact spinach chloroplasts at a rate of 30 micromoles per mg chlorophyll per hour in the presence of 100 micromolar H₂O₂. The oxidation rate was increased by about 20% in a hypotonic reaction mixture. The thylakoid fraction also oxidized the flavonol in the presence of H₂O₂, and the rate was about 25% of that by intact chloroplasts. The oxidation of quercetin was inhibited by KCN and NaN₃. Ascorbate, which permeates slowly across chloroplast envelope, only slightly suppressed the initial rate of quercetin oxidation by intact chloroplasts, while the oxidation by ruptured chloroplasts was suppressed by ascorbate by about 60%. Quercetin glycosides, quercitrin and rutin, were also oxidized by chloroplasts in the presence of H₂O₂. These results suggest that flavonols are oxidized by peroxidase-like activity in chloroplasts and that externally added flavonols can permeate into the stroma through the envelope of intact chloroplasts.     

In vivo-Microspectrophotometric Characterization of Flavonol Glycosides in Vicia faba Guard and Epidermal Cells

Cytological observations by fluorescence and U.V.-absorptionmicroscopy together with in vivo spectrophotometric analysesof stomata, guard cell protoplasts and epidermal cells of Viciafaba have shown that kaempferol 3,7-O-glycosides are localizedin the vacuoles. The alkaline-induced emission spectra recordedwith guard and epidermal cells after NH₄OH-treatment were identical,exhibiting an emission maximum at 525 nm; the spectra correlatedwith that of reference flavonols after exposure to NH₄OH Theexcitation spectra of both cell types are typical of these flavonolsshowing two maxima at 290 nm and 390 nm. In agreement, two absorptionmaxima were recorded for guard cells at 270 nm and 330 nm, withoutalkali, which shifted bathochromically to 275 nm and 380 nm,respectively, after NH₄OH treatment. The fluorescence intensitymeasured at 525 nm demonstrates a photostability in epidermalcells whereas it increases by a factor of about five with theexcitation time up to 30 min in guard cells. For the latter,several possible processes are discussed. Key words: Alkaline-induced fluorescence, emission, excitation, U.V.-absorption spectra, kaempferol glycosides, cell specificity     

Photosynthetic Properties of Guard Cell Protoplasts from Vicia faba L.

Guard cell protoplasts were isolated enzymatically from theepidermis of Vicia faba L. and their photosynthetic activitieswere investigated. Time courses of light-induced changes inthe chlorophyll a fluorescence intensity of these protoplastsshowed essentially the same induction kinetics as found formesophyll protoplasts of Vicia. The transient change in thefluorescence intensity was affected by DCMU, an inhibitor ofphotosystem II; by phenylmercuric acetate, an inhibitor of ferredoxinand ferredoxin NADP reductase; and by methyl viologen, an acceptorof photosystem I. Low temperature (77 K) emission spectra ofthe protoplasts had peaks at 684 and 735 nm and a shoulder near695 nm. A high O₂ uptake (175 µmol mg^–1 Chl hr^–1)was observed in guard cell protoplasts kept in darkness, whichwas inhibited by 2 mM KCN or NaN₃ by about 60%. On illumination,this O₂ uptake was partially or completely suppressed, but itssuppression was removed by DCMU, which indicates that oxygenwas evolved (150 µmol mg^–1 Chl hr^–1) photosynthetically.We concluded that both photosystems I and II function in guardcell chloroplasts and that these protoplasts have high respiratoryactivity. (Received January 30, 1982; Accepted May 15, 1982)     

Hydrogen peroxide generated by copper amine oxidase is involved in abscisic acid-induced stomatal closure in Vicia faba

H₂O₂ is an essential signal in absicic acid (ABA)-induced stomatalclosure. It can be synthesized by several enzymes in plants.In this study, the roles of copper amine oxidase (CuAO) in H₂O₂production and stomatal closure were investigated. ExogenousABA stimulated apoplast CuAO activity, increased H₂O₂ productionand [Ca²⁺]_cyt levels in Vicia faba guard cells, and inducedstomatal closure. These processes were impaired by CuAO inhibitor(s).In the metabolized products of CuAO, only H₂O₂ could inducestomatal closure. By the analysis of enzyme kinetics and polyaminecontents in leaves, putrescine was regarded as a substrate ofCuAO. Putrescine showed similar effects with ABA on the regulationof H₂O₂ production, [Ca²⁺]_cyt levels, as well as stomatal closure.The results suggest that CuAO in V. faba guard cells is an essentialenzymatic source for H₂O₂ production in ABA-induced stomatalclosure via the degradation of putrescine. Calcium messengeris an important intermediate in this process. Key words: Abscisic acid, calcium, copper amine oxidase, hydrogen peroxide, putrescine, stomatal closure, Vicia faba Received 13 October 2007; Revised 16 December 2007 Accepted 20 December 2007     

