Expression of Ornithine Decarboxylase Is ... - Plant Physiology

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in these processes remains unclear (Evans and Malmberg,. 1989; Walden et al., 1997). The synthesis of putrescine from Orn, catalyzed by ODC, is the initial key ...
Plant Physiol. (1998) 118: 323–328

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Expression of Ornithine Decarboxylase Is Transiently Increased by Pollination, 2,4-Dichlorophenoxyacetic Acid, and Gibberellic Acid in Tomato Ovaries1 David Alabadı´ and Juan Carbonell* Departamento de Biologı´a del Desarrollo, Instituto de Biologı´a Molecular y Celular de Plantas, Universidad Polite´cnica de Valencia-Consejo Superior de Investigaciones Cientı´ficas, Camino de Vera 14, 46022-Valencia, Spain 2,4-D, the latter inducing more effective and rapid changes (Alabadı´ et al., 1996). The effect of expression of heterologous ODC genes has previously been studied in plants. Expression of the yeast ODC gene in tobacco resulted in altered putrescine biosynthesis and nicotine accumulation (Hamill et al., 1990), whereas the mouse ODC cDNA caused a high degree of somatic embryogenesis when introduced into carrot cells (Bastola and Minocha, 1995). Recently, the effect on the metabolism of PAs in these transgenic carrot cells has been studied in more detail (Andersen et al., 1998). Genes that encode enzymes of PA biosynthesis in plants have been cloned (Kumar et al., 1997; Walden et al., 1997). An ODC cDNA clone from thorn-apple (Michael et al., 1996) and one partial ODC cDNA clone from tobacco (Malik et al., 1996) have been described. In this report we describe the isolation of a tomato ODC cDNA clone. The amino acid sequence derived from the cDNA shows a high degree of identity with the two ODCs reported in plants and highly conserved motifs present in all the eukaryotic ODCs described. ODC gene expression in unpollinated ovaries and in young fruits was induced by pollination, 2,4-D, and GA3. ODC gene expression in other tissues is also discussed.

A cDNA encoding for a functional ornithine decarboxylase has been isolated from a cDNA library of carpels of tomato (Lycopersicon esculentum Mill.). Ornithine decarboxylase in tomato is represented by a single-copy gene that we show to be up-regulated during early fruit growth induced by 2,4-dichlorophenoxyacetic acid and gibberellic acid.

The PAs putrescine, spermidine, and spermine are small molecules, charged at a physiological pH, that have been implicated in a wide range of growth and developmental processes, such as floral and fruit development, as well as senescence and stress responses, although their exact role in these processes remains unclear (Evans and Malmberg, 1989; Walden et al., 1997). The synthesis of putrescine from Orn, catalyzed by ODC, is the initial key step in the PA biosynthetic pathway in eukaryotic cells (Heby and Persson, 1990). Putrescine in plants is also synthesized from Arg via ADC. ODC has been described as the enzyme controlling PA biosynthesis in tissues undergoing cell division, whereas ADC has been associated with tissues that grow by cell expansion, with responses to stress, and with secondary metabolism (Tiburcio et al., 1990). Despite the fact that early fruit growth in tomato (Lycopersicon esculentum Mill.) is sustained by cell division, both ODC and ADC activities have been detected (Egea-Cortines and Mizrahi, 1991). However, ODC appears to be the main enzyme regulating PA levels during early fruit growth, as indicated by treatments with a-dldifluoromethylornithine, a specific ODC inhibitor (Cohen et al., 1982). Fruit set in tomato can be induced artificially by treatment of unpollinated ovaries with growth regulators (Sawhney, 1984). A transient increase in both ODC and ADC activities was shown after treatment with GA3 and

MATERIALS AND METHODS Plant Material Tomato (Lycopersicon esculentum Mill. cv Rutgers) plants were grown as previously described (Alabadı´ et al., 1996). The first five flowers in the first two inflorescences from each plant were used. In some experiments, self-pollination was allowed in one of the five flowers (usually the third), and the others were emasculated. Experiments with plantgrowth regulators were carried out as described in Alabadı´ et al. (1996). All samples were collected, weighed, and immediately frozen in liquid nitrogen and stored at 270°C until used.

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This work was supported by grant no. PB95-0029-C02-01 from Direccio´n General de Investigacio´n Cientı´fica y Te´cnica, Spain. D.A. was the recipient of a fellowship from Direccio´n General de Investigacio´n Cientı´fica y Te´cnica of Spain. * Corresponding author; e-mail [email protected]; fax 34 – 96 –3877859.

