Propagation of photoinduced signals with the cytoplasmic flow along Characean internodes: evidence from changes in chloroplast fluorescence and surface pH

Emerging evidence suggests that cytoplasmic streaming can regulate the plasma-membrane H⁺ transport and photosynthetic electron flow. Microfluorometric and surface pH measurements on Chara corallina internodes revealed the transmission of photoinduced signals by the cytoplasmic flow for a distance of few millimeters from the site of stimulus application. When a 30-s pulse of bright light was locally applied, the downstream cell regions responded with either release or enhancement of non-photochemical quenching of chlorophyll fluorescence, depending on the background irradiance of the analyzed cell area. Under dim background irradiance (<20 μmol m^?2 s^?1), the arrival of the distant signal from the brightly illuminated 400-μm-wide zone elevated the maximal fluorescence F _m ^′ in the analyzed downstream area, whereas at higher background irradiances it induced strong quenching of F _m ^′ . At intermediate irradiances the increase and decrease in F _m ^′ appeared as two successive waves. The transition between the F _m ^′ responses of opposite polarities occurred at a narrow threshold range of irradiances. This indicates that inevitable slight variations in irradiance at the bottom chloroplast layer combined with the cyclosis-transmitted signals may contribute to the formation of a photosynthetic activity pattern. The rapid cyclosis-mediated release of non-photochemical quenching, unlike the delayed response of opposite polarity, was associated with opening of H⁺ (OH^?)-conducting plasma membrane channels, as evidenced by the concurrent alkaline pH shift on the cell surface. It is proposed that the initial increase in F _m ^′ after application of a distant photostimulus is determined, among other factors, by the wave of alkaline cytoplasmic pH.     

Operation of the xanthophyll cycle in the seagrass Zostera marina in response to variable irradiance

Changes in the photobiology and photosynthetic pigments of the seagrass Zostera marina from Chesapeake Bay (USA) were examined under a range of natural and manipulated irradiance regimes. Photosynthetic activity was assessed using chlorophyll-a fluorescence, and photosynthetic pigments were measured by HPLC. Large changes in the violaxanthin, zeaxanthin, and antheraxanthin content were concomitant with the modulation of non-photochemical quenching (NPQ). Photokinetics (F_v/F_m, rapid light curves (RLC), and non-photochemical quenching) varied as a result of oscillating irradiance and were highly correlated to xanthophyll pigment content. Zeaxanthin and antheraxanthin concentrations increased under elevated light conditions, while violaxanthin increased in darkened conditions. Unusually high concentrations of antheraxanthin were found in Z. marina under a wide range of light conditions, and this was associated with the partial conversion of violaxanthin to zeaxanthin. These results support the idea that xanthophyll intermediate pigments induce a photoprotective response during exposure to high irradiances in this seagrass.     

Monitoring cashew seedlings during interactions with the fungus <Emphasis Type="Italic">Lasiodiplodia theobromae</Emphasis> using chlorophyll fluorescence imaging

The chlorophyll (Chl) fluorescence imaging technique was applied to cashew seedlings inoculated with the fungus Lasiodiplodia theobromae to assess any disturbances in the photosynthetic apparatus of the plants before the onset of visual symptoms. Two-month-old cashew plants were inoculated with mycelium of L. theobromae isolate Lt19 or Lt32. Dark-adapted and light-acclimated whole plants or previously labelled, single, mature leaf from each plant were evaluated weekly for Chl fluorescence parameters. From 21 to 28 days, inoculation with both isolates resulted in the significantly lower maximal photochemical quantum yield of PSII (F_v/F_m) than those for control samples, decreasing from values of 0.78 to 0.62. In contrast, the time response of the measured fluorescence transient curve from dark-acclimated plants increased in both whole plants and single mature leaves in inoculated plants compared with controls. The F_v/F_m images clearly exhibited photosynthetic perturbations 14 days after inoculation before any visual symptoms appeared. Additionally, decays in the effective quantum yield of PSII photochemistry and photochemical quenching coefficient were also observed over time. However, nonphotochemical quenching increased during the evaluation period. We conclude that F_v/F_m images are the effective way of detecting early metabolic perturbations in the photosynthetic apparatus of cashew seedlings caused by gummosis in both whole plants and single leaves and could be potentially employed in larger-scale screening systems.     

