mRNAs encoding ribulose-1,5-bisphosphate carboxylase ... - NCBI

Proc. Nail. Acad. Sci. USA Vol. 85, pp. 4190-4194, June 1988 Biochemistry

mRNAs encoding ribulose-1,5-bisphosphate carboxylase remain bound to polysomes but are not translated in amaranth seedlings transferred to darkness (light regulation/sucrose-gradient analysis/dot blot analysis/in vitro polysome run-off analysis/translational elongation arrest)

JAMES 0. BERRY, JOHN P. CARR, AND DANIEL F. KLESSIG Waksman Institute of Microbiology, Rutgers, The State University of New Jersey, Piscataway, NJ 08854

Communicated by C. S. Levings III, February 19, 1988

ABSTRACT When light-grown seedlings of amaranth are transferred to total darkness, synthesis of the large subunit (LS) and small subunit (SS) of ribulose-1,5-bisphosphate carboxylase [RbuP2Case; 3-phospho-D-glycerate carboxylase (dimerizing), EC 4.1.1.39] is rapidly depressed. This reduction in RbuP2Case synthesis occurs in the absence of any corresponding changes in levels of functional mRNA for either subunit. Four hours after light-to-dark transition little, if any, changes in the distribution of LS and SS mRNAs on polysomes could be detected. The association of these mRNAs with polysomes was authenticated by treatment with RNase A or puromycin. Furthermore, polysomes were able to synthesize LS and SS precursor in cell-free translation systems supplemented with inhibitors of 'initiation. Therefore, during a light-to-dark transition LS and SS mRNAs remained bound to polysomes but were not translated in vivo, suggesting that control is exercised, in part, at the translational elongation step.

SS polypeptides occur in response to changes in illumination without corresponding changes in mRNA levels. Because the stability of these polypeptides and the functionality in vitro of their respective mRNAs were not affected by alterations in illumination, the expression of these genes appears to be regulated, in part, at the translational level. Light-mediated translational regulation has also been observed in other plant species including Volvox (16), pea (17), barley (18), and Lemna (19). In this report we have examined the association of LS and SS mRNAs with polysomes during the rapid repression of RbuP2Case synthesis that occurs when seedlings are transferred to darkness. This study indicates that during a lightto-dark transition, regulation may occur during translational elongation as well as during initiation.

MATERIALS AND METHODS

Ribulose-1,5-bisphosphate carboxylase [RbuP2Case; 3phospho-D-glycerate carboxylase (dimerizing), EC 4.1.1.39] is found in the chloroplasts of all higher plants and is a principal enzyme in photosynthetic carbon fixation. This enzyme has a molecular mass of -555,000 and consists of eight large (51-58 kDa) and eight small (12-18 kDa) subunits (LS and SS, respectively) (1), with the active site located on the LS (2, 3). The LS is encoded on the chloroplast genome and translated on 70S chloroplast ribosomes (4, 5). The SS is encoded in the nucleus and translated on free, cytoplasmic ribosomes as a 20-kDa precursor (6-8). The precursor is processed to its final size during transport into the chloroplast, where it assembles with LS polypeptides to form the active holoenzyme. The control of RbuP2Case production and activity in a number of plant species is very complex, with regulation occurring at many levels. Control at the level of LS or SS mRNA accumulation has been well documented (9). In several cases alterations in transcriptional activity have been shown to be responsible for these changes in mRNA levels (9). At the other end of the spectrum, regulation at the posttranslational level via turnover of the protein (10) as well as by activation (1, 11) or inhibition (12) of the enzyme's activity has also been reported. We have previously described the effects of environmental (light) (13, 14) or developmental (13, 15) signals on the expression of LS and SS genes in the C4 dicotyledonous plant Amaranthus hypochondriacus. The expression of these genes is regulated not only at the level of mRNA accumulation but also posttranscriptionally. In amaranth cotyledons rapid and dramatic alterations in the synthesis of the LS and

