Photosynthesis Research 11:141-151 (1987) © Martinus Nijhoff Publishers, Dordrecht - - Printed in the Netherlands

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Regular paper

Evidence for a light-harvesting chlorophyll a-protein complex in a chlorophyll b-less barley mutant JACQUES DURANTON and JEANETTE BROWN Service de Biophysique, C.E.N. Saclay, 91191 Gif-sur-Yvette, France Carnegie Inst. Washington, Stanford, CA 94305, USA* (Received: 23 January 1986; accepted: 26 March 1986) Key words: chlorophyll a.b proteins, Chlofina-f2 mutant Abstract. Chloroplasts of a chlorophyll (Chl) b-less barley mutant were solubilized with digitonin and fractionated by polyacrylamide gel electrophoresis with sodium deoxycholate in the running buffer. By this procedure, in contrast to using sodium dodecylsulfate (SDS) for solubilization, a Chl a-protein analogous to the major lightharvesting Chl a-b protein complex from wildtype chloroplasts was recovered. This mutant Chl a-protein comprises about fifty percent of the total Chl a, and is very similar in carotenoid, amino acid, protein and polypeptide composition to the major wildtype antenna Chl a-b protein. The only major differences we have found is its instability in the presence of SDS and sensitivity to protease action. Even with deoxycholate, the mutant Chl a complex often dissociates during electrophoresis into two green bands. The lack of Chl b appears to affect the normal organization of Cht a and protein in such a way as to render the complex more unstable. Introduction

The Chl b-less, Chlofina-f2 mutant o f barley has provided a rich source of material for study of many aspects of chloroplast composition and development [5, 6, 11, 13]. With respect to Chl.protein complexes, several laboratories reported that Chl b-less mutants apparently lacked the green protein band equivalent to the Chl a-b protein complex (LHC) isolated from wildtype plants after solubilization with sodium dodecyl sulfate (SDS) [10, 12, 15, 20, 21 ]. Subsequent studies showed that the polypeptides of the Chl a-b protein are formed in the mutant, but they appeared not to bind chlorophyll [2, 3, 9, 18]. SDS was used in all of these studies to solubilize the membranes and during electrophoresis. Here, we demonstrate that a Chl b-less barley mutant does indeed contain Cht a-protein complexes analogous to the LHC o f wildtype plants. When the mutant chloroplasts are dissociated by digitonin and subjected to gel electrophoresis with deoxycholate (DOC)[17], two green bands containing about 50% o f the total Chl a appear at a similar position in the gel as the major LHC from wildtype plants. The characteristics of these mutant antenna Chl a-protein complexes are described. * CIW-DPB No. 917.

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Methods

Isolation of pigment-protein complexes Seedlings of wildtype and the Chlorina-f2 mutant (originally isolated by Highkin [13]) of barley (Hordeum vulgate) were grown in vermiculite in a glasshouse for about 10 days. Chloroplasts were prepared as in [17] and washed with 50mM Tris-HC1 buffer, pH 8, 5 mM EDTA. The following protease inhibitors (1 mM final concentration) were added to all of the buffers used for chloroplast preparation and electrophoresis: E-amino-ncaproic acid, p-aminobenzamidine, phenylmethylsulfonyl fluoride. Solubilization of chloroplasts and preparative gel electrophoresis also were performed as in [17]. The only essential modification was to increase the digltonin to chlorophyll ~eight ratio from 40 with wildtype to 80 for the mutant. The green bands were cut from the gels and ground finely in a mortar. Distilled water was added to the gel mixture, and the pigment-protein complexes were extracted overnight at 4 °C. The green complexes in the DOCTris-glycine buffer were separated from the gel by centrifugation and f'dtration through glass wool. If necessary, the gels were extracted with more water until they were colorless. The complexes were either sedimented by centrifugation at 100,000g overnight or were concentrated with Centriflo membrane cones (Amicon, CF25).

