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)     

Oxidation of Flavonols by Hydrogen Peroxide in Epidermal and Guard Cells of Vicia faba L.

Epidermal strips of Vicia faba were found to contain kaempferoland quercetin glycosides. These flavonols were oxidized by H₂O₂and oxidation was inhibited by KCN (3.5 nM). Quercetin glycosideswere more sensitive to H₂O₂ than kaempferol glycosides. Oxidationcould be detected in epidermal strips even at 30 µM H₂O₂.Flavonol oxidation by H₂O₂ was observed in both guard and epidermalcells. In guard cells, oxidation appeared as the bleaching ofabsorption bands of flavonols. Epidermal cells could be roughlydivided into two types on the basis of their absorption characteristicsin the UV-light region. In one type, only flavonol oxidationwas observed; in the other, both flavonol and 3,4-dihydroxyphenylalanine(DOPA) oxidation were observed. Oxidation of flavonols and DOPAby H₂O₂ was also observed in cell-free extracts of the epidermalstrips, even at 10µ H₂O₂. Oxidation was inhibited by 1mM KCN, suggesting the participation of peroxidase in the reactions.The data obtained in this study indicate the cellular specificdistribution of phenolic compounds in the epidermis and thepossibility of their oxidation by H₂O₂ generated in epidermaland guard cells. (Received August 24, 1987; Accepted January 21, 1988)     

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)     

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)     

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     

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)     

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 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)     

Growth and nitrogen assimilation in nodules in response to nitrate levels in Vicia faba under salt stress

This study analyses the effects of salt on the effective symbiosisof faba bean (Vicia faba L. var. minor cv. Alborea) and salt-tolerantRhizobium leguminosarum biovar. viciae strain GRA19 grown withtwo KNO₃ levels (2 and 8 mM). The addition of 8 mM KNO₃ to thegrowth medium increases plant tolerance to salinity even witha concentration of 100 mM NaCl. This KNO₃ level in control plantsreduced the N₂ fixation. For 2 and 8 mM KNO₃ the plants treatedwith NaCl reduced N₂ fixation to identical values. The activityof the enzymes mediating ammonium assimilation in nodules (GS,NADH-GOGAT and NADH-GDH) was decreased by high KNO₃ levels.The results show that NADH-GOGAT activity was more markedlyinhibited than was GS activity by salinity, therefore NADH-GOGATlimits the ammonium assimilation by nodules in V. faba undersalt stress. The total proline content in the nodule was notrelated to salt tolerance and thus does not serve as a salttoleranceindex for V. faba. Key words: Glutamate synthase, glutamine synthetase, N₂ fixation, nitrate, salinity     

Accumulation of Hydrogen Peroxide in Chloroplasts of SO2-Fumigated Spinach Leaves

Illuminated chloroplasts isolated from SO₂-fumigated spinachleaves accumulated more H₂O₂ than those from non-fumigated ones.This H₂O₂ formation was dependent on light and was inhibitedby DCMU. It also was depressed by cytochrome c and superoxidedismutase (EC 1.15.1.1 [EC] ). The addition of sulfite to rupturedchloroplasts isolated from non-fumigated leaves caused an H₂O₂accumulation that accompanied O₂ uptake. Spinach leaves losttheir catalase (EC 1.11.1.6 [EC] ), ascorbate peroxidase and glutathionereductase (EC 1.6.4.2 [EC] ) activities at the beginning of SO₂ fumigation,when H₂O₂ was accumulated. These results suggest that the accumulationof H₂O₂ in SO₂-fumigated spinach leaves is caused by the increasein O₂^–production, the precursor for H₂O₂, with a sulfite-mediatedchain reaction at the reducing site of photosystem I, and byinactivation of the H₂O₂ scavenging system. (Received October 7, 1981; Accepted June 16, 1982)     

Inhibition of Photosynthesis by Sulfite in Mesophyll Protoplasts Isolated from Vicia faba L. in Relation to Intracellular Sulfite Accumulation

