S-adenosylmethionine decarboxylase that is circadian-clock-regulated ... of this ORF shows a high degree of similarity with dicot S-adenosylmethionine decar-.
Plant Molecular Biology 30: 1021-1033, 1996. © 1996 Kluwer Academic Publishers. Printed in Belgium.
1021
Isolation and characterization of a Tritordeum cDNA encoding S-adenosylmethionine decarboxylase that is circadian-clock-regulated Thomas Dresselhaus 1, Pilar Barcelo 2, Christine Hagel 1, Horst LOrz 1 and Klaus Humbeck 3'* 1lnstitutfiir Allgemeine Botanik, AMP H, Ohnhorststrasse 18, D-22609 Hamburg, Germany,"2Biochemistry and Physiology Department, IARC-Rothamsted, Harpenden, Hertfordshire AL5 2JQ, UK; 3Botanisches Institut der Universitdt zu Krln, Gyrhofstrasse 15, D-50931 Krln (*author for correspondence) Received 10 July 1995; accepted in revised form 19 January 1996
Key words: S-adenosylmethionine decarboxylase, circadian rhythm, ethylene, polyamines, Tritordeum, wounding
Abstract
Sequence analysis of the two cDNA clones 47/11 and 50A which were isolated by differential screening of an explant cDNA library obtained from the monocot Tritordeum (hexaploid hybrid of diploid wild barley and tetraploid wheat lines) reveals that both clones include the same open reading frame (ORF). The sequence of this ORF shows a high degree of similarity with dicot S-adenosylmethionine decarboxylase (SAMDC) gene sequences and contains regions highly conserved in all known SAMDC sequences. It is further shown that the sequence represented by the cDNA clones 47/11 and 50A is derived from the wild barley (Hordeum chilense) genome, where it is present as a single-copy gene. Northern analyses indicate the corresponding transcript to accumulate in response to wounding and the transcript level changes with a circadian rhythm, having a peak in the middle of the light period. The periodicity continues in constant light, but is changed in constant darkness.
Introduction
Polyamines such as spermidine, spermine and their precursor putrescine are polybasic aliphatic amines. Despite their ubiquitous presence in all living organisms their physiological functions remain unclear [8]. In plants, as in animals and bacteria, polyamines seem to be related to growth and development. Numerous investigations indicate a role of polyamines in cell division, embryo-
genesis, floral and fruit development, root formation and stress responses [8, 29]. However, the exact molecular functions of polyamines in these processes are still a matter of debate. The polyamine biosynthetic pathways have been well characterized. In plants, putrescine is either synthesized from ornithine via ornithine decarboxylase (ODC) or from arginine via arginine decarboxylase (ADC) [31 ]. The formation of the triamine spermidine from the diamine pu-
The nucleotide sequence data reported will appear in the EMBL, GenBank and DDBJ nucleotide sequence databases under the accession number X83881.
1022 trescine and also the formation of the tetraamine spermine from spermidine is mediated by the enzyme S-adenosylmethionine decarboxylase (SAMDC; EC 4.1.1.50). The substrate for this enzyme is S-adenosylmethionine (SAM) and the product is decarboxylated SAM which provides the aminopropyl moiety for spermidine and spermine biosynthesis. The activity of SAMDC has been shown to be rate limiting in the biosynthesis of these polyamines [28]. In plants, SAM is not only a precursor for spermidine and spermine synthesis, but also for ethylene biosynthesis via 1-aminocyclopropane-l-carboxylic acid. Both pathways have been shown to be interdependent
[8]. Up to now several genes encoding SAMDC have been characterized from mammals, yeast and bacteria [ 14, 16, 32]. Despite a striking conservation of amino acid sequences among the mammalian SAMDCs, there is little similarity between the sequences of mammalian, yeast and Escherichia coli enzymes [ 14, 20, 22, 30, 34]. Although SAMDC activity could be demonstrated in a variety of plant species [8] only a few fulllength plant sequences have been reported until now (e.g. potato [33], spinach [4], Catharanthus roseus [26]). While the deduced amino acid sequences of potato and spinach SAMDCs show a similarity of about 72 ~o, they exhibit only a low degree of sequence similarity to other eucaryotic SAMDC genes. Despite the low overall sequence similarity some regions are highly conserved in all SAMDC genes. These regions include a proenzyme cleavage site [14, 19] and a putative PEST sequence [24] characteristic of polypeptides that are rapidly turned over. In this study we describe the isolation of two clones (47/11 and 50A)by differential screening of a cDNA library from Tritordeum leaf tissue induced towards embryogenesis by the action of cutting and auxin treatment. Both clones contain the same complete ORF which is characterized by a high degree of overall similarity to the SAMDC sequences of potato and spinach and by highly conserved regions characteristic of all SAMDCs. We conclude that this ORF represents the first monocot SAMDC sequence re-
ported. Southern analyses with genomic DNA from Tritordeum, barley and wheat lines were performed to investigate gene origin and copy number. The expression was studied by northern experiments.
Materials and methods
Plant material
Plants of Tritordeum (line HT 28) [fertile amphiploid between Hordeum chilense and Triticum turgidum var. durum, obtained by [ 17] were grown in soil under 16 h photoperiod and 18/14 °C day/ night temperature cycle. Likewise, plants of H. chilense (lines H 1 and H7), of T. turgidum var. durum (lines T22 and T24) and of Triticum aestivum ssp. sphaerococcum (line T59) were grown in vermiculite under the same conditions. Plants of Hordeum vulgate were grown under the same conditions but slightly higher temperature (20/ 16 ° C) day/night regime.
