Jasmonate perception regulates jasmonate biosynthesis and JA-Ile metabolism: the case of COI1 in Nicotiana attenuata

CORONATINE INSENSITIVE 1 (COI1) is a well-known key player in processes downstream of jasmonic acid (JA) biosynthesis: silencing COI1 in Nicotiana attenuata (ir-coi1) makes plants insensitive to JA, prevents the up-regulation of JA-mediated defenses and decreases the plant's resistance to herbivores and pathogens. In agreement with previous studies, we observed that regulation of several JA biosynthesis genes elicited by Manduca sexta oral secretions (OS) is COI1 dependent. In response to wounding and application of OS ir-coi1 plants accumulate 75% less JA compared with wild-type plants (WT), resembling JA levels found in plants silenced in the key enzyme in JA biosynthesis LIPOXYGENASE 3 (as-lox). However, while OS-elicited as-lox plants also accumulated lower levels of the JA-conjugate JA-isoleucine (JA-Ile) than did WT plants, JA-Ile accumulation in ir-coi1 was higher, prolonged and peaked with a delay of 30 min. In vivo substrate feeding experiments of N. attenuata demonstrate that the increased and prolonged JA-Ile accumulation pattern in ir-coi1 is not the result of altered substrate availability, i.e. of JA and/or Ile, but is due to an approximately 6-fold decrease in JA-Ile turnover. These results provide the first evidence for a second, novel regulatory feedback function of COI1 in enhancing JA-Ile turnover. Hence, in addition to its control over JA biosynthesis, COI1 might fine-tune the dynamics of the jasmonate response after induction by herbivore elicitors.     

Symbiosis between Nicotiana attenuata and Glomus intraradices: ethylene plays a role, jasmonic acid does not

Phytohormones are thought to mediate plant–arbuscular mycorrhizal (AM) interactions. To explore the role of phytohormones in the interaction between Nicotiana attenuata and Glomus intraradices , we analysed levels of jasmonic acid (JA) and its amino acid conjugate JA–isoleucine/JA–leucine (JA–Ile), salicylic acid (SA) and ethylene in either infected or non-infected N. attenuata wild-type (WT) plants growing in soils that mimic the nutrient supply rates found in the plant's native environment. Under these conditions, the infection decreases plant growth and reproductive performance. Levels of JA, JA–Ile and SA did not change upon infection, but ethylene release was slightly decreased. Transgenic N. attenuata plants defective in JA signalling (as lox3 and ir coi1 ) did not differ significantly in growth or reproductive performance compared with infected WT. Furthermore, no difference in infection rates could be observed. Transgenic plants unable to produce (ir aco ) or perceive ( etr1 ) ethylene showed significantly larger decreases in growth and number of seed capsules produced between infected and non-infected plants compared with WT plants. We conclude that ethylene, rather than JA, signalling plays a role in the interaction between N. attenuata and AM, from which the plant does not realize a fitness benefit.     

The jasmonate receptor COI1 plays a role in jasmonate-induced lateral root formation and lateral root positioning in Arabidopsis thaliana

Jasmonic acid (JA) regulates a broad range of plant defense and developmental responses. COI1 has been recently found to act as JA receptor. In this report, we show that low micromolar concentrations of JA inhibited primary root (PR) growth and promoted lateral root (LR) formation in Arabidopsis wild-type (WT) seedlings. It was observed that the coi1-1 mutant was less sensitive to JA on pericycle cell activation to induce lateral root primordia (LRP) formation and presented alterations in lateral root positioning and lateral root emergence on bends. To investigate JA-auxin interactions important for remodeling of root system (RS) architecture, we tested the expression of auxin-inducible markers DR5:uidA and BA3:uidA in WT and coi1-1 seedlings in response to indole-3-acetic acid (IAA) and JA and analyzed the RS architecture of a suite of auxin-related mutants under JA treatments. We found that JA did not affect DR5:uidA and BA3:uidA expression in WT and coi1-1 seedlings. Our data also showed that PR growth inhibition in response to JA was likely independent of auxin signaling and that the induction of LRP required ARF7, ARF19, SLR, TIR1, AFB2, AFB3 and AXR1 loci. We conclude that JA regulation of postembryonic root development involves both auxin-dependent and independent mechanisms.     

