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.     

How to correctly determine the different chlorophyll fluorescence parameters and the chlorophyll fluorescence decrease ratio R<Subscript>Fd</Subscript> of leaves with the PAM fluorometer

This contribution is a practical guide to the measurement of the different chlorophyll (Chl) fluorescence parameters and gives examples of their development under high-irradiance stress. From the Chl fluorescence induction kinetics upon irradiation of dark-adapted leaves, measured with the PAM fluorometer, various Chl fluorescence parameters, ratios, and quenching coefficients can be determined, which provide information on the functionality of the photosystem 2 (PS2) and the photosynthetic apparatus. These are the parameters F_v, F_m, F₀, F_m′, F_v′, NF, and ΔF, the Chl fluorescence ratios F_v/F_m, F_v/F₀, ΔF/F_m′, as well as the photochemical (q_P) and non-photochemical quenching coefficients (q_N, q_CN, and NPQ). q_N consists of three components (q_N = q_E + q_T + q_I), the contribution of which can be determined via Chl fluorescence relaxation kinetics measured in the dark period after the induction kinetics. The above Chl fluorescence parameters and ratios, many of which are measured in the dark-adapted state of leaves, primarily provide information on the functionality of PS2. In fully developed green and dark-green leaves these Chl fluorescence parameters, measured at the upper adaxial leaf side, only reflect the Chl fluorescence of a small portion of the leaf chloroplasts of the green palisade parenchyma cells at the upper outer leaf half. Thus, PAM fluorometer measurements have to be performed at both leaf sides to obtain information on all chloroplasts of the whole leaf. Combined high irradiance (HI) and heat stress, applied at the upper leaf side, strongly reduced the quantum yield of the photochemical energy conversion at the upper leaf half to nearly zero, whereas the Chl fluorescence signals measured at the lower leaf side were not or only little affected. During this HL-stress treatment, q_N, q_CN, and NPQ increased in both leaf sides, but to a much higher extent at the lower compared to the upper leaf side. q_N was the best indicator for non-photochemical quenching even during a stronger HL-stress, whereas q_CN and NPQ decreased with progressive stress even though non-photochemical quenching still continued. It is strongly recommended to determine, in addition to the classical fluorescence parameters, via the PAM fluorometer also the Chl fluorescence decrease ratio R_Fd (F_d/F_s), which, when measured at saturation irradiance is directly correlated to the net CO₂ assimilation rate (_{P N}) of leaves. This R_Fd-ratio can be determined from the Chl fluorescence induction kinetics measured with the PAM fluorometer using continuous saturating light (cSL) during 4–5 min. As the R_Fd-values are fast measurable indicators correlating with the photosynthetic activity of whole leaves, they should always be determined via the PAM fluorometer parallel to the other Chl fluorescence coefficients and ratios.     

Arabidopsis plants lacking PsbS protein possess photoprotective energy dissipation

It is commonly accepted that the photosystem II subunit S protein, PsbS, is required for the dissipation of excess light energy in a process termed ‘non‐photochemical quenching’ (NPQ). This process prevents photo‐oxidative damage of photosystem II (PSII) thus avoiding photoinhibition which can decrease plant fitness and productivity. In this study Arabidopsis plants lacking PsbS (the npq4 mutant) were found to possess a competent mechanism of excess energy dissipation that protects against photoinhibitory damage. The process works on a slower timescale, taking about 1 h to reach the same level of NPQ achieved in the wild type in just a few minutes. The NPQ in npq4 was found to display very similar characteristics to the fast NPQ in the wild type. Firstly, it prevented the irreversible light‐induced closure of PSII reaction centres. Secondly, it was uncoupler‐sensitive, and thus triggered by the ΔpH across the thylakoid membrane. Thirdly, it was accompanied by significant quenching of the fluorescence under conditions when all PSII reaction centres were open (F_o state)_. Fourthly, it was accompanied by NPQ‐related absorption changes (ΔA535). Finally, it was modulated by the presence of the xanthophyll cycle carotenoid zeaxanthin. The existence of a mechanism of photoprotective energy dissipation in plants lacking PsbS suggests that this protein plays the role of a kinetic modulator of the energy dissipation process in the PSII light‐harvesting antenna, allowing plants to rapidly track fluctuations of light intensity in the environment, and is not the primary cause of NPQ or a direct carrier of the pigment acting as the non‐photochemical quencher.     