Transient depletion of transported metabolites in the streaming cytoplasm of Chara upon shading the long-distance transmission pathway

Export of reducing power from chloroplasts to cytoplasm serves to balance the NADPH/ATP ratio that is optimal for CO₂ assimilation. Rapid cytoplasmic streaming in characean algae conveys the exported metabolites downstream towards the shaded plastids where envelope transporters may operate for the import of reducing power in accordance with the direction of concentration gradients. Import of reducing equivalents by chloroplasts in the analyzed area transiently enhances the pulse-modulated chlorophyll fluorescence F′ controlled by the redox state of photosystem II acceptor Q_A. When the microfluidic pathway was transferred to darkness while the analyzed cell area remained in dim background light, the amplitude of cyclosis-mediated F′ changes dropped sharply and then recovered within 5–10 min. The suppression of long-distance signaling indicates temporal depletion of transmitted metabolites in the streaming cytoplasm. The return to overall background illumination induced an exceptionally large F′ response to the first local light pulse admitted to a remote cell region. This indicates the appearance of excess reductants in the streaming cytoplasm at a certain stage of photosynthetic induction. The results suggest highly dynamic exchange of metabolites between stationary chloroplasts lining the microfluidic pathway and the streaming cytoplasm upon light–dark and dark–light transitions. Evidence is obtained that slow stages of chlorophyll fluorescence induction in algae with rapid cytoplasmic streaming directly depend on cyclosis-mediated long-distance delivery of metabolites produced far beyond the analyzed cell area.     

Leaf orientation,photorespiration and xanthophyll cycle protect young soybean leaves against high irradiance in field

In order to fully understand the adaptive strategies of young leaves in performing photosynthesis under high irradiance, leaf orientation, chloroplast pigments, gas exchange, as well as chlorophyll a fluorescence kinetics were explored in soybean plants. The chlorophyll content and photosynthesis in young leaves were much lower than that in fully expanded leaves. Both young and fully expanded leaves exhibited down-regulation of the maximum quantum yield (F_V/F_M) at noon in their natural position, no more serious down-regulation being observed in young leaves. However, when restraining leaf movement and vertically exposing the leaves to 1200 μmol m⁻² s⁻¹ irradiance, more pronounced down-regulation of F_V/F_M was observed in young leaves; and the actual photosystem II (PS II) efficiency (Ф_PSII) drastically decreased with the significant enhancement of non-photochemical quenching (NPQ) and ‘High energy’ quenching (q_E) in young leaves. Under irradiance of 1200 μmol m⁻² s⁻¹, photorespiration (P_r) in young leaves measured by gas exchange were obviously lower, whereas the ratio of photorespiration/gross photosynthetic rate (P_r/P_g) were higher than that in fully expanded leaves. Compared with fully expanded leaves, young leaves exhibited higher xanthophyll pool and a much higher level of de-epoxidation components when exposure to high irradiance. During leaf development, the petiole angle gradually increased all the way. Especially, the midrib angle decreased with the increasing of irradiance in young leaves; however, no distinct changes were observed in mature leaves. The changes of leaf orientation greatly reduced the irradiance on young leaf surface under natural positions. In this study, we suggested that the co-operation of leaf angle, photorespiration and thermal dissipation depending on xanthophyll cycle could successfully prevent young leaves against high irradiance in field.     

