Time-resolution of the antheraxanthin- and delta pH-dependent chlorophyll a fluorescence components associated with photosystem II energy dissipation in Mantoniella squamata.

The electronic excited-state behavior of photosystem II (PSII) in Mantoniella squamata, as influenced by the xanthophyll cycle and the transthylakoid pH gradient (delta pH), was examined in vivo. Mantoniella is distinguished from other photosynthetic organisms by two main features namely (1) a unique light-harvesting complex that serves both photosystems I (PSI) and II (PSII); and (2) a violaxanthin (V) cycle that undergoes only one de-epoxidation step in excess light to accumulate the monoepoxide antheraxanthin (A) as opposed to the epoxide-free zeaxanthin (Z). The cells were treated first with high light to induce the delta pH and A accumulation, followed by herbicide-induced closure of PSII traps and a chilling treatment, to sustain and stabilize the delta pH and nigericin-sensitive fluorescence level in the dark. De-epoxidation was controlled with subsaturating concentrations of dithiothreitol (DTT) and was 5-10 times more sensitive to DTT than higher plant thylakoids. The PSII energy dissipation involved two steps: (1) the pH activation of the xanthophyll binding site that was associated with a narrowing and slight attenuation of the main 2 ns (ns = 10(-9) s) fluorescence lifetime distribution; and (2) the concentration-dependent binding of A to the activated binding site yielding a second distribution centered around 0.9 ns. Consistent with the model of Gilmore et al. (1998) (Biochemistry 37, 13,582-13,593), the fractional intensity of the 0.9 ns component depended almost entirely on the A concentration and correlated linearly with the decrease of the steady-state chlorophyll alpha fluorescence intensity.

Xanthophyll cycle-dependent nonphotochemical quenching in Photosystem II: Mechanistic insights gained from Arabidopsis thaliana L. mutants that lack violaxanthin deepoxidase activity and/or lutein.

This study compares Photosystem II (PS II) chlorophyll (Chl) a fluorescence yield changes of Arabidopsis thaliana L. nuclear gene mutants, thoughtfully provided by the authors of Pogson et al. (1998 Proc Natl Acad Sci USA 95: 13324-13329). One single mutant (npq1) inhibits the violaxanthin deepoxidase that converts violaxanthin to antheraxanthin and zeaxanthin. A second single mutant (lut2) inhibits the in-cyclization enzyme step between lycopene and beta,in-carotene causing accumulation of beta,beta-carotene derivatives, primarily the violaxanthin cycle pigments, at the expense of lutein. The double mutant (lut2-npq1) incorporates both lesions. PS II Chl a fluorescence was characterized in leaves and thylakoids using both steady state and time-resolved methods, the intrathylakoid pH was estimated by 9-aminoacridine fluorescence quenching and chloroplast pigments were determined by HPLC. Under maximal PS II Chl a fluorescence intensity conditions without intrathylakoid acidification, the main 2 nanosecond (ns) fluorescence lifetime distribution mode parameters were similar for the WT and mutants both before and after illumination. The light and ATPase mediated intrathylakoid pH levels were also similar and caused similar changes in the fluorescence lifetime distribution widths and centers for the WT and each mutant. The npq1 exhibited low antheraxanthin and zeaxanthin and high violaxanthin levels and the uncoupler-sensitive amplitudes of short (< 1 ns) PS II Chl a fluorescence distribution modes were strongly inhibited compared to the WT. Lutein deficiency coincided with pleiotropic effects on PS II energy dissipation and probably altered conformations of PS II carotenoid-chlorophyll binding proteins. The lut2 exhibited separate active and inactive pools of antheraxanthin and zeaxanthin with respect to all deepoxidation, epoxidation and fluorescence quenching activities. The active xanthophyll cycle pool in lut2 exhibited a lower ( approximately 35% of WT) concentration efficiency, for a given intrathylakoid pH, to increase the sub-nanosecond distribution amplitudes, which predicts and explains inhibited induction kinetics and fluorescence quenching. The lut2-npq1 mutant exhibited a constant pool of antheraxanthin and zeaxanthin, no deepoxidation and little or no pH-reversible fluorescence decrease. It is concluded that in addition to intrathylakoid acidification, a certain level of zeaxanthin and antheraxanthin (or lutein) is absolutely required for the major reversible component of PS II Chl a fluorescence quenching.

