Report. Arabidopsis Touch-Induced Morphogenesis Is Jasmonate Mediated and Protects against Pests

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1 Current Biology 22, , April 24, 2012 ª2012 Elsevier Ltd All rights reserved DOI /j.cub Arabidopsis Touch-Induced Morphogenesis Is Jasmonate Mediated and Protects against Pests Report E. Wassim Chehab, 1 Chen Yao, 1 Zachary Henderson, 1 Se Kim, 1 and Janet Braam 1, * 1 Department of Biochemistry & Cell Biology, Rice University, Houston, TX , USA Summary Plants cannot change location to escape stressful environments. Therefore, plants evolved to respond and acclimate to diverse stimuli, including the seemingly innocuous touch stimulus [1 4]. Although some species, such as Venus flytrap, have fast touch responses, most plants display more gradual touch-induced morphological alterations, called thigmomorphogenesis [2, 3, 5, 6]. Thigmomorphogenesis may be adaptive; trees subjected to winds develop less elongated and thicker trunks and thus are less likely damaged by powerful wind gusts [7]. Despite the widespread relevance of thigmomorphogenesis, the regulation that underlies plant mechanostimulus-induced morphological responses remains largely unknown. Furthermore, whether thigmomorphogenesis confers additional advantage is not fully understood. Although aspects of thigmomorphogenesis resemble ethylene effects [8], and touch can induce ethylene synthesis [9, 10], Arabidopsis ethylene response mutants show touch-induced thigmomorphogenesis [11]; thus, ethylene response is nonessential for thigmomorphogenesis. Here we show that jasmonate (JA) phytohormone both is required for and promotes the salient characteristics of thigmomorphogenesis in Arabidopsis, including a touchinduced delay in flowering and rosette diameter reduction. Furthermore, we find that repetitive mechanostimulation enhances Arabidopsis pest resistance in a JA-dependent manner. These results highlight an important role for JA in mediating mechanostimulus-induced plant developmental responses and resultant cross-protection against biotic stress. Results and Discussion The Jasmonate Biosynthesis Pathway Is Required for Touch-Induced Growth Alterations Plants perceive touch stimulation and undergo developmental alterations. For example, Arabidopsis plants whose rosette leaves are gently moved back and forth repeatedly over the course of their development exhibit delayed transition to flowering, decreased flowering stem (inflorescence) elongation, and shorter petioles that contribute to the development of smaller rosettes [3, 6]. Touch-induced developmental alterations, called thigmomorphogenesis [2], are thought to be conserved among most, if not all, higher plants [2]; however, the regulatory signals that mediate touch-induced developmental changes were undefined. We hypothesized that the phytohormone jasmonate (JA) might be important for thigmomorphogenesis because of the overlap between genes whose *Correspondence: braam@rice.edu expression is induced by touch [4] and wound-responsive genes induced by JA [12], evidence that wound-induced inhibition of cell division and growth retardation is JA dependent [13, 14], and the role of JA in secondary growth induction [15]. To test the hypothesis that JA is required for thigmomorphogenesis, we assessed the ability of aos, an Arabidopsis mutant lacking functional allene oxide synthase (AOS) and therefore defective in biosynthesis of JA and other oxylipins [16], to undergo thigmomorphogenesis. gl-1, the genetic background for aos, exhibited typical morphogenetic responses to twice-daily touch treatments over 4 weeks (Figure 1; see also Figure S1 available online). In comparison to untreated gl-1, touch-stimulated gl-1 developed shorter inflorescences (Figure 1A), displayed a 28% decrease in average rosette radius (Figure 1C), and flowered with a 1- to 2-day delay (Figure 1D). In striking contrast, similar repetitive touch treatments had no detectable effect on aos inflorescence elongation (Figure 1B), average rosette radius (Figure 1C), or the timing of flowering (Figure 1D). Therefore, AOS function is required for Arabidopsis thigmomorphogenesis. Furthermore, aos, whether touched repeatedly or left untouched, flowered earlier than