Root jasmonic acid synthesis and perception regulate folivore-induced shoot metabolites and increase Nicotiana attenuata resistance

Authors


Summary

  • While jasmonic acid (JA) signaling is widely accepted as mediating plant resistance to herbivores, and the importance of the roots in plant defenses is recently being recognized, the role of root JA in the defense of above-ground parts remains unstudied.
  • To restrict JA impairment to the roots, we micrografted wildtype Nicotiana attenuata shoots to the roots of transgenic plants impaired in JA signaling and evaluated ecologically relevant traits in the glasshouse and in nature.
  • Root JA synthesis and perception are involved in regulating nicotine production in roots. Strikingly, systemic root JA regulated local leaf JA and abscisic acid (ABA) concentrations, which were associated with differences in nicotine transport from roots to leaves via the transpiration stream. Root JA signaling also regulated the accumulation of other shoot metabolites; together these account for differences in resistance against a generalist, Spodoptera littoralis, and a specialist herbivore, Manduca sexta. In Nattenuata's native habitat, silencing root JA synthesis increased the shoot damage inflicted by Empoasca leafhoppers, which are able to select natural jasmonate mutants. Silencing JA perception in roots also increased damage by Tupiocoris notatus.
  • We conclude that attack from above-ground herbivores recruits root JA signaling to launch the full complement of plant defense responses.

Introduction

Plants have evolved refined signaling mechanisms capable of inducing responses specifically according to different stimuli. These signals can act locally, but can also travel to distal systemic parts within the plant, fine-tuning the whole-plant metabolism and maximizing its performance and Darwinian fitness. An interesting case of these systemic events is the inducible responses of plants when attacked by leaf feeders: within minutes, defense mechanisms are triggered locally in attacked leaves and also in unattacked remote parts of the plant (Wu & Baldwin, 2010). In maize, transcriptional changes in roots in response the attack of leaves by Spodoptera littoralis larvae were shown to exceed the responses observed in the infested leaves (Erb et al., 2009). Also, in the wild tobacco species Nicotiana attenuata, transcriptional and metabolic reorganization after mimicked herbivore attack were more pronounced in roots than in local leaves (Gulati et al., 2013). Interestingly, in both cases, minor or no overlap in induced genes was found between the local and the systemic tissues, suggesting that the responses elicited by herbivore attack are triggered in a tissue-specific manner. However, the signaling mechanisms that integrate these below-ground events with the defense responses observed in the shoots remain unknown.

Considerably more is known about the defense responses observed in above-ground plant parts, in which jasmonic acid (JA) and its derivatives, collectively known as jasmonates, play a well-described regulatory role (Howe & Jander, 2008). In attacked leaves, the initial step of JA biosynthesis is the production of linolenic acid, a precursor that, through the 13-lipoxigenase (13-LOX) pathway, culminates in the formation of 12-oxo-phytodienoic acid (OPDA) by the action of an allene oxidase cyclase (AOC; Stenzel et al., 2003). OPDA is then reduced and subjected to cycles of β-oxidation, which finally leads to the production of JA. JA can be subsequently conjugated to isoleucine (JA-Ile) by jasmonate-resistant 1 (JAR1; Staswick & Tiryaki, 2004). JA-Ile, the bioactive conjugated form of JA, interacts with coronatine-insensitive 1 (COI1) and triggers the degradation of jasmonate ZIM domain (JAZ) proteins, which releases downstream positive regulators of the JA-mediated responses (Xie et al., 1998; Chini et al., 2007). In N. attenuata, plants silenced for NaAOC (irAOC; Kallenbach et al., 2012) or for NaCOI1 (irCOI1; Paschold et al., 2007) are more vulnerable to herbivore attack. In addition, wound-treated leaves of irAOC completely lack the capacity to induce jasmonate concentrations, while those of irCOI1 present a delayed and more pronounced accumulation of JA-Ile compared with wildtype (WT) plants, as a consequence of a lower JA-Ile catabolism in leaf tissues of irCOI1 plants (Paschold et al., 2008).

Following the JA burst observed in local attacked leaves, JA signaling is also required in the systemic responses of distal leaves. Grafting experiments have dissected the local vs systemic requirements of the JA signaling components in the shoots of tomato (Li et al., 2002, 2005). However, for the systemic events of the below-ground plant parts, JA has been suggested to mediate root responses to shoot elicitation. One of the best-studied examples of this JA-dependent shoot–root interplay is given by nicotine induction in tobacco plants. Although nicotine is synthesized in roots, it is the most abundant alkaloid found in the leaves. It is highly toxic to most herbivores (Glendinning, 2002), and is effective in thwarting compensatory leaf consumption of generalists, such as Spodoptera exigua, as well as specialists, like Manduca sexta (Steppuhn et al., 2004). Upon leaf damage, concentrations of nicotine in shoots dramatically increase (up to 10-fold) as a consequence of induced root-specific expression of the putrescine N-methyltransferase (NaPMT) gene (Winz & Baldwin, 2001). These events are tightly associated with increased JA pools in roots that follow the rapid increase in locally damaged leaves (Zhang & Baldwin, 1997). When 14C-labeled JA was applied to wounded leaves, this labeled compound was recovered in roots, at similar rates of endogenous root JA, suggesting that leaf-derived JA transported to roots could alone account for the systemic increase in root JA after leaf wounding. In a later study, Wang et al. (2008) showed that, after a treatment that mimicked herbivore attack combined with the application of 13C-labeled Ile, newly synthesized 13C-labeled JA-Ile was only detected in elicited leaves, not in roots. However, these results should be interpreted with caution, because it is possible that labeled compounds are not metabolized or transported in the same way as plant-derived compounds. And although a plant's nicotine investment is elicited in the roots through enhanced JA concentrations in roots (Baldwin, 1996b), it remains unknown whether shoot-derived JA alone can account for nicotine induction.

