OPDA-Ile – a new JA-Ile-independent signal?

ABSTRACT

Expression takes place for most of the jasmonic acid (JA)-induced genes in a COI1-dependent manner via perception of its conjugate JA-Ile in the SCF^COI1-JAZ co-receptor complex. There are, however, numerous genes and processes, which are preferentially induced COI1-independently by the precursor of JA, 12-oxo-phytodienoic acid (OPDA). After recent identification of the Ile-conjugate of OPDA, OPDA-Ile, biological activity of this compound could be unequivocally proven in terms of gene expression. Any interference of OPDA, JA, or JA-Ile in OPDA-Ile-induced gene expression could be excluded by using different genetic background. The data suggest individual signaling properties of OPDA-Ile. Future studies for analysis of an SCF^COI1-JAZ co-receptor-independent route of signaling are proposed.

Ten years ago, the new family of JASMONATE ZIM DOMAIN (JAZ) proteins with 12 members was occasionally identified in Arabidopsis in 3 different labs.^1-3 This was a breakthrough in JA research, since JAZ proteins were characterized as repressors of JA-Ile-induced gene expression. With identification of these proteins, it became clear that JA-Ile is perceived by the SCF^COI1-JAZ co-receptor complex. Components of this complex are the F-box protein called CORONATINE INSENSITIVE 1 (COI1), the Skp1/Cullin/complex with E3 ubiquitin ligase activity and the targets of the F-box protein, the JAZ proteins. JAZ proteins repress in the absence of JA-Ile positively acting transcription factors (TFs), such as MYC2, which bind to JA responsive elements (G-boxes) in the promoters of JA-Ile-regulated genes. Upon stress or developmentally regulated processes, an increase in JA-Ile level occurs leading to its binding in the SCF^COI1-JAZ co-receptor complex followed by proteasomal degradation of JAZ. Consequently, the free, positively acting MYC2 can switch on JA-Ile-responsive gene expression.⁴ This basic scenario could be explained mechanistically upon crystallization of the complex, where an inositol-pentakisphosphate (IP₅) activates JA-Ile binding.⁵ Interaction of the F-box protein COI1 and one of the JAZ proteins requires the C-terminal jas domain of JAZ.⁶ This interaction is promoted by JA-Ile. Among the various stereo-isomeric forms of JA-Ile, the (+)-7-iso-JA-Ile has the highest activity.⁷ JA-Ile perception via the SCF^COI1-JAZ-co-receptor complex was confirmed in the last couple of years including identification of new co-repressors and adaptors (for reviews, see refs.^8-10).

There is, however, one open question due to the structural specificity in binding of the ligand JA-Ile. Among 40 different jasmonate compounds the stereoisomer (+)-7-iso-JA-Ile, carrying a ring conformation similar to coronatine, was most active.⁷ Even coronatine was 10-fold more active then JA-Ile, although this bacterial toxin does not occur in plants. For the methyl ester of JA, for 12-oxo-phytodienoic acid (OPDA, the precursor of JA biosynthesis), and for OPDA-Ile (Fig. 1) no detectable promotion of JAZ3-COI1 interaction was recorded.⁷ Furthermore, in initial interaction experiments with COI1 and JAZ1 or JAZ3, no activity of OPDA was recorded.^2,11 Modeling studies excluded interaction of OPDA with the complex.¹² Alternatives were discussed such as (i) a repertoire of different receptor properties with individual JAZ proteins,^4,13 (ii) an unidentified COI1 substrate¹⁴ or (iii) promotion of COI1-JAZ interaction by non-conjugated jasmonates.⁶ Most of these alternatives were excluded in subsequent studies.

Figure 1.

Biosynthesis of JA-Ile and OPDA-Ile and mutants used in the study by Arnold et al. (2016).

There are, however, numerous OPDA-specific responses suggesting OPDA perception via an alternative route. This route might be a COI1-independent pathway. Indeed, there is an increasing number of COI-independent processes (for reviews, see refs.^15,16). Among such processes in Arabidopsis thaliana are seed germination,¹⁷ glutathione conjugation,¹⁸ drought stress responses,¹⁹ and other plant defense reactions.²⁰ Additionally, seed formation in tomato is regulated by OPDA.²¹ Stress responses in JA-deficient but OPDA-containing lower plant species, such as Marchantia polymorpha,²² Selaginella martensii²³ and Physcomitrella patens,²⁴ point to a signaling function of OPDA. These data correspond to a proteomic analysis of about 6.000 proteins in A. thaliana showing that nearly 5% of the wound-inducible proteins are synthesized JA-independently, that means they are induced COI1-independently.²⁵ In a transcriptomic analysis following OPDA-treatment more than 150 genes were found to be preferentially up-regulated by OPDA.²⁶ Such transcriptomics data, indicating an OPDA-specificity, as well as proteomics data on OPDA-specific upregulated proteins, have been subsequently collected several times.^18,27,28

The reason for inactivity of OPDA in promoting interaction of COI1-JAZ1 or COI1-JAZ3^2,11 could be that – similar to JA – the conjugation with Ile is required. In subsequent interaction studies, however, of COI1 and JAZ1 or JAZ3, also OPDA-Ile did not induce the interaction.⁷ In such interaction studies, however, the detection limit might hamper a positive result. Since OPDA-Ile has been recently identified as a naturally occurring compound in wounded leaves of flowering Arabidopsis plants,²⁹ the question came up again on its putative biological activity.

