Gene-specific trans-Regulatory Functions of Magnesium for Chloroplast mRNA Stability in Higher Plants
2000; Elsevier BV; Volume: 275; Issue: 45 Linguagem: Inglês
10.1074/jbc.m005622200
ISSN1083-351X
Autores Tópico(s)Plant Micronutrient Interactions and Effects
ResumoIn higher plant chloroplasts the accumulation of plastid-encoded mRNAs during leaf maturation is regulatedvia gene-specific mRNA stabilization. The half-lives of chloroplast RNAs are specifically affected by magnesium ions.psbA mRNA (D1 protein of photosystem II),rbcL mRNA (large subunit of ribulose-1,5-bisphosphate carboxylase), 16 S rRNA, and tRNAHis gain stability at specific magnesium concentrations in an in vitrodegradation system from spinach chloroplasts. Each RNA exhibits a typical magnesium concentration-dependent stabilization profile. It shows a cooperative response of the stability-regulatedpsbA mRNA and a saturation curve for the other RNAs. The concentration of free Mg2+ rises during chloroplast development within a range sufficient to mediate gene-specific mRNA stabilization in vivo as observed in vitro. We suggest that magnesium ions are a trans-acting factor mediating differential mRNA stability. In higher plant chloroplasts the accumulation of plastid-encoded mRNAs during leaf maturation is regulatedvia gene-specific mRNA stabilization. The half-lives of chloroplast RNAs are specifically affected by magnesium ions.psbA mRNA (D1 protein of photosystem II),rbcL mRNA (large subunit of ribulose-1,5-bisphosphate carboxylase), 16 S rRNA, and tRNAHis gain stability at specific magnesium concentrations in an in vitrodegradation system from spinach chloroplasts. Each RNA exhibits a typical magnesium concentration-dependent stabilization profile. It shows a cooperative response of the stability-regulatedpsbA mRNA and a saturation curve for the other RNAs. The concentration of free Mg2+ rises during chloroplast development within a range sufficient to mediate gene-specific mRNA stabilization in vivo as observed in vitro. We suggest that magnesium ions are a trans-acting factor mediating differential mRNA stability. polynucleotide phosphorylase The efficiency of gene expression depends on the stability of mRNAs by determining the pool of templates available for synthesis of the respective gene products. Gene regulation in many systems is accomplished by changing the stability of a certain message during the course of a developmental program or in response to environmental changes (1Atwater J.A. Wisdom R. Verma I.M. Annu. Rev. Genet. 1990; 24: 519-541Crossref PubMed Scopus (212) Google Scholar). In chloroplasts of higher plants differential mRNA stability is responsible for controlling mRNA accumulation during chloroplast maturation (2Gruissem W. Tonkyn J.C. Crit. Rev. Plant Sci. 1993; 12: 19-55Crossref Scopus (139) Google Scholar, 3Gruissem W. Schuster G. Brawerman G. Belasco J. Control of mRNA Stability. Academic Press, New York1993: 329-365Google Scholar). In spinach as well as in barley it has been shown that during leaf development mRNAs are stabilized in a gene-specific manner (4Klaff P. Gruissem W. Plant Cell. 1991; 3: 517-529Crossref PubMed Google Scholar, 5Kim J. Klein P.G. Mullet J.E. J. Biol. Chem. 1994; 269: 17918-17923Abstract Full Text PDF PubMed Google Scholar). The mRNA that is stabilized to the highest extent encodes the D1 reaction center protein of photosystem II (psbA). In recent years considerable progress has been made in elucidating the mRNA degradation mechanism in chloroplasts (6Schuster G. Lisitsky I. Klaff P. Plant Physiol. 1999; 120: 937-944Crossref PubMed Scopus (69) Google Scholar, 7Hayes R. Kudla J. Gruissem W. Trends Biochem. Sci. 1999; 24: 199-202Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar). In higher plants plastid mRNA degradation is initiated by endonucleolytic cleavages (8Klaff P. Nucleic Acids Res. 1995; 23: 4885-4892Crossref PubMed Scopus (32) Google Scholar). The resulting proximal fragments are polyadenylated as a tag for rapid exonucleolytic decay (9Lisitsky I. Klaff P. Schuster G. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 13398-13403Crossref PubMed Scopus (124) Google Scholar, 10Lisitsky I. Klaff P. Schuster G. Plant J. 1997; 12: 1173-1178Crossref Scopus (29) Google Scholar). This mechanism implies that initiation of mRNA decay is the crucial process by which the stability of a certain message is regulated. The accessibility of the primary cleavage sites for endonucleases determines the proportion of molecules to be degraded. However, only limited information is available so far about the molecular mechanism mediating the regulation of degradation initiation, the cis-regulatory elements and trans-regulatory factors involved. Up to now, only a few cis-regulatory elements of mRNAs have been described using genetic approaches in higher plants and in the green alga Chlamydomonas reinhardtii. In Chlamydomonasseveral nuclear mutants have been isolated that affect the stability of a variety of chloroplast-encoded mRNAs; those mutations each interfere with the accumulation of a single defined chloroplast mRNA (11Rochaix J.-D. Annu. Rev. Cell Biol. 1992; 8: 1-28Crossref PubMed Scopus (129) Google Scholar). In the nuclear mutation nac2–26, for example, the stability of the chloroplast psbD mRNA is dramatically decreased (12Kuchka M.R. Goldschmidt-Clermont M. Dillewijn J.v. Rochaix J.-D. Cell. 1989; 58: 869-876Abstract Full Text PDF PubMed Scopus (120) Google Scholar). Chloroplast transformation with constructs of the psbD leader fused to a reporter gene showed a destabilized chimeric transcript in the mutant background and normal accumulation in the wild type, indicating that the 74-nucleotide leader of the mRNA includes a determinant for psbDmRNA degradation (13Nickelsen J. Dillewijn J.v. Rahire M. Rochaix J.-D. EMBO J. 1994; 13: 3182-3191Crossref PubMed Scopus (128) Google Scholar, 14Nickelsen J. Fleischmann M. Boudreau E. Rahire M. Rochaix J.D. Plant Cell. 1999; 11: 957-970Crossref PubMed Scopus (103) Google Scholar). Chloroplast lysates from wild type and mutant cells as an in vitro degradation system for the analysis of synthetic RNA transcripts reflect the observations madein vivo. The primary cleavage sites could be detected on these RNA transcripts. cis-Regulatory elements for the degradation of rbcL mRNA in Chlamydomonashave also been analyzed (15Salvador M.L. Klein U. Bogorad L. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 1556-1560Crossref PubMed Scopus (62) Google Scholar). The 63 nucleotides of the rbcL5′ leader fused to the Escherichia coli β-glucuronidase gene (gus) as a reporter confer instability to the chimeric transcripts in the light. The addition of the 257 nucleotides from the adjacent coding region prevented this destabilization. InChlamydomonas the role of the 5′ untranslated region of thepetD mRNA for RNA stability and translation was studied by extensive mutational analysis. It was demonstrated that sequences essential for translation, as well as sequences that directly or indirectly affect RNA stability, reside within the 5′ untranslated region of the petD mRNA. The finding that in all mutants where translation was compromised petD mRNA accumulated to a lower level than in wild type strains indicated that mRNA stability may be linked to translatability (16Sakamoto W. Kindle K.L. Stern D.B. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 497-501Crossref PubMed Scopus (91) Google Scholar). In chloroplasts of higher plants a correlation of polysome association and mRNA stability was suggested for rbcL mRNA from studies of nuclear mutants in maize. Mutants in which many chloroplast mRNAs are associated with abnormally few ribosomes showed that the level of rbcL mRNA was reduced 4-fold, indicating that the rbcL mRNA is destabilized as a consequence of its decreased polysome association (17Barkan A. Plant Cell. 1993; 5: 389-402Crossref PubMed Google Scholar). Further indications thatcis-regulatory elements in higher plant chloroplast mRNAs are involved in RNA accumulation come from studies with transformed chloroplasts of tobacco. Fusions of different 3′ untranslated regions to the E. coli β-glucuronidase gene, which in these constructs is preceded by the psbA promoter and the psbA 5′ untranslated region, suggested that the accumulation of the chimeric RNA is not changed dramatically by the different 3′ ends but is more likely influenced by other elements of the RNA (18Staub J.M. Maliga P. Plant J. 1994; 6: 547-553Crossref