18953. 2 National Institutes of Health Predoctoral Fellows supported by NIH ..... BAR-NUN S, R SCHANTZ, I OHAD 1977 Appearance and composition of chloro-.
Plant Physiol. (1980) 65, 823-827 0032-0889/80/65/0823/05/$00.50/0
A Developmental Study of Photosystem I Peripheral Chlorophyll
Proteins' Received for publication June 4, 1979 and in revised form November 26, 1979
JOHN E. MULLET, JOHN J. BURKE"', AND CHARLES J. ARNTZEN United States Department of Agriculture, Science and Education Administration, Department of Botany, University of Illinois, Urbana, Illinois 61801 ImL plants to continuous illumination provides a "signal" which allows the nuclear-coded Chl a/b LHC to be incorporated into An isolated 'native" photosystem I (PSI complex) contains three spec- thylakoid membranes (5, 12). Here, we report that ImL chlorotral populations of chlorophyll a antennae (Mullet, Burke, Arntzen 1980 plasts exhibit a far red 77 K fluorescence emission maximum at Plant Physiol 65: 814-822). It was hypothesized that nearly one-half of 724 nm as compared to 736 nm in fully greened plants. Our these antennae (=45 ChWP700) are associated with polypeptides of 21,500 previous investigations on PSI suggest that 736 nm fluorescence to 24,500 daltons. The present study utilizes two developmental systems to emission arises from the peripheral antennae of PSI (19). These verify this association. data indicate that part of the antennae of PSI, in addition to the Chloroplasts were isolated from a Chi b-less barley mutant and from Chl a/b LHC, is deficient in ImL plants. partially-developed cucumber cotyledons (greened under intermittent illuA Chl b-less barley mutant has been used to investigate the mination lImLl chloroplasts) and were compared to control chloroplasts composition of higher plant Chl antennae (8, 11, 15, 23). This isolated from wild-type barley and mature cucumber. Both the mutant and nuclear mutant has been shown to contain a modified Chl a/b ImL chloroplasts exhibited a long wavelength fluorescence maximum at LHC (11). Here, we report that the Chl b-less barley mutant is 724 nanometers at 77 K as compared to 735 to 738 nanometers emission also deficient in Chl antennae of PSI. These data and evidence maximum in the respective controls. Both the mutant and ImL chloroplasts gathered from plants grown in ImL will be used to provide further were deficient in polypeptides of 21,500 to 24,500 daltons which were evidence for the hypothesis that the peripheral antennae of PSI is present in control membranes and in PSI fractions isolated from control composed of Chl bound to polypeptides of 21,500-24,500 daltons. membranes. In light-induced maturation of the ImL cucumbers, the synthesis of polypeptides in the 21,500 to 24,500 molecular weight range MATERIALS AND METHODS ABSTRACT
paralleled the appearance of PSI Chl species fluorescing at long wavelength (=735 nm). The PSI spectral properties of the control membranes were retained in isolated PSI particles containing 100 to 120 ChI/P700 (PSI-110). Detergent extraction of PSI-110 removed polypeptides of 21,500 to 24,500 daltons plus = 45 Chl/P7oo. The antennae-depleted PSI particle mimics PSI properties exhibited by incompletely differentiated mutant or ImL chloroplasts.
The major components involved in the two light reactions of green plant photosynthesis are embedded in the chloroplast thylakoid membrane. In addition to enzymes which catalyze electron transport between PSI and II, each photosystem is comprised of a Chl antennae, which serves to absorb incident radiant energy, a reaction center which mediates charge separation, and components which stabilize the charge separation and transform it into useful biochemical intermediates. Seedlings grown in ImL4 develop normal PSI and PSII activity but are deficient in Chl a/b LHC of PSII (1, 2, 12). Exposure of
'Supported in part by National Science Foundation Grant PCM 7718953. 2 National Institutes of Health Predoctoral Fellows supported by NIH Grant GM 7283-1 to the University of Illinois. 3 Present address: Plant Science Department, North Carolina State University, Raleigh, North Carolina 27607. 'Abbreviations: ImL: intermittent illumination; LHC: light-harvesting complex. 823
Chloroplasts were isolated from cucumber (Cucumis sativus L.) or barley (Hordeum vulgare) seedlings as previously described (4). Chl concentrations were determined as described by Arnon (3). Cucumber and barley seedlings were grown as previously described (12). Chromatography. Purified PSI particles containing 100-120 Chl/P700 (PSI- lIO) were isolated as previously described (19). To test the purity of these particles, PSI-l IO was solubilized in 0.2% digitonin, 25 mm Tris-HCl (pH 7.0), and loaded on a DE-52 column (2.5 x 4.5 cm). After loading, the column was washed with 250 ml 0.2% digitonin, 50 mm Tris-HCl (pH 7.0). The PSI complexes were eluted using 0.2% digitonin, 50 mm Tris-HCl (pH 7.0), and 100 mm NaCl. Spectral Analysis. Absorption spectra, P700 determinations, and fluorescence emission spectra were obtained as previously described (19). Polypeptide Analysis. Analysis of membrane polypeptides using SDS-polyacrylamide gel electrophoresis was carried out using the discontinuous buffer system of Laemmli as described previously (19).
