The distribution of the photosynthetic pigments of the chlorophyll-binding proteins or photosystem-II membranes, isolated from dark-adapted maize leaves was determined. Most (80%) of a xanthophyll, violaxanthin, was found in the three minor chlorophyll-ulb proteins CP24, CP26 and CP29 whose function is unknown. Violaxanthin is the precursor of zeaxanthin, which is involved in dissipating excess excitation energy into heat [Demmig-Adams, B. (1991) . We propose that a role for the minor photosystem-II chlorophyll-ulb proteins is the regulation of energy transfer to the reaction centre. It was also confirmed that the photosystem I1 reaction centre (Dl-D2-~ytochrome b5.59) contains pcarotene as the only carotenoid. However, the two other chlorophyll-a-binding proteins of photosystem 11, CP47 and CP43, bind not only p-carotene, but also the xanthophyll lutein, previously thought to be restricted to chlorophyll-ulb proteins.In green plants, light harvesting and photosynthetic electron transport occur in the thylakoids, which are the predominant membranes in plant leaf cells representing up to 80% of the total membrane [l] in a mesophyll cell. Thylakoids are organized into two major functional regions : the appressed or grana membranes and the non-appressed or stroma membranes, which contain different electron-transport components and pigment-binding complexes participating in the photosynthetic process.In both membrane domains, the photosynthetic pigments have four functions : light harvesting, energy transfer, energy dissipation and charge separation [2-41. Charge separation is restricted to the primary chromophore chlorophyll a, while photoprotection through energy dissipation is a carotenoid function, owing to the low energy levels of their triplet states with respect to those of chlorophyll. The role of the pigments in each of the functions listed above depends on the different features of their molecular structures. For example, energy transfer from carotenoids to chlorophylls occurs through singlet-singlet excitation-energy transfer while triplet-triplet interactions are involved in photoprotection. The characteristics of the energetic transitions involved are determined by the covalent molecular structure of the pigments and by their interactions with the polypeptides to which they are bound. The latter can be regarded as a fine tuning process to modify their function. The properties of the photosynthetic pigments in vivo are different from those in solution, the absorbance Abbreviations. PSII core, chlorophyll a complex containing D1, D2, cytochrome b559, CP43, CP47 ; Cab, chlorophyll-u/b proteins; CP, chlorophyll-protein complex; PSII, photosystem I1 ; LHCII, the major light-harvesting complex of PSII; 0. E. E., oxygen evolving enhancer.ste 75, 1-35121 Padova, Italy maxima being red-shifted up to about 30 nm, probably due to the influence of charged residues in the polypeptide(s) [5]; however, shifts due to a transmembrane electric field caused by a pH gradient have also been reported, accounting for a poss...

Although the changes in organization of the lightharvesting antenna upon state transitions are well documented, possible changes in the organization of the photosynthetic electron transfer chain have not been directly investigated. Cytochrome b6/f (cyt b6/f), a major protein complex of this electron-transfer chain, has, however, been implicated in state transitions through its role in LHCIIkinase activation. State transitions are abolished in cyt b6/f mutants of green algae and higher plants due to the absence of LHCII reversible phosphorylation (4-8). Gal et al. (9) recently reported that the LHCII-kinase was, indeed, associated with cyt b6/f complexes.Whereas the PSII and PSI centers are well separated between the stacked and unstacked regions of the thylakoid membranes, cyt b6/f complexes are found in significant amounts in both membrane domains (10-13). The identity of the long-distance carrier between PSIl in the grana regions and PSI in the SL regions has been a matter of debate (14). It has been recently argued that the rapid diffusion ofplastoquinones, which transfer electrons between PSII and cyt b6/f complexes, is limited to small domains containing less than eight PSII centers (15,16). Therefore linear electron flow should be sustained by plastocyanin diffusing in the luminal space from its binding site on cyt b6df complexes in the stacked regions to PSI in the unstacked regions. The fraction of cyt b6/f complexes located in the unstacked regions next to PSI would then serve cyclic electron flow around PSI.There is a growing body of evidence that the ATP requirement of the photosynthetic cell controls state transitions (17)(18)(19) 8262The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

