Endoplasmic reticulum-associated inactivation of the hormone jasmonoyl-L-isoleucine by multiple members of the cytochrome P450 94 family in Arabidopsis

Abstract

The plant hormone jasmonate (JA) controls diverse aspects of plant immunity, growth, and development. The amplitude and duration of JA responses are controlled in large part by the intracellular level of jasmonoyl-L-isoleucine (JA-Ile). In contrast to detailed knowledge of the JA-Ile biosynthetic pathway, little is known about enzymes involved in JA-Ile metabolism and turnover. Cytochromes P450 (CYP) 94B3 and 94C1 were recently shown to sequentially oxidize JA-Ile to hydroxy (12OH-JA-Ile) and dicarboxy (12COOH-JA-Ile) derivatives. Here, we report that a third member (CYP94B1) of the CYP94 family also participates in oxidative turnover of JA-Ile in Arabidopsis. In vitro studies showed that recombinant CYP94B1 converts JA-Ile to 12OH-JA-Ile and lesser amounts of 12COOH-JA-Ile. Consistent with this finding, metabolic and physiological characterization of CYP94B1 loss-of-function and overexpressing plants demonstrated that CYP94B1 and CYP94B3 coordinately govern the majority (>95%) of 12-hydroxylation of JA-Ile in wounded leaves. Analysis of CYP94-promoter-GUS reporter lines indicated that CYP94B1 and CYP94B3 serve unique and overlapping spatio-temporal roles in JA-Ile homeostasis. Subcellular localization studies showed that CYP94s involved in conversion of JA-Ile to 12COOH-JA-Ile reside on endoplasmic reticulum (ER). In vitro studies further showed that 12COOH-JA-Ile, unlike JA-Ile, fails to promote assembly of COI1-JAZ co-receptor complexes. The double loss-of-function mutant of CYP94B3 and ILL6, a JA-Ile amidohydrolase, displayed a JA profile consistent with the collaborative action of the oxidative and the hydrolytic pathways in JA-Ile turnover. Collectively, our results provide an integrated view of how multiple ER-localized CYP94 and JA amidohydrolase enzymes attenuate JA signaling during stress responses.

Keywords: Cytochrome P450; Fatty Acid Oxidation; Jasmonate; Plant Biochemistry; Plant Hormone; Stress Response.

FIGURE 1.

CYP94B1 expressed in yeast catalyzes ω-oxidation of JA-Ile. A, schematic summary of reactions catalyzed by CYP94s and JA amidohydrolases, ILL6 and IAR3. Assignment of enzymes for each step is based on in vitro and in vivo data described in the text and literatures. B, LC chromatogram of products obtained in an in vitro JA-Ile hydroxylation assay. Microsomal preparations from yeast transformed with either CYP94B1 or empty vector control were incubated with 50 μm JA-Ile for 2 h. Reaction products were analyzed by LC-MS/MS to detect 12OH-JA-Ile and 12COOH-JA-Ile. Precursor and product ions used for MS/MS detection are shown next to the chromatograms.

FIGURE 2.

Jasmonate profile in wounded rosette leaves of CYP94 T-DNA insertion mutants and overexpressing lines. Rosette leaves were wounded twice across the midrib with a hemostat. Damaged leaves were harvested for JA extraction at indicated times after wounding and analyzed by LC-MS/MS. A and B, JA-Ile and 12OH-JA-Ile levels in wounded (2 h) and unwounded (0 h) leaves of WT, cyp94b3-1, cyp94b1-1, and cyp94b1-1cyp94b3-1 plants. *, p < 0.001; Student's t test. C and D, time course of JA-Ile and 12OH-JA-Ile accumulation in wounded leaves of plants overexpressing CYP94B3 (94B3OE) or CYP94B1 (94B1OE). *, p < 0.01; Student's t test. E and F, relative abundance of 12COOH-JA-Ile in wounded leaves of WT, cyp94b1-1cyp94b3-1, 94B3OE, and 94B1OE at various times after wounding. *, p < 0.05 between WT and 94B3OE; Student's t test. Each data point represents the mean ± S.D. of three biological replicates.

FIGURE 3.

Interaction between two major JA-Ile turnover pathways mediated by CYP94B3 and ILL6. A–E, time course of jasmonate accumulation in wounded rosette leaves of WT, cyp94b3-1, ill6-1, and cyp94b3-1ill6-1 plants. Asterisks denote significant difference between WT and mutant at p < 0.01 (*) or p < 0.001 (**); Student's t test. Each data point represents the mean ± S.D. of three biological replicates.

FIGURE 4.

CYP94B1-OE plants display JA-resistant phenotypes in roots and flowers. A, photographs showing silique development in WT (left panel) and CYP94B1-OE (right). B, photographs of representative WT (top panel) and CYP94B1-OE (bottom) flowers at three developmental stages. Scale bar, 2 mm. C, developing stamens and pistils of WT (left) and CYP94B1-OE (right) at time of pollination. Petals and sepals were removed to expose the interior parts of flowers. Scale bar, 1 mm. D, pollen viability of WT and two independent homozygous lines of CYP94B1-OE (B1OE-10 and B1OE-20). Pollen viability was assessed in four independent trials. E - G, root growth inhibition assays. WT, CYP94B1-OE (B1OE), and coi1-1 were grown on MS medium containing either 10 μm jasmonic acid (E, F) or 0.1 μm coronatine (COR) (G). Root length was determined on 10-d (E, F) or 8-d (G) old plants. Data show the mean ± S.D. (n > 20).

FIGURE 5.

Tissue-specific expression of CYP94B1 and CYP94B3. A and B, X-gluc staining of control unwounded (UW) or wounded (W) (for 24 h) leaves of CYP94B1promoter:GUS (B1pro:GUS) and CYP94B3promoter:GUS (B3pro:GUS) plants. C, B1pro:GUS (left panel) and B3pro:GUS (right) leaves treated with MeJA (for 24 h). Several droplets (10 μl) of a solution containing 50 μm MeJA were spotted onto the leaf surface. D and E, X-gluc staining of B1pro:GUS and B3pror:GUS seedlings 2∼3-days after germination. Bar, 1 mm. F-I, influorescence and flowers of B1pro:GUS and B3pro:GUS. Bars, 2 mm (G) and 1 mm (I).

FIGURE 6.

Subcellular localization of CYP94B3 and CYP94C1. Confocal images of tobacco leaf epidermal cells co-expressing the GFP-HDEL ER marker (left panel of A and B), and either CYP94B3 (94B3-mRFP) or CYP94C1 (94C1-mRFP) fused to the monomeric red fluorescent protein (middle panel of A and B). Merge between GFP and mRFP indicates co-localization (right panel of A and B). Bars, 5 μm.

FIGURE 7.

12COOH-JA-Ile does not promote assembly of COI1-JAZ receptor complexes. A, schematic of in vitro pull-down assays. Ligand-dependent interaction between COI1 and (B) JAZ2, (C) JAZ10, (D) JAZ12, and (E) JAZ8. Pull-down assays were performed using recombinant JAZ-His protein and crude leaf extract from Arabidopsis plants expressing ³⁵S:COI1-Myc in the presence of the indicated concentrations of JA-Ile, 12COOH-JA-Ile or coronatine. Reactions with JAZ8 contained 25 μm of 12COOH-JA-Ile and JA-Ile or 2.5 μm coronatine. Equal recovery and loading of JAZ-His protein is indicated by a Coomassie Blue-stained acrylamide gel.