Recently, Grebner et al. (2013) showed that Arabidopsis roots, when wounded, can synthesize JA independently of the shoots by the action of a single member of the LOX family (LOX6) that is putatively activated by a rapidly propagated electrical signal. Acosta et al. (2013) reported that after wound treatment applied to cotyledons of Arabidopsis seedlings, the activity of JAR1 and NINJA, a member of the JA transcriptional repression complex, are indispensable to induce the roots' systemic expression of JAZ10-GUSPlus, a JA-responsive reporter. However, whether root JA de novo synthesis and perception are involved in ecologically relevant traits, which in turn affect plant performance under herbivore pressure, remains an open question.

Here we dissected the function of systemic JA synthesis and perception in roots of N. attenuata in response to shoot elicitation. To test whether the disruption of root JA signaling has ecological consequences, we generated chimeric plants consisting of shoots of transgenic plants harboring an empty vector (EV) grafted on to roots of transgenic lines impaired in JA synthesis (irAOC) or perception (irCOI). We then evaluated resistance traits of these grafts in both glasshouse and field experiments. The results reveal that jasmonate synthesis and JA-Ile perception of both shoot and roots are required to induce nicotine production in the roots as well as its transport to the shoots. Strikingly, root JA signaling systematically regulates leaf concentrations of JA and ABA after leaf wounding. Finally, we show that root JA synthesis and perception contribute to the metabolic profile of leaves, which in turn influence above-ground herbivore preference in both the glasshouse and nature.

Materials and Methods

Plant material and treatments

All lines were derived from seeds originally collected in a natural population of N. attenuata Torrey ex Watson at the DI Ranch, near Santa Clara, CA, USA. Seed germination and plant growth are described in Kruegel et al. (2002). WT or transgenic plants harboring an EV construct were used as controls; all transformed and WT plants were from the same inbred generation of the same original accession. Silenced (ir) stably transformed plants were used to knock down JA-Ile perception (irCOI1; Paschold et al., 2007), JA synthesis (irAOC; Kallenbach et al., 2012), or nicotine production (irPMT; Steppuhn et al., 2004). Seven-day-old seedlings were grafted as described in Fragoso et al. (2011), with an average rate of grafting success of 77%, which did not differ significantly among all graft combinations (= 0.1377; c. 300–400 seedlings were grafted for each graft combination).

Glasshouse plants were grown in sand or in soil, and kept at 26–28°C under a light : dark 16 : 8 h photoperiod. In order to induce nicotine concentrations in a standardized way, 5-wk-old plants had three of their rosette leaves punctured with a pattern wheel, run four times on each side of leaf, parallel to the midrib (wounding treatment). After designated time points, systemic tissues (roots and pooled stalk leaves) and local leaves of control and wounded plants were sampled and stored at −80°C until analysis. In order to normalize leaf damage among plants for global metabolic analysis, herbivore attack was mimicked by wounding, as previously described, and applying 20 μl per leaf of a 1 : 5 dilution of oral secretions in water directly to the freshly created puncture wounds. Oral secretions were collected from third- to fourth-`instar larvae of M. sexta or S. littoralis reared on WT plants. To detected differences in metabolite profiles in response to mimicked herbivory, 3 d after treatments, undamaged systemic leaves were sampled and stored at −80°C until analysis.

For field experiments, seeds were imported under US Department of Agriculture Animal and Plant Health Inspection Service (APHIS) notification number 11-350-101r, and planted in a randomized manner to an experimental plot at the Lytle Ranch Preserve (UT, USA) in 2012.

Nicotine extraction and quantification by high-performance liquid chromatography/photodiode array (HPLC-PDA) detector

To test whether disrupting root JA signaling influences nicotine induction, c. 150 mg of leaf or 300 mg of root tissue of control or wound-treated grafts was extracted for nicotine quantification as described by Onkokesung et al. (2012). Plant tissue was ground with two 4 mm steel balls by Genogrinder 2000 (SPEX CertiPrep, Metuchen, NJ, USA). Samples were extracted with 1 ml of methanol: water (40 : 60, v/v) acidified by 0.1% (v/v) acetic acid and homogenized by vortex for 10 min. Supernatants were subjected to two rounds of centrifugation at 16 100 g at 4°C for 20 min. Sample aliquots of 1 μl were analyzed by an Agilent HPLC 1100 Series device (http://www.chem.agilent.com) in a Chromolith FastGradient RP-18e column (endcapped 50 × 2 mm; Merck, Darmstadt, Germany) attached to a precolumn (Gemini NX RP-18e, 3 μm, 2 × 4.6 mm; Phenomenex, Aschaffenburg, Germany) as described in Oh et al. (2012). For quantification, an external standard curve of an authentic and purified nicotine standard was used, and peaks were identified based on their retention time and spectra, and samples were spiked with purified compounds to estimate matrix effects. All extractions were performed with at least six biological replicates, and the resulting nicotine amount was expressed per g fresh mass plant material.

RNA extraction and real-time reverse transcription polymerase chain reaction (RT-PCR)

To check whether NaAOC and NaCOI1 expression in EV shoots was silenced when grafted on to rootstocks of irAOC and irCOI1, respectively, and to check expression levels of NaPMT in roots, which encodes the first enzyme committed to nicotine biosynthesis, total RNA was extracted from leaf or root tissues with the Trizol reagent. After normalizing total RNA concentration of all samples to 500 ng μl−1, cDNA was synthesized as described in Fragoso et al. (2011). All primers were previously described (Paschold et al., 2007; Kallenbach et al., 2012), and for NaPMT a new pair of primers was designed and tested for their ability to amplify a 93-bp-long consensus cDNA fragment of both NaPMT1 and NaPMT2 genes (NaPMT12-for 5′- TCATTGGACCAAGATCGAG-3′ and rev 5′-TGGAAATTATGATAATTACTGCAGA-3′; Winz & Baldwin, 2001). The efficiency of the primers and the estimated initial amount of template were calculated as described in Fragoso et al. (2011) and relativized to N. attenuata's elongation factor1A (NaEF1A). All reactions used qPCR Core Kit for SYBR Green I (Eurogentec, Seraing, Belgium, http://www.eurogentec.com), and performed with at least five biological replicates.