untouched gl-1 (Figure 1D), indicating that the AOS-dependent JA biosynthesis pathway not only is required for the touch-induced delay in flowering but also contributes to the timing of flowering in untouched Arabidopsis. The JA Signaling Pathway Is Required for Touch-Induced Growth Alterations To assess whether the established canonical JA pathway is required for thigmomorphogenesis, we examined the role of two additional JA pathway-relevant functions. JASMONATE RESISTANT 1 (JAR1) conjugates JA to isoleucine (JA-Ile) and therefore is required to generate the bioactive form of the hormone [17, 18]. CORONATINE INSENSITIVE 1 (COI1) encodes the JA-Ile coreceptor [19]. The jar1 and coi1 mutants manifest phenotypes indicative of defective JA pathway function [20 22]. To verify the requirement for the JA signaling pathway in touch-induced thigmomorphogenesis in Arabidopsis and to establish roles for JA-Ile and COI1-mediated responses, we assessed the ability of jar1 and coi1 to undergo thigmomorphogenesis. Repetitive touch treatment of coi1 and jar1 had no significant effect on inflorescence elongation, average rosette radius, or the timing of flowering (Figure S1), similar to that observed with aos (Figure 1). Therefore, JA-Ile and COI1 are essential for thigmomorphogenesis, confirming a required role for JA and the JA-dependent signaling pathway in touch-induced growth alterations in Arabidopsis. Transgenic Overexpression of OPR3 Results in Thigmomorphogenetic-like Alterations Next, we investigated whether elevated JA is sufficient to drive thigmomorphogenetic-like changes using a previously described and characterized Arabidopsis homozygous transgenic line (OPR3-OE) with constitutive expression of the JAbiosynthetic gene, OXOPHYTODIENOATE REDUCTASE 3 [23]. OPR3-OE accumulates w30% more JA than Col-0 [23]. As expected, Col-0, the control genetic background for OPR3-OE, shows robust thigmomorphogenesis, with touch-stimulated

2 Current Biology Vol 22 No Figure 1. JA-Deficient Mutants Are Nonresponsive to Touch (A and B) Representative untouched and repetitively touched gl-1 and aos plants after 4 weeks of twice-daily touch treatment. (C) Average rosette radius of untouched and touched gl-1 and aos plants. Means 6 SD are shown; letters over bars indicate significant differences (p < 0.005, Tukey s test). (D) Percent fraction of both untouched and touched gl-1 and aos plants that develop inflorescence stems over time (days after seed sowing). (C) and (D) are the result of compiling three biological replicates with n > 45. Similar results were obtained with at least two other independent experiments. See also Figure S1. Figure 2. Transgenics that Overproduce JA Show Thigmomorphogenetic Characteristics and Have Enhanced Response to Touch (A and B) Representative untouched and repetitively touched Col-0 and OPR3-OE plants after 4 weeks of twice-daily touch treatment. (C) Average rosette radius of untouched and touched Col-0 and OPR3-OE plants. Means 6 SD are shown; letters over bars indicate significant differences (p < 0.005, Tukey s test). (D) Percent fraction of both untouched and touched Col-0 and OPR3-OE plants that develop inflorescence stems over time (days after seed sowing). (C) and (D) are the result of compiling three biological replicates with n > 45. Similar results were obtained with at least two other independent experiments. plants developing less elongated inflorescence stems (Figure 2A) and smaller average rosette radii (Figure 2C) and displaying a delay in flowering (Figure 2D). Remarkably, untouched OPR3-OE resembled touched Col-0, in that they had shorter inflorescences (Figure 2B), a 33% decrease in average rosette radius (Figure 2C), and an w2-day delay in flowering (Figure 2D) as compared to untouched Col-0. Thus, constitutive expression of OPR3 is sufficient to promote thigmomorphogenetic-like alterations even in the absence of deliberate touch stimulation. These results suggest that constitutive JA overproduction can promote thigmomorphogenetic-like alterations, although it is also possible that overproduction of JA can lead to nonspecific phenotypic changes. These results are consistent with previous studies demonstrating that JA overproduction or exogenous JA treatments can