ALBERT

Notes: Higher plants must dissipate absorbed light energy that exceeds the photosynthetic capacity to avoid molecular damage to the pigments and proteins that comprise the photosynthetic apparatus. Described in this minireview is a current view of the biochemical, biophysical and bioenergetic aspects of the primary photoprotective mechanism responsible for dissipating excess excitation energy as heat from photosystem II (PSII). The photoprotective heat dissipation is measured as nonphotochemical quenching (NPQ) of the PSII chlorophyll a (Chl a) fluorescence. The NPQ mechanism is controlled by the trans-thylakoid membrane pH gradient (ΔpH) and the special xanthophyll cycle pigments. In the NPQ mechanism, the de-epoxidized endgroup moieties and the trans-thylakoid membrane orientations of antheraxanthin (A) and zeaxanthin (Z) strongly affect their interactions with protonated chlorophyll binding proteins (CPs) of the PSII inner antenna. The CP protonation sites and steps are influenced by proton domains sequestered within the proteo-lipid core of the thylakoid membrane. Xanthophyll cycle enrichment around the CPs may explain why changes in the peripheral PSII antenna size do not necessarily affect either the concentration of the xanthophyll cycle pigments on a per PSII unit basis or the NPQ mechanism. Recent time-resolved PSII Chi a fluorescence studies suggest the NPQ mechanism switches PSII units to an increased rate constant of heat dissipation in a series of steps that include xanthophyll de-epoxidation, CP-protonation and binding of the xanthophylls to the protonated CPs; the concerted process can be described with a simple two-step, pH-activation model. The xanthophyll cycle-dependent NPQ mechanism is profoundly influenced by temperatures suboptimal for photosynthesis via their effects on the trans-thylakoid membrane energy coupling system. Further, low temperature effects can be grouped into either short term (minutes to hours) or long term (days to seasonal) series of changes in the content and composition of the PSII pigment-proteins. This minireview concludes by briefly highlighting primary areas of future research interest regarding the NPQ mechanism.

Notes: Abstract The effects of varying the steady-state rate of non-cyclic photosynthetic electron transport on the leaf adenylate energy charge and the epoxidation state of the xanthophyll-cycle pigments were determined in leaves of cotton (Gossypium hirsutum L.) and the mangrove (Aegialitis annulata R.Br.). Different photosynthetic rates were obtained by varying the intercellular CO2 concentration and/or the leaf temperature, and in some cases, by changing the leaf conductance to CO2 diffusion. Also determined were the effects of these treatments on the changes in the adenylate energy charge and the epoxidation state of the xanthophyll-cycle pigments that occur after darkening of the leaves. The leaf adenylate pool remained close to equilibrium with the adenylate kinase both in the light at steady state and during dark relaxation. The adenylate energy charge increased as the photosynthetic rate decreased and maximal levels were obtained when CO2 assimilation and, therefore, non-cyclic electron flow were maximally inhibited. This implies that, in nature, photophosphorylation may provide energy needed for ion-pumping and biosynthetic and repair processes, even under stress conditions that severely restrict or prevent photosynthetic gas exchange. High levels of de-epoxidized violaxanthin in the light did not necessarily indicate or depend on a high adenylate energy charge. Dithiothreitol, an inhibitor of the violaxanthin de-epoxidase a nd ascorbate peroxidase, did not inhibit the adenylate energy charge in the light. Thus we conclude that coupled electron transport during inhibited CO2 fixation was not driven by a dithiothreitol-sensitive Mehler ascorbate-peroxidase reaction. The changes in the adenylate energy charge and xanthophyll re-epoxidation that follow when leaves were darkened are strongly affected by the preceding photosynthetic rate. Postillumination fluctuations in adenylate energy charge, both at 15 ° and 27 °C, were most pronounced when the preceding photosynthetic rate was minimal and least pronounced when this rate was maximal. Temperature had a considerably greater influence in the dark on xanthophyll re-epoxidation than on the pattern of adenylate relaxation.

