Elongating roots for efficient nitrogen foraging via altered brassinosteroid and auxin synthesis and signaling

植物通过感知有效的营养形态和调节其根系构型来维持足够的营养，从而不断适应土壤中的时空养分波动。这种动态的根部结构反应使植物能够优化空间上确定的土壤探索，以限制养分。当植物生长在导致轻度缺氮的次优外部氮素水平下时，它们会增加主根和侧根的伸长。在轻度缺氮的情况下，这种对根膨胀的刺激特别令人感兴趣，因为它增加了根可以探索的土壤体积，以获得养分。在我们以前的工作中，我们在模式植物拟南芥的天然材料中发现了油菜素类固醇和生长素生物合成或信号基因(YUC8和BSK3)的等位基因变异，这些基因在轻度缺氮情况下改变了根的伸长。这项为期6年的项目的最终目标是了解植物根系对低氮的觅食反应的激素调节，并利用这一知识来改善大麦的土壤探索-最终提高氮素吸收效率。前3年的两个主要目标是：1)在拟南芥中确定依赖于YUC8的生长素生物合成的作用及其与油菜素内酯类化合物在轻度缺氮反应中的关系；2)用翻译的方法探索大麦中油菜素类固醇以及生长素生物合成和信号基因的等位变异对轻度缺氮引起的根系觅食反应的贡献。首先，我们将在拟南芥中验证YUC8基因中已识别的等位基因变异背后的分子机制及其在调节根觅食反应中的作用。我们将测定单个和多个YUC突变系以及带有不同YUC8等位基因的品系的侧根伸长反应。利用这些和其他突变体和报告系，我们将研究油菜素类固醇和生长素之间的串扰在根伸长对低氮的反应中。在翻译方法中，我们将把来自拟南芥的“强”BSK3等位基因导入大麦，它在所有迄今研究的等位基因中只携带一个“弱”的BSK3等位基因。为此，我们将结合Cas内切酶介导的弱BSK3内源基因的敲除和强转基因的互补作用。最后，我们将通过RNA测序的方法创建大麦根中N缺乏调控基因的清单，作为进一步筛选大麦基因的资源，这些基因将被CRISPR-Cas介导的基因缺失和使用根特异启动子的异位表达所靶向。所有品系都将接受对轻度缺氮的根构型反应的表型分析。

Plants adapt continuously to spatiotemporal nutrient fluctuations in soils by sensing available nutrient forms and modulating their root system architecture to sustain adequate nutrition. Such dynamic architectural responses of roots allow plants to optimize spatially defined soil exploration for limiting nutrients. When plants grow under suboptimal external nitrogen (N) levels that induce mild deficiency, they increase the elongation of primary and lateral roots. This stimulation of root expansion under mild N deficiency is of particular interest as it increases the soil volume that can be explored by roots for nutrient acquisition. In our previous work, we found in natural accessions of the model species Arabidopsis allelic variation in brassinosteroid and auxin biosynthesis or signalling genes (YUC8 and BSK3), which alters root elongation under mild N deficiency. The ultimate goal of the 6-years project is to understand the hormonal regulation of the root foraging response to low N in plants and to exploit this knowledge for improving soil exploration – and ultimately N uptake efficiency – in barley. The two major objectives of the first 3 years are i) to determine in Arabidopsis the role of YUC8-dependent auxin biosynthesis and its relation to brassinosteroids in the root foraging response to mild N deficiency, and ii) to explore in a translational approach in barley the contribution of allelic variation in brassinosteroid as well as auxin biosynthesis and signaling genes to the root foraging response to mild N deficiency. First, we will verify in Arabidopsis the molecular mechanism behind the identified allelic variation in the YUC8 gene and its role in regulating the root foraging response. We will determine the lateral root elongation response in single and multiple yuc mutant lines and in lines complemented with different YUC8 alleles. Using these and other mutant and reporter lines, we will investigate the crosstalk between brassinosteroids and auxin in the root elongation response to low N. In a translational approach, we will introduce the "strong" BSK3 allele from Arabidopsis into barley, which carries in all accession investigated so far only a "weak" BSK3 allele. For this purpose, we will combine Cas endonuclease-mediated knockout of the weak BSK3 endogene with complementation by the strong transgene in just one step. Finally, we will create an inventory of N deficiency-regulated genes in barley roots by an RNA sequencing approach, which will serve as resource for the selection of further barley genes that will be targeted by CRISPR-Cas-mediated gene deletion and by ectopic expression using a root-specific promoter. All lines will be subjected to phenotypic analysis of root architectural responses to mild N deficiency.