Definition of Tritordeum flag leaf regions 1 (R 1) and 2 (n2)
Region 1 is defined as the basal part of the flag leaf containing cells which are able to re-enter mitosis as a response to cutting and auxin exposure in vitro. Region 2 is defined as the upper part of the flag leaf containing cells not able to respond to the in vitro treatment and therefore unable to proliferate [2].
Construction and differential screening of Tritordeum explant cDNA library
Leaf material for cDNA library construction was collected by harvesting tillers ranging from those with flag leaves at a very early stage of development (0.3 cm) to those with flag leaves about to emerge from the sheath (ca. 25 cm). Sterilization of the tillers and dissection out of flag leaves was done according to Barcelo et al. [2]. Flag leaf
1023 region 1 (see above) was cut into 1 m m segments which then were placed on medium and cultured for 64 h. The medium used in these experiments was composed of L3 macro- and micronutrients, L2 vitamins, amino acids and maltose [3]. The medium was supplemented with 2 mg/1 of the auxin 2,4-D (2,4-dichlorophenoxy acetic acid). Total RNA was extracted as described [5] and poly(A) + RNA isolated using mAP paper (Amersham International). 2 #g poly(A) + RNA was used for cDNA synthesis. Double-stranded cDNA was prepared, quantified and cloned in Uni-ZAPII vector arms using Z A P - c D N A Synthesis Kit (Stratagene) according to the manufacturer's instructions. Total c D N A was run in 2% NuSieve G T G Agarose and cDNA smaller than 350 bp and excess of adaptors were removed. Recombinant phage were in vitro packaged using Gigapack II Gold packaging extract (Stratagene). The titer of the primary library was approximately 2.5 × 10 6 plaque-forming units. Differential screenings were performed using single-stranded cDNA probes after first-strand synthesis from poly(A) + R N A isolated either from Tritordeum leaf segments immediately after cutting or after incubation for 64 h on auxincontaining medium in the dark (see also Fig. 5). About 3500 plaques were plated onto Petri dishes. Duplicate nitrocellulose replicas were lifted from each plate. Hybridization conditions were as described for genomic Southern blots (see below). cDNAs of plaques hybridizing preferentially with cDNA derived from Tritordeum explants after the 64 h auxin treatment in the dark were PCR (polymerase chain reaction) amplified using primers ER I and X I (see [7]) and the PCR products re-screened during two additional screening rounds.
DNA sequence analys~ In vivo excisions, yielding pBluescript plasmids containing the c D N A clones 47/11 and 50A, were performed according to the manufacturer's specifications using the ExAssist helper phage (Stratagene). D N A sequences were determined using
the Taq Dye Primer Cycle Sequencing Kit and the 373A automated D N A sequencer (both Applied Biosystems). D N A and amino acid sequence data were further processed using the PC D N A S I S (Hitachi Software Engineering) software package. D N A and protein sequence data were compiled and compared with EMBL, GenBank, Swiss-Prot and PIR databases using the FASTA and BLAST algorithms [20]. Protein alignments were carried out with the C L U S T A L program [9]. Genomic Southern analysis
Genomic D N A was extracted by the procedure of [6] and digested with EcoRI, H&dIII and MspI. D N A fragments were separated in a 1.2~o (HindIII/EcoRI) or 1.5~o (MspI) agarose gel, transferred onto Hybond N + membranes (Amersham International) by capillary transfer overnight with 0.4 M NaOH. The blots were prehybridized in 7~o SDS, 0.5 M sodium phosphate pH 7.2, 1 mM disodium EDTA and 100 #g/ml salmon sperm DNA at 65 °C. After 2 h the radiolabelled probe (1 x 10 6 counts per minute per ml) (Prime It Kit, Stratagene) was added and further hybridized overnight. The filters were washed in decreasing concentrations of SSC (pH 7.0) [25] with a final washing step in 0.1 x SSC (1 x SSC is 8.8 g sodium chloride, 4.4g sodium citrate, pH 7.0)/0.1~o SDS for 15 rain at 68 °C and exposed to Kodak X-Omat AR films at -70 °C.
PCR amplifications
For primer localizations, see Fig. 1. Primers Sam 1 ( 5 ' - C C A C C G C A G C C A G A T A A G A A C 3') and Sam2 ( 5 ' - C T G G T C G A T T A T C G T G A A G C A G - 3 ' ) were used for amplification of the 5'-untranslated region of the longer S A M D C clone 47/11. Primers Sam l and Sam3 (5'CTCAAACCCGATCGCTGAGAC-3'), both flanking the first two MspI restriction sites, were used for intron amplification. Primers Sam I and Sam 4 ( 5 ' - G T T G T G C C A A C T C T T C T C C A A -
1024 AC-3') were used to amplify the whole genomic sequence. The PCR (polymerase chain reaction) was performed in a final volume of 50/~1 with 200 ng genomic D N A or 1 ng plasmid as template. Final concentrations of 200 #M of each dNTP, 500 nM of the primers each, 1.5 m M magnesium sulfate, 20 mM Tris-HC1 pH 8.4, 50 mM potassium chloride and 1.25 U Taq-DNA-polymerase (GIBCO-BRL) were used for amplifications. The reactions were heated for 5 min at 96 °C and the D N A polymerase added afterwards at 75 °C (hot start PCR). The 35 following PCR cycles were set as follows: 94 °C for 1 min, 62 °C for 1.5 min and 72 °C for 3 min, with a final extension of 5 min at 72 ° C.
