Jasmonate (JA) and its amino acid conjugate, jasmonoyl-isoleucine (JA-Ile), play important roles in regulating plant defense responses to insect herbivores. Recent studies indicate that JA-Ile promotes the degradation of JASMONATE ZIM-domain (JAZ) transcriptional repressors through the activity of the E₃ ubiquitin-ligase SCF^COI1. Here, we investigated the regulation and function of JAZ genes during the interaction of Arabidopsis (Arabidopsis thaliana) with the generalist herbivore Spodoptera exigua. Most members of the JAZ gene family were highly expressed in response to S. exigua feeding and mechanical wounding. JAZ transcript levels increased within 5 min of mechanical tissue damage, coincident with a large (approximately 25-fold) rise in JA and JA-Ile levels. Wound-induced expression of JAZ and other CORONATINE-INSENSITIVE1 (COI1)-dependent genes was not impaired in the jar1-1 mutant that is partially deficient in the conversion of JA to JA-Ile. Experiments performed with the protein synthesis inhibitor cycloheximide provided evidence that JAZs, MYC2, and genes encoding several JA biosynthetic enzymes are primary response genes whose expression is derepressed upon COI1-dependent turnover of a labile repressor protein(s). We also show that overexpression of a modified form of JAZ1 (JAZ1Δ3A) that is stable in the presence of JA compromises host resistance to feeding by S. exigua larvae. These findings establish a role for JAZ proteins in the regulation of plant anti-insect defense, and support the hypothesis that JA-Ile and perhaps other JA derivatives activate COI1-dependent wound responses in Arabidopsis. Our results also indicate that the timing of JA-induced transcription in response to wounding is more rapid than previously realized.

Summary: Jasmonic acid (JA) and its biologically active derivatives (bioactive JAs) perform a critical role in regulating plant responses to wound stress. The perception of bioactive JAs by the F‐box protein COI1 triggers the SCF^COI1/ubiquitin‐dependent degradation of JASMONATE ZIM‐DOMAIN (JAZ) proteins that repress the expression of JA‐response genes. JA is required for many wound‐inducible systemic defense responses, but little is known about the role of the hormone in long‐distance signal relay between damaged and undamaged leaves. Here, we show that the wounding of Arabidopsis thaliana leaves results in the rapid (<5 min) accumulation of jasmonoyl‐l‐isoleucine (JA‐Ile), the bioactive form of JA, in leaves distal to the wound site. The rapid systemic increase in JA‐Ile preceded the onset of early transcriptional responses, and was associated with JAZ degradation. Wound‐induced systemic production of JA‐Ile required the JA biosynthetic enzyme 12‐oxo‐phytodienoic acid (OPDA) reductase 3 (OPR3) in undamaged responding leaves, but not in wounded leaves, and was largely dependent on the JA‐conjugating enzyme JAR1. Interestingly, the wound‐induced synthesis of JA/JA‐Ile in systemic leaves was correlated with a rapid decline in OPDA levels. These results are consistent with a model in which a rapidly transmitted wound signal triggers the systemic synthesis of JA, which, upon conversion to JA‐Ile, activates the expression of early response genes by the SCF^COI1/JAZ pathway.

Detection of 3-nitrotyrosine has served as an in vivo marker for the production of the cytotoxic species peroxynitrite (ONOO⁻). We show here that reaction of nitrite (NO⁻₂), the autoxidation product of nitric oxide (·NO), with hypochlorous acid (HOCl) forms reactive intermediate species that are also capable of nitrating phenolic substrates such as tyrosine and 4-hydroxyphenylacetic acid, with maximum yields obtained at physiological pH. Monitoring the reaction of NO⁻₂ with HOCl by continuous flow photodiode array spectrophotometry indicates the formation of a transient species with spectral characteristics similar to those of nitryl chloride (Cl-NO₂). Reaction of synthetic Cl-NO₂ with N-acetyl-L-tyrosine results in the formation of 3-chlorotyrosine and 3-nitrotyrosine in ratios that are similar to those obtained by the NO⁻₂/HOCl reaction (4:1). Tyrosine residues in bovine serum albumin are also nitrated and chlorinated by NO⁻₂/HOCl and synthetic Cl-NO₂. The reaction of N-acetyl-L-tyrosine with NO⁻₂/HOCl or authentic Cl-NO₂ also produces dityrosine, suggesting that free radical intermediates are involved in the reaction mechanism. Our data indicate that while chlorination reactions of Cl-NO₂ are mediated by direct electrophilic addition to the aromatic ring, a free radical mechanism appears to be operative in nitrations mediated by NO⁻₂/HOCl or Cl-NO₂, probably involving the combination of nitrogen dioxide (·NO₂) and tyrosyl radical. We propose that NO⁻₂ reacts with HOCl by Cl⁺ transfer to form both cis- and trans-chlorine nitrite (Cl-ONO) and Cl-NO₂ as intermediates that modify tyrosine by either direct reaction or after decomposition to reactive free and solvent-caged Cl· and ·NO₂ as reactive species. Formation of Cl-NO₂ and/or Cl-ONO in vivo may represent previously unrecognized mediators of inflammation-mediated protein modification and tissue injury, and offers an additional mechanism of tyrosine nitration independent of ONOO⁻.