A quantitative description of fluorescence excitation spectra in intact bean leaves greened under intermittent light

We present a simple approach for the calculation of in vivo fluorescence excitation spectra from measured absorbance spectra of the isolated pigments involved. Taking into account shading of the pigments by each other, energy transfer from carotene to chlorophyll a, and light scattering by the leaf tissue, we arrive at a model function with 6 free parameters. Fitting them to the measured fluorescence excitation spectrum yields good correspondence between theory and experiment, and parameter estimates which agree with independent measurements. The results are discussed with respect to the origin and the interpretation of in vivo excitation spectra in general.     

Correlation of absorbance changes and thylakoid fusion with the induction of oxygen evolution in bean leaves greened by brief flashes

Dark-grown bean leaves (Phaseolus vulgaris) which had been greened for several days in a repetitive series of brief xenon flashes were studied during the initial induction period when O(2) evolution first appears. The induction of O(2) evolution requires actinic irradiation (e.g. 2 mw/cm(2) of red light) and goes to completion in about 8 minutes with a half-time just under 3 minutes. Absorbance measurements on the intact leaves showed that a change of a carotenoid pigment, monitored at 505 nm, was closely correlated with the rate of O(2) evolution during the induction period. Inhibitor studies, however, showed that the absorbance change persisted in the presence of a number of inhibitors which blocked O(2) evolution. Electron microscopy revealed that the primary thylakoids which were unfused in the flashed leaves before induction became fused in pairs or groups of three during the 8-minute induction period. It is postulated that the 505-nm absorbance change of the carotenoid pigment is correlated more directly with the fusion process than with O(2) evolution. Heat treatment (45 C for 5 min) or infiltration with 0.8 m tris, which prevented the fusion process, also prevented the absorbance change.If the leaves were preilluminated for 8 minutes with very weak red light (20 muw/cm(2)) which induced no O(2) evolution, absorbance change, or thylakoid fusion, there was an immediate burst of O(2) evolution at the onset of actinic irradiation and the induction period, as noted by O(2) evolution or by the 505-nm absorbance change, was reduced to 2 minutes (half-time of 40 seconds). It is concluded that the electron transport system in the flashed leaves is blocked at the Mn site between water and photosystem II and that the photoactivation of Mn into the thylakoid membranes occurs during the low light, photoactivation process. After the electron transport chain is thus repaired, ion-pumping mechanisms driven by actinic light may lead to steady-state photosynthesis as well as to thylakoid fusion.     

Greening of etiolated bean leaves in far red light

Eight-day-old dark-grown bean leaves were greened by prolonged irradiation with far red light. Growth, chlorophyll content, oxygen-evolving capacity, photophosphorylation capacity, chloroplast structure (by electron microscopy), and in vivo forms of chlorophyll (by low temperature absorption and derivative spectroscopy on intact leaves) were followed during the greening process. Chlorophyll a accumulated slowly but continuously during the 7 days of the experiment (each day consisted of 12 hours of far red light and 12 hours of darkness). Chlorophyll b was not detected until the 5th day. The capacity for oxygen evolution and photophosphorylation began at about the 2nd day. Electron microscopy showed little formation of grana during the 7 days but rather unfused stacks of primary thylakoids. The thylakoids would fuse to give grana if the leaves were placed subsequently in white light. The low temperature spectroscopy of intact leaves showed that the chlorophyll a was differentiated into three forms with absorption maxima near 670, 677, and 683 nanometers at −196 C during the first few hours and that these forms accumulated throughout the greening process. Small amounts of two longer wavelength forms with maxima near 690 and 698 nanometers appeared at about the same time as photosynthetic activity.     

Photoinactivation of photosystem II in leaves of Capsicum annuum

Leaf discs of Capsicum annuum L. were illuminated in air enriched with 1% CO₂ in the absence or presence of lincomycin, an inhibitor of chloroplast-encoded protein synthesis. The loss of functional photosystem (PS) II complexes with increase in cumulative light dose (photon exposure), assessed by the O₂ yield per single-turnover flash, was greater in leaves of plants grown in low light than those in high light; it was also exacerbated in the presence of lincomycin. A single exponential decay can describe the relationship between the loss of functional PSII and increase in cumulative photon exposure. From this relationship we obtained both the maximum quantum yield of photoinactivation of PSII at limiting photon exposures and the coefficient k, interpreted as the probability of photoinactivation of PSII per unit photon exposure. Parallel measurements of chlorophyll fluorescence after light treatment showed that 1/F_o−1/F_m was linearly correlated with the functionality of PSII, where F_o and F_m are the chlorophyll fluorescence yields corresponding to open and closed PSII reaction centers, respectively. Using 1/F_o−1/F_m as a convenient indicator of PSII functionality, it was found that PSII is present in excess; only after the loss of about 40% functional PSII complexes did PSII begin to limit photosynthetic capacity in capsicum leaves.     

