Plant Physiol. (1 997) 115: 81 7-826

Jasmonic Acid-Dependent and hdependent Signaling Pathways Control Wound-Induced Gene Activation in Arabidopsis fhaliana’

Elena Titarenko, Enrique Rojo, José León, and Jose J. Sánchez-Serrano* Centro Nacional de Biotecnología, Consejo Superior de lnvestigaciones Científicas, Campus de Cantoblanco Universidad Autónoma de Madrid, 28049 Madrid, Spain

The mechanisms by which plants regulate wound- Plant response to mechanical injury includes gene activation both induced gene expression are not well understood. It has at the wound site and systemically in nondamaged tissues. The been shown that wounding triggers an increase in the model developed for the wound-induced activation of the protein- endogenous levels of the plant growth regulator JA (Creel- ase inhibitor II (Pin.2) gene in potato (Solanum tuberosum) and man et al., 1992; Albrecht et al., 1993; Laudert et al., 1996), tomato (Lycopersicon esculentum) establishes the involvement of and this increase is required for gene activation upon the plant hormones and jasmonic acid (JA) as key wounding (Pefia-Cortés et al., 1993). Application of exog- components of the wound signal transduction pathway. To assess in enous JA or its methyl ester at physiological concentrations Arabidopsis tbaliana the role of these plant hormones in regulating can induce a variety of wound-responsive genes, including wound-induced gene expression, we isolated wound- and JA- Pin2 and Vsp (Mason and Mullet, 1990; Farmer et al., 1992). inducible genes by the differential mRNA display technique. Their In potato and tomato proteinase inhibitor genes can also patterns of expression upon mechanical wounding and hormonal be activated by oligosaccharide fragments generated from treatments revealed differences in the spatial distribution of the transcripts and in the responsiveness of the analyzed genes to both plant and pathogen cell walls (Bishop et al., 1981) and abscisic acid and IA. A correlation can be established between by the 18-amino acid polypeptide (Pearce et al., sensitivity to JA and the accumulation of the transcripts in systemic 1991). Systemin was found to act before JA in the wound tissues upon wounding. A comparative study of the wound response signal transduction chain (PeÍía-Cortés et al., 1995). How- in wild-type and JA-insensitive coil mutant plants indicated that in ever, a role for systemin, or any related peptide hormone, A. tbaliana wound signals are transmitted via at least two different in the transmission of the wound signal has been shown so pathways. One of them does not involve JA as a mediator and is far only in potato and tomato. The phytohormone ABA has preferentially responsible for gene activation in the vicinity of the also been postulated to participate in wound signaling in wound site, whereas the other requires JA perception and activates these plant species (PeÍía-Cortés et al., 1989, 1995). gene expression throughout the aerial part of the plant. Since JA, but not systemin, is able to activate Pin2 expres- sion in the ABA-deficient tomato (sitiens) and potato (droopy) mutants, in which Pin2 genes are not induced by mechanical Plants react to wounding and pest attack by activating a wounding, the site for ABA action has been located between variety of genes. Some of them are expressed only in the systemin and JA (PeÍía-Cortés et al., 1996). These compo- vicinity of the wound site, whereas others are also system- nents would therefore link mechanical injury with an intra- ically activated in the nondamaged parts of injured plants cellular increase in JA concentration, which results in gene (Bowles, 1990). Well-characterized examples of systemi- activation. Recently, the requirement of in mediat- cally inducible genes are the proteinase inhibitor I1 (Pin2) ing wound-induced gene activation has been demonstrated gene family of potato (Solanum tuberosum) and tomato (Ly- in tomato (ODonnell et al., 1996), in which it acts together with JA to regulate Pin2 gene expression. copersicon esculentum; PeÍía-Cortés et al., 1988; Farmer and Arabidopsis tkaliana has served as a model plant to study Ryan, 1990) and two vegetative storage protein genes different hormone signal transduction pathways. A num- (VspA and VspB)of soybean (Glycine max; Mason and Mul- ber of ethylene-response mutants have been isolated, de- let, 1990). fining severa1 loci in the ethylene signal transduction path- way (Kieber et al., 1993; Chang, 1996).Arabidopsis mutants This research was supported in part by the European Com- affected in their responses to ABA (Leung et al., 1994; munities’ Biotech Program, as part of the Project of Technological Meyer et al., 1994), (Hobbie et al., 1994), and cyto- Priority 1993-1996. Financia1 support was also provided by the kinins (Kakimoto, 1996) have also been characterized. Spanish Comisión Interministerial de Ciencia y Tecnología (grant Three independent JA-insensitive mutants have already nos. BI093-0678-C02-02, BI094-1502-CE, and BI096-0532-C02- been identified. jarl was isolated by phenotypic screening 01). J.L. was the recipient of a postdoctoral contract from the for plants with root growth insensitive to inhi- Spanish Ministerio de Educación y Ciencia. * Corresponding author; e-mail jjssQsamba.cnb.uam.es; fax 34- 1-585-4506. Abbreviations: DD, differential display; JA, jasmonic acid. 81 7 81 8 Titarenko et al. Plant Physiol. Vol. 115, 1997

