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.     

Acclimation of Photosystem 2 Function of Norway Spruce Induced during First Season under Elevated CO2 in Lamellar Domes

Since July 28^th, 1997 the two experimental mini-stands of young Norway spruce [Picea abies (L.) Karst.] have been grown in lamellar domes at ambient (AC) and elevated concentrations of CO₂ [EC, i.e., ambient + 350 µmol(CO₂) mol^–1]. Before the start of exposure to EC (June 1997) the dependencies of photosystem 2 (PS2) quantum yield (Y) on irradiance, estimating the efficiency of PPFD utilisation in PS2 photochemistry, were the same for AC and EC shoots. After one month of EC simulation (August 1997), Y values were higher for EC needles as compared with the AC ones (by 1–42 %), whereas two months later (October 1997) an opposite effect was observed (decrease of Y by from 1 to 33 %). By chlorophyll a (Chl a) fluorescence induction the effects of EC on PS2 function were further characterised. During the first month a moderate improvement of PS2 function was estimated for EC needles from slightly higher potential yield of PS2 photochemistry (F_V/F_M, by 1 %) and reduced amount of inactive PS2 reaction centres (relative F_p1 level, by 15 %). However, the prolonged exposure to EC led firstly to a slight but significant decrease of F_V/F_M (by 3 %), secondly to a reduction of half time of fluorescence rise (t_1/2, by 14 %), and finally to pronounced accumulation of inactive PS2 reaction centres (by 41 %). From the gradual response of individual Chl a fluorescence parameters we suggest a probable sequence of events determining the stimulation and subsequent depression of PS2 function for Norway spruce during the first season under EC.     

Analysis of changes in minimal and maximal fluorescence yields with irradiance and o(2) level in tobacco leaf tissue

The responses of minimal and maximal fluorescence yields of chlorophyll a to irradiance of actinic white light were determined by pulse modulated fluorimetry in leaf discs from tobacco, Nicotiana tabacum, at 1.6, 20.5, and 42.0% (v/v) O₂. Steady-state maximal fluorescence yield (F_m′, measured during a saturating light pulse) declined with increasing irradiance at all O₂ levels. In contrast, the steady-state minimal fluorescence yield (F_o′, measured during a brief dark interval) increased with irradiance relative to that recorded for the fully dark-adapted leaf (F_o) or that observed after 5 minutes of darkness (F_o^*). The relative magnitude of this increase was somewhat greater and extended to higher irradiances at the elevated O₂ levels compared with 1.6% O₂. Suppression of F_o′ was only observed consistently at saturating irradiance. The results are interpreted in terms of the occurrence of photosystem II units possessing exceedingly slow turnover times (i.e. “inactive” units). Inactive units play an important role, along with thermal deactivation of excited chlorophyll, in determining the response of in vivo fluorescence yield to changes in irradiance. Also, a significant interactive effect of O₂ concentration and the presence or absence of far red light on oxidation of photosystem II acceptors in the dark was noted.     

Comparison of the effect of excessive light on chlorophyll fluorescence (77K) and photon yield of O2 evolution in leaves of higher plants

