Alfons Radunz

Localization of NADPH-protochlorophyllide reductase in plastids of barley at different greening stages

The localization of protochorophyllide (Pchlide) and of NADPH-protochlorophyllide oxidoreductase (POR, EC 1.6.99.1) within (etio)chloroplasts has been investigated at selected stages of greening of barley seedlings. Pchlide pigment and POR protein contents were evaluated in different plastid membrane fractions by fluorescence spectroscopy and immunoblot analysis using a monospecific polyclonal antibody raised against the purified enzyme. Fluorescence analysis showed the presence of Pchlide in both the envelope and thylakoid membranes. During greening, the Pchlide content, expressed on a total protein basis, decreased in thylakoid membranes, whereas it increased in the envelope membranes. POR proteins were detected mainly in thylakoid membranes at early greening stages. In contrast, the weak amount of POR proteins was associated more specifically with envelope membranes of mature chloroplasts. Whatever the greening stage, thylakoid-bound Pchlide and POR proteins were more abundant in the thylakoid regions which remained unsolubilized after mild Triton treatment used as standard procedure to prepare PS II particles. This suggests the preferential association of Pchlide and POR to the appressed regions of thylakoids.

Function of plastoquinones B and C as electron acceptors in Photosystem II and fatty acid analysis of plastoquinone B

We have found that in petroleum-ether extracted tobacco thylakoids, plastoquinone A (PQ-A) and plastoquinone C (PQ-C) had similar efficiency in restoration of oxygen-evolving activity, while plastoquinone B (PQ-B), which is a fatty acid ester of PQ-C, was about 50% less effective. This indicates that apart from PQ-A, PQ-C and to a smaller extent PQ-B may function as electron acceptors of Photosystem II (PS II). The DCMU inhibition curves for PQ-C and PQ-B were biphasic and an initial slow decline was followed by a sharp decrease in oxygen evolution yield with a 50% inhibition (I50) at 0.25 μM DCMU. In the case of PQ-A (I50 = 0.20 μM DCMU), the activity decreased gradually without the sharp transition. The corresponding inhibition curve for unextracted thylakoids, where all the native prenylquinones are present, shows an intermediate shape between PQ-A and PQ-C but with a higher I50, equal to 0.32 μM, suggesting that the contribution of PQ-C as an electron acceptor of Photosystem II might be significant in thylakoid membranes with natural prenyllipid composition. α-Tocopherol quinone showed no activity in the restoration of oxygen evolution in extracted thylakoids, indicating that it cannot accept electrons from PS II. The fatty acid composition of PQ-B isolated from maple leaves showed a high degree of saturated fatty acids like myristic and palmitic acid, and its unique composition indicates that it is a natural component of the thylakoid membrane.

Organisation of Xanthophyll-Lipid Membranes Studied by Means of Specific Pigment Antisera, Spectrophotometry and Monomolecular Layer Technique Lutein versus Zeaxanthin

Abstract The structure of the xanthophyll pigments lutein and zeaxanthin differs in the position of one double bond and refers to one of the ionon rings. Specific antibodies to zeaxanthin were used to analyse the localisation and orientation of these two xanthophyll pigments in lipid membranes formed with egg yolk lecithin. Bimolecular and monomolecular layers were used. Antibody-antigen interaction was demonstrated and analysed by the bathochromic shift of the absorption spectra of both pigments and by the increase of light-scattering of the pig- mented liposome suspension. It appeared that the extent of the spectral effects accompanying the interaction of the antiserum to zeaxanthin, injected to the liposome suspension which was pigmented with either zeaxanthin or lutein, was different in spite of their similar molecular structures. The results are interpreted in terms of a localisation and distribution of lutein, in the hydrophobic phase of liposomes within two essentially different pigment pools, one oriented horizontally and the other vertically with respect to the membrane plane. This inter- pretation is supported by the analysis of isotherms of the compression of monomolecular layers of lutein and zeaxanthin formed at the air-water interface and of mixed xanthophyll- lipid monolayers as well as by analysis of the penetration of antibody proteins dissolved in the subphase into the mixed xanthophyll-lipid films.