Co(i)-ordinating defenses: NaCOI1 mediates herbivore- induced resistance in Nicotiana attenuata and reveals the role of herbivore movement in avoiding defenses

Arabidopsis and tomato plants mutated in the F-box protein COI1 mediating jasmonate (JA) responses are more susceptible to herbivores in laboratory trials, but the exact mechanisms of COI1-mediated resistance are not known. We silenced COI1 by transformation with an inverted repeat construct (ir-coi1) in Nicotiana attenuata, a plant the direct and indirect defenses of which against various herbivores have been well studied. ir-coi1 plants are male sterile and impaired in JA-elicited direct [nicotine, caffeoylputrescine and trypsin proteinase inhibitor (TPI) activity] and indirect (cis-alpha-bergamotene emission) defense responses; responses not elicited by JA treatment (ethylene production and flower TPI activity) were unaffected. Larvae of Manduca sexta, a common herbivore of N. attenuata, gained three times more mass feeding on ir-coi1 than on wild-type (WT) plants in glasshouse experiments. By regularly moving caterpillars to unattacked leaves of the same plant, we demonstrate that larvae on WT plants can grow and consume leaves as fast as those on ir-coi1 plants, a result that underscores the role of COI1 in mediating locally induced resistance in attacked leaves, and the importance of herbivore movement in avoiding the induced defenses of a plant. When transplanted into native habitats in the Great Basin Desert, ir-coi1 plants suffer greatly from damage by the local herbivore community, which includes herbivores not commonly found on N. attenuata WT plants. Choice assays with field-grown plants confirmed the increased attractiveness of ir-coi1 plants for both common and unusual herbivores. We conclude that NaCOI1 is essential for induced resistance in N. attenuata, and that ir-coi1 plants highlight the benefits of herbivore movement for avoiding induced defenses.     

BAK1 regulates the accumulation of jasmonic acid and the levels of trypsin proteinase inhibitors in Nicotiana attenuata's responses to herbivory

BAK1 is a co-receptor of brassinosteroid (BR) receptor BRI1, and plays a well-characterized role in BR signalling. BAK1 also physically interacts with the flagellin receptor FLS2 and regulates pathogen resistance. The role of BAK1 in mediating Nicotiana attenuata's resistance responses to its specialist herbivore, Manduca sexta, was examined here. A virus-induced gene-silencing system was used to generate empty vector (EV) and NaBAK1-silenced plants. The wounding- and herbivory-induced responses were examined on EV and NaBAK1-silenced plants by wounding plants or simulating herbivory by treating wounds with larval oral secretions (OS). After wounding or OS elicitation, NaBAK1-silenced plants showed attenuated jasmonic acid (JA) and JA-isoleucine bursts, phytohormone responses important in mediating plant defences against herbivores. However, these decreased JA and JA-Ile levels did not result from compromised MAPK activity or elevated SA levels. After simulated herbivory, NaBAK1-silenced plants had EV levels of defensive secondary metabolites, namely, trypsin proteinase inhibitors (TPIs), and similar levels of resistance to Manduca sexta larvae. Additional experiments demonstrated that decreased JA levels in NaBAK1-VIGS plants, rather than the enzymatic activity of JAR proteins or Ile levels, were responsible for the reduced JA-Ile levels observed in these plants. Methyl jasmonate application elicited higher levels of TPI activity in NaBAK1-silenced plants than in EV plants, suggesting that silencing NaBAK1 enhances the accumulation of TPIs induced by a given level of JA. Thus NaBAK1 is involved in modulating herbivory-induced JA accumulation and how JA levels are transduced into TPI levels in N. attenuata.     

Silencing NOA1 elevates herbivory-induced jasmonic acid accumulation and compromises most of the carbon-based defense metabolites in Nicotiana attenuata(F)

Nitric oxide-associated protein 1 (NOA1) is involved in various abiotic stress responses and is required for plant resistance to pathogen infections. However, the role of NOA1 in plant-herbivore interactions has not been explored. We created NOA1-silenced Nicotiana attenuata plants (irNaNOA1). Compared with wild-type (WT) plants, irNaNOA1 plants had highly decreased photosynthesis rates. We further examined various traits important for plant defense against its specialist herbivore Manduca sexta by treating WT and irNaNOA1 plants with mechanical wounding and M. sexta oral secretions (OS). NOA1-silenced plants showed elevated levels of herbivory-induced jasmonic acid (JA), but decreased JA-isoleucine conjugate (JA-Ile) levels. The decreased JA-Ile levels did not result from compromised JAR (jasmonic acid resistant) activity in irNOA1 plants. Moreover, nitrogen-rich defensive compounds, nicotine and trypsin proteinase inhibitors, did not differ between WT and irNaNOA1 plants. In contrast, concentrations of most carbon-based defensive compounds were lower in these plants than in WT plants, although the levels of chlorogenic acid were not changed. Therefore, silencing NOA1 alters the allocation of carbon resources within the phenylpropanoid pathway. These data suggest the involvement of NOA1 in N. attenuata's defense against M. sexta attack, and highlight its role in photosynthesis, and biosynthesis of jasmonates and secondary metabolites.     