Petiole-feeding experiment

To test whether disruption in root JA signaling influences nicotine uptake by leaves, a petiole-feeding assay was performed. At 1 d after leaf wounding, when systemic leaves of nongrafted WT plants start to differentially increment nicotine in response to wounding treatment, younger unwounded systemic leaves from control or wound-treated plants were carefully excised at the base of their petioles, weighed and transferred to vials with water or a 1 mM nicotine-containing solution. After 1 d of feeding, leaf laminas were dissected and stored at −80°C until HPLC analysis for their nicotine content, as described earlier. All tubes containing solutions were weighed before and after the petiole feeding, so that the volume of solution transpired by the leaf could be estimated. In the nicotine treatment, the remaining solution was also analyzed for its nicotine content, so in comparing initial and final nicotine solution concentrations, nicotine uptake was calculated. Treatments were performed with at least eight biological replicates.

Phytohormone analysis

Phytohormones were quantified in order to evalutate whether root-silencing of NaAOC (EV/irAOC) or NaCOI1 (EV/irCOI1) resulted in impaired root jasmonates and ABA accumulation in response to leaf wounding, or whether, alternatively, these molecules would be transported from EV shoots to the silenced roots. Damaged leaves of EV/EV, EV/irAOC, and EV/irCOI1 grafts, along with their respective undamaged roots, were sampled before and at 1, 2 and 3 h after wounding treatment. Approx. 100 mg leaf or 200 mg root frozen tissue material was ground, as described earlier. Phytohormones were extracted with 1 ml of ethyl acetate spiked with internal standards (200 ng of [2H2]JA and 40 ng each of JA-[13C6]-Ile, [2H4]SA, and [2H6]ABA). After extraction by vortexing for 10 min, 500 μl of the organic phase was obtained by centrifugation at 16 100 g for 15 min at 4°C. Samples were evaporated almost to dryness in a vacuum concentrator (Eppendorf, Hamburg, Germany) under reduced pressure at 30°C. Leaf samples were then diluted in 200 μl of methanol: water (70 : 30, v/v), while 100 μl was used for root samples, once roots accumulate smaller amounts of phytohormones. Analysis was performed with a Varian 1200 HPLC-MS/MS system as described in Vadassery et al. (2012), with a modified shortened chromatographic gradient. Sample-derived phytohormones were calculated by the ratios of their ion intensity and of their respective internal standards; for cis-OPDA, [2H2]JA was used as an internal standard, applying an experimentally determined response factor of 0.5. All quantifications were corrected according to the sample dilution, and extractions were performed with six biological replicates. The resulting amount of different phytohormones was then expressed per g fresh mass plant material.

Extraction and unbiased analysis of metabolites

In order to evaluate whether root JA disruption influences leaf accumulation of metabolites other than nicotine, an unbiased metabolomics screen was performed (Gaquerel et al., 2010). Plants were elicited by mimicked and standardized herbivore attack and, 3 d after treatment, systemic leaves were sampled and ground as described earlier. Metabolites were extracted from frozen ground tissue with 1 ml of 50 mM acetate buffer (40 mM acetic acid plus 44 mM ammonium acetate; 4.8 pH) in methanol (60 : 40, v/v). Samples were homogenized, and supernatant was recovered after two rounds of centrifugation. After separation by an Agilent HPLC 1100 Series device (http://www.chem.agilent.com), the eluted compounds were positively charged by electrospray ionization (ESI) and had their masses detected by MS, carried out with a MicroToF (Time-of-Flight; Bruker Daltonik, Bremen, Germany). Extractions used four to five biological replicates for analysis.

Raw data files were converted to netCDF format and processed by XCMS (http://fiehnlab.ucdavis.edu/staff/kind/Metabolomics/Peak_Alignment/xcms/) and CAMERA (http://bioconductor.org/packages/devel/bioc/html/CAMERA.html) R packages according to Kim et al. (2011). All peaks from 40–450 s of ions in the mass range m/z 90–1400 were selected and normalized by the exact amount of plant material used. Only peaks that were found in at least 75% of the replicates with absolute intensities higher than 5 megacounts s–1 of the total ion count within same graft kind were analyzed by principal component analysis (PCA) using MetaboAnalyst (http://www.metaboanalyst.ca/MetaboAnalyst/faces/Home.jsp), following a normalization by the median value and Pareto scaling.

Herbivore assays

To explore whether disruption of JA signaling in roots influence herbivore mass gain or preference, herbivore assays were performed with EV/EV, EV/irAOC and EV/irCOI1 plants under glasshouse or open field conditions. Eggs of M. sexta were obtained from Carolina Biological Supply and were derived from an in-house colony; and S. littoralis eggs were obtained from Syngenta Crop Protection AG (Stein, Switzerland). All eggs were kept in growth chambers (Snijders Scientific, Tilburg, the Netherlands) at 22–26°C under a 16 : 8 h, light : dark photoperiod until hatching. For glasshouse experiments, four freshly hatched neonates were placed on the rosette leaves of each grafted plant (n = 10); owing to a high mortality rate in the first days of life, after 3 d, only two neonates per plant were used for the performance assays. Larvae performance was estimated by their mass gain, measured on the 12th day of M. sexta and the 10th day of S. littoralis feeding. Under field conditions, insect damage was measured on 4 June 2012, on plants between the fifth and sixth week after transplanting to the plot. Insect-specific damage signatures were identified according to Gaquerel et al. (2013), and quantified in standardized units of leaf area consumed relative to insect size (i.e. 5 units of mirid damage ≈ 5 units of leafhopper damage ≈ 1 cm2 of heavily attacked leaf area).

