Proc. Nati. Acad. Sci. USA Vol. 89, pp. 4713-4717, May 1992 Plant Biology Induction by fungal elicitor of S-adenosyl-L- synthetase and S-adenosyl-L- mRNAs in cultured cells and leaves of Petroselinum crispur (activated methyl cycle/ELI cDNAs/pathogen defense/plant S-adenosylhomocysteinase cDNA) PETRA KAWALLECK, GUNNAR PLESCH, KLAUS HAHLBROCK, AND IMRE E. SOMSSICH* Max-Planck-Institut for Zfichtungsforschung, Abteilung Biochemie, D-5000 K6ln 30, Federal Republic of Germany Communicated by Eric E. Conn, January 30, 1992 (receivedfor review November 14, 1991)

ABSTRACT Treatment of cultured parsley (Petroselinum 48-kDa subunits (9-11), whereas the plant is either a crispum) cells with fungal elicitor rapidly activates ftransrip- dimer or a tetramer with subunits of 55 kDa (12, 13). By tion of many genes encoding specific steps in pathogen defense- contrast, little is known about the genes encoding these related pathways. We report evidence that three cDNAs cor- and about their modes of expression. To date, responding to such genes represent two key enzymes of the cDNA or genomic clones for SMS have been isolated from E. activated methyl cycle. Two cDNAs are derived from distinct coli, Saccharomyces cerevisiae, rat, and Arabidopsis members of the S-adenosyl-L-methionine synthetase gene fam- thaliana (6, 15, 17, 18), and only two cDNA clones encoding ily, based on extensive similarity of the deduced polypeptides a rat and a Dictyostelium discoideum SHH have been re- with authentic enzymes from Arabidopsis thaliana, rat, yeast, ported (19, 20). and Escherichia coi. The third cDNA exhibits large sim it Here we describe the identification of parsley cDNAs for with a functionally related gene, encoding S-adenosyl-L- SMS and SHHt and demonstrate that fungal elicitor induces homocysteine hydrolase, from rat and a slime mold. Marked the corresponding mRNAs in cultured cells as well as leaves differences in the mRNA levels occurred in different organs of of this plant. parsley plants. Elicitor treatment strongly induced both mRNAs in cultured cells as well as intact leaves and led to marked increases in S-adenosyl-L-homocysteine hydrolase en- MATERIALS AND METHODS zyme activity. These results suggest a close metabolic link Cell Culture/Plants/Elicitor Treatment. Cell suspension between pathogen defense and an increased turnover of acti- cultures of parsley (Petroselinum crispum L.) were grown in vated methyl groups. the dark for 6 days and were then treated with elicitor at 50 ,ug/ml derived from the fungus Phytophthora megasperma f. In all organisms investigated, S-adenosyl-L-methionine sp. glycinea (21, 22). (AdoMet) serves as the major methyl-group donor for nu- Nondetached leaves of greenhouse-grown parsley plants merous highly specific methyl- reactions. These were treated with elicitor solution (50 jug/ml) using a 1-ml reactions involve a large variety of acceptor molecules, such syringe for pressure infiltration via the stomata. as phenylpropanoid derivatives, cyclic fatty acids, proteins, RNA Isolation and Blot Hybridization. Total RNA from polysaccharides, and nucleic acids (1). In addition, AdoMet parsley plants and cell cultures was prepared according to has regulatory functions-e.g., in the allosteric stimulation of Lois et al. (23). Ten micrograms oftotal RNA was denatured synthase (2). In plants, AdoMet has been studied and separated on formaldehyde-agarose gels. Conditions for primarily in relation to the biosynthesis of various phenyl- denaturing, electrophoresis, and capillary blotting were as propanoid derivatives and as an intermediate in the biosyn- described (24). The RNA was transferred to Hybond-N nylon thesis of the phytohormone ethylene (2, 3). membrane (Amersham) and cross-linked by UV light. Pre- AdoMet is synthesized by transfer of the adenosyl moiety hybridization (30 min) and hybridization (16 hr) were carried from ATP to the sulfur atom of methionine. This reaction is out at 650C in 1 M NaCl/1% SDS/10% dextran sulfate catalyzed by AdoMet synthetase (SMS; ATP:L-methionine containing salmon sperm DNA at 100 ,ug/ml. Blots were S-adenosyltransferase, EC 2.5.1.6). Subsequent transfer of washed twice for 30 min with 2x standard saline citrate/0.5% the activated methyl group in transmethylation reactions SDS and once for 30 min with 0.5 x standard saline citrate/ causes the formation of S-adenosyl-L-homocysteine 0.5% SDS at 650C. The cDNA probes were 32P-labeled by (AdoHcy), which is hydrolyzed to and homocys- random priming (25). Total RNA from diverse parsley organs teine by AdoHcy hydrolase (SHH; S-adenosyl-L-homocys- was provided by B. Weisshaar (MPI, Koln, F.R.G.). teine hydrolase, EC 3.3.1.1.). Methionine is then regenerated Construction and Screening of cDNA Library. A random- by transfer of a methyl group from N5-methyltetrahydrofo- primed Agtll cDNA library was constructed with a mixture late to homocysteine. of poly(A)+ RNA prepared from cultured parsley cells that Because of their central role in cellular , the had been treated for 0.5, 1.5, and 3 hr with elicitor. Con- enzymatic reactions constituting this activated methyl cycle struction was done by using the Pharmacia cDNA synthesis have been studied extensively in bacteria, yeast, plants, and kit and following the instructions ofthe manufacturer. cDNA animals (1, 2). Various SMS complexes from Escherichia coli, yeast, and rat liver appear to be composed of several, Abbreviations: AdoMet, S-adenosyl-L-methionine; AdoHcy, S-ad- partly identical polypeptides with molecular masses between enosyl-L-homocysteine; SHH, S-adenosyl-L-homocysteine hydro- 43 and 48 kDa (4, 6, 8). SHH usually consists of several lase; SMS, S-adenosyl-L-methionine synthetase. identical subunits; the mammalian enzyme is a tetramer of *To whom reprint requests should be addressed at: Max-Planck- Institut fur Zdchtungsforschung, Carl-von-Linn6-Weg 10, D-5000 K6ln 30, Federal Republic of Germany. The publication costs of this article were defrayed in part by page charge tIhe nucleotide sequences ofthe proteins reported in this paper have payment. This article must therefore be hereby marked "advertisement" been deposited in the GenBank data base (accession nos. M62756, in accordance with 18 U.S.C. §1734 solely to indicate this fact. M62757, M62758, and M81885). 