lead to growth alterations [24 28]. OPR3-OE is also capable of robust thigmomorphogenesis when repetitively touch stimulated; in comparison to untouched OPR3-OE, repeatedly touched OPR3-OE exhibits shorter inflorescences (Figure 2B), a 35% decrease in average rosette radius (Figure 2C), and a 2- to 3-day delay in flowering (Figure 2D). The ability of OPR3-OE to display touch-induced growth alterations suggests that the JA-dependent responses are not saturated in untouched OPR3-OE. JA Levels Increase in Response to Touch and Correlate with Thigmomorphogenesis We have shown that the ability to generate JA is required for touch-induced thigmomorphogenesis (Figure 1) and that the ability to constitutively produce JA can result in thigmomorphogenetic-like changes (Figure 2). It is well established that mechanical stress, like wounding, results in JA accumulation in diverse plants, and that touch increases oxylipins in tendrils [29 32]. To verify whether JA accumulation correlates with thigmomorphogenesis, we measured JA levels in the different genotypes. A single touch stimulation causes w1.5-fold more JA accumulation in shoots of both gl-1 and Col-0 (Figure 3A). Furthermore, gl-1 and Col-0 plants that were touched twice daily for 4 weeks and are thigmomorphogenetic (Figures 1 and 2) have JA levels that are w2.5-fold higher than comparable untouched plants (Figure 3A). Repetitively touch-treated and thigmomorphogenetic gl-1 and Col-0 adult plants subjected to an additional single touch, however, do not further increase their overall JA levels (Figure 3A), suggesting that 4 weeks of touch stimulation leads to a saturation of the touch-induced JA production response. As expected, aos accumulates no detectable JA whether left untouched, subjected to a single touch, or treated with repetitive touch throughout development (Figure 3A). Finally, as shown previously [23], OPR3-OE accumulates w30% more JA than untouched Col-0 (Figure 3A). However, OPR3-OE JA levels are further increased 1.5-fold upon a single touch stimulation (Figure 3A), indicating that OPR3-OE expression alone does not saturate JA accumulation. Repetitively touch-stimulated OPR3-OE accumulates w20% more JA than thigmomorphogenetic wild-type (Figure 3A). The findings that JA levels in OPR3-OE increase following touch stimulation and that thigmomorphogenetic OPR3-OE has even higher JA accumulation than thigmomorphogenetic Col-0 are consistent with the existence of a correlation between thigmomorphogenesis characteristics and JA accumulation. Finally, JA levels may saturate by the repetitive touch of OPR3-OE, because an additional

3 Jasmonates Mediate Thigmomorphogenesis 703 Touch Stimulation Delays Flowering by Slowing Leaf Generation A delay in flowering is another prominent characteristic of thigmomorphogenesis [33]. The timing of Arabidopsis flowering is determined by rosette leaf number [34]; variations in this timing are due either to a regulatory change in number of leaves to trigger the transition or to leaf generation rate. All four genotypes, whether touched or untouched, generated approximately eight rosette leaves before flowering (Figure 3B). Therefore, touch delays flowering because leaf production is retarded. It is likely that JA slows leaf emergence, because untouched aos and OPR3-OE generate the same number of rosette leaves before flowering, yet aos flowers early (Figure 1D) and OPR3-OE flowers late (Figure 2D). Figure 3. Touch Treatment Increases JA Accumulation, and Leaf Number Is Relatively Constant Despite Differences in Timing of Flowering (A) JA (methyl jasmonate and jasmonic acid) levels in leaves of 4-week-old plants. Plants were either untouched (untouched) or touched twice daily throughout their development (touched) and then subjected either to no additional stimulus or to a single touch stimulus (single touch) and then harvested 30 min later. Means 6 SD of three independent biological replicates are shown. (B) Rosette leaf number at flowering of untouched and repetitively touched plants. Means 6 SD are shown; letters over bars indicate significant differences (p < 0.005, Tukey s test). Results were obtained by compiling three biological replicates with n > 45. Similar results were obtained with at least two