bition (Staswick et al., 1992). The coil mutant was isolated murine leukemia virus reverse transcriptase (GIBCO-BRL). because of its resistance to the structurally related, One-tenth of the reverse-transcription mixture was used as chlorosis-inducing bacterial toxin coronatine (Feys et al., a template in a PCR reaction containing a given T,,MN 1994). Upon JA treatment, both of these mutants showed primer in combination with one arbitrary 10-base primer reduced accumulation of 29- and 31-kD proteins, which (DD 10-mer kit, Operon Technologies, Alameda, CA). were subsequently identified as VSPs that are immunolog- PCR was performed using Taq polymerase (Perkin- ically related to soybean VspA and VspB (Staswick et al., Elmer), and reaction conditions were as follows: 94°C for 1992; Benedetti et al., 1995). The jasmonate-insensitive mu- 30 s; 40°C for 2 min; 72°C for 30 s for 40 cycles, followed by tants jinl and jin4 exhibited a reduction in the expression of extension at 72°C for 5 min. Aliquots of PCR reactions were the jasmonate-responsive gene AtVsp, which was induced run through a 6% sequencing gel. On the same gel, reac- in the leaves of seedlings upon treatment with methyl tions obtained from two different batches of mRNA were jasmonate (Berger et al., 1996). jin4 and jarl mutated loci compared. Only bands consistently found to be differen- may be allelic. tially amplified were selected and analyzed further. The To further understand the wound signal transduction bands of interest were eluted in 100 FL of sterile water. pathway and the role of JA in this process, a search for Three microliters of each elution were reamplified using nove1 wound- and JA-inducible genes was undertaken in the appropriate pair of primers. The amplified fragments A. tkaliana to (a) obtain molecular markers to monitor were purified from low-temperature melting point agarose wound-induced gene activation, (b) elucidate the role of JA (SeaPlaque FMC, Rockland, ME) using a purification kit in both local and systemic wound-induced gene activation, (Qiaex, Qiagen, Düsseldorf, Germany), cloned in pUC18 and (c) characterize wound- and JA-responsive genes and vector, and sequenced on both strands using the Sequenase subsequently use their promoters in a transgenic approach kit (version 2.0, Amersham). Partia1 cDNA clones (3’) were for the isolation of new signaling mutants. used as probes for northern and Southern hybridization analyses.

MATERIALS AND METHODS Nucleic Acid lsolation and Analysis Plant Material and Treatments Total RNA was isolated as described previously (Loge- Arabidopsis tkaliana ecotype Landsberg erecta plants were mann et al., 1987).RNA samples were separated on agarose- grown in soil in a greenhouse at 22°C with a 12-/12-h formaldehyde gels and transferred onto Hybond-N nylon light / dark period. For wounding experiments one-half of membranes (Amersham) in 20x SSC using standard proce- the rosette leaves of 4- to 6-week-old plants were wounded dures (Sambrook et al., 1989). Equal RNA loading was vi- with forceps, and the injured leaves (local), unwounded sualized by staining the rRNAs with ethidium bromide (40 rosette leaves (systemic), and cauline leaves (upper) were ng/ pL). Replica blots were prepared and sequentially hy- harvested at different times after wounding (0.5,1, 1.5,2,4, bridized to at most three probes. Probes were labeled with 8, 24, and 48 h). A. thaliana seeds from an F2 population [cx-~~P]~CTPusing the Rediprime kit (Amersham). The Vsp segregating for the coil mutant were kindly provided by cDNA (clone 108811) used as probe was obtained from the Dr. J.G. Turner (University of East Anglia, Norwich, UK) Arabidopsis Biological Resource Center (The Ohio State and were grown and selected as described previously University, Columbus). Hybridization was at 42°C in 0.25 M (Benedetti et al., 1995). Wounding of coil mutant plants phosphate buffer, pH 7.2,0.25 M NaCl, 7% SDS, 1mM EDTA, was done as described above for wild-type plants. pH 8,10% PEG 6000,100 Fg/mL denatured salmon sperm To examine hormone action, plants were grown in the DNA, and 50% formamide (Amasino, 1986). After hybrid- greenhouse as described above and were sprayed with a 50 ization radioactive probes were stripped by washing the PM solution of ABA (mixed isomers, Sigma) or JA (mixed blots twice with 0.5% SDS in distilled water at 65°C. isomers, Apex Organics, Devon, UK). Rosette leaves were Total DNA was isolated from 1 g of frozen tissue as collected 2, 4, 8, 24, and 48 h after treatment. Tissue from described previously (Dellaporta et al., 1983). Ten micro- different A. thaliana organs (roots, stems, different types of grams of DNA was digested with restriction enzymes and leaves, flowers, and green siliques) was harvested from 4- subsequently separated on a 0.8% agarose gel. DNA trans- to 6-week-old plants grown in soil. Plants used for DNA fer and hybridization were done according to standard purification were grown from sterilized seeds on agar procedures (Sambrook et al., 1989). Sequence comparison plates with 0.5 x Murashige-Skoog medium (Murashige was performed using the GCG package (version 8, Genetics and Skoog, 1962) supplemented with 1%SUC. Upper parts Computer Group, University of Wisconsin, Madison). of the plants were harvested after 21 d of growth at 22°C under continuous light. RESULTS DD Technique Cloning of A. thaliana Wound- and JA-lnducible genes DD was performed as described previously (Liang and DD is a powerful tool for the detection of differentially Pardee, 1992). Total RNA (1 pg) was reverse-transcribed expressed genes (Liang and Pardee, 1992). We have used using T,,MN 3’ primers (where M stands for a mixture of this technique to identify genes with expression induced by G, A, and C, and N stands for A, C, G, or T) using Moloney mechanical damage. To this end, we have compared the Wound Signaling in Arabidopsis 819

