Journal of Photochemistry

J. Photochem. Photobiol. B: Biol. 49 (1999) 50-60 ELSEVIER

Multiple light-harvesting II polypeptides from maize mesophyll chloroplasts are distinct gene products

Claudio De Luca ap1, Claudio Varotto bp2, Ib Svendsen ‘, Patrizia Polverino De Laureto d, Roberto Bassi b**

a Dipartimento di Biologia, Universitri di Padova, Padua, Italy b Dipartimento di Biotecnologie Vegetali, Universitir di Verona, Strada L.e Grazie, 1-37143 Verona, Italy

’ Carlsberg Research Centre, Gamle Carlsbergweg, DK-2500 Copenhagen-Valby, Denmark ’ Centro di Ricerca Interdipartimentale per le Biotecnologie Innovative (CRIBI), Universitri di Padova, via Bassi, I-35121 Padua, Italy

Received 28 July 1998; accepted 18 January 1999

Abstract

The major light-harvesting complex of photosystem II in higher plants is known as LHCII. It is composed of a number of chlorophyll- binding proteins sharing epitopes with each other. The number of apoproteins resolved by fully denaturing sodium dodecylsulfate polyacryl- amide gel electrophoresis varies in different species. In order to know if this heterogeneity is caused by the expression of a number of homologous genes or if it is the product of post-translational modifications, we have resolved the six major apoproteins of Zeu mays LHCII. Each protein is purified to homogeneity, subjected to direct protein sequencing and the sequences compared with those deduced from Zhcb genes in maize and other organisms. All of the six proteins are distinct gene products, since they show differences in their primary structure. Three apoproteins are identified as products of type I lhcb genes and one each as type II and type III gene products. A sixth protein does not fit the requirements for any of the Zhcb genes so far cloned and is therefore probably the product of an Zhcb gene type not yet described. Our results clearly show that the major source of LHCII protein heterogeneity is the expression of many lhcb genes. Fractionation of maize LHCII by non-denaturing flat-bed isoelectric focusing resolves at least five major isoforms showing distinct differences in their polypeptide com- position and also differing in their spectroscopic properties, thus suggesting that individual Lhcb gene products have distinct pigment-binding properties. 0 1999 Elsevier Science S.A. All rights reserved.

Keywords: Chlorophyll-proteins; lhcb genes; Photosynthesis

1. Introduction

In photosystem II (PSII) of higher plants, the light- harvesting function is ensured by several different pigment- proteins which bind approximately 230 chlorophyll mole- cules per PSI1 reaction centre [ 1,2]. These are the chloro- plast-encoded PsbA,B,C,D gene products which bind around 50 chlorophyll (Chl) a molecules, and the nuclear-encoded Lhcb1,2,3,4,5,6 gene products, which bind both Chl a and b in different proportions [ 3-61. While Ihcb4,.5,6 genes code for proteins (respectively CP29, CP26 and CP24) which are present in small amounts in photosynthetic membrane, at least

* Corresponding author. Tel: + 39-45-809-8916; Fax: + 39-49-809-8929; E-mail: bassiQsci.univr.it

’ Present address: Fidia spa, via Ponte della Fabbrica 3a. I-35031 Abano Terme, (PD), Italy. * Present address: ZIGIA. Max-Plack Institut fiir Ziichtungsforschung, Carl

von Linne Weg 10, D-50829 Cologne, Germany.

60% of the chlorophyll is bound to LHCII complex (the product of lhcb1,2,3 genes) which is peripherally located in PSI1 and is mostly organized into trimers [4,6,7 1.

LHCII is heterogeneous in its composition and a number of closely migrating polypeptides can be resolved by SDS- PAGE, ranging from three in spinach to six in maize and up to eight in petunia [ 8,9], which appear to have higher immu- nological relatedness to each other than to other Zhcb proteins [ lo]. Nonetheless, physiologically distinct roles have been reported for the different LHCII polypeptides; in particular, the fastest-migrating band in urea gels was shown to remain unphosphorylated during State I-State II transitions and to be exclusively located in grana partitions, in conditions where other LHCII polypetides undergo migration into stroma lamelleae [ 11 I. Important differences in both the extent of phosphorylation and lateral migration have also been described for other LHCII polypeptides [ 12,131. The LHCII proteins are encoded by a family of nuclear genes: it was first

101 l-1344/99/$ - see from matter 6 1999 Elsevier Science S.A. All rights reserved. PIISIOII-1344(99)00016-O

C. De Luca et al. /J. Photochem. Photobiol. B: Bid. 49 (1999) SO-60 51

shown in petunia that up to 16 genes falling into five sub- families can be identified [ 141.