Protection and storage of chlorophyll in overwintering evergreens.

How evergreen species store and protect chlorophyll during exposure to high light in winter remains unexplained. This study reveals that the evergreen snow gum (Eucalyptus pauciflora Sieb. ex Spreng.) stores and protects its chlorophylls by forming special complexes that are unique to the winter-acclimated state. Our in vivo spectral and kinetic characterizations reveal a prominent component of the chlorophyll fluorescence spectrum around 715 nm at 77 K. This band coincides structurally with a loss of chlorophyll and an increase in energy-dissipating carotenoids. Functionally, the band coincides with an increased capacity to dissipate excess light energy, absorbed by the chlorophylls, as heat without intrathylakoid acidification. The increased heat dissipation helps protect the chlorophylls from photo-oxidative bleaching and thereby facilitates rapid recovery of photosynthesis in spring.

Quantitative analysis of the effects of intrathylakoid pH and xanthophyll cycle pigments on chlorophyll a fluorescence lifetime distributions and intensity in thylakoids.

The xanthophyll cycle-dependent dissipation of excitation energy in higher plants is one of the most important regulatory and photoprotective mechanisms in photosynthesis. Using parallel time-resolved and pulse-amplitude modulation fluorometry, we studied the influence of the intrathylakoid pH and the xanthophyll cycle carotenoids on the PSII chlorophyll (Chl) a fluorescence yield in thylakoids of Arabidopsis, spinach, and barley. Increases in concentrations of dithiothreitol in thylakoids, which have a trans-thylakoid membrane pH gradient and are known to have decreased conversion of violaxanthin (V) to zeaxanthin (Z), lead to (1) decreases in the fractional intensity of the approximately 0.5 ns Chl a fluorescence lifetime (tau) distribution component and simultaneous increases in a 1.6-1.8 ns fluorescence component and (2) increases in the maximal fluorescence intensity. These effects disappear when the pH gradient is eliminated by the addition of nigericin. To quantitatively explain these results, we present a new mathematical model that describes the simultaneous effects of the chloroplast trans-thylakoid membrane pH gradient and xanthophyll cycle pigments on the PSII Chl a fluorescence tau distributions and intensity. The model assumes that (1) there exists a specific binding site for Z (or antheraxanthin, A) among or in an inner antenna complex (primarily CP29), (2) this binding site is activated by a low intrathylakoid pH (pK approximately 4.5) that increases the affinity for Z (or A), (3) about one Z or A molecule binds to the activated site, and (4) this binding effectively "switches" the fluorescence tau distribution of the PSII unit to a state with a decreased fluorescence tau and emission intensity (a "dimmer switch" concept). This binding is suggested to cause the formation of an exciton trap with a rapid intrinsic rate constant of heat dissipation. Statistical analysis of the data yields an equilibrium association constant, Ka, that ranges from 0.7 to 3.4 per PSII for the protonated/activated binding site for Z (or A). The model explains (1) the relative fraction of the approximately 0.5 ns fluorescence component as a function of both Z and A concentration and intrathylakoid pH, (2) the dependence of the ratio of F'm/Fm on the fraction of the 0.5 ns fluorescence tau component (where F'm and Fm are maximal fluorescence intensities in the presence and the absence of a pH gradient), and (3) the dependence of the ratio of F'm/Fm on the concentration of Z and A and the intrathylakoid pH.

Comparative time-resolved photosystem II chlorophyll a fluorescence analyses reveal distinctive differences between photoinhibitory reaction center damage and xanthophyll cycle-dependent energy dissipation.

