Exploration of novel phytochemicals using mammalian hepatoma cells Regulation of glucosinolate biosynthesis and beyond R2R3 MYBs regulate the biosynthesis of aliphatic and indolic glucosinolates Incoming signal: touch, wounding, selective activation by MeJa, SA, ABA, glucose…. Activation of MYB target genes Activation of glucosinolate biosynthesis genes by MYBs DHS1 ASA1 TSB1 CYP79B2/ CYP79B3 CYP83B1 UGT74B1 C-S lyase AtST5a Induction of TFs: MYB51, MYB122, MYB34 MYB28… 35S:MYB28 I3M 4MOI3M 8MSOO 5MSOP 4MSOB 3MSOP 4MTB 4- Methylpenthyl GLS 5- Methylhexyl GLS Met-GS Trp- GS Leu-GS MYB51 MYB122 MYB34 MYB29 MYB76 MYB28 Protein-protein interaction in the regulation of GS Yeast two hybrid assay At4g19700 bHLH-HFs At3g49570 At3g45900 At4g26930 At1g79280 MYBs:bHLH-HFs bHLH-HF1 bHLH-HF2 bHLH-HF3 bHLH-HF4 Transient expression in N. bent. MYB:SPYNE; bHLH-HF:SPYCE mustard oil Plastidic transporters in GS biosynthesis Intercompartmental metabolite signalling Increased resistance toward plant enemies mustard oil Increased production of gluocosinolates Pull-down experiment demonstrating an interaction of bHLH-HFs with R2R3 MYBs Glucosinolate levels are strongly diminished in the double and triple bhlh-hf1/hf2/hf3 mutants R2R3 MYBs bHLH-HFs Are MYBs regulated posttranslationally? Are they phosphorylated or ubiquitinated ? Do biotic or other stimuli affect this process? How phosphorylation of MYBs affect: - DNA binding activity? - trans-activation potential? - sub-cellular localisation? MYB51-GFP active MYB51-GFP inactive Post-translational regulation of R2R3 MYBs Induction of EpRE:TK:GFP In human hepatoma cells Induction of EpRE:GST:LUX in murine hepatome cells Induction of Phase II Detox. Enzyme activity: QR Screening of extracts of 5000 activation tagged lines for chemoprotective activities SO 4 2- Aldoxime S-Alkyl-thiohydroximate Aci-Nitro-compound Desulfo-glucosinolate Glucosinolate CYP79F1/F2 CYP83A1 C-S lyase AtST5b/c UGT74C1 Methionine a-Keto acid MAM BCAT4 Chain-elongated Met 2-Malat derivative 3-Malat derivative a-Keto acid BCAT3 IMDH IPMI GSH-conjugate GST thiohydroximate GPP +GSH Cytoplasm Vacuole PAPS APS sulfate APK ATPS 2 CYTOPLASMA CHLOROPLAST SO 4 2 - SO 4 2- APS SO 3 2- S 2- Cystein Methionine PAPS APK1 APK2 Sulfation PAPS + AtSt5 a,b, c glucosinolates desulfoglucosinolates OH PAP PAP + A model for the PAPS/PAP antiport in A.thaliana A.The GFP-fusion protein of PAPS/PAP antiporter is localised in envelops; B. The double homozygous A.thaliana knock-out mutant is lethal B A Plastid Mitochond. PAP Nucleus PAP XRN2/3 De-repression of nuclear-encoded stress-responsive genes A model for the intercompartmental PAP signalling in A.thaliana SAL1 FRY1 ALX8 AMP + Pi PAP Gene silencing PAPS APS PAP sulfate APK1 APK2 ATPS3 ATPS1 PAPS PAP AMP + Pi Cytosol Plastid SO 3 2- S 2- Cystein Mitochondrium ATP, ADP PAPT1 PAPT2 XRN2/3 Nucleus SAL1 FRY1 ALX8 A prediction for the subcellular localisation of FRY1 A PAP level is increased in fry1 knock-out mutant fry1 Col-0 Appearance of fry1 knock-out mutant In comparison to wild-type, fry1 is retarded in growth, reveals delayed bolting time, but also increased resistance to high light and drought stresses fry1 Col-0 G l u c o s i n o l a t e s pyk10-1D metabolic profiling Generation and analysis of recapitulation lines 1MOI3M Isolation and analysis of homozygous knock-out line substrate? glycosilated compounds Metabolite profiling of pyk10-1D (51.14) mutant Using UPLC-ESI-QTOF PYK10 700 x T-DNA insertion in 51.14 caused an activation of a PYK10 gene resulting in pym10-1D EpRE EpRE TK/GST LUX/GFP NOVEL PHYTOCHEMICALS Nrf2 Nucleus 3MSOP 4MSOB 5MSOP 8MSOO I3M 4MOI3M 1MOI3M µmol/g TG 0,0 0,5 1,0 1,5 5,0 7,5 10,0 Col-0 bhlh-hf1/2 bhlh-hf1/2/3 nd * nd nd nd nd nd * * * * * * * nd BHLH-HF1 BHLH-HF2 BHLH-HF3 BHLH-HF4 Relative luciferase activity (%) 0 5 10 15 20 25 RLuc-MYB51+ProtA-BHLH(x) RLuc-leer+ProtA-BHLH(x) RLuc-MYB51+ProtA-leer