Quantitative Effects of Pigmentation on the Re‐absorption of Chlorophyll a Fluorescence and Energy Partitioning in Leaves

This study comparatively examined spectroscopic features, photosynthetic parameters and energy partitioning in plants of Fittonia albivenis cv. Agyroneura and Fittonia albivenis cv. Verschaffeltii with different pigmentation. Fittonia albivenis cv. Verschaffeltii, rich in anthocyanins, presented lower values than the green variety (cv. Agyroneura) for several parameters: the ratio chlorophyll a/b, the carotenoid content, the heat dissipation by nonphotochemical quenching (NPQ) and the energy‐dependent component of the quantum yield of NPQ. Additionally, the red plant displayed higher resistance to water shortage. The spectral distribution of the chlorophyll a fluorescence, free from distortions due to light reabsorption processes, was obtained for both varieties by application of a physical model previously developed in our group. From this modeling, a higher ratio photosystem II/photosystem I was inferred for the red variety, in agreement with the screening effect of anthocyanins. From a thorough analysis of the fluorescence, the different operating strategies adopted by these plants with dissimilar pigmentation could be elucidated. These strategies were related to the photosystem stoichiometry, the distribution of the absorbed energy and the dissipation of heat under increasing light intensities.     

Dephasing and Dissipation in a Source–Drain Model of Light‐Harvesting Systems

The energy transport process in natural‐light‐harvesting systems is investigated by solving the time‐dependent Schrödinger equation for a source–network–drain model incorporating the effects of dephasing and dissipation, owing to coupling with the environment. In this model, the network consists of electronically coupled chromophores, which can host energy excitations (excitons) and are connected to source channels, from which the excitons are generated, thereby simulating exciton creation from sunlight. After passing through the network, excitons are captured by the reaction centers and converted into chemical energy. In addition, excitons can reradiate in green plants as photoluminescent light or be destroyed by nonphotochemical quenching (NPQ). These annihilation processes are described in the model by outgoing channels, which allow the excitons to spread to infinity. Besides the photoluminescent reflection, the NPQ processes are the main outgoing channels accompanied by energy dissipation and dephasing. From the simulation of wave‐packet dynamics in a one‐dimensional chain, it is found that, without dephasing, the motion remains superdiffusive or ballistic, despite the strong energy dissipation. At an increased dephasing rate, the wave‐packet motion is found to switch from superdiffusive to diffusive in nature. When a steady energy flow is injected into a site of a linear chain, exciton dissipation along the chain, owing to photoluminescence and NPQ processes, is examined by using a model with coherent and incoherent outgoing channels. It is found that channel coherence leads to suppression of dissipation and multiexciton super‐radiance. With this method, the effects of NPQ and dephasing on energy transfer in the Fenna–Matthews–Olson complex are investigated. The NPQ process and the photochemical reflection are found to significantly reduce the energy‐transfer efficiency in the complex, whereas the dephasing process slightly enhances the efficiency. The calculated absorption spectrum reproduces the main features of the measured counterpart. As a comparison, the exciton dynamics are also studied in a linear chain of pigments and in a multiple‐ring system of light‐harvesting complexes II (LH2) from purple bacteria by using the Davydov D₁ ansatz. It is found that the exciton transport shows superdiffusion characteristics in both the chain and the LH2 rings.     

Photoprotection in the brown alga Macrocystis pyrifera: evolutionary implications

The dissipation of energy as heat is essential for photosynthetic organisms to protect themselves against excess light. We compared Photosystem II florescence changes (non-photochemical quenching, NPQ) in the brown alga Macrocystis pyrifera with that of Ficus sp., a higher plant to examine if the mechanism of heat dissipation (energy-dependent quenching, qE) differs between these evolutionary distant groups of phototrophs. We discovered that M. pyrifera had a slower rise of NPQ upon illumination than the Ficus sp. Further, the NPQ relaxation phase that takes place in the first minutes after light to dark transition is absent in this brown alga. We found that the NPQ induction rate in this alga was 1.5 times faster in preilluminated samples than in dark-adapted samples; this was associated with an increase in the rate of accumulation of the carotenoid zeaxanthin. Therefore, we conclude that NPQ in M. pyrifera is associated only with the formation of zeaxanthin. These results indicate that M. pyrifera lacks the fast component of qE that is related to allosteric changes in the light harvesting complexes of Ficus sp., a representative of higher plants. Although the xanthophyll cycle of this brown alga is similar to that of Ficus sp., yet, the transthylakoid proton gradient (ΔpH) does not influence NPQ beyond the activation of the violaxanthin de-epoxidase enzyme. These findings suggest that NPQ control mechanisms are not universal and we suggest that it may have diverged early in the evolution of different groups of eukaryotic phototrophs.     

