Plant Molecular Biology 28: 871-884, 1995. © 1995 Kluwer Academic Publishers. Printed in Belgium. 871

The Arabidopsis thaliana 4-coumarate:CoA ligase (4CL) gene: stress and developmentally regulated expression and nucleotide sequence of its cDNA

Diana Lee 1, Mary Ellard 1,3, Leslie A. Wanner 2'4, Keith R. Davis 2 and Carl J. Douglas 1,, ~ Department of Botany, University of British Columbia, Vancouver V6T 1Z4, Canada (*author for correspondence); 2 Ohio State Plant Biotechnology Center and Department of Plant Biology, The Ohio State University, Columbus, OH 43210, USA; 3present address: Department of Botany and Plant Pathology, Oregon State University, Corvallis, OR 97331-2902, USA 4present address: Institute for Biology and Geology, Drammsveien 201, N-9037 Tromso, Norway

Received 16 January 1995; accepted in revised form 2 May 1995

Key words: 4-coumarate:CoA ligase, Arabidopsis, lignin, hypersenstive response, phenylalanine ammonia-lyase, plant-pathogen interaction

Abstract

An Arabidopsis cDNA clone encoding 4-coumarate:CoA ligase (4CL), a key enzyme of phenylpropanoid metabolism, was identified and sequenced. The predicted amino acid sequence is similar to those of other cloned 4CL genes. Southern blot analysis indicated that 4CL is single-copy gene in Arabidopsis. Northern blots showed that 4CL expression was activated early during seedling development. The onset of 4CL expression was correlated with the onset of lignin deposition in cotyledons and roots 2-3 days after germination. The timing of the expression of a parsley 4CL1-GUS fusion in transgenic Arabidopsis seedlings was examined in parallel and was very similar to that of endogenous 4CL. In mature plants, highest 4CL expression was observed in bolting stems, where relatively large amounts of lignin accu- mulate. Both 4CL and 4CL1-GUS mRNA accumulation was strongly and transiently activated by wounding of mature Arabidopsis leaves. 4CL expression was specifically activated within 6 h after infiltration of Arabidopsis ecotype Columbia leaves with a Pseudomonas syringae pv. maculicola strain harboring the bacterial avirulence gene avrB, which causes in incompatible interaction. The timing of 4CL activation was identical to the previously observed activation of PAL gene expression in this interaction. No activation of 4CL expression was observed in a compatible interaction caused by a Pseudomonas syringae pv. maculicola strain without avrB.

Introduction

The central pathway of phenylpropanoid meta- bolism, carried out by the sequential action of the

enzymes phenylalanine ammonia-lyase (PAL), cinnamate-4-hydroxylase (C4H) and 4-couma- rate:CoA ligase (4CL) is required for the biosyn- thesis of a large number of natural products. For

The nucleotide sequence data reported will appear in the EMBL, GenBank and DDBJ Nucleotide Sequence Databases under the accession number U18675.

872

example, activated CoA esters of hydroxycin- namic acids, the products of the 4CL reaction, are used for the biosynthesis of fiavonoids and lignin via the enzymes chalcone synthase (CHS) and cinnamoyl-CoA reductase, respectively. Genes encoding phenylpropanoid enzymes have been cloned from a large number of plants. In all an- giosperms examined, PAL is encoded by a multi- gene family [14]. 4CL genes have been cloned from fewer plant species. In both parsley and potato, 4CL is encoded by two highly similar genes [2, 30] while in soybean [42], poplar [1], and tobacco (D. Lee and C. Douglas, in prepa- ration), the enzyme is encoded by divergent mem- bers of a gene family. In Arabidopsis, PAL is en- coded by a family of several genes [34, 45], while CHS is encoded by a single gene [11].

Regulation of phenylpropanoid metabolism occurs primarily via the transcriptional regulation of genes encoding enzymes in the pathway [14, 25 ]. The accumulation of PAL and 4CL mRNAs and the activity of their promoters varies during tissue and cell differentiation [3, 15, 26, 34, 37] when certain cells become specialized for the biosynthesis of phenylpropanoid-derived com- pounds such as lignin, flavonoids, and coumarins. In plants examined in detail, PAL and 4CL ex- pression is coordinately regulated [ 14, 28, 29, 37, 46]. Gene expression is also activated following perception of environmental stresses such as wounding, pathogen attack and UV light irradia- tion, and by ozone treatment [ 14, 24, 39], against which phenylpropanoid compounds may play protective roles. In Arabidopsis, PAL gene expres- sion has been shown to be activated in concert with the accumulation of anthocyanin pigments during seedling development [24], in wounded leaves [ 34], and following infection of plants with pathogenic bacteria which elicit an incompatible (resistant) reaction in the host plant [8, 44]. GUS activity or mRNA accumulates in the roots, de- veloping vascular system of cotyledons and leaves, anthers, and wounded leaves of Arabidop- sis plants transgenic for an Arabidopsis PAL1- GUS gene fusion [34].

In this study, we identified an Arabidopsis cDNA clone encoding 4CL and showed that 4CL

is likely to be encoded by a single gene in this plant. In seedlings grown under light conditions in which anthocyanin pigments do not accumu- late to high levels, 4CL expression was correlated with the development of young root tissues and the onset of lignin deposition in developing coty- ledons. In mature leaves, 4CL expression was strongly activated by wounding. These patterns of Arabidopsis 4CL expression were mirrored by the expression of parsley 4CL1 promoter-GUS fusions in transgenic Arabidopsis, suggesting func- tional conservation of cis elements directing 4CL expression in these two plants. Finally, 4CL ex- pression was rapidly activated in Arabidopsis ecotype Columbia leaves infiltrated with a strain of Pseudomonas syringae pv. maculicola harboring the avrB avirulence gene, with kinetics indistin- guishable from the activation of PAL gene expres- sion previously observed in this interaction [44].

Materials and methods

Plant growth conditions and treatments

An Agrobacterium LBA4404 strain harboring a 1500 bp parsley 4CL1 promoter-GUS fusion on the binary vector BIN19 [15] was used to trans- form Arabidopsis ecotype C24 by the method of Valvekens [43]. Initial analysis of GUS expres- sion in several TO plants showed consistent pat- terns of expression. Arabidopsis lines homozygous for the transgene were generated by self pollina- tion of TO plants, and one line, At204-1-5, was used for detailed analysis of 4CL and 4CL1-GUS expression.

Surface-sterilized Arabidopsis seeds from trans- genic lines were germinated and seedlings grown in Petri plates on MS [33] agar (2~o sucrose and 0.8% agar) supplemented with 100/~g/ml kana- mycin in the absence of hormones in the light. Illumination was by Cool White fluorescent lights at a fluence of 30 #mol m- 2 s - 1 (measured with a Li-Cor quantum photometer, Lincoln, NB), at 24 °C. Mature plants were grown in soil in a growth chamber under light at a fluence of

- 2 - 1 85 ~mol m s

Wounding was carried out by slicing detached mature leaves into 1-2 mm strips and incubating the wounded tissue on filter paper moistened with MS media in the light. For 0 h time points, ex- cised leaves were frozen immediately without fur- ther wounding.