4 °C. Then they were washed twice in distilled water and hydrolysed in 1 M HC1 for 4 min at 60 ° C. After two further washing steps they were stained in SchilTs reagent for ca. 25 min. Small pieces of at least three 1-mm segments were squashed in a droplet of 45~o acetic acid and visualized under the microscope. The number of cells in division was counted in a population of 2000 cells per sample. A score system was developed in which - means no cells, + between 0 and 1 ~ , + + between 1 and 5 and + + + more than 5 ~ of cells were visualized dividing. The division rates of the rest of the samples were already known from previous studies (unpublished data).
Northern experiments Results
Total RNA was isolated from the different plant materials using TRIzol reagent (Gibco-BRL, Eggenstein, Germany) according to the manufacturer's instructions. The RNA (10 #g each lane) was fractionated on 1-1.5~o (w/v) agarose gels containing formaldehyde and transferred overnight by capillary blotting onto positively charged nylon membranes with 10 x SSC, pH 7.0. RNA was fixed to the membranes (Zeta Probe; BioRad, M~inchen, Germany) by UV light. The membranes were prehybridized at 42 ° C for 5 min in a solution consisting of 50 ~o (v/v) deionized formamide, 0.25 M sodium phosphate, pH 7.2, 0.25 M NaCI, 1 mM disodium EDTA, and 7 ~o (w/v) SDS. Hybridization was carried out overnight at 42 ° C in the same solution after addition of radiolabelled probes.
Study of cell div&ion rate Leaf segments were fixed in absolute ethanol/ glacial acetic acid (v/v, 3:1)for at least 24h at
Isolation and sequence analysis of cDNA clones 47/11 and 50A from Tritordeum For construction of a c D N A library we chose leaf tissue from region 1 (basal part, see Materials and methods) of Tritordeum flag leaves which, after cutting, had been in vitro exposed to auxin for 64 h. The auxin treatment induced the cells to divide and to proceed towards embryogenic and regenerative callus formation (unpublished resuits). About 3500 recombinant c D N A clones were analyzed by differential screening with radioactive labeled c D N A obtained from poly(A) ÷ RNA extracted from Tritordeum leaf tissue of region 1 either immediately after cutting or 64 h after cutring and exposure to auxin containing medium in the dark. This experimental system originally set up to screen for cDNAs involved in competence for in vitro regeneration resulted in the isolation of 4 cDNA clones, two of which, clones 47/11 and 50A, were selected for further analysis.
Fig. i. Nucleotide and deduced amino acid sequence of c D N A clone 47/11. Beginning and end of c D N A clone 50A are indicated by arrows and open circles. Putative polyadenylation signals are underlined. The ORF is followed by two stop codons. Five MspI restriction sites are indicated revealing 4 internal fragments (A-D) of the longer c D N A clone 47/11 (see also Fig. 4A). Localisation of the primers Sam 1-4 used for polymerase chain reaction is shown by extended arrows.
1025 5'
C C CACC C C CAC C G T C T C G C C° CCGC C A C C G C A G C C A G A T A A D ~ A C A A A G A A A A G A
Msp !
Sam 1
55
--
GAGAC~GAACTCGTCOGGAGA~CTCGA~TC~X~GAGAG~AT~AGATCGGAATCGGA
126
rC A A G C G T G C G C G C T C G ~ A A G G G G T T A C C A T A A C A A A T T T T C C C A G G ~ A T C C T T A G T G A A T G T T C T A A T G
197
GAGTCAAAGGG T G G C A A G A A G T C T A G C A G T A G T A G T T C C C T G A T G T A C G A A G C T C C C C T C G G C T A C A G C A T
268
TOAAGACGTTCGACCAGCTGOAaGCGCCAAGAAG~CTG
339
CTGCATACTCAAACTGCGCGAAGAAG CCAT
CCTGATATCGT~CTTCCCCTTCCCGTAGTTTAGGAT~ATGCAATTTTATTCTGACT
CTTT~C T
410
481
Sam 2 TCTGCTCTGCCTTCTGACTCA~CTGCAACA
Ms? I
ATG G C T GCC C C G G T C ~ T C A G C G A T C G G G T T T
542
Sam 3 M
"~"
G
Y
E
K
R
L
A
T
A
P
F
V
S
A
E
I
G
I
F
10
A
596 28
GAC CCT C A T G G T CGT GGC CTG CGT G C C CTC T C C A G G G C C CAG A T T G A C TCC G T T D P H ° R G L R A L S R A Q I D S V
650 46
C~ L
704 64
GAT CTT GCA CGG T~ D L A R C
