The carotenoid zeaxanthin and ‘high-energy-state quenching’ of chlorophyll fluorescence

The possibility that zeaxanthin mediates the dissipation of an excess of excitation energy in the antenna chlorophyll of the photochemical apparatus has been tested through the use of an inhibitor of violaxanthin de-epoxidation, dithiothreitol (DTT), as well as through the comparison of two closely related organisms (green and blue-green algal lichens), one of which (blue-green algal lichen) naturally lacks the xanthophyll cycle. In spinach leaves, DTT inhibited a major component of the rapidly relaxing high-energy-state quenching' of chlorophyll fluorescence, which was associated with a quenching of the level of initial fluorescence (F₀) and exhibited a close correlation with the zeaxanthin content of leaves when fluorescence quenching was expressed as the rate constant for radiationless energy dissipation in the antenna chlorophyll. Green algal lichens, which possess the xanthophyll cycle, exhibited the same type of fluorescence quenching as that observed in leaves. Two groups of blue-green algal lichens were used for a comparison with these green algal lichens. A group of zeaxanthin-free blue-green algal lichens did not exhibit the type of chlorophyll fluorescence quenching indicative of energy dissipation in the pigment bed. In contrast, a group of blue-green algal lichens which had formed zeaxanthin slowly through reactions other than the xanthophyll cycle, did show a very similar response to that of leaves and green algal lichens. Fluorescence quenching indicative of radiationless energy dissipation in the antenna chlorophyll was the predominant component of high-energy-state quenching in spinach leaves under conditions allowing for high rates of steady-state photosynthesis. A second, but distinctly different type of high-energy-state quenching of chlorophyll fluorescence, which was not inhibited by DTT (i.e., it was zeaxanthin independent) and which is possibly associated with the photosystem II reaction center, occurred in addition to that associated with zeaxanthin in leaves under a range of conditions which were less favorable for linear photosynthetic electron flow. In intact chloroplasts isolated from (zeaxanthin-free) spinach leaves a combination of these two types of rapidly reversible fluorescence quenching occurred under all conditions examined.Abbreviations DTT dithiothreitol - F₀ (or F₀) yield of instantaneous fluorescence at open PS II reaction centers in the dark (or during actinic illumination) - F_M (or F_M) yield of maximum fluorescence induced by a saturation pulse of light in the dark (or during actinic illumination) - F_V (or F_V) yield of variable fluorescence induced by a saturating pulse of light in the dark (or during actinic illumination) - k _D rate constant for radiationless energy dissipation in the antenna chlorophyll - SV Stern-Volmer equation - PFD photon flux density - PS I photosystem I - PS II photosystem II - Q_A acceptor of photosystem II - q_N coefficient of nonphotochemical chlorophyll fluorescence quenching - q_P coefficient of photochemical chlorophyll fluorescence quenching     

Photosystem II chlorophyll a fluorescence lifetimes and intensity are independent of the antenna size differences between barley wild-type and chlorina mutants: Photochemical quenching and xanthophyll cycle-dependent nonphotochemical quenching of fluorescence

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, F_o) and minimal rates of PS II photochemistry (at maximum Chl fluorescence level, F_m). Further, the fluorescence lifetimes and intensity parameters, as affected by the trans-thylakoid membrane pH gradient (pH) and the carotenoid pigments of the xanthophyll cycle, were also similar and independent of the antenna size differences. In the presence of a pH, 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 pH, 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 pH 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 pH 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.Abbreviations Ax antheraxanthin - BSA bovine serum albumin - c_x lifetime center of fluorescence decay component x - CP chlorophyll binding protein of PS II inner antenna - DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea - DTT dithiothreitol - f_x fractional intensity of fluorescence lifetime component x - F_m, F_m maximal PS II Chl a fluorescence intensity with all Q_A reduced in the absence, presence of thylakoid membrane energization - F_o minimal PS II Chl a fluorescence intensity with all Q_A oxidized - F_v=F_m–F_o variable level of PS II Chl a fluorescence - HPLC high performance liquid chromatography - k_A rate constant of all combined energy dissipation pathways in PS II except photochemistry and fluorescence - k_F rate constant of PS II Chl a fluorescence - LHCIIb main light harvesting pigment-protein complex (of PS II) - N_pig mols Chl a+b per PS II - NPQ=(F_m/F_m–1) nonphotochemical quenching of PS II Chl a fluorescence - PAM pulse-amplitude modulation fluorometer - PFD photon-flux density, mols photons m^–2 s^–1 - PS II Photosystem II - P680 special-pair Chls of PS II reaction center - Q_A primary quinone electron acceptor of PS II - V_x violaxanthin - w_x width at half maximum of Lorentzian fluorescence lifetime distribution x - Zx zeaxanthin - pH trans-thylakoid proton gradient - % MathType!MTEF!2!1!+-% feaafiart1ev1aaatCvAUfeBSjuyZL2yd9gzLbvyNv2CaerbuLwBLn% hiov2DGi1BTfMBaeXafv3ySLgzGmvETj2BSbqef0uAJj3BZ9Mz0bYu% H52CGmvzYLMzaerbd9wDYLwzYbItLDharqqr1ngBPrgifHhDYfgasa% acOqpw0xe9v8qqaqFD0xXdHaVhbbf9v8qqaqFr0xc9pk0xbba9q8Wq% Ffea0-yr0RYxir-Jbba9q8aq0-yq-He9q8qqQ8frFve9Fve9Ff0dme% GabaqaaiGacaGaamqadaabaeaafiaakeaacqGH8aapcqaHepaDcqGH% +aGpdaWgaaWcbaGaamOraiaad2gaaeqaaaaa!4989!\[< \tau > _{Fm}\],% MathType!MTEF!2!1!+-% feaafiart1ev1aaatCvAUfeBSjuyZL2yd9gzLbvyNv2CaerbuLwBLn% hiov2DGi1BTfMBaeXafv3ySLgzGmvETj2BSbqef0uAJj3BZ9Mz0bYu% H52CGmvzYLMzaerbd9wDYLwzYbItLDharqqr1ngBPrgifHhDYfgasa% acOqpw0xe9v8qqaqFD0xXdHaVhbbf9v8qqaqFr0xc9pk0xbba9q8Wq% Ffea0-yr0RYxir-Jbba9q8aq0-yq-He9q8qqQ8frFve9Fve9Ff0dme% GabaqaaiGacaGaamqadaabaeaafiaakeaacqGH8aapcqaHepaDcqGH% +aGpdaWgaaWcbaGaamOraiaad+gaaeqaaOGaeyypa0Zaaabqaeaaca% WGMbWaaSbaaSqaaiaadIhaaeqaaOGaam4yamaaBaaaleaacaWG4baa% beaaaeqabeqdcqGHris5aaaa!50D3!\[< \tau > _{Fo} = \sum {f_x c_x }\] average lifetime of Chl a fluorescence calculated from a multi-exponential model under F_m, F_o conditions     

