Downstream metabolites of (+)-cis-12-oxo-phytodienoic acid function as noncanonical bioactive jasmonates in Arabidopsis thaliana.

doi:10.21203/rs.3.rs-5075946/v1

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Downstream metabolites of (+)-cis-12-oxo-phytodienoic acid function as noncanonical bioactive jasmonates in Arabidopsis thaliana.

https://doi.org/10.21203/rs.3.rs-5075946/v1

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(+)-cis-12-oxo-phytodienoic acid (cis-OPDA) is a biosynthetic precursor of the plant hormone (+)-7-iso-jasmonoyl-L-isoleucine (JA-Ile). It functions as an endogenous chemical signal independent of the JA-Ile receptor COI1-JAZ in Arabidopsis thaliana. The bioactive form of cis-OPDA that induces COI1-JAZ-independent gene expression remains unknown.

In this study, we demonstrated that the genuine bioactive forms of cis-OPDA are the downstream metabolites of b-oxidation, which upregulate the expression of the OPDA marker genes such as ZAT10and ERF5 in a COI1- or JA-Ile-independent manner. These downstream metabolites function independently of the JA-Ile-COI1-JAZ-MYCs canonical jasmonate signaling module, and their electrophilic nature is essential for their bioactivity.

Biological sciences/Plant sciences/Plant hormones/Jasmonic acid

Biological sciences/Plant sciences/Plant signalling

Jasmonic acid and related fatty acid-derived oxylipins are collectively referred to as jasmonates. Jasmonate, (+)-7-iso-jasmonoyl-L-isoleucine (JA-Ile; Fig. 1), is a lipid-derived plant hormone that regulates growth, reproduction, and defense responses against pathogens and chewing insects.^{1, 2, 3} In plant cells, JA-Ile is synthesized from α-linolenic acid via the biosynthetic precursor (+)-cis-12-oxo-phytodienoic acid (cis-OPDA) (Fig. 1A), which is reduced to OPC-8 by OPDA reductase 3 (OPR3), followed by oxidation to naturally occurring (3R, 7S)-7-iso-jasmonic acid (JA, a cis-form) through three rounds of β-oxidation and conjugation with L-isoleucine (Ile) by GH3 enzyme jasmonic acid-resistant 1 (JAR1) or AtGH3.10 to produce JA-Ile.^{4, 5, 6, 7, 8} Recently, an OPR3-independent route for JA-Ile biosynthesis from cis-OPDA was reported (Fig. 1B).⁹ In this bypassing route, three rounds of β-oxidation occurred from cis-OPDA to dinor-cis-OPDA (dn-cis-OPDA), tetranor-cis-OPDA (tn-cis-OPDA), and 7-iso-4,5-didehydro JA (4,5-ddh-JA), which is then reduced to JA by OPR2 (Fig. 1B). An increase in JA-Ile levels in plant cells leads to the triggering of protein-protein interactions between the F-box protein CORONATINE INSENSITIVE 1 (COI1) and jasmonate-ZIM domain (JAZ) repressors, causing proteasomal degradation of JAZ to derepress transcription factors, such as MYC2, and activating gene expression.^{10, 11, 12, 13}

Several reports have demonstrated JA-Ile-independent biological activities of the biosynthetic intermediate cis-OPDA in Arabidopsis thaliana, tomato, and maize.^{14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24} Taki et al. compared the gene expression mediated by cis-OPDA and JA to demonstrate cis-OPDA-specific expression of genes, including genes encoding ZINC FINGER OF ARABIDOPSIS THALIANA 10 (ZAT10), ETHYLENE REPONSIVE ELEMENT BINDING FACTOR 5 (ERF5), DRE-BINDING PROTEIN 2A (DREB2A), ZINC FINGER OF ARABIDOPSIS THALIANA 12 (ZAT12), and ARABIDOPSIS FE-DEFICIENCY INDUCED TRANSCRIPTION FACTOR 1 (FIT1).¹⁶ ZAT10 encodes a transcription factor (TF) involved in salt tolerance,¹⁶ and ERF5 encodes an ethylene-responsive TF involved in wounding and cold stress responses.²⁵ DREB2A encodes a TF involved in drought and salt stress.^{26, 27} ZAT12 encodes a TF in cold stress and salt tolerance.^{28, 29} FIT1 encodes a TF in iron acquisition.³⁰ Biochemical, yeast two-hybrid (Y2H), and pull-down assays demonstrated that cis-OPDA is not recognized by Arabidopsis COI1-JAZ1/3/9 co-receptor pairs,^{12, 31} implying that cis-OPDA functions via a COI1-independent mode of action (MOA). However, in previous studies in A. thaliana, the biological activities of applied cis-OPDA have been distinguished from those of the JA-Ile biosynthesized from applied cis-OPDA using the Arabidopsis mutants opr3-1 and jar1-1, in which the biosynthetic genes of JA-Ile (OPR3 or JAR1) are impaired.³² This could be because cis-OPDA was not converted into JA-Ile in opr3-1 and jar1-1. Nevertheless, recent studies have indicated that cis-OPDA can be converted into JA-Ile in opr3-1 or jar1-1 through OPR3-independent (Fig. 1B) ^{9, 33} or JAR1-independent ³⁴ bypassing routes owing to the leaky nature of these mutant lines. As a result, the participation of cis-OPDA in JA-Ile-independent signaling pathways in A. thaliana remains debatable.^{35, 36} To date, the COI1-independent MOA of cis-OPDA has been reliably reported only in the thermotolerance of Marchantia polymorpha, in which the electrophilic a,b-unsaturated cyclopentenone moiety of cis-OPDA plays an essential role.³⁷

In this paper, we investigated the function of cis-OPDA in WT A. thaliana and the non-leaky mutant lines coi1-1 ³⁸ and opr2-1 opr3-3 ⁹ (Fig. 1) with impaired JA signaling and biosynthesis to identify the genuine bioactive form of cis-OPDA in A. thaliana.

