Michel Havaux

A theoretical and experimental analysis of the qP and qN coefficients of chlorophyll fluorescence quenching and their relation to photochemical and nonphotochemical events

The initial (F0), maximal (FM) and steady-state (FS) levels of chlorophyll fluorescence emitted by intact pea leaves exposed to various light intensities and environmental conditions, were measured with a modulated fluorescence technique and were analysed in the context of a theory for the energy fluxes within the photochemical apparatus of photosynthesis. The theoretically derived expressions of the fluorescence signals contain only three terms, X=J2p2F/(1−G), Y=T/(1−G) and V, where V is the relative variable fluorescence, J2 is the light absorption flux in PS II, p2F is the probability of fluorescence from PS II, G and T are, respectively, the probabilities for energy transfer between PS II units and for energy cycling between the reaction center and the chlorophyll pool: F0=X, FM=X/(1−Y) and FS=X(1+(YV/(1−Y))). It is demonstrated that the amplitudes of the previously defined coefficients of chlorophyll fluorescence quenching, qP and qN, reflect, not just photochemical (qP) or nonphotochemical (qN) events as implied in the definitions, but both photochemical and nonphotochemical processes of PS II deactivation. The coefficient qP is a measure of the ratio between the actual macroscopic quantum yield of photochemistry in PS II (41-1) in a given light state and its maximal value measured when all PS II traps are open (41-2) in that state, with 41-3 and 41-4. When the partial connection between PS II units is taken into consideration, 1-qP is nonlinearily related to the fraction of closed reaction centers and is dependent on the rate constants of all (photochemical as well as nonphotochemical) exciton-consuming processes in PS II. On the other hand, 1-qN equals the (normalized) ratio of the rate constant of photochemistry (k2b) to the combined rate constant (kN) of all the nonphotochemical deactivation processes excluding the rate constant k22 of energy transfer between PS II units. It is demonstrated that additional (qualitative) information on the individual rate constants, kN-k22 and k2b, is provided by the fluorescence ratios 1/FM and (1/F0)−(1/FM), respectively. Although, in theory, 41-5 is determined by the value of both k2b and kN-k22, experimental results presented in this paper show that, under various environmental conditions, 41-6 is modulated largely through changes in kN, confirming the idea that PS II quantum efficiency is dynamically regulated in vivo by nonphotochemical energy dissipation.

Short-term responses of Photosystem I to heat stress : Induction of a PS II-independent electron transport through PS I fed by stromal components.

When 23°C-grown potato leaves (Solanum tuberosum L.) were exposed for 15 min to elevated temperatures in weak light, a dramatic and preferential inactivation of Photosystem (PS) II was observed at temperatures higher than about 38°C. In vivo photoacoustic measurements indicated that, concomitantly with the loss of PS II activity, heat stress induced a marked gas-uptake activity both in far-red light (>715 nm) exciting only PS I and in broadband light (350–600 nm) exciting PS I and PS II. In view of its suppression by nitrogen gas and oxygen and its stimulation by high carbon-dioxide concentrations, the bulk of the photoacoustically measured gas uptake by heat-stressed leaves was ascribed to rapid carbon-dioxide solubilization in response to light-modulated stroma alkalization coupled to PS I-driven electron transport. Heat-induced gas uptake was observed to be insensitive to the PS II inhibitor diuron, sensitive to the plastocyanin inhibitor HgCl2 and saturated at a rather high photon flux density of around 1200 μE m−2 s−1. Upon transition from far-red light to darkness, the oxidized reaction center P700+ of PS I was re-reduced very slowly in control leaves (with a half time t1/2 higher than 500 ms), as measured by leaf absorbance changes at around 820 nm. Heat stress caused a spectacular acceleration of the postillumination P700+ reduction, with t1/2 falling to a value lower than 50 ms (after leaf exposure to 48°C). The decreased t1/2 was sensitive to HgCl2 and insensitive to diuron, methyl viologen (an electron acceptor of PS I competing with the endogenous acceptor ferredoxin) and anaerobiosis. This acceleration of the P700+ reduction was very rapidly induced by heat treatment (within less than 5 min) and persisted even after prolonged irradiation of the leaves with far-red light. After heat stress, the plastoquinone pool exhibited reduction in darkness as indicated by the increase in the apparent Fo level of chlorophyll fluorescence which could be quenched by far-red light. Application (for 1 min) of far-red light to heat-pretreated leaves also induced a reversible quenching of the maximal fluorescence level Fm, suggesting formation of a pH gradient in far-red light. Taken together, the presented data indicate that PS I responded to the heat-induced loss of PS II photochemical activity by catalyzing an electron flow from stromal reductants. Heat-stress-induced PS I electron transport independent of PS II seems to constitute a protective mechanism since block of this electron pathway in anaerobiosis was observed to result in a dramatic photoinactivation of PS I.

