Communication Vol. 256. No. 16, Iswe of August 25, pp. 8252-8255, 1981 THE JOURNAL OF BIOLOGICAL CHEMISTRY

Printed in U.S.A.

Light-stimulated Increase of Translatable mRNA for Phosphoenolpyruvate Carboxylase in Leaves of Maize*

(Received for publication, April 13, 1981, and in revised form, June 9, 1981)

Thomas L. Sims and Donald R. Hague$ From the Department of Biology, University of Oregon, Eugene, Oregon 97403

Polyadenylated RNA has been isolated from maize leaves at various times during greening of etiolated seedlings and used to prime the wheat germ cell-free translation system. Levels of translatable messenger RNA for phosphoenolpyruvate carboxylase increase with the length of the illumination period. The pattern of the increase in translatable mRNA for the enzyme is similar to that of the increase in phosphoenolpyruvate carboxylase protein previously observed in intact tis- sues. Phosphoenolpyruvate carboxylase synthesized in vivo migrates as a doublet band on gradient sodium dodecyl sulfate-polyacrylamide gels. A similar doublet has been seen occasionally with the products of cell- free translation.

We have been studying the synthesis of phosphoenolpyru- vate carboxylase (EC 4.1.1.31, orthophosphate:oxaloacetate carboxylyase, phosphorylating) as a model of regulated gene expression in higher plants. Several characteristics of this system recommend it for such a study: 1) phosphoenolpyru- vate carboxylase comprises 10-15% of the soluble protein of the mature maize leaf (1-3); 2) large quantities of this protein are synthesized de nouo on greening of etiolated leaves (1, 2); 3) the synthesis of phosphoenolpyruvate carboxylase is ap- parently cell-type specific, for the protein is found only in the cytosol of the mesophyll cells of the leaf, and not in the contiguous bundle sheath cells' (4). It appears, therefore, that large quantities of this protein are synthesized in a specific cell type in response to illumination of etiolated leaves.

Here we demonstrate that an increase in translatable mRNA for phosphoenolpyruvate carboxylase accompanies the increase in synthesis of this protein during greening of maize seedlings.

MATERIALS AND METHODS

Growth and illumination conditions for corn seedlings (Zea mays L.; Golden Cross Bantam) have been previously described (1).

Total leaf RNA was extracted as described by Chirgwin et al. (5). Greening leaves were frozen in liquid nitrogen, ground to a fine

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* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "aduertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

592413014670 and by National Science Foundation Grant PCM- + Supported by United States Department of Agriculture Grant

8012035. ' N. H. Chua, The Rockefeller University, personal communication.

powder in a precooled mortar and pestle, and homogenized in extrac- tion buffer (4 M guanidine thiocyanate, 0.1 M 2-mercaptoethanol, 25 mM sodium citrate, pH 7.0, 0.5% sodium N-lauroylsarcosinate). The homogenate was centrifuged twice at 15,000 X g for 10 min to remove cell debris, and the clear, green supernatant liquid was layered over 1.2-ml pads of CsCl (5.7 M CsCI, 0.1 M Na2EDTA, pH 7.0) in SW 50.1 tubes (Beckman) and centrifuged at 35,000 rpm for 12 h (5, 6 ) . After removing the supernatant liquid, the opalescent pellets were rinsed with 100% ethanol, dried, dissolved in sterile water, and precipitated with ethanol overnight at -20 "C. RNA precipitates were pelleted, dissolved in sterile water, heat-denatured (2 min at 65 "C, followed by rapid cooling on ice), and applied to oligo(dT)-cellulose for isola- tion of polyadenylated RNA (7). Typically, the polyadenylated frac- tion represented 0.5-1.056 of the total RNA applied to the oligo(dT) column.

Wheat germ extract was prepared after Roberts and Paterson (8) with the following modifications. The preincubation step was omitted, and the extract was centrifuged at 30,000 X g before and after elution from Sephadex G-25. The final protein-synthesizing mixture con- tained 24 mM 4-(2-hydroxyethyl)-l-piperazineetbanosulfonic acid, pH 7.6, 2.5 mM dithiothreitol, 1.2 mM ATP, 0.5 mM GTP, 12 mM creatine phosphate, 140 mM potassium acetate, 10 mM KCI, 2.5 mM Mg(Ac),, 50 p~ CaC12, 10 p~ Na2EDTA, 0.6 mM spermidine, 1.25 units/ml of creatine kinase, and 40 p~ each amino acid except methionine. Translation assays (25 pl) contained 10 pCi of ["'S]me- thionine (Amersham >lo00 Ci/mmol), 0.2-1.0 pg of polyadenylated RNA, 10 pl of wheat germ S-30, and the reagents listed above. Incubations were carried out for 90 min at 23 "C, and duplicate 5-pl samples were removed for determination of hot trichloroacetic acid- insoluble radioactivity (9). Assay volumes for immunoprecipitation experiments were generally 50 or 100 pl. Pbenylmethylsulfonyl fluo- ride was added to some translation assays at 50 pg/ml but had no apparent effect on translational fidelity. Tobacco mosaic virus RNA was used as a standard to assess the ability of the wheat germ system to synthesize high molecular weight products.