Analyses of cis-OPDA-induced gene expression in A. thaliana and the mutant lines on JA signaling

cis-OPDA (Fig. 1) was chemically synthesized using previously reported methods.^{39, 40} The subsequent experiments were performed using synthetic cis-OPDA of naturally occurring stereochemistry. Gene expression analysis was performed based on the information from a previously reported comprehensive DNA microarray analysis¹⁶: the impact of JA (Fig. S1) or cis-OPDA was respectively evaluated on the expression of three JA marker genes, OPR3, JAZ8, and MYC2 (Figure. 2A-C), and five cis-OPDA-specific marker genes, ZAT10, ERF5, DREB2A, ZAT12, and FIT1 (Fig. 2D-2H).^{16, 32}

In all experiments, cis-OPDA affected the expression of JA and OPDA marker genes in Col-0 (Fig. 2A-H). cis-OPDA and JA induced the expression of JA marker genes, OPR3, JAZ8, and MYC2 in Col-0, and their expression was impaired in coi1-1, indicating their COI1-dependence (Fig. 2A-C). In contrast, the expression of OPDA-marker genes, ERF5, DREB2A, ZAT12, and FIT1, by cis-OPDA was not or slightly affected in coi1-1, confirming their COI1-independence (Fig. 2D-G). However complex effects were observed regarding ZAT10 expression (Fig. 2H). ZAT10 expression was induced by all compounds in Col-0. In the coi1-1 mutant, ZAT10 expression was not induced by (-)-JA, whereas moderate ZAT10 expression was observed for cis-OPDA treatment (Fig. 2D). These findings imply that the expression of ZAT10 by JA is COI1-dependent, whereas that of cis-OPDA depends on two distinct pathways, COI1-dependent and COI1-independent. In addition, cis-OPDA showed no affinity for the functional COI1-JAZ1-6/9–12 co-receptor pairs in the pull-down assay (Fig. 2I and S2 and S3), confirming that cis-OPDA is not ligands for functional COI1-JAZ co-receptors.

We also examined the effect of cis-OPDA on the expression of JA and OPDA marker genes in the myc2myc3myc4 triple mutant, in which the master TFs of JA signaling, MYC2/MYC3/MYC4, were impaired⁴¹ (Fig. S4). The expression of the JA marker genes OPR3 and JAZ8 was suppressed in myc2myc3myc4 (Fig. S4A and S4B). Different effects were observed for the five OPDA marker genes, ZAT10, ERF5, DREB2A, ZAT12, and FIT1; expressions of ZAT10 and DREB2A were moderately suppressed, and that of ERF5, ZAT12, and FIT1 were enhanced or not affected (Fig. S4C-G), respectively. The current results demonstrated that cis-OPDA mediated the expression of OPDA marker genes independently of the canonical COI1-JAZ-MYC signaling pathway.

cis -OPDA induces the expression of OPDA-marker genes independent of the conversion into JA-Ile

Given that cis-OPDA is a major biosynthetic precursor of JA-Ile, we examined whether cis-OPDA-induced gene expression depends on the in planta conversion into JA-Ile. This was investigated using the synthesized cis-OPDA-d₅ (Fig. 3A and Scheme S1). The conversion of cis-OPDA into JA-Ile was examined using Arabidopsis opr2-1opr3-3 double mutant in which the conversion of cis-OPDA into JA-Ile is impaired.⁹ UPLC-MS/MS analysis demonstrated that cis-OPDA-d₅ was converted into JA-d₅-Ile within 30 min in Col-0, whereas this conversion was impaired in opr2-1opr3-3 (Fig. 3A).

Then we examined the cis-OPDA- or JA-mediated marker gene expression in opr2-1opr3-3. For the JA marker gene, cis-OPDA-induced expression of JAZ8 in Col-0 was significantly suppressed in opr2-1opr3-3 (Fig. 3B). In contrast, JA-mediated JAZ8 expression in Col-0 was not affected by opr2-1opr3-3 (Fig. 3B). In contrast, the expression of OPDA-marker genes, ZAT10, ERF5, DREB2A, ZAT12, and FIT1, was not affected or suppressed in opr2-1opr3-3 (Fig. 3C-G). And the JA-induced expression of ZAT10 in opr2-1opr3-3 (Fig. 3C) suggested that expression of ZAT10 expression is also depends on canonical JA-signaling pathway, as shown in Fig. 2H. The undetectable level of JA-Ile in cis-OPDA-treated opr2-1opr3-3 in Figure. 3A⁹ implies that cis-OPDA induces the expression of OPDA maker genes independently of their conversion to JA-Ile. The undetectable level of JA-Ile in cis-OPDA-treated opr2-1opr3-3 in Figure. 3A⁹ implies that cis-OPDA induces the expression of OPDA maker genes independently of their conversion to JA-Ile.

The Arabidopsis cis-OPDA transporter mutant and metabolism showed that cis-OPDA is not a genuine bioactive form.