A similar pattern has so far been recognized in several species [ 151; however, the relation between genes and poly- peptides has not been fully established yet. Since the LHCII polypeptides are synthesized as larger precursors, which are imported into the chloroplast and processed to the mature size concomitantly to their insertion into the thylakoids [ 16-181, the electrophoretic heterogeneity of these polypeptides can derive from a number of sources and the different SDS-PAGE or IEF-PAGE bands can thus be: 1. Products of distinct genes [ 191. 2. Post-translational modification of one or few primary

translation products by phosphorylation [ 201, as previ- ously shown for CP29 [ 2 11, or palmitoylation [ 221.

3. Product cleavage of a precursor protein at different mat- uration sites [ 231. In the case of Zea mays, up to six polypeptides can be

clearly distinguished into a single-dimension SDS-urea- PAGE [ 81, while at least 12 genes can be described which have distinct expression patterns [ 241. In an attempt to estab- lish a correlation between lhcb genes and gene products, in this study we have isolated LHCII from maize mesophyll thylakoids and fractionated the complex by high-resolution two-dimensional SDS-PAGE. Six LHCII apoproteins were isolated and a partial amino acid sequence was determined after cleavage with Lys-c endopeptidase and CNBr. Although highly homologous, the six polypeptides have distinct differ- ences in their primary sequences, thus showing they are dis- tinct gene products.

Fractionation of maize LHCII by non-denaturing flat-bed IEF resolved at least five major isoforms showing distinct differences in their polypeptide composition; the isoforms also differed in their spectroscopic properties, thus suggesting that individual Lhcb gene products have distinct pigment- binding properties.

2. Materials and methods

2.1. Plant material and membrane isolation

Zea mays seedlings were grown in vermiculite for two to three weeks at 27/21”C (day/night) with a 12 h photoperiod and 300 FE m-* s-* light intensity. Leaves were homoge- nized and mesophyll chloroplasts were isolated as previously

described [ 31. PSI1 membranes were isolated by the proce- dure of Berthold et al. [ 251 with the modifications described by Dunahay et al. [ 261.

2.2. Isolation of LHCII complex

LHCII was purified by sucrose gradient ultracentrifugation after solubilization with DM as previously described [ 4,5] . Sucrose gradient band 3 was used, which did not contain any

contamination by CP29, CP26 and CP24 as checked by West- em blot analysis [ 51.

2.3. Isolation of individual LHCII apoproteins

The LHCII complex was fractionated by two-dimensional preparative electrophoresis: the complex was denatured by boiling for 1 min in the presence of 50 mM Trls-glycine pH 9.0, 6 M urea, 1% mercaptoethanol and loaded into a 12- 18% acrylamide gradient SDS-6 M-urea-PAGE gel (350 mm x 350 mm X 2mm) including the Tris-sulphate buffer system [ 271. To prevent N-terminal blocking, acrylamide and urea solutions were deionized with mixed-bed Amberlite resin (Bio-Rad) and 1 mM 2dimethylaminoethanethiol hydrocloride was included in the lower buffer. After running at 20 mA for 72 h, the gel was soaked in 20% methanol then stained for 30 min in 0.5% Coomassie brilliant blue R, 10% acetic acid, 30% isopropanol and destained in 5% acetic acid, 16.5% methanol. To avoid comigrations, a further fraction- ation system was applied: individual Coomassie-stained bands were excised from the gel and loaded into a second gel without urea and including the buffer system of Schaeger and von Jagow [28]. Following electrophoresis, staining and destaining, the bands were excised from the second gel, thor- oughly washed with distilled water and the proteins electro- eluted by using an ISCO device according to Hunkapiller et al. [29].

2.4. Proteolysis and blotting into PVDP

After electroelution, the apoproteins were digested with Lys-C endopeptidase (Boehringer) overnight at 37°C or fragmented with CNBr [ 301 and the peptides loaded into a Tris-Tricine SDS-PAGE ( lO-16% acrylamide gradient). After electrophoresis the gel was blotted into polyvinylidene difluoride using the cyclohexylamino propanesulfonic acid buffer, which did not contain glycine. The filter was stained with Poinceau red and the fragments excised [ 3 11.