Quantitative relationships between induced jasmonic acid levels and volatile emission in <Emphasis Type="Italic">Zea mays</Emphasis> during <Emphasis Type="Italic">Spodoptera exigua</Emphasis> herbivory

Jasmonic acid (JA) has long been hypothesized to be an important regulator of insect-induced volatile emission; however, current models are based primarily on circumstantial evidence derived from pharmacological studies. Using beet armyworm caterpillars (BAW: Spodoptera exigua) and intact corn seedlings, we examine this hypothesis by measuring both the time-course of insect-induced JA levels and the relationships between endogenous JA levels, ethylene, indole and sesquiterpenes. In separate Morning and Evening time-course trials, BAW feeding stimulated increases in JA levels within the first 4-6 h and resulted in maximal increases in JA, indole, sesquiterpenes and ethylene 8-16 h later. During BAW herbivory, increases in JA either paralleled or preceded the increases in indole, sesquiterpenes and ethylene in the Morning and Evening trials, respectively. By varying the intensity of the BAW herbivory, we demonstrate that strong positive relationships exist between the resulting variation in insect-induced JA levels and volatile emissions such as indole and the sesquiterpenes. To address potential signaling interactions between herbivore-induced JA and ethylene, plants were pretreated with 1-methylcyclopropene (1-MCP), an inhibitor of ethylene perception. 1-MCP pretreatment resulted in reduced production of ethylene and volatile emission following BAW herbivory but did not alter the insect-induced accumulation of JA. Our results strongly support a role for JA in the regulation of insect-induced volatile emission but also suggest that ethylene perception regulates the magnitude of volatile emission during herbivory.     

Root growth dynamics of Nicotiana attenuata seedlings are affected by simulated herbivore attack

Many studies demonstrate resource-based trade-offs between growth and defence on a large timescale. Yet, the short-term dynamics of this growth reaction are still completely unclear, making it difficult to explain growth-defence trade-offs mechanistically. In this study, image-based non-destructive methods were used to quantify root growth reactions happening within hours following simulated herbivore attack. The induction of wound reactions in Nicotiana attenuata in the seedling stage led to transiently decreased root growth rates. Application of the oral secretion of the specialist herbivore Manduca sexta to the leaves led to a transient decrease in root growth that was more pronounced than if a mere mechanical wounding was imposed. Root growth reduction was more pronounced than leaf growth reduction. When fatty acid-amino acid conjugates (FACs) were applied to wounds, root growth reduction occurred in the same intensity as when oral secretion was applied. Timing of the transient growth reduction coincided with endogenous bursts of jasmonate (JA) and ethylene emissions reported in literature. Simulation of a wound response by applying methyl jasmonate (MeJA) led to more prolonged negative effects on root growth. Increased nicotine concentrations, trichome lengths and densities were observed within 72 h in seedlings that were treated with MeJA or that were mechanically wounded. Overall, these reactions indicate that even in a very early developmental stage, the diversion of plant metabolism from primary (growth-sustaining) to secondary (defence-related) metabolism can cause profound alterations of plant growth performance.     

Root growth is affected differently by mechanical wounding in seedlings of the ecological model species Nicotiana attenuata and the molecular model species Arabidopsis thaliana

