Proc. Natl. Acad. Sci. USAVol. 85, pp. 5546-5550, August 1988Botany

Cinnamyl-alcohol dehydrogenase, a molecular marker specific forlignin synthesis: cDNA cloning and mRNA induction byfungal elicitor

(lignification/plant disease resistance/transcription)

MICHAEL H. WALTER*t, JACQUELINE GRIMA-PETTENATIt, CLAUDE GRAND*t, ALAIN M. BOUDETt,AND CHRISTOPHER J. LAMB**Plant Biology Laboratory, Salk Institute for Biological Studies, P.O. Box 85800, San Diego, CA 92138; and tCentre de Physiologie Vegetale, Universite PaulSabatier, Unite Associee au Centre National de la Recherche Scientifique, no 241, 118 route de Narbonne, F-31062 Toulouse Cedex, France

Communicated by P. K. Stumpf, April 22, 1988 (received for review December 21, 1987)

ABSTRACT Cinnamyl-alcohol dehydrogenase (CAD; EC1.1.1.195) catalyzes the final step in a branch of phenylpropa-noid synthesis specific for production of lignin monomers. Wehave isolated a full-length cDNA clone encoding CAD, as amolecular marker specific for lignification, by immunoscreen-ing a Agtll library containing cDNAs complementary tomRNA from elicitor-treated cell cultures of bean (Phaseolusvulgaris L.). The clone comprises a single long open readingframe of 1767 base pairs, 31 base pairs of 5' leader, and 152base pairs of 3' untranslated sequence. The deduced 65-kDaCAD polypeptide has several features that are strongly con-served in alcohol dehydrogenases. Addition of fungal elicitor tocell cultures stimulates CAD transcription, which leads to aremarkably rapid, but transient, accumulation of CADmRNA, with no detectable lag and maximal levels after 1.5 hr.Southern blot analysis of bean genomic DNA indicates thatelicitor-induced CAD is encoded by a single gene. The regu-latory significance of the rapid activation of this CAD gene andthe possible existence ofa second, divergent CAD gene involvedin lignification during xylogenesis are discussed.

Lignin, the most abundant biopolymer after cellulose, is anintegral cell wall constituent of all vascular plants (1, 2).Lignin imparts mechanical rigidity to plant tissues specializedin solute conductance or structural support. Moreover, ligninis resistant to microbial degradation, and many plants re-spond to infection by deposition of lignin at the point ofattackto provide an effective barrier to pathogen ingress and spread(3, 4). Lignin deposition can also be stimulated by elicitorsderived from fungal cell walls and culture fluids (5).The complex three-dimensional structure of lignin is elab-

orated by oxidative polymerization of the phenylpropanoidmonomers 4-coumaryl alcohol, coniferyl alcohol, and sinapylalcohol, which are derived from 4-coumaroyl-CoA (6-8).This compound is the central intermediate of phenylpropa-noid biosynthesis; it is the precursor of a wide range ofnatural products including, in addition to lignin, hydroxycin-namic acid esters, flavonoid pigments, and isoflavonoid andcoumarin phytoalexins (6-8). The branch pathway specificfor production of lignin monomers involves two reductivesteps catalyzed by cinnamoyl-CoA reductase (E.C. 1.2.1.44)and cinnamyl-alcohol dehydrogenase (CAD; E.C. 1.1.1.195)to give the lignin precursor alcohols, followed by oxidativepolymerization catalyzed by cell wall peroxidases (8).