A C C A T C G T G TCC G A G CTC TCC A A C A A G G A C T T C G A C T I V S E L S N K D F D
TC° TAT GTG C T A TCC GAG T C C A G C TTG TTT A T C T A C T C T CAG A ~ S Y V L S E S S L F I Y S Q K
ATT GTG ATC I V
758 82 812 i00
GCT G A A GAG CTG TGC A T G CCG CTT G C T GCC GTG A A G TAC TCC C G T G G G A T ° T T C A E E L C M P L A A V K Y S R G M F
866 118
~TC TTC C C C G G C G C A C A G CCT G C T CCC CAC AGG A G C TTC TCT G A G G A G G T T G A T I F P G A Q P A P H R 8 F S E E V D
920 136
GTC CTG A A C CGC TAC TTC G G C CAC CTG A A C TCT GGT G G C A A T GCC T A C G T G A T T V L N R Y F G H L N S G G N A Y V
974 154
GGC GAC C C A G C G A A G C C T GGC CAG A A G TGG CAC A T C TAC T A T G C C A C T G A G C A A G D P A K P G Q K W H I Y Y A T E Q
1028 172
CCT G A G C A G CCC A T G GTC ACC CTG GAG A T G TGC A T G A C T GGG T T G G A C A A G A C G P E Q P M V T L E M C M T G L D K T
1082 190
AAG GCC T C T G T C T T C T T C A A G A C T C A T G C T G A T GGC CAC G T T T C C T G T G C C A A G K A S V F F K T H A D G H V S C A K
1136 208
GAG E
A T ° A C A A A G C T C T C T GGT A T C TCC G A C A T C A T C C C T G A G A T G G A G G T C TGC M T K L S G I S D I I P E M E V C
1190 226
GAC TTC G A C T T C G A G CCC TGC G G C TAC TCC A T ° A A C G C C A T C A A C G G A T C T GCC D F D F E P C G Y S M N A I N G S A
1244 244
TTC TCC A C C A T C CAT GTG A C C CCC GAG G A C G G C TTC A G C T A T G C G A G C T A T G A G F S T I H V T P E D G F S Y A S Y E
1298 262
GTC CA° G G C A T G G A C G C T TCC GCC CTG GCC TAT GGC G A C A T C G T C AAG A G G G T T V Q G M D A S A L A Y G D I V K R V
1392 250
CTC CGA T G C TTT GGC C C T T C A GAG TTC T C T GTG GCG GTC A C C A T C T T C G G T G G C L R C F G P 8 E F S V A V T I F G G
1406 298
CGT G G C CAT GCC G C C A C C TGG G G C A A G A A G CTC GAC G C C G A G G C A T A C G A C T G C R G H A A T W G K K L D A E A ¥ D C
1460 316
A A C A A C G T T GTG G A G CAG G A G CTG C C A T G C G G T G G C G T C CTC A T C TAC CAG A G C N N V V E Q E L P C G G V L I Y Q S
1514 334
T T T G C C G C G AAC G A A G A G C T T G C T GTC T C T G C C G G G T C G CCC A G G T C T G T C TTC F A A N E E L A V S A G S P R S V F
1568 352
CAC TGC TTC G A G A A T G T G G A G A G C G G C CAC C C T CTG G T C A A G G A A O G C A A G CTT H C F E N V E S G H P L V K E G K L
1622 370
°CC AAC C T G CTC G C A T ~ A N L L A W
1676 385
~p!
mpm
CGG GCG GAG G A G G A G TCT CTC G A G G A G GGC A C A G G C R A E E E S L E E G T G
GCG T T G CTG TGC G A G T G A T A A G A T G A T T ~ C T G T C G C T G T T C C G T C T G T G T A A T T C G T T C T G A C A L L C E * *
1740 393
Ms ! TGTTGTCGTGCGTCGTTTGGTTACTGTGAAGCAG~CC~CTATTGCTACTCTCa&ATAJL%CTATTAGC
1811
TCTAGGTGGTT~TG
1882
CGTCTG C C A C A A T G A G C A T A C T ~ A T A 3 U % T A A
Sam 4 GCAAA'~T~-~'~
3'
1893
1026 The nucleotide and deduced amino acid sequences of cDNA clones 47/11 and 50A are shown in Fig. 1. The size of the longer clone (47/ 11) is 1893 bp. The potential ORF extends from position 513 with ATG for methionine to two stop codons (TGA and TAA) ending at position
1697. A stop codon at position 399, 114 bp before the first ATG codon and in frame with the full ORF, suggests that the ATG codon at position 513 is the start codon. This ORF codes for a protein of 393 amino acids having a calculated molecular weight of 42.9 kDa. There are two pu-
Fig. 2. Comparison of the deduced amino acid sequence of 47/11 with SAMDC sequences of potato [33], yeast [ 14] and human [ 19]. The alignment was generated using the CLUSTAL program [9]. Alignment parameters were: gap penalty 5, fixed gap penalty 10, floating gap penalty 10, K-tuple 2. Identical amino acids in at least 3 different sequences are outlined in grey. Amino acids being identical in all 4 sequences listed and in SAMDC sequences of spinach [4], mouse [30, 35], rat [22], golden hamster [34] and cow [ 10] are marked by asterisks. The triangle indicates a putative cleavage site of the SAMDC pro-enzyme and the black bar the putative PEST sequence.