4713 Downloaded by guest on September 26, 2021 4714 Plant Biology: Kawalleck et al. Proc. Natl. Acad Sci. USA 89 (1992) ligated into EcoRI-cut and dephosphorylated Agt1l DNA was For product identification 10-gl reaction mixture aliquots packaged with commercially available extracts (Gigapack were applied to silica gel TLC plates (Kieselgel 60 F2S4, Gold, Stratagene). Merck) and developed with the solvent system 1-butanol/ The previously isolated ELI 14 cDNA clone (see ref. 26 acetic acid/water, 12:3:5 (vol/vol/vol). Radioactive-labeled and Fig. 2) was used as probe to screen for full-length SHH substrate and reaction product were identified by commercial cDNA representatives after amplification of the library. standards (Sigma, retention factor Rf: adenosine 2.4, Labeling of the probe and hybridization conditions were as AdoHcy 5.4) and visualized by autoradiography. Quantifi- described above for RNA. Positive clones were plaque cation was by scanning the plates with a TLC linear analyzer purified, subcloned into the vector pBluescript II KS (Strat- LB2832 (Berthold, F.R.G) with a minicomputer Apple II agene), and sequenced as described below. system. DNA Isolation and Blot Hybridization. Genomic parsley Nucleotide Sequence Analysis. The DNA of recombinant DNA was isolated from cultured cells according to Murray plasmid derivatives was sequenced using the dideoxynucle- and Thompson (27). Ten micrograms of DNA was digested otide chain-termination method (29, 30). Comparative se- with the appropriate endonucleases, separated on 0.8% aga- quences were obtained from published reports or from the rose gels, and transferred to Hybond-N nylon membrane. National Biomedical Research Foundation protein sequence Hybridization and washing were as described above for data base, release 3/90. Sequence compilation and analysis RNA. were done by using the Genetics Computer Group software Protein Extraction/Determination. Crude extracts from package, version 6.2 (31). parsley were prepared by grinding frozen cells or plant material to powder with liquid nitrogen and quartz [50%o of RESULTS cell fresh Two milliliters of 0.2 M 2-amino- weight (wt/wt)]. We have recently reported the isolation of 18 independent 2-(hydroxymethyl)-1,3-propanediol TrisHCl, pH 7.8, and parsley cDNA families (ELIs) representing putative defense- 0.15 g ofDowex AG 1 x 2 (equilibrated with the same buffer) related genes (26). All ELI genes responded with rapid were added per gram ofcells, and the solution was stirred for increases in transcriptional activity to treatment of cultured 20 min on ice. After centrifugation for 10 min at 30,000 x g parsley cells with fungal elicitor. In the following, we de- the supernatant was transferred to a fresh tube and recen- scribe the functional identification of three of these cDNAs, trifuged. One milliliter of crude protein extract was concen- previously designated as ELI 14, ELI 18, and ELI 19. trated by ultrafiltration with Centricon-10 (Amicon) and used ELI 18 and 19 eDNAs Represent Two SMS Genes. Sequence for the enzyme assay. analysis of the ELI 18 and 19 cDNAs, in combination with a Protein concentrations were determined with commercial computer-assisted search in the available data banks, re- dye-binding reagent (Bio-Rad) with bovine serum albumin vealed large similarity with DNA encoding SMSs from var- used as standard. ious organisms. Comparison of the sequence Enzyme Assay/TLC. SHH activity was measured in the deduced from the non-full-length ELI 18 (renamed SMS-1) direction of AdoHcy synthesis by using the method of cDNA indicated 91.5% identity with A. thaliana, 65% with Poulton and Butt (28). The standard incubation mixture of0.1 rat, 56% with S. cerevisiae, and 51% with E. coli SMSs (Fig. ml contained 50 mM Tris HCl, pH 8.5/2 mM adenosine/0.1 1). Ifconserved amino acid exchanges are taken into account, ,.Ci of[8-14C]adenosine (50 mCi/mmol, Amersham; 1 Ci = 37 the similarity increases to 96%, 81%, 73%, and 69%6, respec- GBq)/10 mM DL-homocysteine, pH 8/50-150 pug of crude tively. The similarity is not restricted to a specific region but protein extract. The reactions were stopped after 30-min extends over the entire protein. The SMS-1 cDNA is 885 base incubation at 30°C by adding 10 1,u of 50%o (wt/vol) trichlo- pairs (bp) long and contains one open reading frame encoding roacetic acid and left for 10 min on ice. The protein precip- 234 amino acids. itate was removed by centrifugation, and the supernatant was The parsley SMS mRNA has been estimated to be -1.7 stored at -20°C until further use. The assay was linear with kilobases (kb) (26). The N-terminal amino acid ofthe deduced respect to time and protein concentration, and the reaction polypeptide aligns with position 158 oftheA. thaliana protein was substrate-dependent and heat-labile. (see legend to Fig. 1). Therefore, assuming similar size,