Despite the importance of lignin in plant development anddefense, little is known about the molecular regulation oflignin deposition either during xylogenesis or in response tomicrobial attack. We describe here the isolation and charac-

terization of a full-length cDNA clone encoding CAD,f whichrepresents a molecular marker specific for lignification.Previous studies had shown that treatment of bean (Pha-seolus vulgaris L.) cell suspension cultures with fungalelicitor stimulated CAD synthesis correlated with a markedincrease in extractable enzyme activity (9). In the presentpaper, CAD cDNA clones were identified by antibodyscreening of a Agtll library containing sequences comple-mentary to poly(A)+ RNA isolated from elicitor-treatedcells.We show that fungal elicitor stimulates transcription of the

single stress-induced CAD gene within the bean genome,which leads to a remarkably rapid, but transient, induction ofCAD mRNA, with no detectable lag and maximal levels after1.5 hr. This response is much more rapid than that previouslyobserved for the accumulation of transcripts of other defensegenes encoding enzymes of the central pathway of phenyl-propanoid biosynthesis and a branch pathway specific for theproduction of isoflavonoid phytoalexins (10-12). The regu-latory significance of the ultrarapid elicitor activation of thestress-induced CAD gene and the possible existence of asecond, divergent CAD gene involved in lignification duringxylogenesis are discussed.

MATERIALS AND METHODSPlant and Fungal Cultures. Suspension cultures of bean

(Phaseolus vulgaris L.) were grown as described (12). Thesource, maintenance, and growth of Colletotrichum linde-muthianum were as described (13). Elicitor (final concentra-tion of 60 ug per ml of cell culture) was the high molecularweight fraction heat-released from isolated mycelial cell walls(14).

Agtl1 Library Construction and Screening. Total cellularRNA was isolated from cells homogenized directly in aphenol/0.1 M Tris, pH 9 (1:1, vol/vol) emulsion (15), andpoly(A) + RNA was obtained by oligo(dT)-cellulose chroma-tography (16). Poly(A) + RNA isolated from cells 1.5 hr afterelicitor treatment was used to construct a cDNA library (106recombinants) in Agtll as previously described (11). Phage(1.5 x 105) were screened with CAD antiserum (9, 17) asdescribed (18). Antibodies bound on the phage lift filters weredetected by peroxidase-conjugated goat anti-rabbit IgG (Bio-Rad) and the chromogenic substrate 4-chloro-1-naphthol.Positive clones were carried through five rounds of plaquepurification, and inserts were subcloned in plasmid pUC19

Abbreviation: CAD, cinnamyl-alcohol dehydrogenase.tThe sequence reported in this paper is being deposited in theEMBL/GenBank data base (IntelliGenetics, Mountain View, CA,and Eur. Mol. Biol. Lab., Heidelberg) (accession no. J03825).

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The publication costs of this article were defrayed in part by page chargepayment. This article must therefore be hereby marked "advertisement"in accordance with 18 U.S.C. §1734 solely to indicate this fact.

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(Bethesda Research Laboratories) or pBS (Stratagene, SanDiego, CA).DNA Sequencing. DNA was sequenced by the dideoxy

method (19) using heat-denatured plasmid DNA or in vitrotranscripts as templates (20). Reverse transcriptase was usedto sequence from the whole insert or from suitable fragmentscloned in pBS.Hybrid Selection. pCAD4a (see Results) DNA (15 ,ug) was

linearized by cleavage with Pst I, extracted with phenol/chloroform, 1:1 (vol/vol), and precipitated with ethanol. Thepellet was resuspended in 20 A.l of 10 mM Tris HCl (pH 7.5)containing 1 mM EDTA, heated to 650C for 2 min, and appliedto nitrocellulose (1 cm2). The filter was air dried, and DNAwas denatured by laying the filter on filter paper soaked with0.5 M NaOH/1.5 M NaCl. Neutralization was performed inthe same manner with 1 M Tris HCI, pH 7.5/2 x SSC (1 xSSC = 0.15M NaCI/0.015 M sodium citrate, pH 7). The filterwas washed several times with 2 x SSC, air dried, and baked2 hr at 80'C under reduced pressure. Hybridization condi-tions were as described by Miller et al. (21) with 15 1Lg oftwice-selected poly(A) + RNA at 450C for 6 hr. After elution,the selected RNA was ethanol precipitated, and the transla-tion mix was added directly to the dried pellet.In Vitro Transcription and Translation. pBS containing the