High Respiratory Activity of Guard Cell Protoplasts from Vicia faba L.

The rate of O₂ uptake was about 29 times higher in guard cellprotoplasts (GCPs) than in mesophyll protoplasts (MGPs) on aChi basis. The O₂ uptake was inhibited by respiratory inhibitors,but stimulated by respiratory uncouplers. On a Chi basis, theactivities of Cyt c oxidase and NADH-Cyt c reductase, mitochondrialenzymes, were about 27 and 35 times higher in GCPs than in MCPs.On a Chi basis, the ATP content was about 9 times higher inGCPs. The amount of ATP in GCPs was decreased by respiratoryinhibitors, an energy transfer inhibitor, and uncouplers ofoxidative phosphorylation. On a volume basis, GCPs had 8- to10-fold higher respiratory activities than MCPs, but had a lowChi content and lacked the activity of NADP-glyceraldehyde-3-phosphatedehydrogenase (NADP-GAPD), the Calvin cycle enzyme. From theseresults, we concluded that oxidative phosphorylation plays amain role in ATP production in guard cells and that guard cellshave a heterotrophic feature. Salicylhydroxamic acid (SHAM)in combination with KCN or NaN₃ strongly inhibited O₂ uptake,indicating the presence of cyanide-resistant respiration inguard cells. Phenylmercuric acetate (PMA), a potent inhibitorof stomatal opening, reduced the ATP content of GCPs by about90%, whereas it had a relatively small effect on the ATP levelof MCPs. The specific effect of PMA on GCPs is discussed. (Received March 24, 1983; Accepted June 8, 1983)     

Sulfhydryls associated with H2O2-induced channel activation are on luminal side of ryanodine receptors

The mechanism underlying H₂O₂-inducedactivation of frog skeletal muscle ryanodine receptors was studiedusing skinned fibers and by measuring single Ca²⁺-releasechannel current. Exposure of skinned fibers to 3-10 mM H₂O₂ elicited spontaneous contractures.H₂O₂ at 1 mM potentiated caffeine contracture.When the Ca²⁺-release channels were incorporated into lipidbilayers, open probability (P_o) and open timeconstants were increased on intraluminal addition ofH₂O₂ in the presence of cis catalase,but unitary conductance and reversal potential were not affected.Exposure to cis H₂O₂ at 1.5 mM failedto activate the channel in the presence of trans catalase.Application of 1.5 mM H₂O₂ to the transside of a channel that had been oxidized by cisp-chloromercuriphenylsulfonic acid (pCMPS; 50 µM) still led to anincrease in P_o, comparable to that elicited bytrans 1.5 mM H₂O₂ without pCMPS.Addition of cis pCMPS to channels that had been treated with orwithout trans H₂O₂ rapidly resulted inhigh P_o followed by closure of the channel. Theseresults suggest that oxidation of luminal sulfhydryls in theCa²⁺-release channel may contribute toH₂O₂-induced channel activation and musclecontracture.

     

Effect of Oxygen on Photosynthetic 14CO2 Fixation in Chroomonas sp. (Cryptophyta) II. Effects of Inhibitors, Uncouplers and an Artificial Electron Mediator on the Inhibition of 14CO2 Fixation by Anaerobiosis

