An Investigation of the Sustained Component of Nonphotochemical Quenching of Chlorophyll Fluorescence in Isolated Chloroplasts and Leaves of Spinach

The slowly reversible component of nonphotochemical quenching of Chl fluorescence, ql, has been investigated in intact leaves and chloroplasts of spinach (Spinacia oleracea). In leaves, between 50 and 100% of ql (defined as the quenching that remained after at least 10 min of dark adaptation of a previously illuminated leaf) is instantly reversible when leaves were infiltrated with nigericin. Chloroplasts isolated from leaves in which ql had been induced by prior illumination retained the same level of quenching. No pH gradient, as measured by quenching of 9-aminoacridine fluorescence, was present. However, addition of nigericin caused a partial removal of ql, as observed in whole leaves. It is concluded that ql is not related to a persistence of a bulk phase pH gradient in darkness but to a structural change in the thylakoid that can be reversed by addition of nigericin. The relationship between these observations and the hypothesis that nonphotochemical quenching of chlorophyll fluorescence results from protonation of light-harvesting complex of photosystem II components is discussed.     

Kinetic Studies on the Xanthophyll Cycle in Barley Leaves (Influence of Antenna Size and Relations to Nonphotochemical Chlorophyll Fluorescence Quenching)

Xanthophyll-cycle kinetics as well as the relationship between the xanthophyll de-epoxidation state and Stern-Volmer type nonphotochemical chlorophyll (Chl) fluorescence quenching (qN) were investigated in barley (Hordeum vulgare L.) leaves comprising a stepwise reduced antenna system. For this purpose plants of the wild type (WT) and the Chl b-less mutant chlorina 3613 were cultivated under either continuous (CL) or intermittent light (IML). Violaxanthin (V) availability varied from about 70% in the WT up to 97 to 98% in the mutant and IML-grown plants. In CL-grown mutant leaves, de-epoxidation rates were strongly accelerated compared to the WT. This is ascribed to a different accessibility of V to the de-epoxidase due to the existence of two V pools: one bound to light-harvesting Chl a/b-binding complexes (LHC) and the other one not bound. Epoxidation rates (k) were decreased with reduction in LHC protein contents: kWT > kmutant >> kIML plants. This supports the idea that the epoxidase activity resides on certain LHC proteins. Irrespective of huge zeaxanthin and antheraxanthin accumulation, the capacity to develop qN was reduced stepwise with antenna size. The qN level obtained in dithiothreitol-treated CL- and IML-grown plants was almost identical with that in untreated IML-grown plants. The findings provide evidence that structural changes within the LHC proteins, mediated by xanthophyll-cycle operation, render the basis for the development of a major proportion of qN.     

Purification and Molecular Properties of the Thylakoid-Bound Ascorbate Peroxidase in Spinach Chloroplasts

The hydrogen peroxide that is photoproduced in thylakoids isscavenged by the thylakoid-bound ascorbate peroxidase (tAPX)[Miyake and Asada (1992) Plant Cell Physiol. 33: 541]. tAPXwas purified from spinach thylakoids to homogeneity as judgedby SDS-polyacrylamide gel electrophoresis, and its molecularproperties were studied. Spinach tAPX was a monomer with a molecularweight of 40,000, which is about 10,000 higher than that ofthe stromal ascorbate peroxidase (sAPX) from spinach chloroplasts.tAPX cross-reacted with the antibody raised against sAPX fromtea leaves, as determined by Western blotting, which also providedevidence for the higher molecular weight of tAPX from spinachthylakoids than that of tea sAPX. The amino acid sequence ofthe amino-terminal region of tAPX showed a low degree of homologyto those of cytosolic APXs from spinach, pea and Arabidopsisthaliana, but a high degree of homology to that of stromal APXfrom tea. Thus, the amino-terminal region of tAPX seems notto be a domain required for binding of the enzyme to the thylakoidmembranes. tAPX contained protoheme IX, as identified by itspyridine hemochromogen, and gave a Soret peak at 403 nm and433 nm with an a band at 555 nm in its oxidized and reducedforms, respectively. Resembling sAPX but differing from cytosolicAPX, tAPX showed high specificity for ascorbate as the electrondonor. tAPX was inhibited by cyanide, thiol-modifying reagents,thiols and several suicide inhibitors, such as hydroxyurea andp-aminophenol. ¹Present address: Beijing Vegetable Research Centre, PO Box2443, Beijing, China.     