Chloroplast functionality assessment by flow cytometry: Case study with pea plants under Paraquat stress

Photosynthesis is one of the most important processes in plant biology and in the development of new methodologies that allow a better understanding and characterization of the photosynthetic status of organisms, which is invaluable. Flow cytometry (FCM) is an excellent tool for measuring fluorescence and physical proprieties of particles but it has seldom been used in photosynthetic studies and thus the full extent of its potentialities, in this field of research, remains unknown. To determine the suitability of FCM in photosynthesis studies, pea plants were exposed to Paraquat and their status was analyzed during 24 h. FCM was used to evaluate the integrity (volume and internal complexity) and the relative fluorescence intensity (FL) of chloroplasts extracted from those plants. To elucidate which type of information the FL conveys, FL values were correlated with the minimum fluorescence level (F₀), maximum fluorescence level (F_m) and maximum photochemical efficiency of PSII (F_v/F_m), obtained by using Pulse-Amplitude-Modulation (PAM) fluorometry. Results indicate that: (1) the biomarkers used to evaluate the structural integrity of the chloroplasts were more sensitive to Paraquat exposure than the ones related to fluorescence; (2) the variation of the chloroplast??s structure, as time progressed, pointed to a swelling and subsequent burst of the chloroplast which, in turn, compromised fluorescence emission; (3) FL presented a high and significant correlation with the F_v/F_m and to a lesser degree with Fm but not with F₀; (4) pigment content did not reveal significant changes in response to Paraquat exposure and is in agreement with the proposed model, suggesting that the cause for fluorescence decrease is due to chloroplast disruption. In sum, FCM proved to be an outstanding technique to evaluate chloroplastidal functional and structural status and therefore it should be regarded as a valuable asset in the field of photosynthetic research.     

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.     

Role of calcium ion in protection against heat and high irradiance stress-induced oxidative damage to photosynthesis of wheat leaves

Cross stress of heat and high irradiance (HI) resulted in the accumulation of active oxygen species and photo-oxidative damage to photosynthetic apparatus of wheat leaves during grain development. Pre-treatment with calcium ion protected the photosynthetic system from oxidative damage by reducing O^-. ₂ production, inhibiting lipid peroxidation, and retarding electrolyte leakage from cell. Therefore, high F_v/F_m [maximal photochemical efficiency of photosystem 2 (PS2) while all PS2 reaction centres are open], F_m/F₀ (another expression for the maximal photochemical efficiency of PS2), Φ_PS2 (actual quantum yield of PS2 under actinic irradiation), q_P (photochemical quenching coefficient), and P _N (net photosynthetic rate) were maintained, and lower q_NP (non-photochemical quenching coefficient) of the leaves was kept under heat and HI stress. EGTA (a chelant of calcium ion) and LaCl₃ (a blocker of Ca²⁺ channel in cytoplasmic membrane) had the opposite effect. Thus Ca ion may help protect the photosynthetic system of wheat leaves from oxidative damage induced by the cross stress of heat and HI.     

Pulse amplitude modulated fluorescence in the green macrophytes,Codium adherens,Enteromorpha muscoides,Ulva gigantea and Ulva rigida,from the Atlantic coast of Southern Spain

The effect of ambient and enhanced solar radiation on the photosynthetic apparatus in four marine green macroalgae on the Southern coast of Spain (Strait of Gibraltar) was investigated using pulse amplitude modulation (PAM) fluorescence. The dependence of the fluorescence parameters on the irradiance of the actinic light was determined for all four species. It showed that maximal fluorescence after light adaptation (F_m′), photochemical quenching (q_P) and the photosynthetic quantum yield decreased in Enteromorpha muscoides with irradiance while non-photochemical quenching (q_N) rose continuously. In Ulva rigida the photosynthetic quantum yield dropped at irradiances above 4 W m⁻² but q_P did not decrease with increasing light. q_N quenching rose sharply above 37 W m⁻², and maximal fluorescence dropped above 1 W m⁻². In Ulva gigantea the yield dropped to zero at irradiances of 37 W m⁻², as did q_P at 53 W m⁻². q_N started from an intermediate level and increased to a maximum at the highest irradiances. In Codium adherens, the yield and q_P behaved similarly as in U. rigida, while q_N rose at much lower irradiances. All investigated algae suffered from photoinhibition even at their natural sites of growth when the sun is at high angles. The hypothesis that algae with flat thalli suffer more than those with massive ones was confirmed. Photoinhibition was less pronounced in U. rigida and C. adherens than in the other two species. After 1 h of exposure to solar radiation at the surface, the photosynthetic quantum yield decreased substantially in the surface algae E. muscoides and U. rigida. In both macroalgae, recovery of the photosynthetic quantum yield was almost complete after 2–3 h in the shade. Two other green algae from shaded habitats (U. gigantea and C. adherens) did not show complete recovery of the yield from photoinhibition. This confirms the second hypothesis that sun-adapted algae recover faster from photoinhibition than those adapted to shaded sites.     