other independent experiments. See also Figure S2. stimulation of 4-week-old repeatedly touched OPR3-OE does not result in a detectable JA increase. Overall, these data demonstrate that JA increases in Arabidopsis in response to a single touch stimulus, that JA accumulates more so following repetitive touch stimuli, and that JA accumulation levels correlate with the strength of the thigmomorphogenetic phenotype. JA Accumulates in Inflorescence Stems following Touch Stimulation of Rosette Leaves A prominent aspect of Arabidopsis thigmomorphogenesis resulting from repetitive touch stimulation is the decrease in inflorescence elongation (Figures 1 and 2), even though the majority of the touch stimulus is applied to the rosette leaves. The physical separation between the site of the touch stimulus and the site of response led to the question of whether stimulation of rosette leaves leads to systemic JA accumulation. Therefore, we examined the JA levels in both the rosette leaves and the apical portion of the inflorescence from Col-0 plants whose rosette leaves were subjected to a single touch stimulation. Interestingly, 30 min after stimulation, both the touched rosette leaves and the untouched inflorescence showed enhanced JA accumulation, with w1.5- and w2-fold increases, respectively (Figure S2). This systemic accumulation of JA, distal to the site of stimulation, could be due to JA transport and/or the translocation of a JA synthesis-inducing signal from the site of the stimulus to the inflorescence. Touch-Induced Gene Expression Can Be JA Independent In addition to touch stimulation of Arabidopsis leading to developmental changes, touch also induces expression of genes (e.g., [3, 4, 35]). To examine whether JA has a role in touchinduced gene expression, we compared the expression of three representative touch-responsive genes in gl-1 and aos plants that were either untouched or subjected to a single touch stimulus. TCH2 (CML24) and TCH4 (XTH22) were among the first identified touch-inducible genes in Arabidopsis [3], and CML39 was identified as strongly touch inducible by microarray analyses [4]. All three genes maintain touch inducibility of expression in aos (Figure S2); therefore, at least for these three genes, touch-induced gene expression occurs even in the absence of JA, indicating that there are JA-independent mechanisms for transducing touch perception into gene expression regulation. Such JA-independent touch responses may represent the earliest responses following mechanostimulus perception. The mechanistic pathway responsible for this JAindependent touch-inducible response may also be critical for touch-induced JA production. One possibility is that JAindependent touch-inducible genes may have roles in JA production activation. JA-dependent signaling, however, may contribute to at least some of the expression changes that occur after touch because AOS is required for the full touch inducibility of CML39 expression (Figure S2). Consistent with the idea that there are JA-dependent and -independent mechanical force responses in plants, JA and wind were previously shown to differentially affect defense traits [36]. Finding that touch-induced gene expression remains robust even in aos, which fails to display thigmomorphogenetic changes (Figure 1), indicates that the elevated expression of these genes is insufficient to cause thigmomorphogenetic changes. Therefore, the functional significance of these touch-induced changes in gene expression remains unclear. However, diverse functions for TCH2 (CML24) have been identified, including a required role in mechanical stress-induced root growth responses [37 40]. Thigmomorphogenetic Plants Have Enhanced Resistance to Botrytis cinerea Fungus and Trichoplusia ni Cabbage Loopers Because JA is coupled to plant defense against necrotrophic fungi and plant herbivores [23, 40], we hypothesized that mechanically stimulated plants, with elevated JA, may be primed for defense against pests. Consistent with this hypothesis, windblown bean plants show enhanced pest resistance [41]. Therefore, we examined the susceptibility of untouched and repetitively touched Arabidopsis to Botrytis cinerea infection and cabbage looper (Trichoplusia ni) infestation.

