Shi-Gui Yu

Isolation and characterization of vesicles originating from the chloroplast grana margins

Abstract A membrane fraction composed of right-side-out vesicles and deriving from the periphery of the granal stacked region of spinach thylakoid membranes has been isolated by means of sonication and separation in an aqueous two-phase system, and its biochemical and photochemical properties examined. This vesicle population, referred to as the chloroplast granal margins, is enriched in PSI as compared to the central core of the grana, has a Chl a b ratio of between 3.0 and 3.3, and on a per chlorophyll basis contains the least amount of cytochrome f as compared to both the central core of the grana and the stroma lamellae domains. Notably, however, the margins represent the region of the thylakoid most enriched in the 64 kDa kinase. P700 and room temperature fluorescence induction kinetics show that the granal margins contain PS I with a larger antenna size than the PS I from the stroma lamellae and that the PS II of the margins is, with respect to the antenna size, more like the PS II from the stroma lamellae (i.e., PS IIβ). The margin vesicles also contain the ATPase complex as do the other stroma exposed areas of the thylakoid.

Composition of photosynthetic pigments in thylakoid membrane vesicles from spinach

Thylakoid membranes from spinach were fragmented mechanically and separated into vesicles originating from grana and stroma-exposed lamellae (Andreasson et al. (1988) Biochim Biophys Acta 936: 339–350). The grana vesicles were further fragmented and separated into smaller vesicles originating from different parts of the grana (Svensson and Albertsson (1989) Photosynth Res 20: 249–259). All vesicles so obtained were analyzed with respect to chlorophyll and carotenoid composition by reverse phase HPLC. For all fractions the following relations (mole/mole) were found: 1 carotenoid per 4 chlorophyll (a+b), 2 lutein per 5 chlorophyll b and 5 violaxanthin per 100 chlorophyll (a + b). The contents of lutein and neoxanthin were each linearly related to chlorophyll b and β-carotene was linearly related to chlorophyll a.

Heterogeneity among photosystem IIα. Isolation of thylakoid membrane vesicles with different functional antennae size of photosystem IIα

Abstract The functional antenna size of different chloroplast membrane vesicles was determined from ligh-saturation curves of the oxygen evolution using paraphenylbenzoquinone as acceptor. Thylakoids showed a biphasic light-saturation curve indicative of the presence of at least two types of PS II unit, PS II α and PS II β . Inside-out thylakoids were isolated by press treatment and phase partition. These vesicles, which contain only PS II α , were then further fragmented and separated into different classes of vesicles by a sonication-phase partition procedure (Svensson, P. and Albertsson, P.-A. (1987) in Progress in Photosynthesis Research (Biggins, J., ed.), Vol. II, pp. 281–284, Martinus Nijhoff, Dordrecht). Three fractions enriched in PS II α were obtained; these differ in the antenna size of PS II α by up to 20%. It is suggested that the size of the light-harvesting complex of PS II α from different PS II α units changes in increments of about 25–50 chlorophyll molecules depending on localization of the units in the grana or different parts of the leaf.

Localization of cytochrome f in the thylakoid membrane: evidence for multiple domains

Abstract Thylakoids from spinach were fragmented by sonication and the viscles so obtained were separated into different populations by aqueous two-phase partitioning using the dextran-poly(ethylene glycol) system. The different vesicle populations were analyzed with respect to the concentrations of P700 and cytochrome. The P700 content varied between 0.93 (PS-II-enriched grana vesicles named BS) and 4.85 (stroma membrane vesicles named Y100) mmol per mol chlorophyll. The cytochrome f content varied between 1.92 (vesicles originating from the grana periphery, named 120S vesicles) and 3.12 (PS-II-enriched grana vesicles named BS) mmol/mol chlorophyll. A plot of the P700 content against the cytochrome f content of the different vesicle populations was compared with hypothetical models of membrane vesicles. The results show that the thylakoid membrane consists of at least three different domains with respect to cytochrome f. These are suggested to be: (1) the stroma lamellae; (2) the core of the partition region of the grana; and (3) a peripheral annulus of the grana discs including the margins (and perhaps also the end membranes).