Antibodies against phosphoenolpyruvate carboxylase were pre- pared by injection of the purified protein (estimated purity 9576, see Ref. 1) in Freund's complete adjuvant into a female white New Zealand rabbit. The IgG fraction was obtained from the crude anti- serum by affinity chromatography on protein A agarose (10). Staph- yZococcus aureus Cowan strain I was prepared according to Kessler (11) and used in immunoprecipitations as described by Martial et al. (12) with the following modifications. Cell-free translation products or protein synthesized in vivo were first incubated with one-third volume of S. aureus Cowan strain I for 10 min at 23 "C. S. aureus Cowan strain I was removed by centrifugation, and appropriate IgG fractions were added to the supernatants and incubated for 1 h at 23 "C. After a second addition of S. aureus Cowan strain I, and 10- min incubation on ice, the antigen-antibody-9. aureus Cowan com- plexes were removed by centrifugation and washed four times with buffer (150 mM NaC1, 5 mM Na2EDTA, 50 mM Tris-C1, pH 7.4,0.05% Triton X-100, 2 mM methionine, 1 mg/ml of ovalbumin). After the last wash, complexes were resuspended in buffer free of methionine and ovalbumin, transferred to fresh tubes, centrifuged, and then disrupted with SDSZ sample buffer at 100 "C for 3 min. S. aureus Cowan residue was removed by centrifugation and radioactivity in the supernatant liquid was determined as described above.

SDS-gel electrophoresis was performed according to O'Farrell (13) in 5-15% polyacrylamide gradient slab gels. Methods for labeling of leaf protein in vivo with t3'S)rnethionine have been described previ- ously (1). Radioactive gel bands were detected by fluorography (14) using preflashed Kodak X-Omat x-ray film. Peptide mapping was performed as described by Cleveland et al. (15).

RESULTS

The result of cell-free translation of polyadenylated RNA isolated from greening leaves is shown in Fig. 1. The largest evident product of the translation system is a band which CO-

a The abbreviation used is: SDS, sodium dodecyl sulfate.

8252

Cell-free Synthesis of Phosphoenolpyruvate Carboxylase 8253

migrates with phosphoenolpyruvate carboxylase on SDS- polyacrylamide gradient gels (subunit molecular weight of phosphoenolpyruvate carboxylase is approximately 100,OOO (1, 3)).

RNA isolated from etiolated leaves (Fig. 2, lane 1) directs the synthesis of a weak phosphoenolpyruvate carboxylase band. When RNAs isolated from leaves exposed to light for increasing periods are assayed in the translation system, how- ever, an increase in the intensity of the phosphoenolpyruvate carboxylase band is apparent (Fig. 2, lanes 1-4). Scanning of fluorograms of translation assays indicates that the percentage of radioactivity found in the phosphoenolpyruvate carboxyl- ase band increases 3-4-fold over the course of greening (data not shown). We have previously observed low levels of phos- phoenolpyruvate carboxylase protein and enzyme activity in etiolated leaves. These levels increase 3-5-fold following illu- mination (1).

The identity of the phosphoenolpyruvate carboxylase band has been confirmed by immunoprecipitation. Antibodies to phosphoenolpyruvate carboxylase specifically precipitate this band from the mixture of total translation products (Fig. 1, lune 4; Fig. 2, lunes 5-8). Pre-immune serum does not precip- itate the phosphoenolpyruvate carboxylase band; however,