Statistical analysis

Data were verified for assumptions of normal distribution and homogeneity of variances, and log-transformed when adequate. Parametric or nonparametric (Kruskal–Wallis test) ANOVA, followed by Fisher's least significant difference (LSD) or Dunnett's as post hoc tests, were performed using StatView5 (SAS Institute, Cary, NC, USA) or SigmaPlot 12.0 (Systat Software Inc., San Jose, CA, USA).

Results

Full nicotine induction in systemic leaves in response to leaf wounding requires root JA synthesis and perception

To examine the contribution of the below-ground parts of N. attenuata plants to JA-dependent nicotine induction in systemic leaves, we grafted EV scions on to rootstocks of transgenic lines of N. attenuata. We used grafts of the scions and rootstocks of the same genotypes as controls. As previously described (Fragoso et al., 2011), the silencing of target genes of ir lines is only restrained to the transgenic counterpart of the grafts when those are used as rootstocks (Supporting Information, Fig. S1). We first observed that grafted empty vector plants (EV/EV) responded to leaf wounding with amounts of nicotine 10-fold higher than those of unwounded control EV/EV levels (Fig. 1; P < 0.001). EV shoots grafted on to silenced irPMT roots (EV/irPMT) completely lacked nicotine, and did not differ from fully silenced irPMT/irPMT grafted plants (Fig. 1; P > 0.94 for both control and induced nicotine concentrations).

Figure 1.

Jasmonic acid (JA) de novo synthesis and perception in roots contribute to nicotine accumulation induced in systemic leaves in response to leaf wounding in Nicotiana attenuata. Mean ± SE concentrations of nicotine accumulated in undamaged roots and systemic leaves (shaded) of control (open columns) and leaf-wounded (closed columns; leaves with dashed lines) grafts displaying roots impaired in JA synthesis (EV/irAOC), JA perception (EV/irCOI1), and nicotine synthesis (EV/irPMT) at 3 d after leaf wounding. Grafts of the scions and rootstocks of the same genotypes (EV/EV, irAOC/irAOC, irCOI1/irCOI1 and irPMT/irPMT) were used as controls. Bars sharing the same letters do not differ significantly (two-way ANOVA followed by Fisher's least significant difference test, n = 6). Dashed arrows indicate the percentage reduction in nicotine induction compared with wounded EV/EV grafts. EV, empty vector; irAOC, allene oxidase cyclase silenced line; irCOI1, coronatine-insensitive 1 silenced line; irPMT, putrescine N-methyltransferase silenced line.

A 55% decrease in wound-elicited nicotine induction in systemic leaves of plants with impaired JA synthesis in roots (EV/irAOC) was observed compared with the nicotine induction in EV/EV plants (Fig. 1; P < 0.001). An even more pronounced impairment of nicotine induction in systemic leaves was observed in plants lacking JA perception in roots: wounded EV/irCOI1 plants accumulated < 20% of the induced EV/EV nicotine concentrations (Fig. 1; P < 0.001). Both graft combinations accumulated more nicotine in systemic leaves than their respective entirely silenced graft combinations, irAOC/irAOC and irCOI1/irCOI1 (Fig. 1; P < 0.001 and = 0.023, respectively). Moreover, only EV/EV, EV/irAOC and EV/irCOI1 responded to wounding with increased concentrations of nicotine in systemic leaves compared with control nicotine concentrations (P < 0.001, P 0.001, and P = 0.009, respectively). When analyzing nicotine induction in roots, all graft combinations accumulated similar or lower concentrations of nicotine than those found in EV/EV roots (Fig. 1).

Root JA synthesis and perception control the induction of NaPMT expression in roots

To test whether JA synthesis or perception in roots regulates the expression of nicotine biosynthetic genes, we analyzed the accumulation of NaPMT1 and NaPMT2 transcripts, here collectively referred to as NaPMT. One day after the leaf-wounding treatment, NaPMT transcript abundances in roots of both EV/irPMT and irPMT/irPMT were < 5% of the NaPMT transcript abundances of EV/EV grafts (Fig. 2; P 0.001 in both comparisons). Roots of induced EV/irAOC plants accumulated 63% fewer NaPMT transcripts than did the roots of induced EV/EV plants (Fig. 2; P 0.001). An even more pronounced impairment in NaPMT transcript abundance was found in the roots of induced EV/irCOI1 plants compared with those of EV/EV grafts (76% less, Fig. 2; P 0.001). NaPMT levels in EV/EV, EV/irAOC, and EV/irCOI1 were significantly increased in response to wounding relative to their respective untreated levels (P 0.001, P 0.001, and = 0.005, respectively).

Figure 2.

Jasmonic acid (JA) de novo synthesis and perception in roots contribute to induced NaPMT expression in roots in response to leaf wounding in Nicotiana attenuata. Mean ± SE accumulation of NaPMT transcript in undamaged roots of control (open columns) and leaf-wounded (closed columns) grafts displaying roots impaired in JA synthesis (EV/irAOC), JA perception (EV/irCOI1), and nicotine synthesis (EV/irPMT) at 1 d after leaf wounding. Grafts of the scions and rootstocks of the same genotypes (EV/EV, irAOC/irAOC, irCOI1/irCOI1 and irPMT/irPMT) were used as controls. Bars sharing the same letters do not differ significantly (two-way ANOVA followed by Fisher's least significant difference test, n = 6). Dashed arrows indicate the percentage reduction in nicotine induction compared with wounded EV/EV grafts. EV, empty vector; irAOC, allene oxidase cyclase silenced line; irCOI1, coronatine-insensitive 1 silenced line; irPMT, putrescine N-methyltransferase silenced line.

