Photosynthesis Research 11:211 224 (1987) © Martinus Nijhoff Publishers, Dordrecht - Printed in the Netherlands

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Regular paper Chlorophyll-protein complex composition during chloroplast development: A species comparison 1 KENT O. BURKEY United States Department of Agriculture, Agriculture Research Service, and Departments of Crop Science and Botany, North Carolina State University, Raleigh, NC 27695 7631, USA (Received March 7, 1986; in revised form June 3, 1986; accepted June 6, 1986) Key words: barley, chlorophyll-protein complexes, chloroplast development, maize, pea, soybean Abstract. Barley, maize, pea, soybean, and wheat exhibited differences in chlorophyll a/b

ratio and chlorophyll-protein (CP) complex composition during the initial stages of chloroplast development. During the first hours of greening, the chlorophyll a/b ratios of barley, pea, and wheat were high (a/b >~ 8) and these species contained only the CP complex of photosystem I as measured by mild sodium dodecyl sulfate polyacrylamide gel electrophoresis. A decrease in chlorophyll a/b ratio and the observation of the CP complexes associated with photosystem II and the light-harvesting apparatus occurred at later times in barley, pea, and wheat. In contrast, maize and soybean exhibited low chlorophyll a/b ratios (a/b < 8) and contained the CP complexes of both photosystem I and the light-harvesting apparatus at early times during chloroplast development. The species differences were not apparent after 8 h of greening. In all species, the CP complexes were stabilized during the later stages of chloroplast development as indicated by a decrease in the percentage of chlorophyll released from the CP complexes during detergent extraction. The results demonstrate that CP complex synthesis and accumulation during chloroplast development may not be regulated in the same way in all higher plant species. Abbreviations

Chl, chlorophyll; CP, chlorophyll-protein; CPI, P700 chlorophyll-a protein complex of photosystem I; CPa, electrophoretic band that contains the photosystem II reaction center complexes and a variable amount of the photosystem I light-harvesting complex; LHC, the major light-harvesting complex associated with photosystem II; PSI, photosystem I; PSII, photosystem II; SDS, sodium dodecyl sulfate; SDS-PAGE, sodium dodecyl sulfate polyacrylamide gel electrophoresis.

Introduction Chloroplast development has been studied extensively by measurement of biochemical and biophysical changes during the greening of etiolated ICooperative investigations of the United States Department of Agriculture, Agricultural Research Service, and the North Carolina Agricultural Research Service, Raleigh, NC 27695-7601. Paper No. 10335 of the Journal Series of the North Carolina Agricultural Research Service, Raleigh, NC 27695-7601. 2 The use of trade names in this publication does not imply endorsement by the United States Department of Agriculture, Agriculture Research Service, or the North Carolina Agricultural Research Service of the products named, nor criticism of similar ones not mentioned.

212 angiosperm seedlings [3, 9, 10]. During growth of seedlings in the dark, proplastids develop into etioplasts. Synchronization of the plastids at the etioplast stage allows chloroplasts to develop in a more uniform manner when the etiolated seedlings are placed in the light. Etioplasts contain many of the components of the photosynthetic electron transport chain that include cytochrome f, cytochrome b-563, low potential cytochrome b-559, plastocyanin, ferredoxin, and ferredoxin-NADP + reductase [9, 10]. The ATP synthetase is also present in the etioplast [9, 10]. However, the etioplasts do not contain the CP complexes that are the major intrinsic proteins found in thylakoid membranes of mature chloroplasts [9, 10]. The synthesis of the CP complexes occurs during light-induced greening of eitolated seedlings [10]. The accumulation of CP complexes in thylakoid membranes during chloroplast development was first observed in barley [18] and bean [1] by SDS-PAGE methods that resolved two CP complexes. More recently, mild SDS-PAGE procedures that resolve additional CP complexes have been used to analyze the CP complex composition in seedlings that were greened under intermittent [6] or continuous [20, 27] light. These more recent studies did not investigate the initial appearance of the CP complexes. Previous work in this laboratory utilized a mild SDS-PAGE procedure to show that the CP complex of the PSI reaction center accummulates in barley thylakoids before the CP complexes of the PSII reaction center and the LHC complex during the greening of etiolated seedlings under continuous light [12]. The method was used in present study to examine the accumulation of CP complexes in several species of higher plants. Etiolated seedlings of barley, maize, pea, soybean and wheat were germinated and greened under the same conditions to minimize environmental factors that affect chloroplast development [24]. Species differences were observed in the appearance of the CP complexes during the initial stages of chloroplast development. The results demonstrate that CP complex synthesis and accumulation during chloroplast development may not be regulated in the same way in all higher plant species.