The irreversible photoinhibition of the photosystem II complex in leaves of Vicia faba under strong light

Exposure of the photosynthetic machinery to strong light causes the photoinhibition of the photosystem II complex. The recovery from the photoinhibition in vivo was characterized by monitoring the ratio of variable to maximum fluorescence (F_v/F_m) in detached leaves of broad bean (Vicia faba). The changes in the ratio were explained in terms of three components, namely, two saturating exponential components with half rise-times of about 15 and 120 min, respectively, and a non-recovery component. The non-recovery component increased gradually as the exposure to strong light was prolonged. Our results suggest that this irreversible component of the photoinhibition of the photosystem II complex was caused by severe stress due to strong light under which repair of the photosystem II complex was insufficient to allow full recovery. The irreversible photoinhibition is discussed in terms of both the physiology and ecology of plants.     

Protein crystalloids in the stroma of bean plastids

Summary The formation of protein crystalloids in the stroma of bean plastids has been studied. It has been shown that, besides crystallization of the protein already present induced by water loss (which cannot be inhibited by chloramphenicol), the crystalloids also appear when the leaves are fed — but only in light — with very low concentrations of sucrose or glucose for one or two days. In this case their appearance is completely inhibited by simultaneous treatment of the leaves with chloramphenicol but not with cycloheximide. The process of crystalloid formation and disappearance has also been studied. The possibility of their removal from the plastids into the cell vacuoles has been followed and discussed.     

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 F(v)/F(m), 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 F(v)/F(m) 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.     

Light inactivation of functional photosystem II in leaves of peas grown in moderate light depends on photon exposure

To determine the dependence of in vivo photosystem (PS) II function on photon exposure and to assign the relative importance of some photoprotective strategies of PSII against excess light, the maximal photochemical efficiency of PSII (Fv/Fm) and the content of functional PSII complexes (measured by repetitive flash yield of oxygen evolution) were determined in leaves of pea (Pisum satlvum L.) grown in moderate light. The modulation of PSII functionality in vivo was induced by varying either the duration (from 0 to 3 h) of light treatment (fixed at 1200 or 1800 mol photons · m^-2 · s^-1) or irradiance (from 0 to 3000 mol photons · m^-2 · s^-1) at a fixed duration (1 h) after infiltration of leaves with water (control), lincomycin (an inhibitor of chloroplast-encoded protein synthesis), nigericin (an uncoupler), or dithiothreitol (an inhibitor of the xanthophyll cycle) through the cut petioles of leaves of 22 to 24-day-old plants. We observed a reciprocity of irradiance and duration of illumination for PSII function, demonstrating that inactivation of functional PSII depends on the total number of photons absorbed, not on the rate of photon absorption. The Fv/Fm ratios from photoinhibitory light-treated leaves, with or without inhibitors, declined pseudo-linearly with photon exposure. The number of functional PSII complexes declined multiphasically with increasing photon exposure, in the following decreasing order of inhibitor effect: lincomycin > nigericin > DTT, indicating the central role of D1 protein turnover. While functional PSII and Fv/Fm ratio showed a linear relationship under high photon exposure conditions, in inhibitor-treated leaves the Fv/Fm ratio failed to reveal the loss of up to 25% of the total functional PSII under low photon exposure. The loss of this 25% of less-stable functional PSII was accompanied by a decrease of excitation-energy trapping capacity at the reaction centre of PSII (revealed by the fluorescence parameter, 1/Fo-1/Fm, where Fo and Fm stand for chlorophyll fluorescence when PSII reaction centres are open and closed, respectively), but not by a loss of excitation energy at the antenna (revealed by the fluorescence parameter, 1/Fm). We conclude that (i) PSII is an intrinsic photon counter under photoinhibitory conditions, (ii) PSII functionality is mainly regulated by D1 protein turnover, and to a lesser extent, by events mediated via the transthylakoid pH gradient, and (iii) peas exhibit PSII heterogeneity in terms of functional stability during photon exposure.Abbreviations D1 protein psbA gene product - DTT dithiothreitol - Fo chlorophyll fluorescence corresponding to open PSII reaction centres - Fv, Fm variable and maximum fluorescence after dark incubation, respectively - Fs, Fm steady-state and maximum fluorescence during illumination, respectively - P680 reactioncentre chlorophyll and primary electron donor of PSII - PS photosystem Financial support of this work by Department of Employment, Education and Training/Australian Research Council International Research Fellowships Program (Korea) is gratefully acknowledged.     