Protein analyses For analysis of the polypeptides in the pigment-protein complexes by SDSPAGE [1], the sedimented complexes were resuspended in 2% SDS, 50mM DTT, and 50mM Tris-HC1, pH 8 and heated at 100°C for 30s. For comparison, some complexes were first delipidated by making a concentrated extract to 80% with acetone and incubating at 4 °C for 1 h. Then, with the extract in a glass centrifuge tube, ethyl ether was added dropwise until two phases developed. The pigments partitioned to the upper ether phase, and the proteins precipitated at the interface. The tube was centrifuged at 10,O00g for 10 min. After decanting the liquids, N: gas was passed over the sediment to remove all of the organic solvents. The delipidated sediment was taken up in 2% SDS and treated as above. The amino acid content of the pigmentprotein complexes, hydrolysed with 6 N HC1 under vacuum at 110 °C for 24 or 36 h, was measured in an LKB 4400 Amino Acid Analyser.

Pigmen t determination Normally chlorophyll was measured spectrophotometrically using Mackinney's equations [16]. However, for determining the ratios of Chl a to the various carotenoids, High Performance Liquid Chromatography was used. By passing a linear gradient of 85% methanol-H20 to 100% methanol through a RP-8 column in 15 min, 4 xanthophylls, Chl b, Chl a and B-carotene were well

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Figure 1. SDS-polyacrylamide gel electrophoresis profile of barley wildtype (W) and mutant (M) thylakoids after staining with coomassie blue. separated and detected by their absorbance at 445 nm. The instrument (Hewlett-Packard, Model No. 1082b) integrates the area under each peak, and the amount of each pigment was calculated using a coefficient previously determined with a known amount of each purified pigment. The pigments in the isolated Chl-proteins were usually concentrated by transfer from 80% acetone-water extracts to ethyl ether and evaporated under nitrogen gas. P700 concentration was determined from the lightinduced change in absorption at 700nm [14]. Low-temperature (77K) absorption spectra were measured as described in [8].

Results Figure 1 illustrates a typical gel after electrophoresis of chloroplast membranes from wildtype and mutant barley solubilized with SDS. The gels have been stained with coomassie blue. The 23, 25 and 28KD polypeptides attributed to LHC are diminished in the mutant as reported previously [9, 10, 12, 15, 21]. When these same chloroplast membranes were solubilized by digitonin and subjected to gel electrophoresis with DOC in the running buffer, similar green bands were seen from both the wildtype and mutant (Figure 2). The Chl-proteins in each of these bands were extracted quantitatively from the gels and identified by their P700 content and absorption and fluorescence spectra. The spectra of both the photosystem I and II bands (here labeled PS1 and M1, PS2 and M2) were very similar to the spectra of analogous bands from spinach [7], and their polypeptide profiles were also identical (not shown). The spectra of the wildtype barley LHC were also similar to spinach LHCP. However, spectra of bands M3 and M4 were very different (Figure 3). Part of this difference reflects the loss of Chl b absorption at 650 nm, but the fluorescence emission maximum of M3 is near 685 mn while that of M4 is near 680 nm. The emission bands of the mutant LHC complexes are broader than the corresponding wildtype LHC. The light-green band (W4) running ahead of the WT-LHC was also analysed and compared with M4 in some experiments. The proportions of Chl a in each of the electrophoretic bands are shown in Table 1. In some experiments proportionally more Chl a was found in M3 and less in M4, but their sum remained constant. It is remarkable how similar the relative amounts of Chl a are in corresponding complexes when the sum of M3 and M4 (48%) is compared to the wildtype LHC (50%). The molar ratios of Chl a and Chl b, P700, /~-carotene, lutein and violaxanthin in the wildtype and mutant fractions are shown in Table 2. These ratios in wildtype plants are similar to those we have measured in corresponding spinach fractions. In both wildtype and mutant, the reaction center Chl-proteins are enriched in ~-carotene and deficient in lutein and violaxanthin while the reverse is observed in the antenna complexes (LHC, M3 and M4). We compared the relative amounts of Chl a and proteins in the various fractions of wildtype and mutant plants. In the leaves, the amounts of Chl a per gram of fresh weight were similar; 0.8 mg in wildtype and 0.75 mg in mutant leaves. The quantities of protein and Chl a in the unfractionated thylakoid membranes (lameUae) and the three isolated fractions were compared on a weight basis (Table 3). Again, corresponding fractions of wildtype and mutant are remarkably similar. The amino acid composition of barley wildtype LHC and mutant electrophoretic bands M3 and M4 (M3 and M4 were alike) are shown in Table 4 together with that of spinach LHCP reported by Ryrie and Fuad [19].