Notes: Abstract The relationships among the leaf adenylate energy charge, the xanthophyll-cycle components, and photosystem II (PSII) fluorescence quenching were determined in leaves of cotton (Gossypium hirsutum L. cv. Acala) under different leaf temperatures and different intercellular CO2 concentrations (Ci). Attenuating the rate of photosynthesis by lowering the Ci at a given temperature and photon flux density increased the concentration of high-energy adenylate phosphate bonds (adenylate energy charge) in the cell by restricting ATP consumption (A.M. Gilmore, O. Björkman 1994, Planta 192, 526–536). In this study we show that decreases in photosynthesis and increases in the adenylate energy charge at steady state were both correlated with decreases in PSII photo-chemical efficiency as determined by chlorophyll fluorescence analysis. Attenuating photosynthesis by decreasing Ci also stimulated violaxanthin-de-epoxidation-dependent nonradiative dissipation (NRD) of excess energy in PSII, measured by nonphotochemical fluorescence quenching. However, high NRD levels, which indicate a large trans-thylakoid proton gradient, were not dependent on a high adenylate energy charge, especially at low temperatures. Moreover, dithiothreitol at concentrations sufficient to fully inhibit violaxanthin de-epoxidation and strongly inhibit NRD, affected neither the increased adenylate energy charge nor the decreased PSII photo-chemical efficiency that result from inhibiting photosynthesis. The build-up of a high adenylate energy charge in the light that took place at low Ci and low temperatures was accompanied by a slowing of the relaxation of non-photochemical fluorescence quenching after darkening. This slowly relaxing component of nonphotochemical quenching was also correlated with a sustained high adenylate energy charge in the dark. These results indicate that hydrolysis of ATP that accumulated in the light may acidify the lumen and thus sustain the level of NRD for extended periods after darkening the leaf. Hence, sustained nonphotochemical quenching often observed in leaves subjected to stress, rather than being indicative of photoinhibitory damage, apparently reflects the continued operation of NRD, a photoprotective process.

Notes: Abstract The effects of temperature on the dark relaxation kinetics of nonradiative energy dissipation in photosystem II were compared in lettuce (Lactuca sativa L.) chloroplasts and leaves of Aegialitis annulata R. Br. After high levels of violaxanthin de-epoxidation in the light, Aegialitis leaves showed a marked delay in the dark relaxation of nonradiative dissipation, measured as non-photochemical quenching (NPQ) of photosystem II chlorophyll a fluorescence. Aegialitis leaves also maintained a moderately high adenylate energy charge at low temperatures during and after high-light exposure, presumably because of their limited carbon-fixation capacity. Similarly, dark-sustained NPQ could be induced in lettuce chloroplasts after de-epoxidizing violaxanthin and light-activating the ATP synthase. The duration and extent of dark-sustained NPQ were strongly enhanced by low temperatures in both chloroplasts and leaves. Further, the NPQ sustained at low temperatures was rapidly reversed upon warming. In lettuce chloroplasts, low temperatures sharply decreased the ATP-hydrolysis rate while increasing the duration and extent of the resultant trans-thylakoid proton gradient that elicits the NPQ. This was consistent with a higher degree of energy-coupling, presumably due to reduced proton diffusion through the thylakoid membrane at the lower temperatures. The chloroplast adenylate pool was in equilibrium with the adenylate kinase and therefore both ATP and ADP contributed to reverse coupling. The low-temperature-enhanced NPQ quenched the yields of the dark level (Fo) and the maximal (Fm) fluorescence proportionally in both chloroplasts and leaves. The extent of NPQ in the dark was inversely related to the efficiency of photosystem II, and very similar linear relationships were obtained over a wide temperature range in both chloroplasts and leaves. Likewise, the dark-sustained absorbance changes, caused by violaxanthin de-epoxidation (A508nm) and energy-dependent light scattering (A536nm) were strikingly similar in chloroplasts and leaves. Therefore, we conclude that the dark-sustained, low-temperature-stimulated NPQ in chloroplasts and leaves is apparently directly dependent on lumen acidification and chloroplastic ATP hydrolysis. In leaves, the ATP required for sustained NPQ is evidently provided by oxidative phosphorylation in the mitochondria. The functional significance of this quenching process and implications for measurements of photo-protection versus photodamage in leaves are discussed.