Abbreviations: ADC, Arg decarboxylase; DPA, days post-anthesis; ODC, Orn decarboxylase; PA, polyamine; RT-PCR, reverse transcriptase-PCR. 323

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Nucleic Acids Extraction Genomic DNA was isolated from tomato shoot tips and young leaves according to the method described by Dellaporta et al. (1983). Total RNA from shoot tips, leaves, whole flowers, roots, and stems was isolated as described by Jones et al. (1985). Total RNA from unpollinated ovaries and young fruits was extracted with the RNeasy Plant Mini Kit (Qiagen, Hilden, Germany). Poly(A1) RNA was extracted with the QuickPrep Micro mRNA purification kit (Pharmacia).

RT-PCR Poly(A1) RNA was isolated from tomato ovaries harvested at 21 DPA. First-strand cDNA was synthesized from 1 mg of poly(A1) RNA with oligo(dT)17-adaptor as the primer (Frohman et al., 1988), using avian myeloblastosis virus RT (Pharmacia) according to the manufacturer’s instructions. Reverse transcription was stopped by heating to 100°C for 5 min, and diluting to 200 mL with TE (10 mm Tris-HCl, pH 8.0, 1 mm EDTA). Ten microliters of the first-strand cDNA was used for PCR amplification. PCR primers were: ODC-59 (59-GGCGTCTCATTCCACATCGG39) as the forward primer, and ODC-39 (59-GTGTAAGCACCCATATTAGGAA-39) as the reverse primer. PCR was carried out in a total volume of 50 mL, containing 0.2 mm each dNTP (Promega), 1.5 mm each primer, 13 reaction buffer, and 1 unit of Taq DNA polymerase (Pharmacia). PCR conditions were: 94°C for 5 min of initial denaturation followed by 35 cycles of 94°C for 1 min, 55°C for 2 min, and 72°C for 2 min. The last cycle was followed by an additional extension step of 72°C for 7 min. PCR products were gel purified using the Qiaex II gel extraction kit (Qiagen) and ligated to pGEM-T vector using pGEM-T Vector System I (Promega).

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excision of pBluescript SK(2) phagemids was carried out according to the manufacturer’s instructions (Stratagene). DNA Sequencing RT-PCR products were sequenced using Sequenase, version 2.0 (Amersham), according to the manufacturer’s instructions. Primers used were specific to the vector. Selected clones obtained from the library screening were sequenced on an automated DNA sequencer (PRISM 377, ABI) using primers from the vector and clone specific. DNA sequences were analyzed using the GCG software package (Genetics Computer Group, Madison, WI). Southern-Blot Analysis Approximately 30 mg of genomic DNA was digested with DraI, EcoRV, AccI, HincII, or XbaI, electrophoresed through a 0.7% agarose gel, and transferred to a nylon membrane (Hybond N1), as recommended by the manufacturer. The determination of the ODC gene-copy number was performed by loading appropriate amounts of BamHIlinearized pD12 clone, equivalent to one, two, and four copies of the gene-per-haploid genome in 30 mg of DNA, along with 30 mg of genomic DNA digested with DraI. The entire ODC clone was used as a probe. The membranes were hybridized under high-stringency conditions as described above, and the genomic blot was also hybridized at low stringency (55°C). High-stringency washing conditions were: four times in 13 SSC and 0.1% SDS at room temperature, and three times in 0.13 SSC and 0.1% SDS at 65°C. Low-stringency washing conditions were: four times in 13 SSC and 0.1% SDS at room temperature, and twice in 13 SSC and 0.1% SDS at 65°C. Northern-Blot Analysis

cDNA Cloning Poly(A1) RNA from 21 DPA unpollinated ovaries was isolated as described above and used to synthesize doublestranded cDNA using the Zap-cDNA synthesis kit (Stratagene). The cDNAs obtained were size fractionated, and the selected fractions were ligated to the Uni-ZAP XR vector (Stratagene). Recombinant DNA was packaged in vitro using Gigapack III packaging extract (Stratagene). The library obtained was amplified once. Plaque lifts on replicate filters were prepared on nylon membranes (Hybond N1, Amersham). The RT-PCR product was used as a probe. The probe was labeled by random priming with [a-32P]dCTP using the Ready To Go DNA labeling kit (Pharmacia) and purified by Sephadex G50 Quick Spin columns (Boehringher-Mannheim). Filters were hybridized essentially as described by Church and Gilbert (1984), and high-stringency washes were made according to the method of Sambrook et al. (1989). Filters were exposed to Hyperfilm MP (Amersham) with an intensifying screen at 270°C. Positively hybridizing phages were plaque purified in one or two additional rounds. In vivo