Seasonal changes in the excess energy dissipation from Photosystem II antennae in overwintering evergreen broad-leaved trees Quercus myrsinaefolia and Machilus thunbergii

We monitored chlorophyll (Chl) fluorescence, pigment concentration and the de-epoxidation state of the xanthophyll cycle (DPS(1)) in two warm temperate broad-leaved evergreen species (Quercus myrsinaefolia and Machilus thunbergii). Reduction of the maximal quantum yield of Photosystem II (PSII) (calculated from Fv/Fm, variable to maximal Chl a fluorescence) and retention of a high DPS were observed in both species in the winter, and can be interpreted as acclimation to winter. In particular, the acclimation of PSII in these species can be chiefly attributed to thermal dissipation, which is correlated with the retention of high zeaxanthin. Furthermore, we attempted to divide the fate of the absorbed light energy by the PSII antennae into three components: (i) PSII photochemistry (represented by its quantum yield, ΦPSII), (ii) dissipation by down-regulation via non-photochemical quenching (ΦNPQ) and (iii) other non-photochemical processes (ΦONP). The estimated energy allocation of the absorbed light indicated that the proportion of ΦPSII decreased, whereas that of ΦNPQ+ΦONP increased during winter. This result suggests that the excess energy absorbed in the PSII complexes is safely dissipated from the PSII antennae. Based on these results, we conclude that thermal dissipation from the PSII antennae plays an important role in two overwintering broad-leaved evergreen trees growing in Japan.     

Photoprotection in Cyanobacteria: Regulation of Light Harvesting†

To cope with a rapidly fluctuating light environment, vascular plants and algae have evolved a photoprotective mechanism that serves to downregulate the transfer of excitation energy in the light‐harvesting complexes to the photosynthetic reaction centers. This process dissipates excess excitation energy in the chlorophyll pigment bed by a nonradiative pathway. Since this pathway competes with and therefore quenches chlorophyll fluoresence in a nonphotochemical manner, it has been termed Non‐photochemical Quenching (NPQ). For many years, cyanobacteria were not considered capable of performing NPQ as a photoprotective mechanism. Instead, the redistribution of the phycobilisome (PBS) light‐harvesting antenna between reaction centers by a process called state transitions was considered the major means of regulating the utilization of harvested light energy. Recently, it was demonstrated that cyanobacteria are able to use NPQ as one component of their photoprotective strategies. Cyanobacteria exhibit significant NPQ during nutrient‐replete growth, but it becomes a more prominent means of managing absorbed excitation energy when the cells experience iron starvation. Rapid progress in understanding the molecular mechanism of cyanobacterial NPQ has revealed a process that is very distinct from the functionally analogous process in plants and algae. Cyanobacterial NPQ involves the absorption of blue light by a carotenoid binding protein, termed the Orange Carotenoid Protein, and most likely involves quenching in the PBS core. In this study, we summarize work leading to the discovery of NPQ in cyanobacteria and the elucidation of molecular mechanisms associated with this important photoprotective process.     

Mimicking the role of the antenna in photosynthetic photoprotection

One mechanism used by plants to protect against damage from excess sunlight is called nonphotochemical quenching (NPQ). Triggered by low pH in the thylakoid lumen, NPQ leads to conversion of excess excitation energy in the antenna system to heat before it can initiate production of harmful chemical species by photosynthetic reaction centers. Here we report a synthetic hexad molecule that functionally mimics the role of the antenna in NPQ. When the hexad is dissolved in an organic solvent, five zinc porphyrin antenna moieties absorb light, exchange excitation energy, and ultimately decay by normal photophysical processes. Their excited-state lifetimes are long enough to permit harvesting of the excitation energy for photoinduced charge separation or other work. However, when acid is added, a pH-sensitive dye moiety is converted to a form that rapidly quenches the first excited singlet states of all five porphyrins, converting the excitation energy to heat and rendering the porphyrins kinetically incompetent to readily perform useful photochemistry.     

