Paola Dainese

The resolution of chlorophyll a/b binding proteins by a preparative method based on flat bed isoelectric focusing

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.

Cold-Resistant and Cold-Sensitive Maize Lines Differ in the Phosphorylation of the Photosystem II Subunit, CP29

The effects of low temperature on the relative contributions of the reaction center and the antenna activities to photosystem II (PSII) electron transport were estimated by chlorophyll fluorescence. The inhibition of PSII photochemistry resulted from photo-damage to the reaction center and/or a reduced probability of excitation energy trapping by the reaction center. Although chill treatment did not modify the proportion of the dimeric to monomeric PSII, it destabilized its main light-harvesting complex. Full protection of the reaction center was achieved only in the presence of the phosphorylated PSII subunit, CP29. In a nonphosphorylating genotype the chill treatment led to photoinhibitory damage. The phosphorylation of CP29 modified neither its binding to the PSII core nor its pigment content. Phosphorylated CP29 was isolated by flat-bed isoelectric focusing. Its spectral characteristics indicated a depletion of the chlorophyll spectral forms with the highest excitation transfer efficiency to the reaction center. It is suggested that phosphorylated CP29 performs its regulatory function by an yet undescribed mechanism based on a shift of the equilibrium for the excitation energy toward the antenna.

Effects of a non-ionic detergent on the spectral properties and aggregation state of the light-harvesting chlorophyll a/b protein complex (LHCII)

Abstract The dependence of the spectroscopic properties and aggregation state of a highly purified light-harvesting chlorophyll a/b protein complex (LHCII) population on detergent (octyl-β- d -glucopyranoside (OGP)) concentration in the solvent was investigated. The results, including circular dichroism, time-resolved fluorescence, sucrose gradient centrifugation and isoelectrofocusing, indicate that, depending on the detergent concentration, LHCII can be found in two main quaternary states whose minimal order is three. Thus, at high OGP concentrations (3% or greater), a trimer, which is identical with the trimeric form observed in two-dimensional crystals, is stable. At OGP concentrations lower than approximately 0.65%, macroscopic aggregates are formed, accompanied by the appearance of strong pigment—pigment interactions and fluorescence quenching. At intermediate OGP concentrations (0.650%–2%) an oligomeric form of LHCII, possibly a multiple of the trimer, is stable and is similar to the so-called CPII′ oligomeric form resolved by non-denaturing sodium dodecylsulphate polyacrylamide gel electrophoresis (SDS-PAGE).

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.

Biochemistry and Molecular Biology of Pigment Binding Proteins

Chlorophylls and carotenoids are components of every pigment-protein complex in the photosynthetic apparatus of higher plants. They function in light harvesting, photoprotection, and down-regulation of PSII photochemistry. In the following we summarize the present knowledge on the localization and function of chlorophyll a, chlorophyll b and carotenoids within the photosynthetic apparatus of higher plants. This subject was previously reviewed (Bassi et al. 1990, Jansson 1994, Jennings et al. 1995)

The Role of Light Harvesting Complex II and of the Minor Chlorophyll a/b Proteins in the Organization of the Photosystem II Antenna System

The pigment portion of Photosystem (PS)I and II in green plants is composed of a chlorophyll(chl) a containing core complex and of a light harvesting antenna system containing both chl a and chl b.

Reorganization of Thylakoid Membrane Lateral Heterogeneity Following State I — State II Transition

The thylakoid membrane of higher plant chloroplast is organized into two main compartments: the grana and the stroma exposed membranes. This ultrastructural evidence corresponds to a compartmentation of the light harvesting and the electron transport components which are each localized in the granal or in the stroma exposed membranes. Thus Photosystem I (PSI) has been located in stromal membranes together with its light harvesting complex (LHCI) and the ATPase complex, while PSII and its major antenna LHCII has been shown to be present in the partition of grana stacks (1). The last major thylakoid complex, the cytochrome b6/f (cyt b6/f) has been found to be present in both grana and stroma membranes (2), so that it has been proposed that it is mainly placed in the frets, membranes connecting stroma lamellae to grana (3).