Molecular cloning and functional characterization of two apple S-adenosylmethionine decarboxylase genes and their different expression in fruit development, cell growth and stress responses

Gene 350 (2005) 41 – 50 www.elsevier.com/locate/gene

Molecular cloning and functional characterization of two apple S-adenosylmethionine decarboxylase genes and their different expression in fruit development, cell growth and stress responses Yu-Jin Hao1,2, Zilian Zhang1, Hiroyasu Kitashiba, Chikako Honda, Benjamin Ubi3, Masayuki Kita, Takaya MoriguchiT National Institute of Fruit Tree Science, Tsukuba, Ibaraki 305-8605, Japan Received 8 June 2004; received in revised form 1 December 2004; accepted 6 January 2005 Available online 17 March 2005 Received by G. Theissen

Abstract Two full-length S-adenosylmethionine decarboxylase (SAMDC) cDNAs, MdSAMDC1 and MdSAMDC2, were isolated from apple [Malus sylvestris (L.) Mill. var. domestica (Borkh.) Mansf.]. Both cDNAs encoded tiny and small ORFs in addition to the SAMDC ORFs, and genomic sequences of MdSAMDC1 and MdSAMDC2 contained two or three introns in the 5V upstream regions, respectively. Yeast complementation experiment indicated that two MdSAMDCs encoded functional proteins, and that the tiny and small ORFs possibly repressed their translation efficiency. RNA gel blot analysis showed that MdSAMDC1 were differentially regulated in fruits depending on the developmental stage and in cell suspension during the culture period, but MdSAMDC2 did not. In contrast, MdSAMDC2 was positively induced by cold and salt stresses, but MdSAMDC1 was not. These results suggest that MdSAMDC1 is mainly involved in fruit development and cell growth while MdSAMDC2 in stress responses, compared with their respective counterpart. D 2005 Elsevier B.V. All rights reserved. Keywords: Apple (Malus sylvestris var. domestica); Extreme temperature stress; Fruit development; S-adenosylmethionine decarboxylase; Salt stress

1. Introduction Polyamines are a group of small positively charged molecules that are produced ubiquitously in living cells and are involved in many cellular processes such as DNA replication, transcription, translation and cell proliferation. In plants, polyamines have been reported to play a crucial Abbreviations: BA, N 6-benzylaminopurine; DAF, days after flowering; 2,4-D, 2,4-dichlorophenoxyacetic acid; DIG, digoxigenin-dUTP; EtdBr, ethidium bromide; IBA, 3-indolebutyric acid; ORF, open reading frame; SAMDC, S-adenosylmethionine decarboxylase. T Corresponding author. Tel.: +81 29 838 6500; fax: +81 29 838 6437. E-mail address: [email protected] (T. Moriguchi). 1 Co-first authors. 2 Present address: College of Horticultural Science, Shandong Agricultural University, Tai-An, Shandong 271018, PR China. 3 Present address: Department of Genetics and Biotechnology, Faculty of Science, University of Calabar, P.M.B.1115, Calabar, Nigeria. 0378-1119/$ - see front matter D 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.gene.2005.01.004

role in morphogenesis, embryogenesis, early fruit development and senescence (Galston and Sawhney, 1990; Pandey et al., 2000) and in the elicitation of resistance or tolerance responses to some biotic and abiotic stresses (Bouchereau et al., 1999). S-adenosylmethionine decarboxylase (SAMDC), a pyruvoyl-dependent enzyme producing the aminopropyl group for spermidine and spermine, is one of the rate-limiting enzymes in polyamine biosynthesis. Genes encoding SAMDC have been cloned from several organisms including yeast (Kashiwagi et al., 1990) and human (Pajunen et al., 1988). Due to the important role of polyamines in plant development, there has been growing research interest on the function of SAMDC genes in many plant species. Recently, SAMDC cDNAs have been isolated from a variety of plant species such as potato (Mad Arif et al., 1994), pea (Marco and Carrasco, 2002), soybean (Tian et al., 2004), rice (Pillai and Akiyama, 2004) and so on. Generally,