High-light treatments (1750–2000 mol photons m^–2 · s^–1) of leaves from a number of higher-plant species invariably resulted in quenching of the maximum 77K chlorophyll fluorescence at both 692 and 734 nm (F _M, 692 and F _M, 734). The response of instantaneous fluorescence at 692 nm (F _O, 692) was complex. In leaves of some species F _O, 692 increased dramatically in others it was quenched, and in others yet it showed no marked, consistent change. Regardless of the response of F _O, 692 an apparently linear relationship was obtained between the ratio of variable to maximum fluorescence (F _V/F _M, 692) and the photon yield of O₂ evolution, indicating that photoinhibition affects these two variables to approximately the same extent. Treatment of leaves in a CO₂–free gas stream containing 2% O₂ and 98% N₂ under weak light (100 mol · m^–2 · s^–1) resulted in a general and fully reversible quenching of 77K fluorescence at 692 and 734 nm. In this case both F _O, 692 and F _M, 692 were invariably quenched, indicating that the quenching was caused by an increased non-radiative energy dissipation in the pigment bed. We propose that high-light treatments can have at least two different, concurrent effects on 77K fluorescence in leaves. One results from damage to the photosystem II (PSII) reaction-center complex and leads to a rise in F _O, 692; the other results from an increased non-radiative energy dissipation and leads to quenching of both F _O, 692 and F _M, 692 This general quenching had a much longer relaxation time than reported for pH-dependent quenching in algae and chloroplasts. Sun leaves, whose F _V/F _M, 692 ratios were little affected by high-light exposure in normal air, suffered pronounced photoinhibition when the exposure was made under conditions that prevent photosynthetic gas exchange (2% O₂, 0% CO₂). However, they were still less susceptible than shade leaves, indicating that the higher capacity for energy dissipation via photosynthesis is not the only cause of their lower susceptibility. The rate constant for recovery from photoinhibition was much higher in mature sun leaves than in mature shade leaves, indicating that differences in the capacity for continuous repair may in part account for the difference in their susceptibility to photoinhibition.Abbreviations and symbols kDa kilodalton - LHC-II light-harvesting chlorophyll-protein complex - PFD photon flux density (photon fluence rate) - PSI, PSII photosystem I, II - F _O, F _M, F _V instantaneous, maximum, variable fluorescence emission - absorptance - _a photon yield of O₂ evolution (absorbed light) C.I.W.-D.P.B. Publication No. 925     

Estimation of the effect of photoinhibition on the carbon gain in leaves of a willow canopy

The occurrence of photoinhibition of photosynthesis in leaves of a willow canopy was examined by measuring the chlorophyll-a fluorescence ratio of F _V/F _M (F_M is the maximum fluorescence level of the induction curve, and F_V is the variable fluorescence, F _V=F _M–F ₀, where F₀ is the minimal fluorescence). The majority of the leaves situated on the upper parts of peripheral shoots showed an afternoon inhibition of this ratio on clear days. This was the consequence of both a decrease in F _M and a rise in F _O. In the same leaves the diurnal variation in intercepted photosynthetic photon flux density (PPFD) was monitored using leaf-mounted sensors. Using the multivariate method, partial least squares in latent variables, it is shown that the dose of PPFD, integrated and linearly weighted over the last 6-h period, best predicts photoinhibition. Photoinhibition occurred even among leaves that did not intercept PPFDs above 1000 mol·m^–2·s^–1. Exposure of leaves to a standard photoinhibitory treatment demonstrated that the depression in the F _V/F _M ratio was paralleled by an equal depression in the maximal quantum yield of CO₂ uptake and a nearly equal depression in the rate of bending (convexity) of the light-response curve of CO₂ uptake. As a result, the rate of net photosynthesis is depressed over the whole natural range of PPFD. By simulating the daily course in the rate of net photosynthesis, it is estimated that in the order of one-tenth of the potential carbon gain of peripheral willow shoots is lost on clear days as a result of photoinhibition. This applies to conditions of optimal temperatures. Photoinhibition is even more pronounced at air temperatures below 23° C, as judged from measurements of the F_V/F_M ratio on clear days: the afternoon inhibition of this ratio increased in a curvilinear manner from 15% to 25% with a temperature decrease from 23° to 14° C.Abbreviations and Symbols F_O minimum fluorescence - F_V variable fluorescence - F_M maximum fluorescence - PLS partial least squares in latent variables - PPFD photosynthetic photon flux density - VPD water vapour-pressure deficit This study was supported by the Swedish Natural Science Research Council. We are indebted to Dr. Jerry Leverenz (Department of Plant Physiology, University of Umeå, Sweden) for guidance with the modelling of the photosynthesis data.     