Analysis of xanthophyll cycle carotenoids and chlorophyll fluorescence in light intensity-dependent chlorophyll-deficient mutants of wheat and barley

Three light intensity-dependent Chl b-deficient mutants, two in wheat and one in barley, were analyzed for their xanthophyll cycle carotenoids and Chl fluorescence characteristics under two different growth PFDs (30 versus 600 mol photons·m^–2 s^–1 incident light). Mutants grown under low light possessed lower levels of total Chls and carotenoids per unit leaf area compared to wild type plants, but the relative proportions of the two did not vary markedly between strains. In contrast, mutants grown under high light had much lower levels of Chl, leading to markedly greater carotenoid to Chl ratios in the mutants when compared to wild type. Under low light conditions the carotenoids of the xanthophyll cycle comprised approximately 15% of the total carotenoids in all strains; under high light the xanthophyll cycle pool increased to over 30% of the total carotenoids in wild type plants and to over 50% of the total carotenoids in the three mutant strains. Whereas the xanthophyll cycle remained fairly epoxidized in all plants grown under low light, plants grown under high light exhibited a considerable degree of conversion of the xanthophyll cycle into antheraxanthin and zeaxanthin during the diurnal cycle, with almost complete conversion (over 90%) occurring only in the mutants. 50 to 95% of the xanthophyll cycle was retained as antheraxanthin and zeaxanthin overnight in these mutants which also exhibited sustained depressions in PS II photochemical efficiency (F_v/F_m), which may have resulted from a sustained high level of photoprotective energy dissipation activity. The relatively larger xanthophyll cycle pool in the Chl b-deficient mutant could result in part from the reported concentration of the xanthophyll cycle in the inner antenna complexes, given that the Chl b-deficient mutants are deficient in the peripheral LHC-II complexes.Abbreviations A antheraxanthin - Chl chlorophyll - F_o and F_m minimal yield (at open PS II reaction centers) and maximal yield (at closed centers) of chlorophyll fluorescence in darkness - F level of fluorescence during illumination with photosynthetically active radiation - F_m maximal yield (at closed centers) of chlorophyll fluorescence during illumination with photosynthetically active radiation - (F_m–F)/F_m actual efficiency of PS II during illumination with photosynthetically active radiation - F_v/F_m+(F_m–F_o)/F_m intrinsic efficiency of PS II in darkness - LHC_II light-harvesting chlorophyll-protein complex of Photosystem II - PFD photon flux density (between 400 and 700 nm) - PS I Photosystem I - PS II Photosystem II - V violaxanthin - Z zeaxanthin     

Xanthophyll cycle and light stress in nature: uniform response to excess direct sunlight among higher plant species

Photosystem II (PS II) efficiency, nonphotochemical fluorescence quenching, and xanthophyll cycle composition were determined in situ in the natural environment at midday in (i) a range of differently angled sun leaves ofEuonymus kiautschovicus Loesener and (ii) in sun leaves of a wide range of different plant species, including trees, shrubs, and herbs. Very different degrees of light stress were experienced by these leaves (i) in response to different levels of incident photon flux densities at similar photosynthetic capacities amongEuonymus leaves and (ii) as a result of very different photosynthetic capacities among species at similar incident photon flux densities (that were equivalent to full sunlight). ForEuonymus as well as the interspecific comparison all data fell on one single, close relationship for changes in intrinsic PSII efficiency, nonphotochemical fluorescence quenching, or the levels of zeaxanthin + antheraxanthin in leaves, respectively, as a function of the actual level of light stress. Thus, the same conversion state of the xanthophyll cycle and the same level of energy dissipation were observed for a given degree of light stress independent of species or conditions causing the light stress. Since all increases in thermal energy dissipation were associated with increases in the levels of zeaxanthin + antheraxanthin in these leaves, there was thus no indication of any form of xanthophyll cycle-independent energy dissipation in any of the twenty-four species or varieties of plants examined in their natural environment. It is also concluded that transient diurnal changes in intrinsic PSII efficiency in nature are caused by changes in the efficiency with which excitation energy is delivered from the antennae to PSII centers, and are thus likely to be purely photoprotective. Consequently, the possibility of quantifying the allocation of absorbed light into PSII photochemistry versus energy dissipation in the antennae from changes in intrinsic PSII efficiency is explored.Abbreviations A antheraxanthin - F actual level of fluorescence - F_a, F _o minimal fluorescence in the absence, presence of thylakoid energization - F_m, F _m maximal fluorescence in the absence, presence of thylakoid energization - F_m, - F)/F _m actual PSII efficiency ( = percent of absorbed light utilized in PSII photochemistry) - F_v/F_m, F _v /F_m/ PSII efficiency of open centers in the absence, presence of thylakoid energization - NPQ nonphotochemical fluorescence quenching - F_m/F _m - 1; q_p quenching coefficient for photochemical quenching - V violaxanthin - Z zeaxanthin     

