Abrogation of Upstream Open Reading Frame-mediated Translational Control of a Plant S-Adenosylmethionine Decarboxylase Results in Polyamine Disruption and Growth Perturbations
2002; Elsevier BV; Volume: 277; Issue: 46 Linguagem: Inglês
10.1074/jbc.m206161200
ISSN1083-351X
AutoresColin Hanfrey, Marina Franceschetti, Melinda J. Mayer, Crista Illingworth, Anthony J. Michael,
Tópico(s)Amino Acid Enzymes and Metabolism
ResumoS-Adenosylmethionine decarboxylase (AdoMetDC) is a key enzyme in polyamine biosynthesis. We show that the plant AdoMetDC activity is subject to post-transcriptional control by polyamines. A highly conserved small upstream open reading frame (uORF) in the AdoMetDC mRNA 5′ leader is responsible for translational repression of a downstream β-glucuronidase reporter cistron in transgenic tobacco plants. Elimination of the small uORF from an AdoMetDC cDNA led to increased relative translational efficiency of the AdoMetDC proenzyme in transgenic plants. The resulting increased activity of AdoMetDC caused disruption to polyamine levels with depletion of putrescine, reduction of spermine levels, and a more than 400-fold increase in the level of decarboxylated S-adenosylmethionine. These changes were associated with severe growth and developmental defects. The high level of decarboxylated S-adenosylmethionine was not associated with any change in 5′-methylcytosine content in genomic DNA and S-adenosylmethionine levels were more or less normal, indicating a highly efficient system for maintenance ofS-adenosylmethionine levels in plants. This work demonstrates that uORF-mediated translational control of AdoMetDC is essential for polyamine homeostasis and for normal growth and development. S-Adenosylmethionine decarboxylase (AdoMetDC) is a key enzyme in polyamine biosynthesis. We show that the plant AdoMetDC activity is subject to post-transcriptional control by polyamines. A highly conserved small upstream open reading frame (uORF) in the AdoMetDC mRNA 5′ leader is responsible for translational repression of a downstream β-glucuronidase reporter cistron in transgenic tobacco plants. Elimination of the small uORF from an AdoMetDC cDNA led to increased relative translational efficiency of the AdoMetDC proenzyme in transgenic plants. The resulting increased activity of AdoMetDC caused disruption to polyamine levels with depletion of putrescine, reduction of spermine levels, and a more than 400-fold increase in the level of decarboxylated S-adenosylmethionine. These changes were associated with severe growth and developmental defects. The high level of decarboxylated S-adenosylmethionine was not associated with any change in 5′-methylcytosine content in genomic DNA and S-adenosylmethionine levels were more or less normal, indicating a highly efficient system for maintenance ofS-adenosylmethionine levels in plants. This work demonstrates that uORF-mediated translational control of AdoMetDC is essential for polyamine homeostasis and for normal growth and development. S-Adenosylmethionine decarboxylase (AdoMetDC 1The abbreviations used are: AdoMetDC, S-adenosylmethionine decarboxylase; uORF, upstream open reading frame; MES, 4-morpholineethanesulfonic acid; dansyl, 5-dimethylaminonaphthalene-1-sulfonyl; NL, no leader; HPLC, high performance liquid chromatography; GUS, β-glucuronidase; CaMV, cauliflower mosaic virus ; EC 4.1.1.50) is a key enzyme in the biosynthesis of the polyamines spermidine and spermine. Polyamines are multivalent cations implicated in a wide range of cellular physiological processes including chromatin organization, mRNA translation, cell proliferation, and apoptosis (1Cohen S. A Guide to the Polyamines. Oxford University Press, Oxford1998: 1-543Google Scholar). Most plants form putrescine (1,4-diaminobutane) indirectly from arginine and directly from ornithine. Spermidine is formed from putrescine and spermine from spermidine by successive addition of aminopropyl groups derived from decarboxylated S-adenosylmethionine (AdoMet) that is generated from AdoMet by the activity of AdoMetDC. In mouse, the AdoMetDC gene is essential for embryonic development (2Nishimura K. Nakatsu F. Kashiwagi K. Ohno H. Saito T. Igarashi K. Genes Cell. 2002; 7: 41-47Crossref PubMed Scopus (97) Google Scholar) and an AdoMetDC gene deletion mutant of Leishmania donovani has an absolute requirement for exogenously supplied spermidine (3Roberts S.C. Scott J. Gasteier J.E. Jiang Y. Brooks B. Jardin A. Carter N.S. Heby O. Ullman B. J. Biol. Chem. 2002; 277: 5902-5909Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar). Relatively little is known about the function of polyamines in plants (4Malmberg R.L. Watson M.B. Galloway G.L. Yun W. Crit. Rev. Plant Sci. 1998; 17: 199-224Crossref Google Scholar) but recently a spermine synthase mutant ofArabidopsis thaliana was found to be severely affected in growth and cell elongation (5Hanzawa Y. Takahashi T. Michael A.J. Burtin D. Long D. Pineiro M. Coupland G. Komeda Y. EMBO J. 2000; 19: 4248-4256Crossref PubMed Scopus (233) Google Scholar). Plant AdoMetDC mRNAs possess long 5′ leader sequences of at least 500 nucleotides and are remarkable for the presence of a highly conserved pair of overlapping uORFs (6Franceschetti M. Hanfrey C. Scaramagli S. Torrigiani P. Bagni N. Burtin D. Michael A.J. Biochem. J. 2001; 353: 403-409Crossref PubMed Scopus (87) Google Scholar). The upstream tiny ORF and downstream small ORF consist of 3–4 and 50–54 codons, respectively, overlapping by one nucleotide, being the last base of the tiny uORF termination codon and the first base of the small uORF AUG codon. This one base overlapping arrangement predates the origin of flowering plants and the amino acid sequence encoded by the small uORF is conserved between angiosperms and Pinus taeda (6Franceschetti M. Hanfrey C. Scaramagli S. Torrigiani P. Bagni N. Burtin D. Michael A.J. Biochem. J. 2001; 353: 403-409Crossref PubMed Scopus (87) Google Scholar). Although uORFs occur relatively infrequently in eukaryotic mRNAs, their occurrence is more frequent in growth-related genes such as oncogenes where they are present in nearly two-thirds of genes (7Kozak M. Nucleic Acids Res. 1987; 15: 8125-8148Crossref PubMed Scopus (4549) Google Scholar, 8Kozak M. J. Cell Biol. 1991; 115: 887-903Crossref PubMed Scopus (1475) Google Scholar). The role of uORFs in translational regulation is increasingly recognized as an important component of gene expression control (9Morris D.R. Geballe A. Mol. Cell. Biol. 2000; 20: 8635-8642Crossref PubMed Scopus (581) Google Scholar,10Geballe A.P. Sachs M.S. Sonenberg N. Hershey J.W.B. Mathews M.B. Translational Control of Gene Expression. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY2000: 595-614Google Scholar). The mammalian AdoMetDC mRNA contains a single short uORF encoding the hexapeptide MAGDIS located 14 nucleotides downstream of the 5′ cap. MAGDIS-mediated translational regulation of the AdoMetDC mRNA depends on cell type (11Hill J.R. Morris D.R. J. Biol. Chem. 1992; 267: 21886-21893Abstract Full Text PDF PubMed Google Scholar) and cellular polyamine content (12Ruan H. Shantz L.M. Pegg A.E. Morris D.R. J. Biol. Chem. 1996; 271: 29576-29582Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar). Moving the uORF to a position 47 nucleotides downstream of the 5′ cap enhances recognition of the uORF in nonlymphoid cells (13Ruan H. Hill J.R. Fatemie-Nainie S. Morris D.R. J. Biol. Chem. 1994; 269: 17905-17910Abstract Full Text PDF PubMed Google Scholar). When cellular polyamine levels are