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

植物通过感知土壤中有效养分的形态和调节根系结构来持续适应土壤中养分的时空波动，以维持充足的养分。根系的这种动态结构响应允许植物优化空间限定的土壤探索以限制养分。当植物在诱导轻度缺乏的次优外部氮（N）水平下生长时，它们增加了初生根和侧根的伸长。这种刺激下的根扩张轻度缺氮是特别感兴趣的，因为它增加了土壤体积，可以探索的根养分收购。在我们以前的工作中，我们发现在自然加入的模式物种拟南芥的油菜素类固醇和生长素生物合成或信号基因（YUC 8和BSK 3），这改变了根伸长轻度缺氮。这个为期6年的项目的最终目标是了解植物对低氮的根觅食反应的激素调节，并利用这些知识来改善大麦的土壤勘探-并最终提高氮的吸收效率。前3年的两个主要目标是：i）确定拟南芥中YUC 8依赖的生长素生物合成的作用及其与油菜素类固醇在轻度缺氮的根觅食反应中的关系，和ii）以翻译方法探索大麦中油菜素类固醇的等位基因变异以及生长素生物合成和信号传导基因对轻度缺氮的根觅食反应的贡献。首先，我们将在拟南芥中验证YUC 8基因中所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。我们将确定在单一和多个yuc突变株系和补充不同YUC 8等位基因的株系中侧根伸长的反应。利用这些和其他突变体和报告基因系，我们将研究油菜素类固醇和生长素之间的串扰在根伸长响应低氮。在翻译方法中，我们将从拟南芥中引入“强”BSK 3等位基因到大麦中，大麦在迄今为止研究的所有加入物中仅携带“弱”BSK 3等位基因。为此，我们将联合收割机Cas内切酶介导的弱BSK 3内源基因敲除与强转基因的互补结合在一起。最后，我们将通过RNA测序方法创建大麦根中N缺乏调控基因的清单，这将作为选择其他大麦基因的资源，这些基因将通过CRISPR-Cas介导的基因缺失和使用根特异性启动子的异位表达来靶向。将对所有品系进行根构型对轻度缺氮反应的表型分析。

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.