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SAMDC cDNAs showed a temporal and spatial expression pattern, suggesting that SAMDC functions in a developmental and organ/tissue specific manner. Investigation indicated that SAMDC transcript initially produced a proenzyme that was then cleaved at a serine residue via an autocatalytic mechanism to form a functional protein complex containing a and h subunits (Xiong et al., 1997). Furthermore, the translation efficiency of SAMDC proenzyme was negatively regulated by tiny and small open reading frames (ORFs) located in the upstream regions of the encoding ORF (Hanfrey et al., 2002). On the other hand, results from transformation experiments with SAMDC genes indicated that a variety of plants showed enhanced resistance or tolerance to abiotic stresses including ozone, salt and drought (Roy and Wu, 2002), as well as biotic stresses such as fungal wilts (Waie and Rajam, 2003). In addition to stress responses, transgenic potato with antisense SAMDC cDNA showed a reduction in the level of SAMDC transcripts with modified phenotypes (Kumar et al., 1996). Moreover, tomato plants transformed with a SAMDC gene exhibited enhanced fruit phytonutrient content, fruit juice quality and vine life (Mehta et al., 2002). Thus, several lines of evidence suggest an important role of SAMDC not only in plant developmental and physiological processes, but also in eliciting plant responses to environmental stresses. There is, however, a dearth of knowledge on the physiological function of SAMDC in fruit trees because only a peach SAMDC gene has been isolated so far (Ziosi et al., 2003). Therefore, understanding the function of SAMDC in apple, one of the most economically important fruit trees world-wide, may provide novel tools to overcome constraints for the breeding of apples with desirable agronomic traits including fruit quality characteristics, plant architectural types for easy orchard management and the manipulation of tolerance to biotic and abiotic stresses. In this study, to gain insights into molecular features of SAMDC, two full-length SAMDC cDNAs, MdSAMDC1 and MdSAMDC2, were cloned by screening an apple shoot cDNA library and functionally characterized by yeast complementation. RNA gel blot analysis suggested that MdSAMDC1 and MdSAMDC2 were differentially regulated in developmental or physiological processes and in their response to extreme temperature and salt stresses.

2. Materials and methods 2.1. Plant materials Flower buds, young and mature leaves of the apple cultivar dOrinT (Malus sylvestris var. domestica) were collected in the experimental orchard of National Institute of Fruit Tree Science (Tsukuba, Japan). Fruits were collected at 19, 61, 103, 145 and 174 days after full bloom (DAF), from the same cultivar dOrinT at the Apple Research

Center, National Institute of Fruit Tree Science (Morioka, Japan). Fresh leaves, flowers and fruit flesh were immediately frozen in liquid nitrogen and stored at 80 8C for RNA extraction as described by Wan and Wilkins (1994). Apple callus was induced from young fruits and maintained at 1-month intervals on callus subculture medium containing MS-salt (Murashige and Skoog, 1962), Nitsch organic component (Nitsch and Nitsch, 1969), 3% sucrose, 4.5 AM 2,4-dichlorophenoxyacetic acid (2,4-D), 1 AM N 6-benzylaminopurine (BA) and 0.8% agar in the dark at 25 8C. For suspension culture, the callus was transferred into the liquid subculture medium and placed on a rotary shaker at approximately 120 rpm in the dark at 25 8C. The cell suspension was subcultured three times at 2-week intervals before being used for growth experiment. 2.2. cDNA cloning Total RNA was isolated from apple shoots with RNAqueous-Midik (Ambion). One microgram of total RNA was used to synthesize first-strand cDNA with a kit (Amersham Pharmacia Biotech). A partial SAMDC cDNA fragment was obtained by RT-PCR using an amplification profile containing 1 cycle of 10 min at 94 8C; 30 cycles of 30 s at 94 8C, 30 s at 45 8C, and 90 s at 72 8C and 1 cycle of 10 min at 72 8C. Degenerate primers: 5V-GAY TCN TAT GTN CTN TCN GAG TCN AG-3V (upstream) and 5V-CT NGC RTA RCT GAA NCC RTC YTC NGG-3V (downstream) were designed based on the conserved regions of plant SAMDC amino acid sequences DSYVLSESS and PEDGFSYAS, respectively. The amplified fragment was sequenced and compared with the SAMDC cDNAs of other plants to confirm whether it was a SAMDC homologue. The confirmed fragment was labeled with digoxigenin-dUTP (DIG) by PCR (Roche Diagnostics) and used as a probe to screen an apple shoot cDNA library as described by Zhang et al. (2003). Positive plaques were excised as pBluescript clones following the manufacturer’s instructions (Stratagene). The cDNA clones were sequenced on both strands using the BigDyek Terminator Cycle Sequencing Ready Reaction kit (PE Biosystems). DNA sequences were analyzed using the GCG software package (Genetics Computer Group) and GENETYX-MAC 10.1 (Software Development). 2.3. In vitro translation and Western blot analysis of MdSAMDC2 ORF encoding SAMDC in the MdSAMDC2 was amplified by PCR with primers specific to NcoI (upstream primer 5V-CCA TGG CTG TAC CGG TCT CT-3V) and SmaI (downstream primer 5V-CCC GGG GAT CTT TGC CAT AC-3V) sites. The amplified PCR product was ligated into pIVEX2.3d expression vector (Roche Diagnostics) with His-tag. After in vitro translation using Escherichia coli lysate according to the instruction