The photosystem II (PSII) reaction center in higher plants is susceptible to photoinhibitory molecular damage of its component pigments and proteins upon prolonged exposure to excess light in air. Higher plants have a limited capacity to avoid such damage through dissipation, as heat, of excess absorbed light energy in the PSII light-harvesting antenna. The most important photoprotective heat dissipation mechanism, induced under excess light conditions, includes a concerted effect of the trans-thylakoid pH gradient (delta pH) and the carotenoid pigment interconversions of the xanthophyll cycle. Coincidentally, both the photoprotective mechanism and photoinhibitory PSII damage decrease the PSII chlorophyll a (Chl a) fluorescence yield. In this paper we present a comparative fluorescence lifetime analysis of the xanthophyll cycle- and photoinhibition-dependent changes in PSII Chl a fluorescence. We analyze multifrequency phase and modulation data using both multicomponent exponential and bimodal Lorentzian fluorescence lifetime distribution models; further, the lifetime data were obtained in parallel with the steady-state fluorescence intensity. The photoinhibition was characterized by a progressive decrease in the center of the main fluorescence lifetime distribution from approximately 2 ns to approximately 0.5 ns after 90 min of high light exposure. The damaging effects were consistent with an increased nonradiative decay path for the charge-separated state of the PSII reaction center. In contrast, the delta pH and xanthophyll cycle had concerted minor and major effects, respectively, on the PSII fluorescence lifetimes and intensity (Gilmore et al., 1996, Photosynth. Res., in press). The minor change decreased both the width and lifetime center of the longest lifetime distribution; we suggest that this change is associated with the delta pH-induced activation step, needed for binding of the deepoxidized xanthophyll cycle pigments. The major change increased the fractional intensity of a short lifetime distribution at the expense of a longer lifetime distribution; we suggest that this change is related to the concentration-dependent binding of the deepoxidized xanthophylls in the PSII inner antenna. Further, both the photoinhibition and xanthophyll cycle mechanisms had different effects on the relationship between the fluorescence lifetimes and intensity. The observed differences between the xanthophyll cycle and photoinhibition mechanisms confirm and extend our current basic model of PSII exciton dynamics, structure and function.

Carotenoids 3: in vivo function of carotenoids in higher plants.

The function of the long-chain, highly unsaturated carotenoids of higher plants in photoprotection is becoming increasingly well understood, while at the same time their function in other processes, such as light collection, needs to be reexamined. Recent progress in this area has been fueled by more accurate determinations of the photophysical properties of these molecules, as well as extensive characterization of the physiology and ecology of a particular group of carotenoids, those of the xanthophyll cycle, that play a key role in the photoprotection of photosynthesis under environmental stress. The deepoxidized xanthophylls zeaxanthin and antheraxanthin, together with a low pH within the photosynthetic membrane, facilitate the harmless dissipation of excess excitation energy directly within the light-collecting chlorophyll antennae. Evidence for this function as well as current contrasting hypotheses concerning its molecular mechanism are reviewed. In addition, the acclimation of the xanthophyll cycle content and composition of leaves to contrasting environments with different demands for photoprotection is summarized.

Photosystem II chlorophyll a fluorescence lifetimes and intensity are independent of the antenna size differences between barley wild-type and chlorina mutants: Photochemical quenching and xanthophyll cycle-dependent nonphotochemical quenching of fluorescence.