A highly selective and sensitive fluorescence "turn-on" probe for Ag+ in aqueous solution and live cells

A naphthalimide-based fluorescent probe, NPQ, that contains a novel receptor was successfully developed. NPQ exhibited "turn-on" fluorescence and excellent selectivity toward Ag(+) in the presence of various other metal ions in aqueous solution. A series of control compounds were designed and synthesized in order to explore the photoinduced electron transfer (PET) quenching mechanism of NPQ and binding mode of NPQ with Ag(+). Moreover, with the NPQ-Ag(+) complex, I(-) was easily selectively recognized by a marked fluorescence quenching. The live cell imaging experiments demonstrate that NPQ can be used as a fluorescent probe for monitoring Ag(+) in living cells.     

农用稀土对糯玉米幼苗光合变化和生理指标的分析

为了探明农用稀土对糯玉米幼苗光合和生理指标,采用随机区组排列,研究了农用稀土对糯玉米幼苗叶绿素荧光参数、光合参数、根系活力、植株形态和生物量、MDA和抗氧化酶活性的影响.结果表明,施用农用稀土能显著提高荧光参数Fv/Fm,ETR,NPQ,Fv/F0,Yield,qP最大值分别比对照增加了4.8%,23.9%,45.3%,11.01%,14.8%,26.5%;糯玉米幼苗的光合参数、根系活力、植株形态、生物量、抗氧化酶活性等变化趋势是一致的,在低浓度时,随着农用稀土浓度提高,它们逐渐地提高;到一定浓度后,随着农用稀土浓度升高,它们反而降低;MDA的变化趋势与它们是相反的.     

Equilibrium between quenched and nonquenched conformations of the major plant light-harvesting complex studied with high-pressure time-resolved fluorescence

Nonphotochemical quenching (NPQ) of chlorophyll fluorescence plays an important role in the protection of plants against excessive light. Fluorescence quenching of the major light-harvesting complex (LHCII) provides a model system to study the mechanism of NPQ. The existence of both quenched and nonquenched states of LHCII has been postulated. We used time-resolved fluorescence and hydrostatic pressure to study differences between these states. Pressure shifts the thermodynamic equilibrium between the two states. The estimated volume difference was 5 mL/mol, indicating a local conformational switch. The estimated free energy difference was 7.0 kJ/mol: high enough to keep the quenched state population low under normal conditions, but low enough to switch in a controlled way. These properties are physiologically relevant properties, because they guarantee efficient light harvesting, while at the same time maintaining the capacity to switch to a quenched state. These results indicate that conformational changes of LHCII can play an important role in NPQ.     

Identification of two emitting sites in the dissipative state of the major light harvesting antenna

In order to cope with the deleterious effects of excess light, photosynthetic organisms have developed remarkable strategies where the excess energy is dissipated as heat by the antenna system. In higher plants one main player in the process is the major light harvesting antenna of Photosystem II (PSII), LHCII. In this paper we applied Stark fluorescence spectroscopy to LHCII in different quenching states to investigate the possible contribution of charge-transfer states to the quenching. We find that in the quenched state the fluorescence displays a remarkable sensitivity to the applied electric field. The resulting field-induced emission spectra reveal the presence of two distinct energy dissipating sites both characterized by a strong but spectrally very different response to the applied electric field. We propose the two states to originate from chlorophyll-chlorophyll and chlorophyll-carotenoid charge transfer interactions coupled to the chlorophyll exciton state in the terminal emitter locus and discuss these findings in the light of the different models proposed to be responsible for energy dissipation in photosynthesis.     