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provided by the manufacture (Roche Diagnostics), the translated products were separated on a 12.5% SDSPAGE and then transferred onto a polyvinylidene difluoride (PVDF) membrane (Amersham Pharmacia Biotech). The membrane was blocked for 30 min in 5% non-fat dried milk in Tris-buffered saline, pH 7.5. Immuno-reaction was conducted with a 1:200 dilution of anti-His-tag antibody (H-15) (Santa Cruz) and a 1:25000 dilution of anti-rabbit IgG alkaline phosphate-conjugated secondary antibody. Detection of antigen–antibody complexes was carried out using ECL-Plus kit (Amersham Pharmacia Biotech). 2.4. Complementation assay with SAMDC-defective yeast strain The mutant yeast strain YOL052C (clone ID, 11743; Open Biosystems) is defective in the function of SAMDC gene (spe2). The normal growth of mutant cells depends on the application of spermidine, which makes it possible to characterize the functionality of apple SAMDC cDNAs by heterologous complementation. To perform complementation assay, the full-length MdSAMDC cDNAs or the ORF encoding SAMDC in the respective MdSAMDC1 and MdSAMDC2 was amplified by PCR with primers specific to NotI sites. Four kinds of inserts, namely, SAMDC1all, SAMDC2all, SAMDC1orf and SAMDC2orf were obtained. SAMDC1all or SAMDC2all consisted of SAMDC ORF plus tiny and small ORFs while SAMDC1orf or SAMDC2orf contained only SAMDC ORF (Fig. 4A). After sequence confirmation, these inserts were cloned into the NotIdigested pYES2 plasmid (Invitrogen) under the control of a galactose-inducible GAL1 promoter. Resultant four kinds of plasmid constructs, namely, pYES2DSAMDC1all, pYES2::SAMDC1orf, pYES2DSAMDC2all and pYES2D SAMDC2orf, as well as the empty pYES2 plasmid (control) were introduced into a mutant yeast strain YOL052C using a lithium acetate transformation method (Gietz, 1992). Transformants were initially selected in SDmedium without uracil. Positively transformed singlecolonies for each construct were confirmed by PCR and then grown for 24 h in 10 ml SD-medium, with or without spermidine (100 AM). Approximately, 20 Al was then subcultured in 10 ml of fresh medium at intervals of 24 h. Cell proliferation was monitored according to Marco and Carrasco (2002).

