1. Volume 10, Issue 9, Pages 1159-1173 (September 2017)
Jasmonic Acid Oxidase 2 Hydroxylates Jasmonic Acid and Represses Basal Defense and Resistance Responses against Botrytis cinerea Infection 
Ekaterina Smirnova, Valentin Marquis, Laure Poirier, Yann Aubert, Julie Zumsteg, Rozenn Ménard, Laurence Miesch, Thierry Heitz 
Molecular Plant 
Volume 10, Issue 9, Pages (September 2017)
DOI: /j.molp
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2. Figure 1
Evidence for Amidohydrolase-Independent, Regalis-Sensitive Formation of 12OH-JA.
(A) Main metabolic conversions in the jasmonate pathway after a stimulus. JA is conjugated to isoleucine (Ile) by JAR1 enzyme. The bioactive hormone JA-Ile is perceived by the SCFCOI1 complex, leading to target gene derepression and physiological responses. The hormone JA-Ile is inactivated by a two-step oxidation to 12OH-JA-Ile and 12COOH-JA-Ile by the cytochrome P450 enzymes CYP94B3 and CYP94C1, or by conjugate cleavage under the action of the amidohydrolases (AH) IAR3 and ILL6. In addition, these AH also cleave 12OH-JA-Ile to release 12OH-JA. Question marks indicate uncharacterized steps.
(B) 12-OH JA levels at 3 h post-wounding (hpw) or 3 days post-inoculation (dpi) by B. cinerea in WT or iar3-5 ill6-2 double-mutant leaves. Histograms represent the mean ± SD of three biological replicates. Different letters indicate a significant difference between the two genotypes (P < 0.01 for left panel and P < 0.05 for right panel).
(C and D) 12OH-JA levels at 1, 2, and 5 hpw in WT (C) or iar3ill6 (D) leaves treated with Regalis (Reg) or not (Mock). Histograms represent the mean ± SD of three biological replicates. Different letters indicate a significant difference at a given time point between treated and not treated plants (P < 0.05, one-way ANOVA and Tukey post-hoc test).
Molecular Plant  , DOI: ( /j.molp )
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3. Figure 2
JAO Gene Expression upon Mechanical Wounding or Fungal Infection.
(A and B) Real-time RT–qPCR analysis of JAO4, JAO2, JAO3, and JAO4 gene expression after mechanical wounding (A) or after Botrytis infection (B). Expression is represented as relative expression of each JAO gene normalized by expression of EXP and TIP41 reference genes.
(C) Induction of JAO genes at 2 h post-wounding (hpw) in WT and coi1 leaves. Expression is represented as induction fold relative to expression level at time 0, which was set to 1.
(D) Relative expression of JAO genes in non-stimulated WT and coi1 leaves. Mean values ±SD of a representative experiment are shown.
Molecular Plant  , DOI: ( /j.molp )
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4. Figure 3
Catalytic Activities of Recombinant JAO4, JAO2, and JAO3 Proteins.
LC chromatograms of residual JA or 12OH-JA produced in incubation of JA with affinity-purified JAO4, JAO2, or JAO3 proteins. Incubations were performed in the absence (right panels) or presence (left panels) of the co-substrate 2-oxoglutarate. Control reactions were run by incubating JA substrate with BSA. Reaction mixtures were analyzed by LC–MS/MS. Oxidation product matched retention time and mass transition in multiple reaction monitoring mode in negative electrospray (MRM, m/z 225 > 59 for 12OH-JA and m/z 209 > 59 for JA) of authentic 12OH-JA standard.
Molecular Plant  , DOI: ( /j.molp )
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5. Figure 4
Resistance Levels and Expression Profiles of Jasmonate-Dependent Defense Genes in Response to B. cinerea in jao Loss-of-Function Mutants.
(A) Disease symptoms at 3 dpi. Two sites were inoculated across the main vein with 5 μL of spore suspension containing 2.5 × 106 spores mL−1.
(B) Disease-symptom scoring. Top panel, mean lesion diameter at 3 dpi. Histograms represent the mean lesion diameters ± SEM of about 100 lesion sites from 10 to 15 plants for each genotype. Bottom panel, evaluation of fungal growth by real-time qPCR with B. cinerea cutinase-specific primers on genomic DNA extracted from 3-day-infected leaves. Quantification was performed on three biological replicates analyzed in duplicate. Columns labeled with different letters indicate a significant difference as determined by one-way ANOVA with Tukey post-hoc test (P < 0.01).
(C) Expression profiles of jasmonate-dependent defense genes in response to B. cinerea in jao loss-of-function mutants. Expression is represented as the relative expression of each target gene before (0 dpi, top panels) or at 3 dpi (bottom panels). For each experiment, expression of ORA59, PDF1.2, and PR4 was determined by real-time PCR using gene-specific primers and normalized using EXP and TIP41 reference genes. Transcript quantification was performed on three biological replicates analyzed in duplicate. Histograms represent mean expression ± SEM. Columns labeled with different letters indicate a significant difference between genotypes as determined by one-way ANOVA with Tukey post-hoc test, P < 0.05.
