Chlorophyll Bleaching Research Articles

Studies into the action of the diphenyl ether herbicides acifluorfen and oxyfluorfen. Part I: Activation by light and oxygen in leaf tissue

AbstractThe effect of acifluorfen and oxyfluorfen on chlorophyll bleaching, lipid peroxidation and photosynthesis in pea leaf discs was studied. Both her‐ bicides induced light‐dependent bleaching and lipid peroxidation, the level of damage being greater at higher light intensities. Photosynthetic carbon dioxide fixation was only partially inhibited in treated leaf discs incubated in darkness, thus indicating that these herbicides did not inhibit photo‐ synthesis as a primary mode of action. Leaf discs maintained in darkness showed no visible signs of injury, and light‐dependent herbicide‐induced damage was reduced by incubating discs under nitrogen, orpre‐incubating them with the electron‐transport inhibitor monuron. It is suggested that acifluorfen and oxyfluorfen are activated by a light‐dependent process, which requires photosynthetic electron transport.

Effects of Pyridazinone Herbicides during Chloroplast Development in Detached Barley Leaves III. Effects of SAN 6706 on Photosynthetic Activity and Chlorophyll-Protein Complexes

The photosynthetic activities of barley leaves in vivo and of thylakoids, isolated from the leaves, in vitro were studied during greening in the presence or absence of 2 × 10-4 м SAN 6706. The degree of chlorophyll bleaching increased from 32% at 24 h of greening to 60% at the final stage. Treated leaves were unable to carry out 14CO2-fixation, their fluorescence induction properties showed very limited, if any, photosystem II-activity, and the thylakoids isolated from the leaves were also inactive in mediating ferricyanide Hill reaction. The leaves, however, exhibited lightinduced quenching of fluorescence revealed by slow fluorescence induction measurements; and the thylakoids were active in mediating photosystem I-specific in vitro Mehler reaction. Thylakoid membranes of the chloroplasts isolated from treated leaves contained CP 1 and LHCP3 bands as revealed by polyacrylamide gel electrophoresis. From these results it is concluded that i) greening in the presence of SAN 6706 leads to the formation of inactive photosystem -II units; that ii) photosystem-I per se is active in vitro, and can, possibly, mediate cyclic electron transport in vivo coupled to the formation of ATP; and that iii) the presence of xanthophyll pigments is required for the assembly of the light-harvesting complex. The experiments were repeated with the application of SAN 9789, another pyridazinone compound, and yielded practically the same results as those obtained with SAN 6706. For practical reasons, only the results obtained with SAN 6706 are demonstrated throughout this communication.

Linolenic Acid-Dependent «Chlorophyll Oxidaseo»-activity: a Property of Photosystems I and II

Summary Photosystems (PS) I and II isolated from barley thylakoids supported linolenic acid-dependent bleaching of chlorophyll. This «chlorophyll oxidase» activity was virtually absent from the purified light harvesting complex (LHC). PS I and PS II were practically free of cytochromes. On a chlorophyll basis they bleached chlorophyll at rates comparable to the rates measured in whole thylakoids. Thylakoids as well as PSI were able to bleach the chlorophyll of LHC added to them, the reaction following Michaelis-Menten Kinetics with an apparent K M of ca 30μM chlorophyll. Chlorophyll oxidase appears to be a general property of thylakoids (photosystems) as demonstrated by tests with thylakoids isolated from several plant species.

Properties of Linolenic Acid-dependent Chlorophyll Oxidation Activity in Thylakoid Membranes

Summary Bleaching of chlorophyll in isolated thylakoids can be induced by incubation with lipase from Rhizopus arrhizus as also by treatment with free linolenic acid. Oxidation of chlorophyll is inhibited completely by general scavengers of free radicals whereas scavengers of • OH and • O 2 - are ineffective. The bleaching activity does not depend on co-oxidation of linolenic acid and chlorophyll as the reaction proceeds in the presence of linolenic acid peroxide and remains dependent on oxygen. A hypothetical chain reaction comprising linolenate peroxide and O 2 , and yielding bleached chlorophyll is presented. Assessment of kinetics of chlorophyll a hydroxylation to chlorophyll a-1 under various conditions suggest that chlorophyll a-1 may represent an intermediate in a minor diversionary pathway from the main chlorophyll oxidation reaction.