CAD4a inserts were linearized with BamHI prior to tran-scription with T7 polymerase and with Nae I prior totranscription with T3 polymerase as previously described(11). In vitro translation of poly(A)+ RNA or in vitrotranscribed RNA, immunoprecipitation with CAD antise-rum, and NaDodSO4/10o polyacrylamide gel electropho-retic analysis were as described (9).RNA and DNA Filter Hybridization. Total RNA (5 ug) was

denatured with glyoxal, electrophoresed in 1.2% agarose,and blotted onto nitrocellulose. The insert fragment ofpCAD4a was 32P-labeled by nick-translation and was used asa probe in 50% (vol/vol) formamide/5 x SSC/1 x Denhardt'ssolution (1 x = 0.02% polyvinylpyrrolidone/0.02% Ficoll/0.02% bovine serum albumin)/2.5 mM EDTA/0.1% NaDod-S04 with 100 ,ug of carrier DNA per ml. Hybridization wasperformed at 38°C for 24 hr. Filters were washed at the sametemperature in 1 x SSC/0.1% NaDodSO4 followed by 0.4 xSSC/0.1% NaDodSO4. The same conditions were used forgenomic Southern blots except that a wash at 50°C for 1 hrwas included. Genomic DNA was prepared from bean leavesusing the detergent hexadecyltrimethylammonium bromide(CTAB) (22).Nuclear Runoff Transcription. Isolation of nuclei and

analysis of transcripts completed in vitro in the presence of[a-32P]UTP was as described (23).

RESULTSIdentification of cDNA Clones. A cDNA library was con-

structed in Agtll using poly(A)+ RNA isolated from suspen-sion-cultured cells 1.5 hr after treatment with fungal elicitor.Screening of 1.5 x 105 recombinants with an antiserumagainst poplar CAD purified to homogeneity (9, 17) gave 13positive signals. Inserts of plaque-purified clones were sub-cloned into pUC19 or Bluescribe (pBS). Internal EcoRJ sitesresulted in the generation of two subclones designated a andb from a single A clone. ACAD4 had the largest insert (about2.0 kilobases) and was selected for further study.The major CAD polypeptide isolated from elicitor-treated

bean cells has a molecular mass of65 kDa (ref. 9; F. Sarni andC.G., unpublished results). Immobilized pCAD4a hybrid-selected, from total poly(A)+ RNA from elicitor-treatedcells, an mRNA that encoded a 65-kDa polypeptide that wasimmunoprecipitated by CAD antiserum (Fig. 1). RNA simi-larly processed with immobilized vector sequences failed to

A1 2 3 4 5 6 7 kDa

- 92

B1 2 3 4 kDa

_OM Fq

* 6946~~~~~~~~~~~30

* 46

* 30

14 3

FIG. 1. Identification of CAD cDNA sequences. (A) Translationin vitro of hybrid-selected mRNA. Total polypeptide products (lanes1-3) or polypeptides immunoprecipitated with pCAD antiserum(lanes 4-6) were separated on a 10%6 polyacrylamide gel andvisualized by fluorography. Lanes: 1 and 4, poly(A)+ RNA (1 l.g)from elicitor-treated cells; 2 and 5, RNA selected by immobilizedpCAD4a plasmid DNA from 15 pg of poly(A)+ RNA; 3 and 6, RNAselected by immobilized pUC19 vector DNA from 15 jg of poly(A) +RNA. Lane 7 contains molecular size markers. (B) Translation of apartial CAD mRNA transcribed in vitro. RNA (1 Ag) transcribed invitro from recombinant plasmids was translated in vitro for 90 min bya rabbit reticulocyte lysate system. After immunoprecipitation withCAD antiserum, polypeptides were fractionated by gel electropho-resis and visualized by fluorography. Lanes: 1, RNA transcribed inthe sense orientation with 17 RNA polymerase from BamHI-digested pBS-CAD4a DNA; 2, RNA transcribed in the anti-senseorientation with T3 RNA polymerase from Nael-digested pBS-CAD4a; 3, 14C-labeled molecular size markers; 4, control with noadded RNA.