Next, we examined whether cis-OPDA itself is a genuine bioactive form by using the Arabidopsis cis-OPDA transporter mutant. cis-OPDA is converted to JA in the peroxisomes, and the peroxisomal ATP-binding cassette (ABC) transporter COMATOSE (CTS) is involved in the import of cis-OPDA into the peroxisomes in A. thaliana (Fig. 4A, left).³⁶ In the cts1 mutant line, in which CTS is impaired, wound-induced accumulation of JA-Ile was significantly suppressed but not completely abolished.^{42, 43, 44, 45} In the cts1 mutant line, the level of cis-OPDA was significantly increased (Fig. 4A, right), however, to our surprise, cis-OPDA-induced expressions of ZAT10, ERF5, DREB2A, ZAT12, and FIT1 were suppressed in cts1 (Fig. 4B). These results strongly supported that cis-OPDA itself is not a genuine bioactive form.

In the peroxisome, cis-OPDA is converted into JA by the canonical OPR3-dependent or OPR3-independent route via several downstream metabolites (Figs. 1 and 3A). Considering the attenuation of marker gene expressions in cts1 mutant, our current finding indicated that the downstream metabolites of cis-OPDA are the plausible candidates of the genuine bioactive forms.

The downstream metabolites of cis-OPDA, dn-cis-OPDA, tn-cis-OPDA, and (+)-7-iso-4,5-didehydrojasmonic acid, are bioactive forms of cis-OPDA

When the increased expression of ERF5, DREB2A, ZAT12, and FIT1 by cis-OPDA treatment was compared between Col-0 and opr2-1opr3-3, the increase in expression in opr2-1opr3-3 was statistically significantly higher (Fig. 3D-G). This finding implies that metabolites located downstream of cis-OPDA and upstream of JA in the OPR3-independent bypassing route (Fig. 1B) are potential candidates for the bioactive form of cis-OPDA. As a result, we performed UPLC-MS/MS analyses of the downstream metabolites of cis-OPDA in the canonical OPR3-dependent (OPC-4, Fig. 1A) and OPR3-independent routes (dn-cis-OPDA, tn-cis-OPDA, and 4,5-ddh-JA; Fig. 1B) using Col-0 and opr2-1opr3-3 (Fig. 5). For UPLC-MS/MS analysis, Col-0 and opr2-1opr3-3 were treated with cis-OPDA-d₅. Compared to Col-0, cis-OPDA-d₅-treatment enhanced the accumulation of 4,5-ddh-JA-d₅ and tn-cis-OPDA-d₅ in the opr2-1opr3-3 mutant but did not affect the level of dn-cis-OPDA-d₅ (Fig. 5A). The accumulation of tn-cis-OPDA-d₅ (ca.4200 pmol/g FW in opr2-1opr3-3 and ca. 2400 pmol/g FW in Col-0) and 4,5-ddh-JA-d₅ (ca.3700 pmol/g FW in opr2-1opr3-3 and ca. 1400 pmol/g FW in Col-0) was significantly higher than dn-cis-OPDA-d₅ (ca.400 pmol/g FW). In contrast, the accumulation of OPC-4-d₅, a downstream metabolite of the OPR3-dependent route, was below the detection limit in opr2-1opr3-3 (Fig. 5). Considering the enhanced marker gene expressions in opr2-1opr3-3 (Fig. 3C-G), downstream metabolites of cis-OPDA, such as tn-cis-OPDA and 4,5-ddh-JA are candidates of the genuine bioactive form of cis-OPDA because of the enhanced accumulation in opr2-1opr3-3.

The downstream metabolites of cis-OPDA were similarly effective as cis-OPDA on the expression of OPDA-marker genes

Next, we examined the effects of dn-cis-OPDA, tn-cis-OPDA, and 4,5-ddh-MeJA, the methyl ester of 4,5-ddh-JA (Fig. 6A and Scheme S2 and S3), on the expression of OPDA marker genes in A. thaliana. Tn-cis-OPDA and 4,5-ddh-MeJA were equally effective in upregulating expression of ZAT10, ERF5, DREB2A, ZAT12, and FIT1 in the opr2-1opr3-3 mutant (Fig. 6B, 6C, and S5A). Tn-cis-OPDA and 4,5-ddh-MeJA upregulated the expression of OPDA marker genes in a concentration-dependent manner (Fig. 6D and 6E). However, in this experiment, conversion from dn-cis-OPDA to tn-cis-OPDA or 4,5-ddh-JA and tn-cis-OPDA to 4,5-ddh-JA occurred (Fig. 5). Thus, it is necessary to consider the possibility that the bioactivity of dn-cis-OPDA and tn-cis-OPDA may depend on the in planta conversion to their downstream metabolites.