Effects of Winter Stress on Chlorophyll Organization and Function in Scots Pine

To learn more about the effects of winter stress on chlorophyll organization and function in Scots pine (Pinus sylvestris), 77K fluorescence and absorption characteristics were studied in chloroplast thylakoids isolated from winter stressed and summer active pine. Chlorophyll-protein complexes were also isolated using LDS-PAGE. Energy coupling between LHCP and the two photosystems was not significantly affected by winter stress as judged from the unaffected chlorophyll a to b ratio of excitation spectra measurements of PS I and PS II fluorescence. Furthermore, winter stressed pine had a lower chlorophyll/carotenoid ratio due to a preferential bleaching of chlorophyll in the winter. This change rendered a relative increase of β-carotene in the chlorophyll-protein complex of photosystem I, CPI, in winter stressed as compared with summer active pine. It is suggested that this pigment change may increase the probability of energy transfer from excited chlorophyll to β-carotene, thus counteracting photodynamic effects of the photosynthetic apparatus during late winter when the light intensity is high and when photosynthesis is inhibited.

Leaf senescence in a non-yellowing mutant of Festuca pratensis Huds.

A study was made of linolenic acid-dependent oxidative chlorophyll bleaching (CHLOX) by thylakoid membranes from senescing leaf tissue of a normal cultivar (cv. Rossa) and a non-yellowing mutant genotype (Bf 993) of Festuca pratensis Huds. To overcome the problem of variation in levels of endogenous chlorophyll substrate in membranes from different sources, light-harvesting complex (LHC) was used to supplement thylakoid pigment. It was shown that CHLOX is associated with both Photosystem I and LHC-rich thylakoid subfractions but that purified LHC has negligible associated CHLOX activity and stimulates the rate of bleaching by isolated entire chloroplast membranes. Non-senescent tissue of Bf 993 and Rossa had essentially identical thylakoid CHLOX levels, which subsequently declined during senescence in darkness. The half-life of CHLOX from the mutant was three times greater than that of the normal genotype. In both cultivars, the amount of CHLOX assayed in thylakoids isolated at different times during senescence was more than adequate to support the corresponding in-vivo rate of pigment degradation as calculated from the half-life for chlorophyll. It was concluded that the non-yellowing mutation is not expressed through a lack of CHLOX activity. The role of linolenic acid metabolism in the regulation of thylakoid structure and function during senescence, and as a likely site of the non-yellowing lesion, are discussed.

Superoxide dismutase and chilling injury in Chlorella ellipsoidea

The relationship between superoxide dismutase (SOD) and chilling injury was examined in chilling-sensitive and chilling-resistant strains of Chlorella ellipsoidea. The sensitive strain contained less SOD than the resistant strain. Moreover, all of the SOD in the sensitive strain was the H 2O 2-sensitive, iron-containing SOD, whereas most of the SOD in the resistant strain was the H 2O 2-resistant, manganese-containing SOD. Illumination further enhanced the disparity in SOD content between the sensitive and resistant strains since the SOD in the former declined during illumination, whereas the SOD in the latter strain did not. It was possible to elevate the SOD content of the sensitive strain and to increase the proportion of MnSOD by prior growth in the presence of 50 μ m paraquat. The SOD content of the cultures after 5 h of illumination at 4 °C fell in the order sensitive strain < paraquat-induced sensitive strain < resistant strain. The resistance of these cultures to chilling injury was related to SOD content. This was the case whether resistance was assessed in terms of growth rate after chilling, bleaching of chlorophyll during chilling, or loss of viability during chilling. It thus appears likely that O 2 − is an agent of chilling injury.

Methylmercuric iodide modification of lipoxygenase-1: Effects on the anaerobic reaction and pigment bleaching

The effect of methylmercuric iodide modification of sulfhydryl groups in soybean lipoxygenase-1 on linoleate oxidation, carbonyl production and β-carotene and chlorophyll α bleaching were determined under aerobic and anaerobic conditions. Linoleate oxidation at pH 9.0 was strongly inhibited by modification of the enzyme. On the other hand, pigment bleaching was enhanced with the modified enzyme. Unmodified lipoxygenase-1 was not sensitive to chlorophyll inhibition, but activity of modified lipoxygenase-1 was affected. Linoleate oxidation was inhibited up to 70% when 2.2 μM chlorophyll was present in the reaction mixture. Chlorophyll inhibition was similar with affinity chromatography-purified lipoxygenase-2 and modified lipoxy genase-1. Unmodified lipoxygenase-1 exhibited high bleaching activity under anaerobic conditions and relatively low activity under aerobic (oxygen or air) conditions. Modified lipoxygenase-1 showed a significant increase in carotene and chlorophyll bleaching under both anaerobic and aerobic conditions. Under anaerobic conditions in the presence of either pigment, both modified and unmodified lipoxy genase-1 exhibited high 285 nm absorbing material production. Antioxidants (butylated hydroxyanisole, butylated hydroxytoluene, α-tocopherol, propyl gallate and tertiary butylated hydroxyquinone) were powerful inhibitors of pigment bleaching by modified lipoxygenase-L However, only tertiary butylated hydroxyquinone and propyl gallate blocked the increase in the rate of absorbance at 285 nm.