Methods

Growth conditions Seeds were germinated in moist vermiculite at 25 °C in the dark until the elongated shoots were approximately 10cm in length. Barley [Hordeum vulgare (L) var. Boone], maize [Zea mays (L.) Pioneer 3148], and pea [Pisum sativum (L.) var. Progress 9] required a 7 day germination period whereas soybean [Glycine max (L.) Merr. var. Ransom 2] and wheat [Triticum aestivurn (L.) var. Roy] were germinated for 5 days and 9 days, respectively. The etiolated seedlings were transferred to continuous light

213 and greened at 25-27 °C. The illumination was provided by a combination of fluorescent and incandescent lamps that produced an intensity of 250-300/~E-m-2-sec ~ at the top of the seedlings. Control plants were grown in moist vermiculite in a greenhouse for a period of time equal to the germination period of the etiolated seedlings.

Thylakoid membrane isolation Barley, maize, pea, and wheat leaves or soybean cotyledons were harvested at regular intervals. The tissue was cut into 3 to 5 mm sections and homogenized for 10sec in cold buffer A (0.4M sorbitol, 10mMNaC1, 50 mM Tricine-NaOH pH 7.8) with a Brinkman Polytron PT 10-352 equipped with probe PTA 205. Approximately 10 ml of buffer A were used for each g of tissue. The homogenate was filtered through eight layers of cheesecloth. The filtrate was accelerated to 7700 x g (4 °C) and returned to rest within a total time of 2 min. The pellet was washed once in cold buffer A, collected at 4000 x g for 10rain at 4°C, and then resuspended in buffer A. Soybean cotyledon thylakoid membranes were separated from the intact cells and other cellular debris in the preparation by step gradient centrifugation in buffer B (50 mM NaH2PO4, pH 6.9). The gradient consisted of 10ml of 20% (w/v) sucrose layered over 10ml of 60% (w/v) sucrose. The preparation was layered on top of the gradient followed by centrifugation at 9000 x g (4 °C) for 15 min. The membranes were removed from the interface between the 60% and 20% sucrose layers, diluted with buffer B, and collected by centrifugation at 10000 x g (4 °C) for 10 min. The pellet was resuspended in buffer B. In all cases, the final suspension was stored in liquid N>

Mild SDS-PAGE The plastid membranes were extracted two times with 2 M NaBr in buffer B to remove extrinsic proteins that interfere with the mild SDS-PAGE analysis [I2]. The CP complexes were solubilized in SDS and separated by mild SDS-PAGE as previously described [12]. After electrophoresis, the gels were immediately scanned at 675 nm to measure Chl a and at 720 nm to obtain a baseline with a Hitachi 100-80 spectrophotometer and gel scan attachment. Differential areas (675nm-720nm) under recorder peaks were measured with a planimeter.

Chl and protein determination Chl was measured by grinding whole tissue or suspending aliquots of thylakoid preparations in the appropriate solvent. Thylakoid preparations as well as barley, maize, pea, and wheat leaves were analyzed in 80% acetone [7]. Soybean cotyledons were analyzed in dimethylformamide [25]. Protein content of thylakoid preparations was analyzed by the Lowry method [21] with ovalbumin as a standard.

