Giuseppe Zucchelli

The importance of PS I chlorophyll red forms in light-harvesting by leaves

We have investigated the importance of the long wavelength absorbing spectral forms (red forms) of Photosystem I in photosynthetic light harvesting by leaves. To this end leaf spectra were simulated by using a linear combination of absorption (OD) spectra of purified Photosystem I, Photosystem II and LHC II, multiplied by an empirical multiple scattering chloroplast/leaf conversion function. In this way it is demonstrated that while the PS I red forms account for only about 4–5% of light absorption in a normal ‘daylight’ environment, in different ‘shadelight’ environments these long wavelength pigments may be responsible for up to 40% of total photon capture. In the context of maximising the photosynthetic quantum efficiency under the low light conditions of ‘shadelight’, this relative increase in the absorption cross section of PS I can be understood by considering the increased synthesis of the major PS II antenna complex, LHC II, known to occur in plants growing under these light conditions. It is demonstrated that for plants in a moderate to deep ‘shadelight’ regime the PS II cross section needs to increase by 50% to 100% via LHC II synthesis to balance the increased PS I absorption by the red forms. The possibility that under ‘shade light’ conditions the increased PS I cross section may serve in cyclic phosphorylation is also discussed.

Involvement of uncoupled antenna chlorophylls in photoinhibition in thylakoids

Evidence is presented, by means of both fluorescence and action spectroscopy, that a small, spectroscopically heterogeneous population of both Chl a and Chl b molecules is present in isolated spinach thylakoids and is active in photoinhibition. The broadness of the action spectrum suggests that degraded or incompletely assembled pigment–protein complexes may be involved.

Light-induced fluorescence quenching in the light-harvesting chlorophyll a/b protein complex.

SummaryIrradiation of the principal photosystem II light-harvesting chlorophyll-protein antenna complex, LHC II, with high light intensities brings about a pronounced quenching of the chlorophyll fluorescence. Illumination of isolated thylakoids with high light intensities generates the formation of quenching centres within LHC II in vivo, as demonstrated by fluorescence excitation spectroscopy. In the isolated complex it is demonstrated that the light-induced fluorescence quenching: a) shows a partial, biphasic reversibility in the dark; b) is approximately proportional to the light intensity; c) is almost independent of temperature in the range 0–30°C; d) is substantially insensitive to protein modifying reagents and treatments; e) occurs in the absence of oxygen. A possible physiological importance of the phenomenon is discussed in terms of a mechanism capable of dissipating excess excitation energy within the photosystem II antenna.

The presence of long-wavelength chlorophyll a spectral forms in the light-harvesting chlorophyll a/b protein complex II

Abstract Room temperature absorption spectra (in the wavelength range 600–740 nm) of light-harvesting chlorophyll a/b protein complex II (LHCPII) isolated from spinach and pea have been analysed in terms of a linear combination of asymmetric gaussian bands. All the commonly observed chlorophyll spectra bands are found, including significant levels of the components which peak around 684 and 693 nm. The presence of these long-wavelength absorbing forms in LHCPII is also suggested by analysis of the room temperature absorption spectra of thylakoids of the chlorina barley mutant, which lacks LHCPII, and pea grown in intermittent light. From a comparative analysis of room temperature and liquid nitrogen temperature absorption spectra of LHCPII, it is concluded that temperature modifies the spectral bands, narrowing the bands up to 677 nm and markedly depressing the 684 nm band. These observations are not in close agreement with the commonly accepted ideas on the distribution of the chlorophyll spectral species with respect to reaction centres in the photosystem II (PSII) antenna matrix.

