Amide conjugates of the jasmonate precursor cis-(+)-12-oxo-phytodienoic acid regulate its homeostasis during plant stress responses

Abstract

Jasmonates are a family of oxylipin phytohormones regulating plant development and growth and mediating "defense versus growth" responses. The upstream JA biosynthetic precursor cis-(+)-12-oxo-phytodienoic acid (cis-OPDA) acts independently of CORONATIVE INSENSITIVE 1-mediated JA signaling in several stress-induced and developmental processes. However, its perception and metabolism are only partially understood. An isoleucine analog of the biologically active JA-Ile, OPDA-Ile, was detected years ago in wounded leaves of flowering plants, opening up the possibility that conjugation of cis-OPDA to amino acids might be a relevant mechanism for cis-OPDA regulation. Here, we extended the analysis of amino acid conjugates of cis-OPDA and identified naturally occurring OPDA-Val, OPDA-Phe, OPDA-Ala, OPDA-Glu, and OPDA-Asp accumulating in response to biotic and abiotic stress in Arabidopsis (Arabidopsis thaliana). The OPDA amino acid conjugates displayed cis-OPDA-related plant responses in a JA-Ile-dependent manner. We also showed that the synthesis and hydrolysis of cis-OPDA amino acid conjugates are mediated by members of the amidosynthetase GRETCHEN HAGEN 3 and the amidohydrolase INDOLE-3-ACETYL-LEUCINE RESISTANT 1/ILR1-like families. Thus, OPDA amino acid conjugates function in the catabolism or temporary storage of cis-OPDA in stress responses instead of acting as chemical signals per se.

Figure 1.

OPDA-aa accumulate in Arabidopsis plants during stress responses. A) Time-course accumulation of indicated OPDA-aa in Col-0 after leaf wounding. Six-week-old plants were wounded, and damaged leaves were collected after the indicated times. B) Accumulation of indicated OPDA-aa in Col-0 plants infected with B. cinerea 4 d after inoculation (B) or mock-inoculated (M). C) Time-course accumulation of indicated OPDA-aa after exogenous treatment with 50 µM cis-(±)-OPDA. Plants were sampled after the indicated times. Ala, Alanine; Asp, aspartate; Glu, glutamate; Ile, isoleucine; Phe, phenylalanine; Val, valine. OPDA-aa levels are expressed as pmoles per gram of plant FW. Mean ± SD (n = 3). Below the limit of detection, <LOD.

Figure 2.

(±)-OPDA-aa exhibit cis-OPDA-like activity in a JA-Ile-dependent manner. A) Wild-type (Col-0) and gh3.10-2,jar1-11 mutant seedlings grown on vertical plates in the absence (mock) or presence of 10 µM (±)-JA, cis-(±)-OPDA, and indicated (±)-OPDA-aa. Scale bar in the top left inset represents 1 cm and applies to all the insets of the panel. B) Root length of 14 to 15 seedlings was measured 7 d after germination. Data are shown as mean ± SD of 3 biological replicates (n = 3). Letters indicate significant differences, evaluated by 1-way ANOVA/Tukey HSD post hoc test (P < 0.05). C) Representative seedlings of the 35S:JAZ1-GUS line treated with or without 10 µM (±)-JA, cis-(±)-OPDA, and indicated (±)-OPDA-aa for 2 h. Scale bar in the first inset represents 1 mm and applies to all the insets of the panel. Ala, Alanine; Asp, aspartate; Glu, glutamate; Ile, isoleucine; Phe, phenylalanine; Val, valine.

Figure 3.

Analyses of gene expression in response to (±)-OPDA-aa, affinity between OPDA-Ile and COI-JAZs, and conversion of OPDA-aa into cis-OPDA and JA. A) Expression of THI2.1, VSP1, ZAT10, GRX480, JAZ5, and PDF1.2 after 3 h-treatment with or without 50 µM (±)-JA, cis-(±)-OPDA, and indicated (±)-OPDA-aa in Col-0. Gene expression was measured by RT-qPCR. Data are expressed as relative fold change normalized by ACT2. Letters indicate significant differences, evaluated by 1-way ANOVA/Tukey HSD post hoc test (P < 0.05). B) Pull-down assay of GST-AtCOI1 with Fl-AtJAZPs in the presence of JA-Ile (1 µM) or OPDA-Ile (1 or 30 µM). GST-AtCOI1 bound to Fl-AtJAZPs was pulled-down with an antifluorescein antibody and Protein G magnetic beads and analyzed by immunoblotting (anti-GST-HRP conjugate for detection of GST-AtCOI1) (95 kDa). Fl-AtJAZ13 was used as a negative control because JAZ13 had no canonical JAZ degron sequence, which is necessary for JA-Ile perception. This experiment was repeated 3 times with similar results. C, D) Time-course accumulation of stable isotope-labeled derivatives of OPDA-Val and OPDA-Ile in liquid ½ MS medium (pmol/mL) and 7-d-old Col-0 seedlings (pmol/g FW) after feeding with 10 µM d₅-OPDA-Val (C) and d₅-OPDA-Ile (D). Samples were collected at the indicated times. Ala, Alanine; Asp, aspartate; Glu, glutamate; Ile, isoleucine; Phe, phenylalanine; Val, valine. Mean ± Sd (n = 3). Below the limit of detection, <LOD.

Figure 4.

