Husen Jia

Quantifying and monitoring functional photosystem II and the stoichiometry of the two photosystems in leaf segments: approaches and approximations

Given its unique function in light-induced water oxidation and its susceptibility to photoinactivation during photosynthesis, photosystem II (PS II) is often the focus of studies of photosynthetic structure and function, particularly in environmental stress conditions. Here we review four approaches for quantifying or monitoring PS II functionality or the stoichiometry of the two photosystems in leaf segments, scrutinizing the approximations in each approach. (1) Chlorophyll fluorescence parameters are convenient to derive, but the information-rich signal suffers from the localized nature of its detection in leaf tissue. (2) The gross O2 yield per single-turnover flash in CO2-enriched air is a more direct measurement of the functional content, assuming that each functional PS II evolves one O2 molecule after four flashes. However, the gross O2 yield per single-turnover flash (multiplied by four) could over-estimate the content of functional PS II if mitochondrial respiration is lower in flash illumination than in darkness. (3) The cumulative delivery of electrons from PS II to P700+ (oxidized primary donor in PS I) after a flash is added to steady background far-red light is a whole-tissue measurement, such that a single linear correlation with functional PS II applies to leaves of all plant species investigated so far. However, the magnitude obtained in a simple analysis (with the signal normalized to the maximum photo-oxidizable P700 signal), which should equal the ratio of PS II to PS I centers, was too small to match the independently-obtained photosystem stoichiometry. Further, an under-estimation of functional PS II content could occur if some electrons were intercepted before reaching PS I. (4) The electrochromic signal from leaf segments appears to reliably quantify the photosystem stoichiometry, either by progressively photoinactivating PS II or suppressing PS I via photo-oxidation of a known fraction of the P700 with steady far-red light. Together, these approaches have the potential for quantitatively probing PS II in vivo in leaf segments, with prospects for application of the latter two approaches in the field.

Novel effects of methyl viologen on photosystem II function in spinach leaves

Methyl viologen (MV) is a well-known electron mediator that works on the acceptor side of photosystem I. We investigated the little-known, MV-induced inhibition of linear electron flow through photosystem II (PS II) in spinach-leaf discs. Even a low [MV] decreased the (1) average, light-adapted photochemical efficiency of PS II traps, (2) oxidation state of the primary quinone acceptor QA in PS II during illumination, (3) photochemical efficiency of light-adapted open PS II traps, (4) fraction of absorbed light energy dissipated constitutively in a light-independent manner or as chlorophyll (Chl) a fluorescence emission, (5) Chl a fluorescence yield corresponding to dark-adapted open reaction-center traps (Fo) and closed reaction-center traps (Fm), and (6) half-time for re-oxidation of QA− in PS II after a single-turnover flash. These effects suggest that the presence of MV accelerates various “downhill” electron-transfer steps in PS II. Therefore, when using the MV to quantify cyclic electron flow, the inhibitory effect of MV on PS II should be taken into account.

Recovery of photoinactivated photosystem II in leaves: retardation due to restricted mobility of photosystem II in the thylakoid membrane

The functionality of photosystem II (PS II) following high-light pre-treatment of leaf segments at a chilling temperature was monitored as Fv/Fm, the ratio of variable to maximum chlorophyll fluorescence in the dark-adapted state and a measure of the optimal photochemical efficiency in PS II. Recovery of PS II functionality in low light (LL) and at a favourable temperature was retarded by (1) water stress and (2) growth in LL, in both spinach and Alocasia macrorrhiza L. In spinach leaf segments, water stress per se affected neither Fv/Fm nor the ability of the adenosine triphosphate (ATP) synthase to be activated by far-red light for ATP synthesis, but it induced chloroplast shrinkage as observed in frozen and fractured samples by scanning electron microscopy. A common feature of water stress and growth of plants in LL is the enhanced anchoring of PS II complexes, either across the shrunken lumen in water-stress conditions or across the partition gap in larger grana due to growth in LL. We suggest that such enhanced anchoring restricts the mobility of PS II complexes in the thylakoid membrane system, and hence hinders the lateral migration of photoinactivated PS II reaction centres to the stroma-located ribosomes for repair.