Inactivation of Ascorbate Peroxidase in Spinach Chloroplasts on Dark Addition of Hydrogen Peroxide: Its Protection by Ascorbate

Dark addition of hydrogen peroxide to intact spinach chloroplastsresulted in the inactivation of ascorbate peroxidase accompaniedby a decrease in ascorbate contents. This was also the casein reconstituted chloroplasts containing ascorbate, NADP⁺, NAD⁺and ferredoxin. The addition of hydrogen peroxide during light,however, showed little effect on ascorbate contents and ascorbateperoxidase activity in either the intact or reconstituted chloroplasts.In contrast to ascorbate peroxidase, the enzymes participatingin the regeneration of ascorbate in chloroplasts (monodehydroascorbatereductase, dehydroascorbate reductase and glutathione reductase)were not affected by the dark addition of hydrogen peroxide.Ascorbate contents increased again by illumination of the chloroplastsafter the dark addition of hydrogen peroxide. These resultsshow that the inactivation of the hydrogen peroxide scavengingsystem on dark addition of hydrogen peroxide [Anderson et al.(1983) Biochim. Biophys. Acta 724: 69, Asada and Badger (1984)Plant & Cell Physiol. 25: 1169] is caused by the loss ofascorbate peroxidase activity. Ascorbate peroxidase activitywas rapidly lost in ascorbate-depleted medium, and protectedby its electron donors, ascorbate, isoascorbate, guaiacol andpyrogallol, but not by GSH, NAD(P)H and ferredoxin. (Received June 14, 1984; Accepted August 15, 1984)     

Kinetic and Spectral Resolution of Multiple Nonphotochemical Quenching Components in Arabidopsis Leaves