Photoinhibition of photosynthesis in chilled potato leaves is not correlated with a loss of Photosystem-II activity : Preferential inactivation of Photosystem I.

When 23 °C-grown potato leaves (Solanum tuberosum L.) were irradiated at 23 °C with a strong white light, photosynthetic electron transport and Photosystem-II (PS II) activity were inhibited in parallel. When the light treatment was given at a low temperature of 3 °C, the photoinhibition of photosynthesis was considerably enhanced, as expected. Surprisingly, no such stimulation of photoinhibition was observed with respect to the PS II function. A detailed functional analysis of the photosynthetic apparatus, using in-vivo fluorescence, absorbance, oxygen and photoacoustic measurements, and artificial electron donors/acceptors, showed a pronounced alteration of PS I activity during light stress at low temperature. More precisely, it was observed that both the pool of photooxidizeable reaction center pigment (P700) of PS I and the efficiency of PS I to oxidize P700 were dramatically reduced. Loss of P700 activity was shown to be essentially dependent on atmospheric O2 and to require a continued flow of electrons from PS II, suggesting the involvement of the superoxide anion radical which is produced by the interaction of O2 and the photosynthetic electron-transfer chain through the Mehler reaction. Mass spectrometric measurements of O2 exchange by potato leaves under strong illumination did not reveal, however, any stimulation of the Mehler reaction at low temperature, thus leading to the conclusion that O2 toxicity mainly resulted from a chilling-induced inhibition of the scavenging system for O2-radicals. Support for this interpretation was provided by the light response of potato leaves infiltrated with an inhibitor (diethyldithiocarbamate) of the chloroplastic Cu-Zn superoxide dismutase. It was indeed possible to simulate the differential inhibition of the PS II photochemical activity and the linear electron transport observed during light stress at low temperature by illuminating at 23 °C diethyldithiocarbamate-poisoned leaves. The experimental data presented here suggests that (i) the previously reported resistance of PS I to photoinhibition damage in-vivo is not an intrinsic property of PS I but results from efficient protective systems against O2 toxicity, (ii) PS I is photoinhibited in chilled potato leaf due to the inactivation of this PS I defence system and (iii) PS I is more sensitive to superoxide anion radicals than PS II.

Correlation between Heat Tolerance and Drought Tolerance in Cereals Demonstrated by Rapid Chlorophyll Fluorescence Tests

Summary Drought and heat tolerances were measured in intact leaves of a large number of genotypes of durum wheat ( Triticum durum Desf.), barley ( Hordeum vulgare L.) and triticale ( Triticum durum L. × Secale cereale L.) using rapid in vivo chlorophyll fluorescence tests. The high temperature (T p ) corresponding to maximal F o -fluorescence in leaf samples heated at a rate of 1 ưC min −1 was used to estimate the relative heat tolerance. On the other hand, drought tolerance was measured by the reduction of the photochemical quenching of chlorophyll fluorescence (q Q ) after a short desiccation treatment. The data indicate a very wide range of genotypic adaptation to drought and heat in cereals. The two types of tolerance were, however, closely related: the most heat tolerant cereal varieties were also the most drought tolerant ones. The results presented in this paper also provide a good example of the usefulness and the simplicity of modulated chlorophyll fluorescence measurements as rapid screening tests for stress tolerance in crop plants.