produce this immunoprecipitable CAD polypeptide. To con-firm the identity of the ACAD4 clone, we investigatedwhether polypeptide(s) encoded by the pCAD4a insert wererecognized by the CAD antiserum. The pCAD4a insert wassubcloned in the pBS vector allowing both sense and anti-sense transcription through T7 and T3 promoters. Oneorientation of the pCAD4a subclone gave rise to a transcriptthat encoded a 38-kDa polypeptide that was recognized bythe CAD antiserum (Fig. 1). These data taken together withthe hybrid selection experiment established that ACAD4 wascomplementary to CAD mRNA sequences.cDNA Sequence. The sequence of the ACAD4 insert has a

single open reading frame of 1767 base pairs, 31 base pairs of5' leader sequence, and a 3' untranslated region of 152 basepairs (Fig. 2). The ATG at position 32 is likely to be thetranslation initiation codon since (i) there is a TAA stop codonin frame upstream; (ii) there is an adenosine at position 29, inagreement with Kozak's rule for eukaryotic initiation sites(24); and (iii) the 589 amino acids deduced from this openreading frame correspond to a polypeptide of 65 kDa (Fig. 1).Initiation at the first internal methionine of pCAD4a (nucleo-tide 821, Fig. 2) would generate a 38-kDa polypeptide, inagreement with the data from translation of in vitro transcriptsinto partial CAD polypeptides (Fig. 1). The 3' untranslatedregion contains the sequence AATAATA, which, in the lightof the variability of such signals in plants (25), represents aputative polyadenylylation signal for the CAD transcript.mRNA Induction. CAD4a sequences specifically hybrid-

ized with an RNA species of about 2.2 kilobases, which wasabsent from control cells but was very rapidly inducedwithout detectable lag after elicitor treatment (Fig. 3). CADmRNA attained maximum levels 1.5 hr after elicitor treat-ment and then rapidly declined to basal levels within 6 hr ofelicitor treatment. There was a close correlation between the

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1 AATTCCGTAAGAGAAGTTGTTGGCAGTGATCATGTCGAGCATTTCCTTGAAGGAGAACGGTGGTGAGGTTTCTGTGAAGAAGGATTATAGMetSerSerI leSerLeuLysGl uAsnGlyGlyGl uVal SerVal LysLysAspTyrSe

91 CAATGGTGGGGGTGTGAGGGACTTGTATGGCGAGGACAGTGCCACAGAGGATCATCTTATCACTCCATGGACCTTCTCTGTAGCCAGTGGrAsnGlyGlyGlyValArgAspLeuTyrGlyGl uAspSerAl aThrGl uAspHi sLeu I eThrProTrpThrPheSerVal Al aSerGl

181 ATGCAGTTTGCTGAGAGATCCACGTTACAACAAAGGGCTGGCCTTTACTGAGGGAGAGAGAGATGCTCATTACTTGCGTGGCCTTTTGCCyE3jSerLeuLeuArgAspProArgTyrAsnLysG IyLeuAl aPheThrGl uGlyG luArgAspAl aTyrLeuArgG1 yLeuLeu Pr

271 CCCATCTGTCTTCAATCAGGAACTTCAGGAAAAGAGGTTGATGCACAATCTTCGCCAGTATGAGGTTCCTCTGCATAGGTACATGGCCTToProSerVal PheAsnGlnGl uLeuGl nGl uLysArgLeuMetHi sAsnLeuArgGlnTyrGl uVal ProLeuHi sArgTyrMetAl aLe