depleted, AdoMetDC mRNA is more efficiently loaded with ribosomes (14White M.W. Degnin C. Hill J. Morris D.R. Biochem. J. 1990; 268: 657-660Crossref PubMed Scopus (31) Google Scholar) and the uORF is responsible for this polyamine-mediated translational regulation (15Shantz L.M. Viswanath R. Pegg A.E. Biochem. J. 1994; 302: 765-772Crossref PubMed Scopus (31) Google Scholar, 12Ruan H. Shantz L.M. Pegg A.E. Morris D.R. J. Biol. Chem. 1996; 271: 29576-29582Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar). The termination codon of the uORF is absolutely required for MAGDIS repressive activity (12Ruan H. Shantz L.M. Pegg A.E. Morris D.R. J. Biol. Chem. 1996; 271: 29576-29582Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar) and increased spermidine levels cause ribosome stalling at the termination codon as detected by toe-printing and expression in a gel-filtered rabbit reticulocyte lysate system (16Ryabova L.A. Hohn T. Genes Dev. 2000; 14: 817-829PubMed Google Scholar, 17Law G.L. Raney A. Heusner C. Morris D.R. J. Biol. Chem. 2001; 276: 38036-38043Abstract Full Text Full Text PDF PubMed Google Scholar, 18Raney A. Baron A.C. Mize G.J. Law G.L. Morris D.R. J. Biol. Chem. 2000; 275: 24444-24450Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar). At least two transcribed AdoMetDC genes are present in the model plant Arabidopsis,AdoMetDC1 andAdoMetDC2 (6Franceschetti M. Hanfrey C. Scaramagli S. Torrigiani P. Bagni N. Burtin D. Michael A.J. Biochem. J. 2001; 353: 403-409Crossref PubMed Scopus (87) Google Scholar). The sequences of the tiny and small uORFs are highly conserved between the two mRNAs and indicate that they are similarly regulated. Here we characterize the ArabidopsisAdoMetDC1 mRNA 5′ leader small uORF. Our data demonstrate that: (i) plant AdoMetDC activity is subject to post-transcriptional negative feedback regulation by polyamines; (ii) the small uORF is responsible for the translational repression of a downstream cistron in transgenic plants; (iii) abrogation of small uORF-mediated translational regulation in transgenic plants causes an increased translation of the downstream AdoMetDC ORF, resulting in increased enzyme activity and decarboxylated AdoMet levels, polyamine disruption, and severe growth perturbations. ForArabidopsis studies, the Columbia ecotype (Col-0) was grown in a greenhouse with a 16-h light period. TheArabidopsis cell suspension culture (obtained from J. Murray, University of Cambridge, United Kingdom) was grown in a 1-liter flask in liquid medium containing MS salts (19Murashige T. Skoog F. Physiol. Plant. 1962; 15: 473-497Crossref Scopus (55295) Google Scholar), 3% sucrose, 0.5 mg/liter naphthalene acetic acid, 0.05 mg/liter kinetin, pH 5.8, at 25 °C in the dark, shaking at 80 rpm. For in vitrogrowth of tobacco, seeds of Nicotiana tabacum cv. XHFD8 (20Burtin D. Michael A.J. Biochem. J. 1997; 325: 331-337Crossref PubMed Scopus (58) Google Scholar) were surface sterilized and germinated on medium containing MS salts, 2% sucrose, 0.5 g/liter MES, 8 g/liter Bacto-agar, pH 5.7, and grown at 25 °C with 16 h daylength. For leaf disc experiments, young leaves about 8 cm in length were washed in 10% bleach with a few drops of detergent for 15 min and then rinsed three times in sterile water. Leaf discs of 8 mm diameter were cut from the leaves and placed on solid MS medium without hormones in Petri dishes. The various site-directed mutants of the AdoMetDC1 cDNA were produced using the Chameleon double-stranded mutagenesis kit (Stratagene), following the manufacturer's instructions. Mutants were constructed using the SAMDC1 plasmid (which contains the wild type AdoMetDC1 cDNA in a pBluescript KS vector (6Franceschetti M. Hanfrey C. Scaramagli S. Torrigiani P. Bagni N. Burtin D. Michael