NPQ(T): a chlorophyll fluorescence parameter for rapid estimation and imaging of non‐photochemical quenching of excitons in photosystem‐II‐associated antenna complexes

In photosynthesis, light energy is absorbed by light‐harvesting complexes and used to drive photochemistry. However, a fraction of absorbed light is lost to non‐photochemical quenching (NPQ) that reflects several important photosynthetic processes to dissipate excess energy. Currently, estimates of NPQ and its individual components (q_E, q_I, q_Z and q_T) are measured from pulse‐amplitude‐modulation (PAM) measurements of chlorophyll fluorescence yield and require measurements of the maximal yield of fluorescence in fully dark‐adapted material (F_m), when NPQ is assumed to be negligible. Unfortunately, this approach requires extensive dark acclimation, often precluding widespread or high‐throughput use, particularly under field conditions or in imaging applications, while introducing artefacts when F_m is measured in the presence of residual photodamaged centres. To address these limitations, we derived and characterized a new set of parameters, NPQ_(T), and its components that can be (1) measured in a few seconds, allowing for high‐throughput and field applications; (2) does not require full relaxation of quenching processes and thus can be applied to photoinhibited materials; (3) can distinguish between NPQ and chloroplast movements; and (4) can be used to image NPQ in plants with large leaf movements. We discuss the applications benefits and caveats of both approaches.     

Leaf discs floated on water are different from intact leaves in photosynthesis and photoinhibition

Photoinhibition has been often evaluated with leaf discs floated on water or placed on wet papers to prevent desiccation. Under these conditions, there is a possibility that CO₂ diffusion is blocked by water, which may lead to reduction in photosynthetic CO₂ assimilation. Using Chenopodium album L. grown at two irradiances, photosynthesis, quantum yield of Photosystem II (ΔF/F _m′), non-photochemical quenching (qN), and photoinhibition were compared between detached leaves and leaf discs. In low-light-grown plants, photoinhibition was greater in leaf discs than in detached leaves, while in high-light-grown plants, there was little difference. Leaf discs showed lower rates of photosynthesis and ΔF/F _m′, and higher qN. The ΔF/F _m′ in leaf discs increased when leaf discs were exposed to high concentration of CO₂, suggesting that CO₂ diffusion to chloroplasts was limited in leaf discs floated on water. This revised version was published online in June 2006 with corrections to the Cover Date.     

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.     

Diurnal cycle of chlorophyll fluorescence in <Emphasis Type="Italic">Phalaenopsis</Emphasis>

Chlorophyll (Chl) fluorescence of warm day/cool night temperature exposed Phalaenopsis plants was measured hourly during 48 h to study the simultaneous temperature and irradiance response of the photosynthetic physiology. The daily pattern of fluorescence kinetics showed abrupt changes of photochemical quenching (q_P), non-photochemical quenching (NPQ) and quantum yield of photosystem II electron transport (Φ_PSII) upon transition from day to night and vice versa. During the day, the course of Φ_PSII and NPQ was related to the air temperature pattern, while maximum quantum efficiency of PSII photochemistry (F_v/F_m) revealed a rather light dependent response. Information on these daily dynamics in fluorescence kinetics is important with respect to meaningful data collection and interpretation.     

Paraheliotropic leaf movement in Siratro as a protective mechanism against drought-induced damage to primary photosynthetic reactions: damage by excessive light and heat

