Ismael Moya

Early light-induced proteins protect Arabidopsis from photooxidative stress.

The early light-induced proteins (ELIPs) belong to the multigenic family of light-harvesting complexes, which bind chlorophyll and absorb solar energy in green plants. ELIPs accumulate transiently in plants exposed to high light intensities. By using an Arabidopsis thaliana mutant (chaos) affected in the posttranslational targeting of light-harvesting complex-type proteins to the thylakoids, we succeeded in suppressing the rapid accumulation of ELIPs during high-light stress, resulting in leaf bleaching and extensive photooxidative damage. Constitutive expression of ELIP genes in chaos before light stress resulted in ELIP accumulation and restored the phototolerance of the plants to the wild-type level. Free chlorophyll, a generator of singlet oxygen in the light, was detected by chlorophyll fluorescence lifetime measurements in chaos leaves before the symptoms of oxidative stress appeared. Our findings indicate that ELIPs fulfill a photoprotective function that could involve either the binding of chlorophylls released during turnover of pigment-binding proteins or the stabilization of the proper assembly of those proteins during high-light stress.

Steady-state and maximum chlorophyll fluorescence responses to water stress in grapevine leaves: a new remote sensing system.

Abstract A new fluorimeter built at Orsay allowed us to measure at a distance of up to 6 m both the steady-state and the maximum chlorophyll fluorescence. This instrument has been applied continuously during 17 days of water stress development to follow the chlorophyll fluorescence parameters of a potted grapevine. Gas-exchange rates for H 2 O and CO 2 and chlorophyll fluorescence parameters of the same leaf were recorded concurrently. It was shown that: (1) Under well-watered conditions, before noon, a correlation was found between net photosynthetic rate and the rate of electron transport calculated from fluorescence measurements. After several hours of high light exposure, CO 2 assimilation (A) started to decrease more than the rate of electron transport (ETR). Under drought conditions, the above-mentioned correspondence was lost: when A almost vanished due to high stomatal closure, the ETR was still about 50% of the control value. It is suggested that under these conditions, the ratio of photorespiration to CO 2 assimilation increased. (2) Light response of the quantum yield of ETR became increasingly different between morning and afternoon as water stress progressed, thus serving as a good indicator of plant water status. (3) A simple fluorescence parameter, Fs, accurately reflected the plant physiological state. Over the range of light intensities used in this study, this parameter changed in parallel with irradiance in well-watered plants. With increasing water stress, Fs changed in opposite direction to irradiance changes. The response of Fs to rapid changes in irradiance was fast (within seconds). The potential of this parameter for remote sensing of water stress is discussed.

Dualex: a new instrument for field measurements of epidermal ultraviolet absorbance by chlorophyll fluorescence

Dualex (dual excitation) is a field-portable instrument, hereby described, for the assessment of polyphenolic compounds in leaves from the measurement of UV absorbance of the leaf epidermis by double excitation of chlorophyll fluorescence. The instrument takes advantage of a feedback loop that equalizes the fluorescence level induced by a reference red light to the UV-light-induced fluorescence level. This allows quick measurement from attached leaves even under field conditions. The use of light-emitting diodes and of a leaf-clip configuration makes Dualex a user-friendly instrument with potential applications in ecophysiological research, light climate analysis, agriculture, forestry, horticulture, pest management, selection of medicinal plants, and wherever accumulation of leaf polyphenolics is involved in plant responses to the environment.

Intersystem exciton transfer in isolated chloroplasts

The following arguments in favor of exciton transfer between the two photosystems are presented: 1. (1) MgCl2 (1–10 mM range) decreases the intersystem transfer but does not modify the partition of absorbed photons between the photosystems. MgCl2 addition causes a simultaneous increase of excitation life time (τ) and of fluorescence intensity (F). The same linear relationship is obtained with or without added Mg2+. 2. (2) The deactivation of Photosystem II by the Photosystem II to Photosystem I transfer increases with the level of reduced Photosystem II traps. When all Photosystem II traps are closed, half of Photosystem II excitons are deactivated by transfer to Photosystem I. 3. (3) From the relative values of the 685-nm fluorescence yield and System II electron transport rate in limiting light, measured with and without MgCl2, the values of rate constants of Photosystem II deactivation were calculated. 4. (4) The intersystem transfer determines a 715-nm variable fluorescence, which is lowered by MgCl2 addition. When this transfer is decreased by MgCl2 the efficiency of the transfer between Photosystem II-connected units is enhanced, and a more sigmoidal fluorescence rise is obtained. A double-layer model of the thylakoid membrane where each photosystem is restricted to one leaflet is proposed to explain the decrease of the intersystem transfer after adding cations. It is suggested that MgCl2 decreases the thickness of the Photosystem I polar region, increasing the distance between the pigments of the two photosystems.

The Effect of Decreasing Temperature up to Chilling Values on the in vivo F685/F735 Chlorophyll Fluorescence Ratio in Phaseolus vulgaris and Pisum sativum: The Role of the Photosystem I Contribution to the 735 nm Fluorescence Band

Abstract The effect of leaf temperature (T), between 23 and 4°C, on the chlorophyll (Chl) fluorescence spectral shape was investigated under moderate (200 μE m−2 s−1) and low (30–35 μE 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 lifetime measurements support this interpretation. At a critical temperature (about 6°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 spillover 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.

