Regulation der Jasmonsäure-Synthese durch reversible Dimerisierung der Oxophytodiensäurereduktase 3 (OPR3) (Volume 1) (English shop)

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SUMMARY

Jasmonic acid (JA) is a phytohormone which plays an important role both in the regulation of abiotic and biotic stress responses and in various plant developmental processes. 12-oxophytodienoic acid reductase 3 (OPR3) is substantially involved in JA biosynthesis and catalyzes a decisive step in peroxisomes: the conversion of the JA precursor 12-oxophytodienoic acid (OPDA) to OPC-8:0. After wounding or herbivory, JA and its bioactive form JA-Ile accumulate within a few minutes. This rapid and transient increase could be explained by posttranslational activation of the JA biosynthetic enzymes. The observation that OPR3 crystallizes as a self-inhibited dimer led to the hypothesis that the rapid JA increase is enabled by reversible dimerization of OPR3 and that its regulation could occur via phosphorylation/dephosphorylation. It is assumed that the inactive form of OPR3 represents a homodimer that is stabilized by phosphorylation. Furthermore, it is assumed that in response to a stimulus this dimer is converted into an active monomer by posttranslational modification (e. g. dephosphorylation at position Y365) and subsequently, when no longer needed, dimerizes again through phosphorylation.

In this work, dimerization of OPR3 from Arabidopsis thaliana could be observed both in onion epidermal cells and in different organs of Arabidopsis by means of two fluorophore-based methods (BiFC and FRET). OPR3 variants (AtOPR3E292K and AtOPR3Y365F) showed a reduced dimerization potential. Thus, for the first time, homodimerization of OPR3 in vivo and the importance of individual amino acids for dimerization were demonstrated. The hypothesis of reversible OPR3 dimerization and activation of JA synthesis through formation of the OPR3 monomer could be substantiated by two crucial observations. Both after wounding and during the early phases of anther development, in which plants require JA, the monomer/dimer equilibrium of OPR3 shifted toward the monomer. That a posttranslational modification is necessary for OPR3 dimerization is in agreement with the observation that OPR3 is unable to dimerize when expressed in S. cerevisiae. Apparently, dimerization requires additional factors that are not present in yeast cells. A protein kinase responsible for this posttranslational modification of OPR3 was not identified in this work. However, the detection of OPR3 dimerization in the cytosol led to the conclusion that the kinase does not necessarily have to be localized in peroxisomes and that the search can be extended to cytosolically localized protein kinases.

SUMMARY

Jasmonic acid (JA) is a phytohormone which plays an important regulatory role in abiotic and biotic stress responses as well as in several plant developmental processes. 12-oxophytodienoic acid reductase 3 (OPR3) contributes to jasmonic acid biosynthesis and catalyzes a critical step in peroxisomes: the conversion of the jasmonic acid precursor 12-oxophytodienoic acid (OPDA) to OPC-8:0.

After wounding or herbivory JA and its bioactive form JA-Ile accumulate within a few minutes. This fast and transient increase can be explained by posttranslational activation of biosynthetic enzymes. The observation that OPR3 crystallizes as a self-inhibited dimer led to the hypothesis that the rapid JA burst is mediated by reversible dimerization of OPR3, which may be regulated by phosphorylation and dephosphorylation of the enzyme. It is postulated that the inactive form of OPR3 represents a homodimer, stabilized by phosphorylation. Furthermore it is assumed that in response to a stimulus this dimer dissociates into active monomers by posttranslational modification (e. g. dephosphorylation at position Y365) and, when not needed anymore, dimerizes again after re-phosphorylation.

In this work dimerization of OPR3 from Arabidopsis thaliana could be observed in vivo in onion epidermal cells as well as in different organs of Arabidopsis by means of two fluorophor-based methods (BiFC and FRET). OPR3 variants (AtOPR3E292K and AtOPR3Y365F) showed a reduced dimerization potential in vivo, demonstrating the importance of these two residues. Thus, for the first time homodimerization of OPR3 and the importance of specific amino acids for dimerization were demonstrated in vivo. The hypothesis of reversible OPR3 dimerization, and activation of JA biosynthesis via formation of the OPR3 monomer, could be confirmed by two crucial observations. Both after wounding and during early phases of anther development, when plants need JA, the monomer/dimer equilibrium of OPR3 shifted toward the monomer.

The assumption that OPR3 dimerization requires posttranslational modification was strengthened by the inability of OPR3 to dimerize when expressed in S. cerevisiae. Apparently, additional factors are needed for dimerization that are absent from yeast. A protein kinase responsible for posttranslational OPR3 modification, could not be identified in this thesis. However, detection OPR3 dimerization in the cytosol, led to the conclusion that the kinase is not necessarily localized in peroxisomes and that the search needs to be extended to include cytosolic protein kinases.