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2.6. RNA gel blot analysis of MdSAMDCs in different tissues Probes specific to MdSAMDC1 and MdSAMDC2, respectively, were produced by PCR (Roche Diagnostics) with upstream primer 5V-GGG GTA CCT ATT CCT TAG TTG GA-3V and downstream primer 5V-CCG GAT CCA CTA GTC AAT TGT GC-3V for MdSAMDC1, as well as upstream primer 5V-GGG GTA CCG TTG TGT TAA GTC CC-3V and downstream primer 5V-CCG AGC TCA AGA ACA ATA TCA CC-3V for MdSAMDC2. The PCR profile consisted of 1 cycle of 120 s at 95 8C; 40 cycles of 40 s at 95 8C, 40 s at 55 8C, and 90 s at 72 8C and 1 cycle of 10 min at 72 8C. The products for both MdSAMDC1 and MdSAMDC2 were DIG-labeled. In order to confirm their specificity, MdSAMDC1 and MdSAMDC2 cDNAs recovered from plasmid DNA were used for DNA gel blot analysis. After confirming their specificity, these probes were then used for RNA gel blot analysis. Total RNA (8 Ag) isolated from leaves, flower buds and fruit flesh tissues according to the procedure described by Wan and Wilkins (1994) was electrophoresed in 1.2% formaldehyde denatured agarose gel. RNA was blotted onto Hybond N+ membrane (Amersham Pharmacia Biotech) and hybridized with DIG-labeled probes specific to MdSAMDC1 and MdSAMDC2 as described by Zhang et al. (2003). 2.7. RNA gel blot analysis of MdSAMDCs under stress conditions In vitro apple shoots were maintained on the subculture medium (pH 5.9) containing MS salts (Murashige and Skoog, 1962), B5 organic component (Gamborg et al., 1968), containing 3% sucrose, 4.5 AM BA, 0.5 AM IBA and 0.8% agar under a 16-h photoperiod. For low and high temperature stresses, 20-day-old shoots were treated at 4 8C and 35 8C, respectively, in the dark for 0 (control), 6, 24, 72, and 120 h. For salt stress, 20-day-old shoots were transferred into the subculture medium with 0 (control) and 300 mM NaCl, respectively, for 6, 12, 24, 48, 72, 96 and 120 h. Plant materials exposed to different stress treatments were collected and frozen in liquid nitrogen for RNA extraction as described by Wan and Wilkins (1994). RNA gel blot analysis was carried out with MdSAMDC1 or MdSAMDC2 probes.

2.5. PCR amplification of MdSAMDC genomic DNA 3. Results Genomic DNA was isolated from apple leaves as described by Zhang et al. (2003) and then used as template for PCR with primers specific to MdSAMDC1 and MdSAMDC2 cDNAs, respectively. In order to get the intron sequences, the amplified fragments were sequenced and identified sequences were used to design a second set of primers.

3.1. Isolation of two full-length SAMDC cDNAs Two partial SAMDC fragments, 584 and 602 bp in size, were amplified by RT-PCRs using degenerate primers. Both fragments were highly homologous to other plant SAMDC genes registered in Genebank (data not shown). The 602-bp

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Fig. 1. Schematic diagrams of MdSAMDC1 and MdSAMDC2 cDNAs. Starting positions of introns are indicated by arrows followed by their names and sizes, respectively. Tiny, small and SAMDC-encoding ORFs are shown as different boxes characterized with their names and sizes, respectively. Numbers above those boxes indicate the starting positions of each ORF.

fragment had six additional amino acids compared with the 584-bp fragment. When the two fragments were used as probes to screen the cDNA library, 32 positive clones were obtained from about 100,000 plaques with the 584-bp probe, while only three positive plaques were obtained from about 60,000 plaques with the 602-bp probe. Several clones with large size were selected from the two clone groups and sequenced. Two full-length cDNAs obtained from 584-bp and 602-bp probes were designated as MdSAMDC1 and MdSAMDC2, respectively. MdSAMDC1 (1875 bp) consisted of a poly(A) tail, a 3V-untranslated region of 292 bp and a 1074 bp ORF encoding 358 amino acids. Similarly, MdSAMDC2 (1938 bp) composed of a poly(A) tail, a 238 bp 3V-untranslated region and a 1122 bp ORF encoding 374 amino acids. Both MdSAMDC1 and MdSAMDC2 had long 5V regions that contained additional tiny and small ORFs (Fig. 1). The small ORFs of MdSAMDC1 and MdSAMDC2 encoded 53 and 51 amino acids, respectively, while the tiny ORFs of both cDNAs encoded 3 codons. The full-length cDNA sequences of MdSAMDC1 and MdSAMDC2 were deposited in the DDBJ database under the accession numbers AB077441 and AB077442, respectively. 3.2. Analysis of amino acid sequences deduced from MdSAMDCs The main ORFs of MdSAMDC1 and MdSAMDC2 encoded proteins with predicted molecular masses of 39.8

