Analysis of HY5 phosphorylation and upstream signaling using phosphomimetics and double mutant analysis

Abstract

ELONGATED HYPOCOTYL 5 (HY5) is a transcription factor that coordinates nitrogen uptake in the roots with carbon fixation in the shoot in Arabidopsis thaliana. HY5 is regulated by phosphorylation, but much about this process remains unknown. Two receptors involved in nitrogen responses may be involved: the CLAVATA1 (CLV1) receptor, which is involved in regulating lateral root initiation in response to nitrogen, and XYLEM INTERMIXED WITH PHLOEM (XIP1), a receptor that is involved in activating nitrogen uptake. In this study, epistasis analysis determined that XIP1 and HY5 most likely function in interconnected pathways involving sucrose. Once identified, hy5 clv1 double mutants will be used in an additional epistasis test to determine how HY5 interacts with CLV1 and XIP1. In addition, this study has focused on using phosphomimetics to analyze two phosphorylation sites, S23 and S36. I have completed the first step of site-directed mutagenesis by successfully piecing together the gene with the substituted amino acid. In the future, lateral root growth assays of the resulting mutants can be employed in order to determine whether the change had phenotypic effects. This study reveals information about the signaling pathways that regulate HY5, a gene involved in the vital process of nitrogen uptake.