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Chlorophyll fluorescence (ChlF) excitation spectra were measured to assess the UV-sunscreen compounds accumulated in fully expanded leaves of three woody species belonging to different chemotaxons, (i.e. Morus nigra L., Prunus mahaleb L. and Lagerstroemia indica L.), grown in different light microclimates. The logarithm of the ratio of ChlF excitation spectra (logFER) between two leaves acclimated to different light microclimates was used to assess the difference in epidermal absorbance (EAbs). EAbs increased with increasing solar irradiance intercepted for the three species. This epidermal localisation of UV-absorbers was confirmed by the removal of the epidermis. It was possible to simulate EAbs as a linear combination of major phenolic compounds (Phen) identified in leaf methanol extracts by HPLC-DAD. Under UV-free radiation conditions, shaded leaves of M. nigra accumulated chlorogenic acid. Hydroxybenzoic acid (HBA) derivatives and hydroxycinnamic acid (HCA) derivatives greatly increased with increasing PAR irradiance under the low UV-B conditions found in the greenhouse. These traits were also observed for the HCA of the two other species. Flavonoid (FLAV) accumulation started under low UV-A irradiance, and became maximal in the adaxial epidermis of sun-exposed leaves outdoors. A decrease in the amount of HCA was observed concomitantly to the intense accumulation of FLAV for both leaf sides of the three species. Judging from the logFER, under low UV-B conditions, larger amounts of HCA are present in the epidermis in comparison to FLAV for the three species. Upon transition from the greenhouse to full sunlight outdoors, there was a decrease in leaf-soluble HCA that paralleled FLAV accumulation in reaction to increasing solar UV-B radiation in the three species. In M. nigra, that contains large amounts of HCA, the logFER analysis showed that this decrease occurred in the adaxial epidermis, whereas the abaxial epidermis, which is protected from direct UV-B radiation, continued to accumulate large amounts of HCA.

In vitro-cultured plants typically show a low photosynthetic activity, which is considered detrimental to subsequent ex vitro acclimatization. Studies conducted so far have approached this problem by analysing the biochemical and photochemical aspects of photosynthesis, while very little attention has been paid to the role of leaf conductance to CO(2) diffusion, which often represents an important constraint to CO(2) assimilation in naturally grown plants. Mesophyll conductance, in particular, has never been determined in in vitro plants, and no information exists as to whether it represents a limitation to carbon assimilation during in vitro growth and subsequent ex vitro acclimatization. In this study, by means of simultaneous gas exchange and chlorophyll fluorescence measurements, the stomatal and mesophyll conductance to CO(2) diffusion were assessed in in vitro-cultured plants of the grapevine rootstock '41B' (Vitis vinifera 'Chasselas'xVitis berlandieri), prior to and after ex vitro acclimatization. Their impact on electron transport rate partitioning and on limitation of potential net assimilation rate was analysed. In vitro plants had a high stomatal conductance, 155 versus 50 mmol m(-2) s(-1) in acclimatized plants, which ensured a higher CO(2) concentration in the chloroplasts, and a 7% higher electron flow to the carbon reduction pathway. The high stomatal conductance was counterbalanced by a low mesophyll conductance, 43 versus 285 mmol m(-2) s(-1), which accounted for a 14.5% estimated relative limitation to photosynthesis against 2.1% estimated in acclimatized plants. It was concluded that mesophyll conductance represents an important limitation for in vitro plant photosynthesis, and that in acclimatization studies the correct comparison of photosynthetic activity between in vitro and acclimatized plants must take into account the contribution of both stomatal and mesophyll conductance.

Interactions between photosynthesis, mitochondrial respiration (mitorespiration), and chlororespiration have been investigated in the green alga Chlamydomonas reinhardtii using flash illumination and a bare platinum electrode. Depending on the physiological status of algae, flash illumination was found to induce either a fast (t(1/2) approximately 300 ms) or slow (t(1/2) approximately 3 s) transient inhibition of oxygen uptake. Based on the effects of the mitorespiratory inhibitors myxothiazol and salicyl hydroxamic acid (SHAM), and of propyl gallate, an inhibitor of the chlororespiratory oxidase, we conclude that the fast transient is due to the flash-induced inhibition of chlororespiration and that the slow transient is due to the flash-induced inhibition of mitorespiration. By measuring blue-green fluorescence changes, related to the redox status of the pyridine nucleotide pool, and chlorophyll fluorescence, related to the redox status of plastoquinones (PQs) in C. reinhardtii wild type and in a photosystem I-deficient mutant, we show that interactions between photosynthesis and chlororespiration are favored when PQ and pyridine nucleotide pools are reduced, whereas interactions between photosynthesis and mitorespiration are favored at more oxidized states. We conclude that the plastid oxidase, similar to the mitochondrial alternative oxidase, becomes significantly engaged when the PQ pool becomes highly reduced, and thereby prevents its over-reduction.