Molecular Plant  , DOI: ( /j.molp )
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6. Figure 5
Complementation of jao2-2 by Ectopic Expression of JAO4 or JAO2 or JAO3 Under p35S Promoter.
(A) T2 Basta-resistant jao2-2 plants (selected from primary transformants displayed in Supplemental Figure 5) exhibiting single-locus transgene segregation were analyzed for impact of transgene expression on PDF1.2 transcript levels. Histograms show mean expression ± SEM of three independent plants for each genotype.
(B) Lines analyzed in (A) were inoculated with Botrytis and scored for antifungal resistance. Mean lesion diameter at 3 days post-inoculation is shown. Histograms represent mean lesion diameters ± SEM of about 100 lesion sites from 10 to 15 plants for each genotype. Columns labeled with different letters indicate a significant difference between genotypes determined by one-way ANOVA with Tukey post-hoc test, P < 0.01.
Molecular Plant  , DOI: ( /j.molp )
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7. Figure 6
Impact of 12OH-JA on JA-Ile Signaling (A) and Genetic Interaction of jao2-2 Mutation with JAR1 (B) and COI1 (C).
(A) Wild-type seedlings were grown for 7 days in liquid MS medium before adding 15 μM JA-Ile, or 30 μM 12OH-JA, or a combination of both compounds, or no compound (Mock). Seedlings were harvested at the indicated time points before gene expression analysis by real-time qPCR. Relative expression normalized by expression of EXP and TIP41 reference genes is shown. Values are means ± SEM of three biological replicates.
(B and C) Epistasis analysis of jao2 mutations in a coi1 or jar1 background. Impact of jar1 (B) or coi1 (C) mutations on jao2-triggered expression of defense markers ORA59, PDF1.2, and PR4. Relative expression is shown in unstimulated leaves of WT or jao2-2, jar1-1, coi1-1 mutant, or jao2-2jar1-1 or jao2-2coi1-1 double-mutant plants. Histograms represent means ± SEM of five independent plants for each genotype for the jao2-2*jar1-1 cross and three to five independent plants for the jao2-2*coi1-1 cross. Columns labeled with different letters indicate a significant difference between genotypes as determined by one-way ANOVA with Tukey post-hoc test, P < 0.01.
Molecular Plant  , DOI: ( /j.molp )
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8. Figure 7
Jasmonate Profiles in jao Mutants before and after Response to B. cinerea Infection.
Six-week-old plants were drop inoculated on two sites per leaf with a suspension containing 2.5 × 106 fungal spores mL−1. Leaves were harvested at 0 or 3 dpi, and jasmonates shown in Figure 1A were extracted and quantified by LC–MS. (A) JA, (B) JA-Ile, (C) 12OH-JA, (D) 12OH-JA-Ile, (E) 12COOH-JA, (F) 12COOH-JA-Ile levels were expressed in nmol/g fresh weight (FW). Histograms represent the mean ± SEM of three biological replicates. Columns labeled with different letters indicate a significant difference between genotypes determined by one-way ANOVA with Tukey post-hoc test, P < 0.05.
Molecular Plant  , DOI: ( /j.molp )
Copyright © 2017 The Author Terms and Conditions

9. Figure 8
Working Model for Impact of JAO on JA Metabolism and Defense Signaling.
(A) A metabolic switch controlled by JAO2 activity defines flux and signaling through JA-Ile hormone in unchallenged leaves. In WT leaves, in the absence of a JA-triggering stimulus, JAO2 activity ensures a continuous turnover of basal JA by its oxidation to 12OH-JA and potential derivatives. This minimizes JA-Ile synthesis, signaling, and catabolism and maintains repression of JA-Ile responses. In jao2 leaves, reduced JA consumption channels more JA toward JA-Ile synthesis, resulting in constitutive defense signaling and catabolism, and primes leaves for enhanced antimicrobial resistance. For compound structures, see Figure 1A.
(B) Differential contributions of distinct 12OH-JA formation pathways upon wounding and infection stress in Arabidopsis leaves. The thickness of the arrows reflects the importance of metabolic flux in the respective routes. Upon mechanical wounding (left panel), 12OH-JA is formed predominantly via a 12OH-JA-Ile conjugate intermediate generated by JA-Ile catabolism. Upon fungal infection (right panel), the bulk of 12OH-JA accumulated originates from JAO oxidizing activity on JA. For clarity, fluxes in stressed leaves are shown only for WT background, and potential 12OH-JA derivatives are not shown. Dotted arrows represent an uncharacterized pathway generating 12COOH-JA, a further downstream JA catabolite.
Molecular Plant  , DOI: ( /j.molp )
Copyright © 2017 The Author Terms and Conditions