Changes in photosystem II function during senescence of wheat leaves

Analyses of chlorophyll fluorescence were undertaken to investigate the alterations in photosystem II (PSII) function during senescence of wheat ( Triticum aestivum L. cv. Shannong 229) leaves. Senescence resulted in a decrease in the apparent quantum yield of photosynthesis and the maximal CO₂ assimilation capacity. Analyses of fluorescence quenching under steady‐state photosynthesis showed that senescence also resulted in a significant decrease in the efficiency of excitation energy capture by open PSII reaction centers (F'_v/F'_m) but only a slight decrease in the maximum efficiency of PSII photochemistry (F'_v/F'_m). At the same time, a significant increase in non‐photochemical quenching (q_N) and a considerable decrease in photochemical quenching (q_P) were observed in senescing leaves. Rapid fluorescence induction kinetics indicated a decrease in the rate of Q_A reduction and an increase in the proportion of Q_B‐non‐reducing PSII reaction during senescence. The decrease in both F'_v/F'_m and q_P explained the decrease in the actual quantum yield of PSII electron transport ((φ_PSII). We suggest that the modifications in PSII function, which led to the down‐regulation of photosynthetic electron transport, would be in concert with the lower demand for ATP and NADPH in the Calvin cycle which is often inhibited in senescing leaves.     

Regreening of senescent Nicotiana leaves. II. Redifferentiation of plastids

Single senescent leaves attached to decapitated shoots of Nicotiana rustica L. regreened, especially when treated with cytokinin. Regreening caused an increase in leaf thickness, due to cell expansion. Senescent leaf plastids (gerontoplasts) were smaller than green chloroplasts, with degenerated membrane systems and stroma, and larger plastoglobuli. At advanced senescence, micrographs showed disintegrating gerontoplasts, reduced numbers of plastids were counted, and regreening became variable. The redevelopment of grana and stroma in regreening plastids was accelerated by cytokinin. All plastids in regreening leaves were identifiable as redifferentiating gerontoplasts because of their content of plastoglobuli and starch. Immunogold labelling showed significant association of POR with etioplasts in cotyledons, but with mature plastids in regreening leaves. No proplastids or dividing chloroplasts were observed in regreening leaves. Plastids numbers declined during senescence and did not increase again during regreening. It is concluded that the chloroplasts of regreening leaves arose by redifferentiation of gerontoplasts.Keywords: Chloroplasts, cytokinin, Nicotiana, senescence, regreening.      

Diurnal fluctuation in the composition of chlorophyll a/b light harvesting antenna of photosystem II in young wheat leaves

We investigated the diurnal fluctuation in the composition of the light harvesting chlorophyll a/b antenna of photosystem II in young wheat (Triticum aestivum) leaves grown under periodic day/night irradiation. By means of gel electrophoresis of the polypeptides of thylakoid membranes, we determined the amount of 25 kDa and 27 kDa polypeptides, which are the main components of the peripheral and inner antenna subpopulations, respectively. Our data show a preferential fluctuation in the amount of the 25 kDa protein relative to the 27 kDa polypeptide, in parallel to the fluctuation in the amount of chlorophyll a/b antenna of photosystem II, which suggests that the peripheral antenna plays a role in the diurnal adjustment of the antenna size.     

Cost and benefit of the repair of photodamaged photosystem II in spinach leaves: roles of acclimation to growth light