PcSMS lAWlRLTR V AMVPIRVHTIL ETVTNDEIAADLKEHV AraSaml ------V------R------RatSam -vi-V-----V- -bI-LEAMREA---Q- YscSaml RW--Q-ID- DE -TEDLR-Q--SEI EcoMetK L .RV__Y S_ FmA KI-G-..DA -EIDQKSLQEAVM-EI FIG. 1. Comparison of amino acid sequences deduced from the parsley SMS-1 cDNA (PcSMS) and from SMS-encoding DNA IKVVENYD FHLSRVGGPDIYGWAGAFSGK T from A. thaliana (AraSaml; ref. ------1 YFI D ____ \_- A S-----S 18), rat (RatSam; ref. 17), yeast K-IFPDI+-_KD _- -N YFI _---___------I_T- S-s (YscSaml; ref. 6), and E. coli -ILt EW s -F--- R13 ------S (EcoMetK; ref. 15). Multiple se- quence alignments were done by KVDRS;Y hbANSI KS A SI SVYAIGV PRVFVDT KIPDR.EILKIV using the method df Needleman DR---q-F-|-- + "V-N- -- L ------11 L---.K------and Wunsch (32). Boxes indicate I--e - FC- >Lt------W HISIFA-S -KTE-D-L-EV- identical residues lIi all five pro- A -LACK- 4>Q----A-~H- - p-TKS-.E--ID-I teins. Dashes represent amino ac- -DF-EI- ---A M- 8 - @-V-SEQLTI.L- ids identical to those in PcSMS. Gaps are indicated by dots. The N-terminal amino acid of the in- KE RP ISI D RGGNGRFLrGH REDPDFTWEVVWKPI WEK..A* complete parsley polypeptide --S - MT-T----|--D-PQ-*-- aligns to positions 158 in Arabi- NK - -VRDF--I-K... PIYQ---C --- -- SE-P---P-K F* dopsis, 171 in rat, 157 in yeast, and SK e-F--|-PLVKEF-1I-...PFY-P - ..NQEYP--KP-T-F* 155 in E. coli. Asterisks mark the R- PiP~ts-QM. DSL... HPIYKE - --HFPWEKTDKA"" RDA..-GLK* C-terminal ends of the proteins. Downloaded by guest on September 26, 2021 ------V-Ab Plant Biology: Kawalleck et al. Proc. Natl. Acad. Sci. USA 89 (1992) 4715

