For most plant hormones, biological activity is suppressed by reversible conjugation to sugars, amino acids and other small molecules. In contrast, the conjugation of jasmonic acid (JA) to isoleucine (Ile) is known to enhance the activity of JA. Whereas hydroxylation and carboxylation of JA-Ile permanently inactivates JA-Ile-mediated signaling in plants, the alternative deactivation pathway of JA-Ile by its direct hydrolysis to JA remains unstudied. We show that Nicotiana attenuata jasmonoyl-l-isoleucine hydrolase 1 (JIH1), a close homologue of previously characterized indoleacetic acid alanine resistant 3 (IAR3) gene in Arabidopsis, hydrolyzes both JA-Ile and IAA-Ala in vitro. When the herbivory-inducible NaJIH1 gene was silenced by RNA interference, JA-Ile levels increased dramatically after simulated herbivory in irJIH1, compared with wild-type (WT) plants. When specialist (Manduca sexta) or generalist (Spodoptera littoralis) herbivores fed on irJIH1 plants they gained significantly less mass compared with those feeding on wild-type (WT) plants. The poor larval performance was strongly correlated with the higher accumulation of several JA-Ile-dependent direct defense metabolites in irJIH1 plants. In the field, irJIH1 plants attracted substantially more Geocoris predators to the experimentally attached M. sexta eggs on their leaves, compared with empty vector plants, which correlated with higher herbivory-elicited emissions of volatiles known to function as indirect defenses. We conclude that NaJIH1 encodes a new homeostatic step in JA metabolism that, together with JA and JA-Ile-hydroxylation and carboxylation of JA-Ile, rapidly attenuates the JA-Ile burst, allowing plants to tailor the expression of direct and indirect defenses against herbivore attack in nature.
Plants are often attacked by herbivores with a wide range of feeding modes and preferences. In response, plants evolved diverse defense mechanisms, such as constitutive mechanical barriers, thorns and trichomes (Federal, 1988), replete with chemical defenses to fend off attackers. When these barricades are breached, plants produce inducible defense metabolites that directly affect the survival of the attackers or slow their growth and/or reproduction. In addition, plants release blends of volatile organic compounds to summon predators or parasitoids of the attackers (Kessler and Baldwin, 2002; Howe and Jander, 2008; Baldwin, 2010).
Most inducible plant defenses are known to be regulated by the oxylipin signaling cascade and its immediate product, jasmonic acid (JA), which in its interactions with other phytohormones mediates the large transcriptional and metabolic reconfigurations in plants after attack from biotic aggressors (Verhage et al., 2010). Herbivore attack induces a rapid release of polyunsaturated fatty acids (PUFAs; e.g. 18:3 α-linolenic acid and 18:2 α-linoleic acid) from chloroplastic membranes. α-Linolenic acid is enzymatically converted to 13S-hydroperoxyoctadecatrienoic acid (HPOT) by a specific lipoxygenase, which, in turn, is converted to oxophytodienoic acid (OPDA) by allene oxide synthase (AOS) and allene oxide cyclase (AOC). OPDA is transported to peroxisomes, where it is reduced by a peroxisomal OPDA reductase 3 (OPR3). Finally, JA is produced after three rounds of β-oxidation by the enzymes acyl-CoA oxidase 1 (ACX1), multifunctional protein (MFP) and l-3-ketoacyl CoA-thiolase (KAT) (Schaller and Stintzi, 2009). A proportion of JA is conjugated with isoleucine (Ile) by the JAR enzyme(s) to produce bioactive (+)-7-iso-jasmonoyl-l-isoleucine (JA-Ile) in the cytosol (Staswick and Tiryaki, 2004; Kang et al., 2006; Wang et al., 2007; Fonseca et al., 2009). Interestingly, JAR enzymes belong to the well-known GH3 family of acyl acid amidosynthases that conjugate amino acids to indole acetic acid (IAA) (Staswick et al., 2005; Wang et al., 2007; Westfall et al., 2010), suggesting a parallel evolution of JA and IAA metabolic pathways.
JA-Ile associates with an F-box protein COI1 that, as part of SCFCOI1, forms the E3-ubiquitin ligase complex and leads to the degradation of JAZ repressors by the 26S proteasome (Thines et al., 2007). Degradation of the JAZ repressors releases MYC2 transcription factor(s) from repression and activates the transcription of genes involved in plant defense responses (Fonseca et al., 2009; Memelink, 2009). The presence of bioactive JA-Ile, therefore, directly determines the duration of plant defense responses to biotic stresses that elicit JA signaling (Galis et al., 2009).