Effect of Glomus mosseae on chlorophyll content, chlorophyll fluorescence parameters, and chloroplast ultrastructure of beach plum (Prunus maritima) under NaCl stress

The present study was undertaken to investigate the effect of Glomus mosseae on chlorophyll (Chl) content, Chl fluorescence parameters and chloroplast ultrastructure of beach plum seedlings under 2% NaCl stress. The results showed that compared to control, both Chl a and Chl b contents of NaCl + G. mosseae treatment were significantly lower during the salt stress, while Chl a/b ratio increased significantly. The increase of minimal fluorescence of darkadapted state (F₀), and the decrease of maximal fluorescence of dark-adapted state (F_m) and variable fluorescence (F_v) values were inhibited. The maximum quantum yield of PSII photochemistry (F_v/F_m), the maximum energy transformation potential of PSII photochemistry (F_v/F₀) and the effective quantum yield of PSII photochemistry (??_PSII) increased significantly, especially the latter two variables. The values of the photochemical quenching coefficient (q_P) and the nonphotochemical quenching (NPQ) were similar between G. mosseae inoculation and noninoculation. It could be concluded that G. mosseae inoculation could protect the photosystem II (PSII) of beach plum, enhance the efficiency of primary light energy conversion and improve the primitive response of photosynthesis under salinity stress. Meanwhile, G. mosseae inoculation was beneficial to maintain the integrity of thylakoid membrane and to protect the structure and function of chloroplast, which suggested that G. mosseae can alleviate the damage of NaCl stress to chloroplast.     

Responses of photosynthetic parameters of <Emphasis Type="Italic">Mikania micrantha</Emphasis> and <Emphasis Type="Italic">Chromolaena odorata</Emphasis> to contrasting irradiance and soil moisture

Photosynthetic parameters were measured in two invasive weeds, Mikania micrantha and Chromolaena odorata, grown in soil under full, medium, and low irradiance and full, medium, and low water supply. Both species showed significantly higher net photosynthetic rate, quantum yield of PS 2 photochemistry and photochemical quenching coefficient under high than low irradiance. For M. micrantha, low irradiance caused decreased chlorophyll content (Chl), Chl a/b ratio and maximum photochemical efficiency of PS 2 (F_v/F_m), while drought decreased Chl content and F_v/F_m and increased nonphotochemical quenching (NPQ). However, these parameters were much less affected in C. odorata except that Chl content and NPQ slightly increased under drought and high irradiance. High irradiance increased xanthophyll pools in both species, especially M. micrantha under combination with drought.     

Photosynthetic electron transport is differentially affected during early stages of cultivar/race-specific interactions between potato andPhytophthora infestans

The influence of the early stages of fungal infection on chloroplast metabolism was studied in cultivar/race-specific interactions between potato (Solanum tuberosum L. cv. Datura) and the late-blight fungusPhytophthora infestans. The accumulation of several mRNAs encoding components of the photosynthetic apparatus was not affected, either in compatible or in incompatible interactions. However, within 3 h after inoculation of potato leaves with fungal spores, a change in the photochemistry of photosystem II was detectable by measuring chlorophylla fluorescence. Characteristic fluorescence parameters, such as maximum fluorescence yield (F_m), variable fluorescence yield (F_v) and photochemical efficiency (F_v/F_m), were specifically reduced in the compatible host/pathogen interaction. Analyses of photochemical and nonphotochemical fluorescence quenching showed an increase in the photochemical fraction. The amounts of two selected thylakoid membrane proteins and of total chlorophyll remained unchanged during this process, suggesting that the functional modification of the electron-transport system was not correlated with a change in the composition of the photosynthetic apparatus. The alterations of photosynthetic electron transport represent a rapidly detectable and sensitive physiological marker for compatible interactions in the potato/Phytophthora infestans pathosystem.     

