Identification of putative substrates of the PRT6 N-degron Pathway in cereals

Abstract

Crop yields worldwide are significantly impacted by abiotic stresses including drought, high salt concentration, and waterlogging. The Plant Cysteine Oxidase is a part of the PRT6 N-degron pathway, a key component of ubiquitin-mediated proteolysis, regulates plant responses to these stresses through modulate the turnover of proteins involved in stress sensing and tolerance. The N-degron pathway of ubiquitin-mediated proteolysis regulate protein half-life based on Methionine-Cysteine (Met-Cys) in N-terminal domain that give E3 ligase ubiquitin ability to recognize these proteins for degradation by 26S proteasome. The first identified N-degron pathway plant substrates were the Ethylene-Responsive Factor Group VII transcription factors (ERFVII), identified in A. thaliana and were considered as oxygen and Nitric oxide sensors, appearing on the Cys-PRT6 branch of this pathway. This pathway therefore might be a potential to stabilize plant yields on facing abiotic stresses if we understand the key proteins involved in this process and how they are regulated. In this study we focus on identifying two substrates of Cys/Arg branch PRT6 N-degron pathway in barley based on N-terminal conserved motif of these proteins initiated by Methionine-Cysteine (Met-Cys). The first candidate was Hordeum vulgare Ethylene-Responsive Factor 1-like (HvERF1-like) a member of ERFVII (MCGGAIL) and well-known substrate of this pathway. The second novel was Oryza sativa F-box domain-containing protein (OsF-box) and substrate does not belong to ERFVII but initiated with Met-Cys. Both candidates identified in previous work in silico and cloned to performed using the T7 rabbit reticulocyte Lysate system as well as western blot to clarify if these consider a substrate of this branch. In our research both Met-Cys proteins are considering substrates of the Cys/Arg N-degron pathway in vitro. In addition, in vivo analysis using Dual-Glo luciferase assay was optimized to confirm western blot result of OsF-box substate. But still the protocol requires additive optimization to be applied as in vivo assay for PRT6 N-degron pathway. To conclude, if our expectation is truly that these substates belong to Cys/Arg PRT6 N-degron pathway could give potential purpose for phenotypic screening and manipulate to generate germplasm has ability to tolerate abiotic stress.