In "air-grown" Chroomonas sp. cells, low concentrations of DCMU(less than 0.1 µM) could prevent the inhibition of ¹⁴CO₂fixation by anaerobiosis under light-saturating conditions (morethan 40 W.m^–2), with phenazine methosulfate showing asimilar effect. Antimycin A, carbonyl cyanide m-chlorophenylhydrazone(CCCP), and N,N'-dicyclohexylcarbodiimide strongly inhibitedanaerobic photosynthesis at concentrations which did not significantlyinhibit the rate under 2% O₂ at high light intensity (200 W.m^–2),although 0.2 µM CCCP stimulated the rate under 2% O₂ tosome extent. On the other hand, KCN inhibited the rate muchmore strongly under 2% O₂ than N₂, although it inhibited therate very strongly at concentrations above 5 µM both underN₂ and 2% O₂. These results suggest that the inhibition of photosynthetic¹⁴CO₂ fixation by anaerobiosis in this alga result from ATPdeficiency caused by over-reduction of electron carriers ofthe cyclic electron flow and that oxygen can prevent the over-reduction.Cyclic electron flow seems to be necessary to provide additionalATP for CO₂ reduction under anaerobic conditions, although itseems to be less necessary under aerobic conditions. (Received July 21, 1983; Accepted January 23, 1984)     

Hydrogen Peroxide-Dependent Oxidation of 3,4-Dihydroxyphenylalanine in Vacuoles of Mesophyll Cells of Vicia faba L. Participation of Peroxidase in the Oxidation

Peroxidase activity and 3,4-dihydroxyphenylalanine (DOPA) werefound in vacuoles isolated from mesophyll protoplasts of Viciafaba L. A peroxidase isozyme localized in vacuoles migratedto the cathode during electrophoresis at pH 8.7, indicatingthat the vacuole peroxidase was a basic isozyme. When isolatedvacuoles were treated with 2 mM H₂O₂, dopachrome, a productof oxidation of DOPA, was formed in a reaction that was inhibitedby KCN and NaN₃. These results suggest that DOPA can serve asa donor of electrons to the peroxidase in vacuoles. (Received December 25, 1989; Accepted March 22, 1990)     

NADH2- and succinate-dependent O2 uptake in chromatophores from Chromatium vinosum

The O₂ uptake linked to NADH₂ and succinate oxidation was observedin chromatophores from photosynthetically grown Chromatium vinosum. The maximal rate was 60–120 nmoles of O₂ uptake per minper {diaeresis}mole of bacterio-chlorophyll. The rate of O₂uptake linked to NADH₂ oxidation was higher in the neutral-to-acidicpH range than in the alkaline range, whereas that linked tosuccinate oxidation was higher in the alkaline range. The O₂ uptake linked to NADH₂ oxidation was inhibited by rotenone,amytal, antimycin A, KCN and NaN₃, while that linked to succinateoxidation was inhibited by antimycin A, KCN and NaN₃. Malate,citrate, pyruvate, acetate, -ketoglutarate, NADPH₂ and thiosulfatedid not serve as substrates for the O₂ uptake of isolated chromatophores. The rates of the O₂ uptake linked to both NADH₂ and succinateoxidation were not stimulated by adding uncouplers or underphosphorylating conditions. Little or no ATP was synthesizedin the dark, coupled to either NADH₂ or succinate oxidation,in spite of a high activity of photophosphorylation in Chromatiumchromatophores. (Received February 26, 1980; )     

H(2)O(2) and ethanol act synergistically to gate ryanodine receptor/calcium-release channel

We examined theeffect of low concentrations of H₂O₂ on theCa²⁺-release channel/ryanodine receptor (RyR) to determineif H₂O₂ plays a physiological role in skeletalmuscle function. Sarcoplasmic reticulum vesicles from frog skeletalmuscle and type 1 RyRs (RyR1) purified from rabbit skeletal muscle wereincorporated into lipid bilayers. Channel activity of the frog RyR wasnot affected by application of 4.4 mM (0.02%) ethanol. Openprobability (P_o) of such ethanol-treated RyRchannels was markedly increased on subsequent addition of 10 µMH₂O₂. Increase of H₂O₂to 100 µM caused a further increase in channel activity. Applicationof 4.4 mM ethanol to 10 µM H₂O₂-treated RyRsactivated channel activity. Exposure to 10 or 100 µMH₂O₂ alone, however, failed to increaseP_o. Synergistic action of ethanol andH₂O₂ was also observed on the purified RyR1 channel, which was free from FK506 binding protein (FKBP12).H₂O₂ at 100-500 µM had no effect onpurified channel activity. Application of FKBP12 to the purified RyR1drastically decreased channel activity but did not alter the effects ofethanol and H₂O₂. These results suggest thatH₂O₂ may play a pathophysiological, butprobably not a physiological, role by directly acting on skeletalmuscle RyRs in the presence of ethanol.