The Effect of an Antiserum to Plastocyanin on Various Chloroplast Preparations

Abstract A monospecific antiserum to tobacco plastocyanin agglutinates strom a-free sw ellable chloroplasts from wild type tobacco, (Nicotia na tabacum var. John William’s Broadleaf) from the tobacco aurea mutant Su/su2, (Nicotiana tabacum var. Su/su2) from Antirrhinum majus and spinach (Spi-nacia oleracea). In this condition the antiserum inhibits linear photosynthetic electron flow in tobacco and spinach chloroplasts. This inhibition of electron transport as well as the agglutination are not observed if the chloroplasts have been sonicated prior to antiserum addition. This is due to the fact that plastocyanin is removed by ultrasonication. The antiserum stimulates a number of photophosphorylation reactions in tobacco chloroplasts. This stimulation is always larger in the aurea mutant chloroplasts and in chloroplasts from yellow leaf patches of a variegated tobacco mutant (N . tabacum , var. NC95) than in the green type chloroplasts. The stimulation appears to be a consequence of the inhibition of linear electron transport. The antiserum does not affect PMS-mediated cyclic photophosphorylation in tobacco chloroplasts from the wild type whereas the reaction appears stimulated in the tobacco mutant chloroplasts. However, menadione-mediated cyclic photo phosphorylation is inhibited upon addition of the antiserum. The same is true for noncyclic photo phosphorylation coupled to electron transport in the aerobic system diaminodurene/ascorbate → methylviologen in the presence of N-tetraphenyl-p-phenylenediamine in spinach chloroplasts. If the lamellar system of Antirrhinum and spinach has lost its swellability neither agglutination nor inhibition of electron transport is observed. However, also in this state antibodies to plasto cyanin are specifically adsorbed onto the surface of the thylakoid membrane. This state which is characterized by a morphologically well preserved lamellar system is realized in chloroplast prepa rations from Antirrhinum and spinach and is termed stroma-freed, chloroplasts. In both states of the molecular structure of the thylakoid membrane, plastocyanin is located in the outer surface of the thylakoid. However, it cannot be excluded that functioning plastocyanin is also located in the interior of the thylakoid membrane.

COMPARATIVE IMMUNOLOGICAL DETECTION OF LIPIDS AND CAROTENOIDS ON PEPTIDES OF PHOTOSYSTEM I FROM HIGHER PLANTS AND CYANOBACTERIA

Abstract Photosystem I preparations were obtained from wild-type tobacco Nicotiana tabacum var. JWB, three chlorophyll-deficient tobacco mutants: Su/su, Su/su var. Aurea and yellow- green leaf patches of the variegated mutant NC 95, Spinacia oleracea and furthermore from the mesophilic cyanobacterium Synechococcus PCC 7942 and the thermophilic cyanobacterium Synechococcus sp.. Peptides from these preparations were analyzed by SDS polyacrylamide gel electrophoresis and transferred for detection of bound lipids and carotenoids according to the Western blot procedure to nitrocellulose membranes. The PS I preparations from the Nicotiana tabacum species and spinach consist of the core complex and the LHCP I complex, the latter containing, however, traces of the LHCP II polypeptides. The core complex consists of the two core peptides with the apparent molecular mass of 66 kDa each and peptides with molecular masses of 22, 20, 19, 17, 16, 10 and 9 kDa. The LHCP I complex contains 4 subunits with molecular masses of 28, 26, 25 and 24 kDa. The PS I preparations of the two mutants Su/su and Su/su var. Aurea contain as impurities traces of the core peptides (D )/D 2) and the two chlorophyll-binding peptides (CP43/CP47) of photosystem II. The PS I preparation from the mesophilic and thermophilic cyanobacterium consists of the two core peptides with the apparent molecular mass of 66 kDa and peptides with molecular masses of 16, 14 and 10 kDa. The peptides of the PS I preparations were characterized by specific PS I, CP I and LHCP I antisera. The antiserum to the PS I complex reacts in the Western blot with the homologous peptides of PS I from higher plants, but only with the CP I complex from the two cyanobacteria. In comparative studies with PS II from higher plants the PS I antiserum reacts with the LHCP II complex as expected. The antiserum to the CP I complex reacts only with the 66 kDa peptides of PS I from all objects. There is no cross reaction with the 66 kDa peptides (heterodimer of the D1/D2 peptides) of PS II. The antiserum to the LHCP I complex reacts only with the four LHCP I peptides of PS I from higher plants and as expected with the LHCP II of PS II: Because cyanobacteria do not have LHCP complexes, there is no reaction with the LHCP I antiserum. By means of polyclonal monospecific antisera to lipids it was shown by Western blot procedure that only two lipid species are bound to PS I peptides. The galactolipid monogalactosyldiglyceride is bound to the CP I complex of the Nicotiana tabacum species, spinach and the two cyanobacteria as well as to the LHCP I complex of the higher plants. The phospholipid phosphatidylglycerol is only associated with the CP I complex of the analyzed higher plants and cyanobacteria. With polyclonal m onospecific antisera to carotenoids it was demonstrated that β-carotene, lutein, neoxanthin and zeaxanthin are associated with the CP I complex of the higher plants and the cyanobacteria analyzed. Violaxanthin is also bound to the CP I complex of the two cyanobacteria, whereas it is bound together with neoxanthin to the LHCP I complex of the higher plants.