Heterogeneity in photosystem IIα. Evidence from fluorescence and gel electrophoresis experiments

Abstract The antenna size of different thylakoid membrane vesicles originating from grana partitions and enriched in PS IIα was determined by measuring the kinetics of the variable fluorescence and by gel electrophoresis of pigment-protein complexes. Inside-out thylakoid vesicles were fractionated by sonication and phase partition and separated into different subpopulations of PS IIα vesicles. The kinetics of the rise in the variable fluorescence of these subpopulations differed, demonstrating a heterogeneity in functional antenna size among the PS IIα. Analysis by gel electrophoresis showed that the fractions which have a more rapid rise in variable fluorescence, and hence a larger functional antenna size, also had a larger ratio of LHCII/PS II core polypeptides. The relative content of the 25 kDa polypeptide of LHCII increased with antenna size. There is thus a heterogeneity both with respect to the quantitative and the qualitative composition of the antenna of PS IIα. The results suggest that in vivo there are different domains in the partition region of the thylakoid membrane which have different average antenna size of PS IIα. The results lend further support to previous work in which heterogeneity of the antenna size of PS IIα of the same types of vesicles population was demonstrated by light saturation curves of oxygen evolution (Albertsson, P.-A. and Yu S.-G. (1988) Biochim. Biophys. Acta 936, 215–221).

Characterization of a non-detergent PS II-cytochrome b/f preparation (BS)

A non-detergent photosystem II preparation, named BS, has been characterized by countercurrent distribution, light saturation curves, absorption spectra and fluorescence at room and at low temperature (−196°C). The BS fraction is prepared by a sonication-phase partitioning procedure (Svensson P and Albertsson P-Å, Photosynth Res 20: 249–259, 1989) which removes the stroma lamellae and the margins from the grana and leaves the appressed partition region intact in the form of vesicles. These are closed structures of inside-out conformation. They have a chlorophyll a/b ratio of 1.8–2.0, have a high oxygen evolving capacity (295 μmol O2 per mg chl h), are depleted in P700 and enriched in the cytochrome b/f complex. They have about 2 Photosystem II reaction centers per 1 cytochrome b/f complex.The plastoquinone pool available for PS II in the BS vesicles is 6–7 quinones per reaction center, about the same as for the whole thylakoid. It is concluded, therefore, that the plastoquinone of the stroma lamellae is not available to the PS II in the grana and that plastoquinone does not act as a long range electron transport shuttler between the grana and stroma lamellae.Compared with Photosystem II particles prepared by detergent (Triton X-100) treatment, the BS vesicles retain more cytochrome b/f complex and are more homogenous in their surface properties, as revealed by countercurrent distribution, and they have a more efficient energy transfer from the antenna pigments to the reaction center.

Phosphorylation of thylakoids and isolated subthylakoid vesicles derived from different structural domains of the thylakoid membrane from spinach chloroplast

(1) Thylakoids from spinach chloroplasts were phosphorylated, fragmented by sonication, and then fractionated by aqueous two-phase partitioning to yield membrane fragments, deriving from different structural domains of the membrane: grana, grana margins, grana core and stroma lamellae. The photochemical activities of PS I and PS II, located in the grana, and PS I and PS II, located in the stroma lamellae, were compared for phosphorylated and control thylakoids. The antenna size (reflected by the Km value) and maximum activity (Vmax) of PS II declined by 19 and 23%, respectively, while for PS II the antenna size and Vmax decreased by 4 and 12%, respectively. No significant changes in antenna size were detected for either grana PS I or stroma lamellae PS I. Counter-current distribution was used for the quantitative separation of grana and stroma lamellae vesicles. Upon phosphorylation, the stroma lamellae fraction increased from 30% to 35% of the total, based on total absorbance at 680 nm. This increase can be explained by partial unstacking of the grana periphery and appressed membranes near the fret regions. Portions of the previously stacked membranes can therefore break and separate with the stroma exposed membrane. In addition, since the grana margins contain PS I (with 40% larger antennae than PS I), which is functionally connected to LHC II, it is to be expected that some of these PS I units will also enter the stroma lamellae fraction and thus help contribute to a lower chlorophyll a/b ratio and a small increase in the average PS I antenna size of the stroma lamellae fraction from phosphorylated thylakoids. It is concluded that the incidence of partial destacking of the grana, which occurs due to the phosphorylation of LHC II and PS II polypeptides, may promote the exposure of the granal PS I centers to the aqueous stroma and increase cyclic electron flow around Photosystem I and thereby ATP production over NADPH production. (2) Subthylakoid vesicles, representing the different structural domains, were also examined for their properties following an incubation in presence of light and ATP. Phosphorylation of membrane proteins including LHC II and PS II associated polypeptides was observed in membrane fractions deriving from the grana lamellae and, to a lesser extent, the grana core. Three unidentified polypeptides of 15, 20 and 22 kDa were the most abundantly labeled polypeptides in the stroma lamellae fraction. No membrane proteins became phospho-labeled in the grana margin fraction. (Less)