     

Resistance of Photosynthesis to Hydrogen Peroxide in Algae

The effects of H₂O₂ on the photosynthetic fixation of CO₂ andon thiol-modulated enzymes involved in the photosynthetic reductionof carbon in algae were studied in a comparison with those inchloroplasts isolated from spinach leaves. In both systems,H₂O₂-scavenging enzymes were inhibited by addition of 0.1 mMNaN₃ 1 h prior to the addition of H₂O₂. A concentration (10^-4M) of H₂O₂ caused strong inhibition of the CO₂ fixation by intactspinach chloroplasts, as observed by Kaiser [(1976) Biochim.Biophys. Acta 440: 476], but not that by Euglena and Chlamydomonascells. The same results were also obtained with cells of thecyanobacteria Synechococcus PCC 7942 and Synechocystis PCC 6803in the presence of 1 mM hydroxylamine. These results indicatethat algal photosynthesis is rather resistant to H₂O₂. The insusceptibilityto H₂O₂ of thiolmodulated enzymes, namely, fructose-1,6-bisphosphatase,NADP-glyceraldehyde-3-phosphate dehydrogenase, and ribulose-5-phosphatekinase, was also observed in the chloroplasts of Euglena andChlamydomonas and in cyanobacterial cells. It seems likely thatthe resistance of photosynthesis to H₂O₂ is due in part to theinsusceptibility of the algal thiol-modulated enzymes to H₂O₂. (Received April 22, 1995; Accepted June 29, 1995)     

Hydrogen Peroxide Production is a General Property of the Lignifying Xylem from Vascular Plants

Production of hydrogen peroxide (H₂O₂) by the lignifying xylemof several vascular plants has been studied using a new histochemicalmethod based on the H₂O₂-dependent oxidation of 3,5,3'5'-tetramethylbenzidine(TMB) catalysed by cell wall peroxidases. This method allowsH₂O₂to be determined in the range of 5–100 µM, whereother methods, such as the KI/starch reagent, fail. With thismethod, it has been possible to determine H₂O₂production inthe lignifying xylem of a wide range of vascular plants (gymnospermsand angiosperms). The capability of xylem tissues of sustainingH₂O₂production lends support to the hypothesis that cinnamylalcohol polymerization in xylem vessels is caused by an H₂O₂-dependentoxidative coupling process.Copyright 1998 Annals of Botany Company H₂O₂generation, lignification, peroxidase, tetramethylbenzidine, xylem.     

Participation of Peroxidase in the Metabolism of 3,4-Dihydroxyphenylalanine and Hydrogen Peroxide in Vacuoles of Vicia faba L. Mesophyll Cells

When leaves of Vicia faba were treated with H₂O₂ or visiblelight in the presence of methyl viologen (MV), the orange-redcompound dopachrome was formed transiently and melanin was accumulated.With the darkening of leaves, the level of 3,4-dihydroxyphenylalanine(DOPA) decreased and then recovered to the original level uponaddition of 1 mM H₂O₂. However, if leaves were incubated inthe presence of 10 mM H₂O₂, the level of DOPA decreased againafter the increase. The time course of the changes in levelsof DOPA observed during the accumulation of melanin as a resultof illumination in the presence of MV was very similar to thatobserved after the addition of 10 mM H₂O₂. Illumination of leavesin the absence of MV did not result in any accumulation of melanin,but the level of DOPA changed slightly. When isolated mesophyllcells were incubated in the dark, the level of DOPA decreased.Illumination of the cells stimulated this decrease. Tropolone,an inhibitor of phenol oxidase, did not inhibit and actuallystimulated the H₂O₂- and light-induced oxidation of DOPA andaccumulation of melanin in leaves. Tropolone also stimulatedthe decrease in the levels of DOPA both in the dark and in thelight in isolated mesophyll cells. These data suggest that aperoxidase-H₂O₂ system, and not phenol oxidase, participatesin the oxidation of DOPA. When DOPA was oxidized by a basicperoxidase isolated from V.faba leaves, an intermediate, whichwas perhaps dopaquinone and which was reducible by ascorbate,was formed. Based on the data, a discussion is presented ofthe physiological significance of the oxidation of DOPA by peroxidasein vacuoles. (Received March 4, 1991; Accepted May 21, 1991)     

The Effect of Osmotic Stress on the Solutes in Guard Cells of Vicia faba L.

