Fernanda Rigoni

CHLOROPHYLL BINDING PROTEINS WITH ANTENNA FUNCTION IN HIGHER PLANTS and GREEN ALGAE

The absorption of light by antenna pigments creates mobile singlet state excitons which may migrate within the complex array of chlorophylls to either Photosystem I (PSI)* or PSII reaction centers. When an exciton reaches the reaction center, its energy is converted into charge separation, the exciton disappears, and a chain of redox reactions is triggered by which radiant energy is transduced. Excitons reaching the reaction centers are said to be trapped if charge separation occurs. Alternatively, they may return to the antenna pigments. During migration of excitons in the antenna matrix, the probability that they may decay as fluorescence quanta or by means of other non-radiative decay processes is finite. Owing to the high efficiency by which light is converted in the photosynthetic apparatus, energy losses are small and very little energy (approx. 2%) is emitted as fluorescence. As the purpose of photosynthesis is to convert excited state energy into chemical free energy with maximum efficiency, it might seem that any light energy emitted as fluorescence from the photosynthetic apparatus is “wasted”. However, at the present state of knowledge it cannot be excluded that fluorescence emission, at least in part, is the result of molecular control of the amount of energy reaching the reaction centers in order to avoid damage due to overexcitation and photoinhibition. In any case, the small fraction of energy emitted as fluorescence is of great importance in the investigation of excitation energy distribution and exciton migration among chlorophyll-protein complexes. The proportion of energy which is not utilized in photosynthesis and is emitted as fluorescence varies, depending mark-

Light- and pH-dependent structural changes in the PsbS subunit of photosystem II

In higher plants, the PsbS subunit of photosystem II (PSII) plays a crucial role in pH- and xanthophyll-dependent nonphotochemical quenching of excess absorbed light energy, thus contributing to the defense mechanism against photoinhibition. We determined the amino acid sequence of Zea mays PsbS and produced an antibody that recognizes with high specificity a region of the protein located in the stroma-exposed loop between the second and third putative helices. By means of this antiserum, the thylakoid membranes of various higher plant species revealed the presence of a 42-kDa protein band, indicating the formation of a dimer of the 21-kDa PsbS protein. Crosslinking experiments and immunoblotting with other antisera seem to exclude the formation of a heterodimer with other PSII protein components. The PsbS monomer/dimer ratio in isolated thylakoid membranes was found to vary with luminal pH in a reversible manner, the monomer being the prevalent form at acidic and the dimer at alkaline pH. In intact chloroplasts and whole plants, dimer-to-monomer conversion is reversibly induced by light, known to cause luminal acidification. Sucrose-gradient centrifugation revealed a prevalent association of the PsbS monomer and dimer with light-harvesting complex and PSII core complexes, respectively. The finding of the existence of a light-induced change in the quaternary structure of the PsbS subunit may contribute to understanding the mechanism of PsbS action during nonphotochemical quenching.

Light-harvesting chlorophyll a/b proteins (LHCII) populations in phosphorylated membranes

The properties of the light-harvesting chlorophyll a/b-protein complex of Photosystem II (LHCII) have been analyzed, in thylakoids and PS II membrane after phosphorylation. Using a newly developed fractionation method, by flat-bed electrofocusing in granulated gel, seven Chl a/b proteins have been separated from thylakoids. Their phosphorylation level and polypeptide composition have been evaluated. PhosphoLHCII represent only 30% of the total Chl a/b proteins in thylakoids and the ‘mobile’ fraction binds 20% of the total LHCII chlorophyll. Tightly bound LHCII differs from the mobile fraction for the presence of a 26 kDa polypeptide characterized by the absence of the N-terminal LHCII proteolytic fragment which is phosphorylated during state transition.

Characterization of a 41 kDa photoinhibition adduct in isolated photosystem II reaction centres

When isolated reaction centres of photosystem II are subjected to photoinhibitory illumination, a 41 kDa SDS‐PAGE band is observed under all experimental conditions. The same band is also found, together will lower molecular weight fragments of the D1 protein, in whole thylakoids and in all PSII sub‐particles investigated up to now. In the case of isolated reaction centres the 41 kDa band is represented by a heterodimer of the D1 polypeptide and the α‐subunit of cytochrome b 559. The cross‐linkage between D1 and α‐cyl b 559 involves a region on D1 between the N‐terminal residue and Arg·225, and is an early event in photo‐induced damage to the D1 protein.