Root JA synthesis and perception control the transport of de novo synthesized nicotine to leaves

To further investigate whether JA synthesis or perception in roots regulates the transport of wound-induced nicotine from roots to leaves, a petiole-feeding experiment was performed (Fig. 3a). Systemic leaves of control and wounded EV/EV, EV/irAOC and EV/irCOI1 were excised and had their petioles submerged for 1 d in a nicotine-containing solution at a physiologically relevant concentration of 1 mM. We observed that nicotine concentrations in excised systemic leaves of wounded EV/EV plants were 66% higher than those of control EV/EV plants (Fig. 3b; P 0.001), which matches the degree of the nicotine induction observed in intact systemic leaves of nongrafted WT plants between the 24 and 48 h time interval after leaf wounding (Fig. S2). Conversely, nicotine concentrations of excised systemic leaves of both EV/irAOC and EV/irCOI1 after leaf wounding were significantly lower than those of EV/EV, although the genotype of all leaves was the same (Fig. 3b; P 0.001 in both comparisons). In comparison to nicotine-fed undamaged controls, wounding treatment increased nicotine concentrations in excised systemic leaves of EV/irAOC by only 35% (Fig. 3b; = 0.016). Most strikingly, for nicotine-fed EV/irCOI1 systemic leaves, wounding treatment failed to induce nicotine concentrations, and these remained as low as their respective nicotine-fed control values (Fig. 3b; P = 0.963).

Figure 3.

Jasmonic acid (JA) synthesis and perception in roots tightly control induced nicotine transport to systemic leaves after leaf wounding in Nicotiana attenuata. (a) Experimental setup of the petiole-feeding assay: control or leaf-wounded (dashed lines) grafts had systemic leaves (shaded) detached 24 h after treatment and were fed for the following 24 h with a 1 mM nicotine solution. (b) Mean ± SE concentrations of nicotine accumulated in leaf lamina systemic leaves (shaded) of control (open columns) and leaf-wounded (closed columns) grafts. Asterisks refer to comparisons between control and leaf wounding treatment within same graft type (***, P 0.001; *, P 0.05; ns, not significant; two-way ANOVA followed by Fisher's least significant difference test, n = 8). EV, empty vector; irAOC, allene oxidase cyclase silenced line; irCOI1, coronatine-insensitive 1 silenced line.

When leaves were fed a nicotine-free solution, the wounding treatment failed to induce nicotine in leaves of all grafts, including EV/EV, indicating that induced nicotine concentrations in nicotine-fed systemic leaves are entirely derived from the nicotine-containing solution (Fig. S3). Interestingly, the presence of nicotine in the solution did not increase nicotine concentrations of control EV/EV leaves, these remained similar to those found in water-fed control EV/EV leaves (Figs 3b, S3; P = 0.654). However, systemic leaves of EV/irAOC and EV/irCOI1 plants fed with a nicotine-containing solution accumulated more nicotine than did their respective leaves fed only with water (Figs 3b, S3; nicotine vs water-feeding for EV/irAOC control, P = 0.029, and wounding, P = 0.025; for EV/irCOI1 control, P = 0.013, and wounding, P = 0.052). Although the interaction of factors was statistically significant (feeding × treatment: P = 0.047), treatment factor had a stronger effect than feeding factor in explaining how EV/EV leaves incorporated nicotine from the feeding solution (treatment, P 0.001; feeding, P = 0.099). By contrast, taking EV/irAOC and EV/COI1 leaves together, the feeding factor contributed more to explaining how samples varied in leaf nicotine, and made the interaction between factors no longer significant (feeding solution, P < 0.001; treatment, P 0.001; feeding × treatment, P = 0.337, for both grafts analyzed individually). In other words, for EV/irAOC and EV/irCOI1 plants, systemic leaves of either control or wound-treated plants accumulated more nicotine whenever this molecule was offered in the solution, regardless of the treatment.

The size of the leaves did not differ significantly across different grafts (P = 0.104), treatments (P = 0.536), and feedings (P = 0.515; data not shown). As expected, nicotine uptake from the solutions was highly correlated with the volume of solution transpired by the leaf (correlation coefficient = 0.99, P = 7.5 × 10−9), and wounding significantly increased the volume of solution transpired (Fig. S4; wounding vs control nicotine-fed EV/EV, P 0.001).

Impaired JA synthesis and perception in roots systemically up-regulate jasmonates and ABA accumulation in wound-induced leaves

To evaluate whether the impairment in JA synthesis or perception in roots changes phytohormone accumulation in wounded leaves, we measured the concentrations of phytohormones in damaged leaves of EV/EV, EV/irAOC and EV/irCOI1 plants and in their respective undamaged roots after leaf wounding. A burst in JA and JA-Ile accumulation was detected in local leaves of EV/EV 1 h after wounding, and this pattern was also observed in EV/EV roots 2 h after leaf wounding at much lower concentrations (Fig. 4a). However, local tissues of EV/irAOC and EV/irCOI1 attained strikingly higher concentrations of phytohormones than those in the local tissues of EV/EV (Fig. 4a; P 0.001, for all comparisons), while EV/irAOC and EV/irCOI1 roots completely lacked the JA and JA-Ile burst found in EV/EV roots (Fig. 4a; P 0.001, for all comparisons). JA concentrations in local leaves of EV/irAOC and EV/irCOI1 1 h after wounding were 1.7-fold higher than those of EV/EV leaves, and JA-Ile concentrations were fivefold higher than those of EV/EV leaves (Fig. 4a). Concentrations of leaf OPDA were also higher in EV/irAOC and EV/irCOI1 than in EV/EV (Fig. 4a; P = 0.01, for both grafts compared individually to EV/EV). In addition, ABA concentrations of control and treated leaves were also higher in EV/irAOC and EV/irCOI1 when compared with those of EV/EV (Fig. 4b; P 0.001). Initial basal concentrations of JA, JA-Ile, and OPDA in local tissues were similar among all grafts (P > 0.8 for all comparisons against EV/EV, within control time point at 0 h), while EV/irAOC roots had reduced basal concentrations of jasmonates compared with EV/EV (JA, = 0.002; JA-Ile, = 0.011; OPDA, P 0.001).