PubMed Scopus (134) Google Scholar). Direct evidence for cis-regulatory functions of the 5′ region of higher plant chloroplast mRNAs comes from transplastomic plants carrying fusions of the gus gene withrbcL promotor/leader fragments. gus mRNA accumulation is independent of light as long as a certain element of the 5′ untranslated region is included in the construct. Lower rates ofrbcL transcription in the dark were compensated by increased mRNA stability (19Shiina T. Allison L. Maliga P. Plant Cell. 1998; 10: 1713-1722Crossref PubMed Scopus (83) Google Scholar). In tobacco, the 5′ untranslated region ofpsbA mRNA alone seems not to be sufficient to confer the stability of the intact mRNA to a reporter fusion construct (20Eibl C. Zou Z. Beck A. Kim M. Mullet J. Koop H.-U. Plant J. 1999; 19: 333-345Crossref PubMed Scopus (136) Google Scholar). Most chloroplast mRNAs are flanked by a stem-loop structure in their 3′ untranslated region that participates in the processing of the mature 3′ end (3Gruissem W. Schuster G. Brawerman G. Belasco J. Control of mRNA Stability. Academic Press, New York1993: 329-365Google Scholar). In addition, these elements are important for impeding the progress of processive exoribonucleases (21Adams C.C. Stern D.B. Nucleic Acids Res. 1990; 18: 6003-6010Crossref PubMed Scopus (71) Google Scholar). In transformed Chlamydomonas chloroplasts in vivo, it has been shown that partial or complete deletions of the stem-loop of the atpB gene leads to a decrease in mRNA accumulation, whereas the transcription rate of this gene remains unaffected (22Stern D.B. Radwanski E.R. Kindle K. Plant Cell. 1991; 3: 285-297PubMed Google Scholar). The stem-loop structure can be replacedin vivo by a sequence of 18 guanosines, which also serves as a barrier for a 3′→5′ exonuclease in vitro. Strains containing the polyguanosine tract instead of the stem-loop structure within the 3′ untranslated region accumulate nearly wild type levels ofatpB transcripts and the ATPase β-subunit protein (23Drager R.G. Zeidler M. Simpson C.L. Stern D.B. RNA. 1996; 2: 652-663PubMed Google Scholar). The stem-loop structures of the 3′ untranslated regions are known to bind chloroplast proteins. The petD 3′ untranslated region forms a complex with 55-, 41-, and 29-kDa RNA-binding proteins. An 8-nucleotide AU-rich sequence motif downstream of the stem-loop, termed box II, appears to be essential for RNA-protein complex formationin vitro (24Chen Q. Adams C.C. Usak L. Yang J. Monde R.-A. Stern D.B. Mol. Cell. Biol. 1995; 15: 2010-2018Crossref PubMed Scopus (41) Google Scholar). In addition, the stem-loop itself is necessary for protein binding. The AU-rich box is also recognized by a 57-kDa protein, which possibly forms a stable complex together with a 33-kDa protein (25Hsu-Ching C. Stern D.B. Mol. Cell. Biol. 1991; 11: 4380-4388Crossref PubMed Scopus (45) Google Scholar). These proteins may either be involved in mRNA processing or mediate stabilization against degradation. Beyond those proteins, most RNA-binding proteins that so far have been isolated, cloned, and characterized from chloroplasts of higher plants cannot be assigned to specific mRNAs but exhibit general functions. The 28-kDa ribonucleoprotein is involved in 3′ end processing of several mRNAs (26Schuster G. Gruissem W. EMBO J. 1991; 10: 1493-1503Crossref PubMed Scopus (155) Google Scholar); the 100-kDa ribonucleoprotein is the exonuclease polynucleotide phosphorylase (27Hayes R. Kudla J. Schuster G. Gabay P. Maliga P. Gruissem W. EMBO J. 1996; 15: 1132-1141Crossref PubMed Scopus (145) Google Scholar); the ribosomal protein S1 (28Alexander C. Faber N. Klaff P. Nucleic Acids Res. 1998; 26: 2265-2272Crossref PubMed Scopus (44) Google Scholar) and the nuclease CSP41, which had previously been identified as a 3′ end binding protein (29Yang J. Schuster G. Stern D.B. Plant Cell. 1996; 8: 1409-1420PubMed Google Scholar), could also be detected as part of a complex binding to the 5′ untranslated region ofpsbA mRNA. 1P. Klaff, unpublished data. In this work we provide data showing that, in addition to proteins, the divalent cation Mg2+ (as a non-proteinaceous factor) is required not only for the formation of chemically stable and functional RNA in a general manner