361 GATGGATCTTCAGGAGAGGAATGAAAGGCTGTTCTACAAGCTTCTGATCGATAATGTTGCGGAACTGCTTCCTGTTGTTTACACTCCAACuMetAspLeuGlnGluArgAsnGluArgLeuPheTyrLysLeuLeuIleAspAsnValAlaGluLeuLeuProValValTyrThrProTh

451 GGTTGGAGAAGCATGCCAGAAATACGGAAGCATTTTTAGGCGTCCTCAGGGTCTTTATATCAGTTTGAAAGAAAAAGGCAAGATCCTTGArValGlyGluAlaCysGlnLysTyrGlySerIl ePheArgArgProGl nGlyLeuTyrIl eSerLeuLysGl uLysGl yLys Il eLeuGl

541 AGTACTGAAAAACTGGCCAGAAAAGAGTATTCAAGTTATTGTTGTGACCGATGGTGAGCGTATATTAGGACTGGGAGATCTTGGCTGCCAuValLeuLysAsnTrpProGluLysSerIleGlrnVal I1eValValThrAspGlyGluArgI eLeLeuLE3AspLeuM3ysGI

631 GGGAATGGGAATTCCGGTTGGGAAACTTTCTCTCTATACTGCTTTAGGAGGTGTTCGCCCTTCGTCTTGCTTGCCTGTTACCATTGATGTnGlyMetGly I leProValGlyLysLeuSerLeuTyrThrAl aLeuGlyGlyValArgProSerSerCysLeuProVal ThrlI eAspVa

721 TGGCACAAACAATGAGAAGTTGCTGAATGATGAGTTTTACATTGGGCTTAGACAAAGGCGTGCAACTGGGCAGGAATATGCGACGTTTCTlGlyThrAsnAsnGl uLysLeuLeuAsnAspGl uPheTyrIleGlyLeuArgGlnArgArgAlaThrGlyGlnGluTyrAl aThrPheLe

811 AGACGAGTTTATGCGTGCAGTTAAGCAGAACTATGGAGAGAAAGTTCTCGTACAGTTTGAGGATTTCGCCAATCATAATGCATTTGATCTuAspGluPheMetArgAl aVal LysGlnAsnTyrGlyGluLysVal LeuValGlnPheGluAspPheAl aAsnHisAsnAl aPheAspLe

901 GCTGGAAAAATACAGCTCATCTCATCTTGTTTTCAACGATGATATCCAGGGTACAGCGTCTGTTGTATTAGCAGGATTGCTTGCATCCCTuLeuGluLysTyrSerSerSerHisLeuVal PheAsnAspAspIleGlnGlyThrAlaSerValValLeuAlaGlyLeuLeuAlaSerLe

991 GAAATTAGTTGGGGGAACCTTAGCTGACCACACCTTCTTATTCTTGGGTGCTGGAGAGGCTGGAACTGGTATAGCAGAGCTGATTGCTGTuLysLeuValGlyGlyThrLeuAl aAspHi sThrPheLeuPheLeuGlyAl aGlyGl uAl aGlyThrGlyI eAl aGl uLeuIleAl aVa

1081 TGAGGTCTCAAAGCAGACAAAAGCTCCAGTGGAAGAGACCCGCAAGAAGATATGGCTTGTGGATTCAAAGGGACTTATTGTCAGCTCTCGlGluValSerLysGlnThrLysAlaProValGluGluThrArgLysLysIleTrpLeuValAspSerLysGlyLeulleValSerSerAr

1171 TCTGGAATCACTTCAGCAATTCAAAAAGCCTTGGGCTCATGAGCATGAACCTGTGAAGGGACTTCTAGAGGCTGTCAAGGCAATCAAGCCgLeuGluSerLeuGlnGlnPheLysLysProTrpAl aHi sGl uHi sGl uProVal LysGlyLeuLeuGluAl aVal LysAl aIleLysPr