Greening Studies. Cucumber seedlings were grown in Vermiculite for 4 days in darkness and then exposed to ImL (1 18-min dark, 2-min light cycles) for 2 days. Plants were then transferred to a growth chamber at 29 C for continuous illumination. Chloroplasts from this greening system were isolated at various times in the following manner. The cotyledons from 200-300 plants were ground at maximum speed in a Waring Blendor for 7 s in 0.4 M Sorbitol, 0.1 M Tricine-NaOH (pH 7.8), 1 mm Na-ascorbate, 2.0 mg/ml BSA, and 0.5 mm EDTA; squeezed through one layer of Miracloth; and loaded on 0.75-2.0 M sucrose gradients. The gradients contained 5 mg BSA/ml and 50 mm Tricine-NaOH (pH 7.8). The gradients were centrifuged at 4 C for 30 min at 100,000g.
824
MULLET, BURKE, AND ARNTZEN
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Plant Physiol. Vol. 65, 1980
RESULTS Peripheral Chi-Proteins of Isolated PSI. PSI particles containing 100-120 Chl/P700 (PSI-llO) were isolated from cucumber chloroplasts using minimal concentrations of Triton X-100 as previously described (19). The protein complement of these particles was resolved into 12 polypeptides by SDS slab gel electrophoresis (Fig. 1) (mol wt of individual polypeptides are indicated in the margin). The homogeneity of the PSI-l 1O was tested by subjecting the isolated complexes to DEAE-chromatography. The sample eluted as a single pigmented band; protein analysis of this fraction gave a polypeptide pattern identical to that shown in Figure 1 for nonchromatographic material. In a companion paper we have reported a technique which allows depletion of polypeptides of 21,500-24,500 daltons, plus = 45 Chl/P700 (including long wavelength absorbers at 700-710 nm) from PSI-l 10 isolated from peas (19). We have now extended these observations to another plant system to be used below in developmental studies. PSI-I10 (110 Chl/P700) particles and antennae depleted PSI-65 (65 Chl/P700) particles were isolated and compared. PSI-65 particles which were found to be deficient in polypeptides of 21,500-24,500 daltons (Fig. 1), revealed a decrease in 20 C absorption spectra at 700-710 nm, and a shift in 77 K fluorescence emission maximum from 738 nm in PSI-I 10 to 726 nm in PSI-65. Greening Studies. Chloroplasts isolated from cucumber seedlings grown in ImL exhibited a 77 K long wavelength fluorescence emission maximum at 724 nm (Fig. 2; sample illuminated for 0 h in continuous light). Exposure of the plants grown in ImL to subsequent continuous illumination caused a shift in the long
145
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FIG. 1. SDS (6-16%)-polyacrylamide slab gel of cucumber PSI containing I 10 Chl/P700 (PSI-I 10) and PSI containing 65 Chl/P700 (PSI-65). Polypeptides of 21,500, 22,000, and 24,500 daltons were removed concomitant with antennae depletion.
A band of chloroplasts, which was located near midgradient (= 1.2 M sucrose) was collected, resuspended in 5 mM MgCl2, 20 mM Tricine-NaOH (pH 7.8), and then centrifuged in a fixedangle rotor at l0,000g for 10 min. The pellet obtained was resuspended in the same buffer and repelleted at 5,000g. Samples from this preparation were used for spectral and protein analysis. L[35SJmethionine was used to follow protein synthesis during greening. The leaves of plants grown under ImL were painted with a solution of L-[35SJmethionine, (100 ,tCi in 1.2 ml 1% Tween) and the plants were subsequently exposed to ImL or continuous illumination. Identification of L-35S-labeled polypeptides was accomplished by electrophoretic separation on SDS-polyacrylamide gels as described above. These gels were then analyzed by x-ray fluorography by treating with PPO (9), with subsequent drying and exposure of the gels to x-ray film.