Arabidopsis ecotype Columbia plants used for infection with bacterial pathogens were grown in controlled-environment room as described [44]. Overnight cultures (OD6o o 1.6 to 2.2) of Pseudomonas syringae pv. maculicola harboring either the broad host range plasmid vector pDSK519 [20] alone or pDSK519 containing the bacterial avirulence gene avrB [ 19] were collected by centrifugation and resuspended in 10 mM MgC12 to an OD6o 0 of 0.1 (1 x 108 cfu/ml), and then hand-infiltrated into the undersides of leaves of 4 to 5 week old plants, as described [8, 44].

Library screening and eDNA analysis"

An Arabidopsis 2YES expression cDNA library, made with RNA from above-ground parts of plants which varied in size from those which had just opened their primary leaves to plants which had bolted and were flowering (a gift from the lab of R. Davis), was screened with a hybridization probe made from a potato 4CL eDNA clone [2]. Hybridization conditions and washes at low stringency (2 x SSC, 65 °C) were as described [32]. One positive clone, ca. 1.9 kb in size, was identified, and both strands were sequenced as described [40]. Sequence analysis was carried out with Genetics Computing Group (GCG) soft- ware.

Nucleic acid isolation and hybridization

Total genomic DNA was isolated from Arabidop- sis ecotype Columbia by the method of Doyle and Doyle [ 10]. RNA was isolated as described by Logemann et al. [27] or by Wanner et al. [44]. Hybridization probe preparation, Southern blots, and northern blots were as described [40] or done according to standard methods [36]. High-

873

stringency washes after hybridization were at 65 ° C, 0.2 x S SC; low-stringency washes were at 65 °C, 2 x SSC. In some experiments, hybrid- ized northern blot filters were imaged using a Mo- lecular Dynamics Phosphorlmager. Quantitative data were obtained from Phosphorlmager scans using the Molecular Dynamics ImageQuant soft- ware as described [44]. These filters were then stripped by pouring boiling-hot 0.02 x S SC/0.5 ~o SDS over the filters and re-hybridized with a sub- cloned 1.2 kb fragment of the 28S rRNA from pea clone pHA2 [17]. Final total hybridized counts to the 4CL probe were corrected for counts hybridized to the rRNA probe.

GUS assays and cytochemical analysis

GUS enzyme activity was measured using the fluorometric assay described by Jefferson [ 16]. In situ localization of GUS activity using X- GLUC was performed as described [15]. Clear- ing of seedlings and staining of cleared material with 0.01~o basic fuchsin to detect lignin was carried out as described by Dharmawardhana et al. [7]. Stained seedlings were observed using a Zeiss Axioskop under phase contrast or epi- fluorescence (excitation at 395-425 nm and emis- sion above 450 nm).

Results

Identification of an Arabidopsis 4CL eDNA clone

We used a potato eDNA clone encoding 4CL [2] as a hybridization probe to screen an Arabidopsis eDNA library under reduced stringency. Five potential 4CL clones were plaque-purified after screening 2 × 105 recombinants, but subsequent Southern analysis of the eDNA inserts showed that only one insert strongly and reproducibly hybridized to the potato 4CL probe. This clone, At4CL contained an insert of ca. 2 kb, potentially long enough to encode 4CL. The At4CL sequence is shown in Fig. 1 and contains an open reading frame encoding a protein of 562 amino acids.

874

TTACAATGGCGCCACAAGAACAAGCAGTTTCTCAGG TGATGGAGAAACA~GCAACAACAACAACAGTGACGTCATTTTCC GATCAAAGTTACCGGATATTTACATCCCGAACCAC C TAT MetAlaProGlnGluGlnAlaValSerGlnValMe tGluLysGlnSerAsnAsnAsnAsnSerAspVal I i ePheArgSerLysLeuProAspI i eTyr I leProAsnHisLeuS

CTCTCCACGACTACATCTTCCAAAACATCTCC GAAT TCGCCAC TAAGC CTTCq2CTAATCAACGGACCAACCGGCCACGTGT~JCAC TTACTCCGACGTCCACGTCATC TCCC GCCAAATCG erLeuHisAspTyr I i ePheGlnAsnI i eSerGluPheAlaThrLysProCysLeuI i eAsnGlyProThrGlyHisVa iTyrThrTyr SerAspValHisVal I i eSerArgGlnI i eA

CCGC CAATTTTCACAAACTC GGCGTTAACCAAAACGACGTCGTCATGC TCCTCC TC CCAAACTGTCC CGAATTCGTC CTCTCTTTCC TCGCCGCC TCCTTCCGCGGCGCAACCGC CACCG laAlaAsnPheHisLysLeuGlyVa iAsnGlnAsnAspVa lValMet LeuLeuLeuProAsnCys Pr oGluPheValLeuSerPheLeuAl aAlaSerPheArgGlyAlaThrAl aThrA

CCGCAAAC C C TTTCTTCACTCCGGC GGAGATAGCTAAACAAGCCAAAGCCTC CAACACCAAAC TCATAATCACC GAAGC TCGTTACGTCGACAAAATCAAACCACTTCAAAAC GACGAC G laAlaAsnProPhePheThrProAlaGluI leAl aLysGlnAl aLysAlaSerAsnThrL ~ 'sLeuI i eI leThrGluAlaArgTyrValAspLys I leLysPr oLeuGlnAsr~.spAspG

GAGTAGTCATCGTCTGCATCGACGACAACGAATCCGTGCCAATCCCTGAAGGCTGC CTCCGC TTCACCGAGTTGACTCAGTCGACAACCGAGGCATCAGAAGTCATCGACTCGGTGGAGA IyValVal I i eValCys I leAspAspAsnGluSerValPro I lePr oGluGlyCysLeuArgPheThrGluLeuThrGlnSerThrThrGluAlaS erGluVa 11 leAspSerValGluI

TTTCACCGGACGAC GTGGTGGCAC TACC TTAC TCC TC TGGCAC GACGGGATTACCAAAAGGAGTGATGC TGACTCACAAGGGACTAGTCACGAGCGTTGC TCAGCAAG~GACGGCGAGA leS erPr oAspAspValValAIaLeuPr oTyr Ser S erG1yThrThrGlyLeuProLysG lyValMetLeuThrHisLysGlyLeuValThrSerValAlaGlnGlnValAspGlyGluA

ACCCGAATCTTTATTTC CACAGCGATGACGTCATAC TC TGTGTTTTGC CCATGTTTCATATCTAC GCTTTGAACTCGATCATGTTGTGTGGTCTTAGAGTTGGTGCGGCGATTCTGATAA snPr oAsnLeuTyrPheHis SerAspAspValI leLeuCysValLeuPr oMe t PheHis I i eTyrAlaLeuAsnS er I 1 eMet LeuCysGlyLeuArgValGlyAlaAlaI leLeul leM