1261 AACCGTTTTGATTGGATCATCTGGAGCAGGGAAGACATTTACCAAGGAAGTGGTTGAAACCATGGCATCCTTGAATGAGAAACCACTCAToThrVal LeuIleGlySerSerGlyAl aGl yLysThrPheThrLysGl uVal Val Gl uThrMetAl aSerLeuAsnGl uLysProLeu Il

1351 TCTTGCTCTCTCCAACCCAACTTCACAATCTGAGTGCACAGCTGAAGAGGCTTACACATGGAGCAAGGGTAGAGCAATCTTTGCTAGTGGeLeuAl aLeuSerAsnProThrSerGl nSerGl uCysThrAl aGl uGl uAl aTyrThrTrpSerLysGlyArgAl aI lePheAl aSerGi

1441 AAGCCCATTTGACCCrGTTGAATATGAAGGAAAACTTTTTGTTCCTGGACAGGCCAACAATGCTTACATATTTCCCGGTTTTGGCTTGGGySerProPheAspProValGl uTyrGl uGlyLysLeuPheVal ProGlyGInAl aAsnAsnAl aTyrI ePheProGlyPheGlyLeuGI

1531 TTTGATCATGTCTGGTGCAATCCGTGTGCGTGATGAGATGCTCTTGGCAGCCTCTGAAGCTTTGGCTGCACAAGTGTCAGAGGAGAACTAyLeu I leMetSerGlyAl aI leArgValArgAspGl uMetLeuLeuAl aAl aSerGl uAl aLeuAl aAl aGInVal SerGI uGl uAsnTy

1621 TGATAAGGGACTGATTTATCCTCCATTCACAAACATCAGGAAGATATCAGCCAACATTGCTGCTAAAGTGGCGGCTAAGGCATATGATCTrAspLysGlyLeu I leTyrProProPheThrAsn I leArgLys I leSerAl aAsn I leAl aAl aLysVal Al aAl aLysAlaTyrAspLe

1711 AGGTCTGGCTTCTCATCTGAAAAGGCCTAAGGATCTTGTCAAATATGCAGAAAGTTGCATGTACAGCCCAGGCTACCGAAGCTACCGTTGuGlyLeuAl aSerHi sLeuLysArgProLysAspLeuVal LysTyrAl aGI uSerCysMetTyrSerProGlyTyrArgSerTyrArg

1801 AGTTTTTTTTGATACACAATAAGCTTCTTAGGAACAGATTTATTGCATTGCTTGTTAGGCTTGTGGTATAAGGCATATTGCCATTTGAAC

1891 TTTCAATAGTTGCTTATTCTTCAAGGATTTTATTTGTATCGGGGAATAATATATATTCGG 1950

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FIG. 2. Nucleotide sequence of ACAD4 cDNA encoding bean CAD mRNA. An internal EcoRl site (nucleotide 608) separates plasmidsubclones pCAD4a and pCAD4b. Note that the nucleotides immediately adjacent to the two external EcoRI sites correspond to linker sequencesfrom the cloning procedure. The amino acid sequence, deduced from the single open reading frame, is given below the nucleotide sequence.A putative polyadenylylation signal is underlined. Amino acids attributed specific functional significance are boxed.

kinetics for accumulation of hybridizable CAD mRNA,translatable mRNA activity in vitro, and CAD enzymesynthesis in vivo (Fig. 3). Maximal accumulation of CADmRNA coincided with the period of most rapid increase inCAD enzyme activity.

Elicitor Stimulation of Transcription. The effect of elicitoron CAD transcription was monitored by analysis of tran-scripts completed in vitro by nuclei isolated from cells 1 hrafter elicitor treatment or from equivalent untreated cells.cDNA clone H1 contains sequences complementary to anabundant transcript that is unaffected by elicitor treatment(23). Compared to the constitutive transcription of the H1gene as an internal control, elicitor caused a marked induc-tion in CAD transcription at the time of the maximum rate ofaccumulation of CAD mRNA (Fig. 3).Genomic Sequences. pCAD4a sequences were hybridized

to genomic DNA digested with EcoRI, HindIll, and Pst I

(Fig. 4). In each case, hybridization to only a single genomicfragment was observed.