Using novel specially designed instrumentation, fluorescence emission spectra were recorded from Arabidopsis (Arabidopsis thaliana) leaves during the induction period of dark to high-light adaptation in order to follow the spectral changes associated with the formation of nonphotochemical quenching. In addition to an overall decrease of photosystem II fluorescence (quenching) across the entire spectrum, high light induced two specific relative changes in the spectra: (1) a decrease of the main emission band at 682 nm relative to the far-red (750–760 nm) part of the spectrum (Δ F₆₈₂); and (2) an increase at 720 to 730 nm (Δ F₇₂₀) relative to 750 to 760 nm. The kinetics of the two relative spectral changes and their dependence on various mutants revealed that they do not originate from the same process but rather from at least two independent processes. The Δ F₇₂₀ change is specifically associated with the rapidly reversible energy-dependent quenching. Comparison of the wild-type Arabidopsis with mutants unable to produce or overexpressing the PsbS subunit of photosystem II showed that PsbS was a necessary component for Δ F₇₂₀. The spectral change Δ F₆₈₂ is induced both by energy-dependent quenching and by PsbS-independent mechanism(s). A third novel quenching process, independent from both PsbS and zeaxanthin, is activated by a high turnover rate of photosystem II. Its induction and relaxation occur on a time scale of a few minutes. Analysis of the spectral inhomogeneity of nonphotochemical quenching allows extraction of mechanistically valuable information from the fluorescence induction kinetics when registered in a spectrally resolved fashion.One of the most important photoprotective mechanisms against high-light (HL) stress in photosynthetic organisms is the nonphotochemical quenching (NPQ) of excitation energy, which is mostly due to thermal deactivation of pigment excited states in the antenna of PSII. There exist a number of literature reviews on the subject (Demmig-Adams and Adams, 1992; Horton et al., 1996; Horton and Ruban, 1999, 2005; Niyogi, 1999, 2000; Müller et al., 2001; Golan et al., 2004; Krause and Jahns, 2004). Chlorophyll (Chl) fluorescence, and in particular pulse amplitude-modulated (PAM) fluorometry as introduced by Schreiber et al. (1986), has become by far the dominant technique to measure NPQ in leaves, chloroplasts, and intact microorganisms (Krause and Weis, 1991; Govindjee, 1995; Maxwell and Johnson, 2000; Krause and Jahns, 2003; Schreiber, 2004), more recently often combined with specific NPQ mutant studies (Golan et al., 2004; Kalituho et al., 2006, 2007; Dall''Osto et al., 2007). In this technique, periodic saturating light pulses are applied, superimposed on the continuous actinic irradiation applied to induce NPQ, in order to transiently close the PSII reaction centers (RCs). Since the photochemistry contribution (photochemical quenching) is thus brought to zero, the method allows us to follow the dynamics of the NPQ development and relaxation by fluorescence in a relatively simple manner (Krause and Jahns, 2003, 2004).Mostly based on its relaxation kinetics, NPQ has been divided technically into the three kinetic components qE, qT, and qI, for the rapid, middle, and slow phases of relaxation (Horton and Hague, 1988), initially attributed to energy-dependent quenching, state transitions, and photoinhibitory quenching (Quick and Stitt, 1989). The rapidly forming and reversible part of NPQ, qE, is the most thoroughly studied. It is well established that this type of quenching is a finely regulated process in which the main governing factors are the proton gradient across the chloroplast thylakoid membrane, Δ pH (Wraight and Crofts, 1970; Briantais et al., 1979), the xanthophyll cycle (i.e. conversion of violaxanthin to antheraxanthin and zeaxanthin [Zx]; Demmig et al., 1987; Demmig-Adams, 1990; Demmig-Adams and Adams, 1992), and the action of the PsbS protein (Funk et al., 1995; Li et al., 2000, 2004; Niyogi et al., 2005). The actual molecular mechanism is still unknown, although there is no shortage of hypotheses and proposed quencher candidates: energy transfer from Chl to Zx in the major light-harvesting complex (LHCII; Frank et al., 2000); electron transfer from a carotenoid to Chl forming a Zx-Chl or lutein-Chl charge-transfer state (Holt et al., 2005; Avenson et al., 2009); direct or indirect quenching by the PsbS protein (Li et al., 2000; Niyogi et al., 2005); energy transfer from Chl to lutein in LHCII (Horton et al., 1991; Ruban et al., 2007) linked to the aggregation of or a conformational change in LHCII; and last but not least, a far-red (FR) light-emitting quenched Chl-Chl charge-transfer state formed by the aggregation of LHCII (Miloslavina et al., 2008). Quenching in the PSII RC has also been proposed (Weis and Berry, 1987; Finazzi et al., 2004; Huner et al., 2005; Ivanov et al., 2008) as an additional type of Zx-independent quenching. Alternatively, it has been suggested that quenching by lutein can complement the Zx-dependent quenching (Niyogi et al., 2001; Li et al. 2009). Johnson et al. (2009) have recently given support to the notion that both Zx-dependent and Zx-independent quenching originate from the same PsbS-dependent mechanism, which is modulated by Zx (Crouchman et al., 2006).While the rapidly relaxing phase qE is now well characterized in its dependence on the various factors, the much slower qT and qI phases are still controversial, and each of them may have contributions from more than one mechanism. The qI component has been traditionally attributed to photoinhibition of PSII (Somersalo and Krause, 1988), associated with coordinated degradation and repair of the photosystem (Powles and Björkman, 1982; Kyle, 1987; Krause, 1988; Aro et al., 1993; Long et al., 1994; Murata et al., 2007). Lately, though, it is more widely accepted that under most conditions the photoinhibition is low and qI, like qE, is a result of thermal deactivation of excited states. Different hypotheses have been put forward to account for its seeming irreversibility: persistent transmembrane Δ pH (Gilmore and Yamamoto, 1992), stable protonation of proteins (Horton et al., 1994), accumulation of inactive PSII reaction centers (Briantais et al., 1992; Schansker and van Rensen, 1999), or stable