Control of barley root growth angle by ENHANCED GRAVITROPISM 2 and associated genes

根生长角定义了谷物根相对于重力矢量的生长方式。根系生长角度是根系结构最重要的决定因素之一，因为它控制着植物的吸水能力和养分利用效率，从而影响植物的产量。在该项目的第一个资助期，我们克隆了EGT2基因，并证明它编码一个进化保守的无菌α MOTIF结构域蛋白。此外，我们在转录组学实验中证明了EGT2和重力刺激调控的基因有很大的重叠，这表明该基因在控制大麦中决定根角的分子网络中起着核心作用。最后，我们证明了受重力刺激和EGT2转录调节的基因子集编码直接与EGT2相互作用的蛋白质。基于第一个资助期的结果，第二个资助期的总体目标是更深入地了解决定大麦种子根根角的egt2依赖分子过程。在实验上，我们将通过三组实验来解决这个问题。首先，我们将通过培养过表达EGT2的大麦品系，分析EGT2基因表达对种子根角的调控作用，研究EGT2基因的活性和等位基因变异与大麦种子根角的关系。在这种情况下，我们还将确定EGT2的自然等位基因变异如何调节相应大麦单倍型的根角。在第二组实验中，我们将研究生长素在EGT2介导的大麦根角控制中的作用，通过生成三种大麦品系，这些品系含有生长素外排载体HvPIN3和HvPIN4以及生长素内流载体HvLAX1的翻译荧光融合蛋白，这些蛋白均受EGT2和重力刺激调节。本实验的目的是研究这些标记蛋白在重力刺激野生型和突变型egt2根中的活性和组织细胞型特异性积累。此外，我们将分析HvPIN3、HvPIN4和HvLAX1的突变体以及这些基因与egt2的双突变体。最后，我们将以野生型根为参照，研究这些生长素转运基因和EGT2在所有单突变体和双突变体中的表达。在第三组实验中，我们将对OMT、GXM和HMT基因的突变体进行功能表征，这些基因编码直接与EGT2相互作用的蛋白质，并产生和分析这些基因与EGT2的双突变体。最后，我们将调查这些基因和EGT2在EGT2突变体以及参考野生型根产生的单突变体和双突变体中的表达情况。

The root growth angle defines how cereal roots grow relative to the gravity vector. The root growth angle is among the most important determinants of root system architecture because it controls water uptake capacity and nutrient use efficiency and as a consequence plant yield. In the first funding period of this project, we cloned the EGT2 gene and demonstrated that it encodes an evolutionary conserved STERILE ALPHA MOTIF domain containing protein. Moreover, we demonstrated in transcriptomic experiments a substantial overlap of genes that are regulated by EGT2 and gravistimulation, suggesting a central role of this gene in controlling the molecular networks determining the root angle in barley. Finally, we demonstrated, that a subset of the genes transcriptionally regulated by gravistimulation and by EGT2 encode proteins that directly interact with EGT2. Based on the results of the first funding period, the overall objective of the second funding period is to obtain a deeper understanding of the EGT2-dependent molecular processes underlying the determination of the root angle of barley seminal roots. Experimentally, we will tackle this objective by three sets of experiments. First, we will study the relationship of the barley seminal root angle with the activity and allelic variation of the EGT2 gene by generating barley lines overexpressing EGT2 and analyzing how EGT2 gene expression control the seminal root angle. In this context, we will also determine how natural allelic variation of EGT2 modulates the root angle of the corresponding barley haplotypes. In a second set of experiments, we will survey the role of auxin in the EGT2 mediated control of the root angle in barley by generating three barley lines containing translational fluorescent fusion proteins of the auxin efflux carriers HvPIN3 and HvPIN4 and the auxin influx carrier HvLAX1 all regulated by EGT2 and by gravistimulation. Goal of this experiment is to study the activity and tissue and cell-type specific accumulation of these marker proteins in gravistimulated wild type and mutant egt2 roots. Furthermore, we will analyze tilling mutants of HvPIN3, HvPIN4 and HvLAX1 and double mutants of these genes with egt2. Finally, we will study the expression of these auxin-transport genes and EGT2 in all single and double mutants with reference to wild type roots. In the third set of experiments we will functionally characterize mutants of the genes OMT, GXM and HMT which encode proteins that directly interact with EGT2 and generate and analyze double mutants of these genes with egt2. Finally, we will survey the expression of these genes and of EGT2 in the mutant egt2 and the single and double mutants generated with reference to wild type roots.