After the transduction of initial signals into downstream defense responses, plants must reset their signaling cascades to sense new environmental cues. The COI1-dependent upregulation of JAZ repressors was proposed as a negative feedback loop that downregulates JA signaling (Thines et al., 2007). Recently, hydroxylation of JA (Miersch et al., 2008) and hydroxylation and carboxylation of JA-Ile (Kitaoka et al., 2011; Koo et al., 2011; Heitz et al., 2012) was reported to be another efficient means of directly inactivating JA-Ile. Two cytochrome P450 enzymes, CYP94B3 (Kitaoka et al., 2011; Koo et al., 2011; Koo and Howe, 2012) and CYP94C1 (Heitz et al., 2012), were identified and functionally annotated as JA-Ile hydroxylase and carboxylase, respectively. Although expected, reversible attenuation of JA-dependent plant defense signaling by direct cleavage of JA-Ile to JA has not been reported.
Conjugation of hormones and their hydrolysis are common means of modulating the activity of several plant hormones (Staswick, 2009). Auxins are inactivated by conjugation to amino acids, sugars and other small molecules by members of the GH3 family proteins (Bartel and Fink, 1995; Woodward and Bartel, 2005). Conversely, free IAA is released from the conjugates by enzymes belonging to the ILR1-like family of IAA amidohydrolases that, in Arabidopsis thaliana, comprise ILL1, ILL2, ILL3, ILL5, ILL6, ILR1 and IAR3 (Campanella et al., 2003). Considering the common origin of JA- and IAA-conjugating GH3 class enzymes (Staswick et al., 2005), IAA-amidohydrolases have long been considered as possible candidates for JA-Ile hydrolyzing enzymes. Previously, LeClere et al. (2002) expressed IAA-alanine resistant 3 (IAR3; AT1G51760) protein in vitro as a GST fusion protein, and demonstrated its hydrolytic activity against several IAA-amino acid conjugates. Although discussed, the activity of this protein against JA conjugates was not reported (Davies et al., 1999; LeClere et al., 2002). IAR3-like genes identified from Brassica rapa (Savic et al., 2009) and Triticum aestivum (Campanella et al., 2004) have also been shown to cleave IAA-amino acid conjugates in vitro.
In this study, we cloned a herbivory-regulated homologue of IAR3 from Nicotiana attenuata (named NaJIH1) and showed that it is an active hydrolase of JA-Ile in vitro. We demonstrated the contribution of JIH1 to the control of JA-Ile levels in vivo, and its direct role in attenuating defense responses against herbivores in nature and in the glasshouse.
Results and Discussion
Nicotiana attenuata IAR3-like gene transcripts (NaJIH1) are strongly upregulated by herbivory
Heterologously expressed NaJIH1 hydrolyses JA-Ile and IAA-Ala
In previous reports, heterologously-expressed A. thaliana IAR3 protein hydrolyzed IAA-Ala and IAA-Gly with high efficiency. The detectable hydrolysis of other substrates such as IAA-Phe, IAA-Leu and IAA-Val, however, suggested a broad substrate specificity of this enzyme (Davies et al., 1999). Because NaJIH1 transcripts accumulated in response to simulated herbivory (Figure 1a,b), and IAR3 showed wound-inducible expression in Arabidopsis (Titarenko et al., 1997), we hypothesized that NaJIH1 might hydrolyze JA-Ile, the bioactive compound in JA signaling. Interestingly, NaJIH1 showed stronger induction after wounding compared with WOS treatment in local leaves (Figures 1a and S1), which was inversely correlated with the typical higher accumulation of JA-Ile found in WOS-induced N. attenuata leaves (Wu and Baldwin, 2009).