Antibodies to chlorophyll and their reactions with chloroplast preparations.

Antibodies to chlorophyll are specifically adsorbed onto the membrane surface of thylakoids. The antibodies inhibit photosynthetic electron flow from water to NADP⊕. This inhibition is presumably caused by adsorption of the antibodies onto the centre chlorophyll of light reaction II. Fragments of the thylakoid membrane, obtained by ultrasonication and subsequent fractioning centrifugation, exhibit only photosystem-I activity. Conversely, the specific adsorption of antibodies to sensitizer chlorophyll has no inhibitory effect on electron transport. The ferricyanide Hill reaction of chloroplast preparations is inhibited by chlorophyll antibodies. From these observations it is concluded that the centre chlorophyll of light reaction II and at least part of the sensitizer chlorophyll is located on the surface of the thylakoids. As agglutination is sterically inhibited by the membrane protein, it is assumed that the chlorophyll is located in gaps or pores of the protein layer. Two fractions of the lamellar system exhibit photosystem I activity of different characteristic electron donor specificity. These fractions can be further distinguished in terms of their circular dichroism and protein composition.

Antagonistic Effects of α-Tocopherol and α-Tocoquinone in the Regulation of Cyclic Electron Transport around Photosystem II

Abstract α-Tocoquinone (α-TQ ) and α-tocopherol (α-TOC) which cannot substitute for plastoquinone-9 (PQ-A) as an electron acceptor from photosystem II (PS II), influence the oxygen evolution activity of thylakoid membranes under continuous illumination. In the presence of the herbicide DCMU and the protonophore FCCP which stimulate cyclic electron transport around PS II, α-TQ decreased oxygen evolution whereas α-TOC enhanced it. The effects are attributed to a stimulation or an inhibition of cyclic electron transport around PS II by α-TQ and α-TOC, respectively. Results of flash light experiments on PS II preparations show that both α-TQ and α-TOC increased the d-parameter which describes the transition probability from the S3- to the S0-state of the oxygen-evolving complex, although to a smaller extent when PQ-A is added alone to the preparations. The initial S-state distribution in darkadapted samples was changed only upon PQ-A addition and influenced neither by α-TQ nor by α-TO C supplementation. These effects indicate different kinds of interaction of PQ-A, α-TQ and α-TOC with the PS II components. α-TQ increased and α-TOC decreased the “total miss” parameter both in the presence or absence of PQ-A. A possible site of interaction of α-TQ and α-TO C with the cyclic electron transport around PS II is suggested.