Damage to primary photosynthetic reactions by drought, excess light and heat in leaves of Macroptilium atropurpureum Dc. cv. Siratro was assessed by measurements of chlorophyll fluorescence emission kinetics at 77 K (-196°C). Paraheliotropic leaf movement protected waterstressed Siratro leaves from damage by excess light (photoinhibition), by heat, and by the interactive effects of excess light and high leaf temperatures. When the leaves were restrained to a horizontal position, photoinhibition occurred and the degree of photoinhibitory damage increased with the time of exposure to high levels of solar radiation. Severe inhibition was followed by leaf death, but leaves gradually recovered from moderate damage. This drought-induced photoinhibitory damage seemed more closely related to low leaf water potential than to low leaf conductance. Exposure to leaf temperatures above 42°C caused damage to the photosynthetic system even in the dark and leaves died at 48°C. Between 42 and 48°C the degree of heat damage increased with the time of exposure, but recovery from moderate heat damage occurred over several days. The threshold temperature for direct heat damage increased with the growth temperature regime, but was unaffected by water-stress history or by current leaf water status. No direct heat damage occurred below 42°C, but in water-stressed plants photoinhibition increased with increasing leaf temperature in the range 31–42°C and with increasing photon flux density up to full sunglight values. Thus, water stress evidently predisposes the photosynthetic system to photoinhibition and high leaf temperature exacerbates this photoinhibitory damage. It seems probable that, under the climatic conditions where Siratro occurs in nature, but in the absence of paraheliotropic leaf movement, photoinhibitory damage would occur more frequently during drought than would direct heat damage.Abbreviations and symbols PFD photon flux area density - PSI, PSII photosyntem I, II - F _M, F _O, F _V maximum, instantaneous, variable fluorescence emission - PLM paraheliotropic leaf movement; all data of parameter of variation are mean ± standard error     

Changes in Chlorophyll Fluorescence in Maize Plants with Imposed Rapid Dehydration at Different Leaf Ages

A comparison of the effects of a rapidly imposed water deficit with different leaf ages on chlorophyll a fluorescence and gas exchange was performed in maize (Zea mays L.) plants. The relationships between photosynthesis and leaf relative turgidity (RT) and ion leakage were further investigated. Leaf dehydration substantially decreased net photosynthetic rate (A) and stomatal conductance (G _s), particularly for older leaves. With dehydration time, F _v /F _m maintained a relatively stable level for youngest leaves but significantly decreased for the older leaves. The electron transport rate (ETR) sharply decreased with intensifying dehydration and remained at lower levels during continuous dehydration. The photochemical quenching of variable chlorophyll fluorescence (q _P) gradually decreased with dehydration intensity for the older leaves but increased for the youngest leaves, whereas dehydration did not affect the nonphotochemical chlorophyll fluorescence quenching (NPQ) for the youngest leaves but remarkably decreased it for the older leaves. The leaf RT was significantly and positively correlated with its F _v /F _m, ETR, and q _P, and the leaf ion leakage was significantly and negatively correlated with F _v /F _m and NPQ. Our results suggest that the photosynthetic systems of young and old leaves decline at different rates when exposed to rapid dehydration.     

Sensitivity of Photosystem 2 of Antarctic Lichens to High Irradiance Stress: Fluorometric Study of Fruticose (Usnea antarctica) and Foliose (Umbilicaria decussata) Species

Two lichen species collected in maritime Antarctica (King George Island) were exposed under laboratory conditions to excess irradiance to evaluate the response of photosystem 2 (PS2). The response was measured on fully hydrated lichen thalli at 5 °C by means of a modulated fluorometer using chlorophyll (Chl) fluorescence induction curve supplemented with analysis of quenching mechanisms. Chl fluorescence parameters [i.e. ratio of variable to maximum Chl fluorescence (F_V/F_M), quantum yield of PS2 photochemical reactions (₂), quenching coefficients] were evaluated before and several times after exposition to high irradiance in order to characterise the extent of photoinhibition, fast and slow phase of recovery. Strong irradiance (2 000 mol m^–2 s^–1) caused high degree of photoinhibition, particularly higher in fruticose (Usnea antarctica) than in foliose (Umbilicaria decussata) lichen species. Fast phase of recovery from photoinhibition, corresponding to regulatory mechanisms of PS2, was more apparent in U. decussata and ₂ than in U. antarctica and F_V/F_M and ₂ within 40 min after photoinhibitory treatment. It was followed by a slow phase lasting several hours, corresponding to repair and re-synthesis processes. After photoinhibitory treatment, recovery of non-photochemical quenching (NPQ) was faster and more pronounced in U. decussata than in U. antarctica. Significant differences were found between the two species in the rate of recovery in fast-(q_E) and slow-recovering (q_T+I) component of NPQ.     