Chlorophyll fluorescence in the resurrection plant Selaginella lepidophylla (Hook. & Grev.) Spring during high-light and desiccation stress,and evidence for zeaxanthin-associated photoprotection

The function of photosystem (PS)II during desiccation and exposure to high photon flux density (PFD) was investigated via analysis of chlorophyll fluorescence in the desert resurrection plant Selaginella lepidophylla (Hook. and Grev.) Spring. Exposure of hydrated, physiologically competent stems to 2000 mol · m^–2 · s^–1 PFD caused significant reductions in both intrinsic fluorescence yield (F_O) and photochemical efficiency of PSII (F_V/F_M) but recovery to pre-exposure values was rapid under low PFD. Desiccation under low PFD also affected fluorescence characteristics. Both F_V/F_M and photochemical fluorescence quenching remained high until about 40% relative water content and both then decreased rapidly as plants approached 0% relative water content. In contrast, the maximum fluorescence yield (F_M) decreased and non-photochemical fluorescence quenching increased early during desiccation. In plants dried at high PFD, the decrease in F_V/F_M was accentuated and F_O was reduced, however, fluorescence characteristics returned to near pre-exposure values after 24-h of rehydration and recovery at low PFD. Pretreatment of stems with dithiothreitol, an inhibitor of zeaxanthin synthesis, accelerated the decline in F_V/F_M and significantly increased F_O relative to controls at 925 mol · m^–2 · s^–1 PFD, and the differences persisted over a 3-h low-PFD recovery period. Pretreatment with dithiothreitol also significantly decreased non-photochemical fluorescence quenching, increased the reduction state of Q_A, the primary electron acceptor of PSII, and prevented the synthesis of zeaxanthin relative to controls when stems were exposed to PFDs in excess of 250 mol · m^–2 · s^–1. These results indicate that a zeaxanthin-associated mechanism of photoprotection exists in this desert pteridophyte that may help to prevent photoinhibitory damage in the fully hydrated state and which may play an additional role in protecting PSII as thylakoid membranes undergo water loss.Abbreviations and Symbols DTT dithiothreitol - EPS epoxidation state - F_O yield of instantaneous fluorescence at open PSII centers - F_M maximum yield of fluorescence at closed PSII centers induced by saturating light - F_M F_M determined during actinic illumination - F_V yield of variable fluorescence (F_M-F_O) - F_V/F_M photochemical efficiency of PSII - q_P photochemical fluorescence quenching - q_NP non-photochemical fluorescence quenching of Schreiber et al. (1986) - NPQ non-photochemical fluorescence quenching from the Stern-Volmer equation - PFD photon flux density - RWC relative water content This paper is based on research done while W.G.E. was on leave of absence at Duke University during the fall of 1990. We would like to thank Dan Yakir, John Skillman, Steve Grace, and Suchandra Balachandran and many others at Duke University for their help and input with this research. Dr. Barbara Demmig-Adams provided zeaxanthin for standard-curve purposes.     

Temperature-sensitive coupling and uncoupling of ATPase-mediated,nonradiative energy dissipation: Similarities between chloroplasts and leaves

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 (F_o) and the maximal (F_m) 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 (A_508nm) and energy-dependent light scattering (A_536nm) 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.Abbreviations and Symbols A antheraxanthin - Chl chlorophyll - DPS de-epoxidation state of the xanthophyll cycle, ([Z+A]/[V+A+Z]) - F, F steady-state fluorescence in the absence, presence of thylakoid energization - F_o, F_o dark fluorescence level in the absence, presence of thylakoid energization - F_m, F_m maximal fluorescence in absence, presence of thylakoid energization - NPQ nonphotochemical quenching (F_m/F_m)–1 - V violaxanthin - Z zeaxanthin - NRD nonradiative dissipation - PFD photon flux density - [2ATP+ADP] - pH trans-thylakoid proton gradient - S pH-dependent light scattering - _PSII (F_m–F)/F_m, photon yield of PSII photochemistry at the actual reduction state in the light or dark - [ATP+ADP+AMP] We thank Connie Shih for skillful assistance in growing plants and for conducting HPLC analyses. Support from an NSF/USDA/DOE postdoctoral training grant to A.G. is gratefully acknowledged. A.G. also wishes to thank Prof. Govindjee for valuable discussions. C.I.W.-D.P.B. Publication No. 1197.     

