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KURODA, H.

SAKAI, F.

SHIMOMURA, F.

Kyoto University Research Information Repository

Title<Preliminary>0-methyltransferase (OMT) cDNA Clones fromPinus densiflora SeedlingsAuthor(s)SHIMOMURA, F.; SAKAI, F.; KURODA, H.CitationWood research : bulletin of the Wood Research Institute KyotoUniversity (2002), 89: 11-12Issue Date2002-09-30URL http://hdl.handle.net/2433/53125RightType Departmental Bulletin PaperTextversionpublisherKyoto UniversityPreliminaryO-methyltransferase (OMT) eDNA Clonesfrom Pinus densiflora Seedlings*lF. SHIMOMURA*2,*3, F. SAKAI*2 and H. KURODA*2(Received May 31, 2002)Keywords: O-methyltransferases, GMT, cDNA clones, PI-GMT II, Pinus densifloraIntroductionS-adenosyl-L-methionine (SAM) dependent O-methyl-transferase (SAM-OMTs : EC 2.1.1.6x) is ubiquitous, anddistributed in bacteria, fungi, plants and animals.Because the methylation is a critical step in secondarymetabolism of plant kingdom, the numbers of theregistered plant (PI-OMT) genes are rapidly growing.PI-OMTs consist of PI-OMT I and PI-OMT II. Theformer catalyzes only caffeoyl-CoA as a substrate withdivalent cation, i.e. Mg2+, while the latter catalyzesvarious substrates without divalent cation.Pinus taeda OMTs I and II have been discussed inrelation to lignin biosynthesisl,2). However, there are stillambiguous aspects in the conifer OMTs, especially in thelatter OMT type. The specific activity for caffeate in theOMT II was remarkably smaller than the case ofangiosperm3) although they did not show the kineticconstants. They had used a low concentration of thesubstrates for the assay that may not saturate the OMTIII). Using the assay system, they had reported that theformation ratio of syringyl to guaiacyl nuclei was ca. one inboth crude xylem and recombinant extracts. Theydesignated the OMT as AEOMT because it catalyzedmethylation of both corresponding acids and CoA esters.Polymorphism is one of the aspects to be solved beforeunderstanding their respective function. Distinct OMTII clones from AEOMT have also been suggested in theconifer as following reasons. First, the xylem extractremained OMT activity when an antibody for AEOMThad added I). This is quite reasonable because the conifercarries high gene duplication. Secondary, crude pineextract had resolved into two OMT fractions afterelectrophoresis4). Third, another formation ratio ofsyringyl to guaiacyl nuclei by the enzyme had beenreported as ca. 0.1 in Japanese black pine seedlings4).Finally, pinosylvin methylating OMT with broadsubstrate specificity has also reported from Pinussylvestris5,6). Here, we confirmed pine OMT poly-morphism by isolating five OMT clones with fullrespective coding sequences from Japanese red pine (Pinusdensiflora) elicited with sodium salicylate.*1 A part of this work was presented at 52th Annuual Meetingof Japan Wood Research Society in Gifu, April, 2002.*2 Laboratory of Gene Expression.*3 Present Address: Banyu Pharmaceutical Co., Ltd.- 11ExperimentalTotal RNA from the roots of Japanese red pineseedlings, cut offat 16 hours after elicitation with 10 mM ofsodium salicylate, was extracted by using modifiedguanidium extraction method. Then cDNA wassynthesized by using SuperScript™ Plasmid System (LifeTechnologies). The cDNA was fractionated on 1%agarose gel to enrich the portion larger than 1.0 Kb.They were inserted into plasmid vector pSPORTI (LifeTechnologies). The recombinant plasmid DNA wastransformed into Escherichia coli DH lOB Cells (LifeTechnologies) by using Cell-Porater™ ElectroporationSystem (Life Technologies). The library contains thecDNA larger than 1.0 Kb was used for isolation of