Figure 4.

Impaired jasmonic acid (JA) synthesis and perception in undamaged roots up-regulate jasmonates and abscisic acid (ABA) accumulation in damaged leaves in response to wounding in Nicotiana attenuata. Mean ± SE concentrations of phytohormone accumulated in response to wounding of undamaged roots and damaged leaves (shaded with dashed lines) of grafted plants with roots impaired in JA synthesis and perception (***, P 0.001; **, P 0.01; *, P 0.05; two-way ANOVA followed by Dunnett's test, = 6). EV, empty vector; irAOC, allene oxidase cyclase silenced line; irCOI1, coronatine-insensitive 1 silenced line. OPDA, 12-oxo-phytodienoic acid; JA-Ile, jasmonic acid isoleucine.

JA synthesis and perception in roots contribute to plant resistance in glasshouse and nature

To investigate the contribution of root JA synthesis or perception to plant resistance to above-ground herbivore attack, we performed a series of experiments exploring plant–insect interactions under glasshouse and field conditions using EV/EV, EV/irAOC, and EV/irCOI1 plants. Initially, we compared the global metabolic profile of systemic leaves of these grafts 3 d after simulated herbivore attack, which standardizes the induction treatment across plants. Oral secretions of two herbivore species were tested: M. sexta, a specialist; and S. littoralis, a generalist. PCA revealed that metabolic profiles of EV/EV, EV/irAOC, and EV/irCOI1 grafts were more clearly discriminated when treated with regurgitant of S. littoralis than when treated with regurgitant of M. sexta (Fig. 5a,c). When comparing the principal components (PCs) individually across the two herbivores examined, PC1 and PC2 explained similar amounts of the total variance (PC1, M. sexta, 50.5%, and S. littoralis, 49.6%; PC2, M. sexta, 16.3%, and S. littoralis, 12.8%).

Figure 5.

Jasmonic acid (JA) synthesis and perception in roots in Nicotiana attenuata contribute to plant resistance against leaf attackers and are differentially employed depending on the degree of adaptation of the herbivore species. Untargeted principal component analysis (PCA) of metabolic profile of systemic leaves of EV/EV, EV/irAOC and EV/irCOI1 plants 3 d after simulated Manduca sexta (a) or Spodoptera littoralis (c) attack under glasshouse conditions. Mean ± SE mass of M. sexta (b) and S. littoralis (d) after 12 and 10 d, respectively, of feeding on EV/EV, EV/irAOC and EV/irCOI1 plants under glasshouse conditions. Grafts of the scions and rootstocks of the same genotypes were used as controls. Bars sharing the same letters do not significantly differ (one-way ANOVA followed by Fisher's least significant difference test, n ≈ 20). EV, empty vector; irAOC, allene oxidase cyclase silenced line; irCOI1, coronatine-insensitive 1 silenced line.

We further tested whether root JA signaling influences caterpillar performance. Larvae of M. sexta and S. littoralis were reared on EV/EV, EV/irAOC, and EV/irCOI1 grafts and had their mass measured. While larvae of both M. sexta and S. littoralis reared on EV/EV plants showed the smallest mass gain (Fig. 5b,d), larvae reared on irAOC/irAOC and irCOI1/irCOI1 plants attained the largest masses, followed by those reared on EV/irAOC. Larvae of M. sexta fed on EV/irCOI1 gained the same mass as those fed on EV/EV (Fig. 5b; P = 0.150). However, S. littoralis larvae fed on EV/irCOI1 plants gained significantly more mass than those on EV/EV plants (Fig. 5d; = 0.002).

We also measured herbivore preference between EV/EV, EV/irAOC, and EV/irCO1 plants in N. attenuata's natural habitat, the Great Basin Desert of Utah, USA. Under field conditions, EV/irAOC plants were significantly more damaged by leafhoppers (Empoasca spp., P = 0.044), and less attacked by mirids (Tupiocoris notatus, P 0.001) compared with EV/EV plants (Fig. 6a). EV/irCOI1 plants were more heavily damaged by mirids (P = 0.038), but were consumed as much by leafhoppers as EV/EV plants (Fig. 6b; P = 0.096). The silencing of target genes in field-grown grafts was confirmed to be restricted to the transgenic rootstocks (Fig. S1).

Figure 6.

Under field conditions, Nicotiana attenuata plants impaired in jasmonic acid (JA) synthesis (a) and perception (b) in roots are differentially preferred by Empoasca spp. and Tupiocoris notatus herbivores. Mean ± SE amounts of cumulative plant damage over 6 wk after transplantation to the plot. Grafts of the scions and rootstocks of the same genotypes (EV/EV, irAOC/irAOC and irCOI1/irCOI1) were used as controls. Canopy damage was quantified in standardized units of leaf area consumed relative to insect size (i.e. 5 units of mirid damage ≈ 5 units of leafhopper damage ≈ 1 cm2 of heavily attacked leaf area). Bars sharing the same letters do not significantly differ (one-way ANOVA followed by Fisher's least significant difference test, n = 25). EV, empty vector; irAOC, allene oxidase cyclase silenced line; irCOI1, coronatine-insensitive 1 silenced line.