Regulation of Photosynthesis by Radiation Quality in the Lichen Evernia prunastri

In Evernia prunastri, photosynthetic gas exchange was saturated with yellow radiation (SOX) at 400 mol m^–2s^–1, and then red (R), far-red (FR), or blue (B) radiations at irradiance of 15 mol m^–2s^–1 were added. Because of photosynthesis saturation, any stimulation or decay in CO₂ assimilation by any radiation quality could be attributed to the involvement of a non-photosynthetic photoreceptor. Thus CO₂ assimilation, effective quantum yield, and photochemical quenching were enhanced when R was included, and decreased with FR. Blue radiation completely abolished CO₂ fixation. Hence different spectral radiation qualities may activate non-photosynthetic photoreceptors such as phytochrome and blue photoreceptors, which are involved in regulating the photosynthetic activity in E. prunastri.     

A portable multi-flash kinetic fluorimeter for measurement of donor and acceptor reactions of Photosystem 2 in leaves of intact plants under field conditions

A newly-developed field-portable multi-flash kinetic fluorimeter for measuring the kinetics of the microsecond to millisecond reactions of the oxidizing and reducing sides of photosystem 2 in leaves of intact plants is described and demonstrated. The instrumental technique is a refinement of that employed in the double-flash kinetic fluorimeter (Joliot 1974 Biochim Biophys Acta 357: 439–448) where a low-intensity short-duration light pulse is used to measure the fluorescence yield changes following saturating single-turnover light pulses. The present instrument uses a rapid series of short-duration (2 s) pulses to resolve a complete microsecond to millisecond time-scale kinetic trace of fluorescence yield changes after each actinic flash. Differential optics, using a matrix of optical fibers, allow very high sensitivity (noise levels about 0.05% F_max) thus eliminating the need for signal averaging, and greatly reducing the intensity of light required to make a measurement. Consequently, the measuring pulses have much less actinic effect and an entire multi-point trace (seven points) excites less than 1% of the reaction centers in a leaf. In addition, bu combining the actinic and measuring pulse light in the optical fiber network, the tail of the actinic flash can be compensated for, allowing measurements of events as rapidly as 20 s after the actinic flash. This resolution makes practical the routine measurement of the microsecond turnover kinetics of the oxygen evolving complex in leaves of intact plants in the field. The instrument is demonstrated by observing flash number dependency and inhibitor sensitivity of the induction and decay kinetics of flash-induced fluorescence transients in leaves of intact plants. From these traces the period-two oscillations associated with the turnover of the two-electron gate and the period-four oscillations associated with the turnover of the oxygen evolving complex can be observed. Applications of the instrument to extending our knowledge of chloroplast function to the whole plant, the effects on plants of environmental stress, herbicides, etc, and possible applications to screening of mutants are discussed.Abbreviations DCMU 3-(3,4-Dichlorophenol)-1,1-dimethylurea - PS 2 photosystem 2 - PS 1 photosystem 1 - P₆₈₀ primary electron donor of the PS 2 reaction center - Q_A primary acceptor quinone of PS 2 - Q_B secondary acceptor quinone of PS 2 - CCCP carbonyl cyanide-m-chlorophenylhydrazone - Y_z donor to P₆₈₀ ⁺ - F₀ level of fluorescence with all PS 2 centers open - F_max maximum level of fluorescence with all PS 2 centers closed - P₆₈₀Q_A Open reaction centers with P₆₈₀ reduced and Q_A oxidized (low fluorescence) - P₆₈₀Q_A ^- Closed reaction centers, in which P₆₈₀ is reduced (high fluorescence) - P₆₈₀ ⁺Q_A ^- Closed reaction centers, in which P₆₈₀ is oxidized (low fluorescence)     

Photoinhibition in seedlings of <Emphasis Type="Italic">Fraxinus</Emphasis> and <Emphasis Type="Italic">Fagus</Emphasis> under natural light conditions: implications for forest regeneration?

