.1..ta, 990)

Planta 9 Springer-Verlag1990

Immunological studies on chlorophyll-a/b proteins and their distribution in thylakoid membrane domains* Maria Luisa Di Paolo 1, Angelo Dal Belin Peruffo 2, and Roberto Bassi 1 1 Dipartimentodi Biologia,Universitfidi Padova, Via Trieste 75, and 2 Dipartimentodi BiotecnologieAgrarie, Via Gradenigo6, 1-35121 Padova, Italy

Abstract. The immunological relationships between chlorophyll-a/b proteins from higher-plant thylakoid membranes have been studied by assaying purified chlorophyll proteins (CPs) with polyclonal and monoclonal antibodies. Although low levels of cross-reactions were observed between all light-harvesting proteins, the peripheral antennae (LHCII) were largely distinct from the inner antennae (CP 26 and CP 29). Chlorophyll-protein 24 and LHCI-680 have been proposed to have a role in connecting the inner and outer antennae, respectively, in photosystems I and II, and were closely related. The immunological relationships closely corresponded to the spectral properties. Antibodies were also used for locating chlorophyll-a/b proteins in grana, stroma and bundle-sheath membranes showing a strong lateral heterogeneity, which was maintained following State IState II transition. The only exception to this pattern was a specific LHCII population enriched in State-II stroma membranes. Chlorophyll proteins from bundlesheath chloroplasts, that have only cyclic electron flow, had epitopes distinct from those of their mesophyll homologues. Key words: Chloroplast membranes, proteins (lateral heterogeneity, state I-state II transition) Light-harvesting complexes - State I-State II transition - Thylakoid proteins - Z e a (chloroplast proteins)

Introduction The light reactions of photosynthesis are driven by the excitation energy absorbed by the antenna pigments and transferred to the reaction centres. The primary chromo* A preliminary report was presented at the VIII Int. Congr. on Photosynthesis, Stockholm1989 Abbreviations: Chl = chlorophyll;CP = chlorophyllprotein; kDa = kilodalton; LHC =light harvesting complex; MW---molecular weight; PAGE=polyacrylamidegel electrophoresis; PSI=photosystem I ;'PSII= photosystemII; RC = reaction center; SDS = sodium dodecylsulphate; e-CP 29 = antibodies raised against CP 29

phore responsible for light absorption in higher plants and algae is chlorophyll (Chl) a; accessory pigments such as Chl b and carotenoids extend the spectral range of light absorption and transfer energy to Chl a. While photosynthetic organisms containing phycobilins have developed a water-soluble antenna complex overhanging the inner or outer surface of the thylakoids, Chl-a/bcontaining organisms have their light-harvesting complexes (LHC) inserted into the thylakoid membrane (Staehelin 1986). Most of the Chl b in higher-plant thylakoids has been shown to be bound to LHCII; however, improving the analytical techniques has provided evidence that: (i) there are several other Chl-a/b proteins belonging both to the photosystem (PS)I and the PSII antenna systems; (ii) LHCII itself is the product of a multigene family (Dunsmuir 1985) and is composed of more than one complex having different polypeptide compositions, physiological roles (Larsson and Andersson 1985; Bassi et al. 1988a, b ) a n d cell-specific expression (Sheen and Bogorad 1986). At the present time there is evidence for the presence of at least six Chl-a/b complexes in maize mesophyll thylakoids: two PSI Chl-a/b complexes (LHCI-680 and LHCI-730; Bassi and Simpson 1987a), and three minor Chl-a/b proteins which belong to the PSII antenna system, namely chlorophyllprotein (CP) 24 (Dunahay and Staehelin 1986; Bassi and Simpson 1987b), CP29 (Machold etal. 1979; Camm and Green 1980), and CP 26 (Bassi et al. 1987). The complexes indicated above bind a minor proportion of the thylakoid pigments. In fact some 50% of the total Chl is bound to LHCII. This complex, in turn, can be divided into a phosphorylatable (30%) population which is involved in energy-transfer distribution between PSI and PSII, while the remaining LHCII can not be phosphorylated (Islam 1987; Bassi et al. 1988 b). Phospho-LHCII contains a mobile population which can be transferred from grana to stroma membranes and a population tightly bound to PSII (Bassi et al. 1988b). All these chlorophyll-protein complexes have polypeptides of similar molecular weight (MW) and they are likely to be coded by related genes. This is supported by previous reports of antigenic cross-reactions between CP 29 and a polypeptide of the PSI antenna system (LHCI-680) using both polyclonal (White and Green

