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

在征服土地的过程中，植物发展了关键的适应能力，以应对不断变化的陆地栖息地。这些措施包括适应土壤的养分觅食，形成对波动的渗透和离子条件的复原力，以及建立被动防御和固结结构。钙信号在所有这些过程中起着至关重要的作用，并涉及钙调神经磷酸酶B样传感器蛋白(CBL)及其CBL相互作用蛋白激酶(CIPK)。随着植物在陆地上茁壮成长的能力不断增强，CBL/CIPK系统的复杂性也增加了。这种进化扩展始于仍存在于现存藻类中的单一CBL/CIPK对，继续存在于苔藓植物中的简单结构网络，最终达到它们在高等植物中的现存复杂性。然而，这个网络的古老功能一直是个谜。此外，指导这一网络的进化及其功能多样性的分子驱动因素和机制仍有待确定。这种情况定义了该项目的目标。通过对CBL/CIPK/TARGET模块的比较研究，以毛细支原体和多形支原体为模型系统，我们打算解决Madland的三个核心问题。我们将利用酵母、人类细胞系和拟南芥中的异源途径重建系统来描述CBL、CIPK及其靶标之间的功能互连。目前，无法对链藻进行遗传操作在很大程度上阻止了对其基因功能的遗传解剖。我们忠实的异源途径重建分析谱使我们能够成功地弥合这一技术差距，并描绘出根癌分枝杆菌CBL/CIPK复合体的功能。作为补充，我们将使用反向遗传学方法来证实它们在多态分枝杆菌中的生理功能。因此，我们建议(I)确定陆地植物祖先CBL/CIPK模块的分子特性和功能，并(Ii)阐明早期CBL/CIPK网络进化的原理。为此，我们讨论了以下问题：(1)MeCBL1和MeCIPK1是古老的SOS途径的组成部分，以赋予耐盐性？(2)来自毛细管藻的CIPK1是单功能还是多功能激酶？(3)CBL/CIPK网络的最初进化是在钙传感器水平上发生的，它是否涉及网络功能的亚细胞扩展？(4)CBL/CIPK网络进化的初始步骤是如何体现在钙传感器水平上的，Marchantia CBL网络进化的早期步骤是如何表现的？(5)CBL/CIPK网络进化的早期步骤是如何表达在CIPK蛋白水平上，Marchantia CIPK1和CIPK2的功能是什么？通过这种方法，我们打算合成CBL/CIPK在植物陆地化后早期网络形成和扩展过程中功能多样化的进化情景。

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.