Total RNA (20 mg) was run in 1% agarose-formaldehyde gels as described by Sambrook et al. (1989). Gels were transferred to a nylon membrane (Hybond N1) as recommended by the manufacturer. The entire ODC clone was used as a probe. Hybridization and washing conditions were as described above for screening filters and for the DNA gel blots. rRNA bands, visualized by ethidium bromide staining, were used as a loading control. RESULTS AND DISCUSSION Cloning of a Tomato ODC cDNA We have identified a tomato cDNA clone, pD12, that encodes a functional ODC. We used RT-PCR to clone a tomato partial ODC cDNA. The pair of oligonucleotides used as primers corresponded to positions 97 to 116 (ODC59) and 638 to 659 (ODC-39) in the sequence of a partial ODC cDNA from tobacco (Malik et al., 1996). First-strand cDNA synthesized using poly(A1) RNA from unpollinated (21 DPA) tomato ovaries was used as a template. After PCR, a 564-bp band was obtained. This band was cloned

Orn Decarboxylase Gene Expression in Tomato Fruits into pGEM-T and confirmed by sequencing to be very similar to ODC sequences in the databases. To isolate full-length ODC cDNAs, we constructed a cDNA library with poly(A1) RNA obtained from unpollinated (21 DPA) ovaries. Plaque-forming units (120,000) from the amplified library were screened using the RT-PCR fragment as a probe, and the clone pD12 was selected for further analysis. The pD12 cDNA contains an insert of 1524 bp, excluding the poly(A1) tail. The sequence contains an open reading frame of 1293 bp encoding a polypeptide of 431 amino acids, with an estimated molecular mass of 46.6 kD. The first ATG of this open reading frame is preceded by an in-frame stop codon (TGA) at positions 23 to 21, indicating that the first ATG is most likely the starting codon.

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Moreover, nucleotides at position 21, 11, and 12 around the ATG codon (TTGAATGGC) match for the consensus sequence surrounding the starting codons in plants (Joshi et al., 1997). The 59-untranslated region of the cDNA is 39 bp long and the 39-untranslated region is 191 bp long. In the 39-untranslated region there is an AATAAA-like element (AATAAT) at position 218 from the polyadenylation site. The alignment of the deduced amino acid sequence with some of the reported ODCs is shown in Figure 1. The sequence is 93% and 89.8% identical to that of thorn-apple ODC (Michael et al., 1996) and tobacco ODC, reflecting a close evolutionary relationship between them. Lower percentages were observed with human (Hickok et al., 1987), Drosophila melanogaster (Rom and Kahana, 1993), and the

Figure 1. Alignment of the amino acid sequence derived from the clone pD12 with the ODCs from thorn-apple (Datura) (X87847), tobacco (D89984), Saccharomyces cerevisiae (Scerevisiae) (J02777), D. melanogaster (Drosophila) (X66599), and human (M16650). The sequences were aligned using the CLUSTALW program (Thompson et al., 1994). Identical amino acids in at least four different sequences are outlined in black, and conserved changes are outlined in gray.

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Figure 3. Northern analysis of ODC gene expression in naturally pollinated ovaries. Ovaries were collected at 21 (lane U-1), 2 (lane P2), 5 (lane P5), 8 (lane P8), and 12 (lane P12) DPA. Twenty micrograms of total RNA was loaded per lane.

AccI, HincII, and XbaI was hybridized to the entire tomato ODC cDNA (Fig. 2A). The ODC cDNA contains one recognition site for AccI, HincII, and XbaI, but none for EcoRV and DraI. The probe hybridized to two AccI fragments, two HincII fragments, and two XbaI fragments, whereas only one hybridizing EcoRV fragment and one hybridizing DraI fragment were detected. When the genomic blot was washed at low stringency, the same bands were detected (data not shown). In the reconstruction experiment, amounts of the linearized clone pD12, corresponding to one, two, and four copies of the ODC gene per haploid genome, were loaded along with DraI-digested genomic DNA (Fig. 2B). Comparison of the intensity of the DraI band with that of the reconstructs, together with the pattern of hybridizing bands in the genomic blot, suggest the presence of a single copy of the ODC gene in the tomato genome, as seems to be the case for the tomato ADC gene (Rastogi et al., 1993). However, the ODC gene may be represented by more than one copy in thorn-apple (Michael et al., 1996). ODC Transcript Levels during Early Fruit Growth and in Different Organs Figure 2. A, Southern-blot analysis of tomato genomic DNA. Genomic DNA (30 mg) was digested with: lane 1, DraI; lane 2, EcoRV; lane 3, AccI; lane 4, HincII; and lane 5, XbaI. B, Copynumber reconstruction experiment. Thirty micrograms of genomic DNA digested with DraI was loaded in the first lane (DraI); lanes 2 through 4 correspond to different amounts of the linearized pD12 plasmid representing one, two, and four copies per haploid genome in 30 mg of genomic DNA. The blots were hybridized with the entire ODC clone. Size markers are indicated.