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Figure 2. Green protein complexes separated by preparative gel electrophoresis with deoxycholate from wildtype and mutant membranes. Gel slices were cut between the indicated lines for LHC, W4, M3 and M4. Proline and tryptophane were not measured in our analyses. The proteins in the wildtype LHC and W4 and mutant M3 and M4 bands were concentrated either by direct centrifugation or after delipidation with organic solvents, treated with SDS and subjected to gel electrophoresis. The same quantity (20/ag) of protein was placed in each lane, and the gels

146 Table 1. The percentage of Chl a in each of the gel bands of wildtype and mutant barley after eleetrophoresis Wildtype

Mutant

PS1 PS2 LHC

34 14 50

M1 M2 M3 M4

40 12 32 16

Table 2. The molar ratios of Chl a to other pigments in gel bands of wildtype and mutant barley after electrophoresis Chl b P700 WT Mut WT Mut PSI >8 * PS2 8 * LHC(M3)# 1.2 *

110 * *

80 * *

B-carotene WT Mut

lutein WT Mut

Violaxanthin Wt Mut

10 12 > 30

18 * 4

>20 * 9

10 10 > 50

>50 > 50 6

>50 > 50 9

# Mutant bands M3 and M4 had the same relative pigment composition. * No pigment detected (high ratio approaching infinity) Table 3, The weight ratios of protein to Chl a in the lamellae and various fractions of wildtype and mutant barley Wildtype lameUae PS1 PS2 LHC

4.5 3.5 -4.0 4.8 2.2

Mutant M1 M2 M3 M4

5.0 4.0-4.5 4.8-5.0 2.0 2.0

were stained with coomassie blue. The results are shown in Figure 4. As expected, the LHC shows two polypeptide bands at 25 and 28 kD with the former being predominant. These two bands are also present in both M3, M4 and W4, but in different proportions. The M3, M4 and W4 complexes also show polypeptide bands at 35 and 20 kD which are not seen in the WT-LHC.

Discussion Early studies of the Chl b-less barley mutant showed that its photosynthetic capacity is similar to that of wildtype on a Chl a basis, but the m u t a n t requires much higher light fluxes to saturate photosynthesis [5]. The fine structure of the m u t a n t chloroplasts is less organized (fewer grana) than that of wildtype [11]. Although the m u t a n t chloroplasts are more susceptible to disruption when treated with digitonin, centrifugation into large and small particles does not produce a separation of the photosystems as it does with normal Chl b-containing plants [6].

147 I

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Figure 3. Absorption and fluorescence emission spectra of three Chl-protein complexes, wildtype LHC and mutant M3 and M4, all measured at 77 K. The emission spectra were measured with a 3 nm slit-width and were excited by light at 438nm, 10nm slitwidth.

All reported analyses of the mutant by SDS-PAGE have found that the polypeptides of the light-harvesting Chl-protein complex (LHC) are diminished both in amount and number [2, 3, 9, 10, 12, 15, 18, 20, 21], and we have confirmed this here (Figure 1). At first [11], no differences in fine structure between mutant and normal chloroplasts were noted, but later a large reduction in the number of particles on the PFs face and about 12% smaller particles on the EFs face of the mutant were observed [20]. However, considerable stacking were still seen in the mutant chloroplasts. Bellemare et al. [3] found that the Chl b-less barley mutant contains mRNA for LHC membrane polypeptides indistinguishable from their wildtype counterparts. In addition, mutant chloroplasts were capable of importing newly synthesized LHC polypeptides, either from wildtype or the mutant, and incorporating them into thylakoid membranes. Ryrie [8] also presented strong evidence for the presence of LHC polypeptides in the mutant even though diminshed in amounts compared to wildtype. Here, we show that after solubilization with digitonin and electrophoresis