1027 tative polyadenylation signals (underlined in Fig. 1) in the 3'-untranslated region (AATAAA and ATAAAT). Sequence analysis proved cDNA clone 50A to be identical to a portion of 47/11, only lacking the first 463 nucleotides of the 5'-untranslated region. The homologous region of 50A is indicated by arrows in Fig. 1. Both cDNA clones, 47/11 and 50A, therefore contain the same ORF of 393 amino acids as shown in Fig. 1. Although poly(A) + RNA was used to generate the cDNA library, both clones lack a poly(A) tail. However, both clones end at exact the same position with TTTTTT at their 3' end and, in addition, have putative polyadenylation signals in the common distance of the beginning of poly(A) tails [ 12]. The deduced amino acid sequence of the ORF described above was compared with known sequences using different databases (see Materials and methods). The sequence is homologous to SAMDC genes in plants, mammals and yeast with the highest homology to other plant sequences. There is 56~o identity between Tritordeum and potato sequences [33], with homology
extending through a 339 amino acid overlap. A similar degree of homology was found between Tritordeum and spinach sequences [4]. The two dicot SAMDCs share an amino acid identity of about 72~o. The similarity between the plant SAMDCs and those of yeast or mammals is much lower, at about 35~o (e.g. [14, 22, 30]). Figure 2 shows an alignment of the deduced amino acid sequence of the Tritordeum ORF with known SAMDC sequences of potato [33], yeast [ 14] and human [ 19] as an example for mammalian SAMDCs. All SAMDCs isolated from mammals are around 98 ~o identical to each other. The alignment was generated using the CLUSTAL program [9]. Despite the low degree of homology between plant, yeast and mammalian SAMDC sequences the alignment illustrates the presence of highly conserved regions. One of the highly conserved regions is the amino acid sequence between position 66 and 74 (SAMDC of Tritordeum). This region includes a putative pro-enzyme cleavage site marked by a triangle. Non-hydrolytic cleavage in this highly conserved region which resulted in the formation of a short
Fig. 3. Southern hybridization of genomic DNA of hexaploid Tritordeum (HT, 15 #g), its parental diploid Hordeum chilense (H1, H7, 5/~g each), tetraploid Triticum (T22, T24, T59, 10 #g each) lines and diploid Hordeum vulgare (5/~g) with 47/11 as probe. 5 #g genomic DNA per genome was digested each with EcoRI and HindlIl. On the left 5 pg 47/11 cDNA was applied to the gel, giving a strong signal at 1.9 kb. The Southern blot was exposed for 3 days at -70 °C.
1028 r-chain (N-terminal part) and a longer e-chain (c-terminal part) has been demonstrated before [14, 19]. Another highly conserved region is at residues 247-262. This sequence is characteristic of a PEST sequence, generally associated with rapid protein turnover [24].
Southern blot analysis: Southern blots of genomic DNA from parental lines of Tritordeum (i.e. Hordeum chilense H1, H. chilense H7, Triticum turgidum conv. durum T22, T. turgidum conv. durum T24, Triticum aestivum ssp. sphaerococcum T59), Tritordeum itself (HT) and Hordeum vulgare (H.v.) were hybridized with probe 47/11. The genomic DNAs (5 #g per genome) were digested with EcoRI, HindlII (Fig. 3) and MspI (Fig. 4). As shown in Fig. 3, the Hordeum lines and Tritordeum give strong hybridization signals, whereas all Triticum lines show much fainter signals. This could indicate that 47/11 originates from the H. chilense genome where it is coded by a single-copy gene. Following digestion of DNA derived from the Tritordeum and Triticum lines with EcoRI, two hybridizing bands can be seen. In addition to a strongly hybridizing band of 2.4 kb another 8.0 kb band of lower intensity can be detected. A polymorphism is obtained after digestion of the genomic DNAs with MspI (Fig. 4A). An interesting fact is that the MspI restriction fragment A of47/11 at 0.43 kb which corresponds to fragment A in the 5'-untranslated region as shown in Fig. 1 is missing in the H. chilense lines (Fig. 4A). This could either indicate methylation at this restriction site, or the presence of an intron in this region. PCR analysis using genomic DNA as template with primers Sam 1 and Sam 3 (see Fig. 1) flanking this MspI restriction fragment yielded a PCR product of about 1.18 kb (data not shown) which correlates with the band A ÷ (about 1.1 kbp) in Fig. 4A. The difference in size between A (0.43 kb) and A + (or the PCR amplification product) strongly hints at the presence of an intron in the 5'-untranslated region. In order to further investigate the gene origin of
Fig. 4. A. Southern blot analysis as described in Fig. 3, but genomic DNA was digested with MspI. On the left, MspI restriction fragments of 47/11 are ranging from 0.67 to 0.09 kb. The four internal MspI restriction fragments (A-D, see also Fig. 1) of the cDNA clone 47/11 are indicated. The genomic fragment A containing at least one intron is marked A*. The Southern blot was exposed for 7 days at -70 °C, B. PCR amplification products using the primers Sam I and Sam 4 (see also Fig. 1), both derived from the 5'- and the 3'-untranslated regions of the cDNA clone 47/11, respectively, cDNA of the clone 47/11 and genomic DNAs of the lines described above were used as template for PCR reactions. 2 #1 of the cDNA amplification and 10 #1 of the genomic amplifications were separated in a 1To agarose gel.
47/11 either 47/11 cDNA or genomic DNAs of the above described lines were used for PCR amplifications with primers Sam 1 (specific to the 5'-untranslated region of47/11) and Sam 4 (specific to the 3'-untranslated region of 47/11) (see also Fig. 1). The results are shown in Fig. 4B. 47/11 cDNA gave a band at 1.85 kb. Only when
1029 using D N A from H. chilense (H 1 and H7) or Tritordeum (HT) an amplification product at about 2.7 kbp could be detected. The Triticum and the H. vulgare gave no signals. The results clearly show that the 47/11 gene originates from H. chilense.
