Carotenoid photobleaching induced by photosystem II action in the membrane fragments of the blue-green alga Anabaena variabilis : Action of O2

Carotenoid photobleaching induced by photosystem II action wasstudied using membrane fragments of the blue-green alga Anabaenavariabilis. Special attention was paid to the action of O₂. Carotenoid photobleaching elicited by carbonyl cyanide m-chlorophenylhydrazone(CCCP) depended on O₂. However, the addition of H₂O₂, sodiumsilicotungstate or potassium ferricyanide (Ferri), an electronacceptor for reaction center II action, removed the O₂-dependency.These results indicate that O₂ acts as the electron acceptorfor this reaction. When both CGCP and Ferri were present, a short illumination(0.25 sec) caused a rapid photobleaching followed by a slowrecovery in the subsequent dark period. The spectrum of theabsorption decrease in the light was identical with that ofthe absorption increase in the subsequent dark, indicating thata reversible process is involved in the carotenoid photobleaching.The size in the dark recovery relative to the light bleachingbecame larger under anaerobic conditions and smaller under higherpartial pressure of O₂. The reuslts were interpreted as indicatingthat O₂ does not function in the primary process including areversible bleaching step, but is involved in the slow and irreversiblebleaching process. (Received April 3, 1978; )     

Carotenoid photobleaching induced by the action, of photosynthetic reaction center II: DGMU-sensitivity

Carotenoid photobleaching in photosynthetic membrane fragmentsof the blue-green alga Anabaena variabilis was studied withspecial reference to DCMU-sensitivity. Carotenoid photobleaching supported by CCCP is strongly enhancedby Ferri, and, at the same time, becomes less sensitive to DCMU(cf. 5). The DCMU-insensitive reaction was found to show characteristicsvery similar to those of DCMU-sensitive reaction in (i) thedependence on the excitation of pigment system II chlorophylla, (ii) the stimulation by CCCP and NaNa and the suppressionby antimycin A, and (iii) the partial dependence on molecularoxygen. In our membrane fragments Ferri was found to act asan electron acceptor for the photosystem II reaction bypassingthe DCMU-sensitive site. We concluded that (i) carotenoid photobleachinginsensitive to DCMU is also driven by reaction center II, and(ii) in the presence of Ferri, Ferri accepts electrons ejectedby reaction center II bypassing the DCMU-sensitive site. (Received January 20, 1977; )     

Studies on the Hill reaction of membrane fragments of blue-green algae II. The reaction steps inactivated at high water concentration

DPIP-photoreduction by membrane fragments of Anabaena cylindricaand A. variabilis was studied to determine which step(s) ofthe Hill reaction system is inactivated on incubation of themembrane fragments in a medium with a high water concentration(cf. 1). Supplementary experiments were done with Anacystisnidulans and Plectonema boryanum. After inactivation of the Hill system at a high water concentration,DPIP-photo-reducing activity was strongly enhanced in the A.variabilis system but less so in the A. cylindrica system byadding DPC, NH₂OH, Mn⁺⁺ or H₂0₂. The activity supported by theadded electron donor was inhibited by DCMU. The steady statelevel of chlorophyll fluorescence was lowered by the inactivationtreatment. In the A. variabilis system, the fluorescence yieldincreased to the original level on the addition of an electrondonor. In the A. cylindrica system, the yield was not so stronglyenhanced as in the A. variabilis system. We inferred that, in A. variabilis, inactivation occurs in thereaction system before the site which receives electrons fromartificial donors, probably including the water oxidation system.In A. cylindrica, besides this site, a site at or near the photochemicalsystem is also blocked. Similar types of inactivation were observed in DPIP-Hill reactionsusing Anacystis nidulans and Plectonema boryanum preparations.The characteristic stability of the Hill reaction system observedin two Anabaena preparations is probably common to the blue-greenalgae. (Received December 10, 1971; )     

Oxidation-reduction reactions between electron transfer components and hydrogen peroxide in photosystem II of chloroplasts

