THE JOURNAL OF BIOLOGICAL CHEMWIW Vol. 251, No. 9, Issue of May 10, pp. 2848-2853, 1976

Printed in U.S.A.

Protein Synthesis in Plant Leaf Tissue

THE SITES OF SYNTHESIS OF THE MAJOR PROTEINS

(Received for publication, March 24, 1975)

ANTHONY R. CASHMORE

From the Applied Biochemistry Division, D.S.I.R., Palmerston North, New Zealand

Protein synthesis in the leaves of green pea seedlings (Pisum satiuum) is examined by short term

labeling with [35S]methionine and autoradiography of the labeled proteins after fractionation by sodium

dodecyl sulfate-acrylamide gel electrophoresis. The two subunits of ribulose-1,5-diphosphate carboxylase

and the chloroplast lamellar proteins are identified as the major proteins being synthesized.

Three protein.chlorophyll complexes are characterized by sodium dodecyl sulfate-acrylamide gel

electrophoresis; all three complexes are disrupted by heating to 100” in sodium dodecyl sulfate solution.

Studies with inhibitors of protein synthesis indicate that the large subunit of ribulose-1,5-diphosphate

carboxylase is synthesized in the chloroplast, in contrast to the majority of the soluble proteins, including

the small subunit of ribulose-l,&diphosphate carboxylase, which is synthesized in the cytoplasm. PI1

protein, the major lamellar protein associated with photosystem II, is also synthesized on cytoplasmic

ribosomes. However, many of the lamellar proteins are synthesized within the chloroplast.

Integration into the lamellar system of at least one of the chloroplast-synthesized proteins is shown to

be dependent on cytoplasmic protein synthesis.

One of the first requirements in studies concerned with the

biosynthesis of plant proteins is to determine their respective

sites of synthesis. A popular approach to this problem has been

through the use of inhibitors like cycloheximide and chloram-

phenicol (l-5), which selectively inhibit synthesis either on the

cytoplasmic ribosomes, or on the prokaryotic-like ribosomes

found within the plant organelles. Such studies have shown, for

Chlamydomonas reinhardi (I, 2) and Vicia faba (3), that

ribosomes both in the cytoplasm and the chloroplast are

required for the synthesis of chloroplast lamellar proteins. A

similar sharing of function seems to exist for the synthesis of

ribulose-1,5-diphosphate carboxylase. Isolated pea chloro-

plasts have been shown to synthesize the large subunit of this

protein (6) and inhibitor studies with barley (7) show the

cytoplasm to be the likely site of synthesis for the small

subunit. Recently, the partial in oitro synthesis of the small

subunit on polysomes derived from the cytoplasm of Phaseolus uulgaris has been reported (8). Thus, at least in the case of higher plants, the synthesis of the two subunits of ribulose-1,5-

diphosphate carboxylase seems to occur at distinct sites,

although the inhibitor studies concerned with the site of

synthesis of the small subunit are far from unequivocal. For the

green algae, the situation is not clear, the possibility existing

that both subunits are synthesized within the chloroplast (1,

9-12).

As a prelude to studies concerning the regulation of protein

synthesis in the leaf tissue of pea seedlings, it was of interest to

characterize the major proteins being synthesized and their

sites of synthesis. In the course of these studies, the role of

cytoplasmic ribosomes in the synthesis of chloroplast proteins

has been confirmed. In addition, it is shown that cytoplasmic

protein synthesis is also required for the integration into the

chloroplast lamellae of at least one of the chloroplast-synthe-

sized proteins.

EXPERIMENTAL PROCEDURE

MateriaZs_Pea (Pisum satiuum var. Massey) and spinach (5’pinacia oleracea, hybrid 102) seeds were obtained from Yates. Radiochemicals were obtained from The Radiochemical Centre, Amer- sham/Searle. Tris (Sigma), glycine (Ajax Chemicals Ltd., Sydney), and acrylamide (Eastman) solutions were all filtered through a 0.8-p Millipore filter prior to use in gel electrophoresis. Phenylmethylsul- fonylfluoride and Coomassie brilliant blue R were obtained from Sigma, sodium diethyldithiocarbamate and sodium dodecyl sulfate from BDH Chemicals Ltd., Nonidet P-40 from Shell and Miracloth from Calbiochem. Kodak royal blue and Kodirex films were used for autoradiography, and development was with Kodak liquid X-ray devel- oper (type 2) and Kodak liquid fixer.