Analysis of 47/11 expression The northern analysis as shown in Fig. 5 demonstrates that the level of 47/11 m R N A increases 12 h after cutting and subsequent dark treatment on auxin-containing medium. The size of the transcript is around 2 kb. Poly(A) + mRNAs extracted from samples 1 and 9 were used for differential screenings. Leaf segments from region 1 (R1, see Materials and methods) of plants subjected for 22 h to darkness before cutting (sample 12), do not show a higher level of 47/11 mRNA. This indicates that the dark treatment does not
explain 47/11 m R N A accumulation. Neither seems the auxin treatment to be responsible for the induction of 47/11 mRNA. This is demonstrated by sample 13 were high amounts of 47/11 transcript also accumulate in cut leaf segments after 64 h which had not been exposed to auxin. To investigate the role of cell division in SAMDC transcript accumulation, leaf segments of mature leaves (ML) showing no cell division and root tips (RT) with high rates of cell division were compared in the Northern analysis (samples 14 and 15). The amount of 47/11 m R N A was very low in both samples (slightly more in root tips). Furthermore, apical leaf segments (R 2, see Materials and methods) which have lost the ability for cell division were either extracted immediately after cutting (sample 16) or 64 or 96 h after cutting and exposure to auxin containing medium (samples 17 and 18). 47/11 transcript accumulates only in samples 17 and 18. Taken together, these data indicate that neither active cell divi-
Fig. 5. Northern analysis of 47/11 transcript levels in different Tritordeum tissues. The northern blot was hybridized with the 5'-untranslated fragment (see region between primers S a m 1 and S a m 2 in Fig. 1) of the longer cDNA clone 47/11. RNA was
isolated from leaf segments from region 1 (R1) of freshly isolated flag leaves (sample 1) and from leaf segments from region 1 previously exposed to cutting and subsequent auxin treatment in vitro in the dark (D) for various hours (samples 2-11). In addition, RNA was isolated from not induced leaf segments of region 1 subjected for 22 h to darkness before cutting (D*, sample 12) and from leaf segments from region 1 exposed to cutting and to in vitro treatment for 64 h without auxin exposure (sample 13). RNA was extracted from mature leaf tissue (ML, sample 14) and root tips (RT, sample 15). Samples from leaf segments of region 2 (R2, see Materials and methods) from freshly isolated flag leaves and from leaf segments induced in vitro under auxin treatment for 64 h and 96 h were also analysed. Ethidium bromide-stained 25S and 18S rRNA bands are shown as controls for loaded RNA amounts. The northern was exposed at -70 ° C for 4 h. Poly(A) + RNA of sample 9 was used to generate the cDNA library and cDNAs derived from poly(A) + RNA of samples 1 and 9 were used for the differential screenings. The size of the transcript is around 2 kb.
1030
Fig. 6. Circadianoscillationsof 47/11 transcript levelsin primaryfoliageleaves of Hordeum vulgare.A. RNA was extractedfrom plant materialgrownunder a 16 h light (whitebar, 0-16 h) and 8 h dark (black bar, 16-24 h) cycleat differenthours of the cycle at days 7, 8, 9 and 10 after sowing. Additionally,RNA was extractedfrom plants kept either in continuous light (B) or in continuous darkness (C) starting day 10 after sowing. Same amounts of RNA were loaded to each lane as controlledby ethidium bromidestain ofrRNA bands (data not shown).Electrophoreticseparationof RNA samplesand Northernanalysiswereperformed as describedin Materials and methods. Hybridizationswere carried out with radiolabelledprobe prepared from 47/11 cDNA by random primedlabelling. sion, nor dark treatment or exposure to auxin are responsible for the increase in 47/11 transcript level. This m R N A seems to accumulate in wounded tissue. Transcript levels of 47/11 or a homologous sequence of H. vulgare were further studied during different light and dark periods. Fig. 6 demonstrates that the level of47/11 m R N A is subject to a circadian rhythm. Northern analysis of 47/11 m R N A is primary foliage leaves of H. vulgare shows that the level of the transcript increases at the end of the 8 h dark period (Fig. 6A). Maximum levels can be observed during the first 8 h in the light. The 47/11 transcript level then decreases at the end of the light period and lowest m R N A levels can be found in the middle of the dark period (20 h). This periodicity continues in constant light (Fig. 6B). Omission of the dark period at days 10, 11 and 12 after sowing does not significantly change the circadian pattern of 47/11 expression. Maximum levels of47/11 m R N A can be observed in the middle of the former light period and minimal levels in the former dark period. Yet, transfer into continuous darkness at day 10 after sowing changes the periodic pattern of 47/11 expression (Fig. 6C). Under these conditions there seems to be an oscillation with a long period (about 30 h).