TGC C GAAGTTTGAGATCAATC TGCTATTGGAGCTGATCCAGAGGTGTAAAGTGACGGTGGC TCCGATGGTTC CGC CGATTGTGTrGGCCATTGCGAAGTCTTC GGAGACGGAGAAGTATG et ProLys PheGluI 1 eAsnLeuLeuLeuGluLeuI leGlnArgCysLysVa iThrValAlaPr oMe tVal ProPr ol leValLeuAlall eAlaLysSer SerGluThrGluLysTyrA

ATTTGAGC TCGATAAGAGTGGTGAAATC TGGTGCTGCTCC TCTTGGTA~GAAC TTGAAGATGCCGTTAATGCCAAGTTTCCTAATGCCAAAC TC GGTCAGGGATACGGAATGACGGAAG spLeuSer Ser I 1 eArgValValLysSerGlyAlaAl aProLeuGlyLysGluLeuGluAspAlaValAsnAlaLys PhePr oAsnAlaL rsLeuGlyGlnGlyTyrGlyMetThrGluA

CAGGTCCAGTGC TAGCAATGTCGTTAGGT TTTCq2AAAG( AAC C T TTTCC GGTTAAG TCAGGAGCTTGTGGTACTGTTGTAAGAAATGCTGAGATGAAAATAGTTGATCCAGACAC CGGAG laGlyProValLeuAlaMet SerLeuGlyPheAlaLysGluProPhePr oValLys SerGlyAlaCysGlyThrValValArgAsnAlaGluMet Lys I leVa IAspProAspThrGlyA

ATTC TCTTTC GAGGAATCAACCCGGTGAGAT T TGTATTCGTGGTCACCAGATCATGAAAGGTTACC TCAACAATC CGGCAGCTA~GCAGAGAC C ATTGATAAAGAC GGTTGGCTTCATA spS erLeuSerArgAsnGlnPr oG lyGluI leCys I leArgGlyHisGlnI i eMe t LysGlyTyrLeuAsnAsnProAlaAlaThrAlaGluThr I i eAspLysAspGlyTrpLeuHisT

C TGGAGATATTGGATTGATCGATGACGATGAC GAGC TT~CATCGTTGATCGATTGAAAGAACTTATCAAGTATAAAGGTTTTCAGG TAGCTCCGGCTGAGC TAGAGGC TTTGC TCATCG hrGlyAspl i eGlyLeuI leAspAspAspAspGluLeuPhel leValAspArgLeuLysG l uLeuI i e Lys TyrLysGlyPheGlnValAlaPr oAlaGluLeuGluAlaLeuLeuI leG

GTCATCCTGACATTAC TGATGTTGCTGTTGTCGCAATGAAAGAAGAAGCAGC TGGTGAAGTTC C TGTTGCATTTGTGGTGAAATC GAAGGATTCGGAGTTATCAGAAGATGATGTGAAGC I yHi s ProAsp I i eThrAspValAlaValVa I AI aMetLysGl u Gl uAlaAl aGl yGl uValProVa iAlaPheVa I ValLys S erLysAspSerGl uLeuSerGl uAspAspValLysG

AATTCGTGTCGAAACAGGTTGTGTTTTACAAGAGAATCAACAAAGTGTTC TTCAC TGAATC CATTCC TAAAGC TC CATCAGGGAAGATATTGAGGAAAGATCTGAGGGCAAAAC TAGCAA inPheValSerLysGlnValValPheTyrLysArgI leAsnLysValPhePheThrGluS er I lePr ol sAlaPr oSerGlyLys I i eLeuArgLysAspLeuArgAlaLysLe~AlaA

ATGGATTGTGATGGATGAT~CAACCAAAAAGCAAAGATGATT TCAATGTGTATATACATACAAC TGTTTGACC C AACCAAGGAAACAAAC TCATACGAACCATTGTCTTTTGTTGTTGT snGlyLeuEnd

TGTTGTTGTTGTTGTTGCTGTTC TTGC TTGATTCATGTAATGAGC CTTTGTGATGAAGGTGGTTTC TT T A ~ 1889

120

240

360

480

600

720

840

960

1080

1200

1320

1440

1560

1680

1800

Fig. 1. Nucleotide and deduced amino acid sequences ofArabidopsis 4CL. Both strands of the At4CL cDNA clone were sequenced•

This length is similar to that reported for the de- duced amino acid sequences of parsley [30], potato [2] and rice [47] 4CL genes. Since the putative initiator Met codon is located only 5 bp from the 5' end of the cDNA insert, we cannot exclude the possibility that additional in-frame Met codons are located further upstream. How- ever, because this codon is in a favorable sequence context for initiation of translation [18, 23] and because amino acid homology to other 4CL pro- teins begins shortly after this Met (see below), we consider it likely that this is the initiator codon and that the cDNA is missing most of the 5'- untranslated portion of this gene.

Figure 2 shows that the deduced amino acid sequence of At4CL is very similar to those of potato 4CL1, parsley 4CL1, and rice 4CL (71, 69, and 62~o identity, respectively). Significant simi- larity was found throughout the sequences, but was most pronounced in the central and C- terminal portions of the proteins. Beginning at

G4o7, At4CL contains the amino acid motif GE1- CIRG, conserved in all 4CL proteins and postu- lated to be associated with catalytic activity of 4CL and related enzymes [2]. In addition to the conserved Cys in this motif, four other Cys resi- dues, whose conservation in 4CL proteins has been noted [42] are present in At4CL. Also found in At4CL (beginning at Y2o9 within a region highly conserved in all 4CL proteins), is a Gly, Ser, and Thr-rich amino acid motif that may to be involved in AMP binding [42]. Based on these sequence comparisons, we conclude that At4CL is an Ara- bidopsis clone encoding 4CL.

Arabidopsis 4CL is a single-copy gene

In order to estimate the number of 4CL genes in the Arabidopsis genome, we hybridized genomic Southern blots of the At4CL cDNA. A simple hybridization pattern was observed when Arab#

875

At MAPQEQAVSQVMEKQSNNNNS - DVl FRSKL PDIYI PNHL SLHDY I FQNI S E pot ........... MPMDTETKQ G L K P S C E L par ........... M--GDCVAPKE L K P T C E K ric MGSM QQPESAA-PATEASP-E7 Q A T T P R C ERLP

At FATKP~L INGI~GHVYTYS DVItVI SRQL~IkNFHK - - LGVNQNDVVMLLLP pot NSR D ANDRI AE ELT KV VGLN -- IQ K TI I par VGD S A ETF Q ELL KV SGLN -- IQ G TI ric V AR D A G L A DILL RL ALRRAp LRRGG S R

At NC PEFVL SFLAASFRGATATAANPFFTPAE IAKQAKASNTKL I I TEARYV pot FA IG YL IS M L W SA IV Q CFA par S YFFA G Y IS M S VIK KA KL C ric S F AV T M ES AGATVV SM A

At DKIKPLQNDDGVVIVC IDDNESVPI PEGCLRFTELTQSTTEASEVIDSVE pot G V DYAIENDLKVI V ..... SV VH S I DEHE--- PD K par V DYAAEKNIQ I A ..... QD H SK ME--ADE MPEV - ric L- SHSHGALTV L ER ..... RD H WDDLM ED PLAGDED