Thylakoid-associated «Chlorophyll Oxidase»: Distinction from Lipoxygenase

Barley chloroplasts contain a latent chlorophyll-bleaching activity. It is closely associated with the thylakoid, and depends on the presence of free fatty acids such as linolenic acid. Chlorophyll-bleaching is strictly dependent on oxygen. The principle responsible for the linolenate-dependent bleaching effect is tentatively termed «chlorophyll oxidase».The effects of various inhibitors, particularly chelators of Cu and Fe on «chlorophyll oxidase» resemble the effects on the bleaching of chlorophyll by soybean lipoxygenase in the presence of linolenic acid. However, «chlorophyll oxidase» differs clearly from lipoxygenase in that pigment-bleaching is not associated with the production of linolenate hydroperoxide. Moreover, «chlorophyll oxidase» is active in the presence of oleic- and even stearic acid and thus is distinct from lipoxygenase which requires fatty acids containing a 1,4 pentadiene unit.

Variable Fluorescence and Fluorescence Spectra of Algae after Herbicide-Induced Pigment Bleaching

Herbicides like norflurazon, oxadiazon, or oxyfluorfen affect the pigment apparatus of green algae. Their influence on variable (FmaK - F0) and initial fluorescence (F0) as well as on the state of chlorophylls were investigated. Two main modes of action of these herbicides on the photo­synthetic apparatus during growth have been found: First: Scenedesmus cells grown with norflurazon. a carotene-biosynthesis inhibitor, show chlorophyll bleaching concurrently with decrease or inactivation of photosystem-II reaction centers, with small changes in fluorescence spectra. Further, electron transport is blocked at the acceptor side of photosystem II. Disappearance or inactivation of photosystem-II reaction centers after a 50% loss of chlorophyll is accompanied by a sharp increase of the F0 yield, with small changes in the chlorophyll state. The slow-decay phase in fluorescence induction of these cells, enhanced by dithionite, is ascribed to a reversible photobleaching of chlorophyll during the measurement. Second: Oxadiazon and oxyfluorfen alter substantially the state of chlorophylls and cause strong bleaching, but reaction centers of photosystem II, although less than in control cells, are active even after an 80% disappearance of chlorophyll. The yields of F0 and (Fmax - F0) in treated cells are independent of the extent of chlorophyll bleaching. In contrast to norflurazon, the latter two herbicides do not inhibit carotenogenesis. Decrease of photosystem-II reaction centers may be due to non-specific pigment destruction (oxyfluorfen) or interference w'ith chlorophyll biosynthesis.

Catabolism of Chlorophyll: Demonstration of Chloroplast-localized Peroxidative and Oxidative Activities

Summary Chloroplasts isolated from barley mesophyll protoplasts contain two distinct enzyme activities causing the bleaching of the endogenous chlorophyll. Both enzymes are constituents of the thylakoid membrane. The activities are latent. Activation of the H 2 O 2 requiring chlorophyll-bleaching enzyme requires ultrasonication of the thylakoids or the inclusion of a detergent (0.1% SDS) in the assay mixture. The peroxidative chlorophyll bleaching has a pH optimum of 6.8–7.2 and requires a monophenol as cofactor. The other enzyme was activated in the presence of unsaturated fatty acids and depends strictly on O 2 . Ultrasonication of thylakoids is also required for activation but SDS inhibits the oxidative bleaching of chlorophyll in sonicated thylakoids. It is suggested that thylakoids of mature chloroplasts contain a peroxidase and an oxidase which eventually are activated upon the induction of senescence and become involved in the catabolism of chlorophyll.

