Global spectral–kinetic analysis of room temperature chlorophyll a fluorescence from light-harvesting antenna mutants of barley

Abstract

This study presents a novel measurement, and simulation, of the time-resolved room temperature chlorophyll a £uorescence emission spectra from leaves of the barley wild-type and chlorophyll-b-de¢cient chlorina (clo) f 2 and f104 mutants. The primary data were collected with a streak-camera-based picosecond-pulsed £uorometer that simultaneously records the spectral distribution and time dependence of the £uorescence decay. A new global spectral-kinetic analysis programme method, termed the double convolution integral (DCI) method, was developed to convolve the exciting laser pulse shape with a multimodal-distributed decay pro¢le function that is again convolved with the spectral emission band amplitude functions. We report several key results obtained by the simultaneous spectral-kinetic acquisition and DCI methods. First, under conditions of dark-level £uorescence, when photosystem II (PS II) photochemistry is at a maximum at room temperature, both the clo f 2 and clo f104 mutants exhibit very similar PS II spectral-decay contours as the wild-type (wt), with the main band centred around 685 nm. Second, dark-level £uorescence is strongly in£uenced beyond 700 nm by broad emission bands from PS I, and its associated antennae proteins, which exhibit much more rapid decay kinetics and strong integrated amplitudes. In particular a 705^720 nm band is present in all three samples, with a 710 nm band predominating in the clo f 2 leaves. When the PS II photochemistry becomes inhibited, maximizing the £uorescence yield, both the clo f104 mutant and the wt exhibit lifetime increases for their major distribution modes from the minimal 250^500 ps range to the maximal 1500^2500 ps range for both the 685 nm and 740 nm bands. The clo f 2 mutant, however, exhibits several unique spectral-kinetic properties, attributed to its unique PS I antennae and thylakoid structure, indicating changes in both PS II £uorescence reabsorption and PS II to PS I energy transfer pathways compared to the wt and clo f104. Photoprotective energy dissipation mediated by the xanthophyll cycle pigments and the PsbS protein was uninhibited in the clo f 104 mutant but, as commonly reported in the literature, signi¢cantly inhibited in the clo f 2; the inhibited energy dissipation is partly attributed to its thylakoid structure and PS II to PS I energy transfer properties. It is concluded that it is imperative with steady-state £uorometers, especially for in vivo studies of PS II e¤ciency or photoprotective energy dissipation, to quantify the in£uence of the PS I spectral emission.

The Zeaxanthin-Independent and Zeaxanthin-Dependent qE Components of Nonphotochemical Quenching Involve Common Conformational Changes within the Photosystem II Antenna in Arabidopsis

“…The data also suggest that qE is unlikely to arise solely in the minor monomeric antenna proteins of PSII, as proposed many times (Gilmore et al, 1998;Ahn et al, 2008). These conclusions are consistent with the finding that, while no individual Lhcb protein appears to be obligatory for qE, each one may play a role in shaping the PSII macrostructure for the optimal combination of light harvesting and photoprotection and with studies on the chlorophyll b-less mutants with severely reduced PSII antenna size that showed strong reductions in NPQ capacity (Härtel et al, 1996;Gilmore et al, 2000). The disruptive effect of the depletion of Lhcb proteins on the PSII macrostructure is well documented .…”

Analysis of the Chlorophyll a Fluorescence Transient

“…Today, global spectral and kinetic methods are used to separate the PS I and PS II emission bands in leaves (see Gilmore et al, 2000;Chapter 21, Gilmore). Lombard and Strasser (1984) established the following method to obtain information about the excitation rate of the two photosystems at room temperature.…”

(Sub)-Picosecond Spectral Evolution of Fluorescence Studied with a Synchroscan Streak-Camera System and Target Analysis

“…Such single-chromophore systems are generally easier to study than chlorophyll-containing photosynthetic complexes: in photosynthetic systems excitation-energy transfer between chromophores takes place, which in case of too high excitation energies can result in singlet-singlet annihilation, Chapter 12 Streak-Camera Fluorescence a process that can distort the fl uorescence kinetics (Sauer and Debreczeny, 1996). Gilmore et al (2000Gilmore et al ( , 2003a nicely demonstrated the application of the streak camera to obtain timeresolved fl uorescence spectra of leaves. Spectral and kinetic differences between Photosystems I and II could be discerned but relevant spectral evolution was only observed for times longer than 100 ps.…”