Chlorophyll fluorescence induction: A sensitive indicator of water stress in maize plants

SummaryThis investigation shows that the chlorophyll fluorescence induction phenomenon provides a simple non destructive method for investigating effects of drought on plants. Drastic reduction of the maximum (P) to the minimum (0) chlorophyll fluorescence ratio and strong inhibition of the slow fluorescence induction transients were observed in maize submitted to water stress sufficient to dehydrate leaves to 68% of original water content. The P/0 value and the typical PSMT induction sequence were restored following the removal of drought conditions. However, the slow quenching of chlorophyll fluorescence to the steady-state (T) remained noticeably altered, indicating irreversible damage on the chloroplastic membranes.

Temperature Sensitivity of the Photochemical Function of Photosynthesis in Potato (Solanum tuberosum) and a Cultivated Andean Hybrid (Solanum X juzepczukii)

Summary The responses of the photochemical apparatus of photosynthesis to low and high temperatures were compared in leaves of the frost-sensitive Solanum tuberosum (cv. Haig) and of a frost-tolerant Andean potato, Solanum x juzepczukii (cv. Lucki). The main observations and conclusions of this study are that: (i) Photosystem II (PSII) is noticeably more heat-resistant in S. x juzepczukii than in S. tuberosum, indicating an enhanced generalized stress tolerance of the former genotype to extremes of temperature. (ii) The higher thermostability of PSII in S. x juzepczukii leaves is not associated with any enhancement of the sensitivity of PSII photochemistry to chilling temperature. In both species, the chilling-induced inhibition of electron transport through PSII is closely correlated with the inhibition of the PSII-to-PSI electron flow, the rate of which is determined by the reoxidation of reduced plastoquinone. A slowdown of the latter reaction at low temperature can be attributed to the accumulation of protons in the thylakoid lumen associated with the inhibition of the Calvin cycle activity in chilled leaves, as suggested by the strong non-photochemical quenching of chlorophyll fluorescence. (iii) The photochemical activities of both species are similarly impaired by chilling treatments in the light, indicating that frost resistance does not preclude susceptibility to photoinhibition damage at chilling temperature. (iv) A striking difference between S. tuberosum and S. x juzepczukii is the high plasticity of the PSII thermotolerance in the latter species, with low (8 °C) and high (35 °C) temperature treatments respectively decreasing and increasing the heat-tolerance of PSII. These changes are not observed or are very limited in the Haig variety of S. tuberosum. (v) In contrast to the constitutive thermotolerance of PSII (measured in 23 °C-grown plants), 35 °C-induced thermotolerance has a dramatic effect on the photochemical activity at chilling temperature. When placed at 5 °C, the intersystem electron flow of 35 °C-treated leaves is dramatically inhibited as compared with non-treated leaves whereas ΔpH-related quenching of chlorophyll fluorescence is unchanged. These findings indicate independent control of non-acclimated heat-tolerance and thermally induced heat-tolerance of the photosynthetic membranes. Taken together, the presented data show that the photosynthetic apparatus of the cultivated Andean hybrid, S. x juzepczukii, though sensitive to chilling injury in the light, is adapted to the changing temperature conditions prevailing in the natural habitat of its wild progenitor where night frosts are associated with warm and sunny days.

“ENERGY”-DEPENDENT QUENCHING OF CHLOROPHYLL FLUORESCENCE and THERMAL ENERGY DISSIPATION IN INTACT LEAVES DURING INDUCTION OF PHOTOSYNTHESIS

Abstract— Intact leaves, previously adapted to darkness for a prolonged period of time, were suddenly illuminated with a strong, photosynthetically saturating, white light (ca 1500 μmol m−2 s_1), resulting in the rapid establishment of a large energy‐dependent chlorophyll fluorescence quenching (qE) as shown by in vivo fluorescence measurements with a pulse amplitude modulation technique. Two different photothermal methods, photoacoustic spectroscopy and photothermal deflection spectroscopy, were used to monitor thermal deactivation of excited pigments during the dark‐light transitions. The in vivo photothermal signals measured with both techniques were shown to remain constant during induction of photosynthesis under high light conditions, suggesting that, in contrast to current hypotheses, energy‐dependent quenching qE is not associated with significant changes in thermal dissipation of absorbed light energy in the chloroplasts. When photosynthesis was induced with a low‐intensity modulated light, a noticeable decrease in the heat emission yield was observed resulting from the progressive activation of the competing photochemical processes.