A. hypochondriacus var. R103 was grown as described (13). For light-to-dark transitions, plants grown under normal illumination for 6 days were transferred to lightproof boxes in a darkroom for various times before harvesting. Extreme care was taken to avoid any exposure of the dark-shifted seedlings to light. Cotyledons from dark-shifted seedlings were harvested under a Kodak no. 7 green safelight. Analyses of in vivo protein synthesis and mRNA accumulation were as described (13). Polysomes were isolated according to the procedures of Jackson and Larkins (20) except that cycloheximide and chloramphenicol, each at 50 ,g/ml, were added to the isolation buffers. Crude polysomes were purified from initial extracts by pelleting through a 1.75 M sucrose cushion (20) at 60,000 rpm using a Ti8O rotor (Beckman) for either 3 or 18 hr. The crude polysome preparations were resuspended, clarified by centrifugation for 1 min in a Microfuge at 4°C to remove any remaining debris, and stored at -70°C. Three A260 units of the purified polysome preparation was loaded onto a 10-40% sucrose gradient [containing 40 mM Tris-HCI (pH 8.4), 20 mM KCl, 10 mM MgCl2] directly or first pretreated with a low concentration of RNase A (Sigma, 0.0025 Ig/ml, 10 min on ice) or puromycin (21) (Boehringer Mannheim, 500 ,g/ml in 0.5 M KCI, 15 min at 37°C) before loading. The gradients were centrifuged at 39,000 rpm in an SW41 rotor (Beckman) for 150 min at 4°C. The gradient profiles were determined by using a Perkin-Elmer Lambda 3A spectrophotometer with a flow cell. Gradient fractions were collected and immediately diluted with an equal volume of 50 mM Tris-HCI (pH 8.0), 50 mM EDTA, 1% NaDodSO4, then phenol extracted, and ethanol precipitated. The RNA fractions were characterized by dot blot analysis (Bethesda

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Abbreviations: RbuP2Case, ribulose-1,5-bisphosphate carboxylase; LS, large subunit(s) of RbuP2Case; SS, small subunit(s) of RbuP2Case; ATCA, aurin tricarboxylic acid.

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Biochemistry: Berry et al. Research Laboratories Hybri-Dot manifold) on nitrocellulose using LS (pAlsl) and SS (pAssl) clones as probes. Polysomes used in translational run-off experiments were isolated and fractionated on 10-40%o sucrose gradients as described above. The first four fractions from the sucrose gradients (fractions containing mainly polysomes) were collected into an equal volume of 40 mM Tris HCl (pH 8.0), 20 mM KCI, 10 mM MgCI2, and cycloheximide and choramphenicol, each at 20 gg/ml. The purified polysomes were concentrated by centrifugation through a 1.75 M sucrose cushion for 18 hr as described above. The polysome pellet was washed and resuspended in translational run-off buffer (10 mM Hepes-KOH, pH 7.6/100 mM KCI/4 mM MgAc2/1 mM dithiothreitol) (22) and stored at - 70°C. To identify the run-off translation products of cytoplasmic or chloroplast polysomes, polysomes purified on sucrose gradients were incubated in nuclease-treated rabbit reticulocyte lysates (23) (Green Hectares, Oregon, WI) or cell-free extracts of Escherichia coli (24), respectively. Reticulocyte lysates (32 Al, final volume), containing chloramphenicol at 25 ,g/ml to suppress translation by chloroplastic polysomes, were supplemented with 200 ,uM 7-methylguanosine monophosphate (25) and 10 AM edeine (26) to block translational initiation or with an equivalent volume of water (controls). Similarly, E. coli lysates (30 p1, final volume), containing cycloheximide at 50 pg/ml to block translation by cytoplasmic polysomes, were supplemented with the initiation inhibitor aurin tricarboxylic acid (ATCA) (50 ,uM) (27) or an equivalent volume of water. The in vitro translation systems were preincubated (reticulocyte lysates at 24°C, E. coli lysates at 37°C) for 10 min with or without the initiation inhibitors before the addition ofpolysomes (0.1-0.3 A260 unit) followed by incubation for a further 10 min after which the reactions were terminated by adding RNase A (1-5 ,ug). [35S]Methionine-labeled translation products were analyzed by NaDodSO4/PAGE with or without prior immunoprecipitation (13). All drugs were obtained from Sigma except for edeine, which was a gift from P. Walter (University of California, San Francisco).