Cooperation of xanthophyll cycle with water-water cycle in the protection of photosystems 1 and 2 against inactivation during chilling stress under low irradiance

The xanthophyll cycle and the water-water cycle had different functional significance in chilling-sensitive sweet pepper upon exposure to chilling temperature (4 °C) under low irradiance (100 µmol m⁻² s⁻¹) for 6 h. During chilling stress, effects of non-photochemical quenching (NPQ) on photosystem 2 (PS2) in dithiothreitol (DTT) fed leaves remained distinguishable from that of the water-water cycle in diethyldithiocarbamate (DDTC) fed leaves. In DTT-fed leaves, NPQ decreased greatly accompanied by visible inhibition of the de-epoxidized ratio of the xanthophyll cycle, and maximum photochemical efficiency of PS2 (F_v/F_m) decreased markedly. Thus the xanthophyll cycle-dependent NPQ could protect PS2 through energy dissipation under chilling stress. However, NPQ had a slighter effect on photosystem 1 (PS1) in DTT-fed leaves than in DDTC-fed leaves, whereas effects of the water-water cycle on PS1 remained distinguishable from that of NPQ. Inhibiting superoxide dismutase (SOD) activity increased the accumulation of , the oxidation level of P700 (P700⁺) decreased markedly relative to the control and DTT-fed leaves. Both F_v/F_m and NPQ changed little in DDTC-fed leaves accompanied by little change of (A+Z)/(V+A+Z). This is the active oxygen species inducing PS1 photoinhibition in sweet pepper. The water-water cycle can be interrupted easily at chilling temperature. We propose that during chilling stress under low irradiance, the xanthophyll cycle-dependent NPQ has the main function to protect PS2, whereas the water-water cycle is not only the pathway to dissipate energy but also the dominant factor causing PS1 chilling-sensitivity in sweet pepper.This research was supported by the State Key Basic Research and Development Plan of China (G1998010100), the Natural Science Foundation of China (30370854), and the open project from Key Lab of Crop Biology of Shandong Province.     

Close relationship between the state of the xanthophyll cycle pigments and photosystem II efficiency during recovery from winter stress

The potential involvement of the xanthophyll cycle in photoprotection of overwintering evergreen plants was investigated. Leaves from five evergreen species. Pseudotsuga menziesii, Pinus panderosa, Euonyums kiautschovicus. Mahonia repens and Malva neglecta, were collected from the field predawn during winter and transferred to the laboratory where chlorophyll fluorescence emission as well as the chlorophyll and carotenoid composition were ascertained periodically for 4.5 days. Leaves and needles from all species were found to have retained large amounts of the xanthophyll cycle pigments zeaxanthin and antheraxanthin, and they exhibited sustained low values of the intrinsic efficiency of photosystem II (PSII; measured as the ratio of variable to maximal fluorescence, F_v/F_m) upon collection. The increase in PSII efficiency was biphasic, with a rapid phase (requiring several hours) and a slow phase (requiring several days). Changes in the conversion state of the xanthophyll cycle were found to correlate with increases in PSII efficiency in both phases, with the latter phase involving large increases in both F_m (maximal fluorescence) and F_o (minimal fluorescence) throughout the period of recovery. The relationship between F_m quenching (expressed as nonphotochemical or Stern-Volmer quenching [NPQ] of F_m, i.e. F_m/ F_m–1) and F_o quenching (F_o/F_o–1) was linear, as expected for changes in xanthophyll cycle-dependent energy dissipation in the antenna complexes. Furthermore, the relationship between F_v/F_m and NPQ during recovery followed the theoretical relationship predicted for changes in the rate constant for energy dissipation in the antenna complexes. This fit between the theoretical relationship and the actual data indicates that all changes in NPQ or F_v/F_m can be accounted for by changes in this rate constant. The results suggest a role for the photoprotective xanthophyll cycle-dependent dissipation process in the lowered efficiency of PSII observed in coldstressed evergreen plants in the field.     

Photoinhibition and xanthophyll cycle activity in bayberry (Myrica rubra) leaves induced by high irradiance

The effect of high irradiance (HI, photosynthetically active photon flux density of 1 300 μmol m⁻² s⁻¹) on net photosynthetic rate (P _N), chlorophyll fluorescence parameters, and xanthophyll cycle components were studied in fruit tree bayberry leaves. HI induced the photoinhibition and inactivation of photosystem 2 (PS2) reaction centres (RCs), which was characterized by decreased P _N, maximum yield of fluorescence after dark adaptation (F_m), photochemical efficiency of PS2 (F_v/F_m) and quantum yield of PS2 (Φ_PS2), and increased reduction state of Q_A (1-q_P) and non-photochemical quenching (NPQ). Initial fluorescence (F₀) showed a decrease after the first 2 h, and subsequently increased from the third hour exposure to HI. Furthermore, a greater increase in the ratio (F_i-F₀)/(F_p-F₀) which is an expression of the proportion of the Q_B non-reducing PS2 centres, whereas a remarked decrease in the slope of F_i to F_p which represents the rate of Q_A reduction was observed in leaves after HI exposure. Additionally, HI caused an increase in the pool size of the xanthophyll cycle pigments and sustained elevated contents of zeaxanthin (Z), antheraxanthin (A), and de-epoxidation state (DES) at the end of the irradiation period. During HI, decreased F_m, F_v/F_m, Φ_PS2, NPQ, slope of F_i to F_p, V+A+Z, and DES, and increased F₀, 1-q_P, ratio (F_i-F₀)/(F_p-F₀), and V were observed in dithiothreitol (DTT)-fed leaves compared to control ones under the same conditions. Hence photoinhibition caused by HI in bayberry was probably attributed to inactivation of PS2 RCs, and photoprotection from photodamage were mainly related to the xanthophyll cycle-dependent heat dissipation in excess photons.     