binding of Zx to CP29 (Färber et al., 1997). The connection of the qT phase with state transitions has been doubted as well, and in fact it is now thought that the fraction of energy redistributed from PSI to PSII under high-light conditions is negligible (Walters and Horton, 1991, 1993) and that the qT must have a different origin or that it has erroneously been ascribed as NPQ (Schansker et al., 2006).Along with the large amount of contradictory evidence on the nature and location of the NPQ quenching site(s), the question of whether the light-induced reversible NPQ represents one single mechanism of deexcitation located in a single site brought about by the combined action of PsbS and Zx (Johnson et al., 2009) or whether it comprises several parallel and largely independent mechanisms acting on different parts of the PSII antenna has not been finally answered. One way to answer this question might be to carefully examine the spectral properties of NPQ-related fluorescence changes. Quenching in different locations of the PSII antenna or with different mechanisms might give rise to a differential quenching in various parts of the PSII antenna that might affect the PSII fluorescence spectra in different ways. This appears possible, since the various pigment-protein complexes of the photosynthetic apparatus have slightly different absorption and emission spectra (Holzwarth, 1991; Holzwarth and Roelofs, 1992). However, in the vast majority of modulated Chl fluorescence instrumentation, including the most widely used PAM fluorometer (Schreiber et al., 1986), the signal is integrated over a broad wavelength range, usually covering the whole range of 710 nm or greater. This integration over the long-wave part of the spectrum has several undesirable consequences and is associated with the unnecessary loss of available information. For example, the fluorescence of PSII peaks in the region of 680 to 685 nm, whereas beyond 700 nm, the PSII fluorescence intensity drops to less than 20% of its peak intensity. In contrast, the fluorescence of intact PSI complexes is dominant in the region above 710 nm (Haehnel et al., 1982; Karukstis and Sauer, 1983; Holzwarth et al., 1985; Holzwarth, 1986; Slavov et al., 2008). Thus, the widely used instrumentation measures the NPQ parameters in a region with reduced PSII contribution and relatively high PSI contribution to total fluorescence, despite the fact that NPQ is generally considered to be primarily a PSII phenomenon. Only in a few studies has the fluorescence in the red and the FR region been separated in order to evaluate the contribution of PSI and its influence on the NPQ parameters (Genty et al., 1990; Peterson et al., 2001). NPQ might also shift the fluorescence properties of the PSII antenna complexes or give rise to entirely new fluorescing components (Miloslavina et al., 2008). This would remain undetected if the NPQ fluorescence changes are not resolved in the spectral domain. It follows from these considerations that a great deal of insight into the NPQ mechanisms and locations may be gained if the spectral dimension is added to the NPQ fluorescence characterization. Among the many advantages of such an approach, one would then be able to distinguish whether NPQ simply leads to a uniform decrease of PSII fluorescence across the emission range or whether this decrease is nonuniform, localized in specific pigment protein complexes, and/or whether new fluorescing species are actually being produced in the NPQ process.The HL-induced NPQ effects on the leaf fluorescence spectra have often been studied also at low temperature, where the differentiation between pigment sites is better (Krause et al., 1983; Demmig and Björkman, 1987; Ruban and Horton, 1994). However, the possibility to resolve the kinetics of NPQ development and relaxation is largely lost when performing the measurements at low temperatures. The 77 K spectra of leaves and thylakoid membranes are characterized by three main peaks, F₆₈₅, F₆₉₅, and F₇₃₀, believed to originate predominantly from Chl a in CP47 of PSII, a specific Chl in CP43 of PSII, and PSI, respectively (Satoh and Butler, 1978; van Dorssen et al., 1987; Andrizhiyevskaya et al., 2005; Komura et al., 2007). Fluorescence from the major LHCII peaks at 680 nm (Rijgersberg and Amesz, 1978) and from the PSII reaction center Chls at 683 nm (Roelofs et al., 1993; Andrizhiyevskaya et al., 2005). Low-temperature studies on the effects of HL irradiation are confined to the changes in the FR-to-red fluorescence ratio, which are the result of the quenching of PSII fluorescence or energy redistribution between the photosystems (state transitions). Ruban and Horton (1994) have shown that photochemical quenching in Guzmania is maximal at 688 nm, whereas nonphotochemical processes quench preferentially at 683 and 698 nm.In this study, we undertook a detailed investigation of the NPQ-associated spectral changes in the fluorescence spectra of Arabidopsis (Arabidopsis thaliana) measured at room temperature (RT) and at 77 K. It follows from the above discussion that deeper insight into the mechanisms of NPQ processes may be gained by combining the kinetic and the spectral information of the fluorescence changes occurring in NPQ. For this purpose, we developed a multiwavelength spectrometer with parallel detection, allowing us to follow the entire time-dependent fluorescence spectra of leaves during the induction and relaxation phases of NPQ with high sensitivity.Specific questions to be addressed in this study are the following. Are there more than one NPQ processes and NPQ sites? Are these processes occurring in a linked fashion or are they independent? How do they depend on the various cofactors known to affect NPQ, in particular regarding the roles of PsbS and Zx? Using this novel approach of adding the spectral information to the NPQ fluorescence changes, we discovered specific spectral changes associated with different NPQ components. By comparing the effects measured on various NPQ mutants of Arabidopsis, it is possible to assign these NPQ components to specific quenching processes. The results provide evidence that the total NPQ is a combination of several parallel and largely independent processes, likely occurring at different locations in the photosynthetic apparatus.     