Discussion

In this study, we investigated the role of root JA synthesis and perception in the resistance of above-ground plant parts. For this, we used genetically transformed plants disrupted in different components of the JA pathway and dissected the JA-dependent function of roots using micrografted plants. JA synthesis and perception in roots tightly controlled nicotine accumulation (Fig. 1), NaPMT transcript abundances (Fig. 2), and nicotine transport from roots to the shoots (Fig. 3b). Like nicotine, JA signaling in roots also regulated the concentration of other shoot-accumulated metabolites (Fig. 5a,c), and significantly promoted plant resistance against leaf feeders in both glasshouse and natural conditions (Figs 5b,d, 6).

Nicotine and micrografting in N. attenuata: the toolbox for the study of root-dependent JA signaling in systemic responses

Recently, Mousavi et al. (2013) showed that changes in electrical potentials of the leaf surface were triggered by larval feeding. This electrical wave spread throughout unattacked portions of the shoots, inducing jasmonate biosynthesis and defense-responsive gene expression in systemic leaves. However, it remains unknown whether this signal is also inducing systemic responses in the below-ground parts. Regardless of the identity of the systemic signal conveying the information of leaf wounding to roots, we focused on its downstream events, and investigated whether JA synthesis or perception in roots regulate systemic root responses after leaf wounding. We used nicotine induction as a case study to explore the function of JA in roots, because nicotine is synthesized in roots and induced by leaf wounding. EV/EV plants accumulated 10-fold more nicotine in response to leaf wounding than undamaged control grafted EV/EV plants (Fig. 1), and nicotine induction was completely absent in systemic leaves of EV/irPMT and irPMT/irPMT. These observations are in agreement with previous reports of wounding in Nicotiana spp. (Ohnmeiss et al., 1997) and confirm that nicotine biosynthetic genes are required only in roots (Winz & Baldwin, 2001). These data also highlight the value of micrografting to test root responses to leaf induction, and demonstrate that the mobile wound signal is graft-transmissible.

JA de novo synthesis and perception in roots tightly regulate nicotine production in roots and transport to leaf lamina

Nicotine induction in response to leaf wounding was almost fully dependent on JA-Ile perception (COI1) in roots. JA de novo synthesis (AOC) in roots was also required for nicotine induction in shoots (Fig. 1). However, nicotine concentrations of EV/irAOC and EV/irCOI1 leaves were induced in response to leaf wounding, suggesting a JA-independent root signaling pathway involved in nicotine induction. Alternatively, the residual nicotine induction found in leaves of these grafts might be the result of minor expression levels remaining in silenced roots (Fig. S1) or a consequence of the rapid turnover of shoot-derived jasmonate transported to roots.

The expression levels of NaPMT transcripts in roots of all grafts were strongly correlated with the nicotine amounts found in leaves (Fig. 2), and roots did not overaccumulate nicotine (Fig. 1), suggesting that JA signaling in roots controls nicotine induction at the transcriptional level. NaPMT expression and nicotine production are known to be attenuated by ethylene emission (Kahl et al., 2000; Winz & Baldwin, 2001). However, wounding alone did not induce leaf ethylene emission in EV/EV grafts and intact WT plants (Diezel et al., 2011); also, EV/irAOC and EV/irCOI1 showed similar or even reduced ethylene emission compared with EV/EV (data not shown). It is also noteworthy that nicotine induction in Nicotiana species has allometrically determined setpoints that control the amount of nicotine accumulated in the shoots in response to wounding. The allometric nicotine induction is proportional to the biomass of the plant, and it seems to be mainly dictated by the rate of de novo synthesized nicotine in roots, as well as by other factors regulating nicotine storage in the shoots (Baldwin, 1996a, 1999). Hence, a plant's ability to store nicotine above ground is presumably controlled by the same mechanisms as those that control nicotine synthesis below ground.

In a petiole-feeding experiment, we mimicked the endogenous increase in the transport of nicotine via the apoplast from roots to shoot that occurs between the first and second day after leaf wounding (Fig. S2; Baldwin, 1989). As a control, water-fed leaves failed to induce nicotine in systemic leaves after wounding (Fig. S3), indicating that the wound-induced nicotine increment of nicotine-fed leaves was the result of the nicotine loaded into the leaf by transpiring the nicotine-containing solution. Even when nicotine was equally offered, systemic leaves of EV/irAOC and EV/irCOI1 failed to allocate nicotine to the leaf lamina, suggesting that JA signaling in roots regulates physiological changes in shoots required to transport the root-derived nicotine to the leaf lamina (Fig. 3b), as was previously suggested with N. sylvestris (Baldwin & Callahan, 1993; Baldwin, 1996a). It was also observed that leaves of EV/irAOC and EV/irCOI1 have significantly fewer trichomes than EV/EV leaves on their abaxial surfaces (c. 20–30% less, P = 0.007), and trichomes represent one site of nicotine accumulation (Roda et al., 2003). These data add another regulatory step by which root JA signaling fine-tunes the wound-induced nicotine response of shoots (Baldwin & Schmelz, 1994).

Interestingly, as shown in the petiole-feeding experiment, wounding significantly induced solution uptake in excised systemic leaves of EV/EV plants. These data are consistent with a mechanism by which wound-induced nicotine transport via xylem is facilitated through higher rates of transpiration (Baldwin, 1989; Baldwin & Schmelz, 1994). Conversely, nicotine-fed leaves of EV/irAOC and EV/irCOI tended to absorb more solution than did their water-fed leaves, whether wounded or not (data not shown), and basal concentrations of nicotine in these grafts were already reduced compared with EV/EV. Therefore, it is possible that nicotine per se serves as a signal to promote transpiration in shoots of these nicotine-deprived N. attenuata grafts. However these hypotheses require further tests, and changes in transpiration rates are probably one of the mechanisms regulating nicotine induction in tobacco leaves. Taken together, these results suggest that root JA signaling regulates leaf nicotine uptake, directly and/or indirectly. To explore these differences further, the concentrations of phytohormones were analyzed.