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MIGRATION (mm) Figure l. "Densitometer scans of greening barley CP complexes separated by mild SDSPAGE. The gel scans represent samples from 2 h (A), 3 h (B), 4 h (C), 6 h (D), and control (E) seedlings. The 2, 3, 4, and 6 h samples contained 250 #g of protein. The control sample contained 50 pg of protein. The gels were scanned at 675 n m ( ) to measure chl and at 720 nm (- ~ to obtain a baseline. FP, free pigment.

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TIME OF GREENING (h) Figure 4. CP composition of thylakoid membranes isolated from greening barley. The distribution of Chl between the CP complexes was determined from the gel scan areas associated with the Chl bands separated by mild SDS-PAGE. The percentage was calculated from the area of an individual band and the total area associated with the gel scan of interest. The percentages (+ SD) are the average of two samples from the same thylakoid preparation that were solubilized and analyzed independently. The time point C represents control plants. CPI (A), CPa (O), LHC ([]). pea, and wheat leaves. Maize and soybean had low Chl a/b ratios (a/b < 8) at all observation times (Figures 3C and 3D). The low Chl a/b ratios were correlated with the presence o f the Chl b enriched L H C complex in addition to the C P I complex at all observation times (Figures 5C and 5D). Differences between maize and soybean also existed. C P a was observed in soybean at all sampling times, but was not observed until 6 h in maize. After the first 8 h, chloroplast development proceeded in a similar m a n n e r in all species when Chl a/b ratio and C P complex composition were used as criteria. The Chl a/b ratio declined at a slow rate to the final value in all species (Figure 3). The fraction o f total Chl associated with C P I remained constant or increased by a small a m o u n t whereas the levels o f CPa and L H C increased significantly (Figures 4 and 5).

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Changes in the stability of the CP complexes Evidence exists that all of the Chl present in vivo is complexed with protein [22]. The presence of free Chl after mild SDS-PAGE is the result of the removal of Chl from the CP complexes during detergent solubilization of the thylakoid membranes. The fraction of total Chl removed from the CP complexes by SDS was 55-75% during the early stages of chloroplast development (Figure 6). In maize and soybean that contained both CPI and L H C during the initial 4 h, the amount of Chl released by SDS was less than that in barley, pea, and wheat where only CPI was observed. In

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221 all species examined, the amount of Chl released during detergent extraction decreased significantly as chloroplast development proceeded. After 48 h of greening, the amount of Chl released during SDS extraction decreased to 20~30% in all species. Discussion

CP complex synthesis and accumulation during chloroplast development did not follow the same pattern in the higher plant species examined in this study. The CPI complex was observed after 2 h of light-induced chloroplast development in all species examined. The CPa complex was observed after 6-8 h in all cases except for soybean where CPa was present at all observation times. The major difference between the species examined in this study was the accumulation of the LHC complex. Significant levels of LHC were observed in maize and soybean after 2h of greening. In contrast, LHC was not observed in barley and wheat until 6h. The accumulation of LHC in pea began at an intermediate time compared to the other species tested. The species could not be separated into taxonomic groups by comparing the patterns of LHC accumulation. For example, dicots and monocots did not form distinct groups. Therefore, the possibility exists that variation in LHC accumulation during greening may be present in different cultivars of the same species. The observed differences in LHC accumulation may be caused by species differences in the regulation of LHC biosynthesis, the stability of the LHC complex, or a combination of both possibilities. The LHC complex is known to be unstable during early stages of chloroplast development from experiments that demonstrated LHC degradation when greening seedlings were placed in the dark [2, 27]. The stability of CP complexes is an important consideration in mild SDS-PAGE analysis because Chl must remain bound to protein for the CP complexes to be detected. The large amount of Chl released during detergent extraction of thylakoids isolated early in chloroplast development (Figure 6) may have prevented the detection of trace amounts of CPa and LHC complexes at earlier times in barley, pea, and wheat. The decrease in Chl a/b ratio in barley and wheat leaves (Figure 3A) before the LHC complex was detected in these tissues (Figures 4 and 5A) could be related to the stability of the LHC. However, the high initial Chl a/b ratios (a/b > 8) of barley and wheat would support the conclusion that the LHC complex was not present in significant amounts during early stages of chloroplast development. The patterns of Chl a/b ratio presented for barley and wheat (Figure 3A) were reproduced in several independent greening studies although the absolute values of the ratios measured during the first 4 h varied from one study to the next. Such variability would be expected because of the difficulty in measurement of high Chl a/b ratios by spectrophotometric

