PALACKÝ UNIVERSITY OLOMOUC FACULTY OF SCIENCE DEPARTMENT OF BIOCHEMISTRY CYTOKININ METABOLISM AND ITS REGULATION Ph.D. Thesis Mgr. Kateřina Podlešáková OLOMOUC 2012 Acknowledgement I would like to thank my supervisor doc. RNDr. Petr Tarkowski, Ph.D. for his guidance. Further, my thanks belong to prof. Ing. Miroslav Strnad DrSc., doc. Mgr. Galuszka Ph.D. and Mgr. Lukáš Spíchal Ph.D. for giving me a chance to take part in interesting research. My appreciation also goes to my student Bc. Onřej Kotland for working hard and well and to Mgr. Onřej Novák Ph.D. for his valuable advice and suggestions. I wish thank my colleagues from Laboratory of Growth Regulators and Department of Biochemistry, especially Mgr. Aleš Pěnčík Ph.D., Mgr. Radim Simerský Ph.D. and Mgr. Lubor Urbánek Ph.D. for creating friendly atmosphere at the department. I am also grateful to Hlávka foundation and DAAD institute for financial support. Considerable credit of the thesis belogs to my husband Pavel Podlešák who encouraged me throughout my studies. 2 I declare that the Ph.D. thesis has been written entirely by myself. All sources quoted in this work are listed in the “Reference“section. Olomouc, 30.3. 2012 3 Contents: Abbreviations 5 Bibliographical identification 7 1. Introduction 9 2. Cytokinin structure and biological activity 10 3. Practical applications of cytokinins 13 4. Cytokinin biosynthesis 15 5. Cytokinin metabolism 18 6. Cytokinin transport and signalling 21 7. Cytokinin regulation of developmental processes in plants 21 8. Regulation of cytokinin metabolism in bacteria 23 9. New trends in phytohormone analysis 26 10. Cytokinin analysis 27 11. Discussion and Perspectives 30 12. Conclusions 33 Enclosures 34 References 37 4 ABBREVIATIONS ADP adenosine diphosphate AHK histidine kinase receptor AHL N-acyl homoserine lacton AHP histidine phosphotransfer protein AMP adenosine monophosphate ARR Arabidopsis response regulator ATP adenosine triphosphate AtCKX Arabidopsis cytokinin dehydrogenase AtIPT Arabidopsis isopentenyltransferase BAP benzylaminopurine CE capillary electrophoresis CE-MS capillary electrophoresis – mass spectrometry CLV CLAVATA CRE1/AHK4 cytokinin receptor cZ cis-zeatin cZR cis-zeatin riboside DEAE diethylaminoethyl GC-MS gas chromatography-mass spectrometry DMAPP dimethylallyl pyrophosphate HILIC hydrophilic interaction chromatography HMBDP (E)-4-hydroxy-3-methyl-but-2-enyl diphosphate HPLC high performance liquid chromatography IAE immunoaffinity extraction iP isopentenyladenine IPP isopentenyldiphosphate iPR isopentenyladenosine iPMP isopentenyladenosine-5'-monophosphate iPTP isopentenyladenosine-5'-triphosphate IPT isopentenyltransferase IS internal standard LC-MS liquid chromatography-mass spectrometry LC-MS/MS liquid chromatography-tandem mass spectrometry LOD limit of detection LOG lonely guy MCX mixed mode cationic exchanger MEP methyl-erythritol-phosphate 2MeSiP 2-methylthio-isopentenyladenine 2MeSiPR 2-methylthio-isopentenyladenosine 2MeStZ 2-methylthio-trans-zeatin 2MeStZR 2-methylthio-trans-zeatin riboside 2MeScZ 2-methylthio-cis-zeatin 2MeScZR 2-methylthio-cis-zeatin riboside miaA E.coli tRNA-IPT gene mT meta-topolin mTR meta-topolin riboside MTR methyltransferase ORF open reading frame OsCKX2 Oryza sativa cytokinin dehydrogenase 5 oT ortho-topolin oTR ortho-topolin riboside pFiD188 linear plasmid of Rhodococcus fascians strain D188 PT-SPE pipette tip solid phase extraction RIA radioimmunoanalysis SAM shoot apical meristem SCX strong cation exchanger STM shoot meristemless SPE solid phase extraction tRNA transfer ribonucleic acid tRNA-IPT tRNA isopentenyltransferase tZ trans-zeatin tZR trans-zeatin riboside tZMP trans-zeatin riboside-5'-monophosphate tZ9G trans-zeatin-9-gluoside tZOG trans-zeatin-O-glucoside UDP uridine diphosphate UTP uridine triphosphate UHPLC ultra-high performance liquid chromatography WUS WUSCHEL transcription factor ZmCKX maize cytokinin dehydrogenase 6 Bibliographical identification: First name and surname Kateřina Podlešáková Title Mgr. Type of thesis Ph.D. Department Department of Biochemistry Supervisor doc. RNDr. Petr Tarkowski, Ph.D. The year of presentation 2012 Abstract The thesis focuses on the role of cytokinins in plant- pathogen interactions, seed germination, cambium formation, and stress responses. We also explored an alternative pathway of cytokinin biosynthesis and the metabolism of new synthetic cytokinin derivatives and their effect on plants. Because of the very low concentrations of cytokinins in plant tissues, the use of highly sensitive methods such as UHPLC (HPLC)-MS/MS, are needed. Thus, we have developed an HPLC-MS/MS method for the analysis of 2-methylthio-cytokinin derivatives. The method was used to quantify and elucidate the involvement of cytokinins in the