Dynamics of Xanthophyll-Cycle Activity in Different Antenna Subcomplexes in the Photosynthetic Membranes of Higher Plants (The Relationship between Zeaxanthin Conversion and Nonphotochemical Fluorescence Quenching)

The generation of nonphotochemical quenching of chlorophyll fluorescence (qN) in the antenna of photosystem II (PSII) is accompanied by the de-epoxidation of violaxanthin to antheraxanthin and zeaxanthin. The function of zeaxanthin in two mechanisms of qN, energy-dependent quenching (qE) and photoinhibitory quenching (qI), was investigated by measuring the de-epoxidation state in the antenna subcomplexes of PSII during the generation and relaxation of qN under varying conditions. Three different antenna subcomplexes were separated by isoelectric focusing: Lhcb1/2/3, Lhcb5/6, and the Lhcb4/PSII core. Under all conditions, the highest de-epoxidation state was detected in Lhcb1/2/3 and Lhcb5/6. The kinetics of de-epoxidation in these complexes were found to be similar to the formation of qE. The Lhcb4/PSII core showed the most pronounced differences in the de-epoxidation state when illumination with low and high light intensities was compared, correlating roughly with the differences in qI. Furthermore, the epoxidation kinetics in the Lhcb4/PSII core showed the most pronounced differences of all subcomplexes when comparing the epoxidation after either moderate or very strong photoinhibitory preillumination. Our data support the suggestion that zeaxanthin formation/epoxidation in Lhcb1-3 and Lhcb5/6 may be related to qE, and in Lhcb4 (and/or PSII core) to qI.     

Hill Reaction, Hydrogen Peroxide Scavenging, and Ascorbate Peroxidase Activity of Mesophyll and Bundle Sheath Chloroplasts of NADP-Malic Enzyme Type C(4) Species

Intact mesophyll and bundle sheath chloroplasts wee isolated from the NADP-malic enzyme type C₄ plants maize, sorghum (monocots), and Flaveria trinervia (dicot) using enzymic digestion and mechanical isolation techniques. Bundle sheath chloroplasts of this C₄ subgroup tend to be agranal and were previously reported to be deficient in photosystem II activity. However, following injection of intact bundle sheath chloroplasts into hypotonic medium, thylakoids had high Hill reaction activity, similar to that of mesophyll chloroplasts with the Hill oxidants dichlorophenolindophenol, p-benzoquinone, and ferricyanide (approximately 200 to 300 micromoles O₂ evolved per mg chlorophyll per hour). In comparison to that of mesophyll chloroplasts, the Hill reaction activity of bundle sheath chloroplasts of maize and sorghum was labile and lost activity during assay. Bundle sheath chloroplasts of maize also exhibited some capacity for 3-phosphoglycerate dependent O₂ evolution (29 to 58 micromoles O₂ evolved per milligram chlorophyll per hour). Both the mesophyll and bundle sheath chloroplasts were equally effective in light dependent scavenging of hydrogen peroxide. The results suggest that both chloroplast types have noncyclic electron transport and the enzymology to reduce hydrogen peroxide to water. The activities of ascorbate peroxidase from these chloroplast types was consistent with their capacity to scavenge hydrogen peroxide.     

Thylakoid-Bound Ascorbate Peroxidase in Spinach Chloroplasts and Photoreduction of Its Primary Oxidation Product Monodehydroascorbate Radicals in Thylakoids

Ascorbate peroxidase, a key enzyme for the scavenging of hydrogenperoxide in chloroplasts, was found in a thylakoid-bound formin spinach chloroplasts at comparable activity to that in thestroma. The activity of peroxidase was detectable in the thylakoidsonly when prepared by an ascorbate-containing medium, and enrichedin the stroma thylakoids. The thylakoid enzyme was not releasedfrom the membranes by either 2 mM EDTA, 1 M KCl, 2 M NaBr or2 M NaSCN, but was solubilized by detergents. Enzymatic propertiesof the thylakoid-bound ascorbate peroxidase were very similarto those of the stromal ascorbate peroxidase. Thylakoid-bound ascorbate peroxidase could scavenge the hydrogenperoxide either added or photoproduced by the thylakoids. Nophotoreduction of hydrogen peroxide was observed, however, inthe thylakoids whose ascorbate peroxidase was inhibited by KCNand thiol reagents or inactivated by the treatment with ascorbate-depletion.The primary oxidation product of ascorbate in a reaction ofascorbate peroxidase, monodehydroascorbate (MDA) radical, wasphotoreduced in the thylakoids, as detected by the quenchingof chlorophyll fluorescence, disappearance of EPR signals ofthe MDA radicals and the MDA radical-induced oxygen evolution.Thus, ascorbate is photoregenerated in the thylakoids from theMDA radicals produced in a reaction of ascorbate peroxidasefor the scavenging of hydrogen peroxide. (Received March 26, 1992; Accepted April 22, 1992)     