(pI 4.74) and 40.7 kDa (pI 4.82), respectively. Their deduced amino acid sequences showed 70% identity to each other, 53–70% to SAMDC proteins of other plant species (Franceschetti et al., 2001; Li and Chen, 2000; Marco and Carrasco, 2002) and only 34% identity to human SAMDC (Pajunen et al., 1988). An alignment of the amino acid sequences of MdSAMDCs with those of other plant species is illustrated in Fig. 2A. From the deduced amino acid sequence, one conserved region was the LSESSLF that contained a putative proenzyme cleavage site (Stanley et al., 1989) marked by a triangle as shown in Fig. 2A. Theoretically, non-hydrolytic cleavage in this region produced a short h-subunit (Nterminal part) and a longer a-subunit (C-terminal part), i.e. 7.8 and 32.0 kDa for MdSAMDC1 and 7.0 and 33.1 kDa for MdSAMDC2. In this experiment, in vitro expression and Western blot analysis showed that MdSAMDC2 produced a ca. 33 kDa product, which seemingly corresponds to a subunit of the C-terminus (Fig. 3). Another conserved region was TIHVTPEDGFSYASFE (characterized as PEST region) rich in proline (P), glutamic acid (E), serine (S) and threonine (T), which was associated with rapid protein degradation (Rogers et al., 1986). Besides these conserved regions, highly conserved amino acid residues Glu11, Glu14, Ser71, Cys85, Ser234, and His247 existed in MdSAMDC1 and Glu11, Glu 14 , Ser 71, Cys 85 , Ser 240 , and His 253 in MdSAMDC2. Their sites were very similar with Glu8,

Fig. 2. (A) Alignment of the predicted amino acid sequences of MdSAMDCs and those of several other plants including Arabidopsis thaliana (GenBank accession no. U63633), Nicotiana tabacum (U91924), Oryza sativa (Y07766), Pisum sativum (U60592), Solanum tuberosum (Z11680), Spinacia oleracea (X81414), Triticum aestivum (AF117660) and Zea mays (Y07767). Black-highlighted residues are identical, while light gray-highlighted residues are similar in all proteins. Highly conserved sequence LSESSM and PEST motif are marked by triangle and underlined, respectively. (B) Alignment of the predicted amino acid sequences of small ORFs in MdSAMDCs and those of several other plants including A. thaliana (U63633), N. tabacum (AF033100), O. sativa (Y07766), P. sativum (U60592), Brassica juncea (U80916) and Z. mays (Y07767).

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spermidine supplementation (Fig. 4B). These results indicated that MdSAMDCs encoded functional SAMDC proteins. Based on the presence or absence of the tiny and small ORFs, four SAMDC constructs were divided into two types, i.e. all constructs (pYES2DSAMDC1all and pYES2DSAMDC2all) which contained SAMDC ORF plus tiny and small ORFs, and orf constructs (pYES2DSAMDC1orf and pYES2DSAMDC2orf) which contained only SAMDC ORF (Fig. 4A). The results showed that the growth rate of all-type transformants reached their peak within a longer time than that of orf-type transformants (Fig. 4C, D), suggesting that the presence of tiny and small ORFs inhibited growth to some extent. Similarly, based on the SAMDC cDNAs, four SAMDC constructs were also divided into two types, i.e. SAMDC1 constructs (pYES2DSAMDC1all and pYES2DSAMDC1orf) and S A M D C 2 c o n s t r u c t s ( p Y E S 2 DS A M D C 2 a l l a n d pYES2DSAMDC2orf) (Fig. 4A). Growth assay indicated that the SAMDC1-type always showed a higher growth rate than the SAMDC2-type (Fig. 4C, D). 3.4. Genomic organization of MdSAMDCs

Fig. 3. Schematic diagrams of in vitro translation and immuno-blot analysis of MdSAMDC2. T7P, RBS, MCS and T7T show T7 promoter, ribosomal binding site, multiple cloning site and T7 terminator, respectively.