Effects of Inorganic Nitrogen Availability on the Sporophytes of Acrostichum Aureum L.

Plants grown at low irradiance were fertilized with 0, 60, and 600 g m_-3 NH₄NO₃ once every fortnight. Plants treated with high N concentrations showed an increased growth, producing longer and broader fronds with larger areas, and were darker green in colour. Nitrogen also increased the content of chlorophyll (Chl) and carotenoids per leaf area unit. Different N treatments did not affect the photosynthetic efficiency of photosystem 2, as reflected by the high values of Chl fluorescence kinetics F_v/F_m, ranging between 0.81 to 0.84, and F_v/F₀ of 4.30 to 5.10. An increase in photochemical quenching (q_P), accompanied by a decrease in non-photochemical quenching (q_N), was observed in sporophytes fertilized with increased concentrations of NH₄NO₃. Nitrogen availability allowed sporophytes of Acrostichum aureum to become more established under natural condi tions. This revised version was published online in September 2006 with corrections to the Cover Date.     

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     

Response of the Photosynthetic Apparatus of the Diatom Thallassiosira weisflogii to High Irradiance Light

The response of the photosynthetic apparatus to high irradiance illumination (440–2200 W/m²) was studied in the diatom Thallassiosira weisflogii by fluorescence methods. Changes in the photosynthetic apparatus were monitored by measuring characteristics of chlorophyll fluorescence F ₀, F _m, F _v/F _m, and qN for several hours after illumination of the alga with high-intensity light. Incubation of the alga with 2 mM DTT, an inhibitor of de-epoxidase of carotenoids in the diadinoxanthin cycle, led to a decrease in the nonphotochemical quenching of chlorophyll fluorescence and a drop in the F _v/F _m ratio, a characteristic that reflects the quantum efficiency of the functioning of the photosynthetic apparatus. Light-induced absorption changes associated with transformations of carotenoids of diadinoxanthin cycle were recorded in vivo in algal suspensions in the absence and in the presence of DTT. Using the microfluorometric method, we measured cell distribution over the efficiency of the primary processes of photosynthesis (F _v/F _m) after illumination. We found cells with a high tolerance of their photosynthetic apparatus to photooxidative damage. The relatively high tolerance of a portion of the cell population to high-light illumination can be related to light-induced transformation of carotenoids and to the functioning of other protective systems of the photosynthetic apparatus in diatoms.     

Fluorescence parameters of White Sea phytoplankton under different nitrogen sources

We investigated dependence of fluorescence parameters and phytoplankton biomass on the nitrogen source and irradiance in enriched flask studies with White Sea plankton from August-September 2007. Phytoplankton was exposed in situ for 18 d with addition of 180 μM/L of nitrogen in the forms of nitrate, urea, ammonia, and glycine under two levels of irradiance. Maximum quantum efficiency of PS2 (F_v/F_m) was determined in the samples adapted to darkness. Rapid light curves were obtained for each sample with the sequential increase of light intensity (8 levels). The maximal relative electron transport rate (rETR_max), the maximum light utilization coefficient (α), and the nonphotochemical quenching (NPQ) were calculated. The phytoplankton abundance increased on nitrogen addition, and the photosynthetic parameters changed. The values F_v/F_m reached 0.64–0.71, which indicated a good physiological state of algae and lack of nitrogen limitation. The dynamics of rETR_max and NPQ depended of the nitrogen source and irradiance, while α did not depend on nitrogen addition.     