FIG. 1. Cell-free synthesis of phosphoenolpyruvate carbox- ylase. Total RNA was extracted from leaves after 24 h of illumination and passed over oligo(dT)-cellulose to isolate polyadenylated RNA. Polyadenylated RNA (40 pg/ml) was used to prime the wheat germ cell-free translation system. Excised leaves were labeled between 22 and 28 h of illumination with 250 pCi of ["SJmethionine, and the protein fraction precipitating between 40 and 60% ammonium sulfate saturation was isolated (1). Electrophoresis was performed in a 5-15% gradient SDS-polyacrylamide slab gel a t 200 V. The bottom corre- sponds to the anodic pole. The fluorogram was exposed for 24 h a t -70 "C using preflashed Kodak AR-5 x-ray film. Only the upper portion of the gel is shown in the photograph. Lane 1, 40 to 60% ammonium sulfate fraction of extract of leaves labeled in uiuo. Lane 2, immunoprecipitate of extract labeled in uiuo. Lane 3, total trans- lation products of maize polyadenylated RNA assayed in the wheat germ translation system. Lane 4, immunoprecipitate of products synthesized from maize polyadenylated RNA in the wheat germ translation system. The arrow indicates the position of migration of a purified phosphoenolpyruvate carboxylase marker (stained with Coomassie blue) which was run on the same gel.

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FIG. 2. Translation of mRNA from greening leaves. Total RNA was isolated from leaves at various intervals after exposure to light. Polyadenylated RNA was isolated by oligo(dT)-cellulose chro- matography and used to prime the wheat germ cell-free translation system. The concentration of RNA in the translation assays was 20 pg/ml, which was nonsaturating for incorporation into acid-insoluble radioactivity. (Saturating levels for incorporation were 40-50 pg/ml.) Electrophoresis was performed in a 7.5-10.5% gradient SDS polyacryl- amide slab gel. The bottom corresponds to the anodic pole. The fluorogram was exposed for 36 h at -70 "C using Kodak XRP-5 x-ray film. Lanes 1 4 (400,000 cpm/lane), total translation products. Lanes 5-8 (19,OOO cpm/lane), products immunoprecipitated by antibody to phosphoenolpyruvate carboxylase. Lanes 1 and 5, etiolated. Lanes 2 and 6.4-h greening. Lanes 3 and 7.8-h greening. Lanes 4 and 8, 14-h greening. The arrow indicates the position of phosphoenolpyruvate carboxylase in the gel.

pre-immune serum does precipitate the low molecular weight band seen in lanes 5-8 of Fig. 2 (results of pre-immune precipitation not shown). The weak immunoprecipitable band for phosphoenolpyruvate carboxylase seen in lane 5 of Fig. 2 is the product of translation of mRNA from etiolated leaves. In agreement with the results of analysis of the total transla- tion products, the amount of immunoprecipitable phospho- enolpyruvate carboxylase is greater in assays of RNAs isolated from leaves exposed to light for increasing periods of time.

T o compare the cell-free translation products with phos- phoenolpyruvate carboxylase synthesized in viuo, we have performed peptide mapping on products immunoprecipitated from the translation assays, and from extracts of leaves labeled with [:%]methionine during greening. Fig. 3 demonstrates that proteolytic digestion products from the two sources are similar in relative abundance and migrate identically on 15% SDS-polyacrylamide gels. It is apparent from these data that the immunoprecipitable product synthesized in vitro is iden- tical with the immunoprecipitable product synthesized in the leaves in response to illumination.

We have shown previously that phosphoenolpyruvate car- boxylase isolated from mature, etiolated, or greening leaves

8254 Cell-free Synthesis of Phosphoenolpyruvate Carboxylase

1 2 3 4 5 6

FIG. 3. Peptide analysis of phosphoenolpyruvate carboxyl- ase synthesized in vivo and in vitro. Greening plants were labeled in vivo with ['"SS]methionine and phosphoenolpyruvate carboxylase was extracted, immunoprecipitated, and solubilized with SDS sample buffer. An identical preparation was made of unlabeled immunopre- cipitated protein, and a portion of the unlabeled material was added to immunoprecipitated, [:'"S]methionine-labeled products of a cell- free translation system primed with 60-h greening polyadenylated RNA. The two labeled preparations were applied to a 5-15% gradient SDS-polyacrylamide gel. Phosphoenolpyruvate carboxylase bands were identified after electrophoresis by staining with Coomassie blue, and these were excised and placed in the sample wells of a 15% SDS- polyacrylamide gel for digestion with S. aureus protease and electro- phoresis according to Cleveland et al. (15). Lanes 3 and 4, heavily exposed bands at top represent phosphoenolpyruvate carboxylase subunits synthesized in vivo (lane 3) or in vitro (lane 4). Faster migrating bands in these lanes are large cleavage products of the protein caused by protease spillover from adjacent lanes. Lanes 1 and 2, protease treatment of phosphoenolpyruvate carboxylase labeled in uivo. Lane 1, 0.5 pg of protease; lane 2.0.05 pg of protease. Lanes 5 and 6, protease treatment of phosphoenolpyruvate carboxylase poly- peptides synthesized in vitro. Lane 5, 0.05 pg of protease; lane 6, 0.5 pg of protease. 10,000 to 20,000 cpm were applied to each lane of the 15% gel and the fluorogram was exposed for 72 h at -70 "C using Kodak XR-5 x-ray film.