mRNA population present in the rosette leaves of fully them. We have thus isolated nearly full-length cDNAs for developed A. thaliana plants with that expressed upon cDND D e Asomth clones f eo . However, sequencina f go wounding locall,e botth n hi y damagee th d n leavei d an s full-length Wr3 cDNA has not revealed any significant systemic, undamaged ones representativA . e examplf eo similarit databasee th n yi s (not shown). suc n experimena h s showi te lan th n Figur i enn I . 1 e To investigate whether ther lina s eki between wounding corresponding to the wounded tissue, some differentially and JA in A. thaliana, the mRNA population from JA- expressed bands can be seen. They were isolated, se- treated leave s comparewa s d with that from nontreated quenced d use s an probe,a d n northeri s n hybridization plants using the DD technique. Three JA-induced cDNA experiments to determine their transcript levels in control clones were isolated and their relevant characteristics are and wounded plants (see below). Among them, four cDNA listed in Table II. No homologies were found in the data- clones were found to hybridize to transcripts that accumu- bases for the Jrl and ]r2 (jasmonate-responsive 1 and 2) late leaven di s upon wounding, whereas their expression cDNAs obtaine gelsD d D contrastn - I fro . e de mth e th , levels wer r eabsene leaveo th ver w f n controi o ylo st l duced partial Jr3 amino acid sequence was 38% identical to plants. Relevant characteristics of these clones are summa- previousle th y described llrl gene fro . thalianamA (Bartel rize Tabln di . eI Finkd an , 1995) isolatioe Th . sequencind nan nearla f go y Significant sequence similarit geneo yt s from nonplant full-length Jrl cDNA clone did not reveal any homology to sources was found for the wound-induced Tat (tyrosine sequences in the databases. In contrast, the sequence of a amino transferase), Ck (choline kinase), and Aco (acyl CoA nearly full-length }r1 cDNA clone detected significant sim- oxidase) clones. The DD technique used for gene isolation ilarities to aminotransferase sequences (not shown). Nev- r stud e icorrespondinou nth y f o yielde d e 3en 'th d g ertheless, Jr2 is only 50% identical to Tat in their deduced cDNAs (Liang and Pardee, 1992). Because these parts rou- amino acid sequences. tinely exhibit lower conservation than protein-codin- re g In addition cDNa , A clone with homolog previe th o yt - t succeeno d gionsfindinn di di e w , g homologie somr sfo e ously described Vsp from soybean (Glycine max; Masod nan of the genes we isolated, W>3 (wound-responsive 3) among Mullet, 1990; Staswick, 1990) was obtained from the Ara- bidopsis Biological Resource Center and used in our study. This clone corresponds to the previously described wound- and JA-inducibl . thalianaeA Vsp cDNA (Berger et al, 1995). II Southern hybridization experiments were performed to investigate the copy number of the newly identified W C W S . thalianaA cDNAe th n i sgenome e resultTh .f suco s h experiments are presented in Figure 2. According to the data obtaine l analyzedal d wound JA-inducibld -an e genes are present in few copies (most likely a single copy) in the genome of A. thaliana.

Organ-Specific Expression in Noninduced Plants We analyzed the accumulation of transcripts corre- spondin isolatee th o gt d cDNA clone differenn si t organf so . thaliana.A e resultTh s from these experiment e prear s - sente e gene Figurn r mosi dth Fo f so . t e3 teste basala d , albeit low, leve f expressioo l n coul detectee db somn di e part f nontreateso cDNAp dVs plantsd san wer k C . e found to hybridize on northern blots to different transcripts of kb2 1. , respecd an 1 -1. approximatel d k1 ban 2. d an 6 y1. tively transcripk (TableC d e II) s an observeTh . sI wa t d principall thas roots n yJrl.i f wa o t s a Bot, transcriptp hVs s were strongly expresse rootsn di , flowers siliquesd an , e .Th expressio RNAp Vs differenf n si no . thalianaA t organs swa previously described (Berger et al., 1995), but, in contrast to our results, no expression in roots or green siliques was . thalianaA f o FigurD D lea . e1 f mRNAs autoradiograe Th . m shows detected. There are two Vsp genes in A. thaliana, and the duplicate ) preparesampleII d an I sd( from rosette leave f controso l probe use y Berge . db (1995 al s derivet e rwa ) d froma frod plantdamagee man th ) s(C d rosett eunwoundedd leavean ) s(W , different Vsp cDNA; this may explain the slightly different systemic leavef planto ) s(S s harveste 4 dh after woundinge Th . position f differentiallso y expressed cDNA indicatee sar asterisky db s genomic hybridization and gene expression results. Differ- (*). The marked bands shown in this picture are derived from the Wr3 ences in the age of the plants used or in growing conditions transcript. alsy oma accoun discrepancier fo t expression si n patterns. 820 Titarenko et al. Plant Physiol. Vol. 115, 1997

Table I. Relevant characteristics of the cDNA clones corresponding to wound-inducible genes iso- lated by DD Size of 3’ mRNA Highest-Scored Homology (Amino Acid Clone lnduction Fragment Length Sequences) bP kb Tat 489 1.7 Wounding 30% ldentity with Tat of Trypano- IA soma cruzi (Bontempi et al., 1993); 25% identity with human Tat (Rettenmeier et al., 1990); 23% identity with rat Tat (Har- grove et al., 1989).

Wr3 483 1 .o Wounding No significant similarity found. Ck 253 1.6 Wounding 50% ldentity with rat Ck’(Uchida 2.1 and Yamashita, 1992); 46% iden- tity with yeast Ck (Hosaka et al., 1989).

A co 543 2.6 Wounding 55% identity with rat peroxisomal component of Aco (Miyazawa et al., 1987); 52% identity with hu- man Aco (Aoyama et al., 1994); 45% identity with yeast Aco (Dmochowska et al., 1990).

Specific mRNA Accumulation in A. fhaliana ing. According to their expression patterns the analyzed Plants upon Wounding clones could be separated into three major groups. One group with clones Tat, Vsp, Jrl, and Jr2 exhibited Results from DD experiments indicated that mRNA ac- cumulation of the identified genes was higher in damaged similar or stronger induction in the leaves that were not leaves 4 h after wounding than in leaves of control plants directly wounded compared with that observed in locally or systemic (nonwounded) leaves of injured plants. To damaged leaves, although at the latest times after wound- ascertain the time course of induction upon wounding and ing, Jr2 transcript levels were higher in the locally damaged to elucidate whether transcript accumulation was re- tissue. Wr3 and Ck genes formed a second group with a stricted to the locally damaged tissue, northern analysis higher leve1 of transcript accumulation in the locally in- was performed with total RNA from different leaves (local, jured leaves and very little in the systemic leaves, where systemic, and upper) of both control and wounded plants higher transcript levels could be observed only shortly (Fig. 4). For a11 genes analyzed, large, transient increases in after wounding, especially in Wr3. The Aco and Ir3 genes the steady-state mRNA levels were observed upon wound- formed a third group. Soon after wounding, their expres-

Table II. Relevant characteristics of the cDNA clones corresponding to ]A-inducible genes isolated by DD Sequence similarities found in the databases for a nearly full-length Ir2 cDNA clone (discussed in “Results”) are not indicated. The Vsp clone encompasses most of the coding region and was obtained from the Arabidopsis Biological Resource Center. Size of 3’ mRNA Highest-Scored Homology (Amino Acid ‘Ione lnduction Fragment Length Sequences) bp kb Ir1 250 1.7 Wound i ng No significant similarity found. IA Ir2 21 7 1.5 Wounding No significant similarity found. IA ABA Ir3 490 1.7 Wound i ng 38% ldentity with llrl aminohydro- IA lase of A. thaliana (Bartel and Fink, 1995).