Photoinhibition in vivo and in vitro Involves Weakly Coupled Chlorophyll–Protein Complexes‡,¶

Abstract In the present study the analysis of the relation between the excited state population in the photosystem II (PSII) antenna and photoinactivation has been extended from an in vitro system, isolated thylakoids, to an in vivo system, Chlamydomonas reinhardtii cells. The results indicate that the excited state quenching by an added singlet quencher induces maximal protection against photoinhibition of about 30% of that expected on the basis of the observed light intensity–treatment time reciprocity rule. Similar results, obtained previously with thylakoids, have been interpreted in terms of damaged or incorrectly assembled complexes that play an important role in photoinhibition in the thylakoid membranes (Santabarbara, S., K. Neverov, F. M. Garlaschi, G. Zucchelli and R. C. Jennings [2001] Involvement of uncoupled antenna chlorophylls in photoinhibition in thylakoids. FEBS Lett. 491, 109–113.). In an attempt to better define this aspect, the photoinhibition action spectra were determined for mutant barley thylakoids, lacking the chlorophyll (Chl) a–b complexes of the outer antenna, and for its wild type. The results indicate that in both systems the action spectra are significantly blueshifted (2–4 nm) and are broader than the PSII absorption in the membranes. These data are interpreted in terms of a heterogeneous population of outer and inner antenna pigment–protein complexes that contain significant levels of uncoupled Chl.

The Calculated In Vitro and In Vivo Chlorophyll a Absorption Bandshape

The room temperature absorption bandshape for the Q transition region of chlorophyll a is calculated using the vibrational frequency modes and Franck-Condon (FC) factors obtained by line-narrowing spectroscopies of chlorophyll a in a glassy (Rebane and Avarmaa, Chem. Phys. 1982; 68:191-200) and in a native environment (Gillie et al., J. Phys. Chem. 1989; 93:1620-1627) at low temperatures. The calculated bandshapes are compared with the absorption spectra of chlorophyll a measured in two different solvents and with that obtained in vivo by a mutational analysis of a chlorophyll-protein complex. It is demonstrated that the measured distributions of FC factors can account for the absorption bandshape of chlorophyll a in a hexacoordinated state, whereas, when pentacoordinated, reduced FC coupling for vibrational frequencies in the range 540-850 cm(-1) occurs. The FC factor distribution for pentacoordinated chlorophyll also describes the native chlorophyll a spectrum but, in this case, either a low-frequency mode (nu < 200 cm(-1)) must be added or else the 262-cm(-1) mode must increase in coupling by about one order of magnitude to describe the skewness of the main absorption bandshape.

Kinetic Analysis of the Light‐induced Fluorescence Quenching in Light‐harvesting Chlorophyll a/b Pigment‐Protein Complex of Photosystem II

We carried out a kinetic analysis of the light‐induced fluorescence quenching (AF) of the light‐harvesting chlorophyll a/b pigment‐protein complex of photosystem II (LHCII) that was first observed by Jennings et at (Pho‐tosynth. Res. 27, 57–64, 1991). We show that during a 2 min light, 2 min dark cycle, both the light and dark phases exhibit biexponential kinetics; this is tentatively explained by the presence of two types of light‐induced quenchers in different domains of aggregated LHCII. Quantitative analysis could be carried out on the faster kinetic component; the slower component that was not completed during the measurement was not amenable for quantitative analysis. Our analysis revealed that the rate of the light‐induced decrease of the fluorescence yield depended linearly on the light intensity, which shows that the generation of the quencher originates from a reaction that is first order with respect to the concentration of the excited domains. As shown by the estimated rate constant, pho‐togeneration of the quencher is a fast reaction that can compete with other excitation‐relaxation pathways. Both the decay and the recovery time constants of AF depended strongly on the temperature. Thermodynamic analysis showed that the fast light‐induced decline in the fluorescence was determined by a low fraction of the excited states. Recovery was associated with large decrease in the entropy of activation that indicated the involvement of large structural rearrangements. Macroaggregated LHCII exhibited larger ΔF than small aggregates, which is consistent with the proposed role of aggregated LHCII in thy‐lakoid membranes in nonphotochemical quenching.