276 1987) a n d m o n o c l o n a l a n t i b o d i e s ( H o y e r - H a n s e n et al. 1988). C P 24 was also f o u n d to share e p i t o p e s with L H C I - 6 8 0 (Bassi et al. 1987). This w o u l d i n d i c a t e t h a t C h l - a / b p r o t e i n s m a y all be m e m b e r s o f a n e x t e n d e d f a m i l y o f r e l a t e d p o l y p e p t i d e s . T h e k n o w l e d g e o f antigenic c r o s s - r e a c t i o n s b e t w e e n t h y l a k o i d p o l y p e p t i d e s is r e l e v a n t n o t o n l y for t h e i r e v o l u t i o n a r y r e l a t e d n e s s b u t also w i t h r e s p e c t to the use o f i m m u n o l o g i c a l m e t h o d s for the l o c a l i z a t i o n o f i n d i v i d u a l p r o t e i n s in different t h y l a k o i d d o m a i n s (Vallon et al. 1985, 1986; H o y e r H a n s e n et al. 1988) o r m e m b r a n e surfaces ( R y r i e et al. 1985; Vallon et al. 1987). O n the o t h e r h a n d , d i s c r e p a n c i e s exist in the literature c o n c e r n i n g c r o s s - r e a c t i v i t y b e t w e e n L H C I I a n d L H C I . C r o s s - r e a c t i v i t y h a s b e e n r e p o r t e d in b a r l e y a n d s p i n a c h b y using p o l y c l o n a l a n t i b o d i e s ( E v a n s a n d A n d e r s o n 1986) b u t n o c r o s s - r e a c t i o n s were d e t e c t e d b y o t h e r g r o u p s w h i c h u s e d b o t h p o l y c l o n a l ( L a m et al. 1984; W i l l i a m s a n d Ellis 1986) a n d m o n o c l o n a l antib o d i e s ( H o y e r - H a n s e n et al. 1988) in barley, p e a a n d maize. In this s t u d y we h a v e r a i s e d p o l y c l o n a l a n t i b o d i e s to p u r i f i e d C h l - a / b p r o t e i n s a n d s t u d i e d their i m m u n o logical c r o s s - r e a c t i o n s in Z e a m a y s L. O n this basis we h a v e d e t e r m i n e d the d i s t r i b u t i o n o f C h l - a / b p r o t e i n s a n d P S I I r e a c t i o n c e n t e r ( R C ) c o m p o n e n t s in different m e m b r a n e c o m p a r t m e n t s f r o m Z . m a y s leaves, i n c l u d i n g grana and stroma membranes from mesophyll chloroplasts and agranal bundle-sheath thylakoids. Stroma m e m b r a n e s h a v e also b e e n i s o l a t e d f r o m State-I- a n d S t a t e - I I - a d a p t e d leaves to d e t e r m i n e differences in the p r e s e n c e o f P S I I R C a n d i n d i v i d u a l C h l - a / b proteins. T h i s a l l o w e d us to i d e n t i f y m e m b r a n e c o m p l e x e s u n d e r g o i n g m i g r a t i o n after S t a t e I - S t a t e II t r a n s i t i o n . O u r resuits s u p p o r t the c o n c e p t o f e x t r e m e l a t e r a l h e t e r o g e n e ity in the d i s t r i b u t i o n o f m e m b r a n e c o m p l e x e s b e t w e e n grana and stroma membrane domains. The migration o f a n t e n n a c o m p l e x e s f r o m g r a n a to s t r o m a m e m b r a n e s is e x t r e m e l y selective for a L H C I I s u b - p o p u l a t i o n w h o s e p o l y p e p t i d e c o m p o s i t i o n is distinct f r o m t h a t o f the c o m plex in g r a n a m e m b r a n e s .

Material and methods

M.L. Di Paolo et al. : Immunology of Chl-a/b complexes PAGE) of octylglucoside-solubilizedPSII membranes in the presence of glycerol as previously reported (Bassi et al. 1987c). The oligomeric LHCII band was excised from the gel, macerated and the complex eluted in distilled water at 4 ~ C. This preparation, containing five polypeptides with MWs of 30, 29.5, 28.8, 28.5 and 26 kilodaltons (kDa) was further fractionated by preparative electrophoresis (Curioni et al. 1988) into three fractions containing the polypeptides with MWs 30 and 29.5 kDa, 28.8 and 28.5 kDa and 26 kDa, respectively. PSI-200 was prepared by Triton-X-100 solubilization of destacked thylakoids followed by sucrose-gradient ultracentrifugation as described in Mullet et al. (1980). The complex was further purified by solubilization in 1% dodecylmaltoside and sucrose-gradient ultracentrifugation as previously described (Bassi and Simpson 1987a). The lower band with Chl-a/b ratios of 7.3, which contained six polypeptides in the 20- to 25-kDa range, was used as PSI-LHCI antigen. LHCI-680 and LHCI-730 were respectively obtained from the second and third green band in the same sucrose gradient. Owing to the small amount of LHCI complexes contained by the preparation, LHCI-680 was used as antigen while LHCI-730 was used in Western blotting. The PSILHCI complex containing both LHCI-680 and 730, was used in both immunization and Western blotting. CP 29 was obtained by preparative non-denaturing isoelectricfocusing (IEF) in a granulated gel of octylgucoside-solubilized PSII membranes as previously reported (Bassi et al. 1988b). The green band with isoelectric point (pi)=4.62 and Chl-a/b ratio of 2.8 was eluted from a small plastic column and used as antigen. CP 26 and CP 24 were also obtained by IEF and eluted from fractions with pIs=4.35 and 4.50 (Dainese et al. 1989a). Antigens for PSII RC polypeptides were obtained as described by Bassi et al. (1987). Oxygen-evolving enhancer (O.E.E.) polypeptides were obtained by 2-amino-2(hydroxymethyl)-l,3 propanediol (Tris) washing of PSII membranes followed by preparative electrophoresis (Curioni et al. 1988). Preparation ofantisera. Rabbits were given multiple subcutaneous injections with 100 I~g protein in Freund's complete adjuvant. Subsequent injections were given on days 15, 21 and 29 after the first injection with 50 I~g protein in complete adjuvant followed by bleeding at day 35. Clotted blood was cooled to 4~ C. The serum was then decanted and spun at 4000 .g for 5 min to remove erythrocytes. Western blots of denatured thylakoids were used to screen antisera for activity. None of the preimmune sera possessed activity when tested at 50-fold dilutions. Electrophoresis. Analysis by SDS-PAGE was performed under

non-denaturing condition at 4~ as previously described (Bassi et al. 1985a, b) and under denaturing conditions in the presence of 6 M urea (Bassi 1985). In some cases separations were also obtained in the presence of 8 M urea. Gels were fixed in methanol/ water/acetic acid (2:2:0.4, by vol.) including 10% trichloroacetic acid, and stained with Coomassie brilliant blue or with silver (Goerg et al. 1985).