Carotene and chlorophyll bleaching by soybeans with and without seed lipoxygenase-1

Carotene and chlorophyll bleaching by soybeans with and without seed lipoxygenase-1

Effects of norflurazon (San 9789) on light‐increased extractable nitrate reductase activity in soybean [Glycine max (L.) Merr.]seedlings

Abstract. The effects of norflurazon (San 9789) on light‐increased extractable NADH nitrate reductase activity (NRA) in soybean seedlings were studied. Continuous white light (W) increased NRA steadily in root and cotyledonary tissues over a 5 d period. Morflurazon, a pyridazinone herbicide which causes chlorophyll bleaching in W, reduced the initial NRA induction rate in roots and cotyledons. However, in cotyledons of norfiurazon‐treated plants NRA increased at a more rapid rate than in the control after 24 h of W, with activity levels reaching three times those of control seedlings after 5 d. NRA induced by W in control and norflurazon‐treated cotyledons was fluence‐rate dependent. Continuous FR induced equal amounts of NRA in control and norflurazontreated tissues, suggesting that the superinduceable NRA of norflurazon‐treated plants under W is not phytochrome induced. The FR‐induced NRA of control and norflurazon‐treated cotyledons had pH optima of 6.6, but during development under W the pH optimum of control cotyledons changed from 6.3 to between 6.6 and 7.1. The pH optimum of the norflurazon‐induced NRA of the cotyledon under W was about 7.5. The NADH/NADPH NRA ratio after 4 d of W was 1.3 in control and 2.5 in norflurazontreated cotyledons. These data indicate that photosynthelic pigments are involved only secondarily in light‐induction of NRA in this system.

Superoxide-and Ethane-Formation in Subchloroplast Particles: Catalysis by Pyridinium Derivatives

Abstract Oxygen reduction by chloroplast lamellae is catalyzed by low potential redox dyes with E′0 values between -0 .3 8 V and -0 .6 V. Compounds of E′0 values of -0 .6 7 V and lower are inactive. In subchloroplast particles with an active photosystem I but devoid of photosynthetic electron transport between the two photosystems, the active redox compounds enhance chlorophyll bleaching, superoxide formation and ethane production independent on exogenous substrates or electron donors. The activities of these compounds decrease with decreasing redox potential, with one exception: 1-methyl-4,4′-bipyridini urn bromide with an E′0 value of lower -1 V (and thus no electron acceptor of photosystem I in chloroplast lamellae with intact electron transport) stimulates light dependent superoxide formation and unsaturated fatty acid peroxidation in sub­ chloroplast particles, maximal rates appearing after almost complete chlorophyll bleaching. Since this activity is not visible with compounds with redox potentials below -0 .6 V lacking the nitrogen atom at the 1-position of the pyridinium substituent, we assume that 1 -methyl-4,4′-bi-pyridinium bromide is “activated” by a yet unknown light reaction.

Absence of nitrate reductase activity in San 9789 bleached leaves of barley seedlings (Hordeum vulgare cv. Midas)

Abstract San 9789 (norflurazone) blocks carotenoid synthesis which allows chlorophyll bleaching in the light, and has been used recently as a tool to study phytochrome responses without interference from photosynthetic pigments. By using this herbicide, we have found that nitrate reductase activity and light dependent nitrite reduction were lost simultaneously from achlorophyllous areas of barley leaves, with the green areas of the leaf tip still showing high activities. By contrast nitrate reductase is still present in the roots of herbicide treated plants. We suggest that intact chloroplasts are required for the presence of nitrate reductase in barley leaves.

Paraquat-Catalyzed Photodestructions in Subchloroplast Particles are Independent on Photosynthetic Electron Transport

Abstract Light dependent ethane formation and chlorophyll bleaching in isolated chloroplast lamellae are enhanced by either methylviologen or α-linolenic acid. Both ethane formation and chloro­phyll bleaching are also enhanced in chloroplast particles deficient in photosynthetic electron transport, e. g. after aging, heat treatment or digitonin fragmentation. Ethane formation by sub­ chloroplast particles from endogenous substrates in the presence of methylviologen is inhibited by superoxide dismutase or by a penicillamine copper complex exhibiting superoxide dismutase activity whereas chlorophyll bleaching is enhanced by superoxide dismutase - active substances. Maximal rates of ethane formation in subchloroplast particles are observed when more than 50% of the chlorophyll is bleached and continues after 98% chlorophyll bleaching. This result indicated that methylviologen -stimulated ethane production in subchloroplast particles is not de­pendent on photosynthetic electron transport but involves “activated oxygen” - species like the superoxide radical ion, generated by a light receptor derived from the pigmentsystem of photo­ system I or activated after its destruction.

Chlorophyll photobleaching and ethane production in dichlorophenyldimethylurea- (DCMU) or paraquat-treated Euglena gracilis cells.