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Proc. Natl. Acad. Sci. USA 85 (1988)

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FIG. 1. RbuP2Case synthesis and mRNA accumulation in response to a light-to-dark transition. (A) LS and SS polypeptide synthesis. Amaranth seedlings were germinated and grown under normal illumination for 6 days and then transferred to total darkness. The seedlings were incubated for 2 hr with [35S]methionine at the time periods indicated. To determine changes in LS and SS synthesis equal amounts of incorporated radioactivity were immunoprecipitated with anti-LS and anti-SS sera and fractionated by NaDodSO4/ PAGE. (B) LS and SS mRNA accumulation in response to a light-to-dark transition. Total RNA was extracted in parallel from 6-day-old, light-grown cotyledons and from cotyledons transferred to darkness for the time periods indicated and subjected to RNA-blot analysis, with pAlsl and pAssl used as probes.

profiles of polysomes obtained from light-grown cotyledons. Profiles for cotyledons transferred to darkness for up to 10 hr were similar and are not shown. Four hours after the transfer

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RESULTS Effect of Light-to-Dark Transition on LS and SS Gene Expression. When amaranth seedlings grown under normal illumination were transferred to total darkness, general protein synthesis in the cotyledons was reduced moderately (by factors of 2-4 in 4 hr and by factors of 4-6 in 10 hr after transfer to darkness; data not shown and ref. 14). Synthesis of most proteins was moderately reduced, whereas synthesis of some proteins remained constant, and production of at least one protein actually increased. In contrast, synthesis of the LS and SS polypeptides was very dramatically depressed (Fig. LA). LS and SS synthesis declined more than general protein synthesis by a factor of 10-20 by 4 hr and >20-fold by 10 hr after transfer to darkness. The rapid and dramatic reductions in synthesis of the LS and SS polypeptides were not accompanied by similar changes in the levels of the corresponding mRNAs. Equivalent levels of the LS transcripts were found in light-grown cotyledons and in cotyledons transferred to darkness for as long as 10 hr (Fig. 1B), whereas the amount of SS mRNA was reduced by a factor of 2-3 after the shift to dark. In several other experiments no reduction in SS mRNA levels could be detected after transfer into darkness (data not shown). Association of LS and SS mRNAs with Polysomes. Our previous studies indicated that this shutdown of RbuP2Case synthesis may be due to regulation at the translational level (14). Therefore, it was of interest to determine whether these mRNAs were associated with polysomes in light-grown and in dark-shifted cotyledons. Fig. 2 shows sucrose-gradient

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FIG. 2. Association of LS and SS mRNAs with polysomes during a light-to-dark transition. (A) Polysome profile and dot blot analysis of crude polysome preparations isolated by 3 hr of sedimentation of extracts from light-grown or dark-shifted seedlings. (B) Profile and dot blot analysis of crude polysome preparations isolated by 18-hr centrifugation of plant extracts. Polysomes were prepared from light-grown seedlings or from seedlings transferred to darkness for the time periods indicated. Sedimentation is from right to left. Polysomes (cytoplasmic and chloroplastic) are located primarily in fractions 1-4; monosomes and ribosomal subunits are in fractions 6 and 7. Distribution of the LS and SS mRNAs was determined by dot blot analysis using pAlsl and pAssl as probes.

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Biochemistry: Berry et al.