Photoinactivation of photosystem II by cumulative exposure to short light pulses during the induction period of photosynthesis

Photoinactivation of Photosystem (PS) II in vivo was investigated by cumulative exposure of pea, rice and spinach leaves to light pulses of variable duration from 2 to 100 s, separated by dark intervals of 30 min. During each light pulse, photosynthetic induction occurred to an extent depending on the time of illumination, but steady-state photosynthesis had not been achieved. During photosynthetic induction, it is clearly demonstrated that reciprocity of irradiance and duration of illumination did not hold: hence the same cumulative photon exposure (mol m^–2) does not necessarily give the same extent of photoinactivation of PS II. This contrasts with the situation of steady-state photosynthesis where the photoinactivation of PS II exhibited reciprocity of irradiance and duration of illumination (Park et al. (1995) Planta 196: 401–411). We suggest that, for reciprocity to hold between irradiance and duration of illumination, there must be a balance between photochemical (qP) and non-photochemical (NPQ) quenching at all irradiances. The index of susceptibility to light stress, which represents an intrinsic ability of PS II to balance photochemical and non-photochemical quenching, is defined by the quotient (1-qP)/NPQ. Although constant in steady-state photosynthesis under a wide range of irradiance (Park et al. (1995). Plant Cell Physiol 36: 1163–1169), this index of susceptibility for spinach leaves declined extremely rapidly during photosynthetic induction at a given irradiance, and, at a given cumulative photon exposure, was dependent on irradiance. During photosynthetic induction, only limited photoprotective strategies are developed: while the transthylakoid pH gradient conferred some degree of photoprotection, neither D1 protein turnover nor the xanthophyll cycle was operative. Thus, PS II is more easily photoinactivated during photosynthetic induction, a phenomenon that may have relevance for understorey leaves experiencing infrequent, short sunflecks.Abbreviations D1 protein psbA gene product - DTT dithiothreitol - F_v, F_m, F_o variable, maximum, and initial (corresponding to open traps) chlorophyll fluorescence yield, respectively - NPQ non-photochemical quenching - PS Photosystem - Q_A primary quinone acceptor of PS II - qP photochemical quenching coefficient     

Inhibition of Photosynthesis by Shift in the Balance of Excitation Energy Distribution Between Photosystems in Dithiothreitol Treated Soybean Leaves

Chlorophyll fluorescence kinetics was used to investigate the effect of 1,4-dithiothreitol (DTT) on the distribution of excitation energy between photosystem 1 (PS1) and photosystem 2 (PS2) in soybean leaves under high irradiance (HI). The maximum PS2 quantum yield (F_v/F_m) was hardly affected by the presence of DTT, however, photon-saturated photosynthesis was depressed distinctly. Photochemical efficiency of open PS2 reaction centres during irradiation (F_v/F_m) was enhanced by about 30–40 % by DTT treatment, whereas photochemical quenching (q_P) was depressed by about 40 % under HI. DTT treatment caused a 30 % decrease in allocation of excitation energy to PS1 under HI and a 20 % increase to PS2. An obvious shift in the balance of excitation energy distribution between photosystems was observed in DTT-treated leaves. Though high excitation pressure (1 - q_P) resulted from DTT treatment, non-photochemical quenching (q_N) was lower. DTT completely inhibited the formation of zeaxanthin and also distinctly depressed the state transition (q_T). The shift in the balance of excitation distribution between the two photosystems induced by DTT was mainly due to the enhancement of excitation energy capture by PS2 antenna and the inhibition of state transition. It might be the shift in the balance between the two photosystems that mainly induced the depression of photosynthesis. Thus, to keep high utilization efficiency of absorbed photon energy, it is necessary to maintain the balance of excitation distribution between PS2 and PS1.     

Nigericin insensitive post-illumination reduction in fluorescence yield in Dunaliella tertiolecta (chlorophyte)

Cells of the green alga Dunaliella tertiolecta grown in a light/dark cycle were exposed to high light for about 15 min. In light, energy-dependent quenching reduced fluorescence emission and decreased PS II efficiency. Within 3 minutes after darkening fluorescence quenching largely relaxed. However, PS II fluorescence emission decreased again after further darkening. F_o and F_m decreased to the same relative extent and the PS II efficiency was not reduced. This Reduction in Fluorescence yield in Darkness, termed RFD for the purpose of this paper, lasted about 20 min. The deepoxidation state of xanthophylls remained unchanged during and after the 15-min exposure to high light. We show that RFD is insensitive to the uncoupler nigericin and thus unrelated to energy-dependent quenching. RFD correlated with a reduction of the PQ pool after darkening and low levels of far red or blue light (430 nm more than 460 nm) prevented RFD. This is in contrast to observations in higher plants, where a post-illumination reduction of the PQ pool causes and increase in F_o (Groom et al. (1993) Photosynth Res 36: 205–215). Changes in the adenylate energy charge were not correlated with RFD. Antimycin A and cyanide, both inhibitors of the PQ-oxidase, caused an increase in RFD whereas SHAM, an inhibitor of the chloroplastic glycolate-quinone oxidoreductase, caused a decrease. Low CO₂ concentrations, known to increase the oxygenase activity of Rubisco and to generate glycolate and P-glycolate in light, caused an increase in RFD. We propose that accumulated glycolate and P-glycolate reduce the PQ pool in darkness, leading to the formation of RFD. During RFD, 77 K fluorescence emission from PS II was more reduced than that from PS I, thus resembling a state I, state II transition. However, the reduction in fluorescence yield during RFD is much larger than the reduction previously attributed to state transitions and it is unclear whether RFD and state transitions are identical. The formation and relaxation of RFD increased with higher temperatures and the extent of RFD was largest at the growth temperature (25°C). RFD has to be taken into account when fluorescence is measured after darkening as it may be mistaken for energy-dependent quenching.Abbreviations F_o fluorescence, measured when PS II traps are open - F_o difference between F_o and F_o - F_m fluorescence, measured when PS II traps are temporarily closed - F_m difference between F_m and F_m - FR far red - PFD photosynthetically active photon flux density - PQ plastoquinone - RFD reduction in fluorescence in darkness - SHAM salicylhydroxamic acid - Q_A primary quinone acceptor of PS II     