VSP 600 1.1 Wound i ng 47% ldentity with soybean VspA and 1.2 IA VspB (Mason and Mullet, 1990). Wound Signaling in Arabidopsis 821

TAT WR3 ACO CK wherea h afte 8 s r injury onl e mRNth y A e codinth r gfo E 11 B EHB EHB EHB smaller transcript was detectable. The northern experiment with mixed RNAs shown in Figure 5 confirmed the pres- „ o differentialltw enc f o e y expresse p transcriptsVs d . Wounding strongly induced two Ck transcripts in locally 23 - ^MV 9.4 - ^^ ^^ ^^ ^^ w*» damaged leaves, whereas only the smaller band was 6.5 - 4.3 - present in the control (nonwounded) plants (Fig. 4).

2.2- 2.0 - Hormonal Regulation of Wound-lnducible Genes The expression of some wound-inducible genes previ- ously described (suc s proteinasa h e inhibitors, chalcone synthase beeVsp)r s ,o ha n show strongle b o nt y inducey db application of exogenous JA (Creelman and Mullet, 1995). The three genes Jrl, Jr2, and Jr3 obtained from JA-treated plants were shown to be induced by mechanical injury,

JR1 JR2 JR3 VSP EHB EHB EHB EHB R St YLOL CL F Sq

' •

23 - TAT 9.4 - ^^ 9M • m ^^ 6.5 - 4.3 - -

2.2 - » VSP 2.0- f

- JR1

JR2 Figure 2. Southern analysis of the wound- and JA-inducible genes. A. thaliana genomic DNA (10 /xg per lane) was digested with fcoRI (E), H/ndlll subjecte(H)d SamHd an an , ) (B I Southero dt n analysis. JR3 Blots were hybridized with probes frowound-inducibl d JAe man -th e cDNAs. Size markers (in kilobase pairs) are indicated at the left.

ACO sion increased both in the damaged and in the systemically induced leaves to a similar extent. At later times, however, their expressio s restricte wa ne locall th o t dy damaged tissues. WR3 Large difference persistencn i s e wound-induceth f eo d levels of expression of these genes were observed. Whereas Wr3-, Aco-, /r2-induced an d levels were maintaineo t p du CK 48 e expressio hth , injuren ni d plant f Tat,o s Vsp, ]rl, Jr3, and Ck had returned to almost basal levels by 24 h after wounding (Fig. 4B)persistenc e transcripp Th . Vs e th f n ti eo e systemicallth y induced caulin et bee leaveno ns reha s - rRNA producibly observed. Wounding caused a rapid activation of the genes ana- lyzed s showA . n Figuri n , significan4A e t increasen i s transcript levels for Tat, Ck, Wr3, and ]r3 were already Figure 3. Constitutive expression of wound-responsive genes in the detectable 30 min after wounding, whereas Aco, Jrl, Jr2, different organs of A. thaliana plants. Northern analysis was per- transcripp Vs d an t accumulation started s interlaterwa t -.I formed with total RN r laneji5 Ag( pe ) from root (R), stem (St)d ,an rosette leaves from 4-week-old plants (YD, rosette leaves fro- m6 esting thahybridizino tw t g bands were detected wite hth week-old plants (OL) caulind an , e leaves (CL), flowers (F)sild -,an probesk C d Vs,an p which exhibited different temporad an l iques (Sq). Blots were hybridized to radioactive probes from the spatial expression patterns in wounded A. thaliana plants. wound-inducible cDNAs (see text for information concerning the largee Th r Vsp transcrip detectabls t wa peaked an h h ed2 1 cDNAs use probes)s da ethidiue Th . m bromide-stained rRNAe ar s after wounding in systemic leaves of injured plants, show contro a gelsA s na r loadinRN . fo l l al n gi 822 Titarenko et al. Plant Physiol. Vol. 115, 1997

O.Sh Ih 1.5h 2 h 2h 8h 24h 48h cuclsul sulsul s u clsulsu I s u Isu

TAT TAT 'ft'

VSP »•*•• VSP

JR1 JR1

JR2 JR2

JR3 JR3

ACO ACO

WR3 ••ff'fftfifttt WR3

CK CK Iffft •

rRNA rRNA

Figur . eTim4 e cours f wound-induceeo d mRNA accumulatio leaven i . thaliana. A f so Collection times correspon plano dt t tissues harveste dh8 0.5 4 afte d , 1,1.5 ran h 2 woundin, afted 24 , an r8 woundin, 2 gd (B)an .) Resultg(A representative sar e f thoso e obtaine separaten i d , independent experiments, c, Control, rosette leaves from unwounded plants; uc, cauline leaves from unwounded plants , locallI ; y wounded rosette leaves , unwounde;s d (systemic) rosette leaves , caulinu d ;an e (upper) leave f woundeso d plants. Five microgram f totao s l RNloades r Awa land blot pe dean s were hybridizeo t d radioactive probes of the wound-induced cDNAs, as indicated on the left.