A.J. Biochem. J. 2001; 353: 403-409Crossref PubMed Scopus (87) Google Scholar). Mutagenic primers employed were 5′-GCGTGAATGAGATTATTTTGGAGTCGAAAGGTGG-3′ for the MUT construct and 5′-CGAAGCTCCCCTCGGTTAGAGCATTGAAGACG-3′ for the TAG construct. A primer which disrupted the unique ApaI site in pBluescript was used for selection of mutants. Mutations were confirmed by DNA sequencing. The 5′ leader sequences from SAMDC1 and the site-directed mutants were PCR amplified using the primers 5′-TTAAGAGCTCTCAACTTAATCGTTTCTCTC-3′ (SacI site underlined) and 5′-CTCCCATGGCTCGCCTTGTTGTGTGAGCG-3′ (NcoI site underlined). PCR products were checked for errors by sequencing. SacI-NcoI fragments containing the 5′ leaders were then used to replace the tobacco mosaic virus Ω sequence in the pUC118-based vector, pSLJ4D4 (21Jones J.D. Shlumukov L. Carland F. English J. Schofield S.R. Bishop G.J. Harrison K. Transgenic Res. 1992; 1: 285-297Crossref PubMed Scopus (267) Google Scholar). From the resultant plasmids, 4.4-kilobase pair EcoRI-HindIII fragments containing the CaMV 35S RNA promoter, the 5′ leader variants, the Escherichia coli β-glucuronidase (GUS) coding sequence, and the octopine synthase terminator were cloned into theAgrobacterium tumefaciens binary vector, pBin19 for plant transformation. The NL (no leader) construct was produced by removing aSalI-BamHI fragment from SAMDC1, reducing the leader to 58 bp proximal to the AdoMetDC ORF. For overexpression of AdoMetDC in transgenic plants, the EcoRI-HindIII fragment from pSLJ4D4, consisting of the CaMV 35S RNA promoter, GUS sequence , and the octopine synthase terminator, w ere cloned into pBin19. The GUS sequence was subsequently removed from this plasmid by digestion with XhoI and XbaI, and replaced withSalI-XbaI fragments carrying the sequences of the wild-type SAM or NL and TAG mutant AdoMetDC cDNAs. Constructs in pBin19 were introduced into A. tumefaciens strain LBA4404 and used to transformN. tabacum cv. Xanthi XHFD8 using the leaf disc method, as described previously (20Burtin D. Michael A.J. Biochem. J. 1997; 325: 331-337Crossref PubMed Scopus (58) Google Scholar). Transgenic plantlets were selected on kanamycin and once rooted were transferred to soil in a greenhouse and grown at 25 °C with a 16-h light period. Plant tissue was ground to a fine powder in liquid nitrogen, an aliquot was set aside at −70 °C for GUS or AdoMetDC activity assays, and the remainder was used to prepare total RNA as described previously (22Michael A.J. Furze J.M. Rhodes M.J. Burtin D. Biochem. J. 1996; 314: 241-248Crossref PubMed Scopus (121) Google Scholar). For RNA gel blot analysis, 10 μg of total RNA was size-fractionated on 1.2% agarose-formaldehyde denaturing gels, and blotted onto Hybond-N+ membrane (Amersham Biosciences). Blots were probed with the 1.9-kilobase pair NcoI-XbaI fragment of the GUS sequence from pSLJ4D4 (21Jones J.D. Shlumukov L. Carland F. English J. Schofield S.R. Bishop G.J. Harrison K. Transgenic Res. 1992; 1: 285-297Crossref PubMed Scopus (267) Google Scholar), the 1.8-kilobase pairSalI-XbaI fragment containing the AdoMetDC1 cDNA, or the 0.7-kilobase pair NotI fragment containing the PCR amplified N. tabacum ubiquitin sequence. All hybridization conditions were as described previously (22Michael A.J. Furze J.M. Rhodes M.J. Burtin D. Biochem. J. 1996; 314: 241-248Crossref PubMed Scopus (121) Google Scholar). Ground plant tissue was assayed for GUS activity using the GUS-Light assay system (Tropix, Applied Biosystems, Warrington, UK), following the manufacturer's instructions. Tissue extracts were incubated with substrate for 1 h at room temperature, and light signal output was measured using a Lumat LB9501 luminometer (Berthold, Pforzheim, Germany). Protein contents of extracts were measured using the method of Bradford (23Bradford M.M. Anal. Biochem. 