In this study, we report on the composition of a photosystem-I1 antenna preparation which contains three chlorophyll-a/b proteins (CP), CP29, CP24 and light-harvesting complex (LHC) 11 obtained from Zea mays grana membranes as previously described [Dainese, P. & Bassi, R. (1991) J .Biol. Chem. 266, 8136-81421. We demonstrate that the three chlorophyll proteins are present in the preparation with a 3 : 3 : 9 molar ratio and that they form a supramolecular antenna complex which represents one third of the photosystem-I1 antenna system.Phosphorylation experiments show that this complex is involved in the mechanism of regulation of excitation-energy distribution between photosystems : phosphorylation of the membranes induces dissociation of the LHCII moiety from the CP29-CP24 moiety and changes in the aggregation state of LHCII components of the CP29-CP24-LHCII complex. The LHCII subpopulations of the complex are shown to be distinct from the total LHCII population by isoelectrofocusing analysis. On the basis of these data and in the light of the stoichiometry of photosystem-I1 chlorophyll-binding proteins, we propose a model for the organization of photosystem-I1 antenna system.The light reactions of photosynthesis are driven by excitation energy absorbed by the antenna pigments and transferred to the reaction centres. Although the primary chromophore responsible for light absorption in hgher plants and algae is chlorophyll a, accessory pigments such as chlorophyll b and carotenoids extend the spectral range of light absorption and transfer energy to chlorophyll a. In higher plants, as many as 21 0 molecules of chlorophyll are associated with photosystem (PS) I [l] and 230 with PSII [2]. However, little information is available on the organization of the chlorophyll proteins (CP) of reaction centres and antenna complexes to form photosynthetic units.PSII is composed of an inner complex which contains four or five chlorophyll-a molecules, including the photoactive pigments, bound to D1 and D2, cytochrome b559 proteins [3] and of two chlorophyll-a-binding proteins, CP47 and CP43, each binding 25 chlorophyll-a molecules [4] which have an inner antenna function (for a review see [5]). These five proteins are coded by the chloroplast genome and constitute the PSI1 core complex. The remaining 150 chlorophyll molecules are bound to a number of chlorophyll-alb-binding proteins coded by an extended multigene family in the nuclear genome [6,7]. Three of these, called CP29, CP26 and CP24, are present in small amounts in PSII membranes, while a fourth component, the major light-harvesting complex (LHC) 11, constitutes 50% of the thylakoid protein content (for a review see [S] The PSII antenna system is a highly complex structure, capable of adapting to different environmental conditions, such as state-I/state-I1 transitions [ 131, heat stress [14] and cold stress [15]. The understanding of the molecular mechanisms underlying these physiological adaptations requires more information about the composition and supramolecular o...

The resistance of maize plants to cold stress has been associated with the appearance of a new chlorophyll a/b binding protein in the thylakoid membrane following chilling treatment in the light. The cold-induced protein has been isolated, characterized by amino acid sequencing, and pulse labeled with radioactive precursors, showing that it is the product of post-translational modification by phosphorylation of the minor chlorophyll a/b protein CP29 rather than the product of a cold-regulated gene or an unprocessed CP29 precursor. We show here that the CP29 kinase activity displays unique characteristics differing from previously described thylakoid kinases and is regulated by the redox state of a quinonic site. Finally, we show that maize plants unable to perform phosphorylation have enhanced sensitivity to cold-induced photoinhibition.