2.5. Amino acid analysis

Amino acid analyses were performed using the Millipore- waters Pica-Tag workstation and the Nova-Bondapack C 18 column (4.6 X 150 mm) connected to a Pet-kin-Elmer (Nor- walk, CT) model 7700 utilizing the computer program Chrom-3. Purified apoproteins were subjected to acid hydrolysis on the Pica-Tag workstation for 1 h at 150°C using 200 I.L~ of 6N HCl containing 0.1% phenol and then deriva- tized with phenyl isothiocyanate (PITC) .

2.6. Sequence analysis

N-terminal sequencing of intact proteins as well as of peptides obtained with Lys-C endoproteinase or CNBr was carried out according to the method of Edman [32] on an Applied Biosystems sequencer, model 472A, equipped with

52 C. De Luca et al. /.J. Photochem. Photobiol. B: Bid. 49 (1999) 50-60

an on-line PTH-amino acid analyser, model 120A, according to the protocol given by the manufacturer.

2.7. Fractionation of LHCII

For isolation of LHCII isoforms, grana membranes, Tris- washed for removal of extrinsic polypeptides, were solubi- lized with 1% DM and fractionated by flat-bed IEF as previously reported [ 33 1. 12 green bands were resolved in the pH range 4.06-6.5, five of which contained LHCII (pI=4.06,4.09,4.13,4.17 and4.2) as determinedbyabsorp- tion spectra and immunoblotting with a-LHCII antibodies. These preparations were further fractionated by ultracentri- fugation in 0.1-l M sucrose gradients containing 10 mM Hepes pH 7.6 and 0.06% DM. From each IEF fraction two sucrose gradient bands were resolved at 0.25 and 0.45 M sucrose, respectively. The upper band contained monomeric LHCII, as assessed by green gel analysis using a Deriphat PAGE system [ 341, and small contaminations by CP24. The lower band contained trimeric LHCII and did not contain any contamination as assessed by SDS-PAGE and immunoblot- ting using antibodies specific for each Lhcb protein [ lo]. Densitometry of the gel was performed using a Bio-Rad GS- 670 scanning densitometer. Scans were analysed using a Gaussian deconvolution routine by Origin 4.1 data analysis software ( MicrocalTM, Northampton, MA, USA).

2.8. Spectroscopic measurements

Absorption spectra were recorded using an Aminco DW 2000 spectrophotometer operating in the split beam mode using 1 cm pathlength cuvette. The buffer was 10 mM Hepes pH 7.6, 0.06% DM and 0.15 M sucrose. Absorption was about 1 OD at the red peak. Fluorescence emission and excitation spectra were recorded using a Jasco FP-700 spectrofluorimeter.

2.9. Pigment analysis

Chlorophyll determination was according to Porra et al. [ 351. HPLC analysis of LHCII pigments was performed according to previous reports [ 361.

3. Results

3.1. Primary sequence analysis

When fractionated by high-resolution SDS-urea-PAGE, LHCII from Zea mays mesophyll thylakoids was resolved into six distinct Coomassie-stained bands indicated as OL to 5 in Fig. 1. When assayed with o-LHCII antibody not cross- reacting with other Lhc polypeptides [ lo], all the bands were recognized, thus suggesting all of them were LHCII apopro- teins (not shown). When subjected to further SDS-PAGE with a different buffer system, each band from the first dimen-

L B T

Fig. 1. High-resolution Tris-sulphate SDS-urea-PAGE analysis of maize mesophyll thylakoids (T), PSI1 membranes (B) and of the purified LHCII complex (L )

sion yielded a major band in the second dimension and one or more minor bands, which were also recognized by the CK- LHCII antibody, thus suggesting they also were LHCII apo- proteins. However, due to their low abundance, these bands were not further analysed. The six major bands were elec- troeluted and subjected to quantitative amino acid analysis, which showed distinct differences in their composition (not shown), thus suggesting the bands were probably the result of primary sequence differences rather than of post-transla- tional modifications such as phosphorylation or acylation. We then verified whether these differences were due to cleav- age of a common precursor protein into products with decreasing length [ 23,371, or if each band was a distinct gene product. To this end we blotted the isolated proteins into immobilon-P membrane and subjected them to N-terminal sequencing.

All the proteins proved to be N-terminally blocked, except the most fast-migrating one, LHCIIS, which yielded the sequence in Fig. 2. The upper bands, LHCIIa-e, were then treated with Lys-C endopeptidase and the fragments anal-

C. De Luca et al. /J. Photochem. Photobiol. B: Biol. 49 (1999) 50-60 53

Maize LHCIIC (1) GNDLWYGPDRWKYLGPFSAQTPSYLTXE Tomato (2) _-e--m -------------------NG-...