Dynamics of electron transfer within and between PS II reaction center complexes indicated by the light-saturation curve of in vivo variable chlorophyll fluorescence emission

The dynamics of light-induced closure of the PS II reaction centers was studied in intact, dark-adapted leaves by measuring the light-irradiance (I) dependence of the relative variable chlorophyll fluorescence V which is the ratio between the amplitude of the variable fluorescence induced by a pulse of actinic light and the maximal variable fluorescence amplitude obtained with an intense, supersaturating light pulse. It is shown that the light-saturation curve of V is a hyperbola of order n. The experimental values of n ranged from around 0.75 to around 2, depending on the plant material and the environmental conditions. A simple theoretical analysis confirmed this hyperbolic relationship between V and I and suggested that n could represent the apparent number of photons necessary to close one reaction center. Thus, experimental conditions leading to n values higher than 1 could indicate that, from a macroscopic viewpoint, more than one photon is necessary to close one PS II center, possibly due to changes in the relative concentrations of the different redox states of the PS II reaction center complexes at the quasi-steady state induced by the actinic light. On the other hand, the existence of environmental conditions resulting in n noticeably lower than 1 suggests the possibility of an electron flow between PS II reaction center complexes.

Photoacoustic characteristics of leaves of atrazine-resistant weed mutants

The photosynthetic characteristics of leaves of atrazine-resistant and-susceptible biotypes of several weed species (Solanum nigrum, Senecio vulgaris, Epilobium ciliatum and Chenopodium album) were compared using the photoacoustic method. Analysis of the dependence of the photoacoustic signal of the modulation frequency indicated that, in Solanum, Epilobium and Senecio, the relative quantum yield of O2 evolution ϕ (estimated by the ratio of the amplitude of the O2 signal, AOX, to that of the photothermal signal, APT) was substantially reduced in the atrazine-resistant mutant, without any changes in the O2 diffusion characteristics of the leaves. In contrast, in Chenopodium, atrazine-resistance was associated with a concomitant change in ϕ and in the leaf diffusion parameters. This latter change suggests that the leaf internal anatomy was modified in the resistant Chenopodium. Measurements of the Emerson enhancement indicated that the reduction of ϕ observed in the atrazine-resistant mutants was caused by a marked decrease in the photochemical potential of PS II (β). The study of the light intensity dependence of the AOX/APT ratio showed that saturation of O2 evolution occurred at the same light level (around 2000 μmol m-2 s-1) in both types of plants. However, the relative maximal rate of O2 evolution was slightly lower (-10%) in the atrazine-resistant biotype as compared to the wild type. Reduced ϕ and light-saturated rate of O2 evolution were also measured in atrazine-resistant weed biotypes using a conventional Clark-type O2 electrode.

Reversible effects of moderately elevated temperature on the distribution of excitation energy between the two photosystems of photosynthesis in intact avocado leaves

Initial (Fo), maximum (Fm) and steady-state (Fs) levels of modulated chlorophyll fluorescence were measured in intact avocado leaves (Persea americana Mill.) during state 1-state 2 transitions using a combination of modulated and non-modulated lights with synchronized detection. Under normal temperature conditions (20°C), transition from state 2 to state 1 was associated with a substantial increase (about 20%) in Fm and Fo whereas the Fm/Fo ratio remained constant, reflecting increased absorption cross-section of PS II. On the contrary, at moderately elevated temperature (35°C), these fluorescence changes were very limited, indicating marked inhibition of the state regulation. The fraction of light distributed to PS II (β) was calculated from the Fo, Fm and Fs levels for both types of leaves. In control leaves, β varied from 48% (in state 2) to values as high as 58% (in state 1). In contrast, mild heat treatment resulted in β values close to 50% in both states, indicating the inability of heated leaves to reach extreme state 1. The results suggested that avocado leaves under moderately elevated temperature conditions are blocked in a state close to state 2. This effect was shown to occur in a non-injurious temperature range (as shown by the preservation of the (photoacoustically monitored) oxygen evolution activity) and to be rapidly reversed upon lowering of the temperature. Thermally induced development of state 2 (independent on the light spectral quality) could possibly be a protective mechanism to avoid photodamage of the heat-labile PS II by high light intensities which usually accompany heat stress in the field.