Acclimation of leaves to low light produces large grana: the origin of the predominant attractive force at work

Photosynthetic membrane sacs (thylakoids) of plants form granal stacks interconnected by non-stacked thylakoids, thereby being able to fine-tune (i) photosynthesis, (ii) photoprotection and (iii) acclimation to the environment. Growth in low light leads to the formation of large grana, which sometimes contain as many as 160 thylakoids. The net surface charge of thylakoid membranes is negative, even in low-light-grown plants; so an attractive force is required to overcome the electrostatic repulsion. The theoretical van der Waals attraction is, however, at least 20-fold too small to play the role. We determined the enthalpy change, in the spontaneous stacking of previously unstacked thylakoids in the dark on addition of Mg2+, to be zero or marginally positive (endothermic). The Gibbs free-energy change for the spontaneous process is necessarily negative, a requirement that can be met only by an increase in entropy for an endothermic process. We conclude that the dominant attractive force in thylakoid stacking is entropy-driven. Several mechanisms for increasing entropy upon stacking of thylakoid membranes in the dark, particularly in low-light plants, are discussed. In the light, which drives the chloroplast far away from equilibrium, granal stacking accelerates non-cyclic photophosphorylation, possibly enhancing the rate at which entropy is produced.

Decreased Photochemical Efficiency of Photosystem II following Sunlight Exposure of Shade-Grown Leaves of Avocado: Because of, or in Spite of, Two Kinetically Distinct Xanthophyll Cycles?

Summary: Photoprotection in sunlight by two xanthophyll pigment systems is not enough to prevent inactivation of photochemical efficiency in avocado shade leaves. This study resolved correlations between changes in xanthophyll pigments and photosynthetic properties in attached and detached shade-grown avocado (Persea americana) leaves upon sun exposure. Lutein epoxide (Lx) was deepoxidized to lutein (L), increasing the total pool by ΔL over 5 h, whereas violaxanthin (V) conversion to antheraxanthin (A) and zeaxanthin (Z) ceased after 1 h. During subsequent dark or shade recovery, de novo synthesis of L and Z continued, followed by epoxidation of A and Z but not of L. Light-saturated nonphotochemical quenching (NPQ) was strongly and linearly correlated with decreasing [Lx] and increasing [∆L] but showed a biphasic correlation with declining [V] and increasing [A+Z] separated when V deepoxidation ceased. When considering [ΔL+∆Z], the monophasic linear correlation was restored. Photochemical efficiency of photosystem II (PSII) and photosystem (PSI; deduced from the delivery of electrons to PSI in saturating single-turnover flashes) showed a strong correlation in their continuous decline in sunlight and an increase in NPQ capacity. This decrease was also reflected in the initial reduction of the slope of photosynthetic electron transport versus photon flux density. Generally longer, stronger sun exposures enhanced declines in both slope and maximum photosynthetic electron transport rates as well as photochemical efficiency of PSII and PSII/PSI more severely and prevented full recovery. Interestingly, increased NPQ capacity was accompanied by slower relaxation. This was more prominent in detached leaves with closed stomata, indicating that photorespiratory recycling of CO2 provided little photoprotection to avocado shade leaves. Sun exposure of these shade leaves initiates a continuum of photoprotection, beyond full engagement of the Lx and V cycle in the antenna, but ultimately photoinactivated PSII reaction centers.

Separation of Light-induced Linear, Cyclic and Stroma-sourced Electron Fluxes to P700+ in Cucumber Leaf Discs after Pre-illumination at a Chilling Temperature

Pre-illumination of cucumber leaf discs at 4 degrees C with low-irradiance white light (i) led to a marked decrease in the extent of photo-oxidation of P700 (the special chlorophyll pair in the PSI reaction center) in actinic light at room temperature and (ii) hastened the post-illumination re-reduction of P700+. Quantifying the linear, cyclic and stroma-sourced electron fluxes to P700+ in two actinic light regimes, we found that there was no increase in cyclic or linear electron fluxes to account for these changes. Rather, we observed a decrease in the maximum extent of P700 photo-oxidation assayed by a strong flash superimposed on continuous, background light of wavelength 723 nm, which we interpret to represent a loss of stable charge separation in PSI due to enhanced charge recombination as a result of the pre-illumination treatment. The funneling of electrons towards fewer non-damaged PSI complexes could explain the hastened post-illumination re-reduction of P700+, aided by a slight increase in a stroma-sourced electron flux after prolonged pre-illumination at 4 degrees C. Quantifying the separate fluxes to P700+ helps to elucidate the effects of chilling of cucumber leaf discs in the light and the reasons for the hastened post-illumination re-reduction of P700+.