1976; 72: 248-254Crossref PubMed Scopus (223639) Google Scholar), and GUS activity was expressed as relative light units per μg of protein. Ground plant tissue was assayed for AdoMetDC activity as described previously (22Michael A.J. Furze J.M. Rhodes M.J. Burtin D. Biochem. J. 1996; 314: 241-248Crossref PubMed Scopus (121) Google Scholar). Assays were performed at 37 °C for 45 min, and AdoMetDC activity was determined by measurement of 14CO2 release fromS-adenosyl-l-[14C]methionine (Amersham Biosciences). Protein contents of extracts were measured using the method of Bradford (23Bradford M.M. Anal. Biochem. 1976; 72: 248-254Crossref PubMed Scopus (223639) Google Scholar), and enzyme activities were expressed as nanomole of CO2/h/mg of protein. Polyamines were extracted once from frozen leaf powder in 5% (w/v) trichloroacetic acid containing 1 × 10−4m 1,7-diaminoheptane as an internal standard as described previously (20Burtin D. Michael A.J. Biochem. J. 1997; 325: 331-337Crossref PubMed Scopus (58) Google Scholar). Polyamines were dansylated overnight and sample aliquots of 200 μl were incubated with 100 μl of saturated Na2CO3 and 600 μl of dansyl chloride (10 mg/ml in acetone) for 16 h in the dark in open tubes to allow gradual evaporation of the acetone. Excess dansyl chloride was removed by 30 min incubation with 150 μl of proline (300 mg/ml). The reaction was then extracted with 1 ml of toluene, centrifuged for 5 min at 13,000 × g, and 800 μl of the upper phase was dried with nitrogen and resuspended in 500 μl of acetonitrile. Samples were filtered through Acrodisc CR PTFE filters (Gelman Sciences, Northampton, UK). Dansylated polyamines were separated by HPLC using a Sphereclone 5-μm C18 ODS (2Nishimura K. Nakatsu F. Kashiwagi K. Ohno H. Saito T. Igarashi K. Genes Cell. 2002; 7: 41-47Crossref PubMed Scopus (97) Google Scholar) column (250 × 4.6 mm; Phenomenex, Macclesfield, Cheshire, UK) with fluorescence detection (excitation wavelength 340 nm, emission wavelength 510 nm). Solvent A was HPLC-grade water, solvent B was acetonitrile, and the gradient was run for 50 min at a flow rate of 1.2 ml/min with the following concentrations: t = 0 min, 40% A, 60% B; t = 25 min, 0% A, 100% B;t = 40 min, 40% A, 60% B; t = 50 min, 40% A, 60% B. DNA samples (20 μg in 50 μl water) were hydrolyzed for 14 h at 37 °C with 42 units of P1 nuclease (Sigma) that was in 55 μl of 30 mm sodium acetate buffer, pH 5.3, and 20 μl of 10 mm ZnCl2 to form a total volume of 125 μl. The 5′-phosphate of the free nucleotides was removed by hydrolysis with bacterial alkaline phosphatase (Sigma) for 2 h at 37 °C in a total volume of 100 μl of 300 mm Tris-OH buffer, pH 8.7, containing 3.5 units of enzyme. Samples were then filtered through a 0.45-μm PTFE membrane (Gelman Sciences). The filtered samples were loaded onto a Supelcosil LC 18-S (150 × 4.6 mm) reverse phase HPLC column (Phenomenex, Macclesfield, Chesire, UK). Nucleosides were separated on an isocratic gradient and quantified by UV detection: buffer A was 0.05 m KH2PO4, pH 4.0, 8% MeOH and buffer B was 70% methanol. Column temperature was 25 °C and UV acquisition was at 254 and 280 nm. 5′-Methyl-2′-deoxycytidine and 2′-deoxycytidine standards were obtained from Sigma. AdoMet and decarboxylated AdoMet were measured by reverse phase HPLC. For AdoMet measurement it was essential to keep sample extracts frozen until immediately before injection onto the column because of the lability of AdoMet. Samples of frozen leaf powder were extracted with 1 ml of 5% trichloroacetic acid per 400 mg of powder. Extracted samples were centrifuged at 10,000 × g for 15 min to clear cell debris. The supernatant was then recentrifuged at 13,000 × g to