Non-photochemical fluorescence quenching and the diadinoxanthin cycle in a marine diatom

The diadinoxanthin cycle (DD-cycle) in chromophyte algae involves the interconversion of two carotenoids, diadinoxanthin (DD) and diatoxanthin (DT). We investigated the kinetics of light-induced DD-cycling in the marine diatom Phaeodactylum tricornutum and its role in dissipating excess excitation energy in PS II. Within 15 min following an increase in irradiance, DT increased and was accompanied by a stoichiometric decrease in DD. This reaction was completely blocked by dithiothreitol (DTT). A second, time-dependent, increase in DT was detected 20 min after the light shift without a concomitant decrease in DD. DT accumulation from both processes was correlated with increases in non-photochemical quenching of chlorophyll fluorescence. Stern-Volmer analyses suggests that changes in non-photochemical quenching resulted from changes in thermal dissipation in the PS II antenna and in the reaction center. The increase in non-photochemical quenching was correlated with a small decrease in the effective absorption cross section of PS II. Model calculations suggest however that the changes in cross section are not sufficiently large to significantly reduce multiple excitation of the reaction center within the turnover time of steady-state photosynthetic electron transport at light saturation. In DTT poisoned cells, the change in non-photochemical quenching appears to result from energy dissipation in the reaction center and was associated with decreased photochemical efficiency. D1 protein degradation was slightly higher in samples poisoned with DTT than in control samples. These results suggest that while DD-cycling may dynamically alter the photosynthesis-irradiance response curve, it offers limited protection against photodamage of PS II reaction centers at irradiance levels sufficient to saturate steady-state photosynthesis.Abbreviations CAP chloramphenicol - D1 PS II reaction center protein - DD diadinoxanthin - DD cycle-diadinoxanthin cycle - DT diatoxanthin - DTT dithiothreitol - FCP fucoxanthin chlorophyll a-c protein - F_m maximum fluorescence yield in the dark-adapted state - F_o minimum fluorescence yield in the dark-adapted state - F_m and F_o maximum and minimum fluorescence yields respectively in some light adapted state - F_v maximum variable fluorescence yield in the dark-adapted state - I_k Irradiance at the intercept of the initial slope of the photosynthesis-irradiance curve and the maximum photosynthetic rate - k_D first order rate constant for nonradiative de-excitation of excitions in the PS II antenna - k_d first order rate constant for non-radiative de-excitation of excitons in the PS II reaction center - k_F first order rate constant for fluorescence - k_T first order rate constant for exciton transfer to the reaction center - k_t first order rate constant for exciton transfer from the reaction center to the antenna - Rubisco ribulose bisphosphate carboxylase - SV_m Stern-Volmer quenching coefficient of the maximum fluorescence yield - SV_o Stern-Volmer quenching coefficient of the miniximum fluorescence yield - _{PS II} apparent absorption cross-section of PS II - _arr average interval between exciton arrival to the PS II reaction center (ms) - _rem average interval between electron turnover during photosynthesis in the PS II reaction center (ms) - _d the probability that an exciton is non-radiatively dissipated in the reaction center - _T the probability that an exciton in the antenna is transferred to the reaction center - _t the probability that an exciton is transferred back from the reaction center to the antenna     

Reduced levels of cytochrome b 6/f in transgenic tobacco increases the excitation pressure on Photosystem II without increasing sensitivity to photoinhibition in vivo

We have examined tobacco transformed with an antisense construct against the Rieske-FeS subunit of the cytochromeb ₆ f complex, containing only 15 to 20% of the wild-type level of cytochrome f. The anti-Rieske-FeS leaves had a comparable chlorophyll and Photosystem II reaction center stoichiometry and a comparable carotenoid profile to the wild-type, with differences of less than 10% on a leaf area basis. When exposed to high irradiance, the anti-Rieske-FeS leaves showed a greatly increased closure of Photosystem II and a much reduced capacity to develop non-photochemical quenching compared with wild-type. However, contrary to our expectations, the anti-Rieske-FeS leaves were not more susceptible to photoinhibition than were wild-type leaves. Further, when we regulated the irradiance so that the excitation pressure on photosystem II was equivalent in both the anti-Rieske-FeS and wild-type leaves, the anti-Rieske-FeS leaves experienced much less photoinhibition than wild-type. The evidence from the anti-Rieske-FeS tobacco suggests that rapid photoinactivation of Photosystem II in vivo only occurs when closure of Photosystem II coincides with lumen acidification. These results suggest that the model of photoinhibition in vivo occurring principally because of limitations to electron withdrawal from photosystem II does not explain photoinhibition in these transgenic tobacco leaves, and we need to re-evaluate the twinned concepts of photoinhibition and photoprotection.Abbreviations Chl chlorophyll - DCMU 3-(3,4-dichlophenyl)-1,-dimethylurea - Fo and Fo minimal fluorescence when all PS II reaction centers are open in dark- and light-acclimated leaves, respectively - Fm and Fm maximal fluorescence when all PS II reaction centers are closed in dark- and light-acclimated leaves, respectively - Fv variable fluorescence (Fm-Fo) in dark acclimated leaves - Fv variable fluorescence (Fm-Fo) in lightacclimated leaves - NPQ non-photochemical quenching of fluorescence - PS I and PS II Photosystem I and II - P680 primary electron donor of the reaction center of PS II - PFD photosynthetic flux density - Q_A primary acceptor quinone of PS II - q_p photochemical quenching of fluorescence - V+A+Z violaxanthin+antheraxanthin+zeaxanthin     