Ash (Fraxinus excelsior L.) and beech (Fagus sylvatica L.) seedlings were grown in the field under three levels of natural light: (1) open, (2) gap and (3) shade. Light acclimation of photosynthesis was characterized by means of modulated chlorophyll a fluorescence of intact leaves and growth parameters were measured at the end of the growing season. Measurements of maximum photochemical efficiency (F_v/F_m) of dark-adapted leaves at intervals through the day showed that ash had a higher F_v/F_m than beech in open and gap plots but not in shade plots. This indicated a larger build-up of photoinhibition in beech under gap and open conditions. Steady-state light response curves of the operating efficiency of PSII (F_q/F_m), the electron transport rate (ETR) and the photochemical efficiency factor (F_q/F_v) showed greater variability across light treatments in ash than in beech. Both species exhibited similar responses of non-photochemical quenching (NPQ) to light. When the data were normalized to the mean maximum irradiance in the growth environment, all photochemical parameters showed a reduction in variation across treatments, indicating that light acclimation in the two species occurred primarily through adjustments in rates of photochemistry. Adjustments in thermal heat dissipation were small in both species. This pattern was stronger in ash, suggesting a greater degree of phenotypic plasticity in photosynthetic capacity in this earlier successional species. Contrary to our expectations, the build-up of photoinhibition in beech did not appear to have a negative effect on total biomass accumulation relative to ash.Abbreviations ETR Electron transport rate - F_m Maximal fluorescence in the dark-adapted state - F_o Minimal fluorescence in the dark-adapted state - F_s Steady-state fluorescence in actinic light - F_v=F_m–F_o Variable fluorescence in the dark-adapted state - F_v/F_m Maximum photochemical efficiency of photosystem II in the dark-adapted state - F_m Maximal fluorescence in actinic light - F_o Minimal fluorescence in actinic light - F_v=F_m–F_o Variable fluorescence in actinic light - F_q=F_m–F_s; F_q/F_m Operating efficiency of photosystem II in actinic light - F_q/F_v Efficiency factor of PSII photochemistry (also referred to as qP—photochemical quenching) - F_v/F_m Maximum efficiency of PSII under actinic light if all reaction centres were open - NPQ Stern-Volmer non-photochemical quenching - PPFD Photosynthetic photon flux density (mol m^–2 s^–1) refers to photosynthetically active irradiance measured with a cosine-corrected quantum sensor - PPFFR Photosynthetic photon flux fluence rate (mol m^–2 s^–1) refers to photosynthetically active irradiance measured with a spherical quantum sensor. Fluorescence nomenclature follows Oxborough and Baker (2000).     

Analysis of dark-relaxation kinetics of variable fluorescence in intact leaves

The dark-relaxation kinetics of variable fluorescence, F_v, in intact green leaves of Pisum stativum L. and Dolichos lablab L. were analyzed using modulated fluorometers. Fast (t_1/2 = 1 s) and slow (t_1/2 = 7–8 s) phases in f_v dark-decay kinetics were observed; the rate and the relative contribution of each phase in total relaxation depended upon the fluence rate of the actinic light and the point in the induction curve at which the actinic light was switched off. The rate of the slow phase was accelerated markedly by illumination with far-red light; the slow phase was abolished by methyl viologen. The halftime of the fast phase of F_v dark decay decreased from 250 ms in dark-adapted leaves to 12–15 ms upon adaptation to red light which is absorbed by PSII. The analysis of the effect of far-red light, which is absorbed mainly by PSI, on F_v dark decay indicates that the slow phase develops when a fraction of Q_A ^– (the primary stable electron acceptor of PSII) cannot transfer electrons to PSI because of limitation on the availability of P700⁺ (the primary electron donor of PSI). After prolonged illumination of dark-adapted leaves in red (PSII-absorbed) light, a transient. F_v rise appears which is prevented by far-red (PSI-absorbed) light. This transient f_v rise reflects the accumulation of Q_A ^– in the dark. The observation of this transient F_v rise even in the presence of the uncoupler carbonylcyanide m-chlorophenyl hydrazone (CCCP) indicates that a mechanism other than ATP-driven back-transfer of electrons to QA may be responsible for the phenomenon. It is suggested that the fast phase in F_v dark-decay kinetics represents the reoxidation of Q_A ^– by the electron-transport chain to PSI, whereas the slow phase is likely to be related to the interaction of Q_A ^– with the donor side of PSII.Abbreviations CCCP carbonylcyanide m-chlorophenylhydrazone - F_O initial fluorescence level - F_v variable fluorescence - P700 primary electron donor of PSI - PSI, II photosystem I, II - Q_A (Q_A ^–) Q_B (Q_B ^–) primary and secondary stable electron acceptor of PSII in oxidized (reduced) state Supported by grant B6.1/88 DST, Govt. of India.     