further remove debris and the supernatant was filtered. Decarboxylated AdoMet was a kind gift of Dr. B. Blessington, University of Bradford, UK, and Prof. A. E. Pegg, Hershey Medical School, University of Pennsylvania, and AdoMet was obtained from Sigma. AdoMet and decarboxylated AdoMet were quantified by UV detection after separation on a Luna 5μ ODS (2Nishimura K. Nakatsu F. Kashiwagi K. Ohno H. Saito T. Igarashi K. Genes Cell. 2002; 7: 41-47Crossref PubMed Scopus (97) Google Scholar) 150 × 4.6-mm reverse phase HPLC column (Phenomenex). The solvent gradient was formed from buffer A (0.1 m sodium acetate, pH 4.5, 10 mm 1-octanesulphonic acid) and buffer B (0.2 msodium acetate, pH 4.5, acetonitrile (10:3) with 10 mmoctanesulfonic acid). The gradient was formed as follows:t = 0, 100% A, 0% B; t = 30, 40% A, 60% B; t = 40, 0% A, 100% B; t = 55, 100% A, 0% B with a flow rate of 1.5 ml per min. UV acquisition was at 259 nm. To determine the relevance of the conserved overlapping uORFs in the plant AdoMetDC mRNA 5′ leader to translational control, we looked for evidence of post-transcriptional regulation. AdoMetDC is initially synthesized as an inactive proenzyme and is autocatalytically processed to produce the mature form of the enzyme containing a covalently linked pyruvoyl cofactor at the N terminus of the α-subunit. The processing reaction of the potato andArabidopsis proenzymes is very rapid and, unlike the mammalian enzyme, is not regulated by the polyamine precursor putrescine (24Xiong H. Stanley B.A. Tekwani B.L. Pegg A.E. J. Biol. Chem. 1997; 272: 28342-28348Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar). 2C. Hanfrey, M. Franceschetti, M. J. Mayer, C. Illingworth, and A. J. Michael, unpublished results. The plant AdoMetDC activity is therefore likely to be a reliable indication of the amount of AdoMetDC protein. AdoMetDC1 is the more actively expressed of two expressed AdoMetDC genes inArabidopsis (6Franceschetti M. Hanfrey C. Scaramagli S. Torrigiani P. Bagni N. Burtin D. Michael A.J. Biochem. J. 2001; 353: 403-409Crossref PubMed Scopus (87) Google Scholar). Fig.1 A shows the variation in AdoMetDC activity detected in different organs ofArabidopsis. The ratio of AdoMetDC activity to ubiquitin-normalized AdoMetDC1 mRNA levels in leaves, stems, roots, and flowers is 1, 0.8, 0.7, and 1.2, respectively, indicating that spatial activity of AdoMetDC1 is largely correlated with steady-state mRNA levels. It is known that application of polyamines to tobacco suspension culture cells results in decreased AdoMetDC activity (25Hiatt A.C. McIndoo J. Malmberg R.L. J. Biol. Chem. 1986; 261: 1293-1298Abstract Full Text PDF PubMed Google Scholar). We examined AdoMetDC activity and steady-state mRNA levels in stationary phase 10-day-old Arabidopsis suspension culture cells simultaneously treated for 16 h with 0.5 mm spermidine and spermine (Fig. 1 B). Application of polyamines caused a 30-fold decrease in AdoMetDC activity while the ubiquitin-normalized steady-state AdoMetDC1 mRNA level increased by half. Growth in the presence of added spermidine and spermine caused a 1.8-fold increase in putrescine level probably because of inhibition of AdoMetDC, a 4.6-fold increase in spermidine, and a 37.3-fold increase in spermine (results not shown). Similar results for AdoMetDC activity were obtained with 3-day-old Arabidopsis suspension culture cells (results not shown). We extended this analysis to tobacco seedlings grown on solid medium in vitro in the presence of 0.5 mmspermidine and spermine. This concentration caused a 3-fold decrease in AdoMetDC activity without any effect on the ubiquitin-normalized tobacco AdoMetDC1 steady-state mRNA level (Fig. 1 C). These results provide clear evidence for post-transcriptional regulation