The pattern of ODC mRNA steady-state levels was studied in self-pollinated ovaries by northern analysis (Fig. 3). The tomato ODC cDNA hybridized two bands, a prominent band of approximately 1.6 kb, as we expected from the size of the cDNA clone, and a fainter band of smaller size. There was a transient increase of the ODC mRNA levels, which showed a maximum at 8 DPA. We also analyzed the

yeast S. cerevisiae (Fonzi and Sypherd, 1987) ODCs (42.1%, 42.0%, and 41.2%, respectively). The functionality of the clone was verified by its ability to complement the spe1 gene in the yeast strain AB3, which is a null mutant for the ODC gene (Schwartz et al., 1995) (data not shown). Gene Analysis Southern-blot analysis of genomic DNA and copynumber reconstructions were performed to determine the number of copies of the ODC genes in the tomato genome (Fig. 2, A and B). Genomic DNA digested with DraI, EcoRV,

Figure 4. Analysis of ODC mRNA levels in unpollinated ovaries treated with 2,4-D or with GA3 or untreated (control). Treatments were made at 21 DPA. Numbers indicate the age of the ovaries. Twenty micrograms was loaded per lane.

Orn Decarboxylase Gene Expression in Tomato Fruits changes in the ODC mRNA levels during early parthenocarpic fruit growth of unpollinated ovaries induced by 2,4-D or GA3 (Fig. 4). Unpollinated ovaries at 21 DPA showed a relatively high level of ODC mRNA, which gradually decreased with time. However, treatments with 2,4-D or GA3 induced a transient increase of the ODC mRNA levels with a maximum in the expression at 5 DPA for 2,4-D-treated ovaries and at 8 DPA for GA3-treated ovaries. Maximum levels of mRNA correlate with peaks in the ODC activity (Alabadı´ et al., 1996). The delay observed in pollinated ovaries with respect to 2,4-D-treated ovaries could be explained by the time needed for fertilization. However, in the case of GA3, it may be due to the higher effectiveness of auxins compared with GAs as inducers of parthenocarpic development in tomato (Alabadı´ et al., 1996). The relatively high levels of mRNA and enzymatic activity of the ODC in tomato ovaries contrast with its absence in pea ovaries (Pe´rez-Amador and Carbonell, 1995). Therefore, ODC gene expression correlates with the active cell division during early fruit growth in tomato (Heimer et al., 1979) and with the absence of cell division in pea, where increased ADC gene expression is correlated with cell expansion (Pe´rez-Amador et al., 1995). However, further studies using in situ-hybridization experiments are needed to associate definitively the expression of ODC and ADC genes to tissues undergoing cell division and cell expansion, respectively. The expression of the ODC gene in different organs and tissues of the tomato plant is shown in Figure 5. The ODC mRNA was detected in all tissues and organs analyzed. The highest expression was detected in roots, in agreement with findings in thorn-apple (Michael et al., 1996). As ADC expression in pea roots has been shown to be very low in comparison with other tissues (Pe´rez-Amador et al., 1995), ODC may play a key role in the synthesis of putrescine in this organ. The high expression also observed in shoot tips and whole flowers at anthesis may be associated with active cell division. A lower expression was detected in stems, and the lowest corresponded to adult leaves, in correlation with a low rate of growth and, therefore, with very limited cell division. In general, the pattern of expression of the tomato ODC gene supports the hypothesis that ODC activity is related to active cell division (Heimer et al., 1979).

Figure 5. Analysis of ODC mRNA accumulation in different organs and tissues of tomato plants. T, Shoot tips; L, leaves; F, whole flowers at anthesis; R, roots; and S, stem. Twenty-five micrograms of total RNA was loaded per lane.

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Northern analyses (Figs. 3–5) showed two hybridizing bands in tomato ODC cDNA, a 1.6-kb band and a smallersized band. Because Southern blots indicated that ODC is represented by a single-copy gene, the latter band is unlikely to be the product of a second ODC gene. The parallel pattern of expression of both mRNAs would point to another origin, such as alternative splicing or specific degradation of the 1.6-kb transcript. A more detailed analysis is needed to give a definitive explanation.

ACKNOWLEDGMENTS The authors wish to thank Drs. A. Granell, J. Moreno, P. Carrasco, and M.A. Pe´rez-Amador for critical reading of the manuscript, Dr. E. Grau for his help in the sequencing work, J. Gil for his help with computers, R. Martı´nez-Pardo and A. Villar for their help in the greenhouse, A. Argoma´niz for technical assistance in the lab, and finally Donnellan-Barraclough for their help with the English. Received March 9, 1998; accepted May 28, 1998. Copyright Clearance Center: 0032–0889/98/118/0323/06. The GenBank accession number for the sequence reported in this article is AF030292.

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