Carotenoid composition and photon-use efficiency of photosynthesis inGossypium hirsutum L. grown under conditions of slightly suboptimum leaf temperatures and high levels of irradiance

Summary Cotton (Gossypium hirsutum L. var. DP 61) was grown at different temperatures during 12-h light periods, with either 1800–2000 mol photons m^–2 s^–1 (high photon flux density, PFD) or 1000–1100 mol m^–2 s^–1 (medium PFD) incident on the plants. Night temperature was 25°C in all experiments. Growth was less when leaf temperatures were below 30°C during illumination, the effect being greater in plants grown with high PFD (Winter and Königer 1991). Leaf pigment composition and the photon-use efficiency of photosynthesis were analysed to assess whether plants grown with high PFD and suboptimal temperatures experienced a higher degree of high irradiance stress during development than those grown with medium PFD. The chlorophyll content per unit area was 3–4 times less, and the content of total carotenoids about 2 times less, with the proportion of the three xanthophylls zeaxanthin + antheraxanthin + violaxanthin being greater in leaves grown at 20–21°C than in leaves grown at 33–34°C. In leaves from plants grown at 21°C and 1800–2000 mol photons m^–2 s^–1, zeaxanthin accounted for as much as 34% of total carotenoids in the middle of the photoperiod, the highest level recorded in this study. This finding is consistent with a protective role of zeaxanthin under conditions of excess light. At the lower temperatures, the photochemical efficiency of photosystem II, measured as the ratio of variable to maximum fluorescence yield (F _V/F _M) after 12-h dark adaptation, was 0.76 in medium PFD plants and 0.75 in high PFD plants compared with 0.83 and 0.79, respectively, at the higher temperatures. The photon-use efficiency of O₂ evolution () based on absorbed light between 630 and 700nm, decreased with decrease in temperature from 0.102 to 0.07 under conditions of high PFD, but remained above 0.1 at medium PFD. Owing to compensatory reactions in these long-term growth experiments, sustained differences inF _V/F _M and were much less pronounced than the differences in chlorophyll content and dry matter, particularly in plants which had developed at high PFD and low temperature. In fact, in these plants, which exhibited pronounced photobleaching, a largely functional photosynthetic apparatus was still maintained in cells adjacent to the lower leaf surfaces. This was indicated by measurements of photon use efficiencies of photosynthetic O₂ evolution with leaves illuminated first at the upper, and then at the lower surface.Abbreviations F _O yield of dark level fluorescence - F _M maximum yield of fluorescence, induced in a pulse of saturating light - F _V yield of variable fluorescence (=F _M-F _o) - PFD photon flux density - _iw photon use efficiency of O₂ evolution based on white (400–700 nm) incident light - _ir photon use efficiency based on red (630–700 nm) incident light - _aw photon use efficiency based on white absorbed light - _ar photon use efficiency based on red absorbed light     

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.     

Low Temperature Tolerance of Tobacco Plants Transformed to Accumulate Proline,Fructans, or Glycine Betaine. Variable Chlorophyll Fluorescence Evidence

Tobacco (Nicotiana tabacum L.) has been transformed to accumulate different compatible solutes (proline, fructans, or glycine betaine) in order to improve its tolerance to abiotic stress. Photosynthetic activity of wild Type (wt) and transformed tobacco plants before and after freezing stress was studied by measuring chlorophyll (Chl) fluorescence. The JIP test of Chl fluorescence induction was used to analyze in details the functional activity of photosystem 2. No significant differences were found among wild Type and transgenic plants after 12 h of freezing. Both plant Types maintained the same values of the measured parameters [F_V/F_M, PI(CS_M), ABS/RC, TR₀/RC, ET/RC] after recovery of stress. The studied Chl fluorescence parameters decreased only for the wild Type plants, stressed for 24 h at –2 °C. The strong inhibition of photosynthetic reactions in the wt plant after 24 h of freezing could not be restored. The evaluated parameters of transgenic plants did not change significantly after 24 h at –2 °C and successfully survived freezing stress.     