Light dependent (35 Klux) chlorophyll bleaching in autotrophically grown Euglena gracilis cells at slightly acidic pH (6.5-5.4) is stimulated by the photosystem II blockers DCMU and DBMIB (both 10(-5) M) as well as by the autooxidizable photosystem I electron acceptor, paraquat (10(-3) M). Chlorophyll photobleaching is accompanied by the formation of thiobarbituric acid -- sensitive material ("malondialdehyde") and ethane. Both chlorophyll photobleaching and light dependent ethane formation are partially prevented by higher concentrations (10(-4) M) of the autooxidizable photosystem II electron acceptor DBMIB or by sodium bicarbonate (25 mM). In vitro studies with cell free extracts (homogenates) from E. gracilis suggest that alpha-linolenic acid oxidation by excited (reaction center II) chlorophyll represents the driving force for both ethane formation and chlorophyll bleaching. Ethane formation thus appears to be a sensitive and non-destructive "in vivo" marker for both restricted energy dissipation in photosystem II and, conditions yielding reactive oxygen species at the reducing side of photosystem I.

Effects of light and low temperatures on chlorophyll content and metabolism of Chlorella sorokiniana Shihira and Krauss

Chlorophyll bleaching induced by suboptimal temperatures in Chlorella sorokiniana was shown by Marvin and Karlander (5) to require light and molecular oxygen. The present study with various light qualities and quantities showed that bleaching was light intensity dependent and a two-phase process. White fluorescent light was the most efficient in degrading chlorophyll, then in order, blue, incandescent white, and red. Low intensity blue, 2.55–4.0 mW/cm 2, and red light, 2.8 mW/cm 2, produced an initial decline in chlorophyll more rapid than higher intensities of blue or red light. The low intensity blue and red light curve also showed a mid time-course plateau lasting beyond 100 hr. No mid time-course plateau occurred at low intensities of fluorescent or incandescent white light. Oxygen evolution declined with increasing time of incubation at 10°C. Blue light of 4.0 mW/cm 2 was least destructive, extending the duration of oxygen evolution to nearly 4 times that obtained with white light. Cells incubated at temperatures greater than 22°C exhibited patterns of photosynthesis and respiration approaching those of control cells at 39°C. Cells incubated at temperatures less than 22°C showed patterns approaching those of cells subjected to bleaching at 10°C. Hill activity was destroyed during the initial lag phase before much loss of chlorophyll had occurred. Viability of cells was more closely associated with loss of chlorophyll in the accelerated bleaching phase.

On the Action of Chlorosis-Inducing Herbicides in Leaves

Abstract The chlorosis induced by several different herbicides (group 1: aminotriazole, haloxidine; group 2: Sandoz 6706, difunon) in developing leaves of rye seedlings (Secale cereale L.) growing in light of 5,000 lx was, in addition to the absence of chlorophyll and carotenoids, accompanied by specific deficiencies of the 70 S ribosomes and of both chloroplastic and peroxisomal enzymes as previously described (Plant Physiol. 61, 1017 - 1022 (1978)) while growth and mitochondrial activities were little or not impaired. In dim light (10 lx) chlorophyll was formed and the chloroplastic and peroxisomal enzymes reached activities comparable to those in untreated leaves at 10 lx. Upon exposure to 30,000 lx a rapid bleaching of existing chlorophyll and a rapid inactivation of the plastidic enzyme NADP-glyceraldehydephosphate dehydrogenase occured after treatment with herbicides of group 2 but not in the presence of herbicides of group 1. After all treatments catalase was rapidly inactivated after transfer to high light intensity. The bleaching of chlorophyll and the enzyme inactivations occurred also at 0 °C. In the herbicide-bleached leaves grown at 5,000 lx the in vivo synthesis of (δ-aminolevulinic acid was low and in the presence of Sandoz 6706 and difunon the capacity for protochlorophyll (ide) synthesis was largely inactivated.

Ethane Formation and Chlorophyll Bleaching in DCMU-Treated Euglena gracilis Cells and Isolated Spinach Chloroplast Lamellae

Abstract Light dependent chlorophyll bleaching and ethane formation in Euglena gracilis cells at slightly acidic pH values above 5.2 are enhanced by DCMU and other photosystem II blockers. Excited chlorophyll under conditions of limited energy dissipation seems to be the oxidant for ω-3 unsaturated fatty acids, yielding ethane and other (ethylene, “malondialdehyde”) oxidation products, cooxidizing chlorophyll.