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Wavelength (nm) FIG. 2. Fluorescence emission spectra (77K) of chloroplasts isolated from cucumber seedlings grown under ImL and then exposed to 0-240 min of continuous illumination. Multiple spectra were acquired for each sample; these were averaged and normalized at their fluorescence maximum prior to plotting. The baseline of each spectrum is offset for visual clarity. Excitation was at 440 nm (+4.5 nm) and emission was collected through a 1-nm slit (+0.75 nm). When these spectra were corrected for photomultiplier sensitivity, the long wavelength emission maxima were red-shifted 2 nm.
Plant Physiol. Vol. 65, 1980
PHOTOSYSTEM I PERIPHERAL CHLOROPHYLL PROTEINS
wavelength, low temperature fluorescence emission maximum from 724 to 735 nm (Fig. 2). Under our experimental conditions this shift occurred at a rate of 2-3 nm/h, and correlated with an increase in long wavelength fluorescence emission relative to that at 685 nm. The appearance of long wavelength fluorescence emission during greening suggested that the peripheral antennae of PSI was synthesized when these plants were exposed to continuous illumination. The addition of antennae to PSI could have occurred either by addition of Chl to preexisting protein binding sites or by synthesis and insertion of a new Chl-protein complex. To determine if PSI polypeptides were synthesized concomitant with the shift in fluorescence emission, we treated plants with a radiolabeled amino acid (L-[35Sjmethionine), and then isolated chloroplasts at various times of greening. The appearance of newly synthesized polypeptides ([35S]methionine-labeled) was correlated to changes in stainable polypeptides. Figure 3 shows that PSI polypeptides of 21,500-24,500 daltons are deficient in chloroplasts isolated from plants grown in ImL (labeled IML MEMB) and that [35S]methionine is not incorporated into these polypeptides if plants remain in ImL (shown on the autoradiogram, IML ARG). Exposure of similar plants to continuous illumination results in the appearance and 35S-labeling of polypeptides of 21,500 and 24,500 daltons which co-migrate with PSI polypeptides of identical mol wt (Fig. 3, 8 H MEMB, 8 H ARG). Analysis of Chi b-less Barley Mutant. Chloroplasts of a Chl bless barley mutant exhibited 77 K long wavelength fluorescence emission at 724 nm, whereas wild-type barley chloroplasts fluoresced with a long wavelength maximum at 738 nm (Fig. 4). PSI isolated from wild-type chloroplast membranes exhibited a 77 K fluorescence emission peak at 738 nm (Fig. 4). To test whether PSI polypeptides were depleted concomitant with the fluorescence shift from 738 to 724 nm, we compared the polypeptide patterns of wild type membranes, Chl b-less barley membranes and PSI (100 Chl/P700) isolated from wild-type barley (Fig. 5); PSI polypeptides of 22,500, 23,000, and 24,500 daltons are deficient in the Chl b-less barley mutant. It was previously observed that the purified wild-type Chl a/b LHC contained polypeptides of 25,500-29,000 daltons (11). PSI
_N
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-
DISCUSSION PSI may be visualized as a 106 A particle when reconstituted in lipid vesicles (19). The complex mediates electron transport from electron donors such as 2,6-dichlorophenol indophenol-ascorbate to NADP (6). Kinetic evidence obtained from flash-induced A changes indicate the existence of at least four components involved in charge separation to the level of Fe-S centers A and B (22). In addition to these enzymic compounds, PSI contains tightly bound Chl antennae composed of a variety of spectral forms of Chl (10, 13, 14, 19, 21). Several studies aimed at the identification of polypeptides associated with the various activities of PSI have been reported. Recent investigations using antennae-depleted PSI particles suggest that the core of PSI consists of two polypeptides of 66,00070,000 daltons which bind 40 Chl and the primary acceptor of PSI (7). These authors also suggested that three polypeptides of 18,000-6,000 (bands IV, V, VI) resolved using the gel system of Weber and Osborn (24), may be involved in NADP reduction. The use of Laemmli (18) slab gels allows resolution of at least seven polypeptides in the same mol wt range (see Fig. 1, or ref 19) indicating the need for a reevaluation of these subunit identifications. In the present investigation and a previous study (19), PSI particles which contain 110 Chl/P700 were isolated and subsequently depleted of 40-45 peripheral antennae. In both cases the depletion of peripheral antennae from PSI particles isolated from peas (19) or cucumbers (Fig. 1) was correlated with the loss of far red absorption and fluorescence forms of Chl a and polypeptides of 21,500-24,500 daltons (Fig. 1). These observations were extended by the use of plants grown in ImL and a Chl b-less barley mutant.