but also for gene-specific differential stabilization of chloroplast RNAs. Furthermore, we show that the concentration of free magnesium ions rises during chloroplast development within a range sufficient to confer gene-specific mRNA stability regulation. Spinach plants (Spinacea oleraceaL. cv. Monnopa) were grown on soil in the greenhouse with additional illumination during wintertime to result in 12 h of light per day. For magnesium determinations seedlings were grown in a growth chamber under conditions outlined under "Results." DNA oligonucleotides were commercially synthesized by Interactiva (Ulm, Germany). The following oligonucleotides were used for Northern analysis: psbA-1, 5′-ATTCGCTAGAAATAGAAATTGAAAGATTGTTATT-3′ (complementary to positions −86 to −53 of the mRNA); psbA-2, 5′-TGGTTTATTTAATTTAATCATCAGGG-3′ (complementary to positions −36 to −10 of the mRNA); psbA-3, 5′-GGCTTTCGCTTTCGCGTCTC-3′ (complementary to positions 18–37 of the mRNA); rbcL-1, 5′-GGTCTACTCGACATAAATTAGG-3′ (complementary to positions −72 to −50 of the mRNA); rbcL-2, 5′-GGACTTACTCGGAATGCTGCC-3′ (complementary to positions 111–121 of the mRNA); 16 S, 5′-GTCTCAGTCCCAGTGTGGCTGATCA-3′ (complementary to positions 275–299 of the corresponding tobacco RNA); tRNAHis, 5′-GGCGAACGACGGGAATTGAAC-3′ (complementary to positions 55–75 of the genomic sequence). Numeration of the RNA sequences ofrbcL and psbA is according to Refs. 30Zurawski G. Perrot B. Bottomley W. Whitfeld P.R. Nucleic Acids Res. 1981; 9: 3251-3270Crossref PubMed Scopus (277) Google Scholar and 31Zurawski G. Bohnert H.J. Whitfeld P.R. Bottomley W. Proc. Natl. Acad. Sci. U. S. A. 1982; 79: 7699-7703Crossref PubMed Scopus (319) Google Scholar, respectively; GenBankTM accession numbers of 16 S rRNA and tRNAHis are Z00044S54304 and X00795, respectively. 5′ end-labeling of the oligonucleotides with [γ-32P]ATP was performed according to Ref. 32Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. 2nd Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, New York1989Google Scholar. Intact chloroplasts were isolated according to Ref. 33Gruissem W. Greenberg B.M. Zurawski G. Hallick R.B. Methods Enzymol. 1986; 118: 253-270Crossref PubMed Scopus (93) Google Scholar. For preparation ofin vitro degradation extracts, chloroplasts were resuspended in 20 mm HEPES, pH 7.9, 60 mm KCl, 20 mm EDTA, 2 mm dithiothreitol, and 20% (v/v) glycerol and lysed by 10–15 strokes using a potter with pestle S. Extracts were adjusted to approximately 5 mg/ml protein as determined by Bradford assay. The extracts were stored at −70 °C after freezing in liquid nitrogen. Chlorophyll concentrations were determined spectrophotometrically (34Arnon D.I. Plant Physiol. 1949; 24: 1-13Crossref PubMed Google Scholar). In vitro degradation experiments were performed with 100 μl of degradation extract (5 mg/ml protein) per time point. The extract was thawed on ice, and the mixture was transferred to 25 °C for incubation. Reactions were stopped by adding 50 μl of 6.0 m urea, 1.0% SDS, and 200 μl of phenol/chloroform (8Klaff P. Nucleic Acids Res. 1995; 23: 4885-4892Crossref PubMed Scopus (32) Google Scholar). After phenol/chloroform extraction and a subsequent chloroform extraction, nucleic acids were recovered by ethanol precipitation. Concentrations of nucleic acids were determined spectrophotometrically at A 260. For Northern analysis 2 μg of chloroplast RNA per lane were separated on 1.2% agarose-formaldehyde gels according to Ref. 32Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. 2nd Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, New York1989Google Scholar. Hybridization conditions were as published (4Klaff P. Gruissem W. Plant Cell. 1991; 3: 517-529Crossref PubMed Google Scholar) except that oligonucleotide probes were used. Hybridization temperatures were as follows: psbA-3, 60 °C; rbcL-1/rbcL-2 mixture, 50 °C; 16 S, 55 °C; tRNAHis, 50 °C. Filters were washed three times for 30 min at the respective hybridization temperatures in 5× SSC, pH 7.0, 0.1% SDS. Filters were exposed to Kodak X-AR x-ray films. An excess of the probe was always examined by a dilution series of total spinach RNA on each blot to ensure the quantitative Northern analysis. For high resolution Northern analysis 2 μg