Possible Role of Cbr,an Algal Early-Light-Induced Protein,in Nonphotochemical Quenching of Chlorophyll Fluorescence

The unicellular green alga Dunaliella bardawil exhibits typical responses to excessive light when starved for sulfate under normal light (60 [mu]E m-2 s-1) but not under low light (14 [mu]E m-2 s-1). Algae were analyzed during several days of sulfate starvation for nonphotochemical quenching of chlorophyll fluorescence in the absence or presence of the uncouplers SF-6847 (SF) or carbonyl cyanide p- trifluoromethoxyphenyl hydrazone. Parallel analyses followed two light-stress responses: (a) violaxanthin conversion to zeaxanthin and (b) accumulation of Cbr, a protein analogous to plant early-light-induced proteins and implicated in zeaxanthin binding. In cells starved under normal light SF inhibited nonphotochemical quenching during the first 24 h, but not from 40 h onward. In cells starved under low light SF inhibited nonphotochemical quenching throughout the starvation period. Under normal light accumulation of zeaxanthin was nearly maximal by 24 h, but Cbr was fully induced only by 40h. Under low light zeaxanthin accumulated slowly but no Cbr was evident. These results suggest that during exposure to excessive light, the initial pH gradient-dependent, Cbr-independent mode of nonphotochemical quenching is modified to become less dependent on pH gradient and requires Cbr.     

Effects of pH and Oxygen on Photosynthetic Reactions of Intact Chloroplasts

Oxygen inhibition of photosynthesis was studied with intact spinach (Spinacia oleracea L.) chloroplasts which exhibited very high rates of photosynthetic CO₂ reduction and were insensitive to additions of photosynthetic intermediates when CO₂ was available at saturating concentrations. Photosynthetic rates were measured polarographically as O₂ evolution, and the extent of the reduction of substrate was estimated from the amount of O₂ evolved. With CO₂ as substrate, inhibition of photosynthesis by O₂ was dependent on pH. At pH values above 8, rates of O₂ evolution were strongly inhibited by O₂ and only a fraction of the added bicarbonate was reduced before O₂ evolution ceased. The extent of O₂ evolution declined with increasing O₂ concentration and decreasing initial bicarbonate concentration. At pH 7.2, the initial photosynthetic rate was inhibited about 30% at high O₂ levels, but the extent of O₂ evolution was unaffected and most of the added bicarbonate was reduced. Photosynthetic O₂ evolution with 3-phosphoglycerate as substrate was similarly dependent on pH and O₂ concentration. In contrast, there was little effect of O₂ and pH on oxaloacetate-dependent oxygen evolution. Acid-base shift experiments with osmotically shocked chloroplasts showed that ATP formation was not affected by O₂. The results are discussed in terms of a balance between photosynthetic O₂ evolution and O₂ consumption by the ribulose diphosphate oxygenase reaction.     

Purification of Ascorbate Peroxidase in Spinach Chloroplasts; Its Inactivation in Ascorbate-Depleted Medium and Reactivation by Monodehydroascorbate Radical

Ascorbate specific peroxidase in chloroplasts was purified fromspinach leaves. Spinach chloroplast peroxidase was a monomerwith a molecular weight of about 30,000 and showed an absorptionspectrum similar to a hemoprotein. The enzyme lost its activitywithin a minute in the absence of ascorbate under aerobic conditions.In addition to ascorbate, 20% sorbitol was necessary to stabilizethe enzyme. The inactivation of the enzyme in the ascorbate-depletedmedium was protected by other electron donors, pyrogallol, guaiacoland pyrocatechol, whose oxidation rates were very low comparedwith that of ascorbate. The inactivated enzyme recovered itsactivity with monodehydroascorbate radicals generated by theascorbate-ascorbate oxidase system. A mechanism of inactivationand reactivation of ascorbate peroxidase is proposed. (Received August 28, 1986; Accepted November 13, 1986)     

琯溪蜜柚汁胞在发育与粒化过程中APX活性变化及其同工酶分析

对不同发育时期的琯溪蜜柚(Citrus grandis'Guanximiyou')汁胞进行APX活性测定及同工酶分析.结果表明,随着汁胞的发育,APX活性增大;而柚子衰老腐烂时,APX活性迅速降低.APX同工酶随着汁胞发育而发生变化,花后150 d的APX同工酶增加了1个组分(迁移率0.66),且随着汁胞的成熟,其表达量增加.花后230 d时柚子开始衰老腐烂,花后242 d已难以看到大部分APX同工酶酶谱.粒化汁胞的APX活性比正常汁胞大,同工酶酶谱亮度和清晰度也大,推测琯溪蜜柚汁胞在粒化过程中APX可清除活性氧61由基,抵抗氧化损伤.     