Glu11, Ser68, Cys 82, Ser229, and His 243 in human SAMDC, all of which are indispensable to catalytic activity (Pajunen et al., 1988). Lys83 in both MdSAMDCs seemed to correspond to Lys81 in Arabidopsis that is crucial for substrate binding (Park and Cho, 1999). In addition, the amino acid sequences of small ORFs in both MdSAMDCs were also highly conserved like those of other plants (Fig. 2B). 3.3. Yeast functional complementation Growth of the mutant yeast strain YOL052C was arrested in the absence of spermidine. Under the same conditions, however, growth of the four yeast transformants of pYES2DSAMDC1all, pYES2DSAMDC2all, pYES2DSAMDC1orf or pYES2DSAMDC2orf was recovered (Fig. 4C, D), whereas the yeast transformant containing empty pYES2 vector alone still showed arrested growth, which was recovered by 100 AM

Using genomic DNA as template and primers specific to MdSAMDC1 and MdSAMDC2 cDNAs, a series of PCRs produced specific fragments that were then sequenced. The comparison of these sequences with their cDNA counterparts in both genes showed no intron in the SAMDC-encoding ORFs but a total of 3 introns in the 5V upstream regions. Based on the nomenclature described by Franceschetti et al. (2001), these introns were named as intron 1, 2 and 3. MdSAMDC1 contained intron 2 in the 5V untranslated region and intron 3 in the small ORF, while MdSAMDC2 contained intron 1 and intron 2 in the 5V untranslated region, as well as intron 3 in the small ORF (Fig. 1). 3.5. Expression of MdSAMDCs in different apple tissues and suspension cells Both MdSAMDC1 and MdSAMDC2 probes were specifically hybridized to their respective plasmid DNA (Fig. 5A). Two specific probes were used for RNA gel blot analysis. The results indicated that both MdSAMDC genes were expressed in all tissues tested, and generally at a higher level in reproductive organs than in vegetative organs (Fig. 5B). On the other hand, MdSAMDC1 and MdSAMDC2 showed differential expression patterns. Compared with matured leaves, young leaves showed a high expression level of MdSAMDC1 but a low level of MdSAMDC2. In fruits, the expression level of MdSAMDC1 varied with the developmental stages, i.e. showed a high level in the early stages from 19 to 61 DAF, decreased gradually from 103 to 145 DAF, and finally reached another peak at the ripe stage at 174 DAF. In contrast, MdSAMDC2 was constitutively expressed from 19 to 174 DAF.

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Fig. 4. Complementation of the SAMDC-defective yeast strain (YOL052C) with MdSAMDCs. Schematic diagrams of four constructs SAMDC1all (127–1611 bp), SAMDC1orf (321–1611 bp), SAMDC2all (220–1724 bp), SAMDC2orf (411–1724 bp) (A). Optical density at 600 nm of pYES2 plasmid with or without 100AM spermidine (B), pYES2DSAMDC1all and pYES2::SAMDC1orf (C) as well as pYES2::SAMDC2all and pYES2DSAMDC2 (D). The growth for pYES2DSAMDC1orf in (C) was saturated at 5 days, therefore subculturing was not carried out.

The growth curve of apple cell suspension was typical S-shape. Briefly, the growth of cell suspension showed the slowest rate at the initial stage from 0 to 4th day and the fastest rate at the logarithmic stage from 6th to 14th, respectively. Subsequently, the growth rate slowed gradually from the 16th to 20th day, suggesting that the culture entered stationary phase when a subculture was required for further growth to occur (data not shown). Correspondingly, the expression of MdSAMDC1 varied with the culture time (Fig. 5C). The transcripts increased from 2nd to 4th day, and then recovered to a level similar with 2nd day at 6th day, which was retained till 12th day. When the growth entered stationary phase from 16th to 20th day, the expression further decreased to a comparatively low level relative to that at any other stage during the culture period. In contrast, MdSAMDC2 was constitutively expressed during the culture period from 2nd to 20th day. 3.6. Expression of MdSAMDCs under extreme temperature and salt stresses The in vitro shoots obtained at 4 8C maintained normal growth throughout the treatment (data not