As a direct test of this hypothesis, we expressed the N-terminally truncated form of NaJIH1 (Figure S3) as a GST-tagged fusion protein, and examined its hydrolytic activity against JA-Ile and IAA-Ala in vitro (pH 7.5, 37°C and Mn2+, which was determined as the optimal enzyme co-factor). Under these conditions, NaJIH1 hydrolyzed both JA-Ile and IAA-Ala and released free JA and IAA (Figure 2a and b), respectively. Even when JA-Ile and IAA-Ala were supplied as a mixture, the enzyme cleaved both substrates (Figure S5a). When tested against other available JA-amino acid conjugates, the recombinant NaJIH1 enzyme hydrolyzed JA-Val, JA-Met and JA-Glu. In particular, JA-Val cleavage was consistent with the patterns of accumulation of this metabolite in NaJIH1-silenced plants (Figure S5b and S6). However, the enzyme showed no detectable hydrolytic activity against IAA-Asp, an auxin conjugate recently reported to play an important role in plant–pathogen interactions (Gonzalez-Lamothe et al., 2012).
irJIH1 plants accumulate more JA-Ile after simulated herbivory
To test which activity of NaJIH1 observed in vitro is reflected in vivo, we generated transgenic plants with reduced JIH1 transcript levels using RNA interference (RNAi), and chose two homozygous (T2), single insert-harboring lines (irJIH1-443 and irJIH1-445) for further analysis. Both irJIH1 lines had significantly reduced JIH1 transcript levels compared with wild-type (WT) plants before WOS induction (anova, F2,6 = 6.66, P = 0.05) and after 1 h (anova, F2,6 = 323.59, P < 0.0001) or 2 h (anova, F2,6 = 65.34, P < 0.0001) after WOS treatment (Figure 1c).
After WOS induction, irJIH1 plants accumulated significantly more JA-Ile compared with WT plants 1 h after WOS treatment (anova, F2,4 = 20.24, P = 0.008; Figure 3a). The increase in JA-Ile levels in irJIH1 plants did not result from transcriptional activation of JAR genes in N. attenuata or from the increased transcript levels of threonine deaminase (TD ) involved in isoleucine biosynthesis in irJIH plants (Figure S5c). Also, the higher JA-Ile accumulation in irJIH1 plants was not associated with a lower level of JA, as might be expected (Figure 3f). This comes as no surprise given that after simulated herbivory, only a small fraction of the JA burst (<15%) is converted to JA-Ile in N. attenuata. Hence, the contribution of the JIH1-mediated release of JA to the total JA burst may not be readily detectable against the background of a large and very dynamic pool of JA. Alternatively, a compensatory response through positive feedback of higher JA-Ile levels on JA biosynthesis could have contributed to the total pool of free JA, thus eliminating the expected differences between irJIH1 plants and WT. It has been shown that Arabidopsis CYP94B3 mutant plants defective in JA-Ile hydroxylation, and therefore with elevated JA-Ile content, contained more JA (Kitaoka et al., 2011). However, these differences were only observed 3 h after wounding, a relatively late time point at which JA-Ile levels tend to decline to near basal levels in wounded Arabidopsis leaves (Heitz et al., 2012). Unfortunately, no data on JA accumulation have been provided in two other reports demonstrating the function of the CYP94B3 enzyme (Koo et al., 2011; Heitz et al., 2012).
In contrast to JA-Ile, the levels of IAA were not significantly different between WT and irJIH1 plants at early time points following WOS induction (Figure 3c; 0 h, anova, F1,7 = 1.37, P = 0.28; 0.25 h, anova, F1,6 = 0.33, P = 0.58; 0.5 h, anova, F1,6 = 0.027, P = 0.87; 1 h, anova, F1,8 = 2.69, P = 0.13), suggesting that the primary effect of NaJIH1 is on JA-Ile rather than IAA-Ala metabolism. To further test this inference, we re-examined whether the JA-Ile phenotype in irJIH1 plants could result from local changes in IAA content that might be missed in the analysis of samples of entire homogenized leaves (see Experimental procedures). We induced leaves of WT and irJIH1 plants with WOS, and sprayed 1 mL of 1, 10 or 100 μg mL−1 IAA to simulate the potential release of IAA by NaJIH1 during simulated herbivory. When we determined the accumulation of JA-Ile in the leaves after 1 h (i.e. when plants not treated with IAA showed significantly different JA-Ile levels; Figure 3a), JA-Ile levels always remained significantly higher in irJIH1 than in WT plants (0 h, anova, F2,9 = 0.670, P = 0.535; WOS, 1% methanol, anova, F2,9 = 4.834, P = 0.03; 1 μg mL−1, anova, F2,7 = 9.104, P = 0.01; 10 μg mL−1, anova, F2,9 = 24.404, P = 0.0002; 100 μg mL−1 IAA, anova, F2,9 = 8.902, P = 0.007), suggesting that the observed differences in WOS-induced JA-Ile levels among WT and irJIH1 plants were independent of their IAA content (Figure 3b).