suggesting tha y tregulat JAma e wound-inducible gene the identified wound-responsive genes were studied, and expressio . thaliana.A n ni potatn s I d tomatha oan A oAB the results are presented in Figure 6. been shown to be involved in Pin2 activation in response to A grou genef po s including Tat, Vsp,s wa Jrl, 3 Jr Jr2, d an wounding (Pena-Cortes et al., 1995). To assess whether the strongly induce suggestin, JA y db g that this hormony ema model for wound signaling established for potato and to- mediate activation upon wounding applicatioe Th . - ex f no mato holds true in A. thaliana, the effects of ABA or JA ogenous 50 JAM JA caused a more pronounced accumula- treatment accumulatioe th n so specifif no c transcripts from tion of Tat, Vsp, and Jrl transcripts than mechanical dam- age, since their elevated levels were still detectable afte4 2 r and even 48 h (Fig. 6), whereas upon wounding their expression decline controo t d lh (Fig value4 2 . 4B)y b s . These difference reflecy ma s persistenca t f elevateeo A dJ e exogenousllevelth n i s y treated plants compared with more transient increases in endogenous JA concentration in the wounded plants. In contrast, treatment with JA produced no effects on the level of the Ck transcript and only weak and transient increases over the basal levels of Wr3 and Aco transcripts. These data suggest tha thesn i t e genes factors other thar no involvee addition i ar regulatioe A J th o n dt i woundf no - o VspFigurTw . 5 transcripte s accumulate differentially upon induced expression. wounding RNAe Th . s from cauline leaveafteh 8 rf plantd so an s2 The expression patterns observed after ABA treatment wounding wer eithen eru r separately (lane 1, cauline leave afteh s2 r wounding; lane 3, 8 h after wounding; 5 ^g per lane) or mixed (lane were also different among the genes analyzed. Only the Jr2 2, 5 (j.g of RNA from cauline leaves after 2 h and 5 /xg 8 h after gens induceewa similao dt ; JA rd levelan A boty sb hAB wounding). Blot hybridization was done with a Vsp probe. other genes showed increases treatment upoA nAB , which Wound Signaling in Arabidopsis 823

ABA JA mutan s lowewa t r tha wild-typn ni - ob s e plantwa d an s 8 4 4 2 8 4 2 c 8 4 4 2 8 4 2 c serve de locall onlth n yi y wounded leaves (Fig . Thi7) . s suggests that JA perception may be required for attaining TAT maximal level Acof so expression, especiall systeme th n yi - ft' ically induced parts of the plant. VSP

JR1 DISCUSSION Mechanical injury trigger activatioe sth larga f no e array JR2 of genes. Whereas some of them are strongly expressed at the wound site, others accumulate preferentially in distal, JK3 nondamaged tissues. Whether the same signals (including JA) coordinat e wound-induceth e d expressio f theso n e ACO genes has not been elucidated. Although the involvement of JA in regulating gene activation upon wounding has WR3 been firmly establishe severan i d l plant species (Creelman et al., 1992; Hildmann et al., 1992; Laudert et al., 1996)e th , CK processes occurring immediately after wounding are poorly characterized; therefore, other component(s) that rRNA may also participate in wound signaling are still unknown. We have isolated and characterized new A. thaliana wound d JA-induciblan - e genes, which provid a more e Figure 6. Hormonal induction of wound-responsive genes. A. thali- complete pictur plane th f teo respons mechanicao et l dam- ana plants were treated with ABA (50 HM) or JA (50 IIM), as indicated age. The wound response in this plant species involves the "Materialn i Methods.d san " Rosette leaves were collecte , 84 , , d2 activatio f genno e expression tha remarkabls i t y fast com- afteh 8 r4 treatmentd 24an , , Controc , l leaves from untreated plants. Five microgram f totaso l RNloades A wa r lan hybridized pe ean s da pared with other plants studied, such as Vsp in soybean or described in Figure 4. Pin2 in potato and tomato. As in these other cases, activa- tion of certain wound-inducible genes in A. thaliana is not restricte damagee th o dt d tissuesystemia d an , c accumu- for Tat, Vsp, and Jr3 were more transient and below the lation in distal tissues also occurs (Pena-Cortes et al., 1988; levels attained upon treatment with JA (Fig. 6). In Jrl and Maso Mulletd nan , 1990). Accordin e spatiath o gt l distri- Ck no transcript accumulation was observed in ABA- bution of their expression in injured plants, the genes an- treated plants. alyzed can be separated into three groups: the first group

Comparative Study of the Wound Response in Wild-Type coil A. thaliana and |A-lnsensitive coil Mutant Plants _2h_ Jjh_ 24h 4ita 2h 8h Mb 48h nonallelicw Afe , JA-insensitiv . thalianaeA mutants have c Isis Islscls Isls Is been describe (Staswicr fa o ds t al.ke , 1992; Fey t al.se , 1994; TAT Berger et al., 1996). Among them, the coil showed normal growth in the presence of JA and did not express the 29- VSP ••§ and 31-k homologp DVs s upon treatment with (Benedett t al.e i , 1995). JR1