At .... I S PDDWAL PYSSGTTGLPKGVMLTHKGLVTSVAQQVDGENPNLYF pot .... Q A M par .... NS D M ric DEKVFD RS S IGL

T T At H S DDVI LCVLPMFH IYALNS IMLCGLRVGAAIL IMPKFE INLLLEL I QRC pot LM L SL LL L K IAQFL KH par E MI I L S AVLC A VT Q D VPF Y ric AG A S T M W RR D ~ VE H

At KVTVApMVPP IVLAIAKS S ETEKYDLS S I RVVKSGAAPLGKELEDAVNAK pot IG F PLVDN V T M R par IG F pvDq3 V T M R ric R I L V V EAAAAR V M L M DI FM

T At F PNAKLGQGYGMTEAGPVLAMSLGFAKE PF PVKSGACGTVVRNAEMKIVD pot F C A DI par C A YEI ric L G V S C A K L

At PDTGDS L S RNQPGEICIRGHQ IMKGYLNNPAATAET I DKDGWLHTGDI GL pot C P D D E R E E F par E NA P R D D ES RT EE F ric KSLG R Q E KN AE Y

At I DDDDELF IVDRLKEL I KYKGF QVAPAELEALL IGHPD I TDVAVVAMKEE pot N S A P ID par I LT T S A P ID ric V I I R NT S A A GL --

At AAGEVPVAFVVKSKDS ELSEDDVKQFVSKQVVFYKRINKVFFTES I PKAP pot Q R NGS TIT E D I I KR V TV S par K RTNGFTTT EEI FR VDA S ric -F I A TEG A E IY K RE VDK

At SGKILRKDLRAKLANGL pot R A I par RI S ric E KQ QH

5O 4O 38 49

98 88 86 99

148 138 136 149

198 iS0 178 193

244 226 224 243

294 276 274 293

344 326 324 343

394 376 374 393

444 426 424 443

494 476 474 491

544 526 524 540

562 543 540 555

Fig. 2. Comparison of the deduced amino acid sequence ofArabidopsis 4CL with that of 4CL genes from other plants. The pre- dicted Arabidopsis (At) 4CL amino acid sequence is aligned to those of potato 4CL1 (pot), parsley 4CL1 (par), and rice 4CL (tic). Amino acids are only shown when they differ from the Arabidopsis sequence. Dashes indicate gaps introduced to maximize alignment. Conserved Cys residues in 4CL proteins are indicated by arrows above the sequence; amino acids in bold type indi- cate conserved motifs postulated to be involved in 4CL enzyme activity.

dopsis genomic D N A was cut with several differ- ent enzymes (Fig. 3) which, with the exception of Eco RI, did not cut within the cDNA. In each case, a single strongly hybridizing genomic re- striction fragment was observed when the blot was washed under stringent conditions. The c D N A contains two Eco RI sites, approximately 150 and 300 bp from the 5' end of the clone, within the predicted first exon based on the loca- tion of conserved exon-intron boundaries in pars- ley and potato, and rice 4CL genes [2, 30, 47]. It

is likely that a genomic fragment containing flank- ing D N A 5' to the first Eco RI site was not de- tected on the blot due to a weak signal from this small portion of the probe, and that the predicted 150 bp Eco RI fragment was too small to be de- tected on the blot. Very weak hybridization to certain restriction fragments was observed after digestion with some restriction enzymes (for ex- ample, Eco RV lane, Fig. 3). When blots were washed under non-stringent conditions, identical patterns of hybridization were observed and the

876

Fig. 3. Southern blot of Arabidopsis DNA hybridized to At4CL. Arabidopsis Columbia genomic DNA (10 #g) was di- gested with the indicated restriction enzymes, separated on an agarose gel, and a Southern blot hybridized to an At4CL probe. The migration of size standards is shown to the left of the blot.

under which very little anthocyanin pigmentation was visible in hypocotyls and cotyledons. Seed- ling morphology 2, 3, 5, and 10 days after germi- nation is shown in Fig. 6A-D. Figure 4A shows that no 4CL RNA was detectable in whole seed- lings two days after germination, when seedling roots and cotyledons had developed sufficiently to emerge from the seed coat (Fig. 6A). Beginning 3 days after germination, low amounts of 4CL mRNA were detectable in whole seedlings and appeared to remain at a fairly constant propor- tion of the total RNA through 10 days. By 3 days after germination, it was feasible to separate roots from shoots for RNA analysis, and RNA from shoots and roots was examined separately 3, 4, 5, 7, and 10 days after germination (Fig. 4A). 4CL transcript accumulation was higher in roots than in shoots, and the levels of 4CL RNA remained constant in this organ up to 10 days after germi- nation. Low but detectable levels of 4CL RNA

intensity of hybridization to these fragments did not increase significantly relative to hybridization to the prominant bands in Fig. 3 (not shown). Thus, these weakly cross-hybridizing sequences appear to be highly divergent from 4CL, and, con- sistent with previously reported results [41], 4CL appears to be encoded by a single gene in Arab# dopsis.

Developmental regulation ofArabidopsis 4CL and 4CL1-GUS

The developmental regulation of 4CL in seedlings and plants of different ages was investigated using northern blots. In these experiments, we used an Arabidopsis ecotype C24 line homozygous for a parsley 4CL1 promoter-GUS transgene [15], so that the patterns of expression specified by this heterologous 4CL promoter could be compared to the expression of Arabidopsis 4CL. Seedlings were germinated and grown in light conditions

Fig. 4. Northern blot analysis ofArabidopsis 4CL mRNA ac- cumulation. Total RNA (10/~g) was separated on formalde- hyde gels, transferred to nylon membranes, and hybridized to an At4CL probe. A. RNA from seedlings 2-10 days after germination. RNA was extracted either from whole seedlings or from separated seedlings shoots (hypocotyls and cotyle- dons) and roots. B. RNA from mature plant leaves, bolting stems, and flower buds. Bolting stems were separated accord- ing to size, from 3 to 20 cm in length. Gels were stained with ethidium bromide before transfer to nylon membranes to en- sure equal loading of RNA.

had accumulated in seedling shoots by 3 days after germination, and also appeared to remain fairly constant through 10 days after germination. Northern blots of RNA extracted from mature plants (Fig. 4B) showed that 4CL was expressed at low levels in mature leaves from rosette-stage plants and in flowers before anthesis (floral buds), but was strongly expressed in bolting stems from 3 to 20 cm in length.

In order to determine if the heterologous pars- ley 4CL1 promoter specified a similar pattern of reporter gene expression during seedling develop- ment, GUS activity was measured in extracts of the tissues shown in Fig. 4A. Fig. 5 shows that in whole seedlings 2 days after germination, low GUS activity was observed. However, consistent with expression of the Arabidopsis 4CL gene, ac- tivity in whole seedlings was several-fold higher in 3-day old seedlings and remained at a similar level through 10 days. Also consistent with ex- pression of the Arabidopsis 4CL gene, GUS spe- cific activity was much higher in roots 3-10 days after germination than in shoots.