Barley (3) s------------------------NG-... Arabidopsis 14) ----G-----V-------V--SV--

Fig. 2. N-terminal sequence of the LHCIIC apoprotein from maize. For comparison two cDNA-deduced sequences and the N-terminal sequence of a LHCII polypeptide from Arabidopsis thaliana are reported. For sequences (2)) ( 3), (4) only residues different with respect to LHCIIC are shown, identical residues being indicated by dashes. (1) This study; (2) Ref. [44]; (3) Ref. [45]; (4) Ref. [42].

LHCII a LHCII p LHCII y LHCII 6 LHCII E LHCII [ Consensus maize LHCII genes (1)

LHCII a LHCII p LHCII y LHCII 6 LHCII E LHCII c Consensus

LHCII a LHCII p LHCII y LHCII 8 LHCII 8 LHCII c Consensus

LHCII a LHCII p LHCII y LHCII 6 LHCII E

LHCII c Consensus

LHCII a LHCII p LHCII y LHCII 6 LHCII E LHCII r; Consensus

Ll J.20 ***0*+*-o* * * a l oto**t** a*o* *****+*“*** l ***

................................ .KTARQP ..................... _

............................................................

................................. KTAsPKPa .................

........................................ .KSVPQSIWYGPDR .........

......................................... KSVPQSIWYGPDRPKYLGP

............................................ GNDLWYGPDRPKYLGP . MAssTMAlsStAfaGkavnvpsssfgEaRvTMRKTaaKaKpvaaSGSPWYGpDRVlYLGP

aa it r mv tpikgtrglaf g i vg p -a-s a k 1 aa --dv r

d40 &60 &80 044 ***************** l ****************** l **oo ~I*******~*

................................ KNFJELEVIHSRWAMLGALGlJJFPE& ...

............................... AKNgELEVI.....MLGALGQVFPEE ..

............................... AKNEELEVIHS ..................

................ YGWDR ......... ..KN~ELEVIHSRWAMLGALG~VFPE~ LAA F ............................... KNVELEVI ... ..MLGALG ......... FSAQTPSYLTXE......................T ......................... 1SGePPSYLTGEFPGDYGWDTaGLSADPETFAKNRELEVIHsRW~lgaLGCVFPELLaR f s e C iavg S

&loo $120 J.140 l ***t**o* ************* *****~******************~***~~***

... KFGEA\rXF .................................................

.................. SEELDNPXD ................................ --

.................. SEGGLDYLXNPS...................AVEGYRIAGG P NGVKFGEAVWFKAGXQ....,

-- ......................................

............................................................

............................................................ NGVKFGEaVwFKAGSQIFSEGGLdylGNPSLiHAQSILAIWACQWLMGAvEGYriAGGp

99 shp i hv r

J-160 ;I180 &200 ********* **'******~**** * ~'"****"*~*****~****************~* ............................. KVKELflGRL ..................... ............ SFDPLGLADDPEIJF ....... KGRLAMFSMFGFFVQAIVTGKG ..- ... LG ........ ..~FDPLGLADDPE$FAELKVKELI(NGRLAMFSMFGFFVQAI ........ ....... VYPGGSFDPLGLADD ...... ...... KVYPGGAFDPLGL

.KVKELI(NGRLAMFSMFGFFVQAIVTGKGPLE .........................................

.......................................................... LGEWDPLYpGGsFDPLGLaDDPEaFaelKVKElKnGRLAMfSMFGFFVQAIVTGKGPlE

1t g r gdv i k 1 i

d 220 l ****o*****************t0

.........................

.........................

.........................

N ........................

.........................

.........................

NLADKiaDPVNNNAWAYATNFVPGn It k

Fig. 3. Partial protein sequences of the six LHCII apoproteins isolated from maize mesophyll compared to the consensus sequence for Type I LHCII polypeptides deduced from maize cDNA and genes. Residues responsible for Chl coordination are in bold; underlined, amino acids differing in at least one of the proteins sequenced; *, residues fully conserved; ‘, conservative substitutions. Numbering of residues refers to consensus Zhcbl sequence with #1 corresponding to the first residue of the mature protein. Secondary structure obtained from crystal structure [ 601 is as follows: residues 55-89, helix 1; residues 90-122, loop 1; residues 123-143, helix 2; residues 144-169, loop 2; residues 170-199, helix 3; residues 205-214, helix 4.