of chloroplast RNA per lane were separated on denaturating 5% sequencing polyacrylamide gels containing 8m urea, 0.5× TBE (10×: 89 mm Tris, 89 mm boric acid, 1 mm EDTA). Gels were pre-run at 100 W for 20 min to allow heating and run at 100 W for 60 min. RNA samples were dissolved in 90% formamide containing 0.05% bromphenol blue, 0.05% xylenecyanole, and 1 mm EDTA, heated to 85 °C for 5 min, and chilled on ice prior to loading onto the gel. Gels were transferred to a Biodyne A nylon membrane (Pall Europe Limited, Portsmouth, United Kingdom) in 5× SSC overnight using the setup for agarose gels. The membrane was UV-treated to covalently couple the RNA (120 mJ, Stratalinker; Stratagene GmbH, Heidelberg, Germany). The hybridization procedure was performed as described above. The probe for high resolution Northern analysis was the psbA-1/psbA-2 mixture, which was hybridized at 40 °C. Intact chloroplasts were isolated as described above, except using buffers devoid of magnesium and EDTA. Lysis was performed by resuspension in 20 mm HEPES, 60 mmKCl, 15 mm KOH (to pH 7.9), and 20% (v/v) glycerol and 10 strokes each in a potter with first a light pistil and then a tight fitting pistil. The stromal fraction was isolated as the supernatant of a 30-min centrifugation at 20,000 rpm (Beckman JA20.1 rotor). The chloroplast stroma was filtrated through CentriconTM tubes (Amicon/Millipore, Bedford, MA) with an exclusion mass of 30 kDa. The resulting solution was directly subjected to Mg2+determination. The concentration of free Mg2+ was measured using the magnesium-selective macroelectrode ETH 7025 as described (35Zhang W. Truttmann A.C. Luthi D. McGuigan J.A. Magnes. Bull. 1997; 17: 125-130Google Scholar). Briefly, the magnesium electrode was calibrated in the "background buffer" used for chloroplast extract preparation (20 mm HEPES, 60 mm KCl, 15 mm KOH (to pH 7.9), 20% (v/v) glycerol) followed immediately by measuring up to 11 extract samples and a subsequent additional calibration to assay for drift. The calibration curves were linear from 20–0.15 mm Mg2+, with regression coefficients >0.98. Isolated intact chloroplasts were analyzed for their dimensions using a Zeiss Photomikroskop III. Photographic prints were scanned, and the images were evaluated using the ScionImage software (Scion Corp., obtained as freeware). The length l and width w of chloroplasts were determined, and the volume V was approximated by a rotational ellipsoid. V=2π·w3·l22Equation 1 X-ray films were quantified by densitometric analysis using a Hewlett Packard ScanJet 4c/T scanner. Scans were evaluated as TIFF images using the ScionImage software. Linearity of the scanned hybridization signal was ensured by dilution series of total RNA coprocessed with each quantitative Northern analysis. Relative RNA stability for magnesium-dependent stability profiles was determined by normalizing the data points obtained after 180 min of incubation to the "0" time point. To plot the relative RNA stability against concentrations of free Mg2+, the data were fitted using a modification of the Hill algorithm for binding of small ligands to a macromolecule (36Cantor C.R. Schimmel P.R. Biophysical Chemistry. W. H. Freeman and Company, San Francisco1980Google Scholar). Here the mathematics was applied to describe the stabilizing effect of magnesium ions, i.e. binding is replaced by the stabilizing effect in the analysis. The original Hill function,f=[L] nKn+[L] nEquation 2 where f = fraction of binding sites bound,n = Hill coefficient (n > 1 for cooperative binding), [L] = free ligand concentration, andK = apparent dissociation constant for interacting sites, is therefore modified to fit the stabilization profiles,S([Mg2+])=SO+(Smax−SO)·[Mg2+] nKn+[Mg2+] nEquation 3 where S = RNA stability in relative units as a function of magnesium concentration, S 0 = stability at 20 mm EDTA (no magnesium added to correct for the offset), S max = maximal RNA stability,n = Hill coefficient, and K = [Mg2+] of half-maximum RNA stability. Fits were performed using the Origin software (Microcal Corp.). The rbcL mRNA 5′ untranslated region had been cloned as a 216-base pair PCR fragment containing the T7 promotor fused to a BglII restriction site, and the rbcL 5′ untranslated region (−180–6, Ref.30Zurawski G. Perrot B. Bottomley W. Whitfeld P.R. Nucleic Acids Res. 1981; 9: 3251-3270Crossref PubMed Scopus (277) Google Scholar) was cloned into BamHI/HincII sites of pUC18. The insert sequence was verified by sequencing. Radiolabeled RNA transcripts were synthesized using the T7 in vitrotranscription system (37Klaff P. Mundt S.M. Steger G. RNA. 1997; 3: 1468-1479PubMed Google Scholar). For analysis of protein binding, label transfer experiments were performed according to Ref. 38Klaff P. Gruissem W. Photosynth. Res. 1995; 46: 235-248Crossref PubMed Scopus (21) Google Scholar. 