JA and JA-Ile metabolism in irJIH1 plants
The catabolism of JA-Ile into OH-JA-Ile and COOH-JA-Ile has been recently described as a mechanism to downregulate JA signaling (Kitaoka et al., 2011; Koo et al., 2011; Heitz et al., 2012). We reasoned that, if JA-Ile hydrolysis functions as another independent switch in JA signaling, irJIH1 plants should accumulate more OH-JA-Ile and COOH-JA-Ile than WT plants, to compensate for the lack of NaJIH1-mediated hydrolysis. Indeed, 3 h after WOS, irJIH1 plants accumulated significantly more OH-JA-Ile (anova, F2,5 = 10.86, P = 0.015) and COOH-JA-Ile (anova, F2,5 = 5.595, P = 0.05) compared with WT plants (Figure 3d,e). As the same result was obtained in another independent field experiment with empty vector (EV) and irJIH1 plants after multiple WOS leaf treatments (Figure S7; OH-JA-Ile, anova, F1,12 = 9.26, P = 0.01), we conclude that hydroxylation and hydrolysis of JA-Ile are two equally important means of attenuating the JA-Ile burst in plants, a conclusion consistent with the higher accumulation of JA-Ile in CYP94B3 mutant plants deficient in JA-Ile hydroxylation (Kitaoka et al., 2011; Koo et al., 2011). The increase in OH-JA-Ile levels in irJIH1 plants was, most likely, not regulated at the transcriptional level because the transcripts of a tentative CYP94B3 gene homologue from N. attenuata were not differentially regulated in WT and irJIH1 plants (Figure S5c).
Silencing of NaJIH1 suppresses the performance of herbivores
To investigate whether the silencing of NaJIH1 and the higher levels of JA-Ile in these plants directly affected the performance of feeding herbivores, M. sexta (a specialist) and Spodoptera littoralis (a generalist) caterpillars were fed on WT and irJIH1 plants, and the mass gain of herbivores was determined. Compared with caterpillars fed on WT plants, both the specialist (anova, F2,52 = 9.47, P = 0.0003) and the generalist (anova, F2,54 = 8.154, P = 0.0008) herbivores gained significantly less mass when reared on irJIH1 plants (Figure 4). Previously, M. sexta caterpillars performed better on N. attenuata plants deficient in the production of JA-Ile (irJAR4/6), whereas exogenous supplementation of JA-Ile reduced the performance of these caterpillars (Kang et al., 2006; Wang et al., 2008), consistent with the irJIH1 phenotype.
JIH1 attenuates multiple herbivore-induced direct defenses
Plants accumulate a number of JA-mediated secondary metabolites that directly suppress the performance of caterpillars (Kessler and Baldwin, 2004; Wang et al., 2007; Heiling et al., 2010). To investigate the role of NaJIH1 in the regulation of secondary metabolism, we compared the accumulation of several herbivory-induced defense metabolites in WT and irJIH1 plants grown in the glasshouse, and found that 24 h after WOS induction irJIH1 plants accumulated significantly higher levels of nicotine (anova, F2,6 = 9.31, P = 0.01), 17-hydroxygeranyllinalool diterpene glycosides (HGL-DTGs; anova, F2,6 = 8.91, P = 0.01) and protease inhibitors (PIs; anova, F2,11 = 4.02, P = 0.04) than WT plants (Figure 5). When we examined the WOS-induced accumulation of individual HGL-DTGs in irJIH1 plants, we found that they accumulated significantly more mono- and di-malonylated HGL-DTGs than did wild-type plants (Figure 5d). Because protease inhibitors (Zavala et al., 2004) and HGL-DTGs (Heiling et al., 2010) play very important roles in the defense of N. attenuata plants against herbivores, the over-accumulation of these metabolites in irJIH1 plants is sufficient to explain the reduced performance of caterpillars on irJIH1 plants.
Performance of irJIH1 plants in nature
Taking advantage of irJIH1 plants and their limited ability to regulate active jasmonate levels after WOS treatment, we decided to examine the ecological importance of this regulation in nature and planted EV and irJIH1 plants in a field plot at the field station in Santa Clara (UT, USA) in 2011. After establishment, the plants were WOS-induced to determine the robustness of the NaJIH1-silencing phenotype in nature. Whereas EV plants accumulated progressively more JA-Ile and its catabolites after repeated simulated herbivory (see Experimental procedures), irJIH1 contained significantly more JA-Ile 4 h after the first induction and following the third WOS treatment (anova; F1,12 = 6.01, P = 0.03) (Figure S7), showing that JIH1-mediated hydrolysis of JA-Ile, similar to glasshouse conditions, was required to attenuate the JA-Ile burst in nature.