sequences coding for >150 amino acids are missing on the estingly, the parsley polypeptide contains one additional SMS-1 cDNA. stretch of41 amino acids (positions 150-190) absent in the rat Sequencing revealed that ELI 19 is a chimaeric construct, and D. discoideum proteins. containing some 700 bp of SMS cDNA ligated head-to-head Genomic Complexity of the Parsley SMS and SHH Genes. to an unrelated cDNA. The SMS-specific portion (SMS-2) The parsley SMS-1 and SHH cDNAs were used to probe has 80%o nucleotide sequence identity with SMS-1. However, blots containing genomic parsley DNA restricted with sev- the deduced amino acid sequences are >98% identical, eral different endonucleases. Fig. 3 shows that both probes indicating that the two cDNAs represent distinct SMS genes detected a limited number of restriction fragments. No ad- with closely related functions. ditional fiagments were observed under conditions of low- ELI 14 cDNA Represents an SEIH Gene. Using the deduced ered stringency for hybridization with either the SMS-1 or the amino acid sequence ofthe ELI 14 cDNA for a similar search SHH probe. Hybridization with the SMS-2 probe under for related proteins within the data bank, we discovered large high-stringency conditions revealed a subset of the same similarity with two polypeptides that have been functionally fragments as detected with the SMS-1 probe, possibly indi- identified as SHHs in rat and D. discoideum. The 1-kb ELI cating a tight chromosomal linkage of the two SMS genes 14 cDNA (renamed SHH) contains one open reading frame (data not shown). Although an exact quantification was not encoding 227 amino acids, which represents the C-terminal done, the number and intensity of the observed fragments part of the complete SHH polypeptide (Fig. 2, arrow). As no indicate that SMS as well as SHH comprise small gene plant SHH sequences have been reported to date, we used families within the parsley genome. the ELI 14 clone as probe to screen a parsley cDNA library SMS and SHH mRNA Levels in Different Organs and in for full-length SHH representatives. Numerous candidates Elicitor-Stimulated Leaves. The expression patterns ofSMS-1 were isolated. The longest cDNA clone of 1.8 kb contains the and SHH in different organs of parsley plants were deter- complete coding region consisting of485 amino acid residues mined at the mRNA level (Fig. 4 Left). Large amounts of with a calculated molecular mass of 53,181 Da (Fig. 2). The SMS mRNA were detected in floral buds and roots, consid- deduced polypeptide is 64% and 62% identical to the rat and erably smaller amounts were detected in stems, and only D. discoideum SHH proteins, respectively. The similarity traces were detected in mature leaves. SHH mRNA was most increases to 79% and 76% when conservative amino acid abundant in floral buds and stems; low levels were detected exchanges are taken into account. Furthermore, the in vitro in roots &nd leaves. transcription/translation product of the ELI 14 cDNA spe- Forced infiltration of fungal elicitor solution into leaves cifically reacted with anti-rat SHH antibodies (19). markedly increased both SMS and SHH mRNA levels. Strong sequence conservation is observed within a region Mock-treatment of the tissue with water had a much smaller containing a putative NAD+- (bracket in Fig. 2; effect (Fig. 4 Right). refs. 33 and 34). Furthermore, the two residues Elicitor-Induced SMS and SHH Gene Expression in Cul- identified at the of the rat enzyme (35) are also tured Cells. Rapid and transient increases in the rates of SMS present in the parsley protein (arrowheads in Fig. 2). Inter- and SHH transcription after elicitation of parsley cells were