54 C. De Luco et al. /J. Phorochem. Photobiol. B: Biol. 49 (1999) SO-40

ysed following SDS-PAGE and blotting into immobilon-P. Partial sequences obtained from all the six proteins were compared to the consensus sequence deduced from the .%a mays Zhcbl,2 and 3 genes so far cloned (see Refs. [ 48-521 and Fish and Bogorad, unpublished results). In the N-ter- minal region sequences were obtained from five out of six proteins, showing that LHCI1-y was different from the CK subunit by two substitutions in positions 5 and 6, while posi- tions 10 and 11 made it different from LHCII8 (Fig. 3, under- lined residues). In this region subunits 6 and E had identical sequences while subunit 5, which had a free N-terminal, started at position 13 with four amino acids that were different with respect to subunits 6 and E. Sequences from five subunits were also obtained in the first o-helix region, showing that subunit B, whose sequence was not determined near the N- terminal, differed with respect to 01, B, S and E by respectively 3,1,3 and 1 amino acid positions. Further distinction between B and y subunits came from the sequences in the helix l- helix 2 loop, where the two sequences differed by five posi- tions over a nine amino acid overlapping fragment. Addi- tional differences were determined between subunits E and p, y, 8 at position 161, between I3 and y at position 173 and between B and (Y, y, 6 at position 184.

(4 LHCII IEF 1

3.2. Fractionation and characterization of LHCII isoforms from Zea mays membranes

In the following we addressed the question of whether the presence of distinct gene products in the LHCII complex had any effect on the spectroscopic and pigment-binding prop- erties of the complex. To this end we fractionated PSI1 mem- branes by non-denaturing flat-bed preparative IEF followed by sucrose gradient ultracentrifugation. Five major isoforms were obtained having isoelectric points in the 4.06-4.2 range. Small contaminations by minor chlorophyll-proteins that could affect measurements were eliminated by using only the lower band from the ultracentrifugation step containing tri- meric LHCII. SDS-PAGE analysis showed that the five iso- forms IEFl-5 differed in their polypeptide composition and relative abundance (Fig. 4(a) ) as quantified by densito- metric analysis (Fig. 4(b) and Table 1). The lower band, LHCIIC, for example, was one of the major components of IEFl while it was absent in IEF4 and 5. The trimeric LHCII isoforms were then analysed by absorption spectroscopy. Upon normalization at their red-most peaks, the spectra showed distinct differences both in the Qy transition and in the Soret region (Fig. 5( a,b) ) . As for the Qy region, differ-

IEF?

lEF2 IEF3 IEF4 IEF5

, I I I I 2 I I 1 1 I

5 10 15 20 25 30 35

Electrophoretic mobility (arbitrary unite) Fig. 4. (a) SDS-polyacryhunide gel of five LHCII isoforms obtained by IEF followed by sucrose gradient ultracentrifugation. Each lane was loaded with 2 pg of chlorophyll. (b) Densitometric profile of the gel shown in (a). As an example Gaussian deconvolution is shown for IEF5 (note the almost perfect fit between the original profile (solid) and that obtained as the sum of Gaussian components (broken) ) ,

C. De Luca et al. /J. Photochem. Photobiol. B: Biol. 49 (1999) 50-60 55

IEFI IEF2 IEF3 IEF4 IEF5

I I I I I 1 300 400 500 600 700 800

(a) Wavelength (nm)

0.30 F

0.25 -

-0.05 I I I 1 I 300 400 500 600 700 800

@) Wavelength (nm) Fig. 5. (a) Absorption spectra of LHCII isoforms IEFl-5. Spectra were normalized at their red-most peak. (b) Difference absorption of isoforms IEFl-4 with respect to IEFS. (Spectra were shifted on they axis to avoid superimposition.)

Table 1 Polypeptide composition of the five LHCII isofonns ( IEFl-5) obtained by IEF followed by sucrose gradient ultracenttifugation as calculated by den- sitometry of the gel shown in Fig. 4(a). The amount of each polypeptide has been expressed as a percentage with respect to the whole protein amount of each fraction