INFLUENCE OF CHANGES IN THE PHOTON PROTECTIVE ENERGY DISSIPATION ON RED LIGHT-INDUCED DETRAPPING OF THE THERMOLUMINESCENCE Z-BAND

-Thermoluminescence emission at 110 K (Z-band) was markedly diminished when thylakoid membranes were exposed to red light during or after Z-band charging with blue light. Analysis of this phenomenon showed that deactivation of Z-band-emitting chlorophyll species occurred preferentially on the low temperature side of the glow curve, and red light of670–680 nm was most efficient in the deactivation. In order to test our hypothesis that this detrapping is related to local heating effects caused by dissipation of absorbed energy, we measured thermoluminescence glow curves and Z-band emission spectra from spinach leaf discs and thylakoid membranes during induction of nonphotochemical chlorophyll fluorescence quenching. Pretreatment of the plant material was designed to achieve different levels of (1) de-epoxidized xanthophylls in the photosynthetic apparatus and (2) the proton concentration in the thylakoid lumen. In comparison, measurements were performed in aggregated and trimeric light-harvesting pigment-protein complexes of photosystem II. We observed on all three levels of organization that a higher capacity of excitation energy dissipation was accompanied by a stronger red light-induced detrapping of Z-band thermoluminescence.     

Comparison by PAM fluorometry of photosynthetic activity of nine marine phytoplankton grown under identical conditions

The photosynthetic activity of marine phytoplankton from five algal classes (Phaeodactylum tricornutum, Skeletonema costatum, Thalassiosira oceanica, Thalassiosira weissflogii, Dunaliella tertiolecta, Mantoniella squamata, Emiliania huxleyi, Pavlova lutheri and Heterosigma akashiwo) was investigated under identical growth conditions to determine interspecies differences. Primary photochemistry and electron transport capacity of individual species were examined by pulse amplitude-modulated (PAM) fluorescence. Although few differences were found in maximal photosystem II (PSII) photochemical efficiency between various species, large differences were noticed in their PSII-photosystem I (PSI) electron transport activity. We found that species such as T. oceanica and M. squamata have much lower photochemical activity than H. akashiwo. It appeared that processes involved in electron transport activity were more susceptible to change during algal evolution compared with the primary photochemical act close to PSII. Large variations in the nonphotochemical energy dissipation event among species were also observed. Light energy required to saturate photosynthesis was very different between species. We have shown that M. squamata and H. akashiwo required higher light energy (>1300 micromol m(-2) s(-1)) to saturate photosynthesis compared with S. costatum and E. huxleyi (ca 280 micromol m(-2) s(-1)). These differences were interpreted to be the result of variations in the size of light-harvesting complexes associated with PSII. These disparities in photosynthetic activity might modulate algal community structure in the natural environment where light energy is highly variable. Our results suggest that for an accurate evaluation of primary productivity from fluorescence measurements, it is essential to know the species composition of the algal community and the individual photosynthetic capacity related to the major phytoplankton species present in the natural phytoplankton assemblage.     

LASER SPECTROSCOPY OF MODEL PHOTOSYNTHETIC SYSTEMS

Abstract— The goal of the present work was to create and investigate a model system, using a dye imbedded into polymer structure, and to examine characteristics which would provide low heat dissipation and excitation diffusion characteristics approaching those seen in the "antenna" of the photosynthetic apparatus. Zinc tetraphenoxyphtalocyanine served as a dye, and different types of polyvinyl pyridine polymers and polystyrene were used as polymer matricies. Measurements of the absorption, fluorescence and Raman spectra of the polymeric films with dye molecules show that along with Van der Waals interactions of the dye molecules with the side aromatic groups of the polymer there is a coordination interaction between the metal atoms of Zinc phtalocyanine and the nitrogen atoms of the pyridine group of the polymer. A model system shows low heat losses of excitation energy, when the dye concentration does not exceed 10^-2 M (mean distances between molecules of about 34 Å). Electronic excitation diffusion characteristics appeared to be close to those of the light harvesting antenna of the photosynthetic apparatus, indicating high efficiency of the energy migration in it.     

A T-DNA insertion mutant of AtHMA1 gene encoding a Cu transporting ATPase in Arabidopsis thaliana has a defect in the water–water cycle of photosynthesis