PSII photochemistry, thermal energy dissipation, and the xanthophyll cycle in Kalanchoë daigremontiana exposed to a combination of water stress and high light

Kalanchoë daigremontiana, a CAM plant grown in a greenhouse, was subjected to severe water stress. The changes in photosystem II (PSII) photochemistry were investigated in water‐stressed leaves. To separate water stress effects from photoinhibition, water stress was imposed at low irradiance (daily peak PFD 150 μmol m^?2 s^?1). There were no significant changes in the maximal efficiency of PSII photochemistry (F_v/F_m), the traditional fluorescence induction kinetics (OIP) and the polyphasic fluorescence induction kinetics (OJIP), suggesting that water stress had no direct effects on the primary PSII photochemistry in dark‐adapted leaves. However, PSII photochemistry in light‐adapted leaves was modified in water‐stressed plants. This was shown by the decrease in the actual PSII efficiency (Φ_PSII), the efficiency of excitation energy capture by open PSII centres (F_v′/F_m′), and photochemical quenching (q_P), as well as a significant increase in non‐photochemical quenching (NPQ) in particular at high PFDs. In addition, photoinhibition and the xanthophyll cycle were investigated in water‐stressed leaves when exposed to 50% full sunlight and full sunlight. At midday, water stress induced a substantial decrease in F_v/F_m which was reversible. Such a decrease was greater at higher irradiance. Similar results were observed in Φ_PSII, q_P, and F_v′/F_m′. On the other hand, water stress induced a significant increase in NPQ and the level of zeaxanthin via the de‐epoxidation of violaxanthin and their increases were greater at higher irradiance. The results suggest that water stress led to increased susceptibility to photoinhibition which was attributed to a photoprotective process but not to a photodamage process. Such a photoprotection was associated with the enhanced formation of zeaxanthin via de‐epoxidation of violaxanthin. The results also suggest that thermal dissipation of excess energy associated with the xanthophyll cycle may be an important adaptive mechanism to help protect the photosynthetic apparatus from photoinhibitory damage for CAM plants normally growing in arid and semi‐arid areas where they are subjected to a combination of water stress and high light.     

依赖于叶黄素循环的热耗散对茶树叶片防御强光破坏的相关试验研究

经叶黄素循环抑制剂——二硫苏糖醇(DIT)处理的茶树叶片,以850μmol．m^-2．s^-1的PFD照射120min后,福鼎大白茶的叶黄素循环组分中的环氧玉米黄素(A)和玉米黄素(Z)含量之和降低了76．5％,结果导致非光化学猝灭(NPQ)、光系统Ⅱ(PSⅡ)的光化学效率(Fv／Fm)、光化学猝灭系数(qP)、PSⅡ实际光化学量子效率(ψPSⅡR)和光合电子传递速率(ETR)明显下降,而F0显著上升,暗恢复后Fv／Fm恢复程度小于未经DIT处理的叶片。自然光强下,NPQ与与叶黄素循环的脱环氧化程度(A Z)/(V A Z)比值呈明显的正线性关系(R=0．9488^***)。这些结果充分证明依赖与叶黄素循环的热耗散是茶树叶片光合器官防御强光破坏的主要途径。     

Dithiothreitol,an inhibitor of violaxanthin de-epoxidation,increases the susceptibility of leaves ofNerium oleander L. to photoinhibition of photosynthesis

Leaves ofNerium oleander L. plants, which had been previously kept in a shaded glasshouse for at least two months, were fed 1 mM dithiothreitol (DTT) through their petioles, either for 12h in darkness (overnight) or for 2h in low light (28 μmol photons·m⁻²·s⁻¹), in each case followed by a 3-h exposure to high light (1260 μmol photons·m⁻²·s⁻¹). During exposure to high light, violaxanthin became converted to zeaxanthin in control leaves, to which water had been fed, whereas zeaxanthin did not accumulate in leaves treated with DTT. Total carbon gain was not reduced by DTT during the photoinhibitory treatment. Exposure to high light led to a decrease in the photochemical efficiency of photosystem II, measured as the ratio of variable over maximum fluorescence emission,F _v/F _M, at both 298 K and 77K. The decrease was much more pronounced in the presence of DTT, mainly owing to a sustained increase in the instantaneous fluorescence,F _o. By contrast, in the control leaves,F _o determined immediately after the high-light treatment showed a transient decrease below theF _o value obtained before the onset of the photoinhibitory treatment (i.e. after 12 h dark adaptation), followed by a rapid return (within seconds) to this original level ofF _o during the following recovery period in darkness. Incubation of leaves with DTT led to large, sustained decreases in the photon-use efficiency of photosynthetic O₂ evolution by bright light, whilst the capacity of photosynthetic O₂ evolution at light and CO₂ saturation was less affected. In the control leaves, only small reductions in the photon yield and in the photosynthetic capacity were observed. These findings are consistent with previous suggestions that zeaxanthin, formed in the xanthophyll cycle by de-epoxidation of violaxanthin, is involved in protecting the photosynthetic apparatus against the adverse effects of excessive light.     