The downstream metabolites of cis-OPDA caused gene expression through their electrophilic property

Monte et al. reported that cis-OPDA and dn-cis-OPDA upregulate the expression of HSP genes in A. thaliana and M. polymorpha in a COI1-independent manner.³⁷ In addition, the upregulated gene expression depends on the electrophilic properties of cis-OPDA and dn-cis-OPDA because dn-iso-OPDA, which is an isomer of dn-cis-OPDA and less reactive as an electrophile, is significantly less effective than dn-cis-OPDA. Therefore, we compared the effects of tn-cis-OPDA and tn-iso-OPDA (Fig. 7A), 4,5-ddh-MeJA and 3,7-ddh-MeJA (Fig. 7A), corresponding to an iso-isomer of 4,5-ddh-MeJA, on the expression of ZAT10, ERF5, and HSP genes. The effect on the expression of DREB2A, ZAT12, and FIT1 genes was also investigated (Fig. S6). As shown in Fig. 7 and S6, tn-cis-OPDA and 4,5-ddh-MeJA upregulated the expression of ZAT10 (Fig. 7B), ERF5 (Fig. 7C), DREB2A (Fig. S5A), ZAT12 (Fig. S5B), and FIT1 (Fig. S5C) whereas tn-iso-OPDA and 3,7-ddh-MeJA did not. Similarly, cis-OPDA, tn-cis-OPDA, and 4,5-ddh-MeJA upregulated the expression of HSP, whereas tn-iso-OPDA and 3,7-ddh-MeJA did not (Fig. 7D-F). This suggests that the electrophilic nature of tn-cis-OPDA and 4,5-ddh-JA might be responsible for upregulating ZAT10, ERF5, DREB2A, ZAT12, FIT1, and HSPs. These results suggest their function as protein modifier in the plant cell.

Previous studies reported that cis-OPDA is a bioactive jasmonate in A. thaliana.^{14, 15, 16, 17, 18, 19, 20, 21} Two cis-OPDA derivatives such as cis-OPDA amino acid conjugates (cis-OPDA-AAs) including cis-OPDA-Ile (Fig. 1)^{32, 46, 47, 48} or modified OPDA (mo-OPDA)⁴⁹ which is an unidentified metabolites of cis-OPDA generated by dioxygenase JID1 are previously reported as possible bioactive form of cis-OPDA. However, cis-OPDA-AA was shown as a storage form of cis-OPDA and the genuine bioactive form of cis-OPDA-AA is cis-OPDA,⁵⁰ and mo-OPDA was identified as an artifact produced through Michael addition of cis-OPDA⁵¹. Thus, genuine bioactive form of cis-OPDA remains unknown.

Here, we reexamined the bioactivities of cis-OPDA by combining bioassays using the complete loss-of-function Arabidopsis mutant lines, coi1-1, and opr2-1opr3-3, and in vitro biochemical assays to assess the affinity between cis-OPDA and COI1-JAZs, and concluded that cis-OPDA is not the genuine bioactive form responsible for the expression of OPDA-marker genes. Our findings revealed that the downstream metabolites of cis-OPDA in the OPR3-independent biosynthetic route of JA-Ile (Fig. 1) are responsible for cis-OPDA-induced expression of OPDA-marker genes.

The finding that the expression of OPDA marker genes was suppressed in the cts1 mutant line,⁴⁴ in which cis-OPDA transport to peroxisomes was inhibited, strongly supports our conclusion (Fig. 4). This finding demonstrated that the downstream metabolites of cis-OPDA, and not cis-OPDA itself, were genuine bioactive forms. Next, we focused on the fact that cis-OPDA caused higher expression of ERF5, DREB2A, ZAT12, and FIT1 in opr2-1opr3-3 mutant compared to Col-0 (Fig. 3D-G). This result indicates that metabolites of cis-OPDA are responsible for the upregulation of these genes in opr2-1opr3-3. Chini et al. reported that the OPR3-independent JA biosynthetic pathway operates weakly in Col-0 and is predominant in opr2-1opr3-3/opr3-3 mutants.⁹ In opr2-1opr3-3, the downstream metabolites of the OPR3-independent route, tn-cis-OPDA and 4,5-ddh-JA, accumulated more than in Col-0, whereas little accumulation was observed for the downstream metabolites of cis-OPDA in the canonical OPR3-dependent route (Fig. 5). The accumulation of metabolites in opr3-3 was previously reported using deuterium-labeled α-linolenic acid,⁹ however, we used deuterium-labeled cis-OPDA-d₅ of naturally occurring stereochemistry to exclude the effect of metabolites from the branched metabolic routes of α-linolenic acid, such as hydroperoxide lyase/isomerase, epoxyalcohol synthase, divinyl ether synthase, and 9-LOX routes.^{4, 52} As a result, downstream metabolites of cis-OPDA in the OPR3-independent route are expected to be bioactive forms of cis-OPDA. Unfortunately, we could not identify one of them or all of them function as endogenous chemical signals. Identification of the genuine endogenous signal must await the target identification and subsequent evaluation of ligand-receptor binding affinity. Considering that UPLC-MS/MS analysis revealed that the accumulation of tn-cis-OPDA and 4,5-ddh-JA was increased in opr2-1opr3-3 compared to Col-0, while the accumulation of dn-cis-OPDA was unchanged (Fig. 5), suggesting that tn-cis-OPDA and 4,5-ddh-JA are plausible endogenous signaling molecule to induce enhanced expression of OPDA-marker genes in opr2-1opr3-3 (Fig. 3C-G). However, our current data do not allow us to completely rule out the possibility that dn-cis-OPDA functions as an endogenous chemical signal. Dn-cis-OPDA is converted into tn-cis-OPDA and then 4,5-ddh-JA through b-oxidation in the peroxisome and this conversion cannot be impaired because of the lack of a mutant line (Fig. 1). Thus, the information on the localization of these downstream metabolites will help the identification of genuine bioactive form of cis-OPDA. On this point, previous report have shown that the conversion of 4,5-ddh-JA to JA occurs in the cytosol by cytosol-localized OPR2 reductase, suggesting the localization of 4,5-ddh-JA in cytosol (Fig. 1).⁹ In addition, Mekkaoui et al. clearly demonstrated that 4,5-ddhJA is released from peroxisomes into the cytosol by using a trans-organellar complementation approach⁵³ (Mekkaoui et al. accompanying manuscript). A previous study demonstrated the difference in substrate specificity between OPR1 and OPR2: OPR1 could reduce cis-OPDA with low efficiency and could not reduce 4,5-ddh-JA, whereas OPR2 could not reduce cis-OPDA and could reduce 4,5-ddh-JA.⁹ Based on this difference, they expressed OPR1 or OPR2 linked to localization signal peptides targeted to cytosol, to the peroxisome, and to the mitochondria in opr2-1opr3-3 background. Major jasmonate responses, fertility and normal flower development, are impaired in opr2-1opr3-3,⁵⁴ thus, the restored biosynthesis of JA-Ile in these mutant lines will restore the fertility and normal flower development. Complementation experiments showed that OPR2 in the opr2-1opr3-3 background could restore fertility and normal flower development, suggesting that 4,5-ddh-JA might be the compound released from peroxisome and translocated between different cell organelles. In contrast, OPR1 in the opr2-1opr3-3 background could restore these traits when localized in peroxisomes, suggesting the plausible compartmentation of endogenously generated cis-OPDA in plastids and peroxisomes. This conclusion strongly reinforces our conclusions with cts1 mutant line (Fig. 4). Combining with our results, the exogenously applied cis-OPDA is either detoxified by conjugation to GSH⁵⁵ or rapidly converted to 4,5-ddh-JA of which level reaches to ca.3700 pmol / g FW, and released into cytosol to function as signaling molecule. Thus, it is plausible to conclude that the OPDA marker gene expressions by exogenously applied cis-OPDA, which have been reported in the previous reports, is attributed to the accumulation of the downstream metabolite, such as 4,5-ddh-JA.