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.     

Photosynthesis performance in sweet almond [<Emphasis Type="Italic">Prunus dulcis</Emphasis> (Mill) D. Webb] exposed to supplemental UV-B radiation

Due to anthropogenic influences, solar UV-B irradiance at the earth’s surface is increasing. To determine the effects of enhanced UV-B radiation on photosynthetic characteristics of Prunus dulcis, two-year-old seedlings of the species were submitted to four levels of UV-B stress, namely 0 (UV-B_c), 4.42 (UV-B₁), 7.32 (UV-B₂) and 9.36 (UV-B₃) kJ m⁻² d⁻¹. Effects of UV-B stress on a range of chlorophyll (Chl) fluorescence parameters (FPs), Chl contents and photosynthetic gas-exchange parameters were investigated. UV-B stress promoted an increase in minimal fluorescence of dark-adapted state (F₀) and F₀/F_m, and a decrease in variable fluorescence (F_v, F_v/F_m, F_v/F₀ and F₀/F_m) due to its adverse effects on photosystem II (PSII) activity. No significant change was observed for maximal fluorescence of dark-adapted state (F_m). Enhanced UV-B radiation caused a significant inhibition of net photosynthetic rate (P _N) at UV-B₂ and UV-B₃ levels and this was accompanied by a reduction in stomatal conductance (g _s) and transpiration rate (E). The contents of Chl a, b, and total Chl content (a+b) were also significantly reduced at increased UV-B stress. In general, adverse UV-B effects became significant at the highest tested radiation dose 9.36 kJ m⁻² d⁻¹. The most sensitive indicators for UV-B stress were F_v/F₀, Chl a content and P _N. Significant P<0.05 alteration in these parameters was found indicating the drastic effect of UV-B radiation on P. dulcis.     

Effect of Irradiance, Sugars and Nitrogen on Leaf Size of In Vitro Grown Ceratonia Siliqua L.

Carob (Ceratonia siliqua L.) has compound pinnate leaves consisting of 4 – 6 pairs of leaflets. However, in conditions of in vitro culture only one pair of leaflets develops. With increasing irradiance from 9.3 to 74.1 µmol m^–2 s^–1, leaf area increased 5-fold. Sucrose also significantly increased leaf area and the maxima were at concentration 147 mM at high irradiance and 233.6 mM at low irradiance. Sucrose was superior to fructose, glucose and combination of both in increasing leaf area. Decreasing concentration of KNO₃ and NH₄NO₃ caused a 3-fold decline of leaf area.     

A portable, microprocessor operated instrument for measuring chlorophyll fluorescence kinetics in stress physiology

A portable instrument for measuring chlorophyll fluorescence induction kinetics is described and examples of measurements are given. The instrument is centered around a statistically-mixed bifurcated optical fiber. One fiber branch guides the actinic light to the sample, whereas the other branch carries the emitted chlorophyll fluorescence to the photodetector. Scattered actinic light is cut out from the detector by a red interference filter. The instrument measures fast as well as slow fluorescence induction kinetics, but is particularly well designed for analyzing fast kinetics. The high time resolution and strong, variable actinic light mean that both F_o (non-variable fluorescence) and F_m (maximal fluorescence at the P-peak) are well defined. A built in microprocessor unit with attached memory stores the fluorescence induction curve and calculates key fluorescence parameters such as F_o, F_m, F_v (variable fluorescence equals F_m?F_o), F_v/F_m (the photochemical efficiency of photosystem II) and t_1/2 (half rise time from F_o, to F_m). These values are digitally displayed after each recording and they (or the whole induction curve) can be stored in a memory and later retrieved. Because of a flexible setting of the instrument it can be used with high accuracy both for optically thick leaves and for diluted suspensions of algae or chloroplasts. A simple, light weight clamp cuvette for dark adaptation of leaves has been developed. It is equipped with a gate allowing the optical fiber to be inserted without daylight reaching the dark adapted portion of the leaf. The instrument has been developed for rapid monitoring of changes in activities and organization of the photosynthetic apparatus in vivo when plants are exposed to environmental stress both in the field and in the laboratory. Examples of measurements are given for differently treated leaves of Pinus sylvestris, Salix sp., Betula verrucosa, Zea mays, Epilobium angustifo-lium and for chloroplast thylakoids isolated from Spinacia oleracea.     

