Madita Sophie Knieper (University Bielefeld)
CYP20-3, a peptidyl-prolyl-cis/trans-isomerase, is an important regulatory hub in the redox-regulatory network of chloroplasts, controlling for example thiol synthesis and detoxification of ROS. The oxylipin 12-oxophytodienoic acid, which is generated from α-linolenic acid, has been shown to exert a redox-dependent regulatory effect on the activity of CYP20-3. Protein model structures were generated using SWISS MODEL based on the templates 1f8n.1.A (LOX2), 1zvc.1.A (AOC2), 3.cli.1.A (AOS), 4vrf.1.A (Cyp20-3), 6jvu.1.A (SAT1) and 5zte.1.A (2-CP). Graph: Knieper
Madita Sophie Knieper's Master thesis was awarded with the Prize for the Best Plant Science Master Thesis, which was carried out at Bielefeld University in the year 2021
Title: The role of cyclophilin CYP20-3 in the redox regulatory network of chloroplasts
A redox-dependent modulation of the PPIase activity of cyclophilin CYP20-3, an important regulatory hub in the redox network of chloroplasts, by the oxylipin 12-oxophytodienoic acid was determined, implying a tight coupling of different stresses (e.g. wounding) and defense mechanisms such as enhanced thiol synthesis.
The chloroplast is a metabolically active compartment of plant cells and, in addition, acts as sensor and transducer of environmental cues, e.g. by coordinating redox and diverse hormonal signaling pathways. The cyclophilin CYP20-3 is localized in the chloroplast stroma and displays at least four functions as target of thiol redox regulation, binding partner of 2-cysteine peroxiredoxin, receptor of oxylipins and regulator of the cysteine synthase complex. Thereby, CYP20-3 plays a major role in plant responses to oxidative stress.
The α-β-unsaturated cyclic ketone 12-oxophytodienoic acid (12-OPDA) is synthesized by a short enzyme cascade in the chloroplast, combining 13-lipoxygenases, allene oxide synthase and allene oxide cyclase starting with α-linolenic acid as substrate. 12-OPDA synthesis is strongly stimulated in high light, upon wounding and various stresses. 12-OPDA is independent regulator and in addition precursor of jasmonic acid.
To deeper explore the regulatory role of 12-OPDA on the CYP20-3 activity, 12-OPDA was synthesized with recombinantly produced enzymes and purified via rp-HPLC. CYP20-3 wild type protein and two cysteineàserine variants were incubated with 12-OPDA, and the peptidyl prolyl cis/trans isomerase activity measured photometrically. Surprisingly, the incubation with 12-OPDA had an ambivalent effect on the catalytic activity of CYP20-3. 12-OPDA inhibited reduced CYP20-3 but activated oxidized protein. This pattern was observed for wild type CYP20-3 and its cysteineàserine variant (C129S), whereas the activity of the variant C176S was unaffected by 12-ODPA. It is concluded that the cysteinyl residue 176 is essential for binding 12-OPDA to CYP20-3.
These and additional results from the thesis deepen our understanding of the crosstalk between 12-OPDA and thiol redox regulation using the example CYP20-3. CYP20-3 is a node of signal integration under diverse stress conditions.
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Madita Sophie Knieper conducted this work at the faculty of biology of Bielefeld University in the research group of Prof. Dr. Karl-Josef Dietz.