DISCUSSION

We have identified a cDNA clone comprising the wholecoding sequence of an mRNA encoding CAD, an enzymespecific to the synthesis of lignin monomers, and have usedthis clone to show that fungal elicitor stimulates transcriptionof the CAD gene leading to a very rapid, marked, buttransient, accumulation of CAD mRNA. The cDNA clonewas identified on the basis of antibody recognition of a

polypeptide encoded by hybrid-selected RNA or by in vitrotranscribed RNA. The hybrid-selected mRNA translated intoa polypeptide of 65 kDa, and the single open reading frame ofACAD4 encodes a protein of the same size. These results

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Time (h) 0 0.5 1 1.5 2 2.5 3 4 5 6 8 10 12 24

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A CONTROL ELICITOR

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FIG. 3. Elicitor induction of CAD. (A) Timecourse of CAD mRNA accumulation in suspen-sion cultured cells in response to elicitor. TotalRNA was isolated at various times after additionof elicitor, separated on an agarose gel (5 ,tg perlane), and blotted onto nitrocellulose. The blotwas hybridized with pCAD4a insert. (Inset)Elicitor induction of CAD transcription. Auto-radiogram of filters bearing cloned DNA se-quences following hybridization with labeledtranscripts produced in vitro by nuclei isolatedfrom cells 1 hr after elicitor treatment or control,untreated cells are shown. Immobilized se-quences were CAD, pCAD4a insert; H1, insertof pHi containing cDNA sequences of a genewhose transcription is unaffected by elicitor;and SP6, vector sequences. (B) Kinetics ofaccumulation of hybridizable CAD mRNA inresponse to fungal elicitor ( e ). The kinetics forelicitor stimulation of CAD synthesis as mea-sured by translatable mRNA activity in vitro(-.-.); CAD enzyme activity (----); and theaccumulation of mRNAs encoding phenylala-nine ammonia-lyase, chalcone synthase, andchalcone isomerase (. ) are presented forcomparison (10-12).

agree with a size estimate of 65 kDa for authentic bean CADsubunits (ref. 9; F. Sarni and C.G., unpublished results).CAD has cofactor requirements similar to alcohol dehy-

drogenase (26). The deduced bean CAD polypeptide containsseveral glycine residues at the same position as strictlyconserved glycines of alcohol dehydrogenases in the foldinvolved in coenzyme binding (27). Likewise, cysteine and

4 $Z'

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m FIG. 4. Genomic blot analysis.Genomic DNA samples (5 ,ug)from bean leaves were digestedwith the restriction enzymes indi-cated, and the products were sep-arated by electrophoresis on 0.9%oagarose gels. The gels were blot-ted onto nitrocellulose, and thefilters were hybridized with 32P-labeled pCAD4a insert. Molecularsizes were calibrated by referenceto the migration ofA HindI11 DNAfragments.

histidine residues 21 residues apart involved in Zn2 + bindingin alcohol dehydrogenase are also found in identical respec-tive positions in the deduced CAD polypeptide. Thesesimilarities with other dehydrogenases are a further confir-mation that ACAD4 contains CAD cDNA sequences.The present data show that elicitor induction of CAD

transcription and mRNA accumulation underlie the stimula-tion of translatable mRNA activity and enzyme synthesis (9).A particularly striking feature is the very rapid accumulationof CAD mRNA, with no detectable lag and maximum levels1.5 hr after elicitation. In contrast, maximum accumulation oftranscripts encoding phenylalanine ammonia-lyase, the firstenzyme of the central phenylpropanoid pathway, and chal-cone synthase and chalcone isomerase, the first two enzymesof the flavonoid/isoflavonoid branch pathway, is not ob-served until about 4 hr after addition of elicitor (Fig. 4).Transcripts encoding cell wall hydroxyproline-rich glycopro-teins do not reach maximum levels until at least 12 hr afterelicitor treatment (10-12, 28). These different kinetics mayreflect distinct stimuli or a single stimulus leading to eithersequential effects or divergent signal transduction pathways.The lignin precursors, dehydrodiconiferyl glucosides, have