Members of the Group II GH3 family conjugate cis-OPDA with amino acids in planta. A) Analysis of (±)-OPDA-aa synthesized by recombinant Arabidopsis GH3.1, GH3.2, GH3.3, AtGH3.4, GH3.5, and GH3.6 in the bacterial assay. The cell lysate was incubated with or without 0.1 mmcis-(±)-OPDA and GH3 cofactor mixture for 5 h at 30 °C. The bacterial assay carried out with cell lysate from GFP-producing bacteria was used as a negative control. Cell lysate without cis-(±)-OPDA and cofactor mixture was used as a mock sample. (±)-OPDA-aa level is expressed as pmol/mL. The conjugation assay was performed in triplicate and repeated 3 times with similar results. B) Formation of OPDA-Asp after feeding of 7-d-old Arabidopsis Col-0 and gh3 sextuple mutant (gh3.1,gh3.2,gh3.3,gh3.4,gh3.5,gh3.6) with or without 50 µM cis-(±)-OPDA for 3 h. OPDA-Asp level is expressed as pmol/g FW. Horizontal lines in the box plots are medians, boxes show the upper and lower quartiles, and whiskers show the entire data range. C) Accumulation of indicated OPDA-aa in Col-0 and gh3 sextuple mutant after leaf wounding. Six-week-old plants were wounded, and damaged leaves were collected 2 h postwounding. D, E) GH3.3 has dual cytoplasmic/nuclear localization. D) Subcellular colocalization of GFP–GH3.3 fusion protein (left panel) with marker for cytosol/nucleus (mCherry; middle panel) in Arabidopsis root culture protoplasts. The right panel shows the overlay of the GFP–GH3.3 and mCherry signals. E) Confocal images of roots from 7-d-old Arabidopsis transgenic UBQ10-GFP–GH3.3 seedlings, showing cortex cells (left) and epidermal cells in the mature root region (right). Confocal microscopy was performed on the third generation of at least 8 stable transgenic lines derived from different first-generation plants, with a consistent subcellular localization pattern observed across all lines. Scale bars in D) and E) represent 20 µm. F) Western Blot analysis of GFP–GH3.3 fusion protein in GFP–GH3.3 overexpression Arabidopsis plants using anti-GFP antibody. The predicted molecular weight of the GH3.3 fusion protein is 84.5 kDa (GFP: 27 kDa; GH3.3: 67.5 kDa). Ala, Alanine; Asp, aspartate; Glu, glutamate; Ile, isoleucine; Phe, phenylalanine; Trp, tryptophan; Val, valine. Mean ± Sd (n = 3). Below the limit of detection, <LOD.

Figure 5.

ILR1/ILL enzymes are involved in cis-OPDA release from OPDA-aa in Arabidopsis upon wounding. A) Analysis of release of cis-(±)-OPDA by recombinant IAR3, ILL2, ILR1, and ILL6 in the bacterial assay. The cell lysate was incubated with or without 0.1 mm (±)-OPDA-aa and 1 mm MgCl₂ for 5 h at 30 °C. The bacterial assay carried out with the cell lysate from GFP-producing bacteria was used as a negative control. Cell lysate without (±)-OPDA-aa and MgCl₂ was used as a mock sample. cis-(±)-OPDA level is expressed as pmol/mL. The hydrolysis assay was performed in triplicate and repeated 3 times with similar results. B) Time-course accumulation of indicated OPDA-aa in wild-types (Col-0, Ws), ill6-2 single (Col-0 background), and ilr1-1,iar3-2,ill2-1 triple (Ws background) knockout mutants after leaf wounding. Six-week-old plants were wounded, and damaged leaves were collected after the indicated times. Asterisk indicates statistically significant differences, as determined by Student's t-test (Col-0 vs ill6-2, Ws vs ilr1-1,iar3-2,ill2-1; P < 0.05). OPDA-aa concentrations are given as pmoles per gram FW. Ala, Alanine; Asp, aspartate; Glu, glutamate; Ile, isoleucine; Phe, phenylalanine; Val, valine. Mean ± Sd (n = 3). Below the limit of detection, <LOD.

Figure 6.

In situ visualization of OPDA-aa from wounded leaves and model for cis-OPDA and OPDA-aa subcellular pathways. A) DESI-MSI images showing the localization of OPDA-aa in Col-0 and ill6-2 knockout mutant in wounded (W) and unwounded (UW) leaves. Three-week-old plants were wounded, and damaged leaves were collected after 4 h. Annotated images of the JA, OPDA, OPDA-Ala, OPDA-Glu, OPDA-Ile, OPDA-Phe, and OPDA-Val distribution in control (top) and wounded leaves (bottom) are reported. The wounded region in damaged leaves is delimited with a dashed line. The peak intensity levels are displayed on the scale at the right side of the panels. Scale bars in the insets represent 2 mm. Ala, Alanine; Asp, aspartate; Glu, glutamate; Ile, isoleucine; Phe, phenylalanine; Val, valine. B) Proposed model for cis-OPDA and OPDA-aa subcellular pathways. Once synthesized in the chloroplast, cis-OPDA is mainly reduced and β-oxidized to produce OPC-8 and JA in the peroxisome. Part of the chloroplast-derived cis-OPDA reaches the cytosol, where GH3 enzymes conjugate it with amino acids to form OPDA amino acid conjugates (OPDA-aa). OPDA amide conjugates are hydrolyzed back to cis-OPDA in the ER by ILR1/ILL enzymes and cis-OPDA is then released in the nucleus. Nuclear cis-OPDA can activate the expression of cis-OPDA-responsive genes or be conjugated with amino acids by GH3s. According to this model, cis-OPDA amino acid conjugation might be an essential step in regulating nuclear cis-OPDA levels through the ER. Unknown transporters likely mediate OPDA-aa fluxes among the cytosol, the ER, and the nucleus. Jasmonic acid, JA; 3-oxo-2-(2-(Z)-pentenyl)-cyclopentane-1-octanoic, OPC-8.