Two independent approaches by us and Mekkaoui et al. reached the same conclusion that not cis-OPDA itself, but the downstream metabolite function as the genuine bioactive signal to induce the expressions of OPDA-marker genes. Mekkaoui et al. examined the wound-induced induction of some of cis-OPDA-marker genes in opr2-1opr3-3 mutant (Mekkaoui et al. accompanying manuscript), while our results are based on the experiments by exogenous application of cis-OPDA which have been discussed in previous studies on the JA-independent functions of cis-OPDA. Mekkaoui et al. also reported that all these OPDA-marker genes were also wound-induced in aos, in which OPDA and JA are deficient. Considering with our results, it is plausible to conclude that the application of electrophilic downstream metabolites of cis-OPDA, such as 4,5-ddh-JA, which are released into the cytosol, leads to the same JA-independent response as wounding itself. These responses may include oxidative stress, such as the production of reactive oxygen species (ROS)⁵⁶ and γ-aminobutyric acid (GABA)⁵⁷, which mediate at least part of the wound response, resulting in the induction of general stress-responsive genes. Mekkaoui et al. discussed that low level of endogenous 4,5-ddh-JA in the wounded opr2-1opr3-3 mutant (500 pmol/g FW⁹) is insufficient to induce the expression of marker genes. However, dn-cis-OPDA and tn-cis-OPDA, in addition to 4,5-ddh-JA, can function as similar signaling molecules, although it is unclear whether they are localized in the cytosol. Definitive conclusions can be made once the unidentified non-COI1 target involved in the expression of OPDA marker genes is identified.

In addition, we have another evidence suggesting that downstream metabolites of cis-OPDA is the genuine bioactive signal. Our findings indicated that the electrophilic reactivity of 4,5-ddh-JA and tn-cis-OPDA may be responsible for the expression of OPDA marker genes ZAT10 and ERF5. Monte et al. demonstrated that cis-OPDA and dn-cis-OPDA upregulate MpCOI1-independent expression of HSP genes in M. polymorpha through electrophilic reactivities.³⁷ They concluded that electrophilic reactivity is essential for HSP expression because it is not induced by dn-iso-OPDA, an isomer of dn-cis-OPDA with significantly lower electrophilic reactivity, due to the tetra-substituted a,b-unsaturated ketone moiety.^{37, 58} Similarly, tn-iso-OPDA and 3,7-ddh-JA, iso-isomers of tn-cis-OPDA and 4,5-ddh-JA with little electrophilic reactivity, did not induce the expression of OPDA maker genes (Fig. 7 and S6). These results suggest that these downstream metabolites of OPDA may act as protein modifiers through Michael addition.

Downstream metabolites of cis-OPDA function as chemical signals that cause the upregulation of OPDA marker genes independently of COI1 and MYCs (Fig. 2 and S4). COI1-independent gene upregulation may be attributed to a non-COI1-target, such as cyclophilin 20 − 3 and glutathione S-transferase 19, which are proposed putative targets of cis-OPDA (Fig. 1).^{18, 59} Further studies identifying the non-COI1-target will enable detailed studies of the unknown MOA of cis-OPDA.

In this study, we examined the effects of cis-OPDA on coi1-1 and opr2-1opr3-3 mutants in which JA signaling and biosynthesis were impaired. In addition, we demonstrated that cis-OPDA had no affinity for any of the functional COI1-JAZ co-receptor pairs. Combining the results of gene expression and UPLC-MS/MS analyses, downstream metabolites of cis-OPDA, especially 4,5-ddh-JA, is responsible for the expression of OPDA marker genes through a non-COI1 target.