The light-induced oxygen evolution, photoreduction of 2,6-dichlorophenolindophenol (DPIP) and carotenoid photobleaching induced by carbonylcyanide m-chlorophenylhydrazone (CCCP) were investigated withspinach chloroplast fragments in the presence of H₂O₂. Oxygenevolution in the presence of H₂O₂ was not inhibited by CCCPand was only partially inhibited by 5 µM 3-(3,4-dichlorophenyl)-1,1-dimethylurea(DCMU) which completely inhibited the Hill reaction with DPIP.The degree of inhibition by DCMU was decreased by a simultaneousaddition of CCCP. Carotenoid photobleaching in the presenceof CCCP was stimulated by H₂O₂. The CCCP-induced carotenoidphotobleaching was completely inhibited by DCMU. However, itwas only partially inhibited by DCMU in the presence of H₂O₂.These data indicate that H₂O₂ donates electrons at a site betweenthe CCCP-sensitive site and the reaction center of photosystemII and is reduced at a site between the DCMU-blocked site andthe reaction center of photosystem II. ¹Present address: Department of Biology, Kyushu Dental College,Kitakyushu 803, Japan. (Received June 20, 1974; )     

Oxidation of Quercetin by Carotenoid Radical Generated in Illuminated Spinach Chloroplasts: The Effect of Ascorbate on Quercetin Oxidation

Carotenoid photobleaching in the presence of carbonylcyanidem-chlorophenylhydrazone (CCCP) was suppressed by quercetin,but not by ascorbate. When quercetin suppressed carotenoid photobleaching,quercetin was oxidized. The oxidation of quercetin was inhibitedby ascorbate with half-inhibition at about 10 µM. Ascorbatewas oxidized by CCCP-poisoned chloroplasts upon illumination.The rate of ascorbate oxidation in the presence of both ascorbateand quercetin was lower than that in the presence of ascorbatealone. Based on the present results, the physiological significanceof quercetin as an antioxidant and the redox reaction betweenascorbate and oxidized quercetin are discussed. (Received March 9, 1984; Accepted July 12, 1984)     

Ferricyanide-induced absorbance change of carotenoid in chromatophores of Rhodopseudomonas spheroides correlated to the oxidation of bacteriochlorophyll 885

Addition of high concentrations (e.g., 1–100 mM) of ferricyanideto a chromatophorc suspension of Rhodopseudomonas spheroidescaused a change in the absorption spectrum of carotenoid (spheroidene),which was completely reversed by adding reducing reagents suchas ferrocyanide and ascorbate. The spectral change is representedby a shift in the absorption spectrum of carotenoid by 2 to2.5 nm towards the longer wavelength side. The presence of piericidinA, o-phenanthroline or Cl-CCP in the reaction mixture did notaffect the ferricyanide-induced absorbance change. Triton X-100markedly suppressed the magnitude of the change. The additionof ferricyanide also caused simultaneous absorbance changeswith maxima at 590 and 885 nm. These are ascribed to oxidationof the (bulk) bacteriochlorophyll, BChl 885. There was no absorptionchange at other peaks of bacteriochlorophyll in the infraredregion (i.e., 800 and 855 nm). Therefore, the ferricyanide-inducedabsorbance change of carotenoid did not represent an oxidation-reductionreaction of carotenoid but was intimately correlated with oxidationof BChl 885 in the chromatophores, as judged from similaritiesobserved with respect to the time course patterns, midpointpotential (545–555 mv) in the ferriferrocyanide reactionsystem, as well as behavior towards various reagents and inhibitorsadded. A similar change of carotenoid (i.e., 2–2.5 nmshift of absorption spectrum) was caused by addition of MgCl₂to the chromatophores, but this did not induce any change inthe absorption spectrum of bacteriochlorophyll. The nature ofthe spectral change of carotenoid in chromatophores is discussed. (Received April 16, 1970; )     

Studies on the Hill reaction of membrane fragments of blue-green algae III. Fluorescence characteristics of membrane fragments of Anabaena variabilis and Anabaena cylindrica

Fluorescence spectra of the pigment system at –196°Cin membrane fragments of Anabaena variabilis and A. cylindricawere investigated. The fluorescence spectra of membrane fragments having four emissionbands at 645–655, 685, 695 and 725 nm were basically similarto those reported for intact cells of blue-green algae, thoughthe emission from phycocyanin (645–655 nm) was far strongerwith membrane fragments than with intact algal cells. Incubation of membrane fragments of A. variabilis in a dilutebuffer (10^–2M, pH 7.5) caused an increase in the 645 nmfluorescence and slight decreases in the 685 and 695 nm fluorescences,but had no influence on the 725 nm fluorescence. The decreasein the 685 and 695 nm fluorescences of A. cylindrica was moremarked and had the same kinetics as the inactivation of photosystemII reaction measured by DPIP-photoreduction. When membrane fragments of A. cylindrica were incubated in thebuffer solution at room temperature or in the presence of MgCl₂(10^–3M) at 0°C; phycobilin aggregates, which emittedthe 655 and 685 nm fluorescence, were solubilized. This solubilizationwas not observed with membrane fragments of A. variabilis. (Received August 31, 1972; )     