Fluorescence quenching in four unicellular algae with different light-harvesting and xanthophyll-cycle pigments

We examined the relationship between non-photochemical quenching (NPQ) and xanthophyll de-epoxidation in the unicellular algae Euglena gracilis, Ochromonas danica, Phaeodactylum tricornutum, and Dunaliella tertiolecta. Generally, low-light-grown algae had a smaller pool of xanthophyll-cycle pigments per chlorophyll than medium-light-grown grown cells, but they developed more NPQ during exposure to high light. Thus, lumen acidification was apparently lower in medium-light-grown cells in spite of the exposure to a photon flux density (PFD) three times the growth PFD. In darkness Dunaliella maintained a relatively large content of de-epoxidized xanthophylls, and NPQ developed without concomitant de-epoxidation in response to a 5-min exposure to high light. Violaxanthin de-epoxidation that occurred during longer exposures to light did not cause a further rise in NPQ in Dunaliella. In Ochromonas, NPQ and xanthophyll de-epoxidation increased simultaneously during a 15-min exposure to high light. A further rise in NPQ was not accompanied by xanthophyll de-epoxidation. In Phaeodactylum, the rise in NPQ and de-epoxidation were nearly linearly related during a 60-min exposure to high light. NPQ recovered quickly after darkening in these three algae and no significant photodamage occurred. In Euglena no xanthophyll-conversions and no quickly reversible NPQ occured in response to high light, suggesting that photodamage occurred. Dunaliella has similar light-harvesting and xanthophyll-cycle pigments as higher plants but the relationship between NPQ and DPS during the exposure to high light was different from the linear relationship that is commonly observed in plants. Conversely, Phaeodactylum, which has different light-harvesting and xanthophyll-cycle pigments, had a relationship similar to that in plants.     

Oxygen dependence of photoinhibition at low temperature in intact protoplasts of Valerianella locusta L.

Photoinhibition of photosynthesis in vivo is shown to be considerably promoted by O₂ under circumstances where energy turnover by photorespiration and photosynthetic carbon metabolism are low. Intact protoplasts of Valerianella locusta L. were photoinhibited by 30 min irradiation with 3000 mol photons · m^–2 · s^–1 at 4° C in saturating [CO₂] at different oxygen concentrations, corresponding to 2–40% O₂ in air. The photoinhibition of light-limited CO₂-dependent photosynthetic O₂ evolution increased with increasing oxygen concentration. The uncoupled photochemical activity of photosystem II, measured in the presence of the electron acceptor 1,4-benzoquinone, and maximum variable fluorescence, F_v, were strongly affected and this inhibition was closely correlated to the O₂ concentration. The effect of O₂ did not saturate at the highest concentrations applied. An increase in photoinhibitory fluorescence quenching with [O₂], although less pronounced than in protoplasts, was also observed with intact leaves irradiated at 4° C in air. Initial fluorescence, F_o, was slightly (about 10%) increased by the inhibitory treatments but not influenced by [O₂]. A long-term cold acclimation of the plants did not substantially alter the O₂-sensitivity of the protoplasts under the high-light treatment. From these experiments we conclude that oxygen is involved in the photoinactivation of photosystem II by excess light in vivo.Abbreviations and Symbols Chl chlorophyll - F_o initial fluorescence - F_M maximum fluorescence - F_v maximum variable fluorescence - PCO photorespiratory carbon oxidation - PCR photosynthetic carbon reduction - PFD photon flux density - q_N non-photochemical quenching - q_P photochemical quenching - _S quantum efficiency of electron transport of photosystem II This study was financially supported by the Deutsche Forschungs-gemeinschaft (SFB 189) and the Foundation for Fundamental Biological Research (BION), which is subsidised by the Netherlands Organization for the Advancement of Pure Research (NWO).     

The effects of temperature acclimation on the photoinhibitory responses of Ulva rotundata Blid.