Polypeptide IEFl llSF2 IEF3 IEF4 IEFS

LHCIIu 23.32 20.32 25.52 13.40 22.65 LHCIIp + y 25.39 25.58 30.38 36.62 64.38 LHCII8 16.13 28.48 22.95 28.58 5.56 LHCIk 12.11 7.31 15.74 21.40 7.41 LHCIIC 23.05 18.31 5.42 0 0

ence spectra showed distinct components in the 652-672 nm range as well as at around 684 nm. In the Soret region com-

ponents were resolved at 431,454,473 and 488 nm, indicat- ing that both differences in chlorophyll and xanthophyll absorptions were involved. I-PLC pigment analysis of IEF l-5 shows that, although very similar, they differ in pigment content. In particular, the Chl a/b ratio ranged from 1.38 in IEF5 to 1.47 in IEF4, while the Chl to xanthophyll molar ratio was between 3.75 and 4.35 (Table 2). Most evident was the difference in violaxanthin content, three-fold higher in IEF4 with respect to IEFl. We finally performed fluorescence analysis in order to verify the functional properties of LHCII in the different isoforms. Fluorescence emission spectra showed a single peak at 685 nm, whose shape was identical irrespective of whether it arose from Chl a (440 nm) or Chl b (475 nrn) excitation, while the fluorescence excitation spectra closely matched the absorption spectra in the 350-

56 C. De Luca et al. /J. Photochem. Photobiol. B: Biol. 49 (1999) 50-60

Table 2 Pigment content of LHCII isoforms IEFl-5. HPLC analysis of isoforms IEFl-5 was performed as described in Ref. [ 361. Data are expressed in moles of pigment per 100 moles of CM a. Values are the average of five determinations. Deviation was lower than 5%

Isoform Chlalb ChlT/CarT Neoxanthin Violaxanthin Lutein Chl b

IEFl 1.45 4.30 12.73 1.15 25.33 68.76 IEF2 1.46 4.35 12.81 1.57 24.44 68.72 IEF3 1.40 4.02 14.22 2.35 26.09 7 1.42 IEF4 1.47 3.75 13.20 3.32 28.35 68.04 IEFS 1.38 4.04 12.30 2.55 27.90 72.59

500 nm region (not shown). Measured fluorescence emission spectra for all isoforms closely corresponded to those calcu- lated on the basis of the Stepanov relation [ 38,391, suggest- ing that excitation energy was delocalized over the whole trimeric complex (not shown).

4. Discussion

The nuclear encoded Chl a/b proteins of PSI1 are organized into two groups depending on both their topological location within the PSI1 unit and their relative abundance with respect to the reaction-centre complex. Thus the minor Chl a/b pro- teins (CP29, CP26 and CP24) are closely connected to the PSI1 core [ 5-7,401 where they together bind 10% of the total PSI1 chlorophyll versus 43% of the peripherally locatedmajor LHCII complex [4]. The minor chlorophyll-proteins have been correlated with their corresponding genes and are named Lhcb4, Lhcb5 and Lhcb6, respectively, for CP29, CP26 and CP24 [ 411. The case of the major LHCII is not clear yet: a number of genes have been cloned and sequenced and a wide number of protein bands can be resolved from LHCII prep- arations, depending on the species studied. A first attempt to correlate genes and proteins showed that the lhcbl and lhcb2 genes can be respectively correlated with the 27 and 25 bands, which can be obtained from a number of species using low- resolution gels without urea [ 191. However, a third LHCII protein, having lower apparent molecular mass, was soon discovered [42,43] that did not undergo phosphorylation. The corresponding gene was cloned [44,45]. More recently, the use of high-resolution urea gels allowed resolution of up to six or even eight LHCII bands [ 8,9]. The different bands may arise from expression of independent genes or can be produced by post-translational modification such as phos- phorylation or palmitoylation [ 20,221, or cleavage of a com- mon precursor to successive maturation sites [ 16,23,37,46]. The latter mechanism has been shown to produce multiple forms of fully active chloroplastic carbonic anhydrase in pea [ 471. None of the multiple LHCII polypeptides with decreas- ing apparent molecular weight analysed in this study showed up as being the product of processing at different sites; rather, they all showed distinct primary sequences. Our results here- fore show that the major source of LHCII apoprotein

heterogeneity is the expression of distinct genes, although we cannot discard the hypothesis that further differentiation (in a lower scale) may be due to post-translational modifications, since we have only analysed the most abundant LHCII polypeptides and discarded the electrophoretic fractions of low abundance.

In Fig, 6 we compare the partial sequences of the six LHCII maize polypeptides with the consensus sequences for the three LHCII gene types so far described with the aim of establishing a relation between genes and gene products. This was an easy task in the case of the 4 subunit, which could be recognized as a type III gene product on the basis of its lack of the first 12 amino acids as well of its almost complete identity with the two Zhcb3 sequences so far reported over the first 26 residues (Fig. 2).