Root JA synthesis and perception systemically tune local leaf JA and ABA accumulation in response to leaf wounding

As expected from a previous study with WT intact plants of N. attenuata (Von Dahl & Baldwin, 2004), leaf wounding induced neither JA nor methyl jasmonate in systemic leaves of EV/EV (data not shown). As AOC enzyme activity leads to OPDA production that serves as a substrate for JA and JA-Ile formation, basal concentrations of all these jasmonates were reduced in EV/irAOC roots (Fig. 4a). Although basal concentrations of root jasmonates in EV/irCOI1 plants were similar to those of EV/EV, roots of both EV/irAOC and EV/irCOI1 did not show the systemic wound-induced burst of OPDA, JA, and JA-Ile observed in EV/EV roots. Surprisingly, the reduced jasmonate concentrations of EV/irAOC, and EV/irCOI1 roots were associated with a hyper-responsive JA accumulation in induced leaves, suggesting the existence of a novel shoot–root–shoot loop in regulating the JA response. The impaired accumulation of systemic root jasmonates possibly boosts jasmonate responses in the local leaf as a compensatory effect. We ruled out the possibility that the result was an artifact (i.e. silencing of JA components in shoots) by checking gene expression in both shoot and roots of these grafted plants (Fig. S1).

Moreover, concentrations of ABA were found to be surprisingly higher in leaves of EV/irAOC and EV/irCOI1 when compared with those of EV/EV (Fig. 4b), suggesting a crosstalk between JA and ABA regulating shoot–root–shoot interplay in plant defenses. This agrees with our petiole-feeding data, and also with the notion of facilitated nicotine transport through higher transpiration rates: higher ABA concentrations in EV/irAOC and EV/irCOI1 leaves would inhibit transpiration, ultimately leading to reduced nicotine contents. Furthermore, ABA-regulated water stress, rather than ABA-induced defenses, has already been suggested to be involved in leaf resistance induced by root herbivory in maize plants (Erb et al., 2010). However, the involvement of ABA in JA-dependent responses to wounding and herbivore attack is shown to be beyond the control of guard cells and transpiration rates, and JA–ABA signaling crosstalk probably regulates a more complex range of processes. For instance, reduced concentrations of ABA in leaves, and its consequent augmented transpiration rates, were recently associated with reduced emission of defensive organic volatile compounds in N. attenuata plants silenced for a novel protein that suppress ABA catabolism after herbivore attack (Dinh et al., 2013).

JA synthesis and perception in roots enhance plant resistance against above-ground herbivores

The treatment using regurgitation of a generalist herbivore caused EV/irAOC and EV/irCOI1 to be metabolically more distinct from EV/EV when compared with the weak grouping found for these graft combinations when elicited with M. sexta regurgitant (Fig. 5a,c). These data suggest that other defensive metabolites in addition to nicotine are dependent on JA signaling in roots. This result is consistent with the notion of a herbivore-induced carbon sequestration to roots as a resistance mechanism regulating defensive metabolites, rather than solely a tolerance mechanism (Schwachtje et al., 2006; Machado et al., 2013).

The more distinct pattern of grouping found in PCA of plants induced by S. littoralis (generalist) oral secretion was also reflected in more pronounced effect on S. littoralis larval performance between EV/EV and EV/irCOI1 grafts compared with those of M. sexta, a specialist (Fig. 5). Under field conditions, COI1 activity in roots accounted for enhanced plant resistance against mirids, while it likely had no influence on the feeding choice of Empoasca leafhoppers (Fig. 6). On the other hand, the lack of AOC activity only in roots enhanced the vulnerability of plants to these leafhopper species. Empoasca spp. are able to identify in native populations natural N. attenuata JA-mutants with impaired capacity to mediate JA signaling (Kallenbach et al., 2012). Despite its hyper-response in JA accumulation after wounding (Fig. 4a), EV/irAOC plants were preferably attacked by Empoasca, suggesting that the function of AOC in roots profoundly influences JA-mediated responses of the shoots. In addition, our data support the notion that JA dependent responses are employed in a herbivore-specific way (Hettenhausen et al., 2013), and suggest a COI1-independent JA signaling in the roots.

In addition, we observed that damage caused by mirids was negatively correlated with damage inflicted by Empoasca leafhoppers in irAOC grafts. It would be interesting to test whether the density of these herbivores is tailored directly, simply by their presence/absence, or indirectly, through plants' responses mediated by JA signaling (Kessler & Baldwin, 2004; Kallenbach et al., 2012). Mirids are specialist herbivores, and might be more adapted to N. attenuata defense metabolites than to the presence of other generalist herbivores; this only becomes apparent in irAOC plants. In other words, mirids might prefer plants that are better defended against generalists. Moreover, the negative correlation between damage inflicted by mirids and leafhoppers found in irAOC grafts was not found in grafts using irCOI1. This suggests that the interaction between these herbivores is very likely plant-mediated and CO1-dependent.

The revival of the root-brain theory originally proposed by Charles and Francis Darwin (Baluska et al., 2009) has renewed attention to the function of roots as a regulatory organ of plants. How changes in roots affect shoot responses, and vice versa, is the subject of current intense study. Here, we focused on the above-ground changes induced by leaf attack that engage roots in a more comprehensive shoot–root–shoot loop. Based on the dramatic changes observed in how leaves respond to attack when roots are depleted of JA signaling, we conclude that roots play a central role in orchestrating above-ground processes.

Acknowledgements

This work is supported by the European Research Council advanced grant ClockworkGreen (no. 293926) to I.T.B., the Global Research Lab program (2012055546) from the National Research Foundation of Korea, Human Frontier Science Program (RGP0002/2012), and the Max Planck Society. We thank the Brigham Young University for the use of Lytle Ranch Preserve; Danny Kessler, Youngjoo Oh, and Felipe Yon for help with field plants; Michael Reichelt with phytohormone measurements; and Youngsung Joo for help with glasshouse experiments.

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