Suppression of Carotenoid Photobleaching by Kaempferol in Isolated Chloroplasts

The effects of kaempferol on carotenoid photobleaching wereexamined using chloroplasts poisoned by carbonylcyanide m-chlorophenylhydrazone(CCCP). Kaempferol suppressed carotenoid photobleaching withoutaffecting electron transfer reactions. Half-maximal suppressionwas observed at about 10 µM. Kaempferol was photooxidizedby CCCP-poisoned chloroplasts, as observed by its bleachingat 380 nm. Ascorbate inhibited the oxidation of kaempferol.Under anaerobic conditions, kaempferol did not affect the photobleachingof carotenoid. Other fiavonols, quercetin and its glycosides,also suppressed the carotenoid photobleaching. The results suggestthat flavonols act as antioxidants in illuminated chloroplastsunder aerobic conditions. (Received February 22, 1982; Accepted May 14, 1982)     

Inhibitors of electron transport associated with photosystem II in chloroplasts

The modes of actions of six inhibitors on the electron transportsystem in the vicinity of system II in chloroplasts were studied. The first group, including piericidin A, ioxynil and broxynil,showed relatively simple modes of action on the Hill reaction,fluorescence of chlorophyll and the photobleaching of photosyntheticpigments, which are similar to the action of DCMU. As compared with inhibitors of the first group, the inhibitoryactions of salicylaldoxime, antimycin A and azide on the Hillreaction were more complicated in that they were influencedmore strongly by reaction conditions, i.e. duration of incubation,pH of the reaction mixture and illumination of chloroplasts.Inhibitors of the second group suppressed the rise in fluorescencein the induction period. However, this effect was not observedin the presence of DCMU or dithionite. Salicylaldoxime and azidewere effective in inducing photobleaching of photosyntheticpigments, whereas antimycin A inhibited the photobleaching inducedby ferricyanide or CCCP. Inhibition sites of the inhibitors in the first group are assumedto be similar to that of DCMU, whereas the inhibitors in thesecond group are effective in blocking electron transport onthe oxidizing side of system II between the primary electrondonor of system II and an intermediary electron carrier whichreceives electrons from artificial electron donors for systemII. (Received October 30, 1971; )     

Further investigation of the light-induced cytochrome b-559 reaction in membrane fragments of the blue-green alga Anabaena variabilis

Oxidation-reduction reactions of the low redox potential cytochromeb-559 were studied for membrane fragments of the blue-greenalga Anabaena variabilis. Cytochrome photooxidation was observableat room temperature when the membrane fragments had been preincubatedat room temperature in the dark. A CCCP addition (10^–4M) strongly enhanced the reaction. Oxidation consisted of a DCMU-sensitive and an insensitive reaction.The former depended on actinic illumination of short wavelength.The latter showed a dependency on longer wavelength light. Theformer was assumed to be induced by the action of photosystemII and the latter by that of photosystem I. The photosystem II oxidation was small before preincubation,and was enhanced by added DPIP or Ferro. This was interpretedas photosystem II action inducing oxidation as well as reduction;reduction being inactivated during dark incubation or beingsuppressed by added redox reagents which compete for electronacceptance from photosystem II so that oxidation was apparentlyenhanced. The oxidationreduction reactions of this cytochromewith low redox potential were assumed to be almost identicalwith those of the high redox potential form, at least in themembrane fragments of Anabaena variabilis. (Received June 8, 1975; )     

Studies on the Hill reaction of membrane fragments of blue-green algae V. A correlation between the solubilization of a photochemically active chromoprotein (ACP) and the inactivation of photosystem II activity in membrane fragments of Anabaena cylindrica