Dual-excitation FLIDAR for the estimation of epidermal UV absorption in leaves and canopies

Abstract A new FLIDAR was designed for remote quantitative assessment of epidermal UV absorption of leaves and canopies from chlorophyll (Chl) fluorescence (ChlF) measurements. The dual-excitation fluorescence light detection and ranging (DE-FLIDAR) performs a dual excitation of the Chl present in leaves, in the UV (355 nm) and visible (532 nm) part of the spectrum, the latter being used as a reference excitation not absorbed by the epidermis. Therefore, the epidermal UV absorption of vegetation can be estimated from the Chl fluorescence excitation ratio (FER), ΦF(532)/ΦF(355). Thanks to the alternated excitation by the DE-FLIDAR, the FER is immune to natural conditions in field, such as light-induced variable ChlF and leaf movement (variation of the angle of excitation). The DE-FLIDAR was used to investigate the presence of UV-absorbing compounds in individual leaves and canopies of different plant species, tobacco, pea, barley and wheat. The FER was much larger in outdoor-grown plants, indicating an accumulation of UV-absorbing compounds. We also analysed the epidermal UV absorption of the adaxial and abaxial side of tobacco leaves of different age. The logarithm of the FER showed a good agreement with the absorbance of methanolic extracts obtained from the same leaves. The presented DE-FLIDAR can perform up to three simultaneous fluorescence measurements; therefore, we could compare blue fluorescence (BR) to the epidermal UV absorption. In addition, a dual ratio, the red fluorescence (RF) to far-red fluorescence (FRF) emission ratio, excited at 355 and 532 nm, was shown to be linearly dependent on the Chl content. A mathematical model of leaf absorption and fluorescence, based on the Beer–Lamberts law, was developed to describe and analyse the fluorescence signatures obtained with the DE-FLIDAR.

Chlorophyll fluorescence emission spectrum inside a leaf

Chlorophyll a fluorescence can be used as an early stress indicator. Fluorescence is also connected to photosynthesis so it can be proposed for global monitoring of vegetation status from a satellite platform. Nevertheless, the correct interpretation of fluorescence requires accurate physical models. The spectral shape of the leaf fluorescence free of any re-absorption effect plays a key role in the models and is difficult to measure. We present a vegetation fluorescence emission spectrum free of re-absorption based on a combination of measurements and modelling. The suggested spectrum takes into account the photosystem I and II spectra and their relative contribution to fluorescence. This emission spectrum is applicable to describe vegetation fluorescence in biospectroscopy and remote sensing.

SPECTRA OF FLUORESCENCE LIFETIME AND INTENSITY OF Rhodopseudomonas sphaeroides AT ROOM AND LOW TEMPERATURE. COMPARISON BETWEEN THE WILD TYPE, THE C 71 REACTION CENTER‐LESS MUTANT AND THE B800–850 PIGMENT‐PROTEIN COMPLEX

Abstract— Spectra of the fluorescence lifetime and intensity of chromatophores from the wild type Rhodopseudomonas sphaeroides, from the C 71 reaction center‐less mutant and of the B800–850 light harvesting pigment‐protein complex have been studied by phase fluorimetry techniques at different light modulation frequencies at room and low temperature.

Fluorescence lifetime spectra of in vivo bacteriochlorophyll at room temperature

Fluorescence lifetime spectra of Rhodopseudomonas sphaeroides chromatophores have been measured at room temperature by phase fluorimetry at 82 MHz in order to investigate the heterogeneity of the emission. The total fluorescence was decomposed into two main components. A constant component, Fc, centered at 865 nm, represents about 50% of the total emission from dark-adapted chromatophores (Fo) and has a lifetime of 0.55 ns. A variable component is centered at 890 nm. Upon closing the reaction centers, 5-fold increases take place in both emission yield and lifetime of this component. In the dark-adapted state, its lifetime is about 50 ps and its contribution to the total fluorescence is 70% at 890 nm. In the presence of sodium dithionite, a long-lifetime component (τD ⋍ 4 ns) is observed. This probably arises from radical pair recombination between P+ and I− (P, the primary electron donor, is a dimer of bacteriochlorophyll; I, the primary electron acceptor, is a molecule of bacteriopheophytin). Its spectrum is nearly identical to that of the variable component. This emission seems to be present also under nonreducing conditions, although with a much weaker intensity than when the electron acceptor quinone is prereduced.

Antagonistic effect of mono- and divalent-cations on lifetime (τ) and quantum yield of fluorescence (ψ) in isolated chloroplasts

(maximum) levels [2] . The addition of MgCls (10 mM) at 298°K re-establishes the fluorescence intensity of 685 nm and 695 nm bands (F685 and F695, respectively) compared to the 735 nm band (F735) at 77°K. Attributing F685 and F695 to system II and F735 to System I (see ref. [4] ) it was concluded [l] that a modification in the distribution of the excitation energy occurs in favor of System I, when NaCl is added. On the other hand, the addition of MgCla leads to a decrease of energy received by System I and an increase in energy received by System II [5] . The cations are believed to modify the distribution of excitation energy between the two systems by modifying the ‘spill-over’ of this energy from System II to System I. The object of the experiments described here was to verify whether the variations in fluorescence intensity (fl induced by both the mono- and divalent- cations correspond with the modifications of the quantum yield of fluorescence as calculated from the lifetime measurements yield of fluorescence and TO = intrinsic lifetime fluorescence depending upon the characteristics of the absorption band. This information is essential to the understanding of the salt-effects in terms of the changes in the absorption cross-section or in the energy-transfer (see ref. [6]). A correspondence between the changes in