Plant Materials

A. thaliana ecotype Col-0 and Ler-0 seeds were surface-sterilized in 5% sodium hypochlorite with 0.3% Tween 20 and vernalized for 2–3 d at 4 ℃. All seedlings were grown (118 µmol m^-2 s^-1) under a 16 h light/8 h dark cycle at 22 ℃ in a CL-301 growth chamber (Tomy Seiko Co., Ltd., Japan). Seedlings were grown in 1/2 Murashige and Skoog (MS) liquid medium. The mutant lines used in this study were coi1-1,³⁸ opr2-1opr3-3,⁹ myc2myc3myc4,⁴¹ and cts1 ⁴². The seeds of coi1-1 and opr2-1opr3-3 mutants were kindly gifted from Professor Roberto Solano and Dr. Andrea Chini (CNB-CSIC, Spain). The cts1 mutant was purchased from the Nottingham Arabidopsis Stock Centre (NASC). To select homozygous coi1-1, heterozygous coi1-1 seeds were geminated on a 1/2 MS plate containing 10 µM JA, and well-grown 4 d seedlings (homozygous coi1-1) were transferred to a 1/2 MS liquid medium. After 6 d of culture, the seedling was treated with each compound ((-)-JA, cis-OPDA, cis-OPDA-d₅, dn-cis-OPDA, tn-cis-OPDA, 4,5-ddh-MeJA, tn-iso-OPDA, 3,7-ddh-MeJA). For cts1, surface-sterilized seeds were pipetted onto 1/2 MS plate medium, and seed coats were disrupted using sterile tweezers to stimulate germination.⁴³

Chemical synthesis

Synthesis of (+)-cis-OPDA-d₅

(+)-12-(R)-Hydroxy-phytodienoic alcohol-d₅ was synthesized as described in the Supplementary Text. To a solution of the diol (+)-12-(R)-hydroxyphytodienoic alcohol-d₅ (32.7 mg, 0.11 mmol) in acetone (9.8 mL), Jones reagent (4.0 M solution) at − 20 ℃ was added until the orange color of the reagent persisted (8 drops). After 10 min of stirring at − 20 ℃, i-PrOH was added to quench the remaining reagent. AcOEt/n-hexane (1:1) and H₂O were then added, and the water layer was extracted with AcOEt. The combined organic layers were washed with saturated aqueous NaCl, dried over Na₂SO_4, and concentrated under reduced pressures. The residue was purified by medium-pressure chromatography (Isolera, eluent: 0.1:88:12 AcOH/n-hexane/EtOAc to 0.1:99.9 AcOH/EtOAc) to obtain cis-OPDA-d₅ (14.4 mg, 42%) as a colorless oil. [α]_D²³ +127.2 (c 0.34, CHCl₃). ¹H NMR (400 MHz, CDCl₃) δ_H: 7.74 (dd, J = 6.0, 2.8 Hz, 1H), 6.19 (dd, J = 6.0, 2.0 Hz, 1H), 5.32–5.45 (m, 2H), 2.93–3.02 (m, 1H), 2.40–2.56 (m, 2H), 2.36 (t, J = 7.8 Hz, 2H), 2.08–2.19 (m, 1H), 1.09–1.82 (m, 12H); ¹³C NMR (100 MHz, CDCl₃) δ_C: 211.1, 179.7, 167.3, 132.9, 132.5, 127.0, 49.8, 44.3, 34.0, 30.7, 29.6, 29.1, 28.9, 27.6, 24.6, 23.8, 19.3–20.3 (m), 12.4–13.3 (m); IR (neat) cm^–1: 2929, 2222, 1707, 1213; HRMS (ESI, negative) m/z [M-H]^- calcd for C₁₈H₂₂D₅O₃: 296.2279, found: 296.2268.

Synthesis of tn- cis -OPDA

(+)-8-(R)-hydroxyphytodienoyl alcohol was synthesized as described in the Supplementary Text. To a solution of (+)-8-(R)-hydroxyphytodienoyl alcohol (18.0 mg, 81.1 µmol) in acetone (7 mL), Jones reagent (4.0 M solution) was added at − 20 ℃ dropwise until the color of the reagent persisted (7 drops). i-PrOH and AcOEt/n-hexane (1:1) were then added to quench the remaining reagents. The mixture was extracted using EtOAc. The organic layer was washed with saturated aqueous NaCl, dried over Na₂SO₄, and then filtered. The reaction mixture was purified using medium-pressure chromatography (Isolera, eluent: 88:12:0.1 n-hexane/EtOAc/AcOH to 100:0.1 EtOAc/AcOH) to obtain tn-cis-OPDA as a colorless oil (18.5 mg, 96%). [α]_D²³ +207.2 (c 0.14, CHCl₃); ¹H NMR (400 MHz, CDCl₃) δ_H: 7.75 (dd, J = 5.8, 2.8 Hz, 1H), 6.21 (dd, J = 5.8, 1.8 Hz, 1H), 5.52–5.27 (m, 2H), 3.09–2.92 (m, 1H), 2.65–2.30 (m, 4H), 2.27–1.93 (m, 3H), 1.89–1.59 (m, 3H), 1.34–1.11 (m, 1H), 0.97 (t, J = 7.6 Hz, 3H); ¹³C NMR (100 MHz, CDCl₃) δ_C: 210.56, 178.52, 166.28, 133.21, 132.91, 126.71, 49.67, 44.04, 33.96, 30.19, 23.79, 22.76, 20.80, 14.01; IR (neat) cm^–1: 3050, 1732, 1702, 1109; HRMS (ESI negative) m/z [M-H]^- calcd for C₁₄H₁₉O₃: 235.1340, found: 235.1328.