The minor antenna complexes of an oxygen evolving photosystem II preparation: purification and stoichiometry

A photosystem II complex obtained by partial solubilization of spinach PS II membranes has been analyzed in terms of chlorophyll‐protein complexes. The complex was found to contain the minor antennas CP29 and CP26 and a very small amount of LHC II (less than 3%). No trace of CP24 was found to be associated with the PS II complex. This chlorophyll a/b‐protein was instead found in the fraction containing the main light‐harvesting complex LHC II. The stoichiometry of the minor antennas indicates the values 3:3:1 for the ratios CP29:CP26:P680.

Light-induced degradation of D2 protein in isolated photosystem II reaction center complex.

When isolated photosystem II reaction centers from spinach are exposed to photoinhibitory light in the presence of an electron acceptor, breakdown products of the D2 protein at 28, 25, 23, 18, 9, 5 and 4.5 kDa are detected by immunoblotting with a monospecific anti‐D2 polyclonal antibody. In a time—course experiment the 23 and 4.5 kDa fragments show a transient appearance, whilst the others are photoaccumulated. The regions of the D2 protein containing the cleavage sites for the 28 and 18 kDa photoinduced fragments have been identified. Significant degradation of D2 takes place only in the presence of an electron acceptor, and breakdown of the protein is partially prevented by serine‐type protease inhibitors.

Photoinduced degradation of the D1 protein in isolated thylakoids and various photosystem II particles after donor-side inactivations Detection of a C-terminal 16 kDa fragment

Photoinduced degradation of the photosystem II (PSII) reaction center D1 protein was studied in isolated thylakoids and different PSII subparticles. A 16 kDa fragment corresponding to the C‐terminus of the protein is detected in thylakoids when they are inactivated at the donor side before illumination. The same D1 fragment is found in different types of PSII preparations at different integration levels characterized by different polypeptide compositions so long as they have an inactivated donor side and an active electron acceptor for the reduced pheophytin. However, when the PSII particle is equal to or smaller than the 43‐less PSII core complex, other fragments are observed which are not found in more integrated systems.

Cytochrome b6/f complex from the cyanobacterium Synechocystis 6803: evidence of dimeric organization and identification of chlorophyll-binding subunit.

Fractionation of photosynthetic membranes from the cyanobacterium Synechocystis 6803 by polyacrylamide gel electrophoresis in the presence of Deriphat‐160 allowed the isolation of a number of pigmented bands. Two of them, with molecular masses of 240±20 and 110±15 kDa respectively, showed peroxidase activity and, by means of polypeptide composition, immunoblotting and N‐terminal sequencing, were identified as dimeric and monomeric cytochrome b 6/f complexes, containing 1.3±0.35 chlorophyll molecules per cytochrome f. Further fractionation of monomeric complexes by mild gel electrophoresis in the presence of sodium dodecyl sulfate indicated that it is the cytochrome b 6 polypeptide which provides the actual binding site for the chlorophyll molecule observed in the complex.

Pseudopleurochloris antarctica gen. et sp. nov., a new coccoid xanthophycean from pack-ice of Wood Bay (Ross Sea, Antarctica): ultrastructure, pigments and 18S rRNA gene sequence

A microalga was isolated from the pack-ice of Wood Bay (Antarctica) during austral summer 1993–4. By comparison with an authentic strain obtained from Sammlung von Algenkulturen at the University of Go

Ruthenium red inhibits the mitochondrial Ca2+ uptake in intact bovine spermatozoa and increases the cytosolic Ca2+ concentration

ttingen (SAG 860–3), its ultrastructural details and pigment composition were very similar to those of Pleurochloris meiringensis. However, sequence analysis of the entire 18S rRNA gene revealed substantial genetic divergence (57 nucleotide substitutions and 19 insertions/deletions) between SAG 860–3 and the Antarctic isolate. Phylogenetic analysis of complete 18S rRNA gene sequences from 18 microalgal species, including the two strains studied, rejected the hypothesis that the Antarctic isolate is the sister species to Pleurochloris meiringensis. Molecular evidence indicates that the Antarctic isolate is the sister group of a clade that comprises two other representatives of the Xanthophyceae (synonym Tribophyceae): Pleurochloris meiringensis and Botrydiopsis intercedens. This leads us to conclude that th...