The relationship between a photochemically active chromoprotein(ACP) (cf. ref. 1) and photosystem II was investigated withmembrane fragments of Anabaena cylindrica, A. variabilis andP. boryaman. ACP was solubilized from membrane fragments of A. cylindricabut not from those of A. variabilis or P. boryanum, when themembrane fragments had been incubated in a dilute buffer andhad lost their Hill or photosystem II activity. In A. cylindrica,ACP-solubilization always occurred, independent of photosystemII inactivation, on incubation of the membrane fragments inmedia without PEG. However, the amount of ACP solubilizationaccompanying photosystem II inactivation was twice that withoutphotosystem II inactivation. The increase in ACP solubilizationaccompanying photosystem II inactivation. The kinetics resembledthose for the decrease in 695 nm fluorescence emitted by membranefragments at — 196?C (cf. 2). The ACP solubilized independent of photosystem II inactivationwas assumed to have been released during disruption of intactcells in the preparation of membrane fragments. The slow ACPsolubilization upon the inactivation of photosystem II was attributedto the pigment being bound to membranes. We assume that thephoto-reactive component of ACP, P690 (cf. 3, 4), is releasedfrom the membranes during photosystem II inactivation, and thatP690 is a component of photosystem II which emits the 695 nmfluorescence at — 196?C. (Received March 22, 1974; )     

Effects of Deuterium Oxide and Free Radical Scavengers on Carotenoid Photobleaching in Spinach Chloroplasts

The effect of D₂O on carotenoid photobleaching was examinedin spinach chloroplasts poisoned by carbonylcyanide m-chlorophenylhydrazone.D₂O, which prolongs a life time of singlet molecular oxygen,stimulated carotenoid photobleaching under aerobic conditions,but not under anaerobic conditions. The stimulation became smalleras the intensity of actinic light was lowered. Propyl gallateand (+)-catechin, radical scavengers, suppressed photobleaching.The suppression was greater at a low actinic light intensity.These results suggest that cartoenoid is photobleached by singletmolecular oxygen and radical chain reactions. (Received July 17, 1982; Accepted January 13, 1983)     

Studies on the Hill reaction of membrane fragments of blue-green algae I. Stabilizing effect of various media on the 2,6-dichlorophenol indophenol-Hill activity of membrane fragments obtained from Anabaena cylindrica and Anabaena variabilis

The stability of DPIP-Hill activity of membrane fragments fromblue-green algae was investigated in various suspension media.Two blue-green algae, Anabaena cylindrica and A. variabiliswere used. Activities for the DPIP-Hill reaction were stable in the twoalgal preparations only when preparations were suspended ina medium containing high concentrations of carbohydrates orPEG. Required concentrations of carbohydrates or PEG differedgready in die two preparations; 1.5 M sucrose or 20% PEG forA. cylindrica and 0.3 M sucrose or 6% PEG for A. variabiliswere minimal for maintaining full activity in 2 hr incubationat 0°C, respectively. Below these concentrations, activitiesdecreased rapidly. In both preparations, stabilizing effects,on a molar basis, varied in different kinds of solutes. However,a simple relation was found between the effects and water concentrationsof media. In A. cylindrica, a 50% decay in activity occurredin 2 hr incubation at 0°G in a medium with 43.8 M water,and, in A. variabilis, the water concentration was 53.4 M. In media free of carbohydrate or PEG, Mg++ ion had a moderatestabilizing ability. EDTA acted antagonistically to Mg++. Effectiveconcentrations were the same in two preparations. However, inthe medium containing carbohydrate or PEG, their actions wereinsignificant. Results suggest that the molecular organization in membranesnecessary for Hill reaction is easily destroyed under high waterconcentrations, and that added solutes stabilize the activityby reducing water concentrations. (Received February 13, 1971; )     

Ionophore-sensitive spectral shift of carotenoid induced by ferricyanide in chromatophores from Rhodopseudomonas sphaeroides

In chromatophores from Rhodopseudomonas sphaeroides, ferricyanideinduced a change in the absorption spectrum of carotenoid. Theionophore-sensitive part of the ferri-cyanide-induced changewas similar to that induced by light or by diffusion potential.The ferricyanide-induced change is explained by the electrochromicshift of the carotenoid spectrum by the inside-positive electricalfield change which is probably caused by the electrogenic electronflow from a membrane redox component to ferricyanide in theouter aqueous phase. The ionophore-insensitive part is probablythe response of the carotenoid in another pool to the localfield change by oxidation of bacteriochlorophyll [Okada, M.and A. Takamiya (1970) Plant & Cell Physiol. 11: 713–721]. (Received January 21, 1980; )     

Analysis of photosystem II using particle II preparation. I. Experimental evidence supporting the idea of the involvement of two light reactions in photosystem II of green plant photosynthesis