Characteristics of Photosynthetic Apparatus in Mn-Starved Maize Leaves

The effects of Mn-deficiency on CO₂ assimilation and excitation energy distribution were studied using Mn-starved maize leaves. Mn-deficiency caused about 70 % loss in the photon-saturated net photosynthetic rate (P _N) compared to control leaves. The loss of P _N was associated with a strong decrease in the activity of oxygen evolution complex (OEC) and the linear electron transport driven by photosystem 2 (PS2) in Mn-deficienct leaves. The photochemical quenching of PS2 (q_P) and the maximum efficiency of PS2 photochemistry (F_v/F_m) decreased significantly in Mn-starved leaves under high irradiance, implicating that serious photoinhibition took place. However, the high-energy fluorescence quenching (q_E) decreased, which was associated with xanthophyll cycle. The results showed that the pool of de-epoxidation components of the xanthophyll cycle was lowered markedly owing to Mn deficiency. Linear electron transport driven by PS2 de-creased significantly and was approximately 70 % lower in Mn-deficient leaves than that in control, indicating less trans-thylakoid pH gradient was built in Mn deficient leaves. We suggest that the decrease of non-radiative dissipation depending on xanthophyll cycle in Mn-starved leaves is a result of the deficiency of trans-thylakoid pH gradient.     

Spectroscopy of non-photochemical and photochemical quenching of chlorophyll fluorescence in leaves; evidence for a role of the light harvesting complex of Photosystem II in the regulation of energy dissipation

Dissipation of absorbed excitation energy as heat, measured by its effect on the quenching of chlorophyll fluorescence, is induced under conditions of excess light in order to protect the photosynthetic apparatus of plants from light-dependent damage. The spectral characteristics of this quenching have been compared to that due to photochemistry in the Photosystem II reaction centre using leaves of Guzmania monostachia. This was achieved by making measurements at 77K when fluorescence emission bands from each type of chlorophyll protein complex can be distinguished. It was demonstrated that photochemistry and non-photochemical dissipation preferentially quench different emission bands and therefore occur by dissimilar mechanisms at separate sites. It was found that photochemistry was associated with a preferential quenching of emission at 688 nm whereas the spectrum for rapidly reversible non-photochemical quenching had maxima at 683 nm and 698 nm, suggesting selective quenching of the bands originating from the light harvesting complexes of Photosystem II. Further evidence that this was occurring in the light harvesting system was obtained from the fluorescence excitation spectra recorded in the quenched and relaxed states.Abbreviations pH transthylakoid pH gradient - F_o minimum level of chlorophyll fluorescence when Photosystem II reaction centres are open - F_m maximum level of fluorescence when Photosystem II reaction centres are closed - F_v variable fluorescence F_m minus F_o - F'_o F_o in any quenched state - F_m F_m in any quenched state - LHCII light harvesting complexes of Photosystem II - PSI Photosystem I - PS II Photosystem II - qN non-photochemical quenching of chlorophyll fluorescence - qE non-photochemical quenching of chlorophyll fluorescence that occurs in the presence of a pH     

Two components of onset and recovery during photoinhibition of Ulva rotundata

Short-term (up to 5 h) transfers of shade-adapted (100 mol · m^–2 · s^–1) clonal tissue of the marine macroalga Ulva rotundata Blid. (Chlorophyta) to higher irradiances (1700, 850, and 350 mol · m^–2 · s^–1) led to photoinhibition of room-temperature chlorophyll fluorescence and O₂ evolution. The ratio of variable to maximum (F_v/F_m) and variable (F_v) fluorescence, and quantum yield () declined with increasing irradiance and duration of exposure. This decline could be resolved into two components, consistent with the separation of photoinhibition into energy-dissipative processes (photoprotection) and damage to photosystem II (PSII) by excess excitation. The first component, a rapid decrease in F_v/F_m and in F_v, corresponds to an increase in initial (F_o) fluorescence and is highly sensitive to 1 mM chloramphenicol. This component is rapidly reversible under dim (40 mol · m^–2 · s^–1) light, but is less reversible with increasing duration of exposure, and may reflect damage to PSII. The second (after 1 h exposure) component, a slower decline in F_v/F_m and F_v with declining F_o, appears to be associated with the photoprotective interconversion of violaxanthin to zeaxanthin and is sensitive to dithiothreitol. The accumulation of zeaxanthin in U. rotundata is very slow, and may account for the predominance of increases in F_o at high irradiances.Abbreviations and Symbols CAP chloramphenicol - DTT dithiothreitol - F_o, F_m, F_v initial, maximum, and variable fluorescence - quantum yield - PFD photon flux density - PSII photosystem II To whom correspondence should be addressedWe are grateful to O. Björkman and S. Thayer, Carnegie Institution of Washington, Stanford, Cal., USA, for analysis of xanthophyll pigments reported here. This research was supported by National Science Foundation grant OCE-8812157 to C.B.O. and J.R. Support for G.L. was provided by a NSF-CNRS (Centre National de la Recherche Scientifique) exchange fellowship.