Cytochrome P450 enzymes in the Arabidopsis indole-3-acetonitrile metabolic network: Biological functions and protein-protein interactions

• Applicant: Professor Dr. Erich Glawischnig
• Subject Area: Plant Biochemistry and Biophysics ; Plant Genetics and Genomics
• Term: from 2015 to 2020
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 281071237

• Final Report Year: 2020

Final Report Abstract

Characteristic for cruciferous plants is the synthesis of a complex array of defence-related indolic compounds. In Arabidopsis, these include indole-3-carbaldehyde (ICHO) / indole-3-carboxylic acid (ICOOH) derivatives and camalexin. Key enzymes in the biosynthesis of the inducible metabolites are the cytochrome P450 enzymes CYP71A12, CYP71A13 and CYP71B6 and Arabidopsis Aldehyde Oxidase 1 (AAO1). CYP71A12-specific TALE nucleases (TALENs) were introduced into the cyp71a13 background, multiple mutants were generated and their metabolic phenotypes were analysed. Most strikingly, ICOOH and ICHO derivatives synthesized in response to UV exposure were not metabolically related. For AAO1 we suggest two independent biological functions in glucosinolate degradation and in vitamin B6 metabolism. Camalexin is synthesized efficiently without release of reactive intermediates, suggesting channelling of the biosynthetic pathway involving an enzyme complex. By a comprehensive analysis, involving untargeted and targeted coimmunoprecipitation, FRET-FLIM, and the analysis of enzyme characteristics, it was demonstrated that indeed the camalexin-biosynthetic enzymes form a metabolon. The glutathione transferase GSTU4, which is co-expressed with tryptophan- and camalexin-specific enzymes, was physically recruited to the complex. Surprisingly, camalexin concentrations were elevated in knock-out and reduced in GSTU4 overexpressing plants. This shows that GSTU4 is not directly involved in camalexin biosynthesis but rather has a role in a competing mechanism. Extending our genome editing work to other cytochrome P450 genes in Arabidopsis, we have been focusing on an 83 kb-cluster (CYP71B37 to CYP71B16) of the CYP71B-family. Applying CRISPR/Cas9 technology, this large gene cluster was successfully stably deleted from the genome and phenotyping of these plants is in progress. It is proposed that CRISPR/Cas9-based genome editing is a powerful tool to study the unrevealed functions of clusters of redundant genes.

Publications

• (2015) TALEN- mediated generation and metabolic analysis of camalexin-deficient cyp71a12 cyp71a13 double knockout lines. Plant Physiology 168: 849-858
  Müller TM, Böttcher C, Morbitzer R, Götz CC, Lehmann J, Lahaye T, Glawischnig E
  
• (2019) Dissection of the network of indolic defence compounds in Arabidopsis thaliana by multiple mutant analysis. Phytochemistry, 161:11-20
  Müller T, Böttcher C, Glawischnig E
  
• (2019) The formation of a camalexinbiosynthetic metabolon. Plant Cell, Nov;31(11):2697-2710
  Mucha S, Heinzlmeir S, Kriechbaumer V, Strickland B, Kirchhelle C, Choudhary M, Kowalski N, Eichmann R, Hückelhoven R, Grill E, Kuster B, Glawischnig E
  
• (2020) The role of CYP71A12 monooxygenase in pathogentriggered tryptophan metabolism and Arabidopsis immunity. New Phytol. Jan;225(1):400-412
  Pastorczyk M, Kosaka A, Piślewska-Bednarek M, López G, Frerigmann H, Kułak K, Glawischnig E, Molina A, Takano Y, Bednarek P