4 Current Biology Vol 22 No At 48 and 72 hr postinoculation (hpi), w30% smaller diameter lesions developed on fungus-infected leaves from thigmomorphogenetic gl-1 as compared to untouched gl-1 at 48 and 72 hpi (Figures 4A and 4B). This touch-induced resistance is JA dependent, because touched and untouched aos were comparably and highly susceptible to infection (Figures 4A and 4B). Consistent with the interpretation that OPR3-OE plants may be constitutively thigmomorphogenetic (Figure 2), infected leaves from untouched OPR3-OE had w28% smaller leaf lesions than those on untouched Col-0 leaves at 72 and 96 hpi (Figure 4C). Col-0 behaved similarly to gl-1 (Figures 4A and 4B), with decreased lesion diameters on leaves from thigmomorphogenetic Col-0 relative to those from nonthigmomorphogenetic Col-0 (Figures 4C and 4D). Finally, leaves from repetitively touch-stimulated OPR3-OE develop lesions after 96 hpi that are 32% smaller than those on leaves from untouched OPR3-OE (Figures 4C and 4D). Thus, repetitive touch stimulation results in enhanced B. cinerea resistance in Arabidopsis. T. ni larvae developing on thigmomorphogenetic gl-1 and Col-0 leaves weighed 33% less than larvae fed on untouched gl-1 and Col-0 (Figure 4E). Untouched OPR3-OE resembles thigmomorphogenetic Col-0, in that final weights of T. ni that developed on untouched OPR3-OE were comparable to weights of those fed on thigmomorphogenetic Col-0. T. ni fed on repetitively touched OPR3-OE had the poorest performance, with weights only w70% of their untouched siblings. Finally, the thigmomorphogenetic enhancement of plant resistance to herbivory is dependent upon JA production, because T. ni weight is unaffected by feeding on control versus repetitively touched aos (Figure 4E). Therefore, repetitive mechanical stimulation enhances Arabidopsis resistance to both a necrotrophic fungus and a generalist herbivore. Summary and Implications In summary, this work has shown that JA both is required for and promotes thigmomorphogenetic alterations in Arabidopsis. The lack of touch-induced phenotypes in aos, jar1, and coi1 indicates that thigmomorphogenesis is not a passive consequence of mechanostimulus-induced damage but is instead an active, JA-regulated response. Similarly, an active JA pathway is required for wound-induced growth retardation [13, 14]. How touch leads to increased JA accumulation remains unclear. We would predict that the initial activation of JA production induced by touch likely occurs in a JA-independent manner. The finding that at least some touch-induced changes in gene expression occur in the aos mutant indicates that there are touch-induced responses that are JA independent. Therefore, elucidating the mechanism of touch-induced responses in aos may lead to understanding how touch triggers initial plant responses. Wound-induced JA is known to slow mitosis [13]; this role for JA may be to allocate resources away from growth and toward defense. Touch-induced JA-regulated growth retardation may also be advantageous in environments rich in mechanical perturbation, such as wind. Shorter, stockier plants may be more resistant to mechanical stress [7]. Remarkably, JA has also been implicated in more specialized plant touch responses, including tendril curling of Bryonia dioica [42] and Venus flytrap closure [43]; therefore, the role for JA in plant mechanoresponses may be widespread. Touch-induced JA accumulation also underlies the finding presented here, as well as in previous reports [41, 44], that Figure 4. Thigmomorphogenetic Plants Are More Resistant to B. cinerea and T. ni (A) Leaf lesion diameters of gl-1 and aos 48 and 72 hr postinoculation (hpi) with B. cinerea fungal spores. 