shown). RNA gel blot analysis showed that MdSAMDC1 and MdSAMDC2 responded to low temperature in different ways (Fig. 6A). MdSAMDC1 transcripts dramatically increased in treated shoots at 6 h and then decreased gradually to a lower level from 24 to 120 h than the control (0 h). The induction of MdSAMDC2 expression was much higher than the control and remained nearly constant at 6, 24 and 120 h. At 35 8C, however, the leaf edges of in vitro shoots started to turn brown at 120 h (data not shown). Both MdSAMDC1 and MdSAMDC2 transcripts sharply decreased to a trace level in all the treatments at 35 8C (Fig. 6A). When the in vitro shoots were exposed to 300 mM NaCl stress, their leaves started to turn brown along the veins at 96 h (data not shown). RNA gel blot analysis showed that the expression level of both MdSAMDCs varied with treatment time even in the control, i.e. decreased gradually from 6 to 24 h and then increased to a higher level than the control (0 h) for MdSAMDC1 or decreased gradually from 6 to 24 h and then increased to a level similar to the control (0 h) for MdSAMDC2. Upon 300 mM NaCl stress treatment, the level of MdSAMDC1

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substrate binding (Pajunen et al., 1988; Park and Cho, 1999). The apple SAMDC genes also contained these conserved amino acid residues, and in vitro translation and Western blot analysis suggested the cleavage of proenzyme at the predicted processing site. MdSAMDC1 and MdSAMDC2 also contained conserved small and tiny ORFs in the 5V upstream region that may confer feedback control based on the level of cellular polyamines for the translation of the encoded message in plants (Xiong et al., 1997), as in mammalian genes (Raney et al., 2000). Hanfrey et al. (2002) demonstrated that the small and tiny ORFs in plant SAMDC genes play a repressive role in regulating the translation of the encoding ORF. Here, yeast complementation indicated that the small and tiny ORFs in MdSAMDCs partially inhibited their growth. Therefore, it may be deemed that the upstream ORFs would function to regulate the translation efficiency of SAMDC ORF in apple as proposed by Hanfrey et al. (2002). Hanfrey et al. (2002) also indicated that when the construct with abrogated upstream ORF were transferred

Fig. 5. DNA gel blot analysis of MdSAMDC1 and MdSAMDC2 plasmid DNA with their respective probes (A). RNA gel blot analysis of MdSAMDC1 and MdSAMDC2 RNA with their respective probes in different tissues (B) and in suspension cells (C). Lower panels of each item show the rRNA stained with EtdBr.

transcripts varied with the treatment time as in the control (0 mM NaCl). However, MdSAMDC2 transcript was induced to a high level at 6 h by 300 mM NaCl and retained this level up to 120 h (Fig. 6B).

4. Discussion In this study, we isolated and functionally characterized two apple full-length SAMDC cDNAs in apple. It has been reported that SAMDC proteins have some highly conserved amino acid residues that are crucial for the post-translation processing, regulation and function (e.g. Kashiwagi et al., 1990; Pajunen et al., 1988; Park and Cho, 1999). The amino acid sequences, LSESSLF and TIHVTPEDGFSYASFE were shown to be indispensable for processing of proenzyme (Stanley et al., 1989) and rapid protein degradation (Rogers et al., 1986), respectively, and some residues such as Lys81 in Arabidopsis were found to be crucial for catalytic activity and

Fig. 6. RNA gel blot analysis of MdSAMDC1 and MdSAMDC2 RNA with their respective probes under low and high temperatures (A), and under 0 mM NaCl and 300 mM NaCl (B). Lower panels of each item show the rRNA stained with EtdBr.