We investigated the changes in the levels of solutes in guardcells under osmotic stress. Epidermal strips peeled from Viciafaba L. leaflets were sonicated and incubated in 0.4 M mannitolsolution (osmotic stress) in either light or dark. Stomata wereclosed by osmotic stress. Under osmotic stress, malate accumulatedlight-dependently and sucrose accumulated light-independentlyin the guard cells. The level of K⁺ in guard cells increasedslightly under osmotic stress in the light, although withoutstatistical significance. The levels of all these solutes werereduced by 10 µM ABA treatment. These results suggestthat osmotic stress affects carbon metabolism in guard cells;this metabolic change is different from that caused by ABA alone.Respiratory activity of guard cells decreased under osmoticstress. Therefore, the accumulation of malate and sucrose maybe caused by reduced respiration under osmotic stress. Accumulationof solutes in guard cells by osmotic stress may result in increasedosmotic pressure of guard cells and may play a role in protectionof guard cells from osmotic stress. (Received December 17, 1998; Accepted May 28, 1999)     

Hydrogen peroxide is involved in the acclimation of the Mediterranean shrub, Cistus albidus L., to summer drought

This study evaluated the possible role of hydrogen peroxide(H₂O₂) in the acclimation of a Mediterranean shrub, Cistus albidusL., to summer drought growing under Mediterranean field conditions.For this purpose, changes in H₂O₂ concentrations and localizationthroughout a year were analysed. H₂O₂ changes in response toenvironmental conditions in parallel with changes in abscisicacid (ABA) and oxidative stress markers, together with ligninaccumulation, xylem and sclerenchyma differentiation, and leafarea were also investigated. During the summer drought, leafH₂O₂ concentrations increased 11-fold, reaching values of 10µmol g^–1 dry weight (DW). This increase occurredmainly in mesophyll cell walls, xylem vessels, and sclerenchymacells in the differentiation stage. An increase in ABA levelspreceded that of H₂O₂, but both peaked at the same time in conditionsof prolonged stress. C. albidus plants tolerated high concentrationsof H₂O₂ because of its localization in the apoplast of mesophyllcells, xylem vessels, and in differentiating sclerenchyma cells.The increase in ABA, and consequently of H₂O₂, in plants subjectedto drought stress might induce a 3.5-fold increase in ascorbicacid (AA), which maintained and even decreased its oxidativestatus, thus protecting plants from oxidative damage. Afterrecovery from drought following late-summer and autumn rainfall,a decrease in ABA, H₂O₂, and AA to their basal levels (60 pmolg^–1 DW, 1 µmol g^–1 DW, and 20 µmol g^–1DW) was observed. Key words: Abscisic acid, ascorbate, ascorbate oxidative status, Cistus albidus, hydrogen peroxide, leaf plasticity, lignin, Mediterranean shrubs, oxidative markers, summer drought Received 29 July 2008; Revised 15 September 2008 Accepted 8 October 2008     

Characterisation of Acidic and Basic Apoplastic Peroxidases from Needles of Norway Spruce (Picea abies, L., Karsten) with Respect to Lignifying Substrates

Acidic and basic peroxidases, termed as POD-A and POD-B, wereisolated from the apoplastic space of spruce (Picea abies, L.)needles and purified by acetone precipitation and anion exchangechromatography to apparent homogeneity. The molecular massesof POD-A and POD-B were 39.6 and 29.0 kDa, respectively. ThepH optimum of both isozymes ranged from 4.5 to 6. The apparentK_m values of POD-A and POD-B were 460 and 210 µM for coniferylalcohol. Both isozymes acted also as NADH oxidases with apparentK_m-values of 103 µM (POD-A) and 70 µM (POD-B). NAD⁺but not NADH was found in the apoplastic space of lignifyingneedles. Based on the lignification rate, the contents and kineticproperties of PODs, NADH oxidation by POD is not the major sourceof H₂O₂ required for lignin polymerisation. (Received December 21, 1996; Accepted March 3, 1997)