Labeling and Extraction of Pea Seedlings-Pea seedlings, grown in pumice at 20” with a 12.hour light period, were harvested 9 days after planting by slicing the epicotyl approximately 2.5 cm below the leaves. They were labeled by standing the excised seedling in a small volume of amino acid mixture containing [SsS]methionine. During labeling, the seedlings were irradiated with a 250.watt projector lamp, focused, and cooled through a spherical flask containing water. After labeling, the total leaf tissue, including the meristematic tissue, was homoge- nized in a conical glass tissue grinder (Kontes) with approximately 10 volumes of extraction buffer (10 rn~ Tris; 10 rnM glycine; 1 rn~ phenylmethylsulfonylfluoride, and 1% 2.mercaptoethanol, pH 8.9). After centrifugation through Miracloth, the filtrate was fractionated into soluble and particulate components by centrifugation for 30 min at 27,500 x g. The supernatant was treated with x0 volume of 10% sodium dodecyl sulfate and immediately incubated for 2 min at 100”.

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Protein Synthesis in Plant Leaf Tissue

The pellet was resuspended in one-half the original volume of extraction buffer, heated with sodium dodecyl sulfate as above, and the solubilized proteins clarified by centrifugation for 30 min at 27,500

x g. Sodium Dodecyl Sulfate-Acrylamide Gel Electrophoresis-The slab

gel apparatus described by Reid and Bieleski (13) was used. Acrylam- ide gels (15% acrylamide, 0.085% bisacrylamide; 14 x 11 x 0.15 cm) were prepared in 0.1 M Tris, 0.1 M glycine, and 0.1% sodium dodecyl sulfate (pH 8.9). The gels were pre-electrophoresed for 1 hour at 150 volts using an electrode buffer of 0.1 M Tris, 0.1 M glycine, and 0.1% sodium dodecyl sulfate (pH 8.9). The samples (5 to 20 ~1; previously made 10% in glycerol) were loaded (15 min, 20 volts) and then electro- phoresis was for 3 hours at 150 volts. After staining (3 hours; 0.25% Coomassie blue in acetic acid/methanol/water, l/5/5), gels were de- stained by shaking for several days in acetic acid/methanol/water, l/4/15. The gels were dehydrated onto Whatman 3MM paper accord- ing to the procedure described by Maize1 (14), and autoradiographs were prepared.

Preparation of Chloroplast Lamellar Proteins-Fifteen grams of tissue (12-day pea leaves or the young leaves from 5-week glass-house- grown spinach) were homogenized with a Lourdes homogenizer in 50 ml of 0.1 M Tris-HCl (pH 8.0) and 14 rnM 2-mercaptoethanol. After filtration through Miracloth the extract was centrifuged for 15 min at 106,000 x g. The pellets were resuspended in the extraction buffer (2 ml for pea and 1 ml for spinach), homogenized, and 250 ~1 layered onto a linear sucrose gradient (20 to 50% sucrose in 0.1 M Tris-HCl, pH 8.0, and 14 mM P-mercaptoethanol) and sedimented for 1 hour at 35,000 rpm in a Beckman SW 39 rotor. The green lamellar band (1.16 g/ml) was recovered from the gradient and treated with sodium dodecyl sulfate prior to electrophoresis.

Purification of Fraction I Protein-Leaves from a-week pea seedlings were extracted in a pressure cell (15) with 3 volumes of 50 mM phosphate buffer (pH 7.5) containing 5 rnM 2.mercaptoethanol and 10 mM sodium diethyldithiocarbamate. A 33 to 50% saturated ammonium sulfate cut was obtained, and this was fractionated by sequential gel filtration on Sepharose 6B and Bio-Gel A-(0.5m) in 25 mM Tris-H,SO, (pH 7.5), 5 mM 2-mercaptoethanol and 5 rnM EDTA. Pure Fraction I protein (AZBO:AzBO = 1.90) was obtained as indicated by a single band on gel electrophoresis in the absence of sodium dodecyl sulfate, and two bands (molecular weights equal to 14,000 and 53,000) on gel electrophoresis in the presence of sodium dodecyl sulfate.