I 5 8 PcSHH MALSVEKTAAGRE YK RDM DF ELELREV MPGLS EFGPSQPF rat M-DKLPE---jA_ I A-DI ------N____ YSA-K----- D.disc. MTKLH __-_-_I- _ _K-I-I-_ N_____T 8 Y--A-IL- - 59 rJEJ1..A nGI HTIIMvLL r IET4RIMLA " SFCIFSTUDHAAAAAILDSCWIRWE1 RY~WCI - A------AGI ------TGV --

121 182

HALDWG PD DGG I GVKAEEEYKKSGAIPDPASTDNAEFQIVLSIIR

V TIVF. 4jLN ......

183 244 DGLKSDPHIYHKMKDRLVGVSEETT K Q PiAINVNDSVTKSKFDNLYGCT ...... |PQLLSGIR-IN------H AI-KV ------...... PQFLAGIK------KE ------|

245 W r 306 R ATDVMIAGKVAI_ GYGDVGKGC AMKQ GAR DG VTEI LQ FIG. 2. Comparison of amino _7_ acid sequences deduced from the _ tM: ------__V!------_ QSLSK ______--- E....J_~ parsley SHH cDNA (PcSHH) and 307 36 8 from SHH encoding cDNAs from

yWV &a K ja Al" v J" v_,A &, a--& v 's-S a'''-''F''T'-'-''''-X -a'AV 2TYPGVKR rat liver (rat; ref. 19) and D. dis- E-TTMDEACK-G------V -LGRHFE ------NE.NA-EK coideum (D.disc; ref. 20). The -IVTM-TAAPLS------JR ------A N.. ANA-K bracket encompasses a putative pRGEHFA NAD+-binding site with three in- 369 430 variant residues marked I P4TDFVFPDTGRGI AEG MTHPSVM IR L4EKSSG by large dots. Arrowheads point VN------§LLKNGH-4------i---jM----- M-t --_ H. .PD to the two critical cysteine resi- D------TLANGVH.------L- Aj T.TE dues shown to reside within the 4 31 4 8 5 active site of the rat enzyme (19). Arrow indicates the 5' end of the originally reported incomplete ELI 14 cDNA (26). See legend to E- L L - E --E - ES Fig. 1 for further explanations. Downloaded by guest on September 26, 2021 4716 Plant Biology: Kawalleck et al. Proc. Nad. Acad. Sci. USA 89 (1992) A B C 1 2 3 4 5 6 8 10 12 1416 18 212631 C(h) RI RI/Hill H Iil St I RI RI/Hill Hill RV kb kb 12 - 12- __ 8- - am"' - "-mom 6 I o i to t U 5- SMS 5 -w 4 ...... (1.7kb)

%-am* 3- 3- 2- 2- 1.6.u-.-* 1.6-

I1- 1- _ _SW SHH 40 *-, 400000000"Wo (1.9 kb)

