Towards establishment of model organisms for conjugating green algae – Zygnematophyceae and studies on the evolution of the regulatory network of the plant hormone cytokinin Dissertation with the aim of achieving a doctoral degree at the Institute of Plant Science and Microbiology, University of Hamburg Submitted by M.Sc. Hong Zhou Hamburg, 2020 Day of oral defense: 28.08.2020 The following evaluators recommend the admission of the dissertation: PD Dr. Klaus von Schwartzenberg Prof. Dr. Dieter Hanelt Table of Contents Table of Contents Abstract ............................................................................................................................................. II Zusammenfassung .......................................................................................................................... III Abbreviations ................................................................................................................................... V 1. Introduction ................................................................................................................................... 1 1.1 The occurrence of cytokinins in plants ................................................................................... 1 1.2 Cytokinin metabolism and transport ....................................................................................... 3 1.3 Cytokinin signaling pathway ................................................................................................... 7 1.4 Charophytes: the key aquatic organisms which evolved to land plants ............................... 11 1. 5 Zygnematophyceae: the sister group to land plants ............................................................ 15 1.6 Cytokinin in land plant evolution ........................................................................................... 17 1.7 Cytokinin studies in the basal model organism Physcomitrella patens ................................ 19 1.8 Model organism need to be established in Charophytes ..................................................... 21 1.9 Strains and genetic information available for Charophytes .................................................. 22 1.10 Molecular tools established in Zygnematophyceae ............................................................ 23 1.11 Aim of the project ................................................................................................................ 24 2. Discussion .................................................................................................................................. 27 2.1 The occurrence of cytokinin within the green lineage .......................................................... 28 2.2 Understanding the evolution of the regulatory machinery of cytokinin metabolites ............. 31 2.3 Understanding the evolution of the regulatory machinery of cytokinin signaling ................. 33 2.4 The development of model organisms in Zygenematophyceae ........................................... 37 2.5 The genetic transformation of the potential Zygnematophyceae model strains ................... 41 2.6 Conclusion and future perspective ....................................................................................... 44 3. References ................................................................................................................................. 46 4. Manuscripts ................................................................................................................................ 59 4.1 Searching for the roles of cytokinin regulation in charophyte green algae ........................... 59 4.2 Zygnematophyceae: from living Algae collections to the establishment of future models . 102 4.3 Expanding the molecular toolbox for Zygnamatophyceae ─ transient genetic transformation of the desmid Micrasterias radians var. evoluta ....................................................................... 113 5. Appendix ................................................................................................................................... 124 5.1 Publication with co-authorship: ........................................................................................... 124 5.2 Conference contributions: ................................................................................................... 124 5.3 Vector cards ........................................................................................................................ 125 6. Acknowledgement .................................................................................................................... 139 7. Eidesstattliche Versicherung .................................................................................................... 140 I Abstract Abstract The cytokinins (CKs) are important signaling molecules regulating plant growth and development as well as responses to environmental stresses. Their actions in land plants are executed via a complex regulatory network of CK biosynthesis, metabolism, transport, signal perception, and transduction. In land plants this regulatory network has been well studied for the model organisms Arabidopsis thaliana and the bryophyte Physcomitrella patens. However, knowledge about the origin and evolutionary trajectory of the CK regulatory system is rather limited. In this thesis, the occurrence of CKs and their regulatory network in charophyte green algae (CGA) have been investigated. The CK profiles of five selected CGA species (Cosmarium crenatum var. boldtianum, Micrasterias radians var. evoluta, Spirogyra pratensis, Klebsormidium nitens, and Mesostigma viride) showed a predominance of isopentenyladenine (iP)- and cis-zeatin (cZ)- type CKs in free base, riboside, and phosphate forms, whereas glucosides did not contribute to the algal CK pools. Species from CGA were found to release endogenously produced CKs into the culture medium over the growth period. Correspondingly, BLAST searches of algal and plant genomic and transcriptomic databases indicated the presence of CK biosynthesis and transport across the green lineage, while the CK oxidase/dehydrogenase (CKX) mediated CK breakdown is restricted to land plants. I also found that a complete set of components involved in CK signaling can only be found in CGA and land plants. The CHASE-containing histidine kinases (CHKs) and type-A response regulators seem to first appear in CGA. A domain-based phylogenetic analysis revealed that the CGA genomes encode CHKs related to both, canonical and noncanonical CK receptors. Functional analysis was carried out for two CHKs from Spirogyra pratensis (Transeau) (Zygnematales) (SpCHKs). The SpCHK2, which is closely related to canonical CK receptors, showed weak affinity to iP in a bacterial CK binding assay. However, both SpCHKs were not able to activate a CK response when expressed in a CHK receptor-deficient mutant of the moss Physcomitrella patens. Our study provided hints that the two-component CK signaling pathway has first been acquired in CGA but it is likely to function in a manner independent or less dependent on the CK ligand when compared to land plants. The importance of Zygnematophyceae, the direct sister group of land plants, is discussed with respect to evolutionary and physiological studies, where the urgent need for the establishment of model organisms within this class becomes evident. The current development of model organisms in Zygnematophyceae is reviewed. By own work, two candidates (Micrasterias radians var. evoluta (W.B.Turner) and Spirogyra pratensis) were selected from the Microalgae and Zygnematophyceae Collection Hamburg (MZCH). Both organisms are able to perform their complete life cycle in vitro. Efficient transient transgene expression in M. radians var. evoluta via particle bombardment and polyethylene glycol (PEG)-mediated protoplast transformation is presented. Via protoplast transformation the influence of various promoters on transformation efficiency was compared. Further establishment of transgenesis protocols and genetic resources together with the in vitro control of the life cycle will help to elucidate the processes which enabled the ancestors of extant Zygnematophyceae to adapt to terrestrial environments. II Zusammenfassung Zusammenfassung Cytokinine (CK) sind wichtige endogene Signalmoleküle, die das Wachstum und die Entwicklung von Pflanzen regulieren und ebenfalls Bedeutung für die Anpassung an Umweltbedingungen besitzen. Die Wirkung von CK wird bei Landpflanzen durch ein komplexes regulatorisches Netzwerk bestehend aus CK-Biosynthese, Metabolismus, Transport, Signalperzeption und Signalweiterleitung realisiert. Dieses regulatorische System wurde ausgiebig bei den Modellorganismen für Landpflanzen, (v.a. Arabidopsis thaliana sowie ebenfalls für das Laubmoos Physcomitrella patens) untersucht. Das drzeitige Verständnis des evolutionären Ursprungs sowie der evolutionären Entwicklung des CK-Systems ist lediglich fragmentarisch. Im Rahmen dieser Arbeit wurde das Vorkommen von CK Metaboliten sowie das regulatorische Netzwerk der CK bei charophytischen Grünalgen (CGA) untersucht. Das CK Profil der ausgewählten CGA Species (Cosmarium crenatum var. boldtianum, Micrasterias radians var. evoluta, Spirogyra pratensis, Klebsormidium nitens, and Mesostigma viride) zeigten eine Dominanz von Metaboliten aus den Isopentenyladenin (iP)- und cis-Zeatin (cZ) Familien, die als freie Basen, Riboside und Nukleotide vorlagen. CK-Glykoside wurden nicht bzw.