from light to dark no changes in the association of the LS and SS mRNAs with polysomes had occurred. However, the amount of these transcripts associated with polysomes had begun to decrease by 6 hr and was greatly reduced by 10 hr after the shift to dark. The amount of LS or SS mRNA seen in the profiles was much lower in the 10-hr dark-shifted material than in extracts from light-grown or 4-hr dark-shifted plants (Fig. 2). This was unexpected because little or no change in transcript levels was detected by RNA analysis of total RNA (Fig. 1B). A likely explanation for this discrepancy is the manner of preparation of the crude polysome fraction. A common method of isolating polysomes from plant tissue involves centrifugation of the initial crude extract through a dense sucrose cushion (20, 28, 29). This concentrates the polysomes, monosomes, and free messenger-ribonucleoproteins and separates them from debris that can interfere with their subsequent analysis. A major problem with this sedimentation procedure, especially when using relatively short centrifugation times, is that it tends to enrich for polysomes while leaving monosomes and free messenger-ribonucleoproteins underrepresented in the pellet (28, 29). In the experiment described above (Fig. 2A) crude polysomes were harvested by sedimentation for 3 hr through a 1.75 M sucrose cushion before being resuspended and subjected to sucrose-gradient analysis. Presumably the levels of LS or SS mRNA in the profiles for 10-hr dark-shifted seedlings were reduced because a large proportion of the mRNA was not associated with polysomes and therefore sedimented through the sucrose cushion less rapidly and efficiently than the denser, polysome-associated messages. Increasing the sedimentation time to 18 hr resulted in a more quantitative (but still not complete) recovery of the LS and SS mRNAs in crude polysome pellets from 6- and 10-hr dark-shifted seedlings (Fig. 2B). Therefore, although shorter sedimentation time was adequate for the recovery and identification of polysome-associated RbuP2Case transcripts, longer sedimentation time was required for a more complete isolation and identification of monosome/free messenger-ribonucleoprotein-associated transcripts. Analysis of the crude polysome preparations obtained with the long sedimentation time confirmed that little change occurred in the association of the LS and SS transcripts with polysomes after 4 hr in darkness, but by 10 hr significant dissociation of these mRNAs from polysomes had occurred. These results suggest that the LS and SS mRNAs were still associated with polysomes 4 hr after the shift from light to darkness (Figs. 2 and 3A), even though their translation was dramatically reduced. To insure that the migration of the LS and SS mRNAs in the polysome regions of the gradients was due to authentic association of these mRNAs with ribosomes rather than to fortuitous comigration, the following criteria were used. (i) The crude polysome preparations were subjected to very mild RNase A digestion to cleave the mRNA between the ribosomes. Polysomes from dark-shifted as well as light-grown seedlings were destroyed by this treatment (Fig. 3B), and the LS and SS mRNAs migrated in the monosome region of the gradient. (ii) Pretreatment of polysomes with puromycin (21), under conditions that release mRNAs as well as nascent polypeptides, resulted again in destruction of the polysomes (Fig. 3C) and shifting of these two mRNAs to the top of the gradient. These two sets of experiments confirmed that the LS and SS mRNAs were bound to authentic polysomes in both light-grown and darkshifted seedlings. Translational Run-Off Analysis of Polysomes in Vitro. As a further test to confirm that the LS and SS mRNAs remained on polysomes after shifting into darkness, isolated polysomes were subjected to run-off analysis in vitro. The first four fractions from the sucrose gradient (fractions enriched in polysomes) were collected, concentrated, and used for in


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vitro translational run-off analysis. The polysomes were added to either an E. coli (for LS) or rabbit reticulocyte (for SS) cell-free translation system containing ATCA or edeine/ 7-methylguanosine monophosphate, respectively, to inhibit initiation. In the E. coli system ATCA had little or no effect on polysome-directed protein synthesis but reduced RNAdirected synthesis by >90%, showing that the drug was functioning as a specific inhibitor of initiation (Fig. 4A). Addition of edeine/7-methylguanosine monophosphate to the rabbit reticulocyte system caused a small reduction in polysome-directed protein synthesis, but the block to RNAdirected synthesis was virtually complete (Fig. 4B). This suggested that although this drug combination was highly effective in preventing translational initiation, it might also slightly inhibit elongation. Alternatively, the drug combination may have been preventing reinitiation of polysomal mRNAs. Polysomes isolated from light-grown versus 4-hr darkshifted cotyledons produced similar amounts of either subunit in vitro when translational initiation was inhibited (Fig. 4). Therefore, not only are these transcripts present on polysomes from 4-hr dark-shifted plants, but the polysomes are functional in vitro, even though elongation in vivo is apparently blocked. Polysomes isolated from 6- and 10-hr dark-shifted cotyledons produced less of these polypeptides in run-off translation (data not shown), indicating that at these later times the LS and SS mRNAs had begun to dissociate from functional polysomes. Block to Translational Initiation. If ribosomes in darkshifted cotyledons continued to efficiently initiate translation but failed to complete a round of translation at the normal rate due to the inhibition of elongation, then the LS and SS

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mRNAs might be expected to shift to larger polysomes. Because this shift upon transfer to darkness was not detected (Figs. 2 and 3), we suspect that initiation as well as elongation are inhibited.