Preparation of Antiserum and Immunoprecipitation-Fraction I protein (3.6 mg in 0.5 ml of the above Tris-H,SO, buffer) was emulsified with an equal volume of Freund’s complete adjuvant and injected intramuscularly into a New Zealand white rabbit. The injections were repeated twice, without the adjuvant, at 2.weekly intervals, and after a further 2 weeks, the serum was prepared, fractionated by 40% saturated ammonium sulfate precipitation, and assayed by standard procedures. An excess of antiserum was added to an [SSS]methionine-labeled, unfractionated, seedling extract (in 0.1 M KCl, 50 rnM Tris-HCl (pH 8.0), 0.5% deoxycholate, and 0.5% Nonidet P-40). The mixture was incubated for 30 min at 30” and then 20 hours at 4”. The precipitate was collected, washed three times in the detergent-containing buffer, and then dissolved in sodium dodecyl sulfate-containing.buffer for gel electrophoresis.

RESULTS AND DISCUSSION

Characterization of the Major Proteins-A fractionation by

sodium dodecyl sulfate-acrylamide gel electrophoresis of the peptides derived from the total proteins present in the leaves of g-day light-grown pea seedlings is shown in Fig. 1. By co-electrophoresis with appropriate standards (16), the molec- ular weights of the major peptides have been calculated.

In order to determine which proteins were being actively synthesized, excised pea seedlings were light-irradiated for 2 hours while being labeled with [YS]methionine. The proteins were again fractionated by electrophoresis, and in Fig. 2, the autoradiograph shows which proteins are being synthesized in the leaves of g-day pea seedlings. An essentially identical profile is obtained if the labeling is done with a ‘C-amino-acid mix (Amersham CFB.25), and similarly, an identical profile is obtained if the seedling is light irradiated for 2 hours while

i 9

I I I I

2 I

4 6 8 10 MOBILITY (cm)

FIG. 1. Sodium dodecyl sulfate-acrylamide gel electrophoresis of soluble and particulate proteins from pea seedling leaves. The molecu- lar weights of the major soluble (a) and particulate (b) proteins are indicated in kilodaltons. They have been calculated by co-electro- phoresis with the following standards of indicated molecular weights: bovine serum albumin (BSA, 68,000); pyruvate kinase (PyK, 57,000); lactate dehydrogenase (LDH, 36,000); carbonic anhydrase (CA, 29,000); lysozyme (L, 14,300); and cytochrome c (C, 12,400). The diagram on the left illustrates that in this gel system, a linear relationship is obtained between log (molecular weight) and mobility. The samples for electrophoresis contained 58 /lg of soluble protein and 40 fig of particulate protein.

standing in water prior to the 2-hour labeling with [35S]methio- nine.

It was of interest to characterize the major peptides depicted in Fig. 2. The,large and small subunits of Fraction I protein (17, 18) (ribulose-1,5-diphosphate carboxylase) were expected to form major components of the soluble proteins, and this was confirmed by comparison with purified labeled Fraction I protein prepared by immunoprecipitation (Fig. 2). The inter- nal lamellar structure of the chloroplast contains a significant portion of plant protein, and consequently, it seemed likely that many of the particulate proteins would be derived from the chloroplast lamellae. This comparison is made in Fig. 2, c and d. Quite clearly, the majority of the particulate proteins are seen to correspond to chloroplast lamellar proteins. Not surprisingly then, the predominant protein synthesis in the leaves of young light-grown pea seedlings is associated with the

development of the stromal and lamellar systems of the chloroplast.