Cytosolic Ascorbate Peroxidase in Seedlings and Leaves of Maize (Zea mays)

Ascorbate peroxidase (APX) was purified to homogeneity frommaize (Zea mays L. cv.) coleoptiles. APX was a monomer witha molecular mass of 28 kDa, as determined by gel nitration andSDS-polyacrylamide gel electrophoresis. It contained one protohememoiety per molecule, with the oxidized form giving a Soret peakat 403 nm with small peaks at 502 and 638 nm, and the reducedform giving peaks at 435 and 556 nm. The enzyme was not inactivatedby depletion of ascorbate. Cell fractionation and immunohistochemicalstudies using polyclonal antibodies raised against maize APXrevealed that the enzyme was not located in the chloroplastsof green leaves. It was abundant in the cytoplasm but not inthe vacuoles of cells in the coleoptile, mesocotyl and youngleaves of seedlings. In mature green leaves, small amounts ofthe enzyme were distributed in vascular systems, in particularin the companion cells. The N-terminal amino acid sequence ofmaize APX exhibited high homology to pea cytosolic APX, spinachAPX and Arabidopsis APX, but not to APX from tea chloroplasts. (Received February 15, 1993; Accepted May 6, 1993)     

Quantum Yields and Rate Constants of Photochemical and Nonphotochemical Excitation Quenching (Experiment and Model)

Sunflower (Helianthus annuus L.), cotton (Gossypium hirsutum L.), tobacco (Nicotiana tabacum L.), sorghum (Sorghum bicolor Moench.), amaranth (Amaranthus cruentus L.), and cytochrome b6f complex-deficient transgenic tobacco leaves were used to test the response of plants exposed to differnt light intensities and CO2 concentrations before and after photoinhibition at 4000 [mu]mol photons m-2 s-1 and to thermoinhibition up to 45[deg]C. Quantum yields of photochemical and nonphotochemical excitation quenching (YP and YN) and the corresponding relative rate constants for excitation capture from the antenna-primary radical pair equilibrium system (k[prime]P and k[prime]N) were calculated from measured fluorescence parameters. The above treatments resulted in decreases in YP and K[prime]P and in approximately complementary increases in YN and K[prime]N under normal and inhibitory conditions. The results were reproduced by a mathematical model of electron/proton transport and O2 evolution/CO2 assimilation in photosynthesis based on budget equations for the intermediates of photosynthesis. Quantitative differences between model predictions and experiments are explainable, assuming that electron transport is organized into domains that contain relatively complete electron and proton transport chains (e.g. thylakoids). With the complementation that occurs between the photochemical and nonphotochemical excitation quenching, the regulatory system can constantly maintain the shortest lifetime of excitation necessary to avoid the formation of chlorophyll triplet states and singlet oxygen.     

茶树紫黄素脱环氧化酶基因的体外定点突变及其突变体的表达和活性鉴定

经Swiss Prot蛋白质数据库中查询发现茶树紫黄素脱环氧化酶 (VDE)蛋白结构中含有lipocalin特征区 ,利用重叠延伸法把该特征区中最保守的Gly(GGG)和Trp(TGG)分别定点突变为Leu(TTG)和Tyr(TAG) .构建了表达载体pET 32a G→L和pET 32a W→Y ,在E .coliBL2 1trxB(DE3)进行了诱导表达 ,并对表达蛋白进行His Tag亲和纯化 .结果表明 ,表达蛋白的分子量和预计的相等 ,为 5 8 9kD ,表达量占菌体总蛋白的 4 5 % .体外酶促反应分析得出 ,G→L或W→Y突变都能导致茶树VDE生物活性大幅度降低 ,证明了lipocalin特征区是VDE的主要活性中心之一 .     

草酰乙酸和苹果酸对菠菜叶片和完整叶绿体光合作用的影响

观测了OAA和MA对菠菜叶片和完整叶绿体光合作用的影响.结果显示,当叶片切块在20μmol/L的OAA存在时,其叶片的光合放氧速率增加了89%,经OAA处理的离体完整叶绿体的光合放氧速率增加了72%;当反应体系中存在有较高浓度的NaHCO3时,OAA的作用不明显.叶片经20 μmol/L的MA处理后,叶片光合放氧速率比对照高127%.用CO2分析仪观测了处理后叶片的净光合速率(Pn),结果显示,OAA和MA处理后的叶片Pn值分别是对照的117%和111%.对在C3植物中建立C4微循环系统来提高光合作用效率的可能性进行了讨论.     