Synthesis of 4,5-ddh-MeJA

(+)-Methyl 4,5-ddh-6-epi-cucurbate was synthesized as described in the Supplementary Text. To a solution of (+)-methyl 4,5-ddh-6-epi-cucurbate (9.8 mg, 43.7 µmol) in acetone (4.5 mL), Jones reagent (4.0 M solution) was added at − 20 ℃ until the orange color of the reagent persisted (16 drops). After 10 min of stirring at − 20 ℃, i-PrOH was added to quench the remaining reagent. Subsequently, n-hexane and H₂O were added, and the water layer was extracted with n-hexane. The combined organic layers were washed with saturated aqueous NaCl, dried over Na₂SO_4, and concentrated under reduced pressures. The residue was purified by medium-pressure chromatography (Isolera, eluent: 98:2 n-hexane/EtOAc to 80:20 n-hexane/EtOAc) to obtain 4,5-ddh-cis-MeJA (5.3 mg, 55%) as a colorless oil. Diastereomeric purity of 4,5-ddh-cis-MeJA was > 99% by ¹H NMR spectroscopy (δ_H = 7.71 (dd, J = 5.7, 2.7 Hz, 1H) for 4,5-ddh-cis-MeJA; 7.63 (dd, J = 5.7, 2.4 Hz, 1 H) for the trans isomer). [α]_D²⁴ +127.4 (c 0.26, CHCl₃). ¹H NMR (400 MHz, CDCl₃) δ_H: 7.71 (dd, J = 5.7, 2.7 Hz, 1H), 6.22 (dd, J = 5.7, 2.0 Hz, 1H), 5.45 (dtt, J = 10.9, 7.4, 1.8 Hz, 1H), 5.32 (dddt, J = 10.9, 6.8, 5.0, 1.4 Hz, 1H), 3.72 (s, 3H), 3.55–3.45 (m, 1H), 2.76 (dd, J = 16.4, 5.8 Hz, 1H), 2.57 (dt, J = 6.8, 5.0 Hz, 1H), 2.53 (brdt, J = 15.7, 5.0 Hz, 1H), 2.21 (dd, J = 16.4, 10.1 Hz, 1H), 2.13 (brdt, J = 15.7, 6.8 Hz, 1H), 2.05 (quintet, J = 7.4 Hz, 2H), 0.97 (t, J = 7.4 Hz, 3H); ¹³C NMR (100 MHz, CDCl₃) δ_C: 210.03, 172.34, 165.87, 133.66, 133.19, 126.17, 51.87, 48.32, 40.41, 34.44, 24.39, 20.79, 13.85; IR (film) cm^–1:2961, 1733, 1717, 1198; HRMS (ESI, positive) m/z [M + Na]⁺ calcd for C₁₃H₁₈NaO₃: 245.1154, found: 245.1148.

Gene expression analyses

Surface sterilized A. thaliana seeds were germinated (118 µmol m^-2 s^-1) under a 16 h light/8 h dark cycle at 22 ℃ in a CL-301 growth chamber (TOMY SEIKO Co., Ltd., Japan) after vernalization in the dark at 4 ℃ for 2 d. 10-day-old seedlings (5–6 seedlings/sample) grown in 1/2 MS liquid medium were treated with each compound ((-)-JA, cis-OPDA, dn-cis-OPDA, tn-cis-OPDA, 4,5-ddh-MeJA, tn-iso-OPDA, 3,7-ddh-MeJA). Based on the results of previously reported comprehensive DNA microarray analyses,¹⁶ we performed gene expression analyses by adding cis-OPDA to Arabidopsis WT (Col-0) and the coi1-1 mutant. Arabidopsis WT and coi1-1 plants were treated with (-)-JA (a mixture of cis and trans = 5/95; Figure. S1) or cis-OPDA, to examine their effects on the expression of three JA marker genes, OPR3, JAZ8, and MYC2, as well as cis-OPDA-specific marker genes, ZAT10, ERF5, DREB2A, ZAT12, and FIT1.^{16, 32} Each sample was frozen after treatment for 30 min, and total RNA was extracted using ISOGEN (NIPPON GENE, Japan). First-strand cDNA was obtained using ReverTra Ace^® reverse transcriptase (TOYOBO, Japan) with oligo-dT primers. A StepOnePlus Real-Time PCR System (Life Technologies, USA) was used for quantitative PCR (qPCR). PCR conditions were followed: an initial hold at 95 ℃ for 30 s, followed by a two-step PCR program of 95 ℃ for 5 s and 60 ℃ for 30 s for 40 cycles. Primer sequences used in this study are listed in Supplementary Table S1. Polyubiquitin 10 was used as the reference gene.