An attempt was made to characterize further the lamellar proteins PI and PI1 depicted in the autoradiograph of Fig. 2. In Fig. 3 is shown a stained sodium dodecyl ‘sulfate-acrylamide gel fractionation of the proteins derived from both pea and spinach lamellae, and by comparative electrophoresis, the labeled PI and PI1 proteins have been shown to correspond to the major lamellar proteins in this diagram. An unusual feature of some chloroplast lamellar proteins is that they run as undissociated protein-chlorophyll complexes on electrophoresis in sodium

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2850 Protein Synthesis in Plant Leaf Tissue

FIG. 2 (left). Sodium dodecyl sulfate-acrylamide gel electrophore- sis of [3sS]methionine-labeled proteins from pea seedling leaves. Seedlings were labeled by standing in a 60-J solution of amino acids including [%]methionine (33.9 Ci/mmol, 25 pM; 19 other amino acids, 166 FM). For the preparation of lamellar proteins, 0.5 ml of leaf extract was layered directly onto a linear sucrose gradient and sedimented as described under “Experimental Procedure.” The green lamellar band was recovered (200 ~1) and treated with sodium dodecyl sulfate. The autoradiograph shows a, Fraction I immunoprecipitate (2,704 cpm); b, soluble proteins (53,825 cpm); c, particulate proteins (50,466 cpm) and d, chloroplast lamellar proteins (72,350 cpm).

FIG. 3 (right). Sodium dodecyl sulfate-acrylamide gel electropho- resis of lamellar proteins from pea and spinach chloroplasts. Chloro- plast lamellae were purified by sedimentation through sucrose accord-

dodecyl sulfate (3). In the present investigation, four chloro- phyll-containing bands (PSI, PSIIa, PSII, and Chl) were

observed if lamellar preparations were fractionated by electro- phoresis at 4“ and the prior dissociation in hot sodium dodecyl sulfate was omitted. However, if the lamellar preparations were heated at 100” prior to electrophoresis, which was standard procedure throughout this investigation, then only one chlorophyll band was found (presumably free chloro- phylls). In the heated sample, in contrast to previous claims (19), no protein band was found with electrophoretic properties similar to the photosystem I protein-chlorophyll complex (PSI). When the lamellae are labeled with [YS]methionine and treated according to the procedure in Fig. 3, then, by autoradi- ography, the complexes PSI, PSIIa, and PSI1 are shown to be labeled, indicating that they are indeed protein complexes. In the case of the PSI1 protein .chlorophyll complex, it seems likely that dissociation here gives rise to protein PII, of molecular weight 29 kilodaltons (19-22). Often two proteins are observed in this region (29 and 31 kilodaltons). It has been shown that PSI contains mainly chlorophyll a, whereas PSI1 contains both chlorophyll a and b (3). The nature of the chlorophyll associated with the minor PSIIa complex has not been determined, although from the color of the band, both before and after staining with Coomassie blue, it is similar to PSI1 and distinct from PSI.

From Fig. 3 then, it is concluded that the lamellar system of

ing to the method described under “Experimental Procedure.” The green chloroplast lamellar bands (250 ~1; 1.16 g/ml) were recovered from the gradient and made 1% in sodium dodecyl sulfate and 2-mercaptoethanol. One-half of each sample was kept on ice and the rest incubated for 2 min at 100”. Twenty-microliter samples were fractionated by acrylamide gel electrophoresis as described under “Experimental Procedure” except that electrophoresis was for 6 hours at 4”. After electrophoresis, the chlorophyll bands were marked (PSI, PSIIa, PSII, and Chl) prior to staining in Coomassie blue. a, pea lamellar proteins, not heated in sodium dodecyl sulfate; b, pea lamellar proteins, heated in sodium dodecyl sulfate; c, spinach lamellar proteins, not heated in sodium dodecyl sulfate; d, spinach lamellar proteins, heated in sodium dodecyl sulfate.

the pea chloroplast contains two major proteins, PI and PII, of respective molecular weights 58 and 29 kilodaltons (obtained according to Fig. 1). PI1 appears to be strongly associated with chlorophyll within the chloroplast, and from the work of others (19-22), it forms the major protein of photosystem II. In contrast, the author finds no evidence that PI forms a protein.chlorophyll complex, although it does appear to form the major photosystem I protein (19-22). The nature of the protein moieties associated with the complexes PSI and PSIIa, of apparent molecular weights 96 and 70 kilodaltons, has not been clarified.