of the plant AdoMetDC activity in response to polyamines. TheArabidopsis AdoMetDC1 cDNA used in this study has a 505-nucleotide 5′ leader sequence with 184 bp between the 5′ end of the leader and the AUG intiation codon of the tiny uORF (Fig.2 A). TheArabidopsis tiny uORF UGA termination codon overlaps with the first nucleotide of the second successive AUG of the small uORF (Fig. 2 B) and the small uORF terminates 154 nucleotides upstream of the AdoMetDC proenzyme ORF. To investigate the role of the highly conserved small uORF in regulating AdoMetDC expression, chimeric genes were constructed containing the 5′ leader of theArabidopsis AdoMetDC1 bearing wild type or site-directed mutant forms of the uORF sequences fused to the GUS reporter gene ORF. Chimeric genes were introduced into tobacco leaf discs byAgrobacterium-mediated transformation and transgenic plants were regenerated expressing the reporter cassettes under the control of the constitutive CaMV 35S RNA promoter. GUS activities were determined in leaf tissues of T0 plants (regenerated from tissue culture) for each individual transformant and related to the ubiquitin-normalized GUS reporter transcript levels to provide an indication of the translational efficiency of each construct. There was no detectable correlation between the different constructs and the steady-state levels of the normalized GUS mRNA levels, indicating that the site-directed mutations did not affect mRNA stability (data not shown). The SAM construct contained the wild type AdoMetDC1 5′ leader. The MUT construct contained a leader sequence in which the small uORF was abolished and replaced by the tiny uORF, which was extended downstream to 66 codons (in the +1 reading frame relative to the small uORF and extending 31 nucleotides downstream of the small uORF stop codon). The TAG construct contained a leader sequence with the small uORF C-terminal truncated to 25 codons by the introduction of a UAG nonsense codon (see Fig. 2, B and C, for site-directed mutations). As shown by the results of the MUT construct depicted in Fig.3, elimination of the small uORF caused a 3-fold derepression of GUS translational efficiency in leaves of transgenic plants. The 5-fold translational depression seen with the TAG construct indicates that the C-terminal half of the small uORF peptide or the sequence immediately 3′ of the termination codon is essential for translation inhibition. Together these results suggest that the plant AdoMetDC mRNA is translationally repressed in planta and that the small uORF is responsible for translational repression of the downstream cistron. To investigate the biological significance of the translational control of AdoMetDC expression observed in this study, we produced transgenic tobacco plants expressing either the wild type ArabidopsisAdoMetDC1 cDNA, or AdoMetDC1 cDNA with the 5′ leader truncated from 505 to 58 nucleotides (NL), or with the small uORF C-terminal truncated from 53 to 25 codons by introduction of a premature nonsense codon (TAG). The cDNAs were cloned downstream of a CaMV 35S RNA promoter to allow constitutive expression in transgenic tobacco plants. Transgenic T0 tobacco plants were allowed to flower and self-fertilize. Progeny segregated in a mendelian manner into transgenic progeny contained the transgene and syngenic progeny without the transgene. Transgenic plants from two independent lines for each of the SAM, TAG, and NL cDNAs were analyzed for AdoMetDC activity. Mean AdoMetDC activity recorded in the segregating syngenic siblings was subtracted from the activity in the transgenic siblings to give a measure of activity because of the transgene. TableI shows the relative transgene mRNA, AdoMetDC activity, and relative AdoMetDC translational efficiency