We also determined the accumulation of defense metabolites in the field-grown EV and irJIH1 plants after WOS treatment. As expected, irJIH1 plants contained significantly more HGL-DTGs (anova, F1,8 = 9.81, P = 0.01) and protease inhibitors (PIs; anova, F1,6 = 6.48, P = 0.04) than did EV plants. When we examined the accumulation of individual HGL-DTGs, irJIH1 plants accumulated significantly more mono- and di-malonylated HGL-DTGs than EV plants (Figure S8).
NaJIH1 attenuates indirect defenses in Nicotiana attenuata
When attacked by herbivores, plants release a blend of volatile organic compounds (VOCs) to attract predators, parasitoids or pathogens of the attacking herbivores (Pare and Tumlinson, 1999; Baldwin, 2010). In N. attenuata, the predator Geocoris pallens is attracted by the volatile cues released by attacked plants, and feeds on M. sexta eggs and larvae (Kessler and Baldwin, 2004). To evaluate whether JIH1 contributed to the regulation of indirect defense responses in N. attenuata in a natural ecological setting, we attached M. sexta eggs to the underside of leaves of control or WOS-treated EV and irJIH1 plants in the field, and quantified the eggs predated upon by G. pallens. On control EV and irJIH1 plants, 15.7 and 17.6% of the eggs were predated upon, respectively. However, 24 h after WOS treatment, the percentage predation on EV and irJIH1 plants increased to 31.6 and 52.9%, respectively (Figure 6a). Following simulated herbivory, irJIH1 plants experienced a higher rate of egg predation than EV plants. Previously, N. attenuata plants silenced in the activity of WRKY3/6 transcription factors were less attractive to predators compared with EV plants in the field. Notably, irWRKY plants are impaired in their responses to repeated elicitations, and show lower accumulations of JA and JA-Ile, and consequently less TPIs and HGL-DTGs, and lower emissions of volatile compounds from WOS-induced leaves in the field (Skibbe et al., 2008).
To evaluate if differential egg predation was associated with altered levels of herbivory-elicited emission of VOCs in irJIH1 plants, we analyzed the WOS-induced emission of VOCs from glasshouse-grown plants (Kessler and Baldwin, 2004; Gaquerel et al., 2009). Consistent with observed predation data in the field, irJIH1 plants emitted significantly more trans-α-bergamontene (anova, F1,15 = 8.66, P = 0.01), caryophyllene (anova, F1,22 = 7.60, P = 0.01), α-duprezianene (anova, F1,15 = 8.66, P = 0.01), trans-β-ocimine (anova, F1,22 = 5.75, P = 0.02), β-elemene (anova, F1,22 = 4.41, P = 0.04) and linalool (anova, F1,15 = 16.1, P = 0.01) than did WT plants (Figure 6b), consistent with a role of NaJIH1 (and JA-Ile) in the regulation of herbivory-induced VOC emissions.
Conclusions and perspectives
Focusing on the attack from a single herbivore, and not listening to other warning signals from the environment, could be very harmful for plants in their natural environments. The ability of plants to reset JA signaling (Figure 7) after an initial attack is likely to be important in helping plants tailor their defense responses when repeatedly attacked, or attacked by other aggressors that also elicit JA signaling. In addition, excessive production of defense metabolites is known to be costly for plants (Brown, 1988; Baldwin, 1998), and hence the tight control over defense responses optimizes the fitness of competing plants (Ito and Sakai, 2009). It will be interesting to compare how irJIH1 plants perform in direct competition with other plants in order to address additional roles of rapid JA-Ile catabolism in plants under natural stress conditions.
