How legumes create an environment for nitrogen fixation: control of oxygen permeation into nodules

豆科植物的根瘤结构中存在共生根瘤菌，它们在根瘤中创造了一个适合固氮的环境。了解这种环境如何出现对于优化豆类中的共生关系和其他作物中的工程化种植至关重要。在第一个资助期，我们使用比较转录组学探索了Lotus共生表型的自然多样性。因此，我们确定了在感染的根瘤中特异性上调的基因，从而可能介导这些结构内根瘤菌的适应。系统发育和表达分析的两个根瘤诱导木栓林生物合成基因编码脂肪酰基辅酶A还原酶（法尔斯）揭示了其表达模式的多样性。控制这些和其他木栓质相关基因表达的启动子活性的时空分析显示，在根和根瘤内皮层有明显的激活。根瘤特异性FAR3.2基因的突变株系表现出根瘤渗透性增加、固氮作用受损和芽生长减少。我们的研究结果支持了一个模型，其中结节特异性木栓质相关基因介导的渗透屏障在结节周边的形成。控制氧渗透到根瘤中对保护氧敏感的固氮酶是必不可少的，因此对固氮至关重要。在这个项目中，我们将研究木栓质相关功能基因的功能和调控，因为它们是建立控制氧渗透的结节屏障的有希望的分子标记。为此，我们将通过CRISPR/Cpf 1基因编辑创建突变株系并对其进行表型表征，我们将生成报告株系以确定结节屏障的个体发育，并将在分子水平上研究木栓蛋白相关基因的调控。这项工作将促进我们对结核屏障如何形成的理解，这是使豆类固氮的一个关键适应。

Legumes host symbiotic rhizobia inside root structures called nodules where they create an environment suitable for nitrogen fixation. Understanding how this environment emerges is critical for optimizing this symbiosis in legumes and engineering nodulation in other crops. In the first funding period we explored the natural diversity of the symbiotic phenotypes of Lotus using comparative transcriptomics. Thereby we identified genes that were specifically upregulated in infected nodules and thus might mediate the accommodation of rhizobia inside these structures. Phylogenetic and expression analysis of two nodule induced suberin biosynthesis genes encoding Fatty acyl-CoA reductases (FARs) revealed diversification in their expression pattern. Spatiotemporal analysis of promoter activity controlling the expression of these and other suberin-related genes showed distinct activation in the root and nodule endodermis. Mutant lines in the nodule specific FAR3.2 gene, showed an increase in nodule permeability, impaired nitrogen fixation, and reduced shoot growth. Our results support a model in which nodule-specific suberin related genes mediate the formation of a permeation barrier in the nodule periphery. The control of oxygen permeation into the nodule is essential to protect the oxygen-sensitive nitrogenase and is thus crucial for nitrogen fixation. In this project we will investigate the function and regulation of genes with suberin-related functions, as they are promising molecular markers for the establishment of the nodule barrier, which controls oxygen permeation. To this end we will create mutant lines by CRISPR/Cpf1 gene editing and phenotypically characterize them, we will generate reporter lines to determine the ontogeny of the nodule barrier, and will investigate the regulation of suberin-related genes at a molecular level. This work will advance our understanding of how the nodule barrier is formed, a key adaptation enabling nitrogen fixation in legumes.