Histochemical analysis of 4CL1-GUS expres- sion in seedlings and mature plants is shown in Fig. 6. In 2-day old seedlings, GUS activity was not histochemically detectable (Fig. 6A), but by

x 10 .f° = e 8 u i=. m ~

4 ~ S

i . 2 3 4 5 lO

Days after germination

Fig. 5. GUS enzyme activity in Arabidopsis seedlings trans- genic for a parsley 4CL1-GUS fusion. GUS activity was mea- sured in extracts of whole seedlings (filled bars), seedling shoots (stippled bars) and roots (slashed bars). Extracts were made from seedlings on the days after germination indicated, and activity determined using 4-methylumbeUiferone glucu- ronide (MUG) as a substrate. Specific activity is expressed as the amount of 4-methylumbelliferone (4-MU) produced.

877

three days was detectable in the vascular tissue of cotyledons, stems and roots, as well as in the root cortex (Fig. 6B). Consistent with quantitative assays of GUS activity (Fig. 5) and accumulation of Arabidopsis 4CL mRNA, staining was stron- gest in roots. This pattern of GUS expression was maintained in seedlings up to 10 days after ger- mination (Fig. 6C, D).

Since 4CL activity is required for the biosyn- thesis of lignin, and anthocyanin pigmentation was not observed in seedlings 3-10 days old under our conditions, we determined whether the increase in 4CL mRNA accumulation and 4CL1- GUS expression in Arabidopsis seedlings between 2 and 3 days after germination was correlated with lignin deposition in the developing vascular system. To monitor lignin deposition, we used fluorescence microscopy in combination with basic fuchsin staining because of the sensitivity of this method [7]. Basic fuchsin-stained 2- and 3-day old seedlings were observed using phase contrast microscopy (Fig. 6E, G, I) to visualize cellular detail, and under fluorescence (Fig. 6F, H, J) to visualize sites of lignin deposition. In most 2-day old seedlings (Fig. 6E), no fluores- cence associated with vascular tissues was ob- served in roots or cotyledons (Figure 6F), indi- eating a lack of lignin deposition (the bright signal at the bottom of Fig. 6F is due to autofluores- cence of material in the seed coat). However, in 3-day old seedlings, strong staining was observed in the developing xylem of both roots (Fig. 6G, H) and cotyledons (Fig. 6I, J), indicating that sig- nificant lignin biosynthesis and deposition had occurred. The presence of differentiated but non- lignified tracheary elements in 3-day old cotyle- dons (arrows, Fig. 6I and J) further illustrates that these cotyledons were now active in lignin biosynthesis and deposition. Concomitant with the lignification oftracheary elements in 3-day old seedlings was the appearance of histochemically detectable 4CL1-G US activity in the vascular sys- tem of cotyledons and roots of transgenic seed- lings (Fig. 6K, L), as well as an increase in GUS activity (Fig. 5) and 4CL RNA amount (Fig. 4). In cotyledons, histochemically detectable GUS activity appeared to be confined to the develop-

878

879

ing vascular system, but in roots, activity was not confined to the vascular cylinder.

GUS activity in roots of 4CL1-GUS seedlings was highest in younger portions of roots. In 10- day old seedlings, several lateral roots had differ- entiated, and histochemically detectable activity was much higher in these young, lateral roots than in the older primary root (Fig. 6M). At the sites of recent lateral root differentiation (Fig. 6N), GUS activity was localized to cortical tissue in the primary root where cell divisions had given rise to the lateral root primordium, and to the tip of the young lateral root. At the site of nascent lateral root formation, recognized by its position on the primary root and by the presence of a group of cells dividing to form a root primordium (Fig. 60), increased GUS activity seemed to be associated with the dividing cells of the primor- dium. Thus, 4CL1-GUS expression, and prob- ably endogenous 4CL expression, seems to be highest in young root tissues of both the primary root and lateral roots.

Fig. 7. 4CL and GUS RNA accumulation in wounded leaves from a plant transgenic for a parsley 4CL1-GUS fusion. RNA was isolated at the times indicated after wounding, and ana- lyzed on northern blots (10/~g per lane). Gels were stained with ethidium bromide prior to transfer to ensure even load- ing. A. Northern blot hybridized to an At4CL probe. B. Du- plicate northern blot hybridized to a GUS probe. Estimated size of the hybridizing bands is given in kb.

Stress-induced expression of 4CL

In plants where it has been examined, expression of PAL and 4CL genes is inducible by a variety of stresses including wounding, pathogen infec- tion, and UV light irradiation [9, 28, 34]. We used the transgenic Arabidopsis line described above to examine the wound inducibility of 4CL and the 4CL1-GUS gene fusion. Figure 7A shows that by 2 h after wounding mature Arabidopsis leaves, 4CL transcripts had accumulated to high levels. This RNA accumulation was transient, and was greatly reduced by 24 h after wounding. When a duplicate northern blot was hybridized to a GUS

probe, a similar pattern of GUS RNA accumu- lation was noted (Fig. 7B). Furthermore, strong GUS expression was histochemically detected at the cut surfaces of leaf discs incubated for 24 h prior to staining (Fig. 6P), or at cut surfaces on intact leaves (not shown). In control experiments, no GUS activity at cut surfaces was observed if the tissue was stained immediately after wound- ing (not shown). We conclude that the Arabidopsis 4CL gene is wound responsive and that this gene and the parsley 4CL1 promoter respond similarly to the wound stimulus in these Arabidopsis plants.

In another set of experiments, the inducibility of 4CL expression after infection of Arabidopsis with virulent and avirulent bacterial pathogens

Fig. 6. In situ localization of GUS activity and lignin deposition in Arabidopsis plants transgenic for a parsley 4CL1-GUS fusion. A-D. Seedlings were stained for GUS activity 2 days (A), 3 days (B), 5 days (C), and 10 days (D) after germination. E-J . Lo- calization of lignin in seedlings by staining with basic fuchsin. Whole 2-day old seedlings (E, F) of the roots (G, H) and cotyle- dons (I, J) of 3-day old seedlings were examined. E, G, I. Phase contrast images. F, H, J. The same images viewed under epifluorescence. Arrows indicate differentiated tracheary elements in which lignin deposition is not yet detectable. K, L. Cotyle- dons and roots of 3-day old seedlings stained for GUS activity. M-O. Portions of the primary root of a 10-day old plant stained for GUS activity, showing activity associated with lateral root differentiation. P. Leaf disk from a mature plant incubated 24 h on MS medium before staining for GUS activity.

