Root-preferential OsPUB36 modulates ROS levels and regulates root development

Root development is tightly regulated by spatial gradients of reactive oxygen species (ROS), which coordinate transitions from cell division to elongation and differentiation. Through phylogenetic meta-analysis, we identified a root-preferential subclass of class III PUB E3 ubiquitin ligases ( OsPUB31 – OsPUB37 ). Among them, OsPUB36 was selected as a representative gene for functional characterization. Overexpressing OsPUB36 , an endoplasmic reticulum (ER)-localized protein, resulted in shortened primary roots and elongated root hairs. RNA-seq and proteomic analysis of root hairs from plants overexpressing OsPUB36 revealed significant upregulation of class III peroxidases, key enzymes involved in ROS homeostasis. Histochemical staining confirmed enhanced levels of hydrogen peroxide (H₂O₂) in root hair. However, the loss-of-function mutants ( ospub36 and ospub35 ospub36 ) displayed no obvious phenotypes, suggesting functional redundancy within these gene subclasses. While our yeast two-hybrid screen did not identify direct targets related to ROS or root development, our RNA-seq, proteomic, and histochemical analyses suggest that OsPUB36 and other class III PUBs modulate ROS homeostasis.