Sites of Synthesis of Major Proteins-The sites of protein synthesis have been examined with the aid of the inhibitors cycloheximide and chloramphenicol. In Table I, the effect of these inhibitors on the incorporation of [35S]methionine is shown. Cycloheximide is seen to decrease total protein synthe- sis significantly, and this effect is most pronounced in the case of the soluble proteins. The effect of cycloheximide and chloramphenicol on the synthesis of specific proteins is illus- trated by the autoradiographs in Figs. 4 and 5. It is observed that the synthesis of the large subunit of Fraction I protein is unique among the major soluble proteins in that it is sensitive to chloramphenicol and relatively insensitive to cycloheximide. The small subunit of Fraction I protein, along with all of the other major soluble proteins, is synthesized on ribosomes sensitive to cycloheximide. These observations are consistent

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Protein Synthesis in Plant Leaf Tissue 2851

TABLE I

Effect of chloramphenicol and cycloherimide on protein synthesis in pea seedling leaves

Pea seedlings were light irradiated for 15 min while standing in water (50 Al), chloramphenicol (200 fig/ml), or cycloheximide (20 &ml). To each sample were then added 10 pl of [%]methionine (34.2 Ci/mmol, 0.15 mM; 19 other amino acids, 1.0 mht) and the irradiation continued for a further 2 hours. The leaves were then homogenized in 1 ml of extraction buffer, fractionated into soluble and particulate components, and treated with sodium dodecyl sulfate as described under “Experimental Procedure.” Protein synthesis was determined by trichloroacetic acid precipitation, collection of precipitates on Whatman GFC filters, and liquid scintillation counting.

[3?3]Methionine incorporated/

seedling (cpm x 10-Y

9-c 5% distribution of control

Soluble ‘;;tE- Soluble + Particulate

Control 16.02 9.25 63.4 36.6 Cycloheximide 3.37 5.62 37.5 62.5 35.6 Chloramphenicol 14.11 7.53 65.2 34.8 85.6

with the finding that the large subunit of Fraction I protein is synthesized in isolated pea chloroplasts (6), and the small subunit is synthesized on polysomes derived from the cyto- plasm of bean leaves (P. vulgar-is) (8). It is noted that whereas both inhibitors used show some selectivity, this is not com- plete, particularly in the case of cycloheximide. This presum- ably reflects a rather tight coupling between protein synthesis in the cytoplasm and the chloroplast. These inhibitor studies are extended by the results shown in Fig. 6. Here, Fraction I protein has been purified with the aid of specific antiserum. Clearly, the synthesis of the small subunit of Fraction I protein continues to some extent in the absence of large subunit synthesis. However, in the presence of cycloheximide, where the synthesis of the small subunit is severely restricted, so also is the synthesis of the large subunit, or at least its incorporation into the native molecule. Similar results to those depicted in Fig. 6 have been obtained by purifying Fraction I protein by sucrose gradient centrifugation (1).

The particulate proteins present a very different picture (Figs. 4 and 5), as in this case, the synthesis of many of the proteins is sensitive to chloramphenicol. In the case of the chloroplast lamellar proteins (Fig. 5), this sensitivity to chlor- amphenicol is almost certainly due to their synthesis on chloroplast ribosomes. PI1 protein is exceptional in that its synthesis is sensitive to cycloheximide and insensitive to chloramphenicol. Similar results regarding the sites of synthe- sis of bean (V. fuba) chloroplast lamellar proteins have been reported (3).

All of the results obtained with chloramphenicol (Fig. 4) have been duplicated using lincomycin.

