Adam M. Gilmore

PsbS-dependent enhancement of feedback de-excitation protects photosystem II from photoinhibition

Feedback de-excitation (qE) regulates light harvesting in plants to prevent inhibition of photosynthesis when light absorption exceeds photosynthetic capacity. Although the mechanism of qE is not completely understood, it is known to require a low thylakoid lumen pH, de-epoxidized xanthophylls, and the photosystem II protein PsbS. During a short-term 4-h exposure to excess light, three PsbS- and qE-deficient Arabidopsis thaliana mutants that differed in xanthophyll composition were more photoinhibited than the wild type. The extent of photoinhibition was the same in all of the mutants, suggesting that qE capacity rather than xanthophyll composition is critical for photoprotection in short-term high light, in contrast to longer-term high light conditions (days) when additional antioxidant roles of specific xanthophylls are evident. Plants with a 2-fold increase in qE capacity were generated by overexpression of PsbS, demonstrating that the level of PsbS limits the qE capacity in wild-type Arabidopsis. These results are consistent with the idea that variations in PsbS expression are responsible for species-specific and environmentally induced differences in qE capacity observed in nature. Furthermore, plants with higher qE capacity were more resistant to photoinhibition than the wild type. Increased qE was associated with decreased photosystem II excitation pressure and changes in the fractional areas of chlorophyll a fluorescence lifetime distributions, but not the lifetime centers, suggesting that qE protects from photoinhibition by preventing overreduction of photosystem II electron acceptors. Engineering of qE capacity by PsbS overexpression could potentially yield crop plants that are more resistant to environmental stress.

How Higher Plants Respond to Excess Light: Energy dissipation in photosystem II

In this chapter we present current views concerning the adaptation and acclimation of the higher plant photosynthetic apparatus to excess light levels. The primary focus is at the level of the chloroplast thylakoid membrane which is where the primary events of photosynthetic energy transduction occur. We first summarize our current understanding the molecular composition and macromolecular organization of Photosystem II (PS II) because these factors pertain directly to photosynthetic function during environmental stress. We then discuss the biochemical and biophysical interpretations obtained from the most commonly used tool for probing the photosynthetic function of PS II, namely PS II chlorophyll (Chl) a fluorescence. We explain how PS II Chl a fluorescence yield measurements have provided insights into the dynamic relationships between the primary photosynthetic light-energy transduction processes and the important light adaptation and acclimation strategies utilized by PS II. The basic biochemical and biophysical aspects of the light-energy dissipation, avoidance and damage-repair mechanisms that influence PS II function are discussed in relation to a physiological gradient of increasing environmental stress. The future areas of research interest and importance regarding the optimization and preservation of PS II function during environmental stress are also briefly highlighted.

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.

Diurnal and acclimatory responses of violaxanthin and lutein epoxide in the Australian mistletoe Amyema miquelii

This study investigated the chloroplast pigment content of the Australian mistletoe Amyema miquelii (Lehm. ex Miq.) Tiegh. over diurnal periods in sun- and shade-acclimated leaves. Amyema miquelii exhibited the typical higher plant complement of neoxanthin, the xanthophyll cycle pigments, lutein, chlorophylls a and b and β carotene. Substantial levels of lutein epoxide were also present. Interestingly, diurnal light exposure elicited a decrease in lutein epoxide that paralleled the decrease in violaxanthin. Compared with shade-acclimated leaves, sun leaves exhibited reduced lutein epoxide and violaxanthin levels and higher chlorophyll a/b ratios. It is clear that the pools of violaxanthin and lutein epoxide respond in parallel to both diurnal light changes and sun-shade acclimation, although there seemed to be some differences in the recovery characteristics. These results raise a question as to whether lutein and lutein epoxide cycling may provide an auxiliary means of energy dissipation for some species.