values for representative individuals from each of the transgenic lines. Each of the modified AdoMetDC1 cDNA lines shows an increase of relative translational efficiency of between 5- and 18-fold above that of wild type AdoMetDC1 cDNA overexpressing lines 556 and 557. The higher translational efficiency of the two NL lines compared with the TAG lines is likely because of the inhibitory influence of the long AdoMetDC1 5′ leader sequence. Secondary structure in the maize uORF-containing Lc mRNA leader sequence is responsible for half of the translational repression conferred by the leader sequence (26Wang L. Wessler S.R. Plant Physiol. 2001; 125: 1380-1387Crossref PubMed Scopus (58) Google Scholar). Once translational regulation is removed, AdoMetDC activity is dependent on mRNA levels, which are subject to position effects.Table IAdoMetDC translational efficiency in leaves of T1 transgenic tobacco plantsConstructPlantaIndividuals designated with the same number are from the same transgenic line.Relative AdoMetDC mRNAbAdoMetDC1 mRNA levels were normalized to ubiquitin for each plant. The value for 556-M was set as 1.00.Relative AdoMetDC activitycAdoMetDC activity was measured as nanomole of CO2 released per mg of total protein. For each plant, the mean AdoMetDC activity recorded in the segregating syngenic siblings was subtracted from the total AdoMetDC activity level to give a measure of activity due to the AdoMetDC1 transgene. The value for 556-M was set as 1.00.Relative AdoMetDC translational efficiencydRelative translational efficiency is the relative AdoMetDC activity divided by ubiquitin-normalized AdoMetDC1 mRNA. The value for 556-M was set as 1.00.SAMDC1M1.001.001.00SAMDC1Q0.550.721.31SAMDC1NLM0.9416.0417.06SAMDC1NLT0.6011.2218.70SAMDC1NLP3.4248.5214.19SAMDC1NLR2.7918.936.78SAMDC1TAGC1.099.548.75SAMDC1TAGM0.241.375.71a Individuals designated with the same number are from the same transgenic line.b AdoMetDC1 mRNA levels were normalized to ubiquitin for each plant. The value for 556-M was set as 1.00.c AdoMetDC activity was measured as nanomole of CO2 released per mg of total protein. For each plant, the mean AdoMetDC activity recorded in the segregating syngenic siblings was subtracted from the total AdoMetDC activity level to give a measure of activity due to the AdoMetDC1 transgene. The value for 556-M was set as 1.00.d Relative translational efficiency is the relative AdoMetDC activity divided by ubiquitin-normalized AdoMetDC1 mRNA. The value for 556-M was set as 1.00. Open table in a new tab Plants overexpressing the wild type SAM construct (lines 556 and 557) exhibited a normal morphological phenotype. In contrast, both of the NL lines (754 and 756), and one of the TAG lines (850) exhibited severely abnormal phenotypes, which segregated into two levels of severity for each line and which were clearly visible as growth differences in seedlings (Fig. 4, A–C). The NL756 plants displayed the most extreme morphological phenotype and the NL754 plants the mildest. The phenotype displayed by the NL756 line was usually lethal with the presumed homozygous plants dying before they attained 2 cm and the putative heterozygous plants dying usually before they reached 5 cm. Segregating normal syngenic plants of line NL756 flowered at a height of ∼100 cm. All transgenic plants were stunted with reduced internode length (Fig. 4, D–F) and with wrinkled and curled leaves in the NL754 and NL756 lines. The abnormal morphological and growth phenotype was more marked in the second generation T1 plants that had passed through meiosis than in the first generation T0 plants that had been regenerated from tissue culture. Furthermore, transgenic plants of the T2 generation (resulting from
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