880

was investigated. Arabidopsis ecotype Columbia is susceptible to bacterial leaf spot disease caused by the crucifer pathogen Pseudomonas syringae pv. maculicola (P.s.m.). P.s.m strain 4326 causes strong disease symptoms on race Columbia, in- cluding water-soaked lesions and spreading chlo- rosis (compatible interaction) [8, 44]. The viru- lence of P.s.m is attenuated by the presence of the bacterial avirulence gene avrB, as evidenced by the reduction in disease symptoms, significant re- duction of bacterial multiplication in planta, and increased or earlier expression of defense-related genes caused by P.s.m strain 4326/avrB (incom- patible reaction) [44]. In Columbia plants in- fected with P.s.m strain 4326/avrB, 4CL RNA accumulation was transiently induced within 3 h after inoculation and reached a maximal level within 6 h (Fig. 8). In contrast, there was no in- duction of4CL mRNA accumulation by 30 h after inoculation of Columbia plants with P.s.m. strain 4326 lacking avrB (Fig. 8). Similar results were

I

I -

Hours after infiltration

Fig. 8. 4CL mRNA accumulation in Arabidopsis ecotype Co- lumbia plants inoculated with Pseudomonas syringae pv. ma- culicola strains. Northern blots were prepared from total RNA (10 #g per lane) extracted from leaves harvested at the indi- cated times after bacterial infiltration, and hybridized to an At4CL probe. Open circles, RNA from leaves infiltrated with P. syringae pv. maculicola stain 4326 with an empty pDSK519 vector. Filled circles, leaves infiltrated with P. syringae pv. maculicola strain 4326 containing the cloned avrB gene in pDSK519. The hybridization signals were quantified using a Phosphorlmager and were normalized to hybridization signals obtained in each lane when blots were re-hybridized to an rRNA probe.

obtained with RNA prepared from two indepen- dent sets of plants.

Discussion

We have shown that 4CL is likely to be encoded by a single gene in Arabidopsis, whose expression is activated both developmentally and by such stresses as wounding and pathogen infection. While restriction fragments that very weakly cross-hybridized to 4CL were observed on ge- nomic Southern blots (Fig. 3), these did not cross-hybridize strongly at reduced stringency (not shown). This is in contrast to the behavior of divergent 4CL gene family members in poplar [1] and tobacco (D. Lee and C. Douglas, in preparation) which weakly cross-hybridize at high stringency, but strongly cross-hybridize under the low stringency washing conditions we used here. Thus, we feel it is unlikely that the weakly hy- bridizing bands in Fig. 3 represent additional 4CL gene family members, and that it is likely that 4CL is a single-copy gene in Arabidopsis. Although 4CL has been cloned from several plants, to our knowledge Arabidopsis is the first plant examined in which molecular data suggests that 4CL is present in a single copy. These results are in agreement with those of Trezzini et al. [41], who identified a putative Arabidopsis 4CL genomic clone and showed that a 4CL cross-hybridizing fragment from the clone hybridized with a simple pattern to an Arabidopsis Southern blot.

Given that there is a single 4CL gene in Ara- bidopsis, it follows that there must be a single form of the 4CL enzyme which is capable of utilizing differently substituted cinnamic acid derivatives, for example 4-coumaric acid (4-hydroxy-cin- namic acid) used in the biosynthesis offlavonoids, and ferulic (4-hydroxy, 3-methoxy-cinnamic acid) and sinapic (4-hydroxy, 3,5-di-methoxy-cinnamic acid) acids used in the biosynthesis of lignin. This is in contrast to some plants (e.g., soybean [22] and poplar [ 13]) that appear to have catalytically distinct isoforms of the enzyme encoded by mul- tiple genes [ 1, 42] capable of distinguishing be- tween such substrates. Because 4CL activity

specified by the single Arabidopsis gene is required for the biosynthesis of all classes of phenylpro- panoid-derived natural products requiring CoA esters of substituted cinnamic acids as substrates, the expression of Arabidopsis 4CL at multiple times and places during Arabidopsis growth and development when such diverse compounds ac- cumulate is to be expected.

In the Arabidopsis seedlings and mature plants we examined, 4CL expression was most clearly correlated with lignin deposition during vascular system differentiation. The highest levels of Ara- bidopsis 4CL expression were observed in bolting stems (Fig. 4B), in which stem tissue becomes highly lignified [7]. Relatively low levels of 4CL expression were observed in whole seedlings or seedling shoots (Fig. 4A), which correlates well with the low amount of lignin in 4-day old seed- lings on a fresh weight basis [7]. As well, the appearance of 4CL transcripts in seedlings 3 days after germination (Fig. 4A) was correlated with the beginning of lignin deposition in differentiated tracheids in cotyledons and roots of these seed- lings (Fig. 6E-J). The timing of lignin deposition in these roots and cotyledons is in good agree- ment with that observed by Dharmawardhana et al. [7]. Therefore, 4CL expression appears to be associated with lignin deposition and to be triggered during tracheary element maturation, suggesting that 4CL expression may be useful as a marker for xylem differentiation during vascu- lar system development in Arabidopsis. The higher levels of 4CL expression in seedling roots is likely to be due to the requirement for the biosynthesis of additional phenylpropanoid compounds (e.g., flavonoids) in non-vascular root tissue, where 4CL1-GUS expression (Fig. 6L) and PAL1-GUS expression [34] is also high.

In other studies, high expression of Arabidopsis PAL, CHS and other anthocyanin biosynthetic genes was reported in 3- to 4-day old seedlings, and was correlated with the accumulation of an- thocyanin pigments in the hypocotyls and coty- ledons of these seedlings [24, 34]. Under the light conditions in which we grew seedlings, little an- thocyanin pigmentation was observed, suggesting that gene expression associated with anthocyanin

881

biosynthesis was poorly activated. We would pre- dict that in 4CL expression would parallel that of PAL and CHS in Arabidopsis seedlings grown under higher light intensities.

The patterns of 4CL1-GUS expression during seedling development, measured either fluoro- metrically or histochemically, correlate well with 4CL mRNA accumulation in the same seedling populations. Thus, paralleling 4CL mRNA accu- mulation, GUS specific activity increased mark- edly between two and three days post-germination and was much higher in roots than shoots of seedlings (Fig. 5). Histochemical assay of GUS activity showed that vascular-specific 4CL1-GUS expression appeared 3 days after germination at a time when lignin was initially incorporated into tracheid cell walls. Comparison of the patterns of xylem differentiation (Fig. 6I), lignin deposition (Fig. 6J), and GUS activity (Fig. 6K) in 3-day old cotyledons suggests that 4CL1-GUS expression, like 4CL expression, is a marker for later stages of xylem differentiation. The patterns of 4CL1- GUS expression in seedlings are also similar to those directed by the Arabidopsis PAL1 promoter [34].