UPLC-MS/MS analysis of cis-OPDA metabolites

Surface sterilized A. thaliana seeds were germinated (118 µmol m^-2 s^-1) under a 16 h light/8 h dark cycle at 22 ℃ in a CL-301 growth chamber (TOMY SEIKO Co., Ltd., Japan) after vernalization in the dark at 4 ℃ for 2 d. 10-day-old seedlings (5 seedlings/sample) grown on 1/2 MS liquid medium were treated with cis-OPDA-d₅ (30 µM). The frozen plant materials were homogenized and extracted using 600 µL of ice-cold (pre-cooled at − 20 ℃) 25% MeOH/H₂O. The samples were sonicated for 10 min and extracted for 30 min at 4 ℃ using a rotator. After centrifugation at 15,000 g for 15 min, supernatants were collected. The pooled supernatants were purified by one-step reversed-phase polymer-based solid-phase extraction using Oasis^® HLB cartridges (Waters, USA). Before sample loading, the SPE sorbent was conditioned with 1 mL of 100% MeOH and equilibrated with 1 mL of 0.1% HCOOH/H₂O (v/v). After the samples had been loaded onto the Oasis^® HLB column, interfering compounds were removed by washing with 1 mL of 25% MeOH/H₂O, following which the pre-concentrated analytes on the sorbent were eluted with 2 mL of 50% MeOH/H₂O and 2 mL 100% MeOH. The eluent solution was evaporated to dryness at 30 ℃ under reduced pressure through freeze-drying. The samples were then resuspended in 100 µL of 100% MeOH.

The samples were examined by UPLC-MS/MS using a TripleTOF 5600 system (AB SCIEX, USA) operating in the negative mode. Liquid chromatography separation was performed with an Eclipse Plus C18 RRHD 1.8 µm (Φ1.05 × 50 mm, Waters, USA) at 35 ℃ and a flow rate of 0.3 mL/min. The elution was performed using a gradient of water (solvent A) and MeOH (solvent B), both containing 0.1% formic acid (v/v). The proportion of solvent B in the eluent was increased linearly from 10–60% for 2 min and from 60–90% for 8 min of the elution phase, followed by maintaining a flow of 90% B for 1 min. The column was re-equilibrated with 10% solvent B for 3 min.

Pull-down assay

For the pull-down experiments using fluorescein-tagged JAZ peptides (Fl-AtJAZPs), purified GST-AtCOI1 (5 nM), Fl-AtJAZP (10 nM), and each compound (JA-Ile or cis-OPDA) in 350 µL of incubation buffer (50 mM Tris-HCl buffer, pH 7.8, 100 mM NaCl, 10% glycerol, 0.1% Tween20, 100 nM inositol-1,2,4,5,6-pentakisphosphate (IP5)) were combined with anti-fluorescein antibody (0.2 µL, GeneTex, GTX26644, USA), and incubated for 10–15 h at 4 ℃ with rotation. After incubation, the samples were combined with SureBeads^™ Protein G (10 µL in 50% incubation buffer slurry; Bio-Rad, Hercules, CA, USA). After 3 h of incubation at 4 ℃ with rotation, the samples were washed three times with 350 µL of wash buffer (phosphate-buffered saline containing 0.1% Tween 20). The washed beads were resuspended in 35 µL of SDS-PAGE loading buffer containing dithiothreitol (DTT, 100 mM). After heating for 10 min at 60 ℃, the samples were subjected to SDS-PAGE and analyzed by western blotting. The bound GST-COI1 protein was detected using an anti-GST HRP conjugate (RPN1236, GE Healthcare, USA, 5,000-fold dilution in blocking buffer (Nakalai Tesque, Inc., Japan)).

Statistical analyses

Samples were analyzed in triplicate, and the data are presented as the mean ± standard deviation (SD). An analysis of variance (ANOVA) was used for data analysis. Different letters within a column indicate statistically significant differences by the Tukey-Kramer multiple range test (p < 0.05, CoStat version 6.400). A Student’s t-test was performed to examine differences between the two groups.

Acknowledgments

Seeds of the opr2-1opr3-3 double mutant and coi1-1 mutant were provided by Professor Roberto Solano and Dr. Andrea Chini (CNB-CSIC, Spain). We thank to Dr. Yousuke Takaoka and Ms. Misuzu Nakayama (Tohoku University) for their technical assistance. And we also thank to Prof. Bettina Hause (Leibniz Institute for Plant Biochemistry, Germany) for valuable discussion on our manuscript.

Funding

This work was financially supported by Grants-in-Aid for Scientific Research from JSPS, Japan (Nos. 23H00316, 23K17967, 22KK0076, 21K19037, 20H00402, JPJSBP120229905, and JPJSBP120239903 to MU) and a Grant-in-Aid for Transformative Research Areas (A) “Latent Chemical Space” [JP23H04880 and JP23H04883] for MU from the Ministry of Education, Culture, Sports, Science and Technology, Japan.

Author contributions

Conceptualization, M.U.; methodology, M.U., N.K.; validation, M.U. and N.K..; formal analysis, R.S., Y.N., N.K., T.K.; investigation, R.S., Y.N., N.K., T.K.; resources, R.S., Y.N., N.K., T.K.; data curation, M.U. and N. K.; writing—original draft preparation, M.U.; writing—review and editing, M.U., N.K., Y.N., R.S.; visualization, Y.N., R.S., N.K.; supervision, M.U.; project administration, M.U.; funding acquisition, M.U. All authors have read and agreed to the published version of the manuscript.

Competing interests

The authors declare that they have no competing interests.

Data availability statement

All data needed to evaluate the conclusions in the paper are present in the paper or the Supplementary Materials.

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Downstream metabolites of (+)-cis-12-oxo-phytodienoic acid function as noncanonical bioactive jasmonates in Arabidopsis thaliana.

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Synthesis of 4,5-ddh-MeJA

Gene expression analyses

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