DISCUSSION When light-grown amaranth seedlings were transferred to total darkness, synthesis of the RbuP2Case LS and SS polypeptides was depressed by a factor of 10- to 20-fold more than that of general protein synthesis within the first 4 hr after transfer. This reduction in RbuP2Case synthesis occurred without any corresponding changes in levels of functional mRNA for either subunit. Both the LS and SS mRNAs remained bound to polysomes for several hours after the shift to dark. Furthermore, purified polysomes from 4-hr darkshifted cotyledons (as well as light-grown) could synthesize LS and SS precursors in vitro in the presence of initiation inhibitors, indicating that both mRNAs were associated with functional ribosomes. These results suggest that in the absence of light, translational elongation of the LS and SS polypeptides is inhibited. Alternatively, the two polypeptides might be produced and then rapidly degraded in the dark. However, RbuP2Case in other systems has been shown to be very stable, with half-lives on the order of days (30, 31). In addition, our previous results (14) and those of Ferreira and Davies (32) failed to detect any increased turnover of these polypeptides in the dark. However, we cannot rule out the possibility that two subpopulations of one or both subunits exist-one population that is stable and was detected in our previous pulse-chase experiments and a second population that is rapidly turned over and hence was not detectable. Perhaps both levels of control are used here. For example, in darkness, synthesis of the LS as well as several other chloroplast proteins might be repressed. In the absence of

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LS, which is required for holoenzyme formation, newly synthesized SS might be rapidly degraded. Schmidt and Mishkind (10) observed that in Chlamydomonas mutants defective in chloroplast protein synthesis SS was rapidly turned over. If, as we suspect, translational elongation is blocked in dark-shifted cotyledons, then this block must be readily reversible. When dark-shifted seedlings were transferred back into light, synthesis of both RbuP2Case subunits was resumed within the first 2 hr of illumination (data not shown). In addition, translational run-off analysis indicated that this block could be overcome when dark-shifted polysomes were added to heterologous cell-free translation extracts. If the movement of the ribosomes along these messages in the absence of light were slowed or completely arrested at a specific site(s) on the mRNA located some distance from the initiation codon, then these two mRNAs should be found on larger polysomes after the dark-shift, assuming translational initiation is normal. This situation was not observed, suggesting that initiation as well as elongation are impaired. Similar reports of a block to both initiation and elongation have been described in heat-shocked Drosophila cells (33), in HeLa cells stressed with amino acid analogues (34) or coinfected with influenza virus and adenovirus (35), and in monkey cells abortively infected with adenovirus (36). In these reports nonstress host mRNAs or certain viral RNAs remained attached to cytoplasmic polysomes, even though their translation was blocked. In addition, evidence for light-dependent regulation at the level of translation elongation has been described for Lemna gibba (19). A unique aspect of RbuP2Case regulation in amaranth cotyledons, which is apparent from this study as well as from our previous work, is that rapid light-induced changes in the synthesis of the chloroplast-encoded LS and nuclearencoded SS occur in parallel. Synthesis of both polypeptides appears to be coordinately depressed by darkness and coordinately induced by light. In these different cellular compartments (the chloroplasts, nucleus, and cytoplasm) light-regulated RbuP2Case gene expression is subject to a complex set of control mechanisms, both transcriptional and posttranscriptional. Several of these control mechanisms may be used to maintain coordinate expression of these two genes (37, 38). In dark-shifted cotyledons the synthesis of the LS and SS appears to be regulated, in part, at the level of translational elongation. These results suggest that similar control mechanisms may function in the chloroplasts and in the cytoplasm to regulate the production of RbuP2Case in amaranth cotyledons. We thank Drs. Ellie Ehrenfeld and Donald Summers for their advice on polysome analysis. This work was supported by Grant DCB-8517972 from the National Science Foundation and an Individual Research Award in Plant Biology from the McKnight Foundation. D.F.K. is recipient of Faculty Research Award 270 from the American Cancer Society. 1. Miziorko, H. M. & Lorimer, G. H. (1983) Annu. Rev. Biochem. 52, 507-535. 2. Lorimer, G. H. (1981) Biochemistry 20, 1236-1240. 3. Lorimer, G. H. & Miziorko, H. M. (1980) Biochemistry 19, 5321-5328. 4. Coen, D. M., Bedbrook, J. R., Bogorad, L. & Rich, A. (1977) Proc. Natl. Acad. Sci. USA 74, 5487-5491. 5. Ellis, R. J. (1981) Annu. Rev. Plant Physiol. 32, 111-137. 6. Cashmore, A. R., Broadhurst, M. K. & Gray, R. E. (1978) Proc. Natl. Acad. Sci. USA 75, 655-659. 7. Chua, N.-H. & Schmidt, G. W. (1978) Proc. Natl. Acad. Sci. USA 75, 6110-6114. 8. Highfield, P. E. & Ellis, R. J. (1978) Nature (London) 271, 420-424. 9. Tobin, E. M. & Silverthorne, J. (1985) Annu. Rev. Plant Physiol. 36, 569-593.

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