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 abscisic acid 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 systemin (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 ethylene 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), auxins (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 jasmonate 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.