1. On the move to field (5 min) Lukáš Spíchal Laboratory of Growth Regulators, IEB AS CR & UP Olomouc, Czech Republic Centre of the Region Haná for Biotechnological and Agricultural Research, Olomouc, Czech Republic 1,2 1 2

2. Laboratory of Growth Regulators, IEB AS CR & UP Olomouc : Miroslav Strnad Marek Zatloukal Lucie Szüčová Václav Mik Karel Doležal Markéta Gemrotová Jana Klásková Jaroslav Nisler Jiří Grúz Ondřej Novák Jaromír Mikulík Radoslav Koprna Department of Biochemistry, PřF UP Olomouc: Petr Galuszka David Kopečný Laboratory of Genome Integrity, UP Olomouc & BioPatterns, Ltd., Olomouc: Martin Mistrík Institute of Biology/Applied Genetics, FU Berlin: Tomáš Werner Thomas Schmülling Acknowledgements Grant Support: MSM 6198959216, LC06034, ED0007/01/01, GA522/08/0920 organic synthesis biochemistry/biology analytical chemistry field experiments boss

3. Development of CK-derived PGRs Organic synthesis Testing in CK bioassays Interaction with CK metabolism/perception In vivo applications (temporal/spatial specific) In vivo applications in field conditions SAR Phenotype Gene profiling Hormone levels Testing of toxicity TARGETED SYNTHESIS

4. What to change to gain… BA Cytokinin antagonist CKX inhibitor Spíchal et al., 2009 Nisler et al., 2010 Zatloukal et al., 2008 Spíchal et al., submitted

5. Development of plant growth regulators at Laboratory of Growth Regulators (http://rustreg.upol.cz/) Inhibitors of cytokinin degradation (INCYDE) Inhibitors of cytokinin signalling (PI-55) Inhibitors of cytokinin biosynthesis Cotton defoliants Stimulators of legume nodulation (N 2 -INCYDE) Vigour enhancing compounds and anti-senescence activity development of fluorescent cytokinins development of cytokinin derivatives for affinity purification of CK binding proteins

6. Modulation of cytokinin degradation Ashikari et al., 2005 Bartrina et al., 2011 Petr Galuszka – lecture

7. INCYDE INhibitor of CYtokinin DEgradation INhibitor of CYtokinin oxidase/DEhydrogenase INCYDE

8. INCYDE rescues WT phenotype of CKX overproducing plants

9. INCYDE action INCYDE inhibits CKX activity √ Can we phenocopy the ckx mutants by INCYDE application?

10. Targeted INCYDE application on Arabidopsis WT plants - INCYDE+ INCYDE

11. Targeted INCYDE application on Arabidopsis WT plants

12. INCYDE as a growth regulator for modulation of traits of crops winter rapeseed 2009-2010 INCYDE winter rapeseed 2010-2011 10% increase in field yeild

13. Field trials winter rapeseed 2009-2010, 2010-2011 winter wheat 2009-2010, 2010-2011 barley 2010, 2011 maize 2010, 2011 rice 2010, 2011 different regions different phenological phases different doses different types of application

14. control 28dpi infected INCYDE negatively affects Verticillium longisporum proliferation Col-0 + 10µM INCYDE Col-0 + DMSO Col-0 + DMSO Col-0 + 10 µM INCYDE AB CD E FG *** ** Col-0 + DMSO Col-0 + 10 µM INCYDE ** n.d. T. Teichmann, unpublished

15. INCYDE improves functional properties of edible plants Functional foods can be defined as those providing health benefits beyond basic nutrition and include whole, fortified, enriched or enhanced foods which have a potentially beneficial effect on health when consumed as part of a varied diet on a regular basis at effective levels (Hasler CM, 2000). Shikimate pathwayhydroxybenzoic acids (HBA) L- Phe trans-cinnamic acid hydroxycinnamic acids (HCA) PAL Deikmann et al., 1995

16. INCYDE improves functional properties of edible plants ORAC - radical scavenging TPC - phenolic content Grúz et al., unpublished HBA - hydroxybenzoic acids HCA - hydroxycinnamic acids

17. INCYDE is not cyto- and genotoxic Cytotoxicity: - epithelial and endothelial fibroblasts: NEGATIVE In vivo oral toxicity: MTD > 400 mg/kg Pharmacokinetics - elimination half life ~ 2 h Genotoxicity: - Ames test - NEGATIVE - DNA damage - NEGATIVE

18. Conclusion In the rigth time the right dose of INCYDE application improves functional properties of edible plants protects against biotic stress - strongly reduces symptom development and negatively affects Verticillium proliferation has positive effect on quantitative traits of plants (increased size and number of flowers, fruits and seeds; increased biomass) INCYDE is not cytotoxic, genotoxic and shows very high tolerance in oral toxicity in vivo studies

19. Inhibitors of cytokinin signalling Riefler et al., 2006 Nishimura et al., 2004 Higuchi et al., 2004

20. Inhibitors of cytokinin signalling – PI-55, LGR-991 PI-55 stimulates root branching and increases number of lateral roots PI-55 accelaretes seed germination PI-55 reduces binding of the natural ligand to cytokinin receptors PI-55 antagonizes cytokinin effects in bioassays with different plant species PI-55 protects development of seedlings grown on contaminated soils

21. Development of compounds stimulating nodulation and nitrogen fixation of legumes Regulation of perception and metabolism of cytokinins, important plant hormones, was recently shown to be very important during the nodule formation of legumes (Gonzalez-Rizzo et al., 2006; Murray et al., 2007; Tirichine et al., 2007; Lohar et al., 2004). Preliminary data shows that application of the tested compounds to the roots of pea leads to increment in nodule number and subsequent higher rate of nitrogen fixation. Importantly, in comparison to classical cytokinins the applications of our substances has no negative effects on root and shoot growth and development.

22. 24 h pulsing + 10 days Control (DMSO) Anti-senescent activity and post-harvest storage T-2 (50 µM)T-4 (50 µM) Shimon Meir, Volcani Center, Israel

23. New cotton defoliants derived from cytokinins control TDZ (250 µM) LGR-951 (500 µM) Dropp 4 SC, Free Fall 50 WP, Leafless 4 F, Ginstar 1.5 EC GROSSMANN, K. (1990) Induction of Leaf Abscission in Cotton Is a Common Effect of Urea-and Adenine-Type Cytokinins. Plant Physiol. 234-237. contain thidiazuron as active compound

24. Berlin 2009