148 Table 4. The amino acid composition of LHC from wildtype, M3 or M4 gel bands from mutant barley, and LHCP from spinach [19] in percentage Amino acid

LHC

M3 or M4

LHCP

Asp Thr Ser Gin Pro Gly Ala Val Met lie Leu Tyr Phe His Lys Trp Arg

11.5 4 8 9.3

10.5 5.2 9.5 9.0

17.0 11.0 5.0 0.7 2.7 10.5 2.6 6.2 1.3 6.5

17.0 11.5 4.7 0.7 2.9 10.0 2.6 5.4 1.0 6.9

3.0

3.1

11.0 3.1 5.5 8.8 7.9 13.6 10.2 6.3 1.0 2.6 10.3 2.8 6.4 1.4 5.1 1.0 3.2

with DOC, two green bands are visible from the mutant in the same position as the LHC from wildtype (Figure 2). The proportion of Chl a in the two mutant bands, M3 + M4, was similar to that in wildtype LHC (Table 1). Except for Chl b, the pigment composition and protein content of analogous green bands from mutant and wildtype were also very similar (Tables 2 and 3). On the other hand, the absorption and fluorescence emission spectra (Figure 3) of the mutant bands, M3 and M4, are different from each other as well as from wildtype LHC. Because both wildtype barley and the Chlorina-f2 mutant contain about the same amount of Chl a on a protein basis, the normal Chl b binding-sites on the mutant apoproteins are probably unoccupied. The presence of these vacant sites may affect the molecular organization of Chl a and account for the spectral differences we observe between wildtype and mutant Chl-proteins. Initally, we did not add protease inhibitors to the buffers used for chloroplast isolation and electrophoresis. At that time, although the expected polypeptides were found in all of the wildtype Chl-proteins and in the mutant M1 and M2 green bands after SDS-electrophoresis, we failed to observe any polypeptides in either of the mutant green bands, M3 or M4. It was only after inhibiting protease action that the polypeptides in these latter two mutant green complexes could be recovered (Figure 4). Apparently, not only are the mutant antenna complexes degraded by SDS action, but also they are more susceptible to protease action than are normal Chl-protein complexes. We cannot say from our data whether or not the 35 or 20 kD bands are integral parts of M3, W4 and M4 complexes. Although the wildtype W4 band is relatively light (Figure 2), it runs at nearly the same rate as M4. For this reason, we compared its polypeptide

149

Figure 4. Polypeptide composition of 4 of the green protein bands shown in Figure 2, analysed by SDS-polyacrylamide gel electrophoresis, 20 tzg protein per lane, stained with coomassie blue. pattern with M4 and found it to be very similar (Figure 4). In preliminary experiments with the aid of Marc Lutz at Saclay, resonance Raman spectra of mutant barley thylakoid membranes and the isolated M3 and M4 Chl-proteins were measured. The results show that the binding or association of Chl a with protein is the same in M3 as in the membranes. In M4, the pigments

150 are still attached to protein, but their structure seems less intact. We suggest that with b o t h W4 and M4, the pigment-proteins are beginning to dissociate. The observation that the proportion o f Chl a in M3 and M4 varied somewhat in different experiments, also suggest that small differences in solubilization conditions may cause more or less o f the pigment-proteins to dissociate. Again this dissociation is much greater in the mutant than in wildtype; perhaps because o f the lack o f Chl b and its influence on Chl a-protein binding. The amino acid composition o f either M3 or M4 was very similar to that of wildtype barley or spinach LHC (Table 4). Also wildtype and mutant leaves or pigment.complexes had similar protein contents when compared on a Chl a basis. It does appear that the mutant LHC proteins are relatively unstable; a conclusion also reached by Bennett [4]. However, we conclude that light-harvesting Chl a-proteins can exist in mutant plants which lack Chl b.

Acknowledgements J.D. thanks the Carnegie Institution for Fellowship support, and we b o t h thank Dolores Horvat for technical assistance.