When visible light is excess, the photosynthetic machinery is photoinhibited. The extent of net photoinhibition of photosystem II (PSII) is determined by a balance between the rate of photodamage to D1 and some other PSII proteins and the rate of the turnover cycle of these proteins. It is widely believed that the protein turnover requires much energy cost. The aims of this study are to (1) evaluate the energy cost of PSII repair, (2) measure the benefit in terms of photosynthetic gain realized by the repairing of the photodamaged PSII, and (3) know whether acclimation of photosynthesis to growth light affects the rates of the photodamage and repair. We grew spinach in high-light (HL) and low-light (LL) and measured the rates of D1 photodamage and repair in these leaves. We determined the rate constants of photodamage (k (pi)) and repair (k (rec)) by the PAM fluorometry in the presence or in the absence of lincomycin, an inhibitor of 70S protein synthesis. HL leaves showed smaller k (pi) and greater k (rec) than LL leaves. The energy cost of the repairing of the photodamaged D1 protein was <0.5?% of ATP produced by photophosphorylation at PPFDs ranging from 400 to 1600?μmol?m(-2)?s(-1) and was greater in HL leaves than in LL leaves. The benefits brought about by the repair were more than from 35 to 270 times the cost at PPFDs ranging from 400 to 1600?μmol?m(-2)?s(-1). The benefits of HL leaves were greater than those of LL leaves because of the higher photosynthesis rates in HL leaves. Running a simple simulation of daily photosynthesis using the parameters obtained in this study, we discuss why the plants need to pay the cost of D1 protein turnover to repair the photodamaged PSII.     

Modifications in photosystem II photochemistry in senescent leaves of maize plants

Analyses of CO2 exchange and chlorophyll fluorescence were carried out to assess photosynthetic performance during senescence of maize leaves. Senescent leaves displayed a significant decrease in CO2 assimilatory capacity accompanied by a decrease in stomatal conductance and an increase in intercellular CO2 concentration. The analyses of fluorescence quenching under steady-state photosynthesis showed that senescence resulted in an increase in non-photochemical quenching and a decrease in photo-chemical quenching. It also resulted in a decrease in the efficiency of excitation energy capture by open PSII reaction centres and the quantum yield of PSII electron transport, but had very little effect on the maximal efficiency of PSII photochemistry. The results determined from the fast fluorescence induction kinetics indicated an increase in the proportion of QB-non-reducing PSII reaction centres and a decrease in the rate of QA reduction in senescent leaves. Theoretical analyses of fluorescence parameters under steady-state photosynthesis suggest that the increase in the non-photochemical quenching was due to an increase in the rate constant to thermal dissipation of excitation energy by PSII and that the decrease in the quantum yield of PSII electron transport was associated with a decrease in the rate constant of PSII photochemistry. Based on these results, it is suggested that the decrease in the quantum yield of PSII electron transport in senescent leaves was down-regulated by an increase in the proportion of QB-non-reducing PSII reaction centres and in the non-photochemical quenching. The photosynthetic electron transport would thus match the decreased demand for ATP and NADPH in carbon assimilation which was inhibited significantly in senescent leaves.Key words: Chlorophyll fluorescence, gas exchange, maize (Zea mays L.), photochemical and non-photochemical quenching, photosystem II photochemistry.      

The time course of photoinactivation of photosystem II in leaves revisited

Since photosystem II (PS II) performs the demanding function of water oxidation using light energy, it is susceptible to photoinactivation during photosynthesis. The time course of photoinactivation of PS II yields useful information about the process. Depending on how PS II function is assayed, however, the time course seems to differ. Here, we revisit this problem by using two additional assays: (1) the quantum yield of oxygen evolution in limiting, continuous light and (2) the flash-induced cumulative delivery of PS II electrons to the oxidized primary donor (P700(+)) in PS I measured as a 'P700 kinetics area'. The P700 kinetics area is based on the fact that the two photosystems function in series: when P700 is completely photo-oxidized by a flash added to continuous far-red light, electrons delivered from PS II to PS I by the flash tend to re-reduce P700(+) transiently to an extent depending on the PS II functionality, while the far-red light photo-oxidizes P700 back to the steady-state concentration. The quantum yield of oxygen evolution in limiting, continuous light indeed decreased in a way that deviated from a single-negative exponential. However, measurement of the quantum yield of oxygen in limiting light may be complicated by changes in mitochondrial respiration between darkness and limiting light. Similarly, an assay based on chlorophyll fluorescence may be complicated by the varying depth in leaf tissue from which the signal is detected after progressive photoinactivation of PS II. On the other hand, the P700 kinetics area appears to be a reasonable assay, which is a measure of functional PS II in the whole leaf tissue and independent of changes in mitochondrial respiration. The P700 kinetics area decreased in a single-negative exponential fashion during progressive photoinactivation of PS II in a number of plant species, at least at functional PS II contents ≥6?% of the initial value, in agreement with the conclusion of Sarvikas et al. (Photosynth Res 103:7-17, 2010). That is, the single-negative-exponential time course does not provide evidence for photoprotection of functional PS II complexes by photoinactivated, connected neighbours.     

