Dissecting the role of EPFL9-2 in grapevine root system architecture through rhizotron-based phenotyping

Abstract

Epidermal Patterning Factors (EPFs) constitute a family of small, highly conserved peptides that regulate diverse physiological and developmental processes in plants. In grapevine (Vitis spp.), EPFL9-2 has been identified as a positive regulator of stomatal development and a critical determinant of root system architecture (RSA). Unlike aboveground organs, roots present significant challenges for non-invasive phenotyping. This study aimed to establish a root phenotyping system to deeply characterize the RSA of epfl9-2 knockout (KO) lines of the ‘Kober 5BB’ (Vitis berlandieri x Vitis riparia) rootstock.

Two distinct epfl9-2 KO lines and wild-type (WT) control were cultivated in a greenhouse using a rhizotrons (25 x 50 x 4 cm). Root growth was monitored at three time-points using standardized digital imaging. Furthermore, the RSA was imaged also in grafted plants where an epfl9-2 KO line and Kober WT were grafted with a ‘Syrah’ (WT) scion. Images were processed using RhizoVision Explorer software to quantify key parameters, including total area covered by roots, root length density, and lateral root branching angles.

Our results indicate that epfl9-2 mutants exhibit a more compact RSA compared to the WT, characterized by reduced total length and lateral expansion. Notably, KO lines showed a significant reduction in the gravitropic angle and total root length, while simultaneously displaying increased root diameters and higher density per unit area. Grafting experiments demonstrated that the epfl9-2 root phenotype persists regardless of the presence of a WT scion, suggesting that these altered root traits are independent of the shoot apparatus. This phenotyping approach provides a robust framework for further evaluating the performance of epfl9-2 genotypes under simulated environmental stress. Future research will investigate how these specific root traits may impact scion resilience to abiotic stress in grafted grapevine varieties of economic interest.