ImL plants, which lacked a 77 K fluorescence emission maxi736 nm (Fig. 2), were also deficient in polypeptides which comigrate with PSI polypeptides of 21,500-24,500 daltons. Exposure of ImL plants to continuous illumination caused a shift in the 77 K long wavelength fluorescence maximum from 724 to 735 nm (Fig. 2) with concomitant synthesis of polypeptides of 21,500 and 24,500 daltons (Fig. 3). A correlation between the absence of long wavelength Chl mum at
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FIG. 3. SDS-polyacrylamide gel (14%) of cucumber PSI; Coomassie blue stainable polypeptides of membranes isolated from cucumber chloroplasts grown under intermittent illumination (IML MEMBS) or with 8 h continuous illumination after ImL (8 H MEMBS); autoradiogram of chloroplast isolated from cotyledons which were painted with l['Smethionine and then kept under ImL for 8 h (IML ARG) or exposed to continuous illumination for 8 h (8 H ARG).
_
MULLET, BURKE, AND ARNTZEN
826
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Plant Physiol. Vol. 65, 1980
800
Wavelength (nm) FIG. 4. A: 77 K fluorescence emission spectra of chloroplasts isolated from wild-type barley and a Chl b-less barley mutant. B: 77 K fluorescence emission spectrum of isolated barley PSI. Spectra were obtained as described in Figure 2. species and specific polypeptides was also observed in a barley mutant deficient in Chl b. Chloroplasts isolated from the Chl bless barley mutant exhibited a long wavelength 77 K fluorescence emission maximum at 724 nm (Fig. 4A). This peak is blue-shifted 10-12 nm compared to wild-type barley chloroplasts or PSI-llO particles isolated from wild-type barley which show maxima at 738 nm (Fig. 4, A and B). It was also found that the Chl b-less mutant is deficient in polypeptides of 21,500-24,500 daltons found in PSI isolated from wild-type barley (Fig. 5). Polypeptides of similar mol wt were depleted from PSI complexes concomitant with removal of peripheral antennae (Fig. 1). These data indicate that the Chl b-less barley mutant is deficient in the PSI peripheral antennae complex which is comprised of = 45 Chl bound to polypeptides of 21,500-24,500 daltons. The Chl b-less barley mutant and the ImL greening system provide interesting data concerning the control of Chl antennae synthesis in addition to evidence about the composition of PSI antennae proteins. The information coding for the Chl a/b LHC of PSII resides in the nucleus (17). This is consistent with the fact that the Chl b-less barley mutant, which is a nuclear mutant (8), contains a modified Chl a/b LHC (I 1). The location of information coding for the peripheral antennae of PSI is at present unknown. However, it has been reported that addition of far red Chl a antennae to PSI is blocked by lincomycin, an inhibitor of protein synthesis in chloroplasts (16, 20). We have reported here
_
_
FIG. 5. SDS-polyacrylamide slab gel (14%) of wild-type barley chloroplasts (WT MEMB), PSI isolated from wild-type barley (PSI) and Chl b-less barley chloroplast membranes (Chl b-LESS MEMB). Polypeptides of 22,000, 23,000, and 24,500 daltons were deficient in the Chl b-less mutant which also exhibited blue-shifted fluorescence emission compared to wild-type barley (Fig. 4A).