Plant growth and treatments
The seed germination, growth conditions and Agrobacterium-mediated transformation were previously described in Krügel et al. (2002). Selected NaJIH1 cDNA fragment (277 bp; Figure S3) was used to generate transgenic plants with suppressed NaJIH1 expression by RNAi using pSOL8 transformation vector (Bubner et al., 2006). To simulate herbivory, fully expanded (+1) leaves were wounded with a serrated fabric pattern wheel and 20 μL of either de-ionized water (WW) or diluted M. sexta oral secretions (WOS; diluted in water 1:5, v/v) were applied to the wounds (control samples remained untreated). For herbivore bioassays, wild-type and irJIH1 plants were maintained in the glasshouse until rosette stage, when freshly hatched neonates of the specialist herbivore (M. sexta) were placed on fully elongated (+1) leaves. Neonates of the generalist herbivore S. littoralis were first fed for 6 days on an artificial diet before they were transferred to plants. Caterpillars were allowed to continuously feed for 12 days, whereas their mass was determined every 4 days.
Field experiments were conducted at the Lytle Ranch Preserve research station (Santa Clara, UT, USA). Seeds were imported (APHIS number, 10-004-105m) and released (APHIS number, 06-242-3r-a3) into the field station following Animal and Plant Health Inspection Service (APHIS) regulations. Germination and growth conditions in the field were as described previously (Kessler et al., 2008). irJIH1 and EV plants were planted in randomized pairs and grown until the early elongation stage in the field plot. To determine jasmonate accumulation after multiple elicitations, EV and irJIH1 plants were WOS-treated every hour for 3 h (by making one row of puncture wounds on both sides of the lamina every hour, and applying either water or OS). Samples were collected at the fourth hour and stored on dry ice. To estimate predation rates in the field, one M. sexta egg per plant was glued with a non-toxic glue to the underside of the leaves of 19 pairs of EV and irJIH1 plants, and the percentage of predated eggs by G. pallens (Heteroptera, Geocoridae) was determined visually after 24 h. The adjacent leaves were either left un-induced or induced by WOS to stimulate the release of volatiles.
Total RNA was extracted from deep-frozen leaf material using TRIzol reagent (Invitrogen, http://www.invitrogen.com). After treatment of total RNA with DNase (RQ1 RNase-Free DNase; Promega, http://www.promega.com), cDNA was synthesized using oligo (dT)18 and Superscript II reverse transcriptase (Invitrogen). All qRT-PCR experiments were performed on Mx3005P Multiplex qPCR (Stratagene, now Agilent Technologies, http://www.genomics.agilent.com) with qPCR core kit for SYBR Green I (Eurogentec, http://www.eurogentec.com). Transcript abundances were normalized using N. attenuata elongation factor-1α (EF-1α) as the internal reference. The primer pairs used in qPCRs are listed in Table S1.
For phytohormone analyses, leaves of rosette WT and irJIH1 plants were WOS induced, collected and flash frozen in liquid nitrogen (or dry ice in the field). About 200 mg powder prepared in liquid nitrogen was homogenized in 1 mL ethyl acetate spiked with 200 ng mL−1 of D2-JA and 40 ng mL−1 D6-ABA, D4-SA and JA-[13C6]Ile internal standards. The homogenate was centrifuged for 20 min (16 100 g, 4°C) and the supernatants were transferred into new tubes. The pellets were re-extracted with 0.5 mL ethyl acetate, centrifuged as above, and combined supernatants were dried in a vacuum concentrator (Eppendorf, http://www.eppendorf.com). After dissolving the residue in 0.5 mL 70% methanol in water (v/v) with centrifugation for 10 min (16 100 g, 4°C), cleared supernatants (10 μL) were analyzed on Varian 1200L Triple-Quadrupole-LC-MS (Varian, http://www.varian.com) using a ProntoSIL® column (C18; 5 μm, 50 × 2 mm; Bischoff, http://www.bischoff-chrom.com) attached to a pre-column (C18; 4 × 2 mm; Phenomenex, http://www.phenomenex.com). The mobile phase, consisting of solvent A (0.05% formic acid in water) and solvent B (methanol), was used in a gradient mode with times/concentrations (min/%B) of 0:00/5, 1:02/5, 2:30/5, 5:30/98, 10:30/98, 11:30/5, 15:00/5, and with a flow, time/flow (min/mL), of 0:00/0.4, 1:02/0.2, 2:30/0.2, 5:30/0.2, 10:30/0.4, 11:30/0.4 and 15:00/0.4. The MS was operated in negative ionization mode using multiple reactions monitoring (MRM). The molecular ions, [M–H]−, generated for JA, JA-Ile, OH-JA-Ile, COOH-JA-Ile, D2-JA and JA-13C6-Ile were, respectively, at m/z 209.0, 322.0, 338.0, 352.0, 213.0 and 328.0, and were fragmented under CE 12.0 V (for JA and D2-JA) or 19.0 V (for JA-Ile, OH-JA-Ile, COOH-JA-Ile and JA-13C6-Ile), generating daughter ions at m/z 59.0, 130.0, 130.0, 130.0, 59.0 and 136.0, respectively. The ratios of the ion intensities of endogenous compounds and internal standards were used for quantification after normalizing them with the fresh mass of the samples. Synthesis of the JA-amino acid conjugates used was as described in Wang et al. (2007), and the same instrumental parameters were used to estimate the content of these metabolites in plants. The molecular ions were detected in a negative mode at m/z 308.0, 340.0 and 337.0 for JA-Val, JA-Met and JA-Glu, respectively, fragmented at CE 19.0 V, yielding the respective daughter ions at m/z 116.0, 148.0 and 145.0. D2-JA was used for relative normalization of the contents.