Plant material. Maize seeds (Zea mays L. cv. DF28; Dekalb, Ill.,

USA) were soaked in water for 24 h and grown in a vermiculite-soil mixture under glasshouse conditions in summer, Membrane isolation. Thylakoids were isolated from mesophyll chlo-

roplasts as previously described (Bassi et al. 1985a). The isolation of PSII membranes were carried out according to Berthold et al. (1981) with modifications (Dunahay et al. 1984). Bundle-sheath membranes were purified as previously described (Bassi and Simpson 1986). Stroma membranes were obtained from dark-adapted (State I) and high-light-adapted (State II) plants by French-pressing and differential centrifugation as previously reported (Bassi et al. 1988 a). Protease inhibitors phenylmethylsulfonyl fluoride (200 p.M), benzamidine (200 ~tM) and aminocaproic acid (1 raM) were included in all buffers. Isolation of antigens. LHCII was obtained by non-denaturing sodi-

um dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-

Immuno-blotting. Immediately after the electrophoresis run, the polypeptides were transferred from the SDS-PAGE gels to nitrocellulose filters (Towbin et al. 1979). These were used for immunoblot assays as previously described (Hoyer-Hansen et al. 1985; Burnette 1981). Immobilized antigens were visualized using peroxidase-coupled anti-rabbit immunoglobulins (Sigma, St. Louis, USA), alkaline-phosphatase-coupled anti-rabbit immunoglobulins (Dako Immunoglobulins, Copenhagen, Denmark), or [125I]protein A (Amersham International, Amersham, Bucks., UK). Monoclonalantibody binding was visualized using perioxidase-coupled antimouse immunoglobulins (Sigma). In some cases the antibody binding to antigens was quantified by eluting [125I]protein A from the filters by washing with glycine-HC1 (Evans and Anderson 1984) and counting in an LKB 7-counter (Bromma, Sweden). Other methods. Chlorophyll concentrations and a/b ratios were measured in 80% acetone, according to Arnon (1949), using a Perkin-Elmer lambda 5 spectrophotometer (Uberlingen, FRG).

M.L. Di Paolo et al. : Immunology of Chl-a/b complexes Photosystem-I preparations with a Chl:P700 ratio of 200:1 and 90:1 are indicated as PSI-200 and PSI-90. Both LHCI-680 and LHCI-730 are light-harvesting antennae of PSI which emit fluorescence at low temperature with peaks at 680 nm and 730 nm, respectively. The apparent MW of membrane proteins, as determined by SDS-PAGE, is strongly dependent on the running conditions. This may cause confusion when considering gel figures. Since many polypeptides are indicated with their MWs, we have also used the apparent MW reported in the literature in case the values do not correspond to the relative mobilities obtained in our gel conditions.

Results

277

Assay of thylakoid and grana membranes. When blots of thylakoid and g r a n a - m e m b r a n e polypeptide patterns were assayed with antisera against Chl-a/b proteins, a n u m b e r of bands were detected with M W s between 20 and 31 kDa. Although for each antibody a low n u m b e r of polypeptides was strongly recognized, other weaker reactions were also detectable. These latter reactions can hardly be considered when m e m b r a n e samples are assayed because of both co-migration of the polypeptides and the different amounts of the antigens present. A better evaluation o f the immunological cross-reactions is made possible by using purified antigens (Fig. 2; cf. Bassi and Dainese 1989; Dainese et al. 1989 a).

Cross-reactions between Chl-a/b proteins With the aim of determining immunological cross-reactions between Chl-a/b proteins, we fractionated the purified complexes in S D S - P A G E 6 M urea gels and then transferred them electrophoretically to nitrocellulose sheets. Filters were then assayed with antisera raised against individual Chl-a/b proteins. The following preparations were used in this experiment: P S I - L H C I (containing both LHCI-680 and LHCI-730), LHCI-730, CP 29, CP 26, CP 24 (Fig. 1 A, B) and two L H C I I samples which correspond to the tightly b o u n d and mobile populations, respectively (Bassi et al. 1988b, Figs. I B and 2A). Zea mays L H C I I can be fractionated into at least five subpopulations each containing several polypeptides (Bassi and Simpson 1988) but in this work we have assayed only the two that were shown to be immunologically representative o f the whole L H C I I in a preliminary study.

ct-LHCII. When purified Chl-a/b proteins, whose polypeptide compositions are shown in panel A of Fig. 2, were assayed with ~ - L H C I I antiserum (whole complex), strong reactions were observed only with L H C I I - c o n taining samples (Fig. 2B). However, weaker reactions were also observed with a 25-kDa polypeptide present in b o t h the PSI-200 complex and in its LHCI-730 fraction, with CP 24 and with the polypeptide doublet (28 and 29 kDa) o f the CP 26 complex. The results of Fig. 2 are summarized in Table 1. Figure 3 shows the results of assaying the L H C I I complex with polyclonal antibodies raised against three subfractions of the c o m p o nent polypeptides. It appears that all L H C I I polypeptides are recognized irrespective of the antibody used, indicating that the proteic c o m p o n e n t s o f L H C I I have m a n y epitopes in common. However, the reaction obtained with the antibody (~-3) raised against the lowerM W fraction is stronger at the 26-kDa position

Fig. 1 A, B. Polypeptide compositions (SDSPAGE 6 M urea) of the preparations used in this work as antigens. A Preparations from PSI complex. The samples PSI-LHCI and LHCI680 were used as antigens. The PSI-90 samples is included as a reference, because this preparation only binds Chl a and therefore polypeptides present in this preparation do not belong to LHCI. B Preparations from grana membranes: the samples LHCII, CP 29, CP 26 and CP 24 were used as antigens. The gels were stained with Coomassie blue

278

M.L. Di Paolo et al. : Immunology of Chl-a/b complexes

Fig. 2A-G. Cross-reactivity patterns of purified Chl-a/b proteins from Zea leaves. The Chl-a/b proteins were isolated as detailed in Material and methods, separated by 6 M urea SDS-PAGE and transferred to nitrocellulose. The filters were then assayed with polyclonal antibodies. Antibody binding was revealed by alkalinephosphatase-coupled immunoglobulin G. Weak reactions are indicated by arrows when invisible in the figure but detectable by visual inspection of the nitrocellulose filters. A Coomassie-blue-stained gel. B-G Filters assayed with polyclonal antibodies (B, c~-LHCII; C, c~-CP29; D, e-CP 26; E, c~CP 24; F, c~-LHCI-680; G, ~-PSI-LHCI) to individual Chl-a/b proteins (lanes 2 LHCII; lanes 3, LHCII; lanes 4, CP 29; lanes 5, CP 26; lanes 6, CP 24; lanes 7, LHCI-730; lanes 8, PSI-200). The results are summarized in Table 1