Notes: Abstract This study concerns measurements and interpretations of the trans-thylakoid membrane pH gradient, ΔpH, and xanthophyll cycle-dependent energy dissipation in Photosystem II (PS II). Compared and contrasted are the concentration-dependent inhibitory effects and interactions between two lipophilic tertiary amines, namely, 9-aminoacridine the ΔpH indicator and dibucaine a local anesthetic reported to inhibit both the ΔpH and xanthophyll cycle deepoxidation. Chlorophyll a fluorescence monitored both electron transport efficiency and xanthophyll cycle-dependent energy dissipation, high-performance liquid chromatography monitored deepoxidase and chloroplast ATPase activities and steady-state fluorescence monitored various activities of the amines in solution. Low concentrations (up to 2 μM) of both 9-aminoacridine and dibucaine showed similar fluorescence properties and ΔpH-dependent uptake into thylakoids. Importantly both amines exhibited mutually competitive inhibitory effects with respect to this ΔpH-dependent uptake and fluorescence quenching. The fluorescence yields of both compounds in aqueous solution were strongly quenched by sodium ascorbate, a necessary cofactor for in vitro deepoxidation. Both compounds similarly inhibited several light induced activities including deepoxidation, photosynthetic electron transport and PS II energy dissipation. However, for all these activities 9-aminoacridine was 2 to 5 times more potent. Importantly, 9-aminoacridine inhibited deepoxidation with an I50≈1 μM, a concentration far below that which inhibits the ΔpH, ATP synthesis/hydrolysis or electron transport. The inhibitory effects of both compounds on PS II energy dissipation were exerted at 3 to 5 times lower concentration if added before as opposed to after a saturating level of deepoxidation. This result confirms the important role for deepoxidation in mediating PS II energy dissipation. Compared to 9-aminoacridine and in contrast to similar effects on the light-induced activities, dibucaine exhibited significantly different inhibitory effects on ATPase activity and ATPase mediated PS II energy dissipation. However, we conclude from the more potent inhibition by 9-aminoacridine and the similar inhibitory patterns of all the light-induced activities that neither 9-aminoacridine nor dibucaine possess unique capacities to neutralize the light-mediated ΔpH. DCMU–3-(3,4-dichlorophenyl)-1,1-dimethylurea; DTT–dithiothreitol; fx–fractional intensity of fluorescence lifetime component x; F(′)m–maximal PS II Chl a fluorescence intensity with all QA reduced in the absence (presence) of thylakoid membrane energization; Fo–minimal PS II Chl a fluorescence intensity with all QA oxi dized; Fs–steady state PS II Chl a fluorescence; HPLC–high performance liquid chromatography; I(o)–intensity of fluorescence in the presence (absence) of quencher; Ka–association constant between Z (and A) and protonated PS II units; LA–local anesthetic; NaAsc–sodium ascorbate; NR–neutral red; PAM–pulse-amplitude modulation fluorometer; PFD–photon-flux density, μmols photons m-2 s-1; PS I–Photosystem I; PS II–Photosystem II; [PS II-+]–concentration of PS II units with inactive/deprotonated (active/protonated ) xanthophyll binding sites; [PS IItot]–total concentration of PS II units; [PS II+-Z]–concentration of PS II units with Z or A bound; Q–fraction of fluorescence intensity that is quenched; Qmax–fraction of fluorescence intensity that is quenched under control conditions; QA–primary quinone electron acceptor of PS II; V–violaxanthin; Z–zeaxanthin; 9AA–9-aminoacridine; ΔpH–trans-thylakoid membrane proton gradient; τf–lifetime of Chl a fluorescence

Notes: Abstract 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.

Notes: Abstract 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.