Ancient function and subsequent evolution of CBL/CIPK Ca2+-sensor/kinase complexes during adaptation to land

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During the conquest of land, plants developed crucial adaptations to cope with fluctuating terrestrial habitats. These included adaptation of nutrient foraging to soil, formation of resilience to fluctuating osmotic and ionic conditions as well as the creation of passive defence and consolidation structures. Ca2+ signalling functions crucially in all these processes and involves Calcineurin B-like sensor proteins (CBLs) and their CBL-interacting protein kinases (CIPKs). Coinciding with the increasing ability of plants to thrive on land, the complexity of the CBL/CIPK system also increased. This evolutionary expansion started with singular CBL/CIPK pairs still present in extant algae, continued with simply structured networks in bryophytes to culminate in their extant complexity in higher plants. However, the ancient function of this network has remained enigmatic. Moreover, the molecular drivers and mechanisms that directed the evolution of this network and its functional versatility remain to be identified. This situation defines the objectives of this project. By comparatively studying CBL/CIPK/target modules as subject and using M. endlicherianum and M. polymorpha as model systems, we intend to address three of the core questions of MAdLand. We will employ heterologous pathway reconstitution systems in yeasts, human cell lines and A. thaliana to delineate the functional interconnection of CBLs, CIPKs and their targets. Currently, the inability to genetically manipulate streptophytic algae largely prevents to genetic dissection of the function of their genes. Our spectrum of faithful heterologous pathway reconstitution assays allows us to successfully bridge this technology gap and to delineate the function of M. endlicherianum CBL/CIPK complexes. Complementarily, we will use reverse genetics approaches to corroborate their physiological functions in M. polymorpha. Consequently, we propose to (i) define the molecular properties and function of the ancestral CBL/CIPK module of land plants and to (ii) elucidate principles of early CBL/CIPK network evolution. To this end, we address the following questions: (1) Are MeCBL1 and MeCIPK1 constituents of an ancient SOS pathway to confer salt tolerance? (2) Is CIPK1 from M. endlicherianum a mono-functional or a poly-functional kinase? (3) Did the initial evolution of CBL/CIPK network complexity occur at the Ca2+-sensor level and did it involve subcellular expansion of network function? (4) How are initial steps of CBL/CIPK network evolution manifested on the Ca2+-sensor level and do Marchantia CBLs exhibit functional diversification? (5) How are early steps of CBL/CIPK network evolution manifested on the CIPK kinase level and what are the functions of Marchantia CIPK1 and CIPK2? With this approach, we intend to synthesize an evolutionary scenario for the functional diversification of CBL/CIPKs during early network formation and expansion after plant terrestrialization.