Beyond stomata: a novel role of EPFL9-2 in regulating root system architecture and hydraulics in grapevine

Epidermal Patterning Factors (EPFs) are small plant peptides crucial for various physiological and developmental processes. While EPFL9 positively regulates leaf stomatal formation in crops, its function in other organs remains poorly understood. This study elucidates the role of VviEPFL9-2 in grapevine roots. Morphological and physiological traits were assessed in epfl9-2 knock-out (KO) lines of the rootstock 'Kober 5BB' under well-watered (WW) and water-stressed (WS) conditions, and in grafted plants with combinations of WT and KO scions and rootstocks. A multi-omics and immune-histological analysis of root tissues was also performed. KO root apparatus had a significant reduction in geotropic angle and total length, accompanied by an increase in root diameter and KO stems displayed more xylem vessels with a smaller section area. This phenotype persisted even when KO rootstocks were grafted with a WT scion. KO lines exhibited improved water-use efficiency under both WW and WS conditions. At the molecular level, edited roots showed strong activation of the lignin and salicylic acid pathways, confirmed by the confocal analysis of root tips and by the phytohormone profile. Our results demonstrate for the first time that VviEPFL9-2 loss-of-function induces profound root anatomical, morphological, and physiological modifications that are potentially beneficial for drought tolerance.