Inhibition of photosynthesis by Na₂SO₃ in mesophyll protoplastsisolated from Vicia faba leaves and uptake of sulfite by theprotoplasts were examined at various pH values of the incubationmedium containing Na₂SO₃. As the pH of the incubation mediumlowered, the rate of photosynthesis in the protoplasts decreasedand the amount of sulfite taken up by the protoplasts increased.Most of sulfite accumulated in the protoplasts was not metabolizedduring the dark incubation, as measured with an ion chromatograph.Photosynthetic O₂ evolution by the chloroplasts isolated fromVicia mesophyll protoplasts was inhibited by exogenously-appliedNa₂SO₃ over pH region examined (7.4–9.0). The sulfiteconcentration required for a half inhibition of photosynthesisby the isolated chloroplasts was similar to the intracellularsulfite level required for that by the protoplasts. These resultsindicate that the intracellular sulfite accumulated in the protoplastsin an unmetabolized state is responsible for the inhibitionof protoplast photosynthesis. (Received January 24, 1985; Accepted May 29, 1985)     

Apoplastic pH of intact leaves of Vicia faba as influenced by light

The fluorochrome FITC-dextran was used to measure the effectof light on the apoplastic pH of intact Vicia faba leaves withthe ratio imaging technique. In darkadapted leaves the apoplasticpH varied depending on the leaf between 5.2 and 5.9. Red light(660 nm, 4–12 W m^–2) leads to multiphasic responses:in the first seconds an alkalinization ({small tilde}0.3 pHunits), and thereafter an acidification of the leaf apoplast({small tilde}0.4 pH units) were observed. Both effects couldbe inhibited by DCMU. While variation of CO₂ concentration revealedno effect on light-induced apoplastic pH changes, a decreasein O₂ concentration decreased the effect. On the basis of ourdata it is suggested that the influence of photosynthesis onplasmalemma H⁺ ATPase is responsible for the observed effects,rather than altered CO₂ uptake. Key words: Leaf apoplast, apoplastic pH, light, ratio imaging, pH-sensitive fluorescent dye, Vicia fab     

Detection of Monodehydroascorbic Acid Radical in Sulfite-Treated Leaves and Mechanism of its Formation

The aim of the present study is to detect the monodehydroascorbicacid (MDA) radical in broad bean (Vicia faba L.) leaves whichwere treated by vacuum-infiltration in Na₂SO₃ solution and subsequentcentrifugation (sulfite-treated leaves). When sulfite-treatedleaves were illuminated with white light, the electron spinresonance (ESR) signal of MDA radical was observed. The levelof the MDA radical depended on the concentration of sulfitethat was used for vacuum-infiltration and on the light intensityof illumination. The formation of the MDA radical in sulfite-treatedleaves was inhibited by DCMU or by replacement of air with N₂.Glycolaldehyde also inhibited the formation of MDA radical insulfite-treated leaves. Catalase activity was decreased by thesulfite treatment without affecting significantly the activitiesof ascorbate peroxidase (AA-POX) and of peroxidase which preferentiallyoxidizes phenolics (PhOH-POX). From these results, we concludethat the formation of the MDA radical in sulfite-treated leavesis catalyzed by peroxidases using the H₂O₂ which is generatedby photorespiration and the Mehler reaction. ¹On leave from the Center for Multidisciplinary Studies, Universityof Belgrade, Yugoslavia.     

Differential modulation of voltage-dependent K+ currents in colonic smooth muscle by oxidants

The effect of oxidants on voltage-dependent K⁺ currents was examined in mouse colonic smooth muscle cells. Exposure to either chloramine-T (Ch-T), an agent known to oxidize both cysteine and methionine residues, or the colon-specific oxidant monochloramine (NH₂Cl) completely suppressed the transient outward K⁺ current (I_to) while simultaneously enhancing the sustained delayed rectifier K⁺ current (I_dr). In contrast, the cysteine-specific oxidants hydrogen peroxide (H₂O₂) and 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) exhibited partial and slow suppression of I_to by inducing a shift in channel availability of -18 mV without affecting I_dr. After enhancement by NH₂Cl or Ch-T, I_dr was sensitive to 10 mM tetraethylammonium but not to other K⁺ channel blockers, suggesting that it represented activation of the resting I_dr and not a separate K⁺ conductance. Extracellular dithiothreitol (DTT) partially reversed the effect of H₂O₂ and DTNB on I_to but not the actions of NH₂Cl and Ch-T on either I_dr or I_to. Dialysis of myocytes with GSH (5 mM) or DTT (5 mM) prevented suppression of I_to by H₂O₂ and DTNB but did not alter the effects of NH₂Cl or Ch-T on either I_dr or I_to. Ch-T and NH₂Cl completely blocked I_to generated by murine K_v4.1, 4.2, and 4.3 in Xenopus oocytes, an effect not reversible by intracellular DTT. In contrast, intracellular DTT reversed the effect of H₂O₂ and DTNB on the cloned channels. These results suggest that I_to is suppressed via modification of both methionine and cysteine residues, whereas enhancement of I_dr likely results from methionine oxidation alone. colon; colitis; redox; ion channel     