(Fig. 3 B) thus indicating the presence of epitopes unique to this c o m p o n e n t of L H C I I . This is confirmed by using the a-CP 26 and a - P S I - L H C I polyclonals that recognize higher-MW polypeptides but not the 26-kDa one (Fig. 2 D , lanes 2 and 3, Fig. 2 G , lanes 2 and 3). a - C P 29, ~ - C P 26, ~ - C P 24. Antibodies raised against

the m i n o r Chl-a/b proteins of the grana membranes (CP 29, CP 26 and CP 24) strongly recognized the components of this group, with the partial exception of aCP 29 which detected CP 24 only very weakly (below detection level in Fig. 2 C). Reactions were obtained also

with a 25-kDa polypeptide of PSI-200 and LHCI-730 and, with lower intensity, with a 28-kDa c o m p o n e n t of L H C I I (Fig. 2C, D, E). The polyclonal ~-CP 24 showed the highest level of reaction with L H C I polypeptides and the largest number of c o m m o n epitopes. In fact, in addition to the 25-kDa polypeptide that was also detected by c~-CP 29 and ~-CP 26, it also weakly recognized five other bands in the 20- to 25-kDa M W range. The reaction of one L H C I I apoprotein (28-kDa) with c~CP 29 antiserum clearly indicates the presence of at least a c o m m o n epitope. However, we have never been able to obtain a reaction of CP 29 with ~ - L H C I I antiserum

M.L. Di Paolo et al. : Immunology of Chl-a/b complexes

279

Table 1. Summary of the cross reactions between Chl-a/b proteins of maize thylakoids as observed in Figs. 2 and 5. A semiquantitative evaluation of the strength of the reaction is given. (+ + +), very strong; ( + +), strong; (+), middle, (_+), weak. The reactions whose intensities were very weak and below detection in Fig. 2, are marked with an asterisk in the table and with arrows in Fig. 2. In the case of chlorophyll-proteins containing more than one polypeptide, the MW of the component showing the reaction is given in parenthesis. The monoclonal antibodies were obtained from three different fusions in which the antigens were respectively PSII membranes (CMp Chl a/b P I : I and CMp Chl a/b P1:2), PSI-200 complex (CMpLHCI: 1 and CMpLHCh 11), and a LHCII preparation (MLH1 and MLH2; Darr et al. 1986). C.M., Carlsberg Monoclonals Antibodies

Chl-a/b proteins LHCII

CP29

CP26

CP24

Slot in Fig. 2

2, 3

4

5

6

Polyclonals ~-LHCII

+ + +

--

+

+

~-CP29

4(28)

+++

+++

*

~-CP26

+ (28)

++

+++

+

++

++

~-CP 24 ~-LHCI . 680 o-PSILHCI

+ .

. +

Monoclonals (Fig. 5 A, B) CMp Chl + a/b P1 : 2 MLH1 + . (29, 30) MLH2 + CMpLHCI . . . :11 CMp Chl + a/b P1 : 1 a CMpLHCI :1 ~

* (25) * (25)

* (25) * (25)

+ (25)

+ (25)

+++

+

+++

+

+

+ +

++

+++

+++

+++

+

-

-

-

. _+

LHCI- PSI+ 730 LHCI 7 8

.

. .

. .

. .

.

.

-

-

+ + (20, 21) (20, 21) +

-

+

-

+

d e s p i t e the fact t h a t a n t i s e r a were o b t a i n e d f r o m f o u r different r a b b i t s i m m u n i z e d with L H C I I o f different degrees o f d e n a t u r a t i o n , f r o m the i n t a c t c o m p l e x to S D S urea-denatured apoprotein. ~-LHCI. Antisera raised against LHCI components ( P S I - L H C I a n d L H C I - 6 8 0 ) h a d very different affinties for the o t h e r C h l - a / b p r o t e i n s since P S I - L H C I recognized all C h l - a / b p r o t e i n s , a l t h o u g h w i t h different affinities, while L H C I - 6 8 0 r e a c t e d to a n a p p r e c i a b l e e x t e n t o n l y w i t h C P 24 a n d w e a k l y w i t h L H C I - 7 3 0 (Fig. 2 F , G). It was o f i n t e r e s t to c o m p a r e L H C I - 6 8 0 w i t h C P 24 since these c o m p l e x e s h a v e v e r y similar c h a r a c t e r i s t i c s a l t h o u g h they a r e l o c a t e d in different m e m b r a n e c o m p a r t m e n t s (Bassi et al. 1 9 8 5 a ; D u n a h a y a n d Staehelin 1986). T h i s w a s d o n e b y u s i n g different p o l y c l o n a l antib o d i e s to a s s a y p u r i f i e d C P 24 a n d L H C I , a n d b o t h