12.5% polyacrylamide/SDS gels were used according to Ref. 39Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (212382) Google Scholar. The gels were stained with silver nitrate (40Blum H. Beier H. Gross H.J. Electrophoresis. 1987; 8: 93-99Crossref Scopus (3784) Google Scholar), dried, and exposed to Kodak XAR x-ray films. To study chloroplast mRNA degradation we recently established an in vitro degradation system that faithfully reflects mRNA degradation in terms of cleavages and processing of degradation fragments after cleavage (8Klaff P. Nucleic Acids Res. 1995; 23: 4885-4892Crossref PubMed Scopus (32) Google Scholar, 9Lisitsky I. Klaff P. Schuster G. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 13398-13403Crossref PubMed Scopus (124) Google Scholar, 10Lisitsky I. Klaff P. Schuster G. Plant J. 1997; 12: 1173-1178Crossref Scopus (29) Google Scholar). This system consists of isolated, lysed chloroplasts and allows the observation of the internal mRNA that is complexed with proteins as in the native state as well as the analysis of additionally added transcripts. Lysis of the chloroplasts enables us to vary degradation conditions externally by variation of the buffer constituents. Earlier work already showed the effect of magnesium ions on RNA stability, an effect that is destabilizing in chloroplast extracts from Chlamydomonas (13Nickelsen J. Dillewijn J.v. Rahire M. Rochaix J.-D. EMBO J. 1994; 13: 3182-3191Crossref PubMed Scopus (128) Google Scholar). In degradation extracts of spinach chloroplasts magnesium ions have the opposite effect. They induce stabilization of internal RNAs. In the experiment shown in Fig. 1 a degradation extract was prepared in the presence of 20 mm EDTA to complex the endogenous magnesium ions of the chloroplast. The Mg2+ content was reconstituted by adding MgCl2to a concentration of 25 mm. After incubation of the extract at room temperature for different periods of time as indicated in Fig. 1, total RNA was prepared and subjected to Northern analysis using gene-specific probes. As a control for ionic strength effects NaCl was added to another series of degradation experiments to a final concentration of 50 mm. We analyzed two mRNAs,psbA and the rbcL mRNA (psbA: D1 reaction center protein of photosystem II; rbcL: large subunit of the ribulose-1,5-bisphosphate carboxylase). In spinach,psbA mRNA is stabilized during leaf development, whereas no dramatic changes can be observed in the stability of rbcLmRNA (4Klaff P. Gruissem W. Plant Cell. 1991; 3: 517-529Crossref PubMed Google Scholar). Two structural RNAs, 16 S ribosomal RNA and tRNAHis, which are supposed to be stable in all stages of chloroplast development, were studied. As shown in Fig. 1 all RNAs analyzed are stabilized by the addition of Mg2+, whereas the addition of NaCl has no effect. The finding that NaCl has no stabilizing effect on chloroplast RNAs in vitro indicates that not merely an increase in ionic strength is involved here. Still the stabilizing effect of Mg2+ may be general for all RNAs.Figure 2Magnesium dependence of chloroplast RNA stability. In vitro degradation extracts prepared in the presence of 20 mm EDTA (protein concentration, 5 mg/ml) were incubated at 22 °C for 180 min in the presence of added Mg2+ as labeled and for 0 min as a control. Total RNA was purified, and 2 μg of each sample was analyzed by quantitative Northern hybridization using gene-specific oligonucleotides as indicated. Each of the RNAs exhibits a typical MgCl2concentration where stabilization can be observed.