full-length OMT cDNA (ca. 1.2 Kb) clone.The 629 bp probe for OMT cDNA was prepared from acDNA library of P. densiflora by polymerase chain reaction(PCR). The 21 mer primer pairs were designed from thecorresponding nucleotides sequence of Pinus radiata(Accession No. U70873 in GenBank). This probecontains a conserved SAM binding domain in plant OMT.The probe was fractionated on 1% agarose gelelectrophoresis. After purification, the probe was labeledby ECL™ direct nucleic acid labeling and detectionsystems (Amersham Pharmacia). Out of the cDNAlibrary (5 X 105 clones), 2 X 104 clones were screened byusing the probe describes above. The nucleotides of theOMT clones were sequenced for both strands by thedideoxy chain termination method, by using Big Dye™terminator Cycle Sequencing Ready Reaction Kit (PEApplied Biosystems), Big Dye™ terminator v3.0 ReadyReaction Cycle Sequencing Kit (PE Applied Biosystems),and an ABI PRISM™ 377 DNA sequencer (PE AppliedBiosystems) .For recombinant OMT expression, the wheat germ cell-free protein synthesis core kit Proteios (Toyobo) was usedafter mRNA was synthesized in vitro by Thermo T7 RNApolymerase (Toyobo). After the translated reactionmixture was applied to a spin column, OMT activitieswere monitored with 14C _SAM and phenolic substrates(caffeic acid, 5-hydroxy ferulic acid, pinosylvin,pinocembrin) as described before with slight modifiedreaction mixture4).Results and DiscussionEleven O-methyltransferase cDNA clones (PDOMT)with full-coding lengths were fully sequenced in bothWOOD RESEARCH No. 89 (2002)directions, which had been cloned from an elicited P.densiflora seedlings root cDNA library. All of the PDOMTbelonged to OMT II and, had conserved sequence motifsthat could possibly act as SAM-binding domains. Fivedistinct PDOMTs were recognizes by the sequenceanalyses. Seven PDOMT cDNA clones showed identicalsequences while the other 4 clones are distinct ones.Phylogenetic analyses showed that the 11 clones fall intotwo distinct groups. They are distinct group from P. taedaAEOMT, which was fallen into the same group of P.sylvestiris PSOMT by the phylogenetic classification.Here, we concluded that pine OMT shows polymorphism.The characterization of each PDOMT enzyme was nowgoing on. Preliminary, PDOMT cDNAs weretranscribed in vitro and corresponding PDOMT wereexpressed in the wheat germ cell-free protein synthesissystem. •The translation' products showed methylationactivities for caffeic acids. Because of those isolated OMTcDNA clones and the successful OMT assay system inhand, further research will gain insight into the OMT IIfunction.AcknowledgementAuthors greatly thank to Dr. A. Kodan, RIKENHarima, who had helped a library construction andscreenings of the OMT clones.References1) L. LI,]. L. POPKO, X.-HAI ZHANG, K.OSAKABE, C.-JUI TSAI,C.P. JOSHI and V.L. CHIANG: Proc. Natl. Sci. USA, 94,5461-5466 (1997).2) L. LI, Y. OSAKABE, C.P. JOSHI and V.L. CHIANG: Plant MolBioI, 40, 555-565- (1999).3) L. LI, J.L. POPKO, T. UMEZAWA and V.L. CHIANG: j. BioI.Chern., 275, 6537-6545 (2000).4) H. KURODA, M. SHIMADA and T. HIGUCHI: Phytochem., 14,1759-1763 (1975).5) H. CHIRON,A. DROUET, A.C. CLAUDOT, C. ECKERSKORN, M.TROST, W. HELLER, D. ERNST and H. JR. SANDERMANN:Plant Mol BioI, 44, 733-745 (2000).6) H. CHIRON, A. DROUET,F. bEUTlER, H.D. PAYER, D. ERNSTand H. JR. SANDERMANN: Plant ·Physiol., 124, 865-872(2000).- 12 '-

<Preliminary>0-methyltransferase (OMT) cDNA Clones from Pinus densiflora Seedlings

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<Preliminary>0-methyltransferase (OMT) cDNA Clones from Pinus densiflora Seedlings

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