The water–water cycle is the electron flow through scavenging enzymes for the reactive species of oxygen in chloroplasts, and is proposed to play a role in alternative electron sink in photosynthesis. Here we showed that the water–water cycle is impaired in the T-DNA insertion mutant of AtHMA1 gene encoding a Cu transporting ATPase in chloroplasts. Chlorophyll fluorescence under steady state was not affected in hma1, indicating that photosynthetic electron transport under normal condition was not impaired. Under electron acceptor limited conditions, however, hma1 showed distinguished phenotype in chlorophyll fluorescence characteristics. The most severe phenotype of hma1 could be observed in high (0.1%) CO₂ concentrations, indicating that hma1 has the defect other than photorespiration. The transient increase of chlorophyll fluorescence upon the cessation of the actinic light as well as the NPQ induction of chlorophyll fluorescence revealed that the two pathways of cyclic electron flow around PSI, NDH-pathway and FQR-pathway, are both intact in hma1. Based on the NPQ induction under 0% oxygen condition, we conclude that the water–water cycle is impaired in hma1, presumably due to the decreased level of Cu/Zn SOD in the mutant. Under high CO₂ condition, hma1 exhibited slightly higher NPQ induction than wild type plants, while this increase of NPQ in hma1 was suppressed when hma1 was crossed with crr2 having a defect in NDH-mediated PSI cyclic electron flow. We propose that the water–water cycle and NDH-mediated pathways might be regulated compensationally with each other especially when photorespiration is suppressed.     

Quantification of the effect of nonphotochemical quenching on the determination of in vivo chl a from phytoplankton along the water column of a freshwater reservoir

Nonphotochemical quenching (NPQ) is a well-known collection of different photoprotective mechanisms of plants and algae to avoid photodamage under an excess of light energy. In order to evaluate the overall effect of NPQ processes on the fluorometric determination of in vivo Chl a from a phytoplankton community dominated by diatoms, we compared the results obtained by two different fluorometric field devices with the total concentration of extracted Chl a measured by HPLC ( in vitro Chl a ). A different set of measurements were made to assess the performance of these fluorometers at high, moderate and low irradiance conditions. The Fbbe fluorometer, which is capable of distinguishing different algal groups according to their pigment content, allowed a better determination of in vivo Chl a under high irradiance conditions, with only a 10% mean difference from the in vitro Chl a concentration. In turn, the FMII fluorometer underestimated by as much as 50% the in vitro Chl a concentration under the same light conditions. As data from both fluorometers were in accordance with the in vitro Chl a values at moderate irradiance levels, the differences observed at high irradiances were attributed to the decrease in the yield of Chl a fluorescence caused by photoprotective NPQ processes. Accordingly, we estimated the effect of NPQ processes on the in vivo Chl a determination and the results allow us to provide an equation to correct this effect when in situ fluorometric measurements are carried out under high irradiance regimes. Our results demonstrate that under certain circumstances NPQ seriously compromises the results obtained by in situ fluorometric probes and highlight the need for a cautious interpretation of field data under such environmental conditions.     

Regulation of the excitation energy utilization in the photosynthetic apparatus of chlorina f2 barley mutant grown under different irradiances

Acclimation of the photosynthetic apparatus of chlorophyll b-less barley mutant chlorina f2 to low light (100 micromolm(-2)s(-1); LL) and extremely high light level (1000 micromolm(-2)s(-1); HL) was examined using techniques of pigment analysis and chlorophyll a fluorescence measurements at room temperature and at 77 K. The absence of chlorophyll b in LL-grown chlorina f2 resulted in the reduction of functional antenna size of both photosystem II (by 67%) and photosystem I (by 21%). Chlorophyll fluorescence characteristics of the LL-grown mutant indicated no impairment of the utilization of absorbed light energy in photosystem II photochemistry. Thermal dissipation of excitation energy estimated as non-photochemical quenching of minimal fluorescence (SV(0)) was significantly higher as compared to the wild-type barley grown under LL. Despite impaired assembly of pigment-protein complexes, chlorina f2 was able to efficiently acclimate to HL. In comparison with chlorina f2 grown under LL, HL-grown chlorina f2 was characterized by unaffected maximal photochemical efficiency of photosystem II (F(V)/F(M), doubled content of both beta-carotene and the xanthophyll cycle pigments and considerably reduced efficiency of excitation energy transfer from carotenoids to chlorophyll a. The enormous xanthophyll cycle pool size was however associated with reduced SV(0) capacity. We suggest that the substantial part of the xanthophyll cycle pigments is not bound to the remaining pigment-protein complexes and acts as filter for excitation energy, thereby contributing to the efficient photoprotection of chlorina f2 grown under HL.     