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into plants, the resultant transgenic plants showed severe growth and developmental defects possibly due to the disruption of polyamine homeostasis. In this study, the morphological differences in the yeast among orf-types or all-types were not apparent by microscopic observation (data not shown). The SAMDC1-type constructs showed higher growth rate than SAMDC2-type constructs, although the exact reason is yet unknown. Investigations have indicated the involvement of polyamines in fruit development and senescence (Pandey et al., 2000). MdSAMDC1 was expressed differentially in fruits from 19 to 174 DAF, while MdSAMDC2 showed constitutively expression during fruit development. The highest expression of MdSAMDC1 was at 19, 61 and 174 DAF. It is well known that the expression level of most polyamine biosynthetic genes is higher in actively growing tissues than in mature or senescent tissues (Mad Arif et al., 1994; Marco and Carrasco, 2002). The high level of MdSAMDC1 transcripts at 19 and 61 DAF should result from rapid cell division and expansion at the early stage of fruit development. However, MdSAMDC1 transcripts reached another peak at ripening (174 DAF), which might indicate its involvement in complex ripening-related processes. In addition, the expression patterns of MdSAMDC1 and MdSAMDC2 were different between young and mature leaves. Similarly, the expression level of MdSAMDC1 was positively related with the growth rate during the culture period, while that of MdSAMDC2 did not. In a word, these findings suggest that MdSAMDC1 and MdSAMDC2 may differentially function in developmental and physiological processes, and that MdSAMDC1 seems to be mainly involved in fruit development and cell growth, compared with MdSAMDC2. High temperature has been reported to inhibit pollen germination and growth in tomato by suppressing SAMDC activity (Song et al., 2002). On the other hand, the increase in SAMDC activity induced by low temperature imparted cold-tolerance in cucumber (Shen et al., 2000) and maintained normal photosynthesis in spinach leaves (He et al., 2002). Urano et al. (2003) reported that Arabidopsis AtSAMDC2 mRNAs increased significantly under cold treatment. It has also been reported that rice OsSAMDC transcripts in the cold-resistant rice genotype showed a continued increase for up to 72 h upon exposure to cold stress (5 8C), whereas OsSAMDC transcription in the susceptible rice cultivar remained unchanged under the same conditions from the control (Pillai and Akiyama, 2004). These results indicated that maintenance and/or increase in SAMDC activity and/or gene expression may be a crucial factor for normal plant development under the undesirable temperature stresses. In apple, high level of MdSAMDC1 expression was induced only at the early stage of cold stress treatment, whereas the transcript levels of MdSAMDC2 remained high and nearly constant at 4 8C throughout the cold stress treatment. This finding suggests that MdSAMDC2 might be a cold tolerance-related gene. In

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contrast to the 4 8C treatment, the expression of both MdSAMDCs at 35 8C was significantly reduced to a trace level from 6 to 120 h, suggesting that MdSAMDCs were sensitive to high temperature, so that the transcription of the MdSAMDCs were impaired. Moreover, the leaves of apple shoots at 35 8C started to turn brown at 120 h, unlike those kept at 4 8C which remained normal, indicating that high temperature caused plant damage. The fact that apple trees adapt to cool better than hot climatic regions may indirectly support this observation. Upon salt stress, RNA gel blot analysis showed a high level induction of MdSAMDC2 expression relative to MdSAMDC1, suggesting that MdSAMDC2 may be involved in salt stress tolerance in apple. Li and Chen (2000) showed that salt tolerance in rice was positively related to SAMDC activity. Transgenic rice (Roy and Wu, 2002) and tobacco (Waie and Rajam, 2003) over-expressing SAMDC genes showed an enhanced tolerance to salt stress. More recently, Tian et al. (2004) found that GmSAMDC1 gene expression was induced by salt stress in soybean. Therefore, SAMDC genes can be exploited for enhanced salt tolerance in plant species. In conclusion, this study revealed that both MdSAMDCs encode functional SAMDC proteins and that they play different roles in plant development and stress responses in apple. It can be inferred that MdSAMDC1 is mainly involved in fruit development and cell growth, while MdSAMDC2 is mainly correlated with stress responses and its regulation at transcriptional level should be very important in the acquisition of stress tolerance. However, little is yet known on the molecular mechanism underlying the regulation of MdSAMDC genes. It has been reported that cis-elements DRE/CRT or ABRE are involved in the regulation of stressresponse-related genes (Shinozaki et al., 2003). One DRE/ CRT-related motif, one ABRE motif, three ABRE-related motifs and four G-Box exist in the promoter region of AtSAMDC2, suggesting a putative regulatory mechanism involving these cis-elements (Urano et al., 2003). Further investigations would be needed to confirm whether a similar regulatory mechanism exists in apple SAMDC genes.

Acknowledgements Authors thank Drs. Chun-Gen Hu and Chikako Nishitani for their pieces of advice on experimental techniques. This work was supported in part by Grants-in-Aid from the Ministry of Education, Science, Sports and Culture of Japan and Japan Society for Promotion of Science (JSPS).

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