To elucidate the role of JA as a mediator in wound JR2 signaling in A. thaliana, we performed a comparative study of specific mRNA accumulation upon woundin wildn gi - JR3 type and coil mutant plants using the isolated clones as molecular markers resulte . Th presentee sar Figurn di n I . e7 ACO coil plants part of the wound-responsive genes was not WR3 *• » • ••§-•-•• induced by wounding, either in locally injured leaves or in unwounded, systemic tissues, throughou time th t e course CK § * analyzed. This set of genes coincides with the group of transcripts inducible by JA, further supporting the idea rRNA that this phytohormone mediates their expression upon wounding. s interestinIwa t g tha woune th t d inductio , Wr3,Ck f no Figure 7. Wound-induced gene activation in wild-type (wt) and s similawa o wild-typn ri Ac d an coild ean plants, suggest- JA-insensitive coil . thalianaA plants. Collection times correspondo t ing the presence of a signaling pathway independent afte h o f8 r4 J woundingAd an , 24 , , 2Controlc 8 , , unwounded plants, I ; perceptio r wound-inducefo n d gene activatione Th . locally wounded s,d an systemi; c (unwounded) leave f injureso d wound-induced accumulatio transcripo Ac coile f no th n i t plants. Five microgram loades wa r lanef totaA sdo pe .RN l 824 Titarenko et al. Plant Physiol. Vol. 11 5, 1997 includes Ck and Wr3 and shows a stronger induction in Cortés et al., 1989; Hildmann et al., 1992; Pefia-Cortés et al., locally injured leaves than in systemic leaves; the second 1995), although the role ABA plays in these plant species includes Vsp, Tat, Jrl, and Jr2 and is generally characterized may differ (Pefia-Cortés et al., 1989). The fact that ABA is by higher levels of systemic gene activation; a third group able to activate the expression of the majority of wound- is formed by Aco and Jr3 genes and displays mixed char- inducible genes analyzed here above basal levels suggests acteristics, being expressed to the same extent in both local a role for this hormone in wound signaling in A. thaliana. In and systemic tissues shortly after wounding but later per- most cases, however, only weak activation of gene expres- sisting only in the locally damaged tissue. sion has been observed, which may be due to poor uptake The isolated clones have been used as molecular markers or may be related to side effects from other processes (such to analyze the role of JA in mediating wound responses in as stomatal closure), which are also triggered by ABA plants. Exogenously applied JA was able to induce only a treatment. The involvement of ABA in mediating wound- subset of the characterized genes, which included those inducible gene activation in A. thaliana will certainly re- clones showing a stronger systemic accumulation in quire further analysis. wounded A. thaliana plants. At the same time, for Ck, Wr3, It is known that genes involved in stress responses often and Aco transcripts, low levels (if any, in the case of Ck) of show a complex pattern of organ-specific expression. The accumulation were detected upon JA treatment, suggesting potato Pin2 gene, for example, is constitutively expressed that their induction upon wounding may occur via a JA- in tubers and immature floral buds (Hildmann et al., 1992). independent pathway. The Vsp genes from soybean and A. thaliana are active in Further support for the presence of two distinct wound young, developing organs and flowers (Mason and Mullet, signal transduction pathways was obtained from the study 1990; Berger et al., 1995). The wound-inducible genes de- of the wound response in the JA-insensitive coil mutant. In scribed here have low basal levels of expression in non- coil plants JA perception is impaired; therefore, plants are treated plants. However, Vsp transcripts accumulate to able to grow normally on JA-containing medium, but Vsp high levels in flowers, roots, and green siliques. The Jrl proteins do not accumulate (Feys at al., 1994). In contrast to transcript also has a high leve1 of accumulation in roots wild-type plants, the JA-responsive group of genes, includ- and, to a lesser extent, in flowers. The role of these gene ing Tat, Vsp, Jrl, Jr2, and Jr3, are not induced in coil plants products in the physiology of these organs is not known. upon wounding, thus confirming the role of JA in mediating Vsp may serve as a temporary reservoir for N,, as has their activation in injured plants. It is interesting that tran- already been postulated for its wound-induced presence scripts for the group of non-JA-responsive genes accumulate in leaves (Staswick, 1990). In the case of Jrl, further spec- in coil in response to wounding, revealing the presence of a ulation is hampered by the lack of significant similarities wound-signaling pathway that does not depend on JA per- in the databases, which could give some indication of ception for its activity. The existence of a JA-independent function. pathway is also supported by the wound-induced expres- Some of the wound-induced genes identified show strik- sion of the glutathione S-transferase gene in a JA-deficient ing homologies to housekeeping genes previously de- A. thaliana mutant (McConn et al., 1997). scribed in nonplant species; examples are choline kinase, The levels of Aco transcript attained in wounded coil acyl-COA oxidase, and aminotransferases. In potato and plants are lower than in the wild-type plants and are tomato the expression of severa1 metabolic genes is up- restricted to the damaged tissue, indicating that, although regulated by wounding in a similar way as the Pin2 gene, its wound activation is not dependent on JA as a mediator, but their role in the wound response is still poorly under- the expression of Aco in wild-type plants in response to stood (Hildmann et al., 1992; Bergey et al., 1996). We can wounding may involve both JA-dependent and speculate on the implication of the metabolic genes de- -independent pathways. Wound-induced Ck and Wr3 lev- scribed here in maintaining basal cell functions with addi- els are similar in coil and wild-type plants, both in local tional requirements in the wound response, for instance, in and systemic leaves, indicating that, in contrast to Aco, cell membrane synthesis or maintenance. The Aco enzyme induction of Ck and Wr3 expression is fully independent of catalyzes the first step in the fatty acid /3-oxidation path- JA. Taken together, our results suggest that at least two way and, therefore, may be involved in the last steps of JA signaling pathways are activated upon wounding: one of synthesis. However, in animals Aco activation forms part them does not require JA and is principally responsible for of the response to leukotriene-mediated inflammation gene activation in the vicinity of the wound site, and the (Devchang et al., 1996) by catabolizing these stress-related other involves JA perception and most likely an increase in second messengers. Because the chemical structure and the endogenous levels of this hormone (Laudert et al., biosynthetic pathway of JA are very similar to those of 1996), which would’ activate gene expression throughout prostaglandins and leukotrienes (Vick and Zimmerman, the aerial part of the plant, both locally and systemically to 1987), we speculate that the Aco action in the plant re- the wound site. Some components that are differentially sponse to wounding may include a role in JA catabolism, involved in the JA-dependent and -independent wound- thus temporally limiting the extension of the JA-mediated signaling pathways have been identified (Rojo et al., 1997). wound response. Their mode of regulation suggests cross-talk between the One isolated clone Ur3) has significant homology to IIrl, pathways in the control of the wound response. an amidohydrolase of A. thaliana that releases active IAA The requirement of ABA for wound-induced Pin2 ex- () from conjugates (Bartel and Fink, 1995). Because pression in tomato and potato is well established (Pefia- auxins inhibit the activation of stress-related genes in dif- Wound Signaling in Arabidopsis 825 ferent plant species (Kernan and Thornburg, 1989; DeWald Bomtempi EJ, Búa J, Aslund L, Porcel B, Segura EL, Henriksson et al., 1994; Rojo et al., 1997), a possible role of that enzyme J, Orn A, Pettersson U, Ruiz AM (1993) Isolation and charac- in the damaged plant may be the release of free auxins to terization of a gene from Trypanosomu cruzi encoding a 46- kilodalton protein with homology to human and rat tyrosine check the strength of the wound response. Therefore, Aco aminotransferase. Mo1 Biochem Parasito1 59 253-262 and Ir3 gene products may play a role in feedback regula- Bowles D (1991) The wound response of plants. Curr Biol 1: tion of wound-induced gene expression. 165-167 It is interesting that the induction of Aco and Ir3 upon Chang C (1996) The ethylene signal transduction pathway in wounding appears to be coordinately regulated with re- Arabidopsis: an emerging paradigm? Trends Biochem Sci 21: 129-133 gard to spatial distribution, sensitivity to JA, and duration Creelman RA, Mullet JE (1995) Jasmonic acid distribution and of expression. The generation of transgenic plants bearing action in plants: regulation during development and response to the sense or antisense gene constructions for these genes biotic and abiotic stress. Proc Natl Acad Sci USA 92: 4114-4119 will enable us to establish their role in stress responses. Creelman RA, Tierney ML, Mullet JE (1992) Jasmonic acid/ accumulate in wounded soybean hypocotyls Characterization of the promoter regions of genes belong- and modulate wound gene expression. Proc Natl Acad Sci USA ing to JA-dependent and -independent wound-signaling 89: 49384941 pathways will help us to clarify the role of this phytohor- Dellaporta SL, Wood J, Hicks JB (1983) A plant DNA miniprepa- mone and other regulators in mediating the plant response ration: version 11. Plant Mo1 Biol Rep 3: 19-21 to mechanical wounding. Devchang PR, Keller H, Peters JM, Vazquez M, Gonzalez FJ, Wahli W (1996) The PPARa-leukotriene B, pathway to inflam- mation control. Nature 384: 3943 DeWald DB, Sadka A, Mullet JE (1994) Sucrose modulation of ACKNOWLEDCMENTS soybean Vsp gene expression is inhibited by auxin. Plant Physiol 104 439444 We are indebted to J.G. Turner for kindly providing the coil Dmochowska A, Dignard D, Maleszka R, Thomas DY (1990) seeds and to C. Castresana, S.D. Jackson, and G. Vancanneyt for Structure and transcriptional control of the Saccharomyces cerevi- critica1 reading of the manuscript. The A. thaliana cDNA libraries siue POXl gene encoding acyl-coenzyme A oxidase. Gene 88 used for the isolation of homologs to DD fragments were kindly 247-252 provided by G. Vancanneyt. The excellent technical assistance of Farmer EE, Johnson RR, Ryan CA (1992) Regulation of expression Tomás Cascón is gratefully acknowledged. We also would like to of proteinase inhibitor genes by methyl jasmonate and jasmonic thank Inés Poveda and Angel Sanz for the photographic work. acid. Plant Physiol 98: 995-1002 Farmer EE, Ryan CA (1990) Interplant communication: airborne methyl jasmonate induces synthesis of proteinase inhibitors in Received April21, 1997; accepted July 11, 1997. plant leaves. Proc Natl Acad Sci USA 87: 7713-7716 Copyright Clearance Center: 0032-0889/97/ 115/0817/10. Feys BJF, Benedetti CE, Penfold CN, Turner JG (1994) Arabidop- sis mutants selected for resistance to the phytotoxin coronatine are male sterile, insensitive to methyl jasmonate, and resistant to LITERATURE ClTED a bacterial pathogen. Plant Cell 6: 751-759 Hargrove JL, Scoble HA, Mathews WR, Baumstark BR, Biemann Albrecht T, Kehlen A, Stahl K, Knofel H-D, Sembdner G, Weiler K (1989) The structure of tyrosine aminotransferase. J Biol Chem EW (1993) Quantification of rapid, transient increases in jas- 264 45-53 monic acid in wounded plants using a monoclonal antibody. Hildmann T, Ebneth M, Peiia-CortCs H, Sánchez-Serrano JJ, Planta 191: 86-94 Willmitzer L, Prat S (1992) General roles of abscisic and jas- Amasino RM (1986) Acceleration of nucleic acid hybridisation rate monic acids in gene activation as a result of mechanical wound- by polyethylene glycol. Ana1 Biochem 152: 304-307 ing. Plant Cell 4: 1157-1170 Aoyama T, Tsushima K, Souri M, Kamijo T, Suzuki Y, Shi- Hobbie L, Timpte C, Estelle M (1994) Molecular genetics of auxin mozawa N, Orii T, Hashimoto T (1994) Molecular cloning and and . Plant Mo1 Biol 26: 1499-1519 functional expression of a human peroxisomal acyl-coenzyme A Hosaka K, Kodaki T, Yamashita S (1989) Cloning and character- oxidase. Biochem Biophys Res Commun 198: 1113-1118 ization of the yeast CK2 gene encoding choline kinase and its Bartel B, Fink GR (1995) ILRI, an amidohydrolase that releases expression in Escherichiu coli. J Biol Chem 264 2053-2059 active indole-3-acetic acid from conjugates. Science 268: 1745- Kakimoto T (1996) CKI1, a histidine kinase homolog implicated in 1748 cytokinin signal transduction. Science 274 982-985 Benedetti CE, Xie D, Turner JG (1995) COIZ-dependent expres- Keman A, Thomburg RW (1989) Auxin levels regulate the expres- sion of an Arubidopsis vegetative storage protein in flowers and sion of a wound-inducible proteinase inhibitor II-chloramphenicol siliques and in response to coronatine or methyl jasmonate. acetyl transferase gene fusion in uitro and in vivo. Plant Physiol Plant Physiol 109: 567-572 91: 73-78 Berger S, Bell E, Mullet JE (1996) Two methyl jasmonate- Kieber JJ, Rothenberg M, Roman G, Feldmann KA, Ecker JR insensitive mutants show altered expression of AtVsp in re- (1993) CTR1, a negative regulator of the ethylene response path- sponse to methyl jasmonate and wounding. Plant Physiol 111: way in Arabidopsis, encodes a member of the Raf family of 525-531 protein kinases. Cell 72 427-441 Berger S, Bell E, Sadka A, Mullet JE (1995) Arubidopsis thaliana Laudert D, Pfannschmidt U, Lottspeich F, Hollander-Czytko H, AtVsp is homologous to soybean VspA and VspB, genes encod- Weiler EW (1996) Cloning, molecular and functional character- ing vegetative storage protein acid phosphatases, and is regu- ization of Avabidopsis thalianu allene oxide synthase (CYP 74), the lated similarly by methyl jasmonate, wounding, sugars, light first enzyme of the octadecanoid pathway to jasmonates. Plant and phosphate. Plant Mo1 Biol 27: 933-942 Mo1 Biol 31: 323-335 Bergey DR, Howe GA, Ryan CA (1996) Polypeptide signaling for Leung J, Bouvier-Durand M, Morris P-C, Guerrier D, Chefdor F, plant defensive genes exhibits analogies to defense signaling in Giraudat J (1994) Arabidopsis ABA response gene ABI1: features animals. Proc Natl Acad Sci USA 93 12053-12058 of a calcium-modulated protein phosphatase. Science 264 1448- Bishop PD, Makus DJ, Pearce G, Ryan CA (1981) Proteinase 1452 inhibitor-inducing factor activity in tomato leaves resides in Liang P, Pardee A (1992) Differential display of eukaryotic mes- oligosaccharides enzymically released from cell walls. Proc Natl senger RNA by means of the polymerase chain reaction. Science Acad Sci USA 78: 3536-3540 257: 967-971 826 Titarenko et al. Plant Physiol. Vol. 11 5, 1997