(1) To analyze the photoelectron flow related to photosystemII, particle II preparation, i.e., the chloroplast fragmenthaving only photosystem II activity, proved to be far betterthan the generally used chloroplast preparations having activitiesof both PS-I and PS-II. (2) By simultaneous measurements ofthe activities of O₂ evolution and DPIP- and ferricyanide photoreductionusing variously-treated particle II preparations, it was foundthat a noticeable activity of ferricyanide photoreduction wasstill observed, though the former two activities were completelylost in the course of treatments such as Tris-treatment, pre-illuminationand aging. (3) Besides this, differences were found betweenferricyanide- and DPIP-photoreduction in respect to susceptibilityto CCCP, availability of artificial electron donor, and theeffect of chloride addition. However, both photo-reductionswere equally inactivated by heat-treatment and addition of DCMU.(4) To explain the observed distinctions between DPIP and ferricyanidein their mode of action as electron acceptor for PS-II, a schemesuggesting the involvement of two light reactions in PS-II isproposed and the electron flow near PS-II is discussed. ¹ This work has been supported by Grants from the Ministry ofEducation (Nos. 8425- 70-'71; 4970l4-'69-'71), which are gratefullyacknowledged here. (Received January 12, 1972; )     

THE REACTIONS OF DENATURED EGG ALBUMIN WITH FERRICYANIDE

The following facts have been established experimentally. 1. In the presence of the synthetic detergent, Duponol PC, there is a definite reaction between dilute ferricyanide and denatured egg albumin. 0.001 mM of ferrocyanide is formed by the oxidation of 10 mg. of denatured egg albumin despite considerable variation in the time, temperature, and pH of the reaction and in the concentration of ferricyanide. 2. If the concentration of ferricyanide is sufficiently high, then the reaction between ferricyanide and denatured egg albumin in Duponol solution is indefinite. More ferrocyanide is formed the longer the time of reaction, the higher the temperature, the more alkaline the solution, and the higher the concentration of ferricyanide. 3. Denatured egg albumin which has been treated with formaldehyde or iodoacetamide, both of which abolish the SH groups of cysteine, does not reduce dilute ferricyanide in Duponol PC solution. 4. Cysteine is the only amino acid which is known to have a definite reaction with ferricyanide or which is known to react with dilute ferricyanide at all. The cysteine-free proteins which have been tried do not reduce dilute ferricyanide in Duponol PC solution. 5. Concentrated ferricyanide oxidizes cystine, tyrosine, and tryptophane and proteins which contain these amino acids but not cysteine. The reactions are indefinite, more ferrocyanide being formed, the higher the temperature and the concentration of ferricyanide. 6. The amount of ferrocyanide formed from denatured egg albumin and a given amount of ferricyanide is less in the absence than in the presence of Duponol PC. 7. The amount of ferrocyanide formed when denatured egg albumin reacts with ferricyanide in the absence of Duponol PC depends on the temperature and ferricyanide concentration throughout the whole range of ferricyanide concentrations, even in the low range of ferricyanide concentrations in which ferricyanide does not react with amino adds other than cysteine. The foregoing results have led to the following conclusions which, however, have not been definitely proven. 1. The definite reaction between denatured egg albumin in Duponol PC solution and dilute ferricyanide is a reaction with SH groups whereas the indefinite reactions with concentrated ferricyanide involve other groups. 2. The SH groups of denatured egg albumin in the absence of Duponol PC react with iodoacetamide and concentrated ferricyanide but they do not all react rapidly with dilute ferricyanide. 3. Duponol PC lowers the ferricyanide concentration at which the SH groups of denatured egg albumin react with ferricyanide. The SH groups of denatured egg albumin, however, are free and accessible even in the absence of Duponol PC.     