Simultaneous Time Resolution of the Emission Spectra of Fluorescent Proteins and Zooxanthellar Chlorophyll in Reef-building Corals ¶†

Abstract Light is absorbed by photosynthetic algal symbionts (i.e. zooxanthellae) and by chromophoric fluorescent proteins (FP) in reef-building coral tissue. We used a streak-camera spectrograph equipped with a pulsed, blue laser diode (50 ps, 405 nm) to simultaneously resolve the fluorescence spectra and kinetics for both the FP and the zooxanthellae. Shallow water (<9 m)–dwelling Acropora spp. and Plesiastrea versipora specimens were collected from Okinawa, Japan, and Sydney, Australia, respectively. The main FP emitted light in the blue, blue-green and green emission regions with each species exhibiting distinct color morphs and spectra. All corals showed rapidly decaying species and reciprocal rises in greener emission components indicating Förster resonance energy transfer (FRET) between FP populations. The energy transfer modes were around 250 ps, and the main decay modes of the acceptor FP were typically 1900–2800 ps. All zooxanthellae emitted similar spectra and kinetics with peak emission (∼683 nm) mainly from photosystem II (PSII) chlorophyll (chl) a. Compared with the FP, the PSII emission exhibited similar rise times but much faster decay times, typically around 640–760 ps. The fluorescence kinetics and excitation versus emission mapping indicated that the FP emission played only a minor role, if any, in chl excitation. We thus suggest the FP could only indirectly act to absorb, screen and scatter light to protect PSII and underlying and surrounding animal tissue from excess visible and UV light. We conclude that our time-resolved spectral analysis and simulation revealed new FP emission components that would not be easily resolved at steady state because of their relatively rapid decays due to efficient FRET. We believe the methods show promise for future studies of coral bleaching and for potentially identifying FP species for use as genetic markers and FRET partners, like the related green FP from Aequorea spp.

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 ∈-cyclization enzyme step between lycopene and β,∈-carotene causing accumulation of β,β-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 (≈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.

Sustained downregulation of photosystem II in mistletoes during winter depression of photosynthesis

Cold acclimation by sustained downregulation of PSII was studied in intact leaves of an Australian mistletoe Amyema miquelii (Lehm. ex Miq.) Tiegh. and its host Eucalyptus. The trends were followed from autumn to spring on leaves that had developed in summer and were exposed to different microclimates with progress of the seasons. In sun leaves of mistletoe, efficiency of excitation energy transfer from light-harvesting pigments to Chl a molecules in PSII core complexes was markedly reduced in winter. Concomitantly, a band in 77K fluorescence emission spectra emerged at 715 nm, in the same way as the cold-hard band found in overwintering snow gum seedlings (Gilmore and Ball 2000, Proceedings of the National Academy of Sciences USA 97, 11 098-11 101). Further, a distinct band, which presumably involves Chl-b-containing antennae complexes, appeared at 705 nm in -2°C fluorescence emission spectra with decreasing intensity of the PSII band. Much shorter PSII fluorescence lifetimes measured in sun leaves of mistletoe that were exhibiting sustained downregulation of PSII indicated enhanced thermal dissipation of excitation energy. Winter acclimation symptoms of the photosynthetic apparatus were more striking in mistletoe sun leaves compared with eucalypt sun leaves. Because the light and temperature environments of sun leaves are similar for the parasite and host, we primarily attribute the enhanced light-acclimation symptoms to the limited photosynthetic capacity of A. miquelii in winter.

Time‐resolution of the Antheraxanthin‐ and ΔpH‐dependent Chlorophyll a Fluorescence Components Associated with Photosystem II Energy Dissipation in Mantoniella squamata¶

Abstract The electronic excited-state behavior of photosystem II (PSII) in Mantoniella squamata, as influenced by the xanthophyll cycle and the transthylakoid pH gradient (ΔpH), 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 ΔpH and A accumulation, followed by herbicide-induced closure of PSII traps and a chilling treatment, to sustain and stabilize the ΔpH 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 a fluorescence intensity.

9-Aminoacridine and dibucaine exhibit competitive interactions and complicated inhibitory effects that interfere with measurements of ΔpH and xanthophyll cycle-dependent Photosystem II energy dissipation

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

Quantitative Analysis of Intrathylakoid ph and Xanthophyll Cycle Effects on PSII Fluorescence Lifetime Distributions and Intensity

Dissipation of excess absorbed light energy as heat in the photosynthetic apparatus of higher plants is feedback regulated by limitations in the photosynthetic capacity (1–3). Although, the energy dissipation process depends on both intrathylakoid acidification and xanthophyll cycle deepoxidation (2–4) these relationships have not yet been quantified. Here we summarize a kinetic model, derived from a global analysis, that quantifies the relationships between the intrathylakoid pH, the level of xanthophyll cycle deepoxidation and the PSII chlorophyll (Chl) a fluorescence lifetime distributions and intensity. Supporting experimental results precede the model derivation and application. Details of this summary are in press elsewhere (5).