4mm - 0.5 - 0.5 FIG. 5. Changes in SMS and SHH mRNA concentrations in elicitor-treated parsley cells. Ten micrograms of total RNA per lane was separated by gel electrophoresis, blotted to nylon membrane, SMS SHH and hybridized first with the SMS-1 cDNA probe. After autoradiog- raphy, the probe was removed by washing, and the filter was FIG. 3. Genomic DNA blot analysis of the parsley SMS-1 and rehybridized with the SHH cDNA probe. Time points after elicitor SHH genes. Parsley DNA was digested with EcoRI (RI), EcoRI/ treatment were as indicated. C, untreated control; h, hr. HindIII (RI/HIII), HindIII (HIll), Stu I (Stl), or EcoRI/EcoRV (RV), separated by gel electrophoresis, transferred to nylon mem- nearly all functionally identified elicitor-stimulated genes had branes, and hybridized to the SMS-1 (A) or SHH (B) cDNA probes. been related to secondary metabolism, including specific cell Positions of size markers are as indicated. wall-associated reactions. Typical examples for a close re- recently demonstrated (26). The corresponding time courses lationship to pathogen defense are ammonia- ofelicitor-induced changes in SMS and SHH mRNA amounts (23), formally designated as ELI 4 (26), and two other are shown in Fig. 5. Both mRNAs accumulated transiently, ELIs recently identified as peroxidase and hydroxyproline- although to different extents and with different kinetics. rich glycoprotein cDNAs (P.K., unpublished work). In all Scanning of the hybridization signals revealed about a 3- to cases investigated, rapid gene activation in response to 4-fold increase in the SMS mRNA level, with a maximum of elicitor treatment of cultured parsley cells was paralleled by around 3 hr and a decline to very low levels at later time a similar, rapid and local gene activation around fungal points. A slower, but much stronger and more long-lasting infection sites in parsley leaves (36). By analogy, we con- increase, with a peak between 6 and 10 hr, was seen for SHH clude that the observed elicitor responsiveness of SMS and mRNA. SHH genes in cultured cells likewise indicates a close met- SHH Enzyme Activity Increases Upon Elicitor Treatment. abolic link between the activated methyl cycle and pathogen As an enzymatic activity of the activated methyl cycle, we defense in challenged leaf tissue. This conclusion is further measured SHH, which gave particularly clear-cut increases supported by the observed fungal elicitor-stimulated increase in the mRNA amounts. Fig. 6 shows that elicitor treatment in SHH activity. also resulted in an increase in SHH activity. On average, a Several methylation reactions are known to be associated 2-fold higher enzyme activity was found in cultured cells and with pathogen defense in parsley leaves, most notably the a 1.6-fold increase in infiltrated leaves. In cultured cells, the formation of diverse classes of phenylpropanoid derivatives. increase commenced after 5 hr and a peak in activity around Among these are various methoxylated furanocoumarin phy- 15 hr was followed by a slow decline. toalexins (5, 37). Moreover, AdoMet has an important role as an intermediate in ethylene biosynthesis (2, 37), which is also greatly stimulated in elicitor-treated parsley cells (7). Thus, DISCUSSION high levels of AdoMet synthesis and of activated methyl Identification of three ELI cDNAs as representatives of a group turnover are required for these reactions to proceed at as universally interconnected with primary metab-. pathway B Leaves olism as the activated methyl cycle was a surprise. So far, A Cells C E C W E R S L Fb Lc Lw LE t g * _ -Ado