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 1Ion-dependent chloroplast RNA degradation in vitro. Degradation of internal chloroplast RNAs was observed in extracts of lysed chloroplasts from mature leaves. Extracts of a concentration of 5 mg/ml protein prepared in the presence of 20 mm EDTA were incubated at 22 °C unmodified and after reconstitution with 25 mmMgCl2 or 50 mm NaCl for the times indicated. Total RNA was purified. Two μg of RNA per time point was analyzed by quantitative Northern hybridization using gene-specific oligonucleotides as labeled.View Large Image Figure ViewerDownload Hi-res image Download (PPT) To approach the question of specificity, titration experiments were performed to analyze the stabilization profiles as well as the "midpoint stabilizing" Mg2+ concentration of different RNAs. Chloroplast extract was incubated at room temperature for 180 min in the presence of increasing concentrations of magnesium ions as indicated in Fig. 2 and compared with extract representing the time point 0. Total RNA was prepared from each sample and subjected to quantitative Northern analysis using the same gene-specific probes as described above. The results depicted in Fig. 2 show that magnesium ions have specific effects on the RNAs analyzed. Ribosomal 16 S rRNA and tRNAHis are already stabilized at low added Mg2+ concentrations (2.5–5 mm), whereas psbA mRNA requires concentrations of 10–15 mm Mg2+ added to the 20 mm EDTA extract to gain stability. rbcLmRNA shows an intermediate characteristic, becoming stabilized at concentrations of 5–10 mm added magnesium ions. Besides the specific concentrations of magnesium ions that result in stabilization of RNAs, the stabilization profiles as a function of Mg2+ seem to be different. To gain a more detailed insight into those stabilization profiles that have been described in a rough qualitative manner above, the data were quantitated. However, before a quantitative evaluation could be made, the concentrations of free magnesium ions at each point of the titration experiment had to be determined. This was facilitated using an electropotentiometric procedure with magnesium-specific electrodes (35Zhang W. Truttmann A.C. Luthi D. McGuigan J.A. Magnes. Bull. 1997; 17: 125-130Google Scholar). The advantage of this method compared with atom absorption spectroscopy is that only the concentration of free ionized magnesium is determined, whereas total magnesium would be measured by atom absorption spectroscopy. In chloroplasts, total concentrations of magnesium ions as high as 20–30 mm were determined earlier (41Barber J. Barber J. The Intact Chloroplast. 1. Elsevier Scientific Publishing Co., Amsterdam1976: 89-134Google Scholar). However, it is very likely that only a portion of these ions is present as free ionized Mg2+ and thereby is available for interactions with RNA. To perform the determination of free Mg2+, chloroplast degradation extracts had to be cleared from the membrane fraction by centrifugation, and the resulting supernatant had to be filtered through a CentriconTM tube with a size exclusion mass of 30 kDa. Otherwise, certain components of the extracts induced a drift at the electrode that did not allow for precise measurements. In Fig. 3 A the calibration curve is shown in which measured voltage is plotted against the negative logarithm of [MgCl2]. The calibration curve shows linearity over a broad range of Mg2+ concentration. Fig. 3 B shows the concentration of free Mg2+determined in the extract plotted against [MgCl2]added in the presence of 20 mm EDTA. The first three titration points (2.5, 5.0, 7.5 mm added Mg2+) do not result in more than a micromolar increase of free [Mg2+]. Adding higher concentrations of Mg2+ results in a nearly linear increase of free [Mg2+], showing that EDTA is saturated at [Mg2+]added higher than 7.5 mm. To analyze the magnesium-dependent stabilization profiles of the different RNAs, x-ray films were densitometrically quantified. RNA amounts were expressed as percent of RNA remaining after 180 min compared with the amount of RNA at time 0 set to 100%. The percentage of remaining RNA is plotted against the concentration of free [Mg2+] as shown in Fig. 4. The qualitative observations made before are very much supported by the quantitative evaluati
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