In mature plants, patterns of 4CL1-GUS ex- pression were also similar to those of Arabidopsis 4CL and those reported for an Arabidopsis PAL1- GUS fusion. Histochemical assays showed that 4CL1-GUS expression was high in the xylem of bolting stems (data not shown), where the high- est levels of 4CL mRNA were detected (Fig. 4B). In mature flowers, 4CL1-GUS expression was localized in anthers and at the bases and tips developing siliques (data not shown), sites where PAL1 promoter also directed GUS expression [34]. In flower buds, prior to anthesis, no 4CL1- GUS expression was observed histochemically (data not shown), correlating with the low levels of 4CL mRNA accumulation (Fig. 4B) and lack of expression of the PAL1-GUS expression [34] in immature flowers. Finally, levels of Arabidopsis 4CL and GUS RNA were very low in mature leaves before wounding (Fig. 7), but the accumu- lation of both RNAs was rapidly stimulated by wounding, suggesting coordinate activation of the endogenous 4CL gene and the 4CL1-GUS trans-

882

gene by a wound-generated signal. Similar rapid accumulation of PALl and GUS RNA was re- ported in Arabidopsis plants transgenic for a PAL1-GUS fusion [34]. Taken together, our re- suits suggest conservation of the cis-acting ele- ments which direct developmental expression of Arabidopsis 4CL, parsley 4CL1, and Arabidopsis PAL1 genes during Arabidopsis development.

An interesting pattern of 4CL1-GUS expres- sion was observed in roots, where high GUS ac- tivity was localized in cortical as well as vascu- lar tissue in young primary and lateral roots, but was absent in at the tips of most roots (Fig. 6L, M). Activity seemed to decrease in the older parts of roots (Fig. 6M). This pattern of expression is similar to that directed by the Arabidopsis PAL1 promoter [34]. In contrast to PAL1-GUS expres- sion, however, the 4CL1-GUS fusion was ex- pressed at the sites of lateral root initiation (Fig. 6N) and at the tips of young lateral roots (Fig. 60). This pattern of expression is similar to that specified by the bean CHS8 and 4CL1 pro- moters in transgenic tobacco [15, 38], and could be related to the biosynthesis of cell division- promoting phenolic compounds [35], and/or the biosynthesis of flavonoids which regulate auxin transport, in the early stages of lateral root for- mation and growth.

The coordinate expression of Arabidopsis PAL and 4CL genes extends to their induced expres- sion in an incompatible interaction with a bacte- rial pathogen. The pattern of 4CL mRNA accu- mulation in response to infection of Arabidopsis Columbia plants by a P.s.m. strain containing avrB (Fig. 8) is indistinguishable from the pattern of PAL1 and PAL2 mRNA accumulation in the same plants [44]. Coordinate activation of PAL and 4CL expression has also been documented in several other pathogen-infected plants [12, 29, 37].

While it is apparent that PAL and 4CL genes are coordinately regulated in Arabidopsis and other plants, the mechanisms controlling their coordinate expression have not been defined. The apparent conservation of putative cis-acting ele- ments within the promoters of PAL and 4CL genes [5, 28] has led to the suggestion that the

presence or activity of transcriptional regulators which bind in common to these genes could lead to their coordinate transcriptional regulation. The specific activation of 4CL expression after infec- tion with a bacterial pathogen carrying the avrB gene (Fig. 8), in combination with previously pub- lished results showing similar activation of PAL expression by the same pathogen carrying either the avrB or avrRPT2 gene [8, 44] suggests that coordinate PAL and 4CL gene activation in in- compatible interactions could be mediated by the Arabidopsis resistance genes RPMI/RPS3 [4, 6] and RPS2 [31], which recognize avrB and avr- RPT2, respectively. The Arabidopsis 4CL gene could allow facile testing of the hypothesis that activation of phenylpropanoid metabolism is con- trolled by Arabidopsis resistance genes in incom- patible interactions, similarly to the control of the Arabidopsis ELI3 defense gene expression by RPMI/RPS3 [21 ].

Acknowledgements

This work was supported by a Research Grant from the Natural Science and Engineering Re- search Council (NSERC) of Canada to C.J.D., National Institutes of Health Grant GM45570 to K.R.D., and an NSERC Graduate Fellowship to D.L. We are grateful to Roxanna Moslehi, who helped with Agrobacterium-mediated transforma- tions of Arabidopsis as part of an undergraduate research project.

References

1. Allina SM, Douglas CJ: Isolation and characterization of the 4-coumarate:CoA ligase gene family in a poplar hy- brid. Plant Physiol 105 (Suppl): 154 (1994).

2. Becker-Andr6 M, Schultze-Lefert P, Hahlbrock K: Structural comparison, modes of expression, and putative cis-acting elements of the two 4-coumarate:CoA ligase genes in potato. J Biol Chem 266:8551-8559 (1991).

3. Bevan M, Schuftlebottom D, Edwards K, Jefferson R, Schuch W: Tissue- and cell-specific activity of a phenyl- alanine ammonia-lyase promoter in transgenic plants. EMBO J 8:1899-1906 (1989).

4. Bisgrove SR, Simonich MT, Smith NM, Sattler A, Innes RW: A disease resistance gene in Arabidopsis with speci-

ficity for two different pathogen avirulence genes. Plant Cell 6:927-933 (1994).

5. da Costa e Silva O, Klein L, Schmelzer E, Trezzini GF, Hahlbrock K: BPF-1, a pathogen-induced DNA-binding protein involved in the plant defense response. Plant J 4: 125-135 (1993).

6. Debener T, Lehnackers H, Arnold M, Dangl JL: Identi- fication and molecular mapping of a single Arabidopsis thaliana locos determining resistance to a phytopatho- genic Pseudomonas syringae isolate. Plant J 1:289-302 (1991).

7. Dharmawardhana DP, Ellis BE, Carlson JE: Character- ization of vascular lignification in Arabidopsis thaliana. Can J Bot 70:2238-2244 (1992).

8. Dong X, Mindrinos M, Davis K, Ausubel F: Induction of Arabidopsis defense genes by virulent and avirulent Pseudomonas syringae strains and by a cloned avirulence gene. Plant Cell 3:61-72 (1991).

9. Douglas CJ, Hauffe KD, Ites-Morales ME, Ellard M, Paszkowski U, Hahlbrock K, Dangl JL: Exonic se- quences are required for elicitor and light activation of a plant defense gene, but promoter sequences are sufficient for tissue specific expression. EMBO J 10:1767-1775 (1991).

10. Doyle JJ, Doyle JL: Isolation of plant DNA from fresh tissue. Focus 12:13-15 (1990).

11. Feinbaum RL, Ausubel FM: Transcriptional regulation of the Arabidopsis thaliana chalcone synthase gene. Mol Cell Biol 8:1885-1892 (1988).

12. Fritzemeier K-H, Cretin C, Kombrink E, Rohwer F, Taylor J, Scheel D, Hahlbrock K: Transient induction of phenylalanine ammonia-lyase and 4-coumarate:CoA ligase mRNAs in potato leaves infected with virulent or avirulent races ofPhytophthora infestans. Plant Physio185: 34-41 (1987).

13. Grand C, Boudet A, Boudet AM: Isoenzymes of hy- droxycinnamate: CoA ligase prom poplar stems proper- ties and tissue distribution. Planta 158:225-229 (1983).

14. Hahlbrock K, Scheel D: Physiology and molecular biol- ogy of phenylpropanoid metabolism. Annu Rev Plant Physiol Plant Mol Biol 40:347-469 (1989).