that the Chl b-less barley mutant is deficient in PSI peripheral antennae. These data suggest that the synthesis and/or incorporation of the PSI peripheral antennae may involve both nuclear and chloroplast control. In addition, the fact that the Chl a/b LHC and PSI peripheral antennae require a continuous illumination "signal" for synthesis and/or incorporation into ImL thylakoid suggests that common regulatory points exist for production of antennae for PSI and PSII. Acknowledgments-We thank Dr. Jan M. Anderson, CSIRO, Canberra, Australia for the Chl b-less barley mutant seeds used in this study. The excellent technical assistance of J. Watson and C. Ditto is appreciated. LITERATURE CITED 1. ARMOND PA, CJ ARNTZEN, J-M BRIANTAIS, C VERNOTTE 1976 Differentiation of chloroplast lamellae. Arch Biochem Biophys 175: 54-63 2. ARMOND PA, LA STAEHLIN, CJ ARNTZEN 1977 Spatial relationship of photosystem I, photosystem II and the light-harvesting complex in chloroplast membranes. J Cell Biol 73: 400-418 3. ARNON DI 1949 Copper enzymes in isolated chloroplasts. Polyphenoloxidase in Beta vulgaris. Plant Physiol 24: 1-15 4. ARNTZEN CJ, CL Dirro 1976 Effects of cations upon chloroplast membrane subunit interaction and excitation energy distribution. Biochim Biophys Acta 449: 259-274 5. BAR-NUN S, R SCHANTZ, I OHAD 1977 Appearance and composition of chlorophyll-protein complexes I and II during chloroplast membrane biogenesis in Chiamydomonas reinhardi Y- 1. Biochim Biophys Acta 459: 451-467 6. BENGIS C, N NELSON 1975 Purification and properties of the photosystem I
Plant Physiol. Vol. 65, 1980
PHOTOSYSTEM I PERIPHERAL CHLOROPHYLL PROTEINS
reaction center from chloroplasts. J Biol Chem 250: 2783-2788 7. BENGIS C. N NELSON 1977 Subunit structure of chloroplast photosystem I reaction center. J Biol Chem 252: 4564-4569 8. BOARDMAN NK, HR HIGHKIN 1966 Studies on a barley mutant lacking chlorophyll b. Photochemical activity of isolated chloroplasts. Biochim Biophys Acta 126: 189-199 9. BONNER WM, RA LASKY 1974 A film detection method for tritium-labeled proteins and nucleic acids in polyacrylamide gels. Eur J Biochem 46: 83-88 10. Brown JS 1977 Fluorescence spectroscopy of a P700-chlorophyll-protein complex. Photochem Photobiol 26: 519-525 11. BURKE JJ, KE STEINBACK, CJ ARNrZEN 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 12. DAVis DJ, PA ARMOND, EL GROSS, CJ ARNTZEN 1976 Differentiation of chloroplast lamellae. Onset of cation regulation of excitation energy distribution. Arch Biochem Biophys 175: 64-70 13. GASANOV RA, CS FRENCH 1973 Chlorophyll composition and photochemical activity of photosystems detached from chloroplast grana and stroma lamellae. Proc Nat Acad Sci USA 70: 2082-2085 14. IKEGAMI 1 1976 Fluorescence changes related in the primary photochemical reaction in the P700-enriched particles isolated from spinach chloroplasts. Biochim Biophys Acta 449: 245-258 15. HENRIQUES F, RB PARK.J975 Further chemical and morphological characterization of chloroplast membranes from a chlorophyll b-less mutant of Hordeum vulgare. Plant Physiol 55: 763-767
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16. HILLER RG, TBG PILGER, S GENGE 1977 Effect of lincomycin on the chlorophyll protein complex I content and photosystem I activity of greening leaves. Biochim Biophys Acta 460: 431444 17. KUNG SD. JP THORNBER, SG WILDMAN 1972 Nuclear DNA codes for the photosystem II chlorophyll-protein of chloroplast membranes. FEBS Lett 24: 185-188 18. LAEMMLI UK 1970 Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227: 680-685 19. MULLET JE, JJ BuRKE, CJ ARNTZEN 1980 Chlorophyll proteins of photosystem I. Plant Physiol 65: 814822 20. SARVAI E, G. HALASZ, P NYITRAI, F LANG 1976 Effect of lincomycin treatment on the greening process in bean (Phaseolus vulgaris) leaves. Plant Physiol 36: 187-192 21. SATOH K, WL BUTLER 1978 Low temperature spectral properties of subchloroplast fractions purified from spinach. Plant Physiol 61: 373-379 22. SAUER K, P MATHIS, S ACKER, JA VAN BEST 1979 Electron acceptors associated with P700 in Triton solubilized photosystem I particles from spinach chloroplasts. Biochim Biophys Acta 503: 120-134 23. THORNBER JP, HR HIGHKIN 1974 Composition of the photosynthetic apparatus of normal barley and a mutant lacking chlorophyll b. Eur I Biochem 41: 109116 24. WEBER K, M OSBORN 1969 The reliability of molecular weight determination by dodecyl sulfate-polyacrylamide gel electrophoresis. J Biol Chem 244: 44064412
