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

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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.