222 methods [11]. The decrease in the amount of Chl released during mild SDS-PAGE analysis of thylakoids isolated at later stages of chloroplast development indicated stabilization of the CP complexes. The stabilization may be the result of the formation of CP complex aggregates that are less susceptible to removal of Chl by detergent [20]. CP complex synthesis during chloroplast development depends on the coordinated synthesis of both the apoproteins and Chl [15, 29]. The differences in LHC complex accumulation could be the result of transcription and translation of LHC genes at earlier times in certain species. Although the transcription o f L H C mRNA is induced by light in both pea [15] and barley [16], dark-grown pea seedlings contained low levels of LHC mRNA [15] whereas dark-grown barley contained no LHC mRNA [16]. Therefore, LHC mRNA may accumulate at earlier times during greening in some species. Differences may also exist in the timing of LHC mRNA translation or the processing and insertion of the polypeptide into thylakoid membranes. Indirect evidence suggests that the translation of LHC mRNA is controlled by light [26]. The species differences in LHC accumulation may involve aspects of Chl biosynthesis. High levels of the Chl precursor magnesium protoporphyin methyl ester may inhibit LHC mRNA accumulation [19] and therefore affect the accumulation of LHC apoprotein. In this way, accumulation of Chl biosynthetic intermediates could indirectly affect LHC biosynthesis. Species variation in the rates of Chl synthesis at early times during the greening process could cause differences in LHC accumulation. Etiolated bean leaves illuminated with intermittent light synthesize only small amounts of Chl and incorporate this Chl into reaction center CP complexes and not LHC complexes [6]. When Chl synthesis is limited by transfer of greening seedlings from continuous light into the dark, LHC complexes are degraded and the Chl a from LHC is incorporated into reaction center complexes [2, 27]. Therefore, synthesis of limited amounts of Chl in certain species during the early stages of chloroplast development may favor the assembly of reaction center CP complexes and inhibit LHC accumulation. The increase in Chl a/b ratio that was consistently observed in barley and wheat leaves between 1 h and 2 h (Figure 3A) may indicate degradation of trace amounts of LHC that occurred as a result of limited available Chl in these tissues. The species differences in LHC accumulation may be related to differences in the onset of Chl b synthesis. The chlorina f2 barley mutant cannot synthesize Chl b. The mutant contains active LHC mRNA, but does not accumulate the LHC complex [8]. Experiments with the mutant suggest that LHC accumulation requires the presence of Chl b to stabilize the complex in the thylakoid membranes [8]. Therefore, the species differences in LHC accumulation during chloroplast development may be the result of immediate Chl b synthesis in maize and soybean and delayed Chl

223 b synthesis in barley, pea, a n d wheat. A l t h o u g h the details of Chl b synthesis are still the subject of intense study [14], the regulation of Chl b synthesis could be responsible for the observations reported here. Experim e n t s have been initiated to examine how the factors discussed above m a y be involved in the species differences in CP complex a c c u m u l a t i o n that are observed d u r i n g chloroplast development.

Acknowledgements The a u t h o r wishes to t h a n k Cheryl R o b i n s o n for her excellent technical assistance.

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