Response of xanthophyll cycle and chloroplastic antioxidant enzymes to chilling stress in tomato over-expressing glycerol-3-phosphate acyltransferase gene

Over-expression of chloroplastic glycerol-3-phosphate acyltransferase gene (LeGPAT) increased unsaturated fatty acid contents in phosphatidylglycerol (PG) of thylakoid membrane in tomato. The effect of this increase on the xanthophyll cycle and chloroplast antioxidant enzymes was examined by comparing wild type (WT) tomato with the transgenic (TG) lines at chilling temperature (4 °C) under low irradiance (100 μmol m⁻² s⁻¹). Net photosynthetic rate and the maximal photochemical efficiency of photosystem (PS) 2 (F_v/F_m) in TG plants decreased more slowly during chilling stress and F_v/F_m recovered faster than that in WT plants under optimal conditions. The oxidizable P700 in both WT and TG plants decreased during chilling stress under low irradiance, but recovered faster in TG plants than in the WT ones. During chilling stress, non-photochemical quenching (NPQ) and the de-epoxidized ratio of xanthophyll cycle in WT plants were lower than those of TG tomatoes. The higher activities of superoxide dismutase (SOD) and ascorbate peroxidase (APX) in TG plants resulted in the reduction of O₂ ^−· and H₂O₂ contents during chilling stress. Hence the increase in content of unsaturated fatty acids in PG by the over-expression of LeGPAT could alleviate photoinhibition of PS2 and PS1 by improving the de-epoxidized ratio of xanthophyll cycle and activities of SOD and APX in chloroplast.     

Photosynthetic characteristics of female vegetative tissues of Porphyra katadai var. hemiphylla (Bangiales, Rhodophyta) in culture

In this study, chlorophyll fluorescence parameters (?F/F _m′, F _v/F _m) and oxygen evolution of female vegetative tissues of Porphyra katadai var. hemiphylla in unisexual culture (FV) and in mixed culture with male vegetative tissues (FV-M) were followed at 5–20 °C, 10 and 80 μmol photons m^?2 s^?1. The formation of reproductive tissues was closely correlated with decreasing photosynthetic activities. At the same temperature the tissues cultured under 80 μmol photons m^?2 s^?1 showed a greater extent of maturation than those under 10 μmol photons m^?2 s^?1, and their decrease in photosynthesis was also larger. Under the same light intensity the extent of maturation increased with increasing temperature, and both cultures showed higher values of ?F/F _m′ and F _v/F _m at 10 and 15 °C, while their oxygen evolution became negative at 15–20 °C during the later period. Under the same culture condition the maturation of FV-M culture was relatively faster than that of FV culture, while their photosynthetic activity, especially ?F/F _m′, was lower.     

Photoadaptation in the Green Alga Spongiochloris Sp. A Three-Fluorometer Study

The dark-adapted cells of the green alga Spongiochloris sp. were exposed to "white light" of 1000 μmol(photon) m⁻² s⁻¹ for 2 h and then dark adapted for 1.5 h. Changes of photochemical activities during photoadaptation were followed by measurement of chlorophyll (Chl) fluorescence kinetics, 77 K emission spectra, photosynthetic oxygen evolution, and pigment composition. We observed a build-up of slowly-relaxing non-photochemical quenching which led to a decrease of the F_v/F_m parameter and the connectivity. In contrast to the depression of F_v/F_m (35 %) and the rise of non-photochemical quenching (∼ 1.6), we observed an increase in effective absorption cross-section (20 %), Hill reaction (30 %), photosynthetic oxygen evolution (80 %), and electron transport rate estimated from the Chl fluorescence analysis (80 %). We showed an inconsistency in the presently used interpretation schemes, and ascribe the discrepancy between the increase of effective absorption cross-section and the photosynthetic activities on one side and the effective non-photochemical quenching on the other side to the build-up of a quenching mechanism which dissipates energy in closed reaction centres. Such a type of quenching changes the ratio between thermal dissipation and fluorescence without any effect on photochemical yield. In this case the F_v/F_m ratio cannot be used as a measure of the maximum photochemical yield of PS2. This revised version was published online in August 2006 with corrections to the Cover Date.     

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.