Discussion
By differential screening of a Tritordeum explant c D N A library we were able to isolate the two c D N A clones 47/11 and 50A which both contain the same ORF. On the basis of their sequence homology with two known full-length plant SAMDC sequences of potato [15, 33] and spinach [4] we conclude that this ORF represents an SAMDC m R N A of Tritordeum. This conclusion is further supported by the presence of several highly conserved regions documented in all known SAMDC sequences of plants, mammals and yeast [4, 14, 15, 19, 22, 35] (Fig. 2). One of these regions (amino acids 66-74 of Tritordeum in Fig. 2) represents a putative cleavage site. The cleavage of SAMDC pro-enzymes in this region which yielded two products of 31 and 7 kDa was demonstrated before [ 19, 14, 32]. A second highly conserved region (amino acids 247-262) is a putative PEST sequence [ 16, 24], which is possibly responsible for the rapid degradation of the protein. SAMDC has a relative short half-life of about 1-2 h [ 31 ]. Genomic Southern analyses with EcoRI- and HindlII-digested DNA from Tritordeum, its parental lines and H. vulgare (Fig. 3) shows that 47/11 or homologous sequences are present in
1031 Tritordeum, its parental lines and related species. Digestion of Tritordeum, Hordeum and Triticum lines with MspI, a restriction enzyme which is sensitive to methylation, results in a complex hybridization pattern (Fig. 4A) showing that the S A M D C transcript is derived from the wild barley (H. chilense) genome. This was further proved by P C R analysis using genomic D N A of the different lines as template and primers specific to the 47/11 sequence (Fig. 4B). An interesting point is the absense of a band at 0.43 kb in the H. chilense lines indicating methylation at this restriction site or the presence of an intron in this region. PCR amplification of genomic DNA using primers flanking this MspI fragment strongly supports the idea that an intron is located within the 5'-untranslated region of 47/11. A very long 5'-untranslated sequence of over 500 nucleotides has also been reported for potato [15] and for the highly conserved mammalian S A M D C genes, which also contain an upstream O R F [ 19]. Furthermore, it was shown that the 5' transcript leader sequence of human S A M D C gene plays a regulatory role in cellspecific translation of S A M D C m R N A [ 10, 27]. Similar results have been reported for Catharanthus roseus S A M D C [26]. The authors describe a long 5' leader of 469 bp. This leader contains start and stop codons for a polypeptide suggesting also translational regulation. The observed intron within the 5'-untranslated region may play a role in regulation of S A M D C expression. Based on numerous investigations, polyamines have been assumed to play an important role in processes involved in division and differentiation of cells [8 ]. Consistent with this postulation it has been reported [ 15] that in potato high levels of S A M D C transcripts can be found in actively dividing and differentiating tissue, but much lower levels in mature tissues. However, in spite of the use of Tritordeum explants in the phase of active cell division when embryogenic calli were induced for c D N A library construction, our results do not show a clearcut correlation between S A M D C m R N A levels and cell division activity. The formation of embryogenic calli from the Tritordeum explants was induced by exposure of cut leaf seg-
ments of Tritordeum to auxin containing medium in the dark. Our results (Fig. 5) indicate that neither the process of induction of embryogenic calli which is accompanied by active cell division, nor the exposure to auxin medium nor the dark treatment are sufficient to cause S A M D C m R N A accumulation. The observed increase in S A M D C m R N A level rather seems to be induced by cutting of leaf segments which has to be investigated in more detail in future experiments. Another interesting feature of S A M D C gene expression is that at least in H. vulgare the changes in S A M D C m R N A levels follow a circadian rhythm which is maintained in continuous light (Fig. 6). Circadian regulation of expression has been shown for various plant genes [13, 21]. The periodicity of S A M D C expression is retained in continuous light but seems to change in continuous darkness showing now a long period of about 30 h (Fig. 6C). Similar results have been reported for Lhc gene expression [18]. Both responses, induction by cutting and circadian rhythm, are well documented to play a role in ethylene biosynthesis. A multifold increase in ethylene production within hours after wounding was reported for various plants (e.g. [ 1, 11]). Ethylene production also follows a circadian rhythm with a peak during the middle of the light period and rates of ethylene production are in trace amounts during the night [1, 23]. The periodicity continues in constant light, but stops in darkness [23]. The two biosynthetic pathways, ethylene formation and synthesis of polyamines, share the common precursor SAM (S-adenosylmethionine) and have been shown to be interdependent [8]. On one hand it has been demonstrated that ethylene and polyamines negatively regulate the synthesis of each other and compete for their common precursor SAM [8]. But, on the other hand, counterexamples exist in which ethylene and poly-amines are not mutually antagonistic [8]. In these experimental systems an increase in polyamine levels coincides with an increase in ethylene production. These data indicate a complex coordinated regulation of both pathways with two possibilities: a parallel and a countercurrent mode of regulation of ethylene and polyamine
1032 biosynthesis. Our data indicate that regulation of SAMDC transcript level might play a role in the interaction between these two pathways. Future experiments in which changes in levels of SAMDC mRNA under various conditions are compared with changes in ethylene and polyamine formation are needed to clarify the role of SAMDC expression in the regulation of polyamine and ethylene metabolism.
Acknowledgements We gratefully acknowledge stimulating discussions by K. Krupinska and R. Brettschneider. K. Chalmers is thanked for critical reading of the manuscript. A. Br/tutigam and C. Adami are thanked for drawing the figures and for photography. This research was supported by the Bundesminister for Forschung und Technologic (BEO 031.6601) and the Deutsche Forschungsgemeinschaft (DFG Kr1256/1-3).
References 1. Abeles FB, Morgan PW, Saltveit ME Jr: Ethylene in Plant Biology. Academic Press, San Diego (1992). 2. Barcelo P, Lazzeri PA, Martin A, Lrrz H: Competence of cereal leaf cells. I Patterns of proliferation and regeneration capability in vitro of the inflorescence sheath leaves of barley, wheat and Tritordeum. Plant Sci 77:243-251 (1991). 3. Barcelo P, Lazzeri PA, Martin A, Lrrz H: Competence of cereal leaf cells. II Influence of auxin, ammonium and explant age on regeneration. J Plant Physiol 139:448-454 (1992). 4. Bolle C, Herrmann RG, Oelm~lllerR: A spinach cDNA with homology to S-adenosylmethionine decarboxylase. Plant Physiol 107:1461-1462 (1995). 5. Chirgwin JM, Pryzybyla AE, MacDonald ILl, Rutter WJ: Isolation of biologically active ribonucleic acid from sources enriched in ribonuclease. Biochemistry 18: 52945299 (1979). 6. Dellaporta SL, Wood J, Hicks JB: A plant DNA minipreparation: version II. Plant Mol Biol Rep 1:19-21 (1983). 7. Dresselhaus T, Lrrz H, Kranz E: Representative cDNA libraries from few plant cells. Plant J 5:605-610 (1994). 8. Evans PT, Malmberg RL: Do polyamines have roles in plant development? Annu Rev Plant Physio140:235-269 (1989).