The sequences of the other subunits had to be analysed on the basis of the amino acids present at the type-specific posi- tion as previously indicated by Jansson et al. [ 191. On this basis LHCIIol and /3 can be attributed to a type I group because of a Lys residue in position 2 and a Ser in position 161, respectively. Subunit y can also be identified as the product of a type I gene on the basis of the amino acid residues in three type-specific positions ( Lys-2, Ala- 145 and Ser- 16 1) . LHCIIE, on the other hand, is attributed to type II genes on the basis of residues Gln-14, Ile-16 and Ala-161 (Fig. 6). It was not possible, on the contrary, to attribute LHCIIG to any of the three types so far identified, since this polypeptide showed type II specific amino acids in positions 14 (Gln) and 16 (Ile) but a type I residue (Ser) in position 161. In addition the type-specific position #88 is neither Arg (type I) nor Lys (type II) but Ala.

The analysis of the five c-DNA and gene sequences so far published from maize [48-521 showed that they all fit the requirement of the type 1 lhcb genes consistently with the lack of introns in the gene sequences. Our results show that not only type I but also type II and III LHCII genes are expressed in maize mesophyll chloroplasts which were already shown to contain Lhcb4,5 and 6 gene products [ 31. Moreover, an additional gene product is identified, which has distinctive differences with respect to the Lhcb1,2 and 3 pro- teins and therefore may represent a member of an additional type of lhcb gene. Cloning of the gene corresponding to this protein is needed in order that the complete primary sequence can be compared with that of other Zhc genes. Although many Lhcbl DNA sequences have been reported so far, none of our three type I protein sequences fully fits a DNA sequence. The best alignment was obtained between the lhc1*2 gene 1491 and the LHCIIy protein which, however, differed by four positions over the 86 available in the N-terminal region. This is not surprising in the light of the results of Sheen and Bogorad [241, who reported on the presence of at least 12 independent LHCII genes in maize. It is therefore possible that all Zhcb genes are expressed, although at different rates. In fact, the six independent proteins we have characterized represent the most abundant ones, while others were not

C. De Luca et al. /J. Photochem. Photobiol. B: Biol. 49 (1999) 50-60

LHCII a LHCII y LHCII 6 LHCII E LHCII f

Type I -

Type II -

Type III-

YGP YGP YGP GPFSAQTPSYLTXE

M YGP LGPFSGESPSYLTGEFPGDYGVDTA -50 : : :::::::::::::::::

M YGE LGPFSEQTPSYLTGEFPGDYGVDTA -46 : : ::::: ::: ::::::::::::

YGP LGPFSAQTRSYLNGEFPGDYGVDTA -38

LHCII y KNRELEVIHSRWAMLGALGCVFPEL

Type I - GLSADPETFAKNRELEVIHCRWAMLGALGCVFPEL G-VKFGEAVWFK -100 :::::::::: :::::::::::::::::::::::

Type II - GLSADPETFARNRELEVIHCRWAMLGALGALGCVFPEI ::"::' . . . : : :: ::::: :::: :::::: : : : ::::

Type III- GLSADPEAFAKNRALEVIHGRWAMLGALGCIFPEVL KVDFKEPWFK -89

LHCII y SEGGLDYLXN AVEGYR GPLG

LHCII p LHCII y LHCII 6 LHCII E

FDPLGLADDPEKF FDPLGLADDPEAFAELKVKELKNGRLAMFSMFGFFVQAI

VYPG FDPLGLADD WYPG FDPLGL

Type I - EVVDPLYPG FDPLGLADDPEAFAELKVKEIKNGRLAMFSMFGFFVQAI -200 :: : : : : :::::::::::::::::::::::::::::::::::

Type II - EGLDKIYPG FDPLGLADDPEAFAELKVKEIKNGRLAMFSMFGFFVQAI -196 : : : : :: :::::: :::::::::::::::::::::::::::

Type III- GEGNDLYPG FDPLGLADDPTTFAELKVKEIKNGRLAMFSMFGFFVQAI -190

Type I - VTGKGPLENLADHLADPVNNNAWAFATNFVPGK -233 :::::: ::: :: :::: ::::::::::::::

Type II - VTGKGPIENLSDHIADPVANNAWAFATNFVPGK -229 : : : : ': : ::: : : : : : : :: :: : : : : :

Type III- VTGKGPLENLLDHLDNPVANNAWVYATKFVPGK -223 Fig. 6. Sequence of peptides containing type-specific amino acid position compared to the consensus for types I, II and III Ihcb-deduced sequences. Boxed residues refers to type-specific residues for type I and type II lhcb genes as defined in Ref. [ 191.

sequenced due to their low abundance and N-terminal blocking.

