AbstractWhereas jasmonic acid (JA) and its amino acid conjugates, particularly JA-isoleucine (Ile), are known to play important roles in plant-herbivore interactions, whether other compounds also function as signals independently of JA-Ile and whether conjugates elicit systemic responses are unknown. To answer these questions, we simultaneously silenced JASMONATE-RESISTANT4 (JAR4) and JAR6, two functionally redundant enzymes in Nicotiana attenuata that conjugate JA to amino acids to produce plants (irjar4/6) with low levels of JA-Ile, JA-leucine (Leu), and JA-valine (Val; <16% of wild type). As expected, irjar4/6 plants are more vulnerable to herbivore attack, but only JA-Ile—not JA-Leu or JA-Val—applications restored the resistance of irjar4/6 plants, suggesting that JA-Leu and JA-Val do not mediate herbivore defense responses. Interestingly, the direct defense traits of irjar4/6 plants are significantly higher than those in LIPOXYGENASE3 (LOX3)-silenced (aslox3) plants, which are impaired in JA biosynthesis, and JA-Ile treatment could not fully restore the resistance of aslox3 plants. We thus conclude that JA, its precursors, or other metabolites complement the function of JA-Ile by eliciting a panoply of induced defenses. Similarly, transcriptional profiling of wild-type, irjar4/6, and aslox3 plants with microarrays demonstrated that JA-Ile and JA play overlapping yet distinct roles in herbivore defense. Analysis of transcripts in distal tissues demonstrated that JAR activity is essential in eliciting systemic responses. However, attempts to recover JA-13C6-Ile in systemic leaves and roots after feeding wounded leaves with 13C6-Ile were unsuccessful, suggesting that JA-Ile is not a long-distance signal, but is rather synthesized after the arrival of an unknown mobile signal to systemic tissues.