In the ecological model plant Nicotiana attenuata, leaf wounding or herbivory lead to a reduction of root growth via jasmonic acid (JA) signaling. A single wounding treatment is sufficient to induce this response; multiple wounding does not increase the plant growth reaction. in a recent study, in which JA bursts were elicited in leaves of the molecular model species Arabidopsis thaliana in different ways,¹ we tested whether JA induces the same response there. Root growth reduction was neither induced by foliar application of herbivore oral secretions nor by direct application of methyl jasmonate to leaves. Root growth reduction was observed when leaves were infected with the pathogen Pseudomonas syringae pv. tomato, which persistently induces the JA signaling pathway. Yet, growth analyses of this effect in wild type and JA-signaling mutants showed that it was elicited by the bacterial toxin coronatine which suggests ethylene—but not JA-induced root growth reduction in A. thaliana. Moreover, the growth effects were somewhat masked by a light-induced diurnal decrease of root growth. Overall, we conclude that the reaction of root growth to herbivore-induced JA signaling differs among species, which is related to different ecological defence strategies that have evolved in different species.Key words: coronatine, ethylene, image analysis, phytohormones, Pseudomonas syringae pv. tomato, woundingUpon pathogen or herbivore attack, plants have to meet the decision how much of their resources are invested in growth processes and how much into defense. The ecological model species Nicotiana attenuata increases defence measures and decreases root, but not leaf growth immediately after a single simulated herbivory event.² This reaction is elucidated via jasmonic acid (JA) signaling.³ The intensity of root growth reduction is not amplified when multiple wounding events occur (Fig. 1A). This clearly demonstrates that wounding acts as a signal for the reduction of root growth and that root growth is not reduced due to a lack of growth resources as a consequence of a resource-based trade-off between growth and defence. This hypothesis is further supported by the finding that a surplus of carbohydrates is stored in the root system,⁴ which thereby acts as a safe retreat for future re-growth of the plant after herbivore damage.Open in a separate windowFigure 1Root growth in Nicotiana attenuata and Arabidopsis thaliana seedlings. (A) Root growth dynamics of Nicotiana attenuata seedlings after single and multiple wounding treatments as well as multiple wounding treatments followed by application of oral secretions of Manduca sexta (OS). Wounding treatments were applied at time points 0 h (single treatments) or at the time points 0 h, 2 h and 4 h (multiple treatments). Controls were not treated. (B) Normalized values of velocity of the root tip (v_Tip) of Arabidopsis thaliana seedlings whose roots were exposed to light (control and wounded) and seedlings whose roots were darkened by wrapping aluminium foil around the Petri dish throughout the growth period. Shaded areas indicate the night period. Mean ± SE. N = 4–8.We asked ourselves whether this is a general reaction pattern that is followed in more plant species. To test this, we performed a suite of experiments on the molecular model species Arabidopsis thaliana.¹ Several studies showed that direct application of JA or methyl jasmonate (MeJA), which is commonly used to mimick herbivory-induced signaling, to the cultivation medium decrease root growth of A. thaliana. Yet, in contrast to the situation in N. attenuata, the application of MeJA to leaves did not lead to a decrease in root growth. To exclude the possibility that the MeJA applied to the leaf was not taken up by the plants, we induced plant-internal JA bursts by mechanical wounding and/or application of bacteria. The treatments were performed on Col-0 and Col-6 wild type plants. Additionally, two mutants defective in the JA signaling pathway were used to select for JA-induced effects. coi1-1 (coronatine-insensitive) is known to lack the F-box protein COI1 and shows decreased sensitivity to JA application compared to wild type plants.⁵ The aos mutant, in contrast, is unable to produce JA following mechanical wounding as the biosynthesis of the rate-limiting enzyme allene oxide synthase is blocked.⁶Upon mechanical wounding of two leaves with sterile tweezers, JA concentration in the seedlings increased and root growth decreased rapidly, but only very transiently in all four investigated A. thaliana lines. In contrast to the situation in N. attenuata, root growth in A. thaliana recovered to pre-treatment levels within a few hours (Fig. 1B) and growth was not further decreased upon addition of oral secretions of Spodoptera littoralis larvae. This suggests that the observed short-term growth reduction was caused by hydraulic decrease of the plant growth potential. A slight, but continuous decrease of root growth during the day was noted both in wounded and in control plants that were not completely protected from ambient light in the transparent Petri dishes. When root systems were completely protected from ambient light by shading, root growth was almost steady throughout 24 h (Fig. 1B).In another experimental approach to clarify the connection between JA signaling and root growth reduction, we infected leaves with the avirulent Pseudomonas syringae pv. tomato (Pst) DC3000 avrRpt2 strain. Upon mechanical wounding and application of bacterial suspension in order to facilitate infection, root growth decreased more rapidly than upon mere wounding. In the course of two days after infection, v_Tip was lower in the wild types and the aos mutant suggesting that JA was not the major reason of the decrease of root growth. With Pst DC3000 deficient in coronatine biosynthesis, it was verified that the bacterial toxin was the major reason of the root growth reduction following Pst infection. Using the ethylene reception blocker 1-methyl cyclopropene (1-MCP), ethylene was also figured out to be involved in coronatine-mediated root growth impairment in Arabidopsis. Thus, root growth of Arabidopsis is more sensitive to ethylene than to JA which is very different to observations on N. attenuata.The conclusion has to be drawn that elicitation of JA-bursts in the leaves of A. thaliana does not induce the same root growth reactions as in N. attenuata, although roots of both species react towards MeJA externally applied to the cultivation medium. This in turn demonstrates clearly that the interpretation of the JA signal differs between species. Possibly, this reflects different survival strategies to which the two investigated annual rosette species have evolved. While N. attenuata uses the root as a safe retreat for resources allowing later re-growth after the herbivore threat has passed by, A. thaliana is more successful in its ecological niche if it does not slow down growth in response to herbivory but continues its development as rapidly as possible.     