migrates as a doublet band on 5-15% SDS-polyacrylamide gradient gels (1). We have observed a doublet band in several of our translation assays. Fig. 4, lane 1, demonstrates a trans- lation assay in which the doublet nature of the phosphoenol- pyruvate carboxylase band is clearly seen. Lane 2 of Fig. 4 shows phosphoenolpyruvate carboxylase immunoprecipitated from extracts of leaves labeled in uiuo. The doublet nature of the immunoprecipitated product is evident in the original gel. Fig. 1, lane 1, also shows the doublet nature of phosphoenol- pyruvate carboxylase in a sample labeled in uiuo. Overloading of the protein in lane 2 of Fig. 1 has obscured the doublet.

DISCUSSION

Phosphoenolpyruvate carboxylase, the major protein of the cytosol of mesophyll cells of C-4 plants, in synthesized de novo during the greening of etiolated plants (1, 2). The work re- ported here indicates that this synthesis results largely from an increase in the level of translatable mRNA for this protein.

Earlier studies on greening of etiolated bean leaves (16)

FIG. 4. Doublet nature of phosphoenolpyruvate carboxylase synthesized in vitro. Lane 1, total translation products of polyad- enylated RNA isolated from leaves after 60 h of illumination. Lane 2, phosphoenolpyruvate carboxylase immunoprecipitated from extracts of leaves labeled in vivo between 36 and 40 h of greening. Electro- phoresis was in a 5-15% gradient SDS-polyacrylamide slab gel a t 200 V. The bottom corresponds to the anodic pole. Fluorography was for 24 h at -70 "C using preflashed Kodak XR-5 x-ray film. The arrow indicates the location of phosphoenolpyruvate carboxylase. The dou- blet nature of the band in the in vivo immunoprecipitate was evident in the original film.

concluded that mobilization of ribosomes into polysomes in the absence of mRNA synthesis may account for a degree of increased protein synthesis during greening. Since we have used total leaf RNA as starting material for mRNA prepara- tion for cell-free translation, it is likely that the increase in translatable mRNA we observe results from a change in the rate of transcription, post-transcriptional processing, or mes- senger RNA turnover, and not from mobilization of stored mRNA sequences into polysomes.

In a previous study (1) we reported that etiolated leaves of the same developmental age as those used here did not incorporate ['%]methionine into phosphoenolpyruvate car- boxylase when maintained in the dark during the labeling period. Other proteins were labeled during the dark period, however. Despite the apparent lack of synthesis of phospho- enolpyruvate carboxylase in the dark, measurable quantities of phosphoenolpyruvate carboxylase are present in the dark- grown tissue (1) and, as indicated in this study, low levels of phosphoenolpyruvate carboxylase messenger RNA are also present in these tissues. The dark levels of protein and mRNA for phosphoenolpyruvate carboxylase are not the result of inadvertant light stimulation during the dark growth period rigorous exclusion of light during dark growth gives similar results.:' It appears that low levels of the protein are synthe- sized at one period during growth in the dark, and that such synthesis ceases but low levels of messenger RNA for phos- phoenolpyruvate carboxylase are maintained in the tissues.

Our previous work demonstrated that phosphoenolpyruvate carboxylase synthesized in vivo appears as a doublet band on SDS-polyacrylamide gradient gels. Both bands of the doublet increase in intensity during greening, and proteolytic digestion patterns of the individual bands are very similar (1). As shown here, we sometimes observe the two-band pattern in transla-

Unpublished results.

Cell-free Synthesis of Phosphoenolpyruvate Carboxylase 8255

tion assays (Fig. 4 ) and sometimes do not (Fig. 1). The reason for this discrepancy in different translations is unclear. We are attempting to resolve this question in order to clarify the molecular basis for the carboxylase protein doublet band that we observe consistently with protein synthesized in uiuo.

Acknowledgments-Staphylococcus aureus Cowan strain I was a generous gift of Dr. Edward Herbert. We would like to acknowledge the assistance of Evelyn Hess in cultivation of the plants used in this work.

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