The effect of cumulative over-saturating pulses (OSP) of white light (1 s, >10 000 mumol photons m(-2) s(-1)), applied every 20 min on pea leaves, was investigated during a complete diurnal cycle of 24 h. In dark-adapted leaves, this treatment leads to a progressive decline of the optimum Photosystem II (PS II) quantum yield. Continuous low background light (except far-red light) had a protective effect against this OSP-induced photoinactivation. The lack of far-red effect could be due to its absorption mainly in PS I and not in PS II, but could be also due to the general low absorption in this wavelength region. The photoinactivation was enhanced in leaves that had been previously infiltrated with chloramphenicol. The quantum yield of CO(2) assimilation, but not its maximal capacity, was inhibited by the OSP treatment. The most spectacular effects observed, in addition to an irreversible quenching of Fm, was a strong inhibition of Q(A) (-) reoxidation revealed by a large increase in the Fs level and consequently by a decrease of DeltaF/Fm'. Under such conditions, we observed that the electron flow deduced from DeltaF/Fm' underestimated the real electron flow to CO(2). Time-resolved Chlorophyll a fluorescence measurements showed that the reduced capacity of Q(A) (-) reoxidation in OSP treated leaves was accompanied by the appearance of a 4.7 ns component attributed to PS II charge recombination. We suggest that a modification at the Q(B) site may influence the redox potential of Q(A)/Q(A) (-), facilitating the reversion of the primary charge separation. In addition, a 1.2 ns fluorescence component accumulated, which appeared to be responsible for the underestimation of PS II electron flow. The observed photoinactivation seemed to be different from the photoinhibition often described in the literature, which occurs under continuous light.

Isolated chloroplasts show a light-induced reversible increase in blue-green fluorescence (BGF), which is only dependent on NADPH changes. In the present communication, we report a time-resolved and spectral analysis of this BGF in reconstituted chloroplasts and intact isolated chloroplasts, in the dark and under actinic illumination. From these measurements we deduced the contribution of the different forms of NADPH (free and bound to proteins) to the light-induced variation of BGF and conclude that this variation is due only to the redox change of the NADP pool. A simple model estimating the distribution of NADPH between the free and bound form was designed, that explains the differences measured for the BGF of reconstituted chloroplasts and intact chloroplasts. From the decay-associated spectra of the chloroplast BGF, we also deduced the participation of flavins to the green peak of chloroplast fluorescence emission spectrum, and the existence of excitation energy transfer from proteins to bound NADPH in chloroplasts. In addition, we re-examined the use of chloroplast BGF as a quantitative measure of NADPH concentration, and confirmed that chloroplast BGF can be used for non-destructive, continuous and probably quantitative monitoring of light-induced changes in NADP redox state.

The effect of leaf temperature (T), between 23 and 4 degrees C, on the chlorophyll (Chl) fluorescence spectral shape was investigated under moderate (200 microE m-2 s-1) and low (30-35 microE m-2 s-1) light intensities in Phaseolus vulgaris and Pisum sativum. With decreasing temperature, an increase in the fluorescence yield at both 685 and 735 nm was observed. A marked change occurred at the longer emission band resulting in a decrease in the Chl fluorescence ratio, F685/F735, with reducing T. Our fluorescence analysis suggests that this effect is due to a temperature-induced state 1-state 2 transition that decreases and increases photosystem II (PSII) and photosystem I (PSI) fluorescence, respectively. Time-resolved fluorescence life-time measurements support this interpretation. At a critical temperature (about 6 degrees C) and low light intensity a sudden decrease in fluorescence intensity was observed, with a larger effect at 685 than at 735 nm. This is probably linked to a modification of the thylakoid membranes, induced by chilling temperatures, which can alter the spill-over from PSII to PSI. The contribution of photosystem I to the long-wavelength Chl fluorescence band (735 nm) at room temperature was estimated by both time-resolved fluorescence lifetime and fluorescence yield measurements at 685 and 735 nm. We found that PSI contributes to the 735 nm fluorescence for about 40, 10 and 35% at the minimal (F0), maximal (Fm) and steady-state (Fs) levels, respectively. Therefore, PSI must be taken into account in the analysis of Chl fluorescence parameters that include the 735 nm band and to interpret the changes in the Chl fluorescence ratio that can be induced by different agents.

A newly developed nitrogen laser fluorimeter insensitive to actinic illumination was used to follow simultaneously the light induced changes in red and blue fluorescence of intact isolated spinach chloroplasts and leaf pieces. The recorded variable blue fluorescence was linked to a water soluble component of intact isolated chloroplasts, depended on Photosystem I, and was related to changes in carbon metabolism. From the comparison of changes in intact and broken chloroplasts and from fluorescence spectra under different conditions, it was concluded that the variation in NADPH was the major cause for the changes in blue fluorescence. This study opens a path towards continuous and non-destructive monitoring of NADPH redox state in chloroplasts and leaves.

A new approach to measurements of photosynthesis by isolated chloroplasts has been devised. Intact isolated chloroplasts were trapped in the cavities of membrane filters. The thin layers of chloroplasts so obtained were assayed for O(2) evolution and CO(2) assimilation in leaf-chambers. Photosynthetic gas exchange could be demonstrated to take place either in a closed or a flow-through system. The chloroplasts were morphologically intact as shown by light or scanning electron microscopy and displayed stable rates of photosynthesis in the presence of phosphate and alkaline phosphatase. The methods described open the way to in vitro measurement of photosynthesis, by chloroplasts under conditions more closely resembling those in leaves.