The effect of acclimation to 25, 18, or 10° C on the relationship between photoprotection and photodamage was tested in low-light-grown (80 mol · m^–2 · s^–1) Ulva rotundata Blid. exposed to several higher irradiances at the acclimation temperature. Changes in chlorophyll fluorescence parameters (minimum fluorescence, F₀, and the ratio of variable to maximum fluorescence, F_v/F_m, measured after 5 min darkness) were monitored during 5 h transfers to 350, 850, and 1700 mol · m^–2 · s^–1, and during recovery after 1- or 5-h treatments. At all temperatures, rate of onset and final extent of photoinhibition, measured by a decrease in F_v/F_m, increased with increasing irradiance. At a given photoinhibitory irradiance, rate of onset was most rapid at 10 ° C, but the extent was temperature-independent. Recovery rates from mild light stress were similar at all temperatures, but recovery from the most extreme photoinhibitory treatment lagged 2 h at 10° C. De-epoxidation of xanthophyll-cycle components proceeded faster and to a lower epoxidation status at 25° C, but there was little difference in the pool size among the three growth conditions. Using chloramphenicol to inhibit chloroplast protein synthesis and dithiothreitol to inhibit violaxanthin de-epoxidation, it was shown that at the lowest light treatment given, the extent of photoinhibition could be attributed both to greater amounts of photodamage and to greater zeaxanthin-related photoprotection at 25 than at 10° C. While these two mechanisms for high-light-induced loss of photosynthetic efficiency were operating at 10° C, there was evidence for a relatively greater proportion of zeaxanthin-unrelated photoprotection at the low temperature. This photoprotective mechanism is related to a rapidly reversible increase in F₀ and is insentivite to both chloramphenicol and dithiothreitol.Abbreviations and Symbol CAP chloramphenicol - DTT dihiothreitol - F₀, F_m, F_v minimum, maximum, and variable fluorescence - quantum yield This research was conducted in partial fulfillment of the requirements for the Ph. D. degree in the Department of Botany, Duke University. The author wishes to thank E.-M. Aro, W.J. Henley, G. Levavasseur, C.B. Osmond, and J. Ramus for helpful discussions, and C. Lovelock for pigment standards. Funding was provided by Grants-in-Aid of Research from Sigma Xi and the Phycological Society of America, and a Lynde and Harry Bradley Foundation Fellowship to L.A.F., and National Science Foundation grant OCE-8812157 to C.B.O. and J.R.     

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.     

Simultaneous gas exchange and fluorescence measurements indicate differences in the response of sunflower,bean and maize to water stress

Gas exchange and fluorescence measurements of attached leaves of water stressed bean, sunflower and maize plants were carried out at two light intensities (250 mol quanta m^-2s^-1 and 850 mol quanta m^-2s^-1). Besides the restriction of transpiration and CO₂ uptake, the dissipation of excess light energy was clearly reflected in the light and dark reactions of photosynthesis under stress conditions. Bean and maize plants preferentially use non-photochemical quenching for light energy dissipation. In sunflower plants, excess light energy gave rise to photochemical quenching. Autoradiography of leaves after photosynthesis in ¹⁴CO₂ demonstrated the occurrence of leaf patchiness in sunflower and maize but not in bean. The contribution of CO₂ recycling within the leaves to energy dissipation was investigated by studies in 2.5% oxygen to suppress photorespiration. The participation of different energy dissipating mechanisms to quanta comsumption on agriculturally relevant species is discussed.Abbreviations F_o minimal fluorescence - F_m maximal fluorescence - F_p peak fluorescence - g leaf conductance - P_N net CO₂ uptake - q_N coefficient of non-photochemical quenching - q_P coefficient of photochemical quenching     

Photosynthetic performance and light response of two olive cultivars under different water and light regimes

The olive tree (Olea europaea L.) is commonly grown in the Mediterranean area, where it is adapted to resist periods characterized by severe drought and high irradiance levels. Photosynthetic efficiency (in terms of F_v/F_m and Φ_PSII), photochemical (q_P) and nonphotochemical quenching (NPQ) were determined in two-year-old olive plants (cultivars Coratina and Biancolilla) grown under two different light levels (exposed plants, EP, and shaded plants, SP) during a 21-day controlled water deficit. After reaching the maximum level of drought stress, plants were rewatered for 23 days. During the experimental period, measurements of gas exchange and chlorophyll (Chl) fluorescence were carried out to study the photosynthetic performance of olive plants. The synergical effect of drought stress and high irradiance levels caused a reduction of gas exchange and photosynthetic efficiency and these decreases were more marked in EP. EP showed a higher degree of photoinhibition, a higher NPQ and a lower q_P if compared to SP. Coratina was more sensitive to high light and drought stress but also showed a slower recovery during rewatering, whereas Biancolilla showed a less marked photosynthesis depression during drought and a considerable resilience during rewatering. The results confirm that photoinhibition due to high light intensity and water deficit can be an important factor that affects photosynthetic productivity in this species.     

