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

根生长角度定义了谷物根相对于重力矢量的生长方式。根系生长角是根系构型的最重要的决定因素之一，因为它控制着水分吸收能力和养分利用效率，从而控制着植物产量。在该项目的第一个资助期内，我们克隆了EGT2基因，并证明它编码一个进化保守的含无菌α基序结构域的蛋白质。此外，我们在转录组学实验中证明了EGT2和gravistimulation调控的基因的大量重叠，表明该基因在控制决定大麦根角的分子网络中起着核心作用。最后，我们证明，一个子集的基因转录调控重力刺激和EGT2编码的蛋白质，直接与EGT2相互作用。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的根角的EGT2依赖性分子过程。在实验上，我们将通过三组实验来解决这个问题。首先，我们将通过产生过量表达EGT2的大麦品系并分析EGT2基因表达如何控制种子根角度来研究大麦种子根角度与EGT2基因的活性和等位变异的关系。在这种情况下，我们也将确定如何自然的等位基因变异EGT2调制相应的大麦单倍型的根角。在第二组实验中，我们将调查生长素在EGT2介导的控制大麦根角的作用，通过产生三个大麦系含有翻译荧光融合蛋白的生长素外排载体HvPIN 3和HvPIN 4和生长素内流载体HvLAX 1都调节EGT2和重力刺激。本实验的目的是研究这些标记蛋白在重力刺激的野生型和突变体egt2根中的活性以及组织和细胞类型特异性积累。此外，我们将分析HvPIN 3、HvPIN 4和HvLAX1的分蘖突变体以及这些基因与egt2的双突变体。最后，我们将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的表达与野生型根。在第三组实验中，我们将在功能上表征编码与EGT2直接相互作用的蛋白质的基因OMT、GXM和HMT的突变体，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的表达和EGT2的突变体EGT2和单和双突变体产生的参考野生型根。

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.