Fig. 3A, B. Cross-reactions between polypeptides belonging to the LHCII complex. The whole LHCII was fractionated by preparative denaturing SDS-PAGE and the three fractions used to raise antibodies in rabbits. These antibodies were then used to assay a whole LHCII preparation. A Fractionation of LHCII complex. The gel is stained with Coomassie blue. Lane 1, fraction 1: polypeptides of MWs 30 and 29.5 kDa; lane 2, fraction 2: polypeptides of MWs 28.8 and 28.5 kDa; lane 3, fraction 3: polypeptide of MW 26 kDa; lane 4, whole LHCII complex. B Immuno-blots of whole LHCII assayed with polyclonals ~-1 to ~-3 raised against the fractions 1-3 in A and with MLH1 monoclonal antibody raised against the Nterminal trypsin cleavable peptide of LHCII m o n o c l o n a l s a n d p o l y c l o n a l s to d e t e c t the p r e s e n c e o f the c o m p l e x e s in d i f f e r e n t m e m b r a n e p r e p a r a t i o n s . T h e h i g h r e s o l v i n g p o w e r o f the gel s y s t e m u s e d in the p r e s e n t s t u d y d i s t i n g u i s h e d several p o l y p e p t i d e s in the 20- to 2 1 - k D a M W range. W i t h i n this r a n g e L H C I - 6 8 0 a p o p r o t e i n s were slightly less m o b i l e t h a n the C P 24 a p o p r o teins. M o r e o v e r , C P 24 a n d L H C I - 6 8 0 were l o c a t e d in g r a n a a n d s t r o m a t h y l a k o i d m e m b r a n e s , respectively (Fig. 5 B). T h e r e a c t i o n p a t t e r n s o f C P 24 a n d L H C I - 6 8 0 w i t h ~ - L H C I - 6 8 0 a n d ~ - C P 24 a n t i s e r a were d i f f e r e n t : t w o p o l y p e p t i d e s were d e t e c t e d in the t h y l a k o i d m e m b r a n e s a m p l e b y using ~-CP 24 p o l y c l o n a l (Fig. 5 B, lane T : C P 24) while a single b a n d w a s o b t a i n e d w i t h ~ - L H C I 680 (Fig. 5 B, l a n e T : L H C I - 6 8 0 ) . A c l e a r - c u t result was o b t a i n e d w i t h the C M p L H C I : l l monoclonal that only recognizes L H C I - 6 8 0 (Fig. 5 B, l a n e T : C M p L H C I : 11). T h u s C P 24 a n d L H C I - 6 8 0 h a v e c o m m o n e p i t o p e s ( H o y e r - H a n s e n et al. 1988, Fig. 2 E , lane 6, Fig. 2 F , lane 6) b u t also u n i q u e e p i t o p e s (Fig. 5 B). P r e - a b s o r p t i o n e x p e r i m e n t s . A l t h o u g h the p u r i t y o f the a n t i g e n s g u a r a n t e e s the existence o f c r o s s - r e a c t i o n s bet w e e n C h l - a / b - b i n d i n g p r o t e i n s , we h a v e p r o b e d the p u r ified a n t i g e n with a n t i s e r a a d s o r b e d w i t h p u r i f i e d C h l - a / b proteins. Western blots of ~-LHCII, ~-PSI-LHCI and ~-CP 29 p o l y c l o n a l s p r e a d s o r b e d w i t h L H C I I a n d P S I L H C I c o m p l e x e s a r e s h o w n as a n e x a m p l e in Fig. 4. Absorbing ~-PSI-LHCI with LHCII complex resulted in a d e c r e a s e o f a n t i b o d y b i n d i n g to b o t h L H C I I a n d

280

M.L. Di Paolo et al. : Immunology of Chl-a/b complexes

Fig. 4. Preabsorption experiments. Filters, blotted with purified antigens, either LHCII (lane A) or PSI-LHCI (lane B), were reacted with polyclonal antibodies (~-PSI-LHCI, ~-LHCII and ~-CP 29) that were (+) or were ( - ) not previously incubated with one of the antigens: ~-PSI-LHCI was preincubated with purified LHCII (10 ~tg of Chl), ~LHCII was preincubated with PSI-200 complex (20 ~tg of Chl), ~-CP 29 was preincubated with LHCII (10 ~g of Chl). A decreased intensity of the reaction with both antigens would be expected if the antigens share one or more epitopes. A complete disappearance of the reaction with the sample used in the pre-incubation and no decrease in the sample used to raise the antibody would be expected in the case of proteins carrying distinct epitopes. A small amount of LHCII apoprotein was added to the PSI-200 preparation to be assayed with ~-LHCII (~-LHCII lanes B - and B§ as an internal standard. The position of this LHCII in the filter is indicated with (D) while the position of a major, 25 kDa, LHCI apoprotein is indicated with (A) the higher-MW polypeptides of L H C I . Similar results were obtained by absorbing ~ - L H C I I with P S I - L H C I complex. In this case, because of the low level of crossreaction, we mixed a very small a m o u n t of purified L H C I I protein with the P S I - L H C I complex as an internal standard. This L H C I I preparation whose polypeptide composition is shown in Fig. 3 A lane 1, was recognized by ~ - L H C I I (D in Fig. 4) and the intensity o f the reaction was fainter u p o n preadsorption of the ~ - L H C I I antibody with P S I - L H C I complex. The reaction with the 25-kDa c o m p o n e n t o f L H C I (zx in Fig. 4) was faint and disappeared u p o n preadsorption. The intensity of the reaction with the 26-kDa c o m p o n e n t of L H C I I also decreased. When ~-CP 29 antiserum was saturated with L H C I I we observed a decreased reaction with CP 26 apoprotein. It was not possible to study the effect of the absorption of ~ - L H C I I with CP 29 since none of our L H C I I antisera recognized CP 29 apoprotein. In Table 1 the intensity o f immunological cross-reaction is given in a semiquantitative f o r m ( - to + + + ) from visual inspection of the stained filters and-or autoradiographs. While the amounts of the antigens loaded onto the gels of Fig. 2 A were normalized to their Chl contents, a more precise quantification was very difficult due to changes in the n u m b e r of polypeptides in each complex and to the non-linearity of the signal in the wide range of reaction intensities obtained in this study. With the aim of obtaining a guideline in the evaluation of the reaction levels in Table 1, we eluted [12~I]protein A from the filters and assayed it in a 7-counter. In the case of the assay with ~ - L H C I I polyclonal (Fig. 2B), radioactivity values in the ratio 720: 600: 15: 8 : 1 were obtained for lanes 2, 3, 5, 6 and 7, respectively. This determination was not extended to the reaction with other antibodies.

Distribution o f antigens between grana, stroma and bundle-sheath membranes The study o f the distribution of m e m b r a n e complexes in different thylakoid c o m p a r t m e n t s has been carried

out mainly by immunocytochemical methods (Vallon et al. 1985, 1986; D u n a h a y and Staehelin 1987; HoyerHansen et al. 1988). However, in the case of Chl-a/b proteins this approach cannot be employed because of the c o m m o n epitopes of L H C I and L H C I I proteins present in these complexes that are homogeneously labeled in grana and stroma thylakoid membranes. A better resolution can be obtained by Western blotting since antigens can be further characterized on the basis of their mobility in S D S - P A G E provided that pure m e m b r a n e fractions are obtained. The cross contamination can be evaluated not only from the mutually exclusive presence of m a n y polypeptides in Coomassie stained gels also by the more sensitive antibody reactions. The absence of contamination of the grana fraction by stroma membranes can be appreciated by the lack of any reaction at the position o f P S I - R C I apoprotein (67 k D a ; Fig. 5A, B). The degree of contamination of the stroma lamellae by grana partitions has been evaluated to be less than 0.1% by freeze-fracture analysis (Bassi et al. 1988a). The purity of bundle sheath membranes can be appreciated by taking advantage of the fact that at least two PSI R C proteins (67 k D a and 18 kDa, Fig. 5 B) carry different epitopes with respect to the homologous proteins from mesophyll thylakoids and are not recognized by the ~ - P S I - L H C I antibodies. Figure 5A and B clearly show that no mesophyll PSI R C is detected in bundle sheath preparation by polyclonals directed to mesophyll P S I - L H C I complex. For the study of Chl a/b protein distribution in different chloroplast m e m b r a n e fractions we have assayed blots with the above characterized polyclonal antibodies. These results were integrated by assaying with a series of monoclonal antibodies raised against Chl a/b proteins. The M L H 1 recognizes the higher M W polypeptide of L H C I I (Darr et al. 1986; Bassi et al. 1988b), M L H 2 is specific for CP 29 (Darr et al. 1986; Hoyer-Hansen et al. 1988), C M p L H C I : 11 recognizes LHCI-680, C M p Chl a/b Pl :2 recognizes an epitope c o m m o n to CP 29 and CP 26 (data not shown).