been shown to exert regulatory effects in plant cells exhib-iting cytokinin-like activity (29, 30). Structurally relatedcompounds like acetosyringone and its a-hydroxy derivative,as well as certain flavonoids, play important roles as signalmolecules in plant-soil bacteria interactions (31, 32). Hence,the very rapid elicitor stimulation of CAD may be a compo-nent in the generation of secondary stress signals. Moreover,

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hydroxycinnamic acid intermediates have been shown tomodulate the expression of genes encoding enzymes in thecentral phenylpropanoid pathway and flavonoid branch path-way (33, 34). The rapid stimulation of CAD activity relativeto other enzymes of phenylpropanoid biosynthesis mighttransiently reduce the levels of such pathway intermediates.Hence, the very rapid stimulation ofCAD transcription mightin turn promote the induction of other genes involved inphenylpropanoid biosynthesis.The rapidity of CAD induction implies that this is not an

indirect effect but an early component in the sequence ofevents between elicitor binding to a putative receptor andexpression of inducible defense responses. Therefore, exam-ination of the molecular mechanisms underlying elicitorstimulation of CAD transcription will be a particularly at-tractive system for elucidation of signal pathways in higherplants involved in transduction of environmental stimuli.Moreover, the rapid, marked, but transient, induction ofCAD mRNA in response to elicitor suggests that the CADpromoter should prove useful for engineering stress-inducedexpression of foreign genes.

Blot hybridization of genomic DNA revealed only oneclass of CAD genes. This is in marked contrast to phenylal-anine ammonia-lyase and chalcone synthase, which areencoded by gene families of four and six to eight members,respectively, and are differentially regulated by variousenvironmental stimuli including fungal elicitor, wounding,infection and light (ref. 35; C. L. Cramer, X. Liang, andC.J.L., unpublished results). The single stress-induced CADgene may thus also be regulated by developmental stimuliassociated with lignin deposition during differentiation ofxylem elements, or alternatively, there may be a divergent,developmentally regulated CAD gene. The latter is suggestedby the observation of two CAD isoenzymes of 43 kDa and 69kDa in soybean cell suspension cultures (35). Likewise,purification of CAD from bean cells yields, in addition to themajor, elicitor-induced 65-kDa protein, a second form of 40kDa, which is unaffected by elicitor treatment (M.H.W. andC.J.L., unpublished results). Interestingly, 2 out of the 13 Aclones show no detectable cross-hybridization to the CAD4group ofcDNAs, and one ofthese selects an mRNA encodinga polypeptide of 40 kDa that is weakly recognized by CADantiserum (M.H.W. and C.J.L., unpublished results). Hence,this cDNA may correspond to the transcript of a second,highly divergent CAD gene putatively involved in the depo-sition of lignin during normal development rather than inresponse to microbial attack.

We thank Michael A. Lawton for performing the runoff transcrip-tion assays, Mona C. Mehdy for help with cDNA cloning, David R.Corbin for samples of bean genomic DNA, and Valerie Zatorski forpreparation ofthe manuscript. This research was supported by grantsto C.J.L. from the Samuel Roberts Noble Foundation and to M.H.W.and C.G. from the J. Aron Foundation. M.H.W. and C.G. wererecipients offellowships from the Deutsche Forschungsgemeinschaftand French Government, respectively.

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Physiol. 30, 105-130.7. Hahlbrock, K., Kreuzaler, F., Ragg, H., Fautz, E. & Kuhn,

D. N. (1982) in Biochemistry of Differentiation and Morpho-genesis, ed. Jaenicke, L. (Springer, Berlin), pp. 34-43.

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