Effect of light absorbed by photosystem 1 and photosystem 2 on the fluorescence yield of green leaves]

The influence of strong active light, which mainly excited the photosystem 1 (AL I) and photosystem 2 (AL II), on the fluorescence of weak detecting light under different intensities of active light has been investigated under aerobic and anaerobic conditions. It is shown that an increase or decrease in fluorescence can be observed under the influence of AL 1 according to the experimental conditions.     

Oxygen evolution and chlorophyll fluorescence from multiple turnover light pulses: charge recombination in photosystem II in sunflower leaves

Oxygen evolution and Chl fluorescence induction were measured during multiple turnover light pulses (MTP) of 630-nm wavelength, intensities from 250 to 8,000?μmol quanta m(-2)?s(-1) and duration from 0.3 to 200?ms in sunflower leaves at 22?°C. The ambient O(2) concentration was 10-30?ppm and MTP were applied after pre-illumination under far-red light (FRL), which oxidized plastoquinone (PQ) and randomized S-states because of the partial excitation of PSII. Electron (e ( - )) flow was calculated as 4·O(2) evolution. Illumination with MTP of increasing length resulted in increasing O(2) evolution per pulse, which was differentiated against pulse length to find the time course of O(2) evolution rate with sub-millisecond resolution. Comparison of the quantum yields, Y (IIO)?=?e ( - )/hν from O(2) evolution and Y (IIF)?=?(F (m)?-?F)/F (m) from Chl fluorescence, detected significant losses not accompanied by fluorescence emission. These quantum losses are discussed to be caused by charge recombination between Q (A) (-) and oxidized TyrZ at a rate of about 1,000?s(-1), either directly or via the donor side equilibrium complex Q(A)?→?P (D1) (+) ??TyrZ(ox), or because of cycling facilitated by Cyt b (559). Predicted from the suggested mechanism, charge recombination is enhanced by damage to the water-oxidizing complex and by restricted PSII acceptor side oxidation. The rate of PSII charge recombination/cycling is fast enough for being important in photoprotection.     

Energy dissipation in photosystem 2 complexes of peanut leaves subjected to light pulses

To increase crop yields, intercropping agricultural cultivation approach (IACA) and relay cropping agricultural cultivation approach (RACA) were introduced into cultivation of peanut (Arachis hypogaca L.), one of the most important oil crops in the world. IACA/RACA improves the yield of the crop per unit area, but rather decreases the yield of a single crop compared with mono-cropping agricultural cultivation approach. In peanut IACA/RACA, peanut plants would grow under low light and high light pulses (HLP) before maize/wheat harvest. Energy dissipation in photosystem 2 (PS2) complexes was evaluated in peanut leaves after being induced by light pulses and continuous light with different light intensities. Actual photochemical efficiency of PS2 (ΦPS2) and non-photochemical quenching induced by light pulses in peanut leaves generally were significantly lower compared with those induced by continuous light. In addition, the degree of PS2 reaction center closure (1-qP) induced by light pulses in peanut leaves was significantly higher compared with those induced by continuous light. Different results were obtained only for ΦPS2 and 1-qP induced by 20 s HLP. In methyl viologen (MV) treated samples, ΦPS2 and the quantum yield of light induced thermal dissipation by non-functional PS2 (Φ_NF) were similar to those observed in non-MV treatments. These results implied that light pulses could induce photoinactivation of PS2 reaction centers but not the xanthophyll cycle to dissipate excess energy.     

FtsH-mediated repair of the photosystem II complex in response to light stress

A common feature of light stress in plants, algae, and cyanobacteria is the light-induced damage to the photosystem II complex (PSII), which catalyses the photosynthetic oxidation of water to molecular oxygen. A repair cycle operates to replace damaged subunits within PSII, in particular, the D1 reaction centre polypeptide, by newly synthesized copies. As yet the molecular details of this physiologically important process remain obscure. A key aspect of the process that has attracted much attention is the identity of the protease or proteases involved in D1 degradation. The results are summarized here of recent mutagenesis experiments that were designed to assess the functional importance of the DegP/HtrA and FtsH protease families in the cyanobacterium Synechocystis sp. PCC 6803. Based on these results and the analysis of Arabidopsis mutants, a general model for PSII repair is suggested in which FtsH complexes alone are able to degrade damaged D1.