96 hpi is not included because aos leaves were fully destroyed by infection. Mean lesion diameters 6 SD (n = 40) are shown. Letters over bars indicate significant differences (p < 0.005, Tukey s test) within a treatment. (B) Representative aos and gl-1 leaves showing lesions at 72 hpi. Scale bars represent 1 cm. (C) Leaf lesion diameters of Col-0 and OPR3-OE at 48, 72, and 96 hpi with fungal spores. Mean lesion diameters 6 SD (n = 40) are shown. Letters over bars indicate significant differences (p < 0.005, Tukey s test) within a treatment. (D) Representative Col-0 and OPR3-OE leaves at 96 hpi. Scale bars represent 1 cm. (E) T. ni larva final weights 12 days after release of a newly hatched larva in an arena containing leaves of the indicated genotype and treatment. Means 6 SE of at least 15 arenas per genotype treatment are shown. Asterisk denotes significant difference in larva weight between treatments for a given genotype (p < , t test). Similar results were obtained with at least two other independent experiments. mechanically stimulated and/or thigmomorphogenetic plants may be primed for defense and have enhanced resistance to plant invaders. Perhaps wind, a critical mechanism for

5 Jasmonates Mediate Thigmomorphogenesis 705 fungal spore dispersal [45], prepares plants for potential infection, and the mechanical perturbation caused by alighting insects or passing larger animals triggers JA production to activate antiherbivore defenses in case the interaction becomes an attack. Despite the apparent quiescence of their lifestyle, plants are well equipped to mount defenses to withstand the often violent environments in which they live. Experimental Procedures Plant Growth and Treatment Arabidopsis thaliana plants were grown at 22 C, under 16 hr of light (140 me m 22 s 21 ), as described previously [46]. At approximately 1 week of age, when seedlings had developed the first true leaves, plants were touched twice daily at approximate 8 hr intervals for 4 weeks. Gentle touch was applied by hand so as to bend the leaves back and forth ten times. Plants were considered to have transitioned to flowering when the primary inflorescence reached 0.5 cm in length. B. cinerea and T. ni Development Assays B. cinerea assay was performed on leaves from 5-week-old plants, as described previously [23]. T. ni infestation was performed as described previously with minor modifications [40]. One newly hatched larva was transferred with a fine brush to an arena composed of an agar plate containing leaves from 5-week-old plants. Quantification of JA Metabolites Quantification of JA metabolites was performed as described previously [23]. Quantitative RT-PCR Quantitative RT-PCR was performed as described previously [23]. Primers used were CML39 (forward: 5 0 -GATTGCATTACTCCGGGGAG-3 0 ; reverse: 5 0 -GAGGGCGAACTCATCAAAGC-3 0 ), TCH2 (CML24) (forward: 5 0 -GAGTAAT GGTGGTGGTGCTTGA-3 0 ; reverse: 5 0 -ACGAATCATCACCGTCGACTAA-3 0 ), and TCH4 (XTH22) (forward: 5 0 -GAAACTCCGCAGGAACAGTC-3 0 ; reverse: 5 0 -TGTCTCCTTTGCCTTGTGTG-3 0 ). Supplemental Information Supplemental Information includes two figures and can be found with this article online at doi: /j.cub Acknowledgments This material is based upon work supported by the National Science Foundation under grant MCB to J.B. We are grateful to Seiichi Matsuda for sharing his GC-MS facility and to Braam lab members for feedback on the manuscript. Received: October 25, 2011 Revised: January 20, 2012 Accepted: February 21, 2012 Published online: April 5, 2012 References 1. Darwin, C., and Darwin, F. (1881). The Power of Movement in Plants (New York: D. Appleton and Company). 2. Jaffe, M.J. (1973). Thigmomorphogenesis: the response of plant growth and development to mechanical stimulation. Planta 114, Braam, J., and Davis, R.W. (1990). Rain-, wind-, and touch-induced expression of calmodulin and calmodulin-related genes in Arabidopsis. Cell 60, Lee, D., Polisensky, D.H., and Braam, J. (2005). Genome-wide identification of touch- and darkness-regulated Arabidopsis genes: a focus on calmodulin-like and XTH genes. New Phytol. 165, Telewski, F.W., and Jaffe, M.J. (1986). Thigmomorphogenesis: anatomical, morphological and mechanical analysis of genetically different sibs of Pinus taeda in response to mechanical perturbation. Physiol. Plant. 