Regulation of D1-protein degradation during photoinhibition of photosystem II in vivo: Phosphorylation of the D1 protein in various plant groups

Photoinhibition of PSII and turnover of the D1 reaction-centre protein in vivo were studied in pumpkin leaves (Cucurbita pepo L.) acclimated to different growth irradiances and in low-light-grown moss, (Ceratodon purpureus) (Hedw.) Brid. The low-light-acclimated pumpkins were most susceptible to photoinhibition. The production rate of photoinhibited PSII centres (k_PI), determined in the presence of a chloroplast-encoded protein-synthesis inhibitor, showed no marked difference between the high- and low-light-grown pumpkin leaves. On the other hand, the rate constant for the repair cycle (k_REC) of PSII was nearly three times higher in the high-light-grown pumpkin when compared to low-light-grown pumpkin. The slower degradation rate of the damaged D1 protein in the low-light-acclimated leaves, determined by pulsechase experiments with [³⁵S]methionine suggested that the degradation of the Dl protein retards the repair cycle of PSII under photoinhibitory light. Slow degradation of the D1 protein in low-light-grown pumpkin was accompanied by accumulation of a phosphorylated form of the D1 protein, which we postulate as being involved in the regulation of D1-protein degradation and therefore the whole PSII repair cycle. In spite of low growth irradiance the repair cycle of PSII in the moss Ceratodon was rapid under high irradiance. When compared to the high- or low-light-acclimated pumpkin leaves, Ceratodon had the highest rate of D1-protein degradation at 1000 mol photons m^–2 s^–1. In contrast to the higher plants, the D1 protein of Ceratodon was not phosphorylated either under high irradiance in vivo or under in-vitro conditions, which readily phosphorylate the D1 protein of higher plants. This is consistent with the rapid degradation of the D1 protein in Ceratodon. Screening experiments indicated that D1 protein can be phosphorylated in the thylakoid membranes of angiosperms and conifers but not in lower plants. The postulated regulation mechanism of D1-protein degradation involving phosphorylation and the role of thylakoid organization in the function of PSII repair cycle are discussed.Abbreviations Chl Chlorophyll - D1^* phosphorylated form of D1 protein - F_max and F_v maximal and variable fluorescence respectively - k_PJ and k_REC rate constants of photoinhibition and concurrent recovery respectively - LHCII lightharvesting chlorophyll a/bprotein of PSII - PFD photon flux density Dr. R. Barbato (Dipartimento di Biologia, Universita di Padova, Padova, Italy), Prof. P. Böger (Lehrstuhl fur Physiologie und Biochemie der Pflanzen, Universität Konstanz, Konstanz, Germany), Prof. A. Melis (Department of Plant Biology, University of California, Berkeley, USA), Prof. I. Ohad (Department of Biological Chemistry, Hebrew University, Jerusalem, Israel) and Mr. A. Soitamo (Department of Biology, University of Turku, Turku, Finland) are gratefully acknowledged for the D1-protein-specific antibodies. The authors thank Ms. Virpi Paakkarinen for excellent technical assistance. This work was supported by the Academy of Finland and the Foundation of the University of Turku.     