Secondary metabolite analysis
For secondary metabolite analyses (nicotine, total 17-hydroxygeranyllinalool diterpene glycosides, i.e. HGL-DTGs, individual HGL-DTGs and caffeoylputrescine), samples were flash-frozen in liquid nitrogen (or dry ice in the field) and 100 mg of ground powder was extracted and analyzed by HPLC equipped with a photodiode array detector (nicotine, total HGL-DTGs, caffeoylputrescine), as described in Onkokesung et al. (2012), or LC-MS3 (individual HGL-DTGs), as described in Heiling et al. (2010). The proteinase inhibitor activity in plant extracts was quantified by radial diffusion assay as described previously (Jongsma et al., 1993) after measuring the protein concentrations by standard Bradford assay.
To compare herbivore-induced VOC emissions, fully expanded (+1) leaves of elongated, glasshouse-grown WT and irJIH1 plants were treated with WOS. Induced leaves were immediately clip-caged to trap the volatiles for 3 h, when the volatile traps were replaced and VOCs were collected for an additional 24 h. The trapping and analysis of VOCs were performed as described in Wu et al. (2008).
Cloning, heterologous expression and purification of NaJIH1 protein
The truncated NaJIH1 gene lacking its first 23 N-terminal amino acids was PCR amplified and cloned between BamHI/NotI sites of pGEX-4T-3 expression vector (GST Gene Fusion system; GE Healthcare, http://www.gehealthcare.com) using specific adaptor primers (Table S1). A sequence-verified expression clone of pGEX-JIH1 vector was transformed into BL-21 (DE3) pLysS strains of Escherichia coli (Novagen, now EMD Millipore, http://www.emdmillipore.com), and the production of the JIH1-GST fusion protein was induced in LB media containing ampicillin (100 μg mL−1) at 28°C by adding 0.1 mm isopropyl β-d-1-thiogalactopyranoside (IPTG; Carl Roth, http://www.carlroth.com) to cells at an OD of 0.6–0.8. Protein accumulation was carried out for 24 h at 28°C, cultures were centrifuged at 922 g for 10 min and pellets were stored at −80°C until protein purification using GST SpinTrapTM columns (GE Healthcare), following the manufacturer’s protocols.
Enzyme activity assays
Previously, Arabidopsis IAR3 protein hydrolyzed IAA-Ala in TRIS buffer (pH 7.5 and 8.0) in the presence of Mn2+ co-factor (LeClere et al., 2002). Similar conditions were adopted to test the activity of recombinant NaJIH1-GST against JA-Ile and IAA-Ala in 100-μL enzyme reactions: 50 mm TRIS buffer, 1 mm dithiothreitol, 1 mm MnCl2, 40 μL of purified enzyme and 2 μm IAA-Ala or 2 μm JA-Ile (or both substrates). Control reactions were set without enzyme to monitor non-enzymatic hydrolysis. To test the optimum temperature and co-factor preference, reactions were incubated at 25 or 37°C in the presence of selected bivalent co-factors (Mn2+, Mg2+, Ca2+ and Co2+). To terminate reactions, 100 μL of stop solution (99 : 1; v/v, methanol : acetic acid) spiked with equimolar concentrations of D2-JA or phenyl-13C6-indole-3-acetic acid as internal standards was added to each 100-μL reaction. After brief vortexing and centrifugation (3 min, 13 000 g, 4°C), 10 μL of clear supernatant was injected onto a ProntoSIL® column (C18; 5 μm, 50 × 2 mm; Bischoff) attached to a pre-column (C18; 4 × 2 mm, Phenomenex) on Varian 1200L Triple-Quadrupol-MS (Varian). The MS was run in negative MRM mode with the same gradient used for phytohormone analysis. The molecular ions, [M-H]−, for JA, D2-JA, IAA, [13C6]IAA, JA-Ile and IAA-Ala were at m/z 209.0, 213.0, 174.0, 179.0, 322.0 and 245.0, respectively. The molecular ions were fragmented at CE 10.0, 9.5, 12.0, 12.0, 15.0 and 19.0 V, yielding respective daughter ions at m/z 59.0, 59.0, 130.0, 136.0, 130.0 and 88.0. The intensities of the daughter ions were normalized against that of labeled internal standards.