Photosystem II (PS II) chlorophyll (Chl) a fluorescence lifetimes were measured in thylakoids and leaves of barley wild-type and chlorina f104 and f2 mutants to determine the effects of the PS II Chl a+b antenna size on the deexcitation of absorbed light energy. These barley chlorina mutants have drastically reduced levels of PS II light-harvesting Chls and pigment-proteins when compared to wild-type plants. However, the mutant and wild-type PS II Chl a fluorescence lifetimes and intensity parameters were remarkably similar and thus independent of the PS II light-harvesting antenna size for both maximal (at minimum Chl fluorescence level, Fo) and minimal rates of PS II photochemistry (at maximum Chl fluorescence level, Fm). Further, the fluorescence lifetimes and intensity parameters, as affected by the trans-thylakoid membrane pH gradient (ΔpH) and the carotenoid pigments of the xanthophyll cycle, were also similar and independent of the antenna size differences. In the presence of a ΔpH, the xanthophyll cycle-dependent processes increased the fractional intensity of a Chl a fluorescence lifetime distribution centered around 0.4-0.5 ns, at the expense of a 1.6 ns lifetime distribution (see Gilmore et al. (1995) Proc Natl Acad Sci USA 92: 2273-2277). When the zeaxanthin and antheraxanthin concentrations were measured relative to the number of PS II reaction center units, the ratios of fluorescence quenching to [xanthophyll] were similar between the wild-type and chlorina f104. However, the chlorina f104, compared to the wild-type, required around 2.5 times higher concentrations of these xanthophylls relative to Chl a+b to obtain the same levels of xanthophyll cycle-dependent fluorescence quenching. We thus suggest that, at a constant ΔpH, the fraction of the short lifetime distribution is determined by the concentration and thus binding frequency of the xanthophylls in the PS II inner antenna. The ΔpH also affected both the widths and centers of the lifetime distributions independent of the xanthophyll cycle. We suggest that the combined effects of the xanthophyll cycle and ΔpH cause major conformational changes in the pigment-protein complexes of the PS II inner or core antennae that switch a normal PS II unit to an increased rate constant of heat dissipation. We discuss a model of the PS II photochemical apparatus where PS II photochemistry and xanthophyll cycle-dependent energy dissipation are independent of the Peripheral antenna size.

Xanthophyll cycle-dependent quenching of photosystem II chlorophyll a fluorescence: formation of a quenching complex with a short fluorescence lifetime.

Excess light triggers protective nonradiative dissipation of excitation energy in photosystem II through the formation of a trans-thylakoid pH gradient that in turn stimulates formation of zeaxanthin and antheraxanthin. These xanthophylls when combined with protonation of antenna pigment-protein complexes may increase nonradiative dissipation and, thus, quench chlorophyll a fluorescence. Here we measured, in parallel, the chlorophyll a fluorescence lifetime and intensity to understand the mechanism of this process. Increasing the xanthophyll concentration in the presence of a pH gradient (quenched conditions) decreases the fractional intensity of a fluorescence lifetime component centered at approximately 2 ns and increases a component at approximately 0.4 ns. Uncoupling the pH gradient (unquenched conditions) eliminates the 0.4-ns component. Changes in the xanthophyll concentration do not significantly affect the fluorescence lifetimes in either the quenched or unquenched sample conditions. However, there are differences in fluorescence lifetimes between the quenched and unquenched states that are due to pH-related, but nonxanthophyll-related, processes. Quenching of the maximal fluorescence intensity correlates with both the xanthophyll concentration and the fractional intensity of the 0.4-ns component. The unchanged fluorescence lifetimes and the proportional quenching of the maximal and dark-level fluorescence intensities indicate that the xanthophylls act on antenna, not reaction center processes. Further, the fluorescence quenching is interpreted as the combined effect of the pH gradient and xanthophyll concentration, resulting in the formation of a quenching complex with a short (approximately 0.4 ns) fluorescence lifetime.

Intrathylakoid pH in Isolated Pea Chloroplasts as Probed by Violaxanthin Deepoxidation.