The effects of low temperature acclimation and photoinhibitory treatments on Photosystem 2 studied by thermoluminescence and fluorescence decay kinetics

The effects of low temperature acclimation and photoinhibitory treatment on Photosystem 2 (PS 2) have been studied by thermoluminescence and chlorophyll fluorescence decay kinetics after a single turnover saturating flash. A comparison of unhardened and hardened leaves showed that, in the hardened case, a decrease in overall and B-band thermoluminescence emissions occurred, indicating the presence of fewer active PS 2 reaction centers. A modification in the form of the B-band emission was also observed and is attributed to a decrease in the apparent activation energy of recombination in the hardened leaves. The acclimated leaves also produced slower Q_A ^– reoxidation kinetics as judged from the chlorophyll fluorescence decay kinetics. This change was mainly seen in an increased lifetime of the slow reoxidation component with only a small increase in its amplitude. Similar changes in both thermoluminescence and fluorescence decay kinetics were observed when unhardened leaves were given a high light photoinhibitory treatment at 4°C, whereas the hardened leaves were affected to a much lesser extent by a similar treatment. These results suggest that the acclimated plants undergo photoinhibition at 4°C even at low light intensities and that a subsequent high light treatment produces only a small additive photoinhibitory effect. Furthermore, it can be seen that photoinhibition eventually gives rise to PS 2 reaction centers which are no longer functional and which do not produce thermoluminescence or variable chlorophyll fluorescence.Abbreviations D1 The 32 kDa protein of Photosystem 2 reaction center - F_m maximum chlorophyll fluorescence yield - F₀ minimal chlorophyll fluorescence yield obtained when all PS 2 centers are open - F_i intermediate fluorescence level corresponding to PS 2 centers which are loosely or not connected to plastoquinone (non-B centers) - F_v maximum variable chlorophyll fluorescence yield (F_v=F_m–F₀) - PS 2 Photosystem 2 - Q_A and Q_B respectively, primary and secondary quinonic acceptors of PS 2 - S1, S2 and S3 respectively, the one, two and three positively charged states of the oxygen evolving system - Z secondary donor of PS 2     

Rapid regulation of excitation energy in two pennate diatoms from contrasting light climates

Non-photochemical quenching (NPQ) is a fast acting photoprotective response to high light stress triggered by over excitation of photosystem II. The mechanism for NPQ in the globally important diatom algae has been principally attributed to a xanthophyll cycle, analogous to the well-described qE quenching of higher plants. This study compared the short-term NPQ responses in two pennate, benthic diatom species cultured under identical conditions but which originate from unique light climates. Variable chlorophyll fluorescence was used to monitor photochemical and non-photochemical excitation energy dissipation during high light transitions; whereas whole cell steady state 77 K absorption and emission were used to measure high light elicited changes in the excited state landscapes of the thylakoid. The marine shoreline species Nitzschia curvilineata was found to have an antenna system capable of entering a deeply quenched, yet reversible state in response to high light, with NPQ being highly sensitive to dithiothreitol (a known inhibitor of the xanthophyll cycle). Conversely, the salt flat species Navicula sp. 110-1 exhibited a less robust NPQ that remained largely locked-in after the light stress was removed; however, a lower amplitude, but now highly reversible NPQ persisted in cells treated with dithiothreitol. Furthermore, dithiothreitol inhibition of NPQ had no functional effect on the ability of Navicula cells to balance PSII excitation/de-excitation. These different approaches for non-photochemical excitation energy dissipation are discussed in the context of native light climate.     

On the significance of photoinhibition of photosynthesis in the field and its generality among species

Photoinhibition was examined in naturally exposed willow leaves in the field. In the afternoon on clear and warm days, the quantum yield of electron transport, derived from gas exchange data, was decreased by 28%. Besides this photoinhibition, decreases in the photosynthetic capacity and in the stomatal conductance were also observed. Of these three limitations of carbon assimilation, photoinhibition was the major one at limiting light only.To investigate the generality of photoinhibition, shade- and sun-acclimated leaves of fourteen different species were compared in a laboratory study. Photoinhibition was quantified by fluorescence measurements following exposure to moderate and high light for 30 min. The extent of photoinhibition was inversely related to the photochemical quenching, q_p, during exposure (the proportion of open PS II traps). This relationship was the same independent of the species, the light-acclimation state of the leaf and the light intensity. However, sun- and shade-acclimated leaves occupied opposite sides of the relationship: the sun-leaves showed lower photoinhibition and higher q_p. The sun-leaves were also more competent than shade-leaves by showing faster recovery from a given level of photoinhibition.Abbreviations F₀, F_V, F_M, F_S minimal, variable, maximal and steady-state fluorescence - q_N, q_i total and photoinhibitory non-photochemical quenching of variable fluorescence - q_p photochemical quenching