EFFECTS OF UVB RADIATION ON LIGHT-DEPENDENT AND LIGHT-INDEPENDENT PROTEIN PHOSPHORYLATION IN THYLAKOID PROTEINS

Abstract Protein phosphorylation in thylakoids can be divided into light-dependent and light-independent types. The phosphorylation patterns of these two types of process show about 30 and 40 phosphorylated polypeptides respectively in two-dimensional (2-D) protein separation (molecular mass discrimination in one dimension, isoelectric point (Ip) in the other). It is suggested that each type involves its own signal switch system and kinases responsible for signal transduction from the input domain to the final substrates. A thylakoid protein having a molecular mass of 45 kDa and focusing at Ip 5.0 in the 2-D protein pattern is suggested to be a kinase-like protein and its N-terminal sequence is reported. Freshly prepared spinach thylakoids were exposed to UVB (280–315 nm) radiation emitted from a 4W Philips fluorescent TL12 lamp for 60 min (0.21 W m−2 plant weighted radiation). The proteins were extracted and subjected to 2-D gel electrophoresis. There was no distinct difference in the 2-D protein patterns between UVB-irradiated thylakoids and non-irradiated thylakoids. The experiment was repeated under conditions of light-dependent protein phosphorylation with [γ-32P] ATP as phosphate donor. The phosphorylation of a group of polypeptides having molecular masses around 60–65 kDa and isoelectric points around 5–6, thought to include LHCII kinase, was suppressed by UVB pretreatment. The phosphorylation of LHCII polypeptides and other polypeptides was also decreased by UVB treatment. A similar experiment was performed with phosphorylation taking place in the dark after 1 h UVB (or control) treatment. In this case, the UVB treatment had an even greater impact on the phosphorylation pattern. The phosphorylation of a 45 kDa polypeptide, thought to be a kinase-like thylakoid protein active under oxidizing conditions, was almost eliminated, and that of a 60–65 kDa polypeptide group, thought to include LHCII kinase, was strongly suppressed; however, some polypeptides, e.g. 9 kDa and 10 kDa polypeptides focusing at Ip 5.7 and 7.3 respectively, became strongly phosphorylated in UVB-irradiated thylakoids. They may contribute to the protection against UVB radiation. A preliminary model is proposed for the action mechanism of both the enhanced phosphorylated proteins. We believe that altered kinase activity and protein phosphorylation may be important early steps in the impact of UVB radiation on plants.

Differences in UV-B sensitivity between PSII from grana lamellae and stroma lamellae

UV-B treatment of thylakoid membranes or fractions derived from them accelerates the first rise (Fo---Fi phase) of variable fluorescence and slows down the second (Fi---Fm) rise. This can be observed for all membrane fractions but to different extents. The grana fraction is affected more by the UV-B treatment than are the intact thylakoids and the stroma lamella fraction. An exogenous electron acceptor, 2,6-dichloro-p-benzoquinone, inhibits both the initial and second rises of the fluorescence induction kinetics including the changes induced by UV-B. However, other electron acceptors, such as 2,5-dimethyl-p-benzoquinone and methyl viologen quench only the second rise of the curve. Oxygen evolution from grana-derived membranes is more sensitive to UV-B treatment than oxygen evolution from stroma lamella-derived membranes.

Two dimensional electrophoresis of thylakoid membrane proteins and its application to microsequencing

A procedure of two-dimensional gel electrophoresis adapted for application on membrane proteins from the thylakoids is described. It involves isoelectric focusing in the first dimension and size dependent electrophoresis in the second dimension. About 100 polypeptides are clearly separated with relatively little streaking. About 20 polypeptides are identified by immunoblotting or location in the gel. They are the polypeptides of the PS I core, the 64 kDa protein, the α and β subunits of CF1 ATPase, cytochrome f, Rieske iron-sulfur protein, the 23 kDa and 33 kDa polypeptides of the oxygen evolving complexes, CP29, CP24, CP27 and CP25 (last two proteins belong to LHCII). Some proteins give rise to two or more separate spots indicating a separation of different isoforms of these proteins. Among them, the LHCII polypeptides (27 kDa and 25 kDa) were each resolved into at least three spots in the pH range 4.75–5.90; the Rieske FeS protein, as published elsewhere (Yu et al. 1994), was separated into two forms having different isoelectric points (pI 5.1 and 5.4), each of them was also microsequenced; the 64 kDa protein claimed to be a LHCII-kinase was found to be multiple forms appearing in at least two isoforms with pI 6.2 (K1) and 6.0 (K2) respectively, furthermore, K1 can be resolved into two subpopulations.The lateral distribution of these proteins in the thylakoid membrane was determined by analysing the vesicles originating from different parts of the thylakoids. The data obtained from this analysis can be partially used as markers for different thylakoid domains.This procedure for sample solubilization and 2-D electrophoresis is useful for the analysis of the polypeptide composition of vesicles originating from the thylakoid membrane and for microsequences of individual polypeptides isolated from the 2-D gel.