Multiple Effects of Viscosity,Water Activity and Glass Transition Temperature on Peroxidase Activity in Binary and Ternary Carbohydrate Solutions

The individual and combined effects of water activity (a_w), bulk viscosity and glass transition temperature (Tg’) on the activity of horseradish peroxidase (HRP) in buffered sugars (glucose, trehalose and maltose) and maltodextrin solutions were investigated. Viscosity was the most important factor in the inhibition of HRP activity; however, when Tg’ was changed by the using solutes with different molecular weight, it became a key factor in the modulation of enzyme activity. Viscosity being equal, the sugar addition to maltodextrin solution lowered a_w and lowered Tg’ causing an increase of the enzymatic activity. Nevertheless, an inhibition of the HRP activity occurred when a_w values of 0.87 were reached due to the addition of glucose, which, among the tested sugars, showed the lowest molecular weight. Among disaccharides, maltose was more effective than trehalose in impairing the enzyme activity both in binary and ternary systems, and this is due to a non competitive biochemical inhibition exerted by this sugar on HRP. When compared to glucose, maltose and trehalose were more effective in reducing HRP activity only in the low viscosity range whilst in the high viscosity range (1–4 10^?6 m² s^?1) glucose, despite its lower Tg’ value, was slightly more efficient than disaccharides due to its a_w lowering effect.     

Influence of Water Activity and System Mobility on Peroxidase Activity in Maltodextrin Solutions

Maltodextrins influenced the enzymatic activity in aqueous solutions by affecting the water activity (a_w) and mobility as described by viscosity and T’g. In diluted solutions, viscosity being equal, (1) maltodextrin with dextrose equivalents (DE) of 33 was more effective than glucose in limiting horseradish peroxidase (HRP) activity; (2) an increase in the maltodextrin chain length from DE 33 to DE 8.7 did not further limited enzymatic activity; (3) the maltodextrin with the highest chain length (DE 2.5) determined the highest enzyme inhibition. In general, the increase of molecular weight negatively affected the HRP activity by increasing the viscosity and T’g (decreasing molecular mobility) but it positively affected the a_w and, in some cases, this compensated the HRP activity inhibition. In concentrated solutions (apparent viscosity ≈ 40 mPa s) the HRP activity decreased with the increase of the maltodextrin molecular weight, and it showed a dependence on T’g which could be described by a William, Landel and Ferry (WLF)-type equation. On the contrary, in the solution added with the maltodextrin with the highest chain length (DE 2.5) the HRP activity was much higher than that predicted by the WLF-type equation. The maltodextrin with DE 2.5 contains intact starch fragments and in water forms a suspension. In such a discontinuous system, the viscosity in the vicinity of the protein is lower than the bulk viscosity, and thus, the enzyme activity is higher than expected. Moreover, since T’g is a property of the soluble phase, it does not explain the mobility in discontinuous systems.     

Fluorescence and Photosynthetic Activity of Chloroplasts in Acid and Alkaline Zones of Chara corallina

Continuous profiles of local pH near the cell surface of Chara corallinawere recorded during uniform longitudinal movement of an internodal cell relative to a stationary pH microelectrode. Under illumination, the pH profile consisted of alternating acid and alkaline bands with a pH difference of up to 3 pH units. After darkening, the bands disappeared and pH became uniformly distributed along the cell length. Chlorophyll fluorescence of chloroplasts was measured by microfluorometry at different locations within one cell, and significant differences were observed in close relation to light-dependent pH banding. The chlorophyll fluorescence yield was lower in zones of low external pH than in alkaline zones both under actinic and saturating light. The fluorescence parameters Fand F" _m and the quantum yield of photosystem II (PSII) displayed variations along the cell length in accordance with pH changes in unstirred layers of the medium. The results show that PSII photochemical efficiency and the rate of noncyclic electron transport are higher in the chloroplasts of acid zones (zones of H⁺extrusion from the cell) than in alkaline zones. The dependence of photosynthetic electron transport on local pH near the cell surface may result from different contents of CO₂in acid and alkaline regions. The acid zones are enriched with CO₂that readily permeates through the membrane providing the substrate for the Calvin cycle. Conversely, a poorly permeating form, HCO^– ₃is predominant in alkaline zones, which may restrict the dark reactions and photosynthetic electron flow.