M.L. D i Paolo et al. : Immunology of Chl-a/b complexes

281

Fig. 5A, B. Localization of antigens in different thylakoid membrane fractions from Zea leaves. A Separation using a Tris-HCl/8 M urea SDSP A G E gel. This gives a better separation of CP 29 from LHCII apoproteins. B Separation using a Tris-H2SO4/6 M urea SDS-PAGE gel. This gives a better separation in the 20- to 28-kDa range

282 Figure 5A reports the results obtained by using a Tris-HC1, 8 M urea gel that yields a better separation of CP 29 from LHCII. In the experiment of Fig. 5B, Tris-H2SO4, 6 M urea SDS-PAGE gel was used that allows a better resolution of L H C I polypeptides in the 20- to 25-kDa range. The blots assayed with the polyclonal c~-PSI-LHCI were included in both figures as a reference. CP 29 and CP 26. When filters blotted with electrophoretograms o f different membrane fractions were assayed with the CMp Chl a/b P1:2 monoclonal that recognizes CP 29 and CP 26, it clearly appeared that these two chlorophyll proteins are exclusively located in grana fractions since no trace o f reaction was obtained with stroma membranes. Bundle-sheath membranes contained a polypeptide reacting with this monoclonal but it had a higher mobility in the gel (30 kDa) and was not recognized by another monoclonal against CP 29 (MLH2) (not shown). This pattern is confirmed by the assay with polyclonal antibodies against CP 26 and CP 29. L H C H . The c~-LHCII antiserum strongly reacted with the thylakoid polypeptides in the 26- to 30-kDa range,

M.L. Di Paolo et al. : Immunology of Chl-a/b complexes as was shown using the purified complex (Fig. 2), and did not recognize bands with different mobility in the gel. In the [~25I]protein A autoradiograph shown in Fig. 5 A the relative intensity of ~-LHCII reaction can be appreciated. The strength of the reaction was enhanced in PSII membranes compared with thylakoids but the staining pattern was otherwise identical. In stroma membranes the reaction was very low and absent in the position of the lower-MW polypeptide (26 kDa). A very strong reaction was obtained with bundle-sheath thylakoids (compared with the weak Coomassie stain); however, the relative mobilities of the reacting polypeptides were different from those of L H C I I polypeptides in mesophyll membranes. The absence of the 26 kDa L H C I I polypeptide in stroma membranes was confirmed by using the more sensitive assay with alkaline-phosphatase-conjugated anti-rabbit immunoglobulin (IG)g as secondary antibody. The reaction of the MLH1 monoclonal was more selective. This recognizes higher-MW L H C I I polypeptides and no reaction was detected in both stroma and bundle-sheath membranes, thus showing that L H C I I polypeptides in bundle-sheath membranes are different from their homologues in the mesophyll. It is somehow surprising that L H C I I polypeptides,

Fig. 6. Western-blot assay with polyclonal antibodies directed to various thylakoid complexes of stroma membrane fractions obtained from State-I- and State II-adapted Zea leaves. OEE, oxygen-evolving enhancer; CF1, coupling factor

M.L. Di Paolo et al. : Immunologyof Chl-a/b complexes detected in stroma membranes by the LHCII polyclonal, are not recognized by MLH1; however, it has been reported that this monoclonal is directed to the N-teminal phosphorylatable fragment of LHCII (Darr et al. 1986) and it is thus possible that the actual epitope is the phosphorylation site, modified upon state transition. This is consistent with the lack of reaction to the bundlesheath LHCII that cannot be phosphorylated (Schuster et al. 1985).

L H C I and CP 24. The reaction patterns with the polyclonals ~-LHCI-680 and c~-CP 24 in the different membrane fractions indicate that these chlorophyll proteins are distinct from each other; in fact, the 20 kDa polypeptide doublet of CP 24 is found in grana while the 20to 21 kDa bands of LHCI-680 are detected in stroma membranes (Fig. 5 B). This is confirmed by the results of the assay with monoclonal c~-LHCI (CMpLHCI: 11). This monoclonal does not recognize CP 24 but only LHCI-680 (Fig. 5 B) and shows that the latter is absent in grana membranes. This monoclonal also recognizes a polypeptide of 22 kDa with identical distribution between membrane domains. The higher-MW polypeptides of LHCI (22-25 kDa) are absent from grana partitions as determined by the assay with several cross-reacting antibodies (Fig. 5 A, B). Although LHCIs of bundle sheath and stroma share many epitopes, yet they show different reactivities with polyclonals c~-CP 26 and c~CP 24 (Fig. 5 A, B). Changes in the amounts of Chl-a/b-binding proteins in stroma membranes following State I-State H transition. Current hypotheses on the regulation of light energy distribution between PSI and PSII propose that, mediated by its phosporylation, LHCII undergoes dissociation from PSII in grana stacks and then migrates into stroma lamellae where it can transfer excitation energy to PSI. It has been shown that LHCII polypeptides are enriched in stroma membranes after state transition (Bassi et al. 1988b) but it is still unclear whether other complexes undergo repartition between thylakoid domains upon state transition. To clarify this point we isolated stroma lamellae from dark-adapted (State I) and high-lightadapted (State II) plants as previously reported (Bassi et al. 1988a) and probed blots of their polypeptide patterns with specific antibodies. The results are shown in Fig. 6A, lanes I and II oxygen-evolving enhancer polypeptides are equally represented in State I and State II. This is also true for other PSII RC components, namely CP 47, CP 43, D1, D2 and cytochrome b 559 (Fig. 6B, lanes I and II). The reaction with the antibody against LHCII (Fig. 6C, lanes I and II), is much stronger in the lightadapted sample and the 26-kDa LHCII components is absent from both fractions. No differences were detected in the reaction pattern with ~-PSI-LHCI antiserum (Fig. 6E, lanes I and II) while it was not possible to detect any trace of the CP 26 and CP 29 complexes with the ~-CP 29 antiserum (Fig. 6D, lanes I and II).

