On the move to field (5 min)

1. On themove to field (5 min) Lukáš Spíchal LaboratoryofGrowthRegulators, IEB AS CR & UP Olomouc, CzechRepublic Centre of the Region Hanáfor Biotechnological and Agricultural Research, Olomouc, CzechRepublic 1,2 1 2

2. Acknowledgements LaboratoryofGrowthRegulators, 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 ofBiochemistry, PřF UP Olomouc: Petr Galuszka David Kopečný Laboratoryof Genome Integrity, UP Olomouc &BioPatterns, Ltd., Olomouc: Martin Mistrík Institute of Biology/AppliedGenetics, FU Berlin: Tomáš Werner Thomas Schmülling boss organicsynthesis biochemistry/biology analyticalchemistry fieldexperiments Grant Support: MSM 6198959216, LC06034, ED0007/01/01, GA522/08/0920

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

4. What to change to gain… Cytokinin antagonist Spíchal et al., 2009 Nisler et al., 2010 CKX inhibitor BA BA 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 signalling (PI-55) Inhibitors of cytokinin degradation (INCYDE) Inhibitors of cytokinin biosynthesis Cotton defoliants Stimulators of legume nodulation (N2-INCYDE) Vigourenhancingcompoundsandanti-senescence activity developmentoffluorescent cytokinins developmentof cytokinin derivativesforaffinitypurificationof CK bindingproteins

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

7. INCYDE INCYDEINhibitor of CYtokinin DEgradationINhibitor of CYtokinin oxidase/DEhydrogenase

8. INCYDE rescues WT phenotype of CKX overproducing plants

9. INCYDE action INCYDE inhibits CKX activity√ Canwephenocopytheckxmutants 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 winterrapeseed 2009-2010 winterrapeseed2010-2011 10% increase in fieldyeild INCYDE

13. Fieldtrials winterrapeseed 2009-2010, 2010-2011 winterwheat 2009-2010, 2010-2011 barley 2010, 2011 maize 2010, 2011 rice 2010, 2011 differentregions differenttypesofapplication differentphenologicalphases differentdoses

14. INCYDE negativelyaffectsVerticilliumlongisporumproliferation control 28dpi infected E Col-0 + DMSO A B Col-0 + DMSO Col-0 + 10µMINCYDE *** ** C D Col-0 + 10µM INCYDE F G Col-0 + DMSO Col-0 + 10µMINCYDE ** n.d. n.d. *** *** T. Teichmann, unpublished

15. INCYDE improves functional properties of edible plants Functional foods can bedefined as those providing health benefits beyond basic nutritionand include whole, fortified, enriched or enhanced foods whichhave a potentially beneficial effect on health when consumedas part of a varied diet on a regular basis at effective levels (Hasler CM, 2000). Shikimate pathway hydroxybenzoicacids (HBA) L- Phe PAL trans-cinnamic acid hydroxycinnamicacids (HCA) Deikmannetal., 1995

16. INCYDE improves functional properties of edible plants TPC - phenoliccontent ORAC - radicalscavenging HBA - hydroxybenzoicacids HCA - hydroxycinnamicacids Grúzetal., unpublished

17. INCYDE is not cyto- and genotoxic • Cytotoxicity: • epithelialandendothelialfibroblasts: • NEGATIVE • In vivooral 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 has positive effect on quantitativetraitsofplants (increasedsizeandnumberofflowers, fruitsandseeds; increasedbiomass) improvesfunctionalpropertiesofedibleplants protectsagainstbiotic stress - strongly reduces symptom development and negativelyaffectsVerticilliumproliferation INCYDE isnot cytotoxic, genotoxicandshowsveryhigh tolerance in oral toxicity in vivostudies

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

20. Inhibitorsof cytokinin signalling – PI-55, LGR-991 PI-55 reduces binding of the natural ligand to cytokinin receptors PI-55 stimulates root branching and increases number of lateral roots PI-55 accelaretes seed germination PI-55 antagonizes cytokinin effects in bioassays with different plant species PI-55 protectsdevelopmentofseedlingsgrown on contaminatedsoils

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. Anti-senescentactivityand post-harveststorage 24 h pulsing + 10 days T-2 (50 µM) T-4 (50 µM) Control (DMSO) Shimon Meir, Volcani Center, Israel

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

24. Berlin 2009