References 1. Acker S, Lagoutte B, Picaud A and Duranton J (1982) Protein identification of purified particles isolated from spinach tilylakoids by deoxycholate electrophoresis. Photosyn Res 3:215-225 2. Apel K and Kloppstech K (1980) The effect of light on the biosynthesis of the light-harvesting chloropbyll a/b protein. Planta 150:426-430 3. BeUemare G, Bartlett SG and Chua N-H (1982) Biosynthesis of chlorophyll a/bbinding polypeptides in wild type and the Chlorina f2 mutant of barley. J Biol Chem 257:7762-7767 4. Bennett J (1981) Biosynthesis of the fight-harvesting chlorophyll a/b protein. Eur J Bioehem 118:61-70 5. Boardman NK and Highkin HR (1966) Studies on a barley mutant lacking chlorophyll b I. Photochemical activity of isolated chloroplasts. Biochim Biophys Acta 126:189-199 6. Boardman NK and Thorne SW (1968) Studies on a barley mutant lacking chlorophyll b II. Fluorescence properties of isolated chloroplasts. Biochim Biophys Acta 153:448-458 7. Brown JS (1983) A new evaluation of chlorophyll absorption in photosynthetic membranes. Photosyn Res 4:375 -383 8. Brown JS and Schoch S (1981) Spectral analysis of chlorophyll protein complexes from higher plant chloroplasts. Biochim Biophys Acta 636:201-209 9. Burke J J, Steinback KE and Arntzen CJ (1979) Analysis of the light-harvesting pigment-protein complex of wild type and a chlorophyll-b-less mutant of barley. Plant Physiol 63:237-243 10. Dunkley PR and Anderson JM (1979) The light-harvesting chlorophyll a/b-protein complex from barley thylakoid membranes. Biochirn Biophys Acta 545:174-187 11. Goodchild DJ, Highkin HR and Boardman NK (1966) The fine structure of chloroplasts in a barley mutant lacking chlorophyll b. Exp Cell Res 43:684-688

151 12. Henriques F and Park RB (1975) Further chemical and morphological characterization of chloroplast membranes from a chlorophyll b-less mutant of Hordeum vulgate. Plant Physiol 55:763-767 13. Highkin HR and Frenkel AW (1962) Studies of growth and metabolism of a barley mutant lacking chlorophyll b. Plant Physiol 37:814-820 14. Hiyama T and Ke B (1972) Difference spectra and extinction coefficients of PT00. Biochim Biophys Acta 267:160-171 15. Machold O, Meister A, Sagromsky H, Hoyer-Hansen G and yon Wettstein D (1976) Composition of photosynthetic membranes of wild-type barley and chlorophyll b-less mutants. Photosynthetica 11:200-206. 16. Mackinney G (1940) Criteria for purity of chlorophyll preparations. J Biol Chem 132:91-109. 17. Picaud A, Acker S and Duranton J (1982) A single step separation of Ps 1, Ps 2 and chlorophyll-antenna particles from spinach chloroplasts. Photosyn Res 3:203213. 18. Ryrie IJ (1983) Immunological evidence for apoproteins of the light-harvesting chlorophyll-protein complex in a mutant of barley lacking chlorophyll b. Eur J Biochem 131:149-155 19. Ryrie IJ and Fuad N (1982) Membrane adhesion in reconstituted proteoliposomes containing the light-harvesting chlorophyll a/b-protein complex: the role of charged surface groups. Arch Biochem Biophys 214:475-488 20. Simpson DJ (1979) Freeze-fracture studies on barley plastid membranes. III. Location of the light-harvesting chlorophyll-protein. Carlsberg Res Commun 44: 305-336 21. Thornber JP and Highkin HR (1974) Composition of the photosynthetic apparatus of normal barley leaves and a mutant lacking chlorophyll b. Eur J Biochem 41: 109-116

Evidence for a light-harvesting chlorophyll a-protein complex in a chlorophyll b-less barley mutant.

Chloroplasts of a chlorophyll (Chl) b-less barley mutant were solubilized with digitonin and fractionated by polyacrylamide gel electrophoresis with s...
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