leafy gall syndrome induced by Rhodococcus fascians. The first rate limiting step in cytokinin biosynthesis is catalyzed by isopentenyltransferase. An alternative pathway might be tRNA degradation. We tried to elucidate to what extent tRNA degradation could contribute to the total cytokinin pool in germinating seeds. Furthermore, we explored the role of cytokinins in the formation of a very important plant secondary meristem – the cambium. Furthermore, we followed the metabolism and the effect of synthetic cytokinin derivatives on plant development and identified them as valuable novel compounds to be applied in in vitro micropropagation procedures in the future. Keywords 2-methylthio cytokinins, Rhodococcus fascians, UHPLC (HPLC)-MS/MS, tRNA, cambium, Number of pages 43 Number of appendices 8 Language English 7 Bibliografická identifikace: Jméno a příjmení Kateřina Podlešáková Titul Mgr. Druh práce Ph.D. Pracoviště Katedra biochemie Školitel doc. RNDr. Petr Tarkowski, Ph.D. Rok odevzdání 2012 Abstrakt Tato disertační práce je zaměřená na problematiku cytokininů a to zejména na jejich roli při infekcích rostlin patogeny, tvorbě kambia v rostlinách, alternativní dráhu jejich biosyntézy a nové syntetické deriváty cytokininů. Vzhledem k velmi nízkým hladinám cytokininů v rostlinách jsou pro jejich studium nutné vysoce citlivé a selektivní analytické metody jako např. UHPLC-MS/MS. Jedním z dílčích projektů bylo tedy vyvinout HPLC-MS/MS metodu pro analýzu 2- methylthio-derivátů cytokininů, která byla poté pouţita pro objasnění role 2-methylthio derivátů cytokininů v mechanismu infekce rostlin bakterií Rhodococcus fascians. První klíčový krok v biosyntéze cytokininů je katalyzován isopentenlytransferasou (IPT). Alternativní drahou biosyntézy cytokininů je degradace tRNA. Doposud však nebylo objasněno nakolik degradace tRNA navyšuje celkové hladiny cytokininů. V rámci dalšího podprojektu jsme se zaměřili na roli tRNA degradace při klíčení semen. Kambium je druhotné meristematické pletivo. Jiný podprojekt této práce se zaměřil na roli cytokininů při tvorbě kambia. Součástí práce bylo rovněţ sledování metabolismu nových syntetických derivátů cytokininů, jeţ vykazují vysokou biologickou aktivitu a mohly by být vyuţity při in vitro mikropropagaci. V rámci této práce byla zpracována rešerše na téma: Nové trendy v analýze fytohormonů, kde se zabýváme nejnovějšími přístupy k analýze nejen cytokininů, ale všech rostlinných hormonů a to jak jednotlivě, tak v rámci tzv. hormonomiky, kdy je analyzováno více skupin součastně. Klíčová slova 2-methylthio cytokininy, Rhodococcus fascians, UHPLC (HPLC)-MS/MS, tRNA, kambium, Počet stran 43 Počet příloh 8 Jazyk Angličtina 8 1. Introduction The heterogeneous messengers regulating plant growth and development by acting as signal molecules are called plant hormones. Generally, it is accepted that there are five main classes of phytohormones: auxin, cytokinins (CKs), gibberellins, abscisic acid and ethylene. Nevertheless, several new growth regulators have been identified recently, such as jasmonates, brassinosteroids, salycic acid and strigolactones, and thus the list of plant hormones keeps expanding. This thesis deals mainly with CKs, but since none of the phytohormones act independently (Tanaka et al., 2006; Růţička et al., 2007; Růţička et al., 2009), it is inevitable to discuss the other phytohormones as well. CKs are involved in many aspects of plant development. In particular, they are known to regulate shoot and root morphogenesis, leaf senescence (Gan and Amasino, 1995) and axillary bud growth, they transduce nutritional signals (Takei et al., 2001) and are implicated in apical dominance through cross-talk with auxin (Tanaka et al., 2006). Furthermore, CKs are involved in mediating developmental changes induced by the interaction with symbiotic or pathogenic bacteria (Werner and Schmülling, 2009). All these regulatory features are mediated by a complex signalling network. In the last years, significant progress has been made in understanding the key aspects of CK biosynthesis and breakdown in plants and important signalling pathways have been elucidated (Perilli et al., 2010; Werner and Schmülling, 2009). However, concerning the molecular basis of how CKs control developmental processes as diverse as stress responses, seed germination, cambial development, lateral root emergence and plant-bacteria interactions, there is still ample room for discovery. For our understanding of the physiological role of CKs in the processes mentioned above, the development of sensitive and selective up-to-date analytical methods, such as ultra-high performance liquid chromatography (UHPLC) and tandem mass spectrometry (MS/MS) have become essential. Hence, the main objectives of the work underlying