The chlorophyll-protein complexes that form the antenna system of photosystem II have been purified and analyzed in terms of the commonly observed chlorophyll spectral forms. With the exception of chlorophyll b, which is known to be associated with the complexes comprising the outer antenna (LHCII, CP24, CP26, CP29), the spectral forms occur with similar absorption maxima and are present in rather similar amounts in each of the antenna complexes. On the basis of the published chlorophyll stoichiometries for the complexes in photosystem II antenna, the distribution of the spectral forms in a "reconstituted" antenna has been determined. These data were used to calculate the equilibrium population of excited states within the various chlorophyll-protein complexes within photosystem II. This was compared with the light absorption capacity of each of the complexes in the "reconstituted" antenna. The ratio of these two parameters (excited-state equilibrium distribution/absorption capacity) was determined to be 1.21 for the inner (core) antenna and 0.88 for LHCII. The standard free energy change for exciton transfer from the outer to the inner antenna was calculated to be -0.17 kcal mol-1. It is concluded that the photosystem II antenna is arranged as a very shallow funnel.

We describe a new fractionation method for intrinsic membrane proteins based on flat bed isoelectric focusing (IEF) in granulated gel. The characteristics of the separation in the presence of the non-ionic detergent dodecylmaltoside are considered. The method has been applied to the fractionation of chlorophyll a/b binding proteins from chloroplast grana membranes. Several Light Harvesting Complexes II (LHC II) have been resolved showing differences in their polypeptide composition. Probing with monoclonal and polyclonal antibodies showed that polypeptides belonging to different [EF fractions with the same mobility in denaturing sodium dodecyl sulphate polyacrylamide gel electrophoresis, are immunologically distinct polypeptides. This is the first report of the presence in the thylakoid membrane of a number of LHCII polypeptides that may reflect the genetic complexity of the Cab genes. Moreover preparative amounts have been obtained of the minor chlorophyll a/b proteins CP 29, CP 26 and CP 24 that have been recently described. The analysis of a currently used LHCII preparation by the present method shows that this fraction is actually contaminated by two minor chlorophyll a/b proteins.

Room temperature and 10 K absorption and linear dichroism spectra of the chlorophyll-protein complexes comprising the outer antenna of PSII (LHCII, CP29, CP26, CP24) have been analyzed in terms of a linear combination of asymmetric Gaussian bands. The results demonstrate the following: (a) The absorption and linear dichroism spectra of each sample can be described by nearly the same set of Gaussian bands at room temperature and 10 K. (b) The relative distributions of the transition moments of the major red-absorbing spectral forms seem to be similar in all four outer antenna chlorophyll-protein complexes at room temperature, with the 684-nm band being oriented closest to the particle plane at room temperature and the 677- and 669-nm bands being tilted at progressively greater angles out of the particle plane. The shorter wavelength transitions seem to be oriented close to the magic angle, but interpretation is complicated in this spectral region due to the low linear dichroism values and by overlap with vibrational bands. (c) The 684-nm band, detected in room temperature absorption and linear dichroism spectra of all complexes, vanishes at 10 K.

Myofibrillar proteins (MPs) were extracted from isolated and perfused rat hearts subjected to different periods of ischemia to investigate the occurrence of protein degradation and/or the association of cytosolic proteins with the myofibrillar pellet. A 23-kD band was detected by SDS-PAGE of MPs after 5 minutes of ischemia, with its density gradually increasing to a plateau after 20 minutes. Longer periods of ischemia were associated with the appearance of a 39-kD band. Irrespective of the duration of ischemia, both these bands persisted during reperfusion. A partial proteolytic degradation of troponin T (TnT) and troponin I (TnI) has been claimed to be responsible for the generation of these peptides. However, the N-terminal sequence of the 39-kD band was identical to that of GAPDH, whereas Edman sequencing after pepsin digestion showed that the 23 kD is alpha B-crystallin. The binding of the two cytosolic proteins to myofibrils was confirmed by immunofluorescence analysis on cryosections of ischemic hearts. In vitro studies showed that acidosis was sufficient to induce the binding of alpha B-crystallin, whereas the inhibition of ATP depletion prevented the binding of GAPDH. Thiol oxidation is unlikely to promote GAPDH binding, since perfusion with iodoacetate under aerobic conditions or treatment of homogenates with N-ethylmaleimide or diamide failed to induce GAPDH association with the myofibrils. These changes of the myofibrillar proteins could be considered as intracellular markers of the evolution of the ischemic damage. In addition, the binding of the 23-kD peptide might be involved in alterations of contractility.