Light-driven violaxanthin deepoxidation was measured in isolated pea (Pisum sativum) chloroplasts without ATP synthesis (basal conditions) and with ATP synthesis (coupled conditions). Thylakoids stored in high salt (HS) or low salt (LS) storage medium were tested. In previous experiments, HS thylakoids and LS thylakoids were related to delocalized and localized proton coupling, respectively.Light-driven deepoxidase activity was compared to the pH dependence of deepoxidase activity established in dark reactions. At an external pH of 8, light-driven deepoxidation indicated effective pH values close to pH 6 for all reaction conditions. Parallel to deepoxidation, the thylakoid lumen pH was estimated by the fluorescent dye pyranine.In LS thylakoids under coupled conditions the lumen pH did not drop below pH 6.7. At pH 6.7, no deepoxidase activity is expected based on the pH dependence of enzyme activity. The results suggest that deepoxidation activity is controlled by the pH in sequestered membrane domains, which, under localized proton coupling, can be maintained at pH 6.0 when the lumen pH is far above pH 6.0. The extent of violaxanthin conversion (availability), however, appeared to be regulated by lumenal pH. Dithiothreitol-sensitive nonphotochemical quenching of chlorophyll fluorescence was dependent on zeaxanthin and not related to lumenal pH. Thus, zeaxanthin-dependent quenching[mdash]known to be pH dependent[mdash]appeared to be triggered by the pH of localized membrane domains.

Epoxidation of zeaxanthin and antheraxanthin reverses non-photochemical quenching of photosystem II chlorophyll a fluorescence in the presence of trans-thylakoid delta pH.

The xanthophyll cycle apparently aids the photoprotection of photosystem II by regulating the nonradiative dissipation of excess absorbed light energy as heat. However, it is a controversial question whether the resulting nonphotochemical quenching is soley dependent on xanthophyll cycle activity or not. The xanthophyll cycle consists of two enzymic reactions, namely deepoxidation of the diepoxide violaxanthin to the epoxide-free zeaxanthin and the much slower, reverse process of epoxidation. While deepoxidation requires a transthylakoid pH gradient (delta pH), epoxidation can proceed irrespective of a delta pH. Herein, we compared the extent and kinetics of deepoxidation and epoxidation to the changes in fluorescence in the presence of a light-induced thylakoid delta pH. We show that epoxidation reverses fluorescence quenching without affecting thylakoid delta pH. These results suggest that epoxidase activity reverses quenching by removing deepoxidized xanthophyll cycle pigments from quenching complexes and converting them to a nonquenching form. The transmembrane organization of the xanthophyll cycle influences the localization and the availability of deepoxidized xanthophylls is to support nonphotochemical quenching capacity. The results confirm the view that rapidly reversible nonphotochemical quenching is dependent on deepoxidized xanthophyll.

Linear models relating xanthophylls and lumen acidity to non-photochemical fluorescence quenching. Evidence that antheraxanthin explains zeaxanthin-independent quenching.

Zeaxanthin has been correlated with high-energy non-photochemical fluorescence quenching but whether antheraxanthin, the intermediate in the pathway from violaxanthin to zeaxanthin, also relates to quenching is unknown. The relationships of zeaxanthin, antheraxanthin and ΔpH to fluorescence quenching were examined in chloroplasts ofPisum sativum L. cv. Oregon andLactuca sativa L. cv. Romaine. Data matrices as five levels of violaxanthin de-epoxidation against five levels of light-induced lumen-proton concentrations were obtained for both species. The matrices included high levels of antheraxanthin as well as lumen-proton concentrations induced by subsaturating to saturation light levels. Analyses of the matrices by simple linear and multiple regression showed that quenching is predicted by models where the major independent variable is the product of lumen acidity and de-epoxidized xanthophylls, the latter as the sum of zeaxanthin and antheraxanthin. The interactions of lumen acidity and xanthophyll concentration are shown in three-dimensional plots of the best-fit multiple regression models. Antheraxanthin apparently contributes to quenching as effectively as zeaxanthin and explains quenching previously not accounted for by zeaxanthin. Hence, we propose that all high-energy dependent quenching is xanthophyll dependent. Quenching requires a threshold lumen pH that varies with xanthophyll composition. After the threshold, quenching is linear with lumen acidity or xanthophyll composition.

Dark induction of zeaxanthin-dependent nonphotochemical fluorescence quenching mediated by ATP.