Electron flow from hydrogen peroxide in photosystem II-catalyzed oxidation-reduction reactions of spinach chloroplast fragments

Oxidation-reduction reactions of photosystem II were investigatedin spinach chloroplast fragments. Chloroplast fragments treatedwith 8-hydroxyquinoline sulfate showed only a low activity forthe 2,6-dichlorophenolindophenol (DPIP) Hill reaction, as wasobserved in chloroplast fragments treated with a high-concentrationof Tris buffer. Hydrogen peroxide could donate electrons tophotoreaction center II in chloroplast fragments treated with8-hydroxyquinoline, high-concentration Tris, or ethylene glycol,but water could not serve as an electron donor in these preparations.Electrons from hydrogen peroxide were transferred to DPIP, ferricyanide,and p-benzoquinone viaphotosystem II. (Received May 12, 1971; )     

The oxidation of C-550 by exogenously added oxidants in the presence of dichlorophenyldimethylurea: The localization of the primary electron acceptor of Photosystem II in the thylakoid membrane

The oxidation of C-550 by exogenously added oxidants in spinachchloroplasts and digitonin-treated chloroplasts was studiedin the presence of 3-(3,4-dichlorophenyl)-1,1-dimethylurea inan attempt to elucidate localization of the primary electronacceptor of Photosystem II in the thylakoid membrane. C-550was directly oxidized by various oxidants such as ferricyanide,N-methylphenazonium methosulfate (PMS) and quinones with redoxpotentials higher than that of C-550. Among the oxidants used,dibromothymoquinone was the most effective oxidant of C-550,followed by PMS. In spite of its high redox potential, ferricyanidewas rather a poor oxidant. The rates of C-550 oxidation by quinoneswere linearly proportional to the oxidant concentration, whereasthe rates tapered off with increasing concentrations of polaroxidants within the ranges of concentration used. These resultsindicate that C-550 is located inside the thylakoid membraneand is covered by a lipophilic shield. Addition of cations,especially divalent cations, significantly affected C-550 oxidationby ferricyanide or PMS but not by nonpolar oxidants. C-550 oxidationby ferricyanide was accelerated at low pH. Thus the accessibilityof C-550 to polar oxidants appears to be affected by electrostaticinteractions between oxidant ions and the negative charges ofthe thylakoid surface. When electrostatic interaction was minimized,ferricyanide oxidized C-550 as rapidly as several quinones did.This suggests that C-550 is located close to the surface ofthe membrane. The evidence indicates that the rates of C-550oxidation depend not only on the accessibility of C-550 to addedoxidants, but also on the reactivity between them. Reduction of cytochrome f by added reductants shows featuressimilar to those of C-550 oxidation by added oxidants, indicatingthat properties of the shield covering cytochrome f are similarto those of the shield covering C-550. (Received March 8, 1977; )     

Stimulation of lipid peroxidation and carotenoid bleaching by deuterium oxide in illuminated chloroplast fragments: Participation of singlet molecular oxygen in the reactions

The effects of deuterium oxide (D₂O) on light-induced lipidperoxidation and carbonylcyanide m-chlorophenylhydrazone (CCCP)-inducedcarotenoid photobleaching were examined in isolated chloroplastfragments. D₂O stimulated the lipid peroxidation in the presenceof CCCP or methyl viologen as well as in their absence. Carotenoidphotobleaching was also enhanced by D₂O. These results led tothe conclusion that the lipid peroxidation and part of the carotenoidphotobleaching were induced by the singlet molecular oxygenbecause D₂O prolongs its lifetime. (Received June 23, 1978; )     

Redox Reactions between Kaempferol and Illuminated Chloroplasts

Bleaching of kaempferol by illuminated chloroplasts was observed at 380 nanometers. The photobleaching was stimulated by methyl viologen and suppressed by superoxide dismutase indicating the participation of O₂⁻ in the reaction. An electron transfer inhibitor on the oxidizing side of photosystem II, carbonylcyanide m-chlorophenylhydrazone (CCCP), stimulated the photobleaching and 3-(3,4-dichlorophenyl)-1,1-dimethylurea partially suppressed it. The stimulation by CCCP suggests that kaempferol is also bleached on the oxidizing side of photosystem II. The spectrum of kaempferol bleaching in the presence of methyl viologen was the same as that in the presence of CCCP having a maximum in absorbance decrease at around 380 nanometers. When kaempferol was oxidized by KMnO₂ or KO₂, the oxidized minus reduced difference spectra had also a negative peak at about 380 nanometers. The results suggest that kaempferol was oxidized by illuminated chloroplasts.

The rate of kaempferol photooxidation increased as its concentration was increased from 1 to 100 micromolar. The rate of quercetin photooxidation also increased as its concentration was increased from 1 to 100 micromolar. Concentration of quercetin glycosides higher than 10 micromolar was required to detect their photobleaching by illuminated chloroplasts. From these results, it is postulated that flavonols function as antioxidants in chloroplasts.