15. Hauffe KD, Paszkowski U, Schulzelefert P, Hahlbrock K, Dangl JL, Douglas CJ: A parsley 4CL-1 promoter fragment specifies complex expression patterns in trans- genic tobacco. Plant Cell 3:435-443 (1991).

16. Jefferson RA: Assaying chimeric genes in plants: the GU S gene fusion system. Plant Mol Biol Rep 5:387-405 (1987).

17. Jorgensen A, Cuellar E, Thompson F: Modes and tem- pos in the evolution of nuclear encoded ribosomal RNA genes in legumes. Carnegie Inst of Washington Year Book 81:98-101 (1982).

18. Joshi CP: An inspection of the domain between putative TATA box and translation start site in 79 plant genes. Nucl Acids Res 15:6643-6653 (1987).

19. Keen NT, Buzzell R: New disease resistance genes in

883

soybean against Pseudomonas syringae pv. glycinea: evi- dence that one of them interacts with a bacterial elicitor. Theor Appl Genet 81:133-138 (1991).

20. Keen NT, Tamaki S, Kobayashi D, Trollinger DJ: Im- proved broad host range plasmids for DNA cloning in gram-negative bacteria. Gene 70:191-197 (1988).

21. Kiedrowski S, Kawalleck P, Hahlbrock K, Somssich IE, Dangl JL: Rapid activation of a novel plant defense gene is strictly dependent on the Arabidopsis RPM1 disease resistance locus. EMBO J 11:4677-4684 (1992).

22. Knobloch K-H, Hahlbrock K: Isoenzymes of p-couma- rate: CoA ligase from suspension cultures of Glycine max. Eur J Biochem 52:311-320 (1975).

23. Kozak M: Point mutations define a sequence flanking the AUG initiatior codon that modulates translation by eukaryotic ribosomes. Cell 44:283-292 (1986).

24. Kubasek WL, Shirley BW, McKillop A, Goodman HM, Briggs W, Ausubel FM: Regulation of ftavonoid biosyn- thetic genes in germinating Arabidopsis seedlings. Plant Cell 4:1229-1236 (1992).

25. Lamb CJ, Lawton MA, Dron M, Dixon RA: Signals and tranduction mechanisms for activation of plant defenses against microbial attack. Cell 56:215-224 (1989).

26. Liang X, Dron M, Schmid J, Dixon RA, Lamb CJ: De- velopmental and environmental regulation of a phenyl- alanine ammonia-lyase beta-glucuronidase gene fusion in transgenic tobacco plants. Proc Natl Acad Sci USA 86: 9284-9288 (1989).

27. Logemann J, Schell J, Willmitzer L: Improved method for the isolation of RNA from plant tissues. Anal Biochem 163:16-20 (1987).

28. Lois R, Dietrich A, Hahlbrock K, Schulz W: A phenyl- alanine ammonia-lyase gene from parsley: structure, regu- lation and identification of elicitor and light responsive cis-acting elements. EMBO J 8:1641-1648 (1989).

29. Lois R, Hahlbrock K: Differential wound activation of members of the phenylalanine ammonia-lyase and 4-cou- marate:CoA ligase gene families in various organs of parsley plants. Z Naturforsch C 47:90-94 (1992).

30. Lozoya E, Hoffmann H, Douglas CJ, Schulz W, Scheel D, Hahlbrock K: Primary structure and catalytic prop- erties of isoenzymes encoded by the two 4-coumarate- :CoA ligase genes in parsley. Eur J Biochem 176: 661- 667 (1989).

31. Mindrinos M, Katagiri F, Yu G-L, Ausubel FM: The A. thaliana disease resistance gene RPS2 encodes a nucle- otide-binding site and leucine-rich repeats. Cell 78: 1089- 1099 (1994).

32. Moniz de Sfi. M, Subramaniam R, Williams FE, Douglas CJ: Rapid activation of phenylpropanoid metabolism in elicitor-treated hybrid poplar (Populus trichocarpa Torr and Gray × Populus deltoides Marsh) suspension- cultured cells. Plant Physiol 98:728-737 (1992).

33. Murashige T, Skoog F: A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol Plant 15:473-497 (1962).

884

34. Ohl S, Hedrick SA, Chory J, Lamb CJ: Functional prop- erties of a phenylalanine ammonia-lyase promoter from Arabidopsis. Plant Cell 2:237-248 (1990).

35. Orr JD, Lynn DG: Biosynthesis of dehydrodiconiferyl alcohol glucosides: implications for the control of tobacco cell growth. Plant Physiol 98:343-352 (1992).

36. Sambrook J, Fritsch EF, Maniatis T: Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Press, Cold Spring Harbor, NY (1989).

37. Schmelzer E, Kruger-Lebus S, Hahlbrock K: Temporal and spatial patterns of gene expression around sites of attempted fungal infection in parsley leaves. Plant Cell 1: 993-1001 (1989).

38. Schmid J, Doerner PW, Clouse SD, Dixon RA, Lamb CJ: Developmental and environmental regulation of a bean chalcone synthase promoter in transgenic tobacco. Plant Cell 2:619-631 (1990).

39. Sharma YK, Davbis KR: Ozone-induced expression of stress-related genes in Arabidopsis thaliana. Plant Physiol 105:1089-1096 (1994).

40. Subramaniam R, Reinold S, Molitor EK, Douglas CJ: Structure, inheritance, and expression of hybrid poplar (Populus trichocarpa x Populus deltoides) phenylalanine ammonia-lyase genes. Plant Physiol 102:71-83 (1993).

41. Trezzini GF, Horrichs A, Somssich IE: Isolation of pu-

tative defense-related genes from Arabidopsis thaliana and expression in fungal elicitor-treated cells. Plant Mol Biol 21:385-389 (1993).

42. Uhlmann A, Ebel J: Molecular cloning and expression of 4-coumarate:coenzyme A ligase, an enzyme involved in the resistance of soybean (Glycine max L.) against patho- gen infection. Plant Physiol 102:1147-1156 (1993).

43. Valvekens D, Van Montagu M, Van Lijsebettens M: Agrobacterium tumefaciens-mediated transformation of Arabidopsis thaliana root explants by using kanamycin selection. Proc Natl Acad Sci USA 85:5536-5540 (1988).

44. Wanner LA, Mittal S, Davis KR: Recognition of the avirulence gene avrB from Pseudomonas syringae pv. gly- cinea by Arabidopsis thaliana. Mol Plant-Microbe Interact 6:582-591 (1993).

45. Wanner LE, Li G, Ware DH, Somssich I, Davis KR: The phenylalanine ammonia-lyase gene family in Arabidopsis thaliana. Plant Mol Biol 27:327-338 (1995).

46. Wu SC, Hahlbrock K: In situ localization of phenylpro- panoid-related gene expression in different tissues of light- grown and dark-grown parsley seedlings. Z Naturforsch C 47:591-600 (1992).

47. Zhao Y, Kung SD, Dube SK: Nucleotide sequence of rice 4-coumarate:CoA ligase gene, 4-CL.1. Nucl Acids Res 18:6144 (1990).