Intermolecular Interactions of Polypeptides and Lipids in the Thylakoid Membrane

Abstract Intermolecular interactions between chloroplast lipids and a polypeptide fraction from thylakoids were investigated by far ultraviolet circular dichroism. The polypeptide fraction was isolated from dodecyl sulfate-containing buffers. It exhibits an average molecular weight of 24 000. The circular dichroism of this polypeptide fraction measured as mean residue ellipticity is greater in the presence of sodium dodecyl sulfate than in the absence of this detergent. This effect is reversible. Addition of sulfoquinovosyl diglyceride to the dodecyl sulfate-free solution of the polypeptide also causes an increase of the circular dichroism. This increase was only observed in the pH-range between 6.9 and 7.4. The effect of dodecyl sulfate or sulfolipid on the circular dichroism is interpreted to indicate an increase of α-helix content. Monogalactosyl diglyceride, digalactosyl diglyceride and phosphatidyl glycerol gave no reaction. The attempt to obtain a conformational analysis of the polypeptide in the different states did not yield an entirely satisfactory result. Antisera to sulfolipid inhibit photosynthetic electron transport of stroma-freed chloroplasts in the region of light reaction I. This inhibition is restricted to the same pH-range as the non-covalent binding of sulfolipid to the polypeptides. It appears that in the cell membrane-bound metabolic processes are regulated by this pH-dependence of the sulfolipid-polypetide interactions.

Reactions of a monospecific antiserum to ferredoxin-NADP+-reductase with chloroplast preparations.

Abstract A monospecific antiserum to ferredoxin-NADP+-reductase inhibits the diaphorase activity of soluble ferredoxin-NADP+-reductase from chloroplasts. Two states of the molecular structure of the lamellar system have been observed, one of which is the state described earlier by Berzborn. Stroma-freed chloroplasts in this condition are not agglutinated by the antiserum, although a specific adsorption of antibodies to reductase onto the lamellar system was demonstrated by the Coombs test. However, a second type of chloroplast preparations gives direct agglutination upon addition of the antiserum. Apparently, agglutination in this state is not sterically hindered by neighboring protein structures. This type of chloroplast preparations appears swollen under the light micro scope, but exhibits high rates of electron transport. Chloroplasts of three types of tobacco have been used which differ in the morphology of their lamellar systems. The green type contains a normal ratio of grana and intergrana regions whereas the other two types have extended intergrana regions with either only small grana or no partitions at all. Comparison of the maximal degree of inhibition of the NADP+-reduction in chloroplasts from these types of tobacco by the antiserum, leads to the conclusion that ferredoxin-NADP+-reductase is located in the grana and the intergrana regions of the lamellar system, in the outer surface of the thylakoid membrane.

Binding of Antibodies onto the Thylakoid Membrane. VI. Asymmetric Distribution of Lipids and Proteins in the Thylakoid Membrane

The maximal binding of antibodies out of monospecific antisera to proteins and lipids onto three different chloroplast preparations is compared. In these preparations different parts of the thylakoid membrane surface are accessible to antibodies. Whereas stroma-freed chloroplasts bind antibodies only at the outer surface, also the inner membrane surface is exposed in the two chloroplast preparations which were obtained by ultrasonication and subsequent fractionating centrifugation. In the ultrasonic sediment the inner surface is prevailing. In the ultrasonic supernatant antibodies do not only react with the inner and outer surface but also with considerable parties of the interior of the thylakoid membrane. It was found that the thylakoid membrane surface exposed to the stroma consists preponderantly of proteins whereas the surface directed towards the interior of the thylakoids consists mainly of lipids. All proteins involved in electron transport such as ferredoxin, ferredoxin- NADP+-reductase, plastocyanin, cytochrome f and the coupling factor of photophosphorylation are detectable in the outer surface. The molecules of the coupling factor span the thylakoid membrane from the outside to the inside. They hinder the binding of antibodies to mono- galactosyl diglyceride and to a polypeptide with the apparent molecular weight 24000. The polypeptide 24000 is a major component of the membrane proteins, and is detected on the outer and inner surface of the membrane. The major part of this polypeptide, however, is located in the interior of the thylakoid membrane. The lipid mixture has a different composition in the outer surface than on the inside face of the membrane. The sulfoquinovosyl diglyceride and the phosphatides, phosphatidyl glycerol, phosphatidyl cholin and phosphatidyl inositol are stronger concentrated in the outer surface than in the inside face. The neutral monogalactosyl diglyceride and di- or trigalactosyl diglyceride, however, occur in the inner surface in higher concentrations than in the outer surface. The major part of the sulfoquinovosyl diglyceride and of the monogalactosyl diglyceride are located in the interior of the membrane.