Activity of ascorbate-dependent H2O2-scavenging enzymes and leaf chlorosis are enhanced in magnesium- and potassium-deficient leaves, but not in phosphorus-deficient leaves

The effect of phosphorus (P), potassium (K), and magnesium (Mg)deficiency on the development of leaf symptoms (chlorosis andnecrosis) and activities of ascorbate-dependent H₂O₂ scavengingenzymes (ascorbate peroxidase, monodehydroascorbate reductase,dehydroascorbate reductase, and glutathione reductase) was studiedin bean (Phaseolus vulgans) plants over a 12 d period of growthin nutrient solution. With increasing plant age Mg- and K-deficientleaves developed severe interveinal chlorosis and, accordingly,chlorophyll concentrations were reduced. However, in P-deficientleaves neither chlorosis nor necrosis appeared; the leaves remaineddark green and even at an advanced stage of P deficiency, chlorophyllconcentrations were still higher than those of control plants.In K- and, particularly, Mg-deficient leaves with an increasein severity of leaf chlorosis, activity of ascorbate-dependentH₂O₂- scavenging enzymes was progressively increased. In contrast,in P-deficient leaves, as in leaves of the control plants, activityof H₂O₂-scavenging enzymes remained at a low level over the12 d period. Accordingly, compared with P-deficient and controlplants, Mg- and K-deficient leaves with elevated anti-oxidativepotential showed much higher resistance to chlorophyll destructionby the herbicide paraquat. Elevated levels of H₂O₂-scavengingenging enzymes in Mg- and K-deficient leaves indicate a higherproduction of H₂O₂ and related toxic O₂ species. It Is suggestedthat in Mg- and K-deficient leaves, utilization of photoreductantsin CO₂ fixation is restricted because of impaired export andthus accumulation of photosynthates. This disturbance mightlead to enhanced photoreduction of molecular O₂ to toxic O₂species causing chlorophyll destruction (chlorosis), a processwhich is not important in P-deficient leaves where export ofsucrose is not affected. Key words: Bean, hydrogen peroxide detoxification, leaf chlorosis, magnesium nutrition, oxygen activation, phosphorus nutrition, potassium nutrition     

Attachment of CuZn-Superoxide Dismutase to Thylakoid Membranes at the Site of Superoxide Generation (PSI) in Spinach Chloroplasts: Detection by Immuno-Gold Labeling After Rapid Freezing and Substitution Method

In chloroplasts O^–₂ is photoproduced via the univalentreduction of O₂ in PSI even under conditions that are favorablefor photosynthesis. The photogenerated O^–₂ is disproportionatedto H₂O₂ and O₂ in a reaction that is catalyzed by superoxidedismutase (SOD). The H₂O₂-scavenging ascorbate peroxidase isbound to the thylakoid membranes at or near the PSI reactioncenter [Miyake and Asada (1992) Plant Cell Physiol. 33: 541],and the primary product of oxidation in the peroxidase-catalyzedreaction, the monodehydroascorbate radical, is photoreducedto ascorbate in PSI in a reaction mediated by ferredoxin [Miyakeand Asada (1994) Plant Cell Physiol. 35: 539]. Therefore, SODshould be localized at or near the PSI complex. We report herethe microcompartmentalization of the chloroplastic CuZn-SODon the stromal-faces of thylakoid membranes where the PSI-complexis located. Spinach leaves were fixed and substituted by a rapidfreezing and substitution method that allows visualization ofintact chloroplasts. The embedded sections were immuno labeledwith the antibody against CuZn-SOD by the immunogold method.About 70% of the immunogold particles were found within 5 nmfrom the surface of the stromal-faces of thylakoid membranes.Of these particles, about 40% were found at the ends and marginsof the grana thylakoids and 60% were found on the stromal sideof the stromal thylakoids. From these results, the local concentrationof CuZn-SOD on the stroma-facing surfaces of the thylakoid membraneswas estimated to be about 1 mM. The effect of the microcompartmentalizationof CuZn-SOD on the scavenging of superoxide radicals is discussed. (Received November 25, 1994; Accepted February 23, 1995)     

Bacteroid oxalate oxidase and soluble oxalate in nodules of faba beans (Vicia faba L.) submitted to water restricted conditions: possible involvement in nitrogen fixation