SHH -t i -AdoHcy-

FIG. 4. SMS and SHH mRNA levels in different parsley organs FIG. 6. SHH enzyme activity in extracts of parsley cells and and in response to elicitor infiltration into leaves. (Left) Total RNA leaves after elicitor treatment. Enzymatic conversion of "4C-labeled (5 ,ug per lane) from roots (R), stems (S), mature leaves (L), or floral adenosine to "4C-labeled AdoHcy was monitored by using 100 jLg of buds (Fb) were separated by gel electrophoresis, blotted to nylon crude protein extracts from untreated (lane C) and 15-hr elicitor- membrane, and hybridized with the SMS-1 and SHH cDNA probes. treated (lane E) parsley cells (A) or from mature control (lane C), (Right) The same procedure was used for hybridization to total RNA water-infiltrated (lane W), and elicitor-infiltrated (lane E) parsley from mature control (Lc), water-infiltrated (Lw), and elicitor- leaves (B), as described in Fig. 4. After trichloroacetic acid precip- infiltrated (LE) leaves. Water and elicitor treatments were for 24 hr. itation, 10-jl aliquots were analyzed by TLC on a silica gel plate and All three leaves were taken from one main branch ofthe same parsley subsequent autoradiography. The arrow indicates the flow direction plant. during TLC. Downloaded by guest on September 26, 2021 Plant Biology: Kawalleck et al. Proc. Natl. Acad. Sci. USA 89 (1992) 4717 elevated rates, and the basic levels of SMS and SHH gene mitted step in the shikimic acid pathway, in elicitor-treated expression, probably occurring in control tissue for the parsley cells (16) is a strong indication that this may, indeed, various housekeeping functions ofprimary metabolism (1, 2), be the case. may not suffice under stress conditions. The identification of ELI 18 and ELI 19 as SMS and ELI We are especially grateful to Dr. Giulio Cantoni (National Institute 14 as SHH cDNAs seems beyond doubt. In particular, the of Mental Health, Bethesda, MD) for providing us with samples of deduced amino acid sequences of SMS-1 and SMS-2, al- the rat liver SHH enzyme and anti-SHH antibodies. We thank Petra though not full-length, are almost 92% identical to the cor- Robertz for excellent technical assistance, Dr. Erich Kombrink, Dr. responding portion of a recently reported SMS from A. Bermd Weisshaar, and Ursula van de L6cht for critical reading ofthe thaliana (18). The relatively large sequence divergence at the manuscript, and Fonds der Chemischen Industrie for financial sup- nucleotide level (=20%o)-apart from the fact that the original port. ELI 19 cDNA clone turned out to be a chimaeric construct- 1. Tabor, C. W. & Tabor, H. (1984) Adv. Enzymol. 56, 251-282. may explain why ELI 18 (SMS-1) and ELI 19 (SMS-2) were 2. Giovanelli, J., Mudd, S. H. & Datko, A. H. (1980) in The Biochem- initially thought to represent unrelated genes (26). istry of Plants: A Comprehensive Treatise, ed. Miflin, B. J. (Aca- The previously (26) estimated sizes of the hybrid-select in demic, New York), Vol. 5, pp. 453-505. vitro translation products (-45 kDafor SMS and =50 kDafor 3. Adams, D. 0. & Yang, S. F. (1977) Plant Physiol. 60, 892-896. SHH) and the calculated size of 53,181 Da deduced from the 4. Markham, G. D., Hafner, E. W., Tabor, C. W. & Tabor, H. (1980) parsley full-length SHH cDNA agree well with values re- J. Biol. Chem. 255, 9082-9092. 5. Hauffe, K. D., Hahlbrock, K. & Scheel, D. (1986) Z. Naturforsch. ported for the respective enzyme proteins from other orga- C 41, 228-239. nisms, including higher plants (1, 2). In all systems studied, 6. Thomas, D. & Surdin-Kerjan, Y. (1987) J. Biol. Chem. 262, 16704- SHH is a multimeric enzyme, with each subunit binding one 16709. molecule of NAD', which is required for catalytic activity 7. Chappell, J., Hahlbrock, K. & Boller, T. (1984) Planta 161, 475-480. (34). The deduced portion of the parsley SHH protein con- 8. Suma, Y., Shimizu, K. & Tsukada, K. (1986)J. Biochem. 100, 65-75. tains a typical dinucleotide-binding domain (33) containing 9. Richards, H. H., Chiang, P. K. & Cantoni, G. L. (1978) J. Biol. the sequence Gly-Xaa-Gly-Xaa-Xaa-Gly, which is invariably Chem. 253, 4476-4480. 10. Fujioka, M. & Takata, Y. (1981) J. Biol. Chem. 256, 1631-1635. found in NADW-binding proteins. The importance ofthe three 11. Doskeland, S. 0. & Ueland, P. M. (1982) Biochim. Biophys. Acta glycine residues for both NADW-binding and catalytic activ- 708, 185-193. ity has recently been demonstrated for the rat SHH by 12. Guranowski, A. & Pawelkiewicz, J. (1978) Planta 139, 245-247. oligonucleotide-directed mutagenesis (14). Furthermore, 13. Sebestovi, L., Votruba, I. & Holy, A. (1983) Collect. Czech. Chem. Gomi et al. (35) identified two cysteine residues within the Commun. 