Beta and habitat diversity in marine systems: a new approach to measurement, scaling and interpretation

Specialist herbivores are known to alter their host's wound-induced responses but the beneficiaries of these alterations are unknown. Nicotiana attenuata plants release a burst of ethylene specifically in response to feeding by Manduca sexta larvae, which is known to suppress wound- and methyl jasmonate (MeJA)-inducible nicotine accumulation. The ethylene burst may be a mechanism by which M. sexta larvae feed "stealthily" on their host plants or, alternatively, it may allow the plant to optimize its defense response against this specialist herbivore by reducing costs of induction. We examined the impact of the ethylene burst on defense-related fitness costs that are readily observed when plants are treated with MeJA and grown in competition with untreated plants. We elicited nicotine induction (with MeJA), the ethylene burst (with the ethylene releasing compound, ethephon) and inhibited the plant's ability to perceive ethylene (with applications of an antagonist of ethylene receptors, 1-methylcyclopropene, 1-MCP). By simultaneously applying MeJA and ethephon we mimicked the plant's hormonal response to larval attack. We hypothesized that if the ethylene burst benefited the plant, the fitness costs of MeJA induction should be reduced by ethephon and restored if the plants were additionally treated with 1-MCP. In a second experiment, we applied larval oral secretion (OS) to elicit endogenous hormone production and predicted that the 1-MCP treatment should reduce the fitness of OS-treated plants. Our measures of plant fitness, namely the rate of stalk elongation and lifetime capsule production, supported these predictions. We conclude that the ethylene burst elicited by this specialist herbivore can reduce MeJA-induced fitness costs and increase the competitive strength of OS-treated plants. Suppressed nicotine production is likely to contribute to, but is not sufficient to explain, the observed fitness outcomes. The intensity of intra-specific competition and herbivore attack will likely determine the adaptive value of the M. sexta-elicited ethylene response.     

Use of silenced plants in allelopathy bioassays: a novel approach

Volatile phytohormones or other chemicals can affect processes in distal plant parts but may also influence neighboring plants, and thereby function allelopathically. While this hypothesis has been widely discussed, rigorous tests are lacking. Transgenic plants, silenced in the production of an emitted chemical, are ideal tools to test the hypothesis that the release of a chemical can negatively influence the growth of neighbors (allelopathy). We used isogenic wild type (WT) and genetically transformed plants that lacked the ability to produce ethylene (ir-aco), as both “emitters” and “receivers” of this volatile phytohormone in experiments where receiver plants were only exposed to the headspace of WT or ir-aco emitters, in order to evaluate if natural ethylene releases can function allelopathically. Root growth (a proxy of plant fitness) of WT receivers correlated negatively with the number of WT emitters and headspace ethylene concentrations. Reducing ethylene concentrations in the headspace with the ethylene scrubber, KMnO₄, and using ir-aco seedlings as emitters restored root growth of WT receiver seedlings. 1-Aminocyclopropane-1-carboxylic acid (ethylene biosynthesis substrate) supplementation to WT but not ir-aco emitters inhibited root growth of ir-aco, but not WT receivers, suggesting increased sensitivity to exogenous ethylene of ir-aco seedlings. We conclude that plants genetically silenced in the production of a putative allelochemical are useful in determining if the emitted chemical functions allelopathically. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Interactions between jasmonates and ethylene in the regulation of root hair development in Arabidopsis

Root hair formation is an important model with which to study cell patterning and differentiation in higher plants. Ethylene and auxin are critical regulators of root hair development. The role of jasmonates (JAs) was examined in Arabidopsis root hair development as well as their interactions with ethylene in this process. The results have shown that both methyl jasmonate (MeJA) and jasmonic acid (JA) have a pronounced effect on promoting root hair formation. However, the effect of MeJA and JA on root hair formation was blocked by ethylene inhibitors Ag+ or aminoethoxyvinylglycine (AVG). The stimulatory effects of MeJA and JA were also diminished in ethylene-insensitive mutants etr1-1 and etr1-3. Furthermore, the JA biosynthesis inhibitors ibuprofen and salicylhydroxamic acid (SHAM) suppressed 1-aminocyclopropane-1-carboxylic acid (ACC)-induced root hair formation, and decreased the root hairs in seedlings of the ethylene over-producing mutant eto1-1. These results suggested that JAs promote root hair formation, through an interaction with ethylene.