The inhibitory effect exerted by water stress on acetylene reductionactivity (ARA) by nodulated roots of faba beans (Vicia fabaL.) was correlated with a 40% decline in the organic acid poolof nodule cytosol. Oxalate concentration was lowered (–55%)whereas a stimulation of the bacteroid oxalate oxidase concomitantlyoccurred. This enzyme was characterized by an optimal activityat pH 8 but, as in higher plants, exhibited a K_m for oxalateof 1.4 mM and an inhibition by substrate excess. Oxalate providedto bacteroid incubations supported C₂H₂ reduction up to 2.5mM whereas higher concentrations were strongly inhibitory. Incontrast, purified symbiosomes incubated with oxyleghaemoglobinreduced C₂H₂ in the presence of oxalate concentrations up to10 mM. The peribacteroid membrane (PBM), in controlling theoxalate flux to the bacteroids avoided the substrate inhibitionwhich would limit its efficiency. Thus, oxalate present in highconcentration in faba bean nodules could play a role as complementarysubstrate for bacteroids slowing down the nitrogen fixationdecline induced by water restricted conditions. Key words: Faba bean, water stress, oxalate, acetylene reduction, bacteroid     

Simultaneous CO2- and 16O2/18O2-gas exchange and fluorescence measurements indicate differences in light energy dissipation between the wild type and the phytochrome-deficient aurea mutant of tomato during water stress

The CO₂-, H₂O- and ¹⁶O₂/¹⁸O₂ isotopic-gas exchange and the fluorescencequenching by attached leaves of the wild-type and of the phytochrome-deficienttomato aurea mutant was compared in relation to water stressand the photon fluence rate. The chlorophyll content of aurealeaves was reduced and the ultra-structure of the chloroplastswas altered. Nevertheless, the maximum rate of net CO₂ uptakein air by the yellow-green leaves of the aurea mutant was similarto that by the dark-green wild-type leaves. However, less O₂was produced by the leaves of the aurea mutant than by leavesof the wild-type. This result indicates a reduced rate of photosyntheticelectron flux in aurea mutant leaves. No difference in bothphotochemical and non-photochemical fluorescence quenching wasfound between wild-type and aurea mutant leaves. Water stresswas correlated with a reversible decrease in the rates of bothnet CO₂ uptake and transpiration by wild-type and aurea mutantleaves. The rate of gross ¹⁶O₂ evolution by both wild-type andaurea mutant leaves was fairly unaffected by water stress. Thisresult shows that in both wild-type and aurea leaves, the photochemicalprocesses are highly resistant to water stress. The rate ofgross ¹⁸O₂ uptake by wild-type leaves increased during waterstress when the photon fluence rate was high. Under the sameconditions, the rate of gross ¹⁸O₂ uptake by ^aurea mutant leavesremained unchanged. The physiological significane of this differencewith respect to the (presumed) importance of oxygen reductionin photoprotection is discussed. Key words: Water stress, gas exchange, fluorescence quenching, Lycopersicon esculentum, mutant (tomato, aurea), energy dissipation     

Ascorbic Acid-Dependent Regulation of Redox Levels of Chlorogenic Acid and its Isomers in the Apoplast of Leaves of Nicotiana tabacum L.

There is a question whether ascorbic acid (AA) can control redoxlevels of phenolics in the apoplast. The present study was designedto answer this question. AA, dehydroascorbic acid (DHA), chlorogenicacid (CGA) and its two structural isomers were present in theapoplast of leaves of tobacco (Nicotiana tabacum L. cv. BelW3).The levels of AA plus DHA (AA + DHA) and the ratios of AA to(AA + DHA) decreased while the levels of CGA plus its isomersincreased during leaf aging. o-Quinones of CGA plus its isomerswere found in the apoplast only in aged leaves of which apoplasticlevel of AA was nearly zero. In addition, activity of apoplasticperoxidase that could oxidize CGA and its isomers increasedduring leaf aging. From the observations, it is concluded thatAA can regulate the accumulation of the o-quinones of CGA andits isomers in the apoplast. Based on the conclusion, it isproposed that soluble peroxidase in the apoplast has two functions,namely, (i) scavenging of H₂O₂ and/or regulation of the levelof apoplastic H₂O₂ in the presence of AA, and (ii) accumulationof oxidation products of the phenolics in the absence of AA. (Received January 30, 1998; Accepted April 7, 1998)     

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)