Evidence from action and fluorescence spectra that UV-induced violet-blue-green fluorescence enhances leaf photosynthesis

We assessed the contribution of UV-induced violet-blue-green leaf fluorescence to photosynthesis in Poa annua, Sorghum halepense and Nerium oleander by measuring UV-induced fluorescence spectra (280-380 nm excitation, 400-550 nm emission) from leaf surfaces and determining the monochromatic UV action spectra for leaf photosynthetic O2-evolution. Peak fluorescence emission wavelengths from leaf surfaces ranged from violet (408 nm) to blue (448 nm), while excitation peaks for these maxima ranged from 333 to 344 nm. Action spectra were developed by supplementing monochromatic radiation from 280 to 440 nm, in 20 nm increments, to a visible nonsaturating background of 500 mumol m-2 s-1 photosynthetically active radiation and measuring photosynthetic O2-evolution rates. Photosynthetic rates tended to be higher with the 340 nm supplement than with higher or lower wavelength UV supplements. Comparing photosynthetic rates with the 340 nm supplement to those with the 400 nm supplement, the percentage enhancement in photosynthetic rates at 340 nm ranged from 7.8 to 9.8%. We suspect that 340 nm UV improves photosynthetic rates via fluorescence that provides violet-blue-green photons for photosynthetic energy conversion because (1) the peak excitation wavelength (340 nm) for violet-blue-green fluorescence from leaves was also the most effective UV wavelength at enhancing photosynthetic rates, and (2) the magnitude of photosynthetic enhancements attributable to supplemental 340 nm UV was well correlated (R2 = 0.90) with the apparent intensity of 340 nm UV-induced violet-blue-green fluorescence emission from leaves.     

Effects of methanol on photosynthetic processes and growth of Lemna gibba

Effects of methanol on growth and photosynthetic activity of Lemna gibba exposed under continuous illumination were examined. As a higher plant, L. gibba appeared to be much more sensitive to methanol inhibitory effect compared with some algae (Theodoridou et al. [2002] Biochim. Biophys. Acta, 1573, 189-198). We found that stimulatory or inhibitory effects were strongly dependent on the methanol concentration and the time of exposure. When the exposure was up to 0.2% methanol, the growth rate of biomass was improved by 50%. However, stimulatory effect of methanol appeared to be smaller when plants were exposed for 48 h compared with 24 h. Increase in biomass induced by methanol was not based on the increase in primary photosynthetic process but rather on accommodation of energy dissipation during photosynthesis. Inhibitory effect on the growth of L. gibba already observed for 0.5% methanol was strongly associated with the increase in the nonphotochemical energy dissipation. The ratio between biomass and methanol concentration appeared to determine the stimulatory or the inhibitory effect. Suggested explanations for the stimulatory and the inhibitory effects are presented.     

Light‐Harvesting Systems Based on Organic Nanocrystals To Mimic Chlorosomes

We report the first highly efficient artificial light‐harvesting systems based on nanocrystals of difluoroboron chromophores to mimic the chlorosomes, one of the most efficient light‐harvesting systems found in green photosynthetic bacteria. Uniform nanocrystals with controlled donor/acceptor ratios were prepared by simple coassembly of the donors and acceptors in water. The light‐harvesting system funneled the excitation energy collected by a thousand donor chromophores to a single acceptor. The well‐defined spatial organization of individual chromophores in the nanocrystals enabled an energy transfer efficiency of 95 %, even at a donor/acceptor ratio as high as 1000:1, and a significant fluorescence of the acceptor was observed up to donor/acceptor ratios of 200 000:1.     

Synthesis and Functional Reconstitution of Light‐Harvesting Complex II into Polymeric Membrane Architectures

One of most important processes in nature is the harvesting and dissipation of solar energy with the help of light‐harvesting complex II (LHCII). This protein, along with its associated pigments, is the main solar‐energy collector in higher plants. We aimed to generate stable, highly controllable, and sustainable polymer‐based membrane systems containing LHCII–pigment complexes ready for light harvesting. LHCII was produced by cell‐free protein synthesis based on wheat‐germ extract, and the successful integration of LHCII and its pigments into different membrane architectures was monitored. The unidirectionality of LHCII insertion was investigated by protease digestion assays. Fluorescence measurements indicated chlorophyll integration in the presence of LHCII in spherical as well as planar bilayer architectures. Surface plasmon enhanced fluorescence spectroscopy (SPFS) was used to reveal energy transfer from chlorophyll b to chlorophyll a, which indicates native folding of the LHCII proteins.