Cytoplasmic Protein Synthesis Is Required +or Chloroplast Lamellar Assembly-A further interesting piece of information is depicted in Fig. 4. This concerns protein PI, which is quite clearly a chloroplast lamellar protein (Fig. 2), and conse- quently, in Fig. 4, it occurs in the particulate fraction. However, in the presence of cycloheximide, an inhibitor which does not appear to influence its synthesis, approximately 50% of what appears to be PI is found in the soluble fraction. Confirmation of the relationship between this soluble protein

I

FIG. 4. Effect of chloramphenicol (CAM) and cycloheximide (CH) on the synthesis of specific proteins in pea seedling leaves. [s5S]- Methionine-labeled proteins were prepared according to Table I. The samples (10 ~1) were fractionated by electrophoresis, an autoradio- graph was prepared, and this was scanned with a densitometer. a, control, soluble proteins (112,622 cpm); b, chloramphenicol, soluble proteins (73,622 cpm); c, cycloheximide, soluble proteins (27,438 cpm); d, control, particulate proteins (101,358 cpm); e, chloramphenicol, particulate proteins (60,120 cpm); f , cycloheximide, particulate pro- teins (81,808 cpm).

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2852 Protein Synthesis in Plant Leaf Tissue

FIG. 5. Effect of chloramphenicol and cycloheximide on the synthe- sis of chloroplast lamellar proteins. Pea seedlings were labeled with [“Slmethionine according to the procedure described in Table I. Leaf proteins were extracted with 1 ml of extraction buffer per seedling and 0.5 ml of filtered extract was layered directly onto a linear sucrose gradient. After centrifugation, the lamellar band was recovered, treated with sodium dodecyl sulfate, and fractionated by electrophore- sis. The autoradiograph shows proteins synthesized in a, control (28,876 cpm); b, chloramphenicol (21,448 cpm); and c, cycloheximide (17,986 cpm).

and PI protein has been attained by comparative isoelectric focusing.’ It appears that a continuous supply of a cytoplasmi- tally synthesized component is required for the incorporation of PI into the chloroplast lamellae. A possible identity of such d component is PI1 protein, as it represents the major lamellar protein and its synthesis is sensitive to cycloheximide. Similar results pertaining to PI have been obtained with anisomycin, indicating that the phenomenon is indeed a reflection of the inhibition of cytoplasmic protein synthesis.

In this investigation, it has been shown in pea seedlings that the small subunit of Fraction I protein is apparently synthe- sized within the cytoplasm and this finding is in agreement with the work of others (7,B). In Nicotians, the small subunit of Fraction I protein is coded by the nuclear genome (23), and this presumably holds for the pea Fraction I subunit. It has also been shown that the cytoplasm is the likely site of synthesis for the major protein of photosystem II, and this protein is also coded for by the nucleus (24). The cytoplasmic origin of the major photosystem II protein is also found in bean (V. fuba) (3) and in an unicellular green alga (C. reinhardii) (25). These conclusions regarding the site of synthesis of the small subunit of Fraction I protein and PI1 protein have recently been con- firmed by in vitro studies.z

Acknowledgment-The author thanks J. W. Lyttleton for many helpful discussions.

’ A. R. Cashmore, unpublished results. ZA. R. Cashmore, M. K. Broadhurst and R. E. Gray, manuscript in

preparation.

25%

CAM

‘7% 1_-1___ :’

FIG. 6. Immunoprecipitation of Fraction I protein synthesized in the presence of chloramphenicol (CAM) or- cycloheximide (CH). IYSlMethionine-labeled proteins were prepared according to Table I. To &l-~l aliquots of the soluble fractions were added 500 il of Fraction I antiserum. The immunoprecipitates were prepared as described under “Experimental Procedure,” solubilized in 200 ~1 of sodium dodecyl sulfate-containing buffer, and then fractionated by sodium dodecyl sulfate-acrylamide gel electrophoresis. The percentage figures shown on the densitometer tracings of the autoradiograph refer to the amount of [*%]methionine present in the immunoprecipitates as a percentage of the control. In the control sample a, an aliquot of 5 ~1 was used for electrophoresis, whereas in b (chloramphenicol), and c (cvcloheximide), 20-pl aliquots were used.

1. 2. 3.

4.

5.

6.

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A R CashmoreProtein synthesis in plant leaf tissue. The sites of synthesis of the major proteins.

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