This raises the question of what can be the function of such a variety of highly homologous antenna proteins. Our results support the statement, from DNA sequences [ 531, that most of the differences that do occur between LHCII polypeptide sequences are located near the N-terminal domain. This domain can be phosphorylated during a State l-State 2 tran- sition while the kinetics of phosphorylation, as shown by the use of synthetic peptides [54], can be influenced by the presence of basic residues near the phosphorylation site. Crystal structure [55] showed that most of the predicted subunit-subunit interactions of the LHCII monomer will be mediated by residues in the N-terminal domain, while the

conformation of this part of the molecule has been proposed to be modified following phosphorylation, thus interfering with energy transfer between facing opposite LHCII com- plexes in opposite membranes of the thylakoid partitions [ 56,571. It can be hypothesized that differences in the pri- mary structure can thus provide modulation of the physiolog- ical effect of LHCII phosphorylation in different LHCII subpopulations, which have distinct topological location within the PSI1 supramolecular complex [ 5 ] as well as defin- ing the location of individual Lhcb gene products during PSI1 assembly.

Fractionation of maize grana membranes by flat-bed IEF followed by sucrose gradient ultracentrifugation yields five major LHCII isoforms with a trimeric structure, as confirmed

58 C. De Luca et al. /J. Phurochem. i’hatabioi. B: Sol. 49 (1999) 5C-60

by their mobility in sucrose gradient and in green gels. The absorption spectrum of pigment-proteins is determined by their chromophore composition and by pigment-pigment and pigment-protein interactions (see Refs. [58,59] for reviews). If the different LHCII gene products have the same pigment composition and interactions, it should be expected that all LHCII isoforms have the same spectrum. Different LHCII isoforms, nevertheless, show distinct differences in both the absorption spectrum and pigment content, while fluorescence excitation and emission spectra indicate com- plete energy transfer from Chl b to Chl a and equilibration of excitation energy over the trimeric complexes, therefore sug- gesting that these complexes are fully functional and did not undergo denaturation during purification procedures. LHCII binds 12 Chl molecules per polypeptide [ 4,601, for eight of which the residues responsible for coordination have been identified [60] and are conserved in all Zhcbl,2,3 genes so far sequenced as well as in LHCII polypeptides characterized in this work. The possibility remains, however, that changes in the sequence influence pigment-protein interactions around the pigment-binding sites not yet characterized. Although the changes detected in the Chl a/b ratio (Table 2) are consistent with the hypothesis of a different number of pigment molecules perpolypeptide (e.g., some gene products binding 12 Chl and others binding 13 chlorophylls), the alter- native hypothesis can be considered that an individual site can be occupied by either Chl a or Chl b although with different probabilities, as previously shown for the homolo- gous proteins CP29 [61] and CP24 [ 621. In this case small sequence changes could well modify the selectivity of a Chl binding site for Chl a versus Chl b. Similar considerations can apply to the differences in xanthophyll content: two xan- thophyll binding sites have been identified in the LHCII struc- ture [ 601, while the location of a third site (Table 2) is still unknown. On the basis of 12 Chl per polypeptide, slightly less than two lutein molecules and one neoxanthin molecule per polypeptide can be attributed. Violaxanthin might either bind to one of the lutein or neoxanthin sites or bind to a fourth site. Acclimation to high-light conditions yields a threefold increase in violaxanthin content [63] which is bound to LHCII [ 641. The differential expression of different lhcbl- 3 genes could well be, at least in part, responsible for such an adaptive response.

5. Abbreviations

ol-LHCII antibody against LHCII CNBr cyanogen bromide CP chlorophyll-protein complex Chl chlorophyll DM dodecyl P-D-maltoside Hepes (N- [ ZHydroxyethyl] piperazine-N’- [ 2-

ethanesulfonic acid] ) HPLC high-pressure liquid chromatography IEF isoelectrofocusing

LHCII OD PSII PAGE SDS Tris

major light-harvesting complex of PSI1 optical density photosystem II polyacrylamide gel electrophoresis sodium dodecylsulphate tris [ hydroxymethyl] aminomethane

Acknowledgements

We would like to thank Dr Roberta Croce for performing HPLC analysis, Dr D. Dalzoppo (Department of Organic Chemistry, University of Padua) for amino acid analysis and Professor Lawrence Bogorad for sending preprints of his work and sequence data before publication. This work was supported by ‘Progetto Finalizzato Biotecnologie’ of the CNR (National Research Council) and by the Ministry of University and Scientific Research.

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