Zeaxanthin-dependent nonphotochemical fluorescence quenching is a light-induced activity in plants that apparently protects against the potentially damaging effects of excess light. We report a dark-induced nonphotochemical quenching in thylakoids of Lactuca sativa L. cv. Romaine mediated by ATP. This effect is due to low lumen pH from hydrolysis-dependent proton pumping and hence required an active ATPase. The induction was optimal at 0.3 mM ATP, a physiological concentration, and occurred under conditions of little or no reverse electron flow. The properties of ATP-induced quenching were in all respects examined similar to light-induced quenching, including antimycin inhibition of quenching induction but not delta pH. We conclude that zeaxanthin-dependent quenching depends directly on lumen pH and that the role of light is indirect. Although it is known that zeaxanthin and low lumen pH are insufficient for quenching to occur, the results apparently exclude the redox state of an electron-transport carrier or formation of light-induced carotenoid triplets as a further requirement. We propose that a slow pH-dependent conformational change together with zeaxanthin cause static quenching in the pigment bed; possibly antimycin inhibits this change. Furthermore, we suggest from the ability of ATP to sustain quenching in the dark for extended periods that persistent or slowly reversible zeaxanthin quenching often observed in vivo may be due to sustained delta pH from ATP hydrolysis.

Zeaxanthin Formation and Energy-Dependent Fluorescence Quenching in Pea Chloroplasts under Artificially Mediated Linear and Cyclic Electron Transport.

Artificially mediated linear (methylviologen) and cyclic (phenazine methosulfate) electron transport induced zeaxanthin-dependent and independent (constitutive) nonphotochemical quenching in osmotically shocked chloroplasts of pea (Pisum sativum L. cv Oregon). Nonphotochemical quenching was quantitated as Stern-Volmer quenching (SV(N)) calculated as (F(m)/F'(m))-1 where F(m) is the fluorescence intensity with all PSII reaction centers closed in a nonenergized, dark-adapted state and F'(m) is the fluorescence intensity with all PSII reaction centers closed in an energized state. Reversal of quenching by nigericin and electron-transport inhibitors showed that both quenching types were energy-dependent SV(N). Under light-induced saturating DeltapH, constitutive-SV(N) reached steady-state in about 1 minute whereas zeaxanthin-SV(N) continued to develop for several minutes in parallel with the slow kinetics of violaxanthin deepoxidation. SV(N) above the constitutive level and relative zeaxanthin concentration showed high linear correlations at steady-state and during induction. Furthermore, F(o) quenching, also treated as Stern-Volmer quenching (SV(O)) and calculated as (F(o)/F'(o))-1, showed high correlation with zeaxanthin and consequently with SV(N) (F(o) and F'(o) are fluorescence intensities with all PSII reaction centers in nonenergized and energized states, respectively). These results support the view that zeaxanthin increases SV(N) above the constitutive level in a concentration-dependent manner and that zeaxanthin-dependent SV(N) occurs in the pigment bed. Preforming zeaxanthin increased the rate and extent of SV(N), indicating that slow events other than the amount of zeaxanthin also affect final zeaxanthin-SV(N) expression. The redox state of the primary electron acceptor of photosystem II did not appear to determine SV(N). Antimycin, when added while chloroplasts were in a dark-adapted or nonenergized state, inhibited both zeaxanthin-SV(N) and constitutive-SV(N) induced by linear and cyclic electron transport. These similarities, including possible constitutive F(o) quenching, suggest that zeaxanthin-dependent and constitutive SV(N) are mechanistically related.

The reliability of 'expert judgement' in the marking of state nursing examinations.

A pilot study was undertaken to investigate the reliability of the 'expert judgement' exercised in marking state examination papers. Estimates of inter-rater reliability had a median coefficient of 0.76. Intra-rater reliability was marginally higher overall, with a median coefficient of 0.81. While marker reliability is only one source of variation in the assessment of extended answers, it is important the level be known. This study provides useful information and guidelines for improvements.