Entropy and biological systems: Experimentally-investigated entropy-driven stacking of plant photosynthetic membranes

According to the Second Law of Thermodynamics, an overall increase of entropy contributes to the driving force for any physicochemical process, but entropy has seldom been investigated in biological systems. Here, for the first time, we apply Isothermal Titration Calorimetry (ITC) to investigate the Mg2+-induced spontaneous stacking of photosynthetic membranes isolated from spinach leaves. After subtracting a large endothermic interaction of MgCl2 with membranes, unrelated to stacking, we demonstrate that the enthalpy change (heat change at constant pressure) is zero or marginally positive or negative. This first direct experimental evidence strongly suggests that an entropy increase significantly drives membrane stacking in this ordered biological structure. Possible mechanisms for the entropy increase include: (i) the attraction between discrete oppositely-charged areas, releasing counterions; (ii) the release of loosely-bound water molecules from the inter-membrane gap; (iii) the increased orientational freedom of previously-aligned water dipoles; and (iv) the lateral rearrangement of membrane components.

Photoinhibition and Active Oxygen Species Production in Detached Apple Leaves During Dehydration

In the course of dehydration, the gas exchange and chlorophyll (Chl) fluorescence were measured under irradiance of 800 μmol m−2 s−1 in detached apple leaves, and the production of active oxygen species (AOS), hydrogen peroxide (H2O2), superoxide (O2−), hydroxyl radical (−OH), and singlet oxygen (1O2), were determined. Leaf net photosynthetic rate (PN) was limited by stomatal and non-stomatal factors at slight (2–3 h dehydration) and moderate (4–5 h dehydration) water deficiency, respectively. Photoinhibition occurred after 3-h dehydration, which was defined by the decrease of photosystem 2 (PS2) non-cyclic electron transport (P-rate). After 2-h dehydration, an obvious rise in H2O2 production was found as a result of photorespiration rise. If photorespiration was inhibited by sodium bisulfite (NaHSO3), the rate of post-irradiation transient increase in Chl fluorescence (Rfp) was enhanced in parallel with a slight decline in P-rate and with an increase in Mehler reaction. At 3-h dehydration, leaf P-rate decrease could be blocked by glycine (Gly) or methyl viologen (MV) pre-treatment, and MV was more effective than Gly at moderate drought time. AOS (H2O2 and O2−), prior to photoinhibition produced from photorespiration and Mehler reaction in detached apple leaves at slight water deficiency, were important in dissipating photon energy which was excess to the demand of CO2 assimilation. So photoinhibition could be effectively prevented by the way of AOS production.

A novel P700 redox kinetics probe for rapid, non‐intrusive and whole‐tissue determination of photosystem II functionality, and the stoichiometry of the two photosystems in vivo

We sought a rapid, non-intrusive, whole-tissue measure of the functional photosystem II (PS II) content in leaves. Summation of electrons, delivered by a single-turnover flash to P700(+) (oxidized PS I primary donor) in continuous background far-red light, gave a parameter S in absorbance units after taking into account an experimentally determined basal electron flux that affects P700 redox kinetics. S was linearly correlated with the functional PS II content measured by the O(2) yield per single-turnover repetitive flash in Arabidopsis thaliana expressing an antisense construct to the PsbO (manganese-stabilizing protein in PS II) proteins of PS II (PsbO mutants). The ratio of S to z(max) (total PS I content in absorbance units) was comparable to the PS II/PS I reaction-center ratio in wild-type A. thaliana and in control Spinacea oleracea. Both S and S/z(max) decreased in photoinhibited spinach leaf discs. The whole-tissue functional PS II content and the PS II/photosystem I (PS I) ratio can be non-intrusively monitored by S and S/z(max), respectively, using a quick P700 absorbance protocol compatible with modern P700 instruments.

The Stoichiometry of Photosystem II to Photosystem I in Higher Plants

The stoichiometry of photosystem II to photosystem I reaction centres in spinach leaf segments was determined by two methods, each capable of monitoring both photosystems in a given sample. One method, based on the fast electrochromic (EC) signal, was applied to leaf segments, thereby avoiding potential artefacts associated with the isolation of thylakoids. Two variations of the EC method were used, either suppression of PSII activity by prior photoinactivation or suppression PSI by photo- oxidation of P700, gave the separate contribution of each photosystem to the fast EC signal. The PSII/PSI stoichiometries obtained by the EC methods ranged from 1.5 to 1.8 for spinach, and 1.5 to 1.9 for two other plant species. A second method, based on electron paramagnetic resonance (EPR), gave comparable values of 1.7–2.1 for spinach. A third method consisting of separate determination of the contents of functional PSII by oxygen yield per single turnover flash and of P700 gave a PSII/PSI stiochiometry consistent with above values. We conclude that the content of functional PSII is greater than that of PSI, and PSII/PSI reaction centre ratios considerably higher than unity in typical higher plants.