66, Braam, J. (2005). In touch: plant responses to mechanical stimuli. New Phytol. 165, Coutand, C., Dupraz, C., Jaouen, G., Ploquin, S., and Adam, B. (2008). Mechanical stimuli regulate the allocation of biomass in trees: demonstration with young Prunus avium trees. Ann. Bot. (Lond.) 101, Jaffe, M.J., and Biro, R. (1979). Thigmomorphogenesis: the effect of mechanical perturbation on the growth of plants, with special reference to anatomical changes, the role of ethylene, and interaction with other environmental stresses. In Stress Physiology of Crop Plants, H. Mussell and R.C. Staples, eds. (New York: John Wiley and Sons), pp Biro, R.L., and Jaffe, M.J. (1984). Thigmomorphogenesis: ethylene evolution and its role in the changes observed in mechanically perturbed bean plants. Physiol. Plant. 62, Takahashi, H., and Jaffe, M.J. (1984). Thigmomorphogenesis: the relationship of mechanical perturbation to elicitor-like activity and ethylene production. Physiol. Plant. 61, Johnson, K.A., Sistrunk, M.L., Polisensky, D.H., and Braam, J. (1998). Arabidopsis thaliana responses to mechanical stimulation do not require ETR1 or EIN2. Plant Physiol. 116, Devoto, A., Ellis, C., Magusin, A., Chang, H.S., Chilcott, C., Zhu, T., and Turner, J.G. (2005). Expression profiling reveals COI1 to be a key regulator of genes involved in wound- and methyl jasmonate-induced secondary metabolism, defence, and hormone interactions. Plant Mol. Biol. 58, Zhang, Y., and Turner, J.G. (2008). Wound-induced endogenous jasmonates stunt plant growth by inhibiting mitosis. PLoS One 3, e Yan, Y., Stolz, S., Chételat, A., Reymond, P., Pagni, M., Dubugnon, L., and Farmer, E.E. (2007). A downstream mediator in the growth repression limb of the jasmonate pathway. Plant Cell 19, Sehr, E.M., Agusti, J., Lehner, R., Farmer, E.E., Schwarz, M., and Greb, T. (2010). Analysis of secondary growth in the Arabidopsis shoot reveals a positive role of jasmonate signalling in cambium formation. Plant J. 63, Park, J.H., Halitschke, R., Kim, H.B., Baldwin, I.T., Feldmann, K.A., and Feyereisen, R. (2002). A knock-out mutation in allene oxide synthase results in male sterility and defective wound signal transduction in Arabidopsis due to a block in jasmonic acid biosynthesis. Plant J. 31, Fonseca, S., Chini, A., Hamberg, M., Adie, B., Porzel, A., Kramell, R., Miersch, O., Wasternack, C., and Solano, R. (2009). (+)-7-iso- Jasmonoyl-L-isoleucine is the endogenous bioactive jasmonate. Nat. Chem. Biol. 5, Staswick, P.E., and Tiryaki, I. (2004). The oxylipin signal jasmonic acid is activated by an enzyme that conjugates it to isoleucine in Arabidopsis. Plant Cell 16, Sheard, L.B., Tan, X., Mao, H., Withers, J., Ben-Nissan, G., Hinds, T.R., Kobayashi, Y., Hsu, F.F., Sharon, M., Browse, J., et al. (2010). Jasmonate perception by inositol-phosphate-potentiated COI1-JAZ co-receptor. Nature 468, Staswick, P.E., Yuen, G.Y., and Lehman, C.C. (1998). Jasmonate signaling mutants of Arabidopsis are susceptible to the soil fungus Pythium irregulare. Plant J. 15, Clarke, J.D., Volko, S.M., Ledford, H., Ausubel, F.M., and Dong, X. (2000). Roles of salicylic acid, jasmonic acid, and ethylene in cprinduced resistance in arabidopsis. Plant Cell 12, Xie, D.X., Feys, B.F., James, S., Nieto-Rostro, M., and Turner, J.G. (1998). COI1: an Arabidopsis gene required for jasmonate-regulated defense and fertility. Science 280, Chehab, E.W., Kim, S., Savchenko, T., Kliebenstein, D., Dehesh, K., and Braam, J. (2011). Intronic T-DNA insertion renders Arabidopsis opr3 a conditional jasmonic acid-producing mutant. Plant Physiol. 156, Ellis, C., Karafyllidis, I., Wasternack, C., and Turner, J.G. (2002). The Arabidopsis mutant cev1 links cell wall signaling to jasmonate and ethylene responses. Plant Cell 14, Hyun, Y., Choi, S., Hwang, H.J., Yu, J., Nam, S.J., Ko, J., Park, J.Y., Seo, Y.S., Kim, E.Y., Ryu, S.B., et al. (2008). Cooperation and functional diversification of two closely related galactolipase genes for jasmonate biosynthesis. Dev. Cell 14, Dathe, W., Rönsch, H., Preiss, A., Schade, W., Sembdner, G., and Schreiber, K. (1981). Endogenous plant hormones of the broad bean, Vicia faba L. (-)-Jasmonic acid, a plant growth inhibitor in pericarp. Planta 153,