CD spectroscopy provides evidence for excitonic interactions involving red-shifted chlorophyll forms in photosystem I

Selective destruction of the strongly dichroic red‐shifted chlorophyll form (C709 nm) in photosystem I (PSI) trimers from Spirulina, by either non‐selective high intensity illumination (photobleaching) or incubation with low concentrations of Triton X‐100 is accompanied by changes in the circular dichroism spectrum of the same amplitude and of opposite sign at 677 nm. The data are interpreted in terms of a dimeric chlorophyll structure with excitonic bands at these two wavelengths. Similar photobleaching experiments with PSI‐200 from maize also suggest the presence of bulk antenna/red form excitonic interactions.

Independent fluorescence emission of the chlorophyll spectral forms in higher plant Photosystem II

Room-temperaure steady-state emission and Q1 absorption spectra of light-harvesting chlorophyll ab protein complex II (LHCII) isolated from spinach have been analysed in terms of a linear combination of asymmetric gaussian bands. To investigate a possible correspondence between the absorption and emission bands, the thermal emission spectrum of each absorption band has been calculated. It is demonstrated that the calculated fluorescence bands correspond closely to those obtained by gaussian deconvolution. It is thus possible to associate an emission band with each absorbing chlorophyll spectral species: Chl653648, Chl663660, Chl672670, Chl680678, Chl687684, Chl697695 (Chlmn is the spectral species that absorbs with maximum at wavelength n and emits with maximum at wavelength m) and to interpret the emission spectrum of LHCII as a linear combination of the emission spectra of each absorbing chlorophyll spectral species. In particular, the correspondence between the 684 nm and 695 nm absorption bands and the emission bands with maximum at 687 nm and 697 nm lends support to the presence of long wavelength Chl a spectra forms in LCHII, the external antenna of PS II. A close correlation between the emission and absorption gaussian bands is also found in the analysis of the room temperature absorption and emission spectra of such membrane preparation as BBY-grana from spinach and thylakoids prepared from barley wild type and the chlorina f2 mutant (lacking LHCII). On the basis of these data, the commonly observed 5–7 nm wavelength difference between the absorption and emission spectra maxima of chlorophyll containing biological membranes is interpreted in terms of (a) the Stokes shifts of the single spectral forms together with (b) the increased emission contribution of the longer wavelength forms to the emission spectrum caused by energy transfer.

A study of Photosystem II fluorescence emission in terms of the antenna chlorophyll-protein complexes

Abstract Fluorescence emission spectra for the seven chlorophyll-protein complexes comprising the antenna system of Photosystem II (PS II) have been measured. All four outer antenna complexes (LHC II, CP24, CP26, CP29) have relatively greater emission near 648 nm and 680 nm with respect to the inner antenna complexes (CP43, CP47, D1/D2/cyt b-559). The emission spectra for both outer and inner antenna were calculated from the measured emission spectra of the single chlorophyll-protein complexes, using as weighting factors the excited state population at thermodynamic equilibrium in the various chlorophyll-protein complexes suggested by Jennings et al. (Jennings, R.C., Bassi, R., Garlaschi, F.M., Dainese, P. and Zucchelli, G. (1993) Biochemistry 32, 3203–3210). Subsequently, the overall emission spectra for the total PS II antenna (i.e., outer plus inner antenna) were calculated for situations in which varying excited state levels were assumed for the inner and outer antenna. In an attempt to determine the steady-state distribution of excited states between outer and inner antenna these calculated fluorescence spectra were compared with those measured for (a), PS II particles prepared from maize and (b), chloroplasts of wild-type barley and the chlorina F2 mutant. From this comparison it is concluded that at steady-state fluorescence emission, between 28% and 38% of the excited states in PS II are associated with the inner antenna and between 62% and 72% with the outer antenna. These results suggest that the PS II antenna is organised as a very shallow energy funnel. This antenna organisation is discussed in terms of the generation of non-photochemical quenching mechanisms which are designed to protect PS II from high light stress.