ROS Bursts in Plant Growth and Fertility: Evolution and Diversification of the NADPH Oxidase Family

陆地植物的进化在世代交替和施肥方式上产生了根本性的变化。苔藓植物具有优势的单倍体，受精发生在鞭毛精子和配子体上的卵母细胞之间，而被子植物的配子体则减少到很少的细胞数量。在这里，受精不再依赖于水，而是需要顶端生长的花粉管与封闭在胚珠内的胚囊相互作用。活性氧（ROS）的局部生成参与了雄性和雌性生殖的细胞壁重塑和信号传递过程。在被子植物中，呼吸爆发氧化酶同源物（RBOHs）已被确定为特异性细胞外ROS生成的来源。最早的植物RBOHs出现在绿藻向陆地过渡之前，在陆地植物进化过程中，蛋白质家族不断扩大，表明功能多样化。从进化的角度来看，RBOH异构体和细胞外ROS的局部产生对植物生长和繁殖的确切作用仍然未知。该项目将研究(1)RBOH植物同源物的祖先生物学功能；(2)RBOH在模式物种内和进化距离较远的植物模式物种之间的亚功能化和新功能化。我们将通过反向遗传学的方法获得多态地苔（Marchantia polymorpha）的两个RBOH同型体和苔藓（Physcomitrium patens）的四个RBOH同源体的零突变体，并量化与植物生长和繁殖有关的结果表型。为了明确和量化ROS依赖性的生育缺陷，我们将使用并进一步开发通过基因编码的氧化还原生物传感器对局部ROS生成的细胞内和细胞外动态传感。此外，我们将通过报告结构研究四种P. patens亚型的表达域和亚细胞定位。在尖端生长和繁殖能力降低的拟南芥RBOH突变体中，与藻类和苔藓植物RBOH同源物的互补研究将使我们能够在大进化距离上得出功能多样化的结论。因此，本项目将明确RBOHs在不同陆地植物系有性生殖过程中外质体局部ROS生成中的作用和功能进化。这将有助于更好地理解被子植物受精过程中发生的复杂氧化还原过程，并揭示植物RBOH功能的进化根源。

Land plant evolution has shaped fundamental changes in the alternation of generations and the fertilization mode. Whereas bryophytes possess a dominant haploid generation and fertilization occurs between flagellated spermatozoids and egg cells in archegonia on gametophytes, gametophytes in angiosperms are reduced to a low number of cells. Here, fertilization has become independent of water, but requires the interaction of tip-growing pollen tubes with the embryo sac enclosed in the ovule. The local generation of reactive oxygen species (ROS) is involved in cell wall remodelling and signalling processes on both the male and female side of reproduction. In angiosperms, Respiratory Burst Oxidase Homologs (RBOHs) have been identified as the source of specific extracellular ROS generation. The first plant RBOHs appeared before the transition to land in green algae and the protein family has continuously expanded during land plant evolution, suggesting functional diversification. The precise role of RBOH isoforms and local generation of extracellular ROS for plant growth and reproduction remains unknown in an evolutionary perspective.This project will investigate (1) the ancestral biological function of RBOH plant homologs and (2) the sub- and neo-functionalisation of RBOH within model species and between evolutionary distant plant model species. We will generate null mutants for the two RBOH isoforms of the liverwort Marchantia polymorpha and the four RBOH homologs of the moss Physcomitrium patens via reverse genetics and quantify the resulting phenotypes regarding plant growth and fertility. In order to specify and quantify ROS-dependent fertility defects, we will use and further develop intracellular and extracellular dynamic sensing of local ROS generation via genetically encoded redox biosensors. In addition, we will investigate expression domains and subcellular localisation of the four P. patens isoforms via reporter constructs. Complementation studies with algal and bryophyte RBOH homologs in A. thaliana rboh mutants with reduced tip growth and fertility will allow us to conclude on functional diversification across large evolutionary distances.Thus, this project will specify the role and functional evolution of RBOHs in local ROS generation in the apoplast during sexual reproduction in different land plant lineages. This will allow a better understanding of the complex redox processes occurring during angiosperm fertilization, as well as reveal the evolutionary roots of RBOH function in plants.