6 Current Biology Vol 22 No Ueda, J., and Kato, J. (1982). Inhibition of cytokinin-induced plant growth by jasmonic acid and its methyl ester. Physiol. Plant. 54, Yamane, H., Sugawara, J., Suzuki, Y., Shimamura, E., and Takahashi, N. (1980). Syntheses of jasmonic acid related-compounds and their structure-activity-relationships on the growth of rice seedlings. Agric. Biol. Chem. 44, Tretner, C., Huth, U., and Hause, B. (2008). Mechanostimulation of Medicago truncatula leads to enhanced levels of jasmonic acid. J. Exp. Bot. 59, Falkenstein, E., Groth, B., Mithofer, A., and Weiler, E.W. (1991). Methyljasmonate and alpha-linolenic acid are potent inducers of tendril coiling. Planta 185, Sembdner, G., and Parthier, B. (1993). The biochemistry and the physiological and molecular actions of jasmonates. Annu. Rev. Plant Physiol. Plant Mol. Biol. 44, Creelman, R.A., and Mullet, J.E. (1995). Jasmonic acid distribution and action in plants: regulation during development and response to biotic and abiotic stress. Proc. Natl. Acad. Sci. USA 92, Biddington, N.L. (1986). The effects of mechanically-induced stress in plants a review. J. Plant Growth Regul. 4, Koornneef, M., Hanhart, C.J., and van der Veen, J.H. (1991). A genetic and physiological analysis of late flowering mutants in Arabidopsis thaliana. Mol. Gen. Genet. 229, Braam, J. (1992). Regulation of expression of calmodulin and calmodulin-related genes by environmental stimuli in plants. Cell Calcium 13, Cipollini, D.F., and Redman, A.M. (1999). Age-dependent effects of jasmonic acid treatment and wind exposure on foliar oxidase activity and insect resistance in tomato. J. Chem. Ecol. 25, Delk, N.A., Johnson, K.A., Chowdhury, N.I., and Braam, J. (2005). CML24, regulated in expression by diverse stimuli, encodes a potential Ca2+ sensor that functions in responses to abscisic acid, daylength, and ion stress. Plant Physiol. 139, Wang, Y., Wang, B., Gilroy, S., Chehab, E.W., and Braam, J. (2011). CML24 is involved in root mechanoresponses and cortical microtubule orientation in Arabidopsis. J. Plant Growth Regul. 30, Tsai, Y.C., Delk, N.A., Chowdhury, N.I., and Braam, J. (2007). Arabidopsis potential calcium sensors regulate nitric oxide levels and the transition to flowering. Plant Signal. Behav. 2, Chehab, E.W., Kaspi, R., Savchenko, T., Rowe, H., Negre-Zakharov, F., Kliebenstein, D., and Dehesh, K. (2008). Distinct roles of jasmonates and aldehydes in plant-defense responses. PLoS One 3, e Cipollini, D.F., Jr. (1997). Wind-induced mechanical stimulation increases pest resistance in common bean. Oecologia 111, Weiler, E.W., Albrecht, T., Groth, B., Xia, Z.-Q., Luxem, M., Liß, H., Andert, L., and Spengler, P. (1993). Evidence for the involvement of jasmonates and their octadecanoid precursors in the tendril coiling response of Bryonia dioica. Phytochemistry 32, Escalante-Pérez, M., Krol, E., Stange, A., Geiger, D., Al-Rasheid, K.A., Hause, B., Neher, E., and Hedrich, R. (2011). A special pair of phytohormones controls excitability, slow closure, and external stomach formation in the Venus flytrap. Proc. Natl. Acad. Sci. USA 108, Cipollini, D.F., Jr. (1998). The induction of soluble peroxidase activity in bean leaves by wind-induced mechanical perturbation. Am. J. Bot. 85, Aylor, D.E. (1990). The role of intermittent wind in the dispersal of fungal pathogens. Annu. Rev. Phytopathol. 28, Chehab, E.W., Raman, G., Walley, J.W., Perea, J.V., Banu, G., Theg, S., and Dehesh, K. (2006). Rice HYDROPEROXIDE LYASES with unique expression patterns generate distinct aldehyde signatures in Arabidopsis. Plant Physiol. 141,