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     

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.     

Photosynthesis in dynamic light: systems biology of unconventional chlorophyll fluorescence transients in Synechocystis sp. PCC 6803

Photosynthetic organisms live in a dynamic environment where light typically fluctuates around a mean level that is slowly drifting during the solar day. We show that the far-from-equilibrium photosynthesis occurring in a rapidly fluctuating light differs vastly from the stationary-flux photosynthesis attained in a constant or slowly drifting light. Photosynthetic organisms in a static or slowly drifting light can be characterized by a steady-state quantum yield of chlorophyll fluorescence emission F′ that is changing linearly with small and slow variations of the incident irradiance I+ΔI(t): F′(I+ΔI(t))≈ F_mean^′(dF)/(dI)·ΔI(t). In Synechocystis sp. PCC 6803, the linear approximation holds for an extended interval covering largely the static irradiance range experienced by the cyanobacteria in nature. The photosynthetic dynamism and, consequently, the dynamism of the chlorophyll fluorescence emission change dramatically when exposing the organism to a fluctuating irradiance. Harmonically-modulated irradiance I+ΔI · sin(2πt/T), T ≈ 1–25 s induces perpetual, far-from-equilibrium forced oscillations that are strongly non-linear, exhibiting significant hysteresis with multiple fluorescence levels corresponding to a single instantaneous level of the incident irradiance. We propose that, in nature, the far-from-equilibrium dynamic phenomena represent a significant correction to the steady-state photosynthetic activity that is typically investigated in laboratory. Analysis of the forced oscillations by the tools of systems biology suggests that the dynamism of photosynthesis observed in fluctuating light can be explained by a delayed action of regulatory agents.     

Acclimation of Two Distinct Plant Species,Spring Barley and Norway Spruce,to Combined Effect of Various Irradiance and CO2 Concentration During Cultivation in Controlled Environment

The short-term acclimation (10-d) of Norway spruce [Picea abies (L.) Karst] to elevated CO₂ concentration (EC) in combination with low irradiance (100 mol m^–2 s^–1) resulted in stimulation of CO₂ assimilation (by 61 %), increased total chlorophyll (Chl) content (by 17 %), significantly higher photosystem 2 (PS2) photochemical efficiency (F_v/F_m; by 4 %), and reduced demand on non-radiative dissipation of absorbed excitation energy corresponding with enhanced capacity of photon utilisation within PS2. On the other hand, at high cultivation irradiance (1 200 mol m^–2 s^–1) both Norway spruce and spring barley (Hordeum vulgare L. cv. Akcent) responded to EC by reduced photosynthetic capacity and prolonged inhibition of F_v/F_m accompanied with enhanced non-radiative dissipation of absorbed photon energy. Norway spruce needles revealed the expressive retention of zeaxanthin and antheraxanthin (Z+A) in darkness and higher violaxanthin (V) convertibility (yielding even 95 %) under all cultivation regimes in comparison with barley plants. In addition, the non-photochemical quenching of minimum Chl a fluorescence (SV₀), expressing the extent of non-radiative dissipation of absorbed photon energy within light-harvesting complexes (LHCs), linearly correlated with V conversion to Z+A very well in spruce, but not in barley plants. Finally, a key role of the Z+A-mediated non-radiative dissipation within LHCs in acclimation of spruce photosynthetic apparatus to high irradiance alone and in combination with EC was documented by extremely high SV₀ values, fast induction of non-radiative dissipation of absorbed photon energy, and its stability in darkness.