283 Discussion

Immunological relations between Chl-a/b proteins The antenna system of higher plants includes several intrinsic membrane proteins that bind both Chl a and Chl b in different ratios and are connected to PSI (LHCI-680 and LHCI-730) (Bassi and Simpson 1987a) and to PSII (CP 29, CP 26, CP 24) (Machold et al. 1979; Camm and Green 1980; Dunahay and Staehelin 1986; Bassi and Simpson 1986; Bassi et al. 1987c), while the major light-harvesting complex (LHCII) may serve PSI or PSII, depending on its phosphorylation state (Allen et al. 1984). Although the common characteristic of binding Chl a and Chl b indicates structural similarities, it is not clear whether these polypeptides have immunological relationships. Cross-reactivity between LHCI and LHCII polypeptides has been described in spinach (Evans and Anderson 1986) and between LHCI and CP 29 in barley (White and Green 1987). On the other hand LHCI and LHCII were shown to be distinct proteins in pea and barley by using both polyclonal and monoclonal antibodies (William and Ellis 1986; Hoyer-Hansen et al. 1985). We have shown here that indeed CP 29, CP 24, CP 26 and LHCI are largely distinct with respect to LHCII since the respective antisera do not recognize heterologous polypeptides unless blots are assayed by very sensitive procedures. It is however possible to detect definite cross-reactions that may yield information on these chlorophyllbinding proteins.

Chlorophyll-binding L H C I polypeptides. Light-harvesting complex I has been shown to exist in a complex distinct from the P-700-containing core complex (Wollman and Bennoun 1982; Mullet et al. 1980) and has been shown to contain at least five polypeptides (Bassi et al. 1985a) belonging to two different Chl a/b complexes (Lam et al. 1984; Bassi and Simpson 1987a). However, at least two reports (Machold 1986; Nechustai et al. 1987) suggest that only the 21 kDa polypeptide binds chlorophyll. Since Lemna LHCI has been shown to contain two copies of the 21 kDa polypeptide (Bruce and Malkin 1988), this poses the problem of how two 21 kDa proteins can bind 100 Chl molecules (Ortiz et al. 1984; Bassi and Simpson 1987a) when each 21 kDa polypeptide contains only a single histidine residue (Nechustai et al. 1987). Our data show that at least five polypeptides aside from the 20-kDa one are recognized in the PSI-LHCI complex by antibodies raised against heterologous Chl-a/b proteins. Since these antibodies do not react with colorless or Chl-a-binding proteins, we suggest that Chl belonging to LHCI is bound to six polypeptides in the 20- to 25-kDa range rather than to the single 21 kDa component. LHCI-680 and LHCI-730. These two Chl-a/b proteins of PSI have been isolated by sucrose-gradient ultracentrifugation or SDS-PAGE from the PSI-LHCI complexes of spinach (Lam et al. 1984), maize (Bassi et al.

284 1985 a) and barley (Bassi and Simpson 1987 a). Although the two complexes have different spectroscopic characteristics, in principle it cannot be excluded that differences in the aggregation state rather than the actual composition of the complex might be responsible for these two LHCI forms. The immunological reactions of the polyclonal e-LHCI-680 with PSI-LHCI (which contains both LHCI-680 and LHCI-730) and with the purified LHCI-730 indicate that although the two LHCI forms have common epitopes (Fig. 2 F) they, nevertheless, have many distinct determinants. An example is given by the 21 kDa component of LHCI-680 that is more closely related to CP 24 than to LHCI-730. On the other hand, the 25-kDa polypeptide of LHCI-730 seems to share epitopes with almost all the others Chl-a/b complexes, namely LHCII, CP 29, CP 26, CP 24 (Fig. 2 B-E), while this is not the case with the others LHCI polypeptides that are only recognized by the homologous antibodies (e-PSI-LHCI and c~-LHCI-680) and by c~-CP 24. It appears that the polypeptides belonging to LHCI are heterogeneous and cannot be included with certainty in a group showing common LHCI features. This is in agreement with the comparison of the sequences of two LHCI genes (Hoffman et al. 1987; Stayton et al. 1987) that are no more homologous to each other than they are to LHCII genes. This is consistent with different roles for LHCI polypeptides in accordance with a model recently proposed by Bassi and Simpson (1987 a).

LHCI-680 and CP 24. Light-harvesting complex 1-680 and CP 24 have been isolated, respectively, from stroma (Bassi et al. 1985a, 1988c; Bassi and Simpson 1987a) and grana membranes (Dunahay and Staehelin 1986; Bassi et al. 1987c). They were shown to have very similar polypeptide compositions and spectral properties and thus it has been suggested that they are the same complex (Green 1988). The results of Figs. 2E, F and 5B show that these complexes, although sharing some epitopes as previously reported (Hoyer-Hansen et al. 1988), also have unique features and mutually exclusive locations in thylakoid membrane compartments. The high resolving power of the gel system used in the present study distinguishes several polypeptides in the 20- to 21-kDa MW range. Within this range LHCI-680 polypeptides are slightly less mobile than CP 24 aproproteins. Moreover, CP 24 and LHCI-680 are located in different membrane compartments. This shows that LHCI-680 and CP 24 are distinct chlorophyll proteins. They have, however, similar characteristics and may possibly play similar roles as connecting antennae in both PSI and PSII light-harvesting systems (Bassi et al. 1987c). Immunological groups in Chl-a/b proteins. Chlorophyll-a/ b proteins are quite different from each other with respect to both their epitope patterns and the strength of the reactions. In general our results are in agreement with previous reports of cross-reactivity between Chl-a/b proteins (Evans and Anderson 1986; White and Green 1987), although we stress that some of these cross-reactions