Extraction and quantification of auxin
Control and WOS-treated WT and irJIH1 leaves were finely ground in liquid nitrogen and 1 g of the powder was extracted overnight in 10 mL of 100% methanol containing 2.5 mm diethyldithiocarbamic acid (Sigma-Aldrich, http://www.sigmaaldrich.com) and 50 ng phenyl-[13C6]IAA (Cambridge Isotope Laboratories, http://www.isotope.com). Extracts were centrifuged for 30 min (3000 g, 4°C), and supernatants were transferred to new tubes. Pellets were re-extracted as before for 30 min in pure methanol, centrifuged and supernatants were combined. The concentration of extracts was adjusted to 50% methanol (v/v) by adding distilled water, and samples were purified by sequentially passing through Supelco Supelclean LC-18 SPE columns (Sigma-Aldrich) and were then adsorbed to activated DEAE Sephadex A25 columns (GE Healthcare) that were pre-equilibrated with 50% methanol. After adsorption of the IAA, columns were rinsed with 50 mL of 50% (v/v) methanol and IAA was eluted by 6% (v/v) formic acid into new Supelco Supelclean LC-18 SPE columns coupled underneath. After briefly drying the columns with a syringe filled with air, IAA was eluted from the column with 5 mL of diethylether (the water phase retained in the samples was immediately removed). The organic phase was evaporated under a stream of nitrogen, and the residue was dissolved in 1.5 mL of 100% methanol. Samples were dried under vacuum, dissolved in 70% methanol, centrifuged for 30 min (16 000 g, 4°C) and measured on Varian 1200 L Triple-Quadrupol-MS (Varian) by injecting 10 μL of the supernatant onto a Prodigy column [3 μm, ODS(3), 100 Å, 150 × 2 mm; Phenomenex] attached to a pre-column (C18, 4 × 2 mm; Phenomenex). The mobile phase consisted of solvent A (0.05% acetic acid) and solvent B (acetonitrile), used in a gradient mode with time/concentration (min/% B) compositions of 0:00/20, 1:30/20, 6:00/97, 17:00/97, 18:00/20 and 25:00/20, with a flow, time/flow (min/mL), of 0:00/0.2, 1:30/0.2, 6:00/0.2, 17:00/0.3, 18:00/0.3 and 25:00/0.2. The MS was run in negative MRM mode and the molecular ions for IAA and phenyl-[13C6]IAA detected, respectively, at m/z 174.0 and 180.0. The daughter ions were generated at collision energy (CE) 10.0 V and detected at m/z 130.0 and 136.0, respectively. The ratios of the signal intensities of the daughter ions were normalized by the fresh mass used for extraction to quantify the WOS-induced auxin content.
Exogenous IAA treatments
To test the effect of exogenous IAA application on WOS-induced JA-Ile accumulation, fully expanded leaves of elongated N. attenuata plants were treated by WOS and immediately sprayed with 1 mL of 1 μg mL−1, 10 μg mL−1 and 100 μg mL−1 IAA (Sigma-Aldrich). Samples were collected 1 h after treatment and the levels of JA-Ile were analyzed as before.
All statistical analyses were performed using statview 5.0 (SAS Institute, http://www.sas.com). We used an alpha level of 0.05 for all statistical tests.
We acknowledge the German Academic Exchange Service (DAAD) and the International Max Planck Research School (IMPRS) for financial support. Manduca sexta eggs and larvae used in field experiments were provided by Dr Carol Miles, Department of Biological Sciences, Binghamton University. We thank Brigham Young University for the use of their field station, the Lytle Ranch Preserve.