49, 1543-1551. active site of each monomer of the rat enzyme, whose 14. Gomi, T., Date, T., Ogawa, H., Fujioka, M., Aksamit, R. R., positions are conserved in the parsley and slime mold poly- Backlund, P. S., Jr., & Cantoni, G. L. (1989) J. Biol. Chem. 264, 16138-16142. peptides. In addition, >60%o of all amino acids were found 15. Markham, G. D., DeParasis, J. & Gatmaitan, J. (1984) J. Biol. identical among the parsley, rat (19), and slime mold (20) Chem. 259, 14505-14507. SHH proteins, and the specific reaction with authentic anti- 16. McCue, K. F. & Conn, E. E. (1989) Proc. NatI. Acad. Sci. USA 86, rat SHH antibodies is taken as further evidence for the 7374-7377. identity of the parsley protein with SHH. 17. Horikawa, S., Ishikawa, M., Ozasa, H. & Tsukada, K. (1989) Eur. The subunits of plant SHH enzymes have been estimated J. Biochem. 184, 497-501. 18. Peleman, J., Boerjan, W., Engler, G., Seurinck, J., Botterman, J., to be larger than those of the mammalian and bacterial Alliotte, T., Van Montagu, M. V. & Inze, D. (1989)Plant Cell 1, 81-93. proteins (19). The larger size of the parsley SHH protein is 19. Ogawa, H., Gomi, T., Mueckler, M. M., Fujioka, M., Backlund, mainly due to an additional peptide region spanning >40 P. S., Jr., Aksamit, R. R., Unson, C. G. & Cantoni, G. L. (1987) amino acids not present in the otherwise very similar rat or Proc. Natl. Acad. Sci. USA 84, 719-723. D. discoideum proteins. However, whether this is a general 20. Kasir, J., Aksamit, R. R., Backlund, P. S., Jr., & Cantoni, G. L. SHH (1988) Biochem. Biophys. Res. Commun. 153, 359-364. characteristic of plant enzymes remains open. 21. Ragg, H., Kuhn, D. N. & Hahlbrock, K. (1981) J. Biol. Chem. 256, Another open question concerns the apparent differences 10061-10065. in timing and magnitude of SMS and SHH mRNA induction, 22. Ayers, A. R., Ebel, J., Finelli, F., Berger, N. & Albersheim, P. as well as clearly detectable differences between the relative (1976) Plant Physiol. 57, 751-759. SMS and SHH mRNA levels in different plant organs. Similar 23. Lois, R., Dietrich, A., Hahlbrock, K. & Schulz, W. (1989) EMBO organ-specific patterns ofSMS mRNA distribution have been J. 8, 1641-1648. 24. Maniatis, T., Fritsch, E. F. & Sambrook, J. (1982) Molecular reported for Arabidopsis plants (18), except that very low Cloning: A Laboratory Manual (Cold Spring Harbor Lab., Cold levels were found in inflorescences, in contrast to the large Spring Harbor, NY). abundance observed here in floral buds of parsley. Thus, 25. Feinberg, A. P. & Vogelstein, B. (1984) Anal. Biochem. 137,266-269. either inflorescences from the two species are not compara- 26. Somssich, I. E., Bollmann, J., Hahlbrock, K., Kombrink, E. & ble in this respect or SMS gene activity varies greatly with Schulz, W. (1989) Plant Mol. Biol. 12, 227-234. floral development as has been demonstrated for SHH en- 27. Murray, M. G. & Thompson, W. F. (1980) Nucleic Acids Res. 8, 6323-6327. zyme activity during ontogenesis in yellow lupin (12). 28. Poulton, J. E. & Butt, V. S. (1976) Arch. Biochem. Biophys. 172, Clear-cut answers to these questions must await more 135-142. detailed studies on the tissue-specific expression of individ- 29. Sanger, F., Nicklen, S. & Coulson, A. R. (1977) Proc. Natl. Acad. ual members of the two small gene families. Apart from that, Sci. USA 74, 5463-5467. AdoHcy is a potent inhibitor of many or all methyltrans- 30. Chen, E. J. & Seeburg, P. H. (1985) DNA 4, 165-170. 31. Devereux, J., Haeberli, P. & Smithies, 0. (1984) Nucleic Acids Res. ferases (2), and it is conceivable that an "overshoot" ofSHH 12, 387-395. activity is required under certain conditions to efficiently 32. Needleman, S. B. & Wunsch, C. D. (1970) J. Mol. Biol. 48,443-453. hydrolyze excess AdoHcy. 33. Wierenga, R. K. & Hol, W. G. J. (1983) Nature (London) 302, In any event, our results suggest a high degree of com- 842-844. plexity in the regulation of SMS and SHH genes in higher 34. Palmer, J. L. & Abeles, R. H. (1979) J. Biol. Chem. 254, 1217-1226. plants. It will be interesting to see whether other genes as 35. Gomi, T., Ogawa, H. & Fujioka, M. (1986) J. Biol. Chem. 261, 13422-13425. closely related to primary metabolism are also regulated 36. Schmelzer, E., Kruger-Lebus, S. & Hahlbrock, K. (1989) Plant Cell according to specific needs for pathogen defense. A recent 1, 993-1001. report on the induction of 3-deoxy-D-arabino-heptulosonate- 37. Hahlbrock, K. & Scheel, D. (1987) in Innovative Approaches to 7-phosphate synthase, the enzyme catalyzing the first com- Plant Disease Control, ed. Chet, I. (Wiley, New York), pp. 229-253. Downloaded by guest on September 26, 2021