9. Higgins DG, Sharp PM: CLUSTAL: a package for performing multiple sequence alignments on a microcomputer. Gene 73:237-244 (1988). 10. Hill JR, Morris DR: Cell-specific translation of Sadenosylmethionine decarboxylase mRNA. Regulation by the 5' transcript leader. J Biol Chem 267: 2188621893 (1992). 11. Hyodo H, Tanaka K, Watanabe K: Wound-induced ethylene production and 1-aminocyclopropane-l-carboxylic acid synthase in mesocarp tissue of winter squash fruit. Plant Cell Physiol 24:963-969 (1983). 12. Joshi CP: Putative polyadenylation signals in nuclear genes of higher plants: a compilation and analysis. Nucl Acids Res 15:9627-9640 (1987). 13. Kay SA, Millar AJ: Circadian regulated cab gene expression in higher plants. In: Young M (ed) The Molecular Biology of circadian Rhythms, pp. 73-89. Marcel Dekker, New York (1992). 14. Kashiwagi K, Taneja SK, Liu TY, Tabor CW, Tabor H: Spermidine biosynthesis in Saccharornyces cerevisiae. Biosynthesis and processing of a proenzyme form of S-adenosylmethionine decarboxylase. J Biol Chem 265: 22321-22328 (1990). 15. MadArif SA, Taylor MA, George LA, Butler AR, Burch LR, Davies HV, Starck MJR, Kumar A: Characterization of the S-adenosylmethionine decarboxylase (SAMDC) gene of potato. Plant Mol Biol 26:327-338 (1994). 16. Maric SC, Crozat A, Janne OA: Structure and organization of the human S-adenosylmethionine decarboxylase gene. J Biol Chem 267:18915-18923 (1992). 17. Martin A, Sanchez-Monge E: Cytology and morphology of the amphiploid Hordeum chilense × Triticum turgidum conv. durum. Euphytica 31:261-267 (1982). 18. Millar AJ, Straume M, Chory J, Chua N-H, Kay S: The regulation of circadian period by phototransduction pathways in Arabidopsis. Science 267: 1163-1166 (1995). 19. Pajunen A, Crozat A, Janne OA, Ihalainen R, Laitinen PH, Stanley B, Madhubala R, Pegg AE: Structure and regulation of mammalian S-adenosylmethionine decarboxylase. J Biol Chem 263:17040-17049 (1988). 20. Pearson WR, Lipman DJ: Improved tools for biological sequence comparison. Proc Natl Acad Sci USA 85: 2444-2448 (1988). 21. Piechulla B: 'Circadian clock' directs the expression of plant genes. Plant Mol Biol 22:533-542 (1993). 22. PulkkaA, Keraenen MR, SalmelaA, Salmikangas P, Ihalainen R, Pajunen A: Nucleotide sequence of rat S-adenosylmethionine decarboxylase cDNA. Comparison with an intronless rat pseudo gene. Gene 86:193-199 (1990). 23. Rikin A, Chalutz E, Anderson JD: Rhythmicity in ethylene production in cotton seedlings. Plant Physiol 75: 493-495 (1984). 24. Rogers S, Wells R, Rechsteiner M: Amino acid sequences
1033
25.
26.
27.
28.
29. 30.
common to rapidly degraded proteins: the PEST hypothesis. Science 234:364-368 (1986). Sambrook J, Fritsch EF, Maniatis T: Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1989). Schrt~der G, SchrOder J: cDNAs for S-adenosylmethioninedecarboxylase from Catharanthus roseus, heterologous expression, identification of the proenzyme-processing site, evidence for the presence of both subunits in the active enzyme, and a conserved region in the 5' leader. Eur J Biochem 228:74-78 (1995). Shantz LM, Viswanath R, Pegg AE: Role of the 5'-untranslated region of mRNA in the synthesis of S-adenosylmethionine decarboxylase and its regulation by spermine. Biochem J 302:765-772 (1994). Slocum RD: Polyamine biosynthesis in plants. In: Slocum RD, Flores HE (eds) The Biochemistry and Physiology of Polyamines in Plants, pp. 23-40. CRC Press, Boca Raton, FL (1991). Smith TA: Polyamines. Annu Rev Plant Physiol 36:117143 (1985). Suzuki T, Sadakata Y, Kashiwagi K, Hoshino K, Kakinuma Y, Shirohata A, Igarashi K: Overproduction of
31. 32.
33.
34.
35.
S-adenosylmethionine decarboxylase in ethylgloxal-bis (guanylhydrazone)-resistant mouse FM3A cells. Eur J Biochem 215:247-253 (1993). Tabor CW, Tabor H: Polyamines. Annu Rev Biochem 53:749-790 (1984). Tabor CW, Tabor H, Xie QW: Spermidine synthase of Escherichia coli: localization of the speE gene. Proc Natl Acad Sci USA 83:6040-6044 (1986). Taylor MA, MadArif SA, Kumar A, Davies HV, Scobie LA, Pearce SR, FlaveUe AJ: Expression and sequence analysis of cDNAs induced during the early stages of tuberisation in different organs of the potato plant (Solanum tuberosum L.). Plant Mol Biol 20: 641-65l (1992). Tekwani BL, Stanley BA, Pegg AE: Nucleotide sequence of hamster S-adenosylmethionine decarboxylase cDNA. Biochim Biophys Acta 1130:221-223 (1992). Waris T, lhalainen R, Keranen MR, Pajunen A: Molecular cloning of the mouse S-adenosylmethionine decarboxylase cDNA: specific protein bindingto the conserved region of the mRNA 5'-untranslated region. Biochem Biophys Res Commun 189:424-429 (1992).