Tests on biological activity of OPDA-Ile were done recently in terms of gene expression by comparing expression of OPDA-induced genes upon treatment with OPDA and OPDA-Ile using 10-days-old seedlings or leaves of 6-week-old flowering plants.³⁰ Expression analyses were performed with the signaling mutant coi1, the JA biosynthesis mutant opr3 affected in the OPDA reductase 3, and the JA-Ile deficient mutant jar1 affected in the JA-Ile synthetase as well as the corresponding wild types Columbia-5 (Col-5), Wassilewskija (WS) and Col-0, respectively. Using deuterated OPDA-Ile sufficient uptake was shown, and putative cleavage of OPDA-Ile during application could be excluded. The opr3 mutant has been often used to detect OPDA specific responses, but identification of an intronic T-DNA insertion showed conditional JA formation.³¹ Therefore, JA-deficiency of opr3 mutant has to be checked for each conditions used. This was done in the above mentioned OPDA/OPDA-Ile treatment experiments by determination of endogenous levels of OPDA, JA and JA-Ile and indicated that opr3 mutant is a reliable control for OPDA-specific responses under the conditions used. Furthermore, marginal conversion of OPDA into OPDA-Ile in WS was shown. To distinguish biological activity of OPDA and OPDA-Ile from the dominant activity of JA-Ile, lack of JA-Ile formation upon treatment with OPDA and OPDA-Ile, respectively, in the opr3 and jar1 mutant background was an absolute prerequisite. Indeed, here JA-Ile formation could be excluded.

Based on these prerequisites, expression of 2 genes, GRX480 and ZAT10 was recorded. ZAT10 encodes a salt-tolerance zinc-finger protein that acts as transcription factor in response of plants to various abiotic stresses.²⁶ GRX480 encodes a GLUTAREDOXIN and is regarded as candidate for an OPDA-associated transcription regulator.¹⁸ Both genes were previously shown to be OPDA-inducible,^18,26 indicating their role as a putative marker for OPDA responses. COI1-independent expression was confirmed for GRX480, since tests with the coi1-16 mutant showed an OPDA-inducible expression of GRX480. The comprehensive expression analysis for GRX480 and ZAT10 showed their clear induction by both, OPDA and OPDA-Ile treatment in all genotypes. Since neither OPDA treatment resulted in enhanced OPDA-Ile levels nor OPDA-Ile was cleaved to OPDA, both compounds acted independently in application experiments.

Preferential expression of genes upon OPDA treatment such as ZAT10 or GRX480 together with about 150 genes have been postulated repeatedly,^18,26,28 and also proteomic approaches showed OPDA-specific responses.²⁷ In all these experiments, however, putative formation of JA upon OPDA treatment was not excluded. In the recent publication summarized above,³⁰ use of different genetic background such as the signaling mutant coi1, the JA biosynthesis mutant opr3 and the JA-Ile deficient mutant jar1, compared to the corresponding wild types, as well as analytical controls by determination of endogenous levels of OPDA, OPDA-Ile, JA and JA-Ile allowed an unequivocal proof for biological activity of OPDA-Ile occurring independently of OPDA, JA or JA-Ile. Due to the minor levels detected so far in late flowering plants of Arabidopsis,²⁹ there seems to be only a minor biological relevance for this compound. But the new property of OPDA-Ile suggests a screening on occurrence of OPDA-Ile in different plant species or various organs and tissues. Such screening is supported by the great differences of up to 3 orders of magnitude among different jasmonate derivatives such as hydroxylated, glucosylated or sulfated compounds detected in various plant species and organs.³²

Furthermore, the so far completely unknown mechanism of OPDA/OPDA-Ile perception suggests that new studies are worthwhile on an alternative route to the well-established JA-Ile perception via the SCF^COI1-JAZ co-receptor complex. Such studies might involve (i) screening of tissues and species on elevated levels of OPDA-Ile due to higher probability of its action, and (ii) a genetic screen on a new SCF-receptor complex.

Disclosure of potential conflicts of interest

No potential conflicts of interest were disclosed.

Funding

This work was supported by the Ministry of Education, Youth and Sports of the Czech Republic under Grant No. LO1204, by the Palacký University Olomouc, Czech Republic, program Interhaná under Grant No. CZ.1.07/2.3.00/20.0165, and by the Czech Science Foundation under Grant No. GA14-34792 S.