Logemann J, Schell J, Willmitzer L (1987) Improved method for (1996) Abscisic acid-deficient plants do not accumulate protein- the isolation of RNA from pIant tissues. Ana1 Biochem 163: ase inhibitor I1 following systemin treatment. Planta 198: 16-20 447-451 Mason HS, Mullet JE (1990) Expression of two soybean vegetative Peiia-Cortes H, Sanchez-Serrano J J, Mertens R, Willmitzer L, storage protein genes during development and in response Prat S (1989) Abscisic acid is involved in the wound-induced to water deficit, wounding, and jasmonic acid. Plant Cell 2: expression of the proteinase inhibitor I1 gene in potato and 569-579 tomato. Proc Natl Acad Sci USA 86 9851-9855 McConn M, Creelman RA, Bell E, Mullet JE, Browse J (1997) Peiia-Cortes H, Sanchez-Serrano JJ, Rocha-Sosa M, Willmitzer L Jasmonate is essential for insect defense in Arabidopsis. Proc Natl (1988) Systemic induction of proteinase inhibitor I1 gene expres- Acad Sci USA 94: 5473-5477 sion in potato plants by wounding. Planta 174: 84-89 Meyer K, Leube MP, Grill E (1994) A protein phosphatase 2C Rettenmeier R, Natt E, Zentgraf H, Scherer G (1990) Isolation and involved in ABA signal transduction in Arabidopsis thaliana. characterization of the human tyrosine aminotransferase gene. Science 264: 1452-1455 Nucleic Acid Res 8: 3853-3861 Miyazawa S, Hayashi H, Hijikata M, Ishii N, Furuta S, Rojo E, Titarenko E, Leon J, Berger S, Jancanneyt G, Sánchez- Kagamiyama H, Osumi T, Hashimoto T (1987) Complete nu- Serrano JJ (1997) Reversible protein phosphorylation regulates cleotide sequence of cDNA and predicted amino acid sequence jasmonic acid dependent and independent wound signal trans- of rat acyl-COA oxidase. J Biol Chem 262: 8131-8137 duction pathways in Arabidopsis thaliana. Plant (in press) Murashige T, Skoog F (1962) A revised medium for rapid growth J and bioassays with tobacco tissue cultures. Physiol Plant 15: Sambrook J, Fritsch EF, Maniatis T (1989) Molecular Cloning: A 493-497 Laboratory Manual, Ed 2. Cold Spring Harbor Laboratory, Cold ODonnell PJ, Calvert C, Atzorn R, Wasternack C, Leyser HMO, Spring Harbor, NY Bowles DJ (1996). Ethylene as a signal mediating the wound Staswick PE 11990) Nove1 regulation of vegetative storage protein response of tomato plants. Science 274: 1914-1917 genes. Plant Cell 2: 1-6 Pearce G, Strydom D, Johnson S, Ryan CA (1991) A polypeptide Staswick PE, Su W, Howell SH (1992) Methyl jasmonate inhibi- from tomato leaves induces wound-inducible proteinase inhib- tion of root growth and induction of a leaf protein are decreased itor proteins. Science 253: 895-898 in an Arabidopsis thaliana mutant. Proc Natl Acad Sci USA 89: Peiia-Cortes H., Albrecht T, Prat S, Weiler EW, Willmitzer L 6837-6840 (1993) Aspirin prevents wound-induced gene expression in to- Uchida T, Yamashita S (1992) Molecular cloning, characterization, mato leaves by blocking jasmonic acid biosynthesis. Planta 191: and expression in Escherichia coli of a cDNA encoding mamma- 123-128 lian choline kinase. J Biol Chem 267: 10156-10162 Peiia-CortCs H, Fisahn J, Willmitzer L (1995) Signals involved in Vick BA, Zimmerman DC (1987) Oxidative systems for modifica- wound-induced proteinase inhibitor I1 gene expression in to- tion of fatty acids: the lipoxygenase pathway. In PK Stumpf, EK mato and potato plants. Proc Natl Acad Sci USA 92: 4106-4113 Com, eds, The Biochemistry of Plants, Vol9: Lipids. Academic Peiia-Cortes H, Prat S, Atzom R, Wasternack C, Willmitzer L Press, New York, pp 53-90