M.L. Di Paolo et al. : Immunologyof Chl-a/b complexes are weak and can be detected only with the most sensitive methods. Thus it is not surprising that the studies with monoclonals did not provide evidence for common epitopes in LHCI and LHCII (William and Ellis 1986; Hoyer-Hansen et al. 1988). From the cross-reaction patterns a tentative grouping of similar Chl-a/b proteins can be made by considering the cross-reactions that are strong enough to be easily detected (+ to + + + in Table 1). (i) A first group of closely cross-reacting polypeptides includes all LHCII-type apoproteins. These are very similar to each other (Fig. 3) and different from the other Chl-a/b proteins since the reactions of ~-LHCII antiserum with the other Chl-a/b complexes are very weak (Fig. 2B). (ii) Chlorphyll-protein 26 and CP 29 are very similar to each other from the immunological point of view according to their absorption spectra which show a strongly redshifted Chl-a peak (677 nm) (Bassi and Dainese 1989; Dainese et al. 1989b). (iii) Chlorophyll-protein 24 and the 21-kDa polypeptide of LHCI-680 are closely related with respect to both immunological reactions (Fig. 2 E, F) and previously described spectral properties (Bassi et al. 1985a, 1987c; Dunahay and Staehelin 1986; Bassi and Simpson 1987a). The distribution of the epitopes in the different LHCI polypeptides does not correspond to their belonging to LHCI-730 and LHCI-680, and the different LHCI apoproteins are heterogeneous, as discussed above. However, the LHCI-730 complex, which absorbs at 677 nm (Bassi and Simpson 1987), shares epitopes with both CP 26 and CP 29, which have similar absorption spectra, while LHCI-680, whose Chl-a absorption peak is at 674 nm, does not.

Location of Chl-a/b proteins It has been shown that LHCII is mainly located in PSII membranes in both Chlamydomonas and spinach (Vallon et al. 1986). Similar results have been reported for CP 29 (Dunahay and Staehelin 1987; Hoyer-Hansen etal. 1988) while immunogold localization with the CMpLHCI:I (c~-LHCI) monoclonal yielded labeling in both grana and stroma membranes (Hoyer-Hansen et al. 1988) due to the common epitope detected by this monoclonal in CP 24 and LHCI-680 (Bassi et al. 1987). Our results indicate a lateral heterogeneity for Chl-a/ b proteins even stronger than that shown for PSII RC, provided that dark-adapted plants are used for the isolation of grana and stroma membranes. Only traces of LHCII polypeptides are absent in dark-adapted stroma and the 26-kDa LHCII component is absent. CP 29 and CP 26 are strictly confined to grana, since these complexes were not revealed in stroma membranes regardless of the sensitivity of the detection method, while LHCI730 was not detectable in grana membranes. A previous report (Dunahay and Staehelin 1987) indicated 85% CP 29 in grana stacks but it is possible that some of the labelling in stroma membranes might be caused by cross-reactions with LHCI. Once LHCI-680 and CP 24 are distinguished on the

M.L. Di Paolo et al. : Immunology of Chl-a/b complexes basis of their immunological and electrophoretic behavior (see section LHCI-680 and CP 24) it is possible to see that they are confined, respectively, to stroma and grana membranes. Polypeptides reacting with each of our polyclonals directed to Chl-a/b proteins were detected in bundlesheath membranes. However, it clearly appears that these proteins are different from those found in both grana and stroma membranes when compared for both reactivity and electrophoretic mobility of the proteic bands. Similar observations can be made about the polypeptides of L H C I I in the 26- to 28-kDa range that are recognized by the c~-LHCII antiserum but not by MLH1. The most surprising feature about the bundle sheath is that PSI R C apoproteins (67 kDa and 18 kDa) are not recognized by the very strong polyclonal directed against PSIL H C I , although it was clearly demonstrated that PSI RC is present in these membranes (Bassi et al. 1985b; Bassi 1985c; Schuster et al. 1985). These findings are consistent with the reports of differential expression of the genes for Chl-a/b-binding protein in maize leaf cell types (Sheen and Bogorad 1986; Bassi and Simpson 1986) and indicate that bundlesheath proteins have undergone a strong diversification in response to the adaptation to exclusive cyclic electron flow around PSI in these chloroplasts.

Chlorophyll-a/b proteins in state transition

The current model of thylakoid membrane reorganization following State I-State II transition proposes that a phosphorylated C h l - a / b - L H C I I complex migrates from grana to stroma membranes. Although it has been shown that the mobile complex is a subpopulation of the L H C I I (Larsson and Andersson 1985; Bassi et al. 1988 a), little is known about the changes in the distribution o f other complexes between appressed and unappressed membrane regions. We have approached this problem by isolating stroma lamellae from dark-adapted (State I) and high-light-adapted (State II) maize leaves and by identifying the presence of individual complexes by Western blotting with specific antibodies. Our results indicate that there are no changes in the a m o u n t of the PSII R C components in stroma lamellae upon state transition. The polypeptide composition of the mobile L H C I I is different from that of the whole L H C I I complex in that it lacks at least a 26 k D a component. This confirms previous reports on the polypeptide composition of the mobile L H C I I population (Bassi et al. 1988a) and shows that the extreme lateral heterogeneity of thylakoid components is maintained upon state transition for all of the complexes except for a very specific L H C I I population. We thank Professor G. Hoyer-Hansen (Carlsberg Research Center, Copenhagen, Denmark) and S. Darr (Department of Biology, Indiana University, Bloomington, USA) for their gift of the monoclonal antibodies. Dr. Roberto Barbato (Dipartimento di Biologia, Padua, Italy) is thanked for his assistance in the preparation of PSI-LHCI. We are grateful to Dr. Paola Dainese (Dipartimento

285 di Biologia, Padua, Italy) for help in the purification of Chl-a/b complexes and to Dr. Francis-Andr6 Wollman (Institut de Biologie Physico-Chimique, Paris, France) for critically reading the manuscript.

References

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