Loss of function of the AP2/ERF transcription factor StGAME9 abolishes activation and induction of steroidal glycoalkaloid biosynthesis in potato plants.

Abstract

Steroidal glycoalkaloids (SGAs) are toxic defense substances present in certain species of the Solanaceae, including major crops such as eggplant, tomato, and potato. GLYCOALKALOID METABOLISM 9 (GAME9) was first identified in tomato and potato as an APETALA 2/ETHYLENE RESPONSE FACTOR (AP2/ERF) transcription factor regulating key genes in SGA biosynthesis. However, the spatial effects of endogenous GAME9 in potato remain largely unexplored, particularly in tubers, highlighting a knowledge gap in understanding activation and induction of SGA biosynthesis in this important staple food crop. Here, we generated StGAME9 knockout mutants in potato via DNA-free CRISPR/Cas9. Compared to the wild type, knockout mutants contained significantly reduced SGA levels in leaves, and were almost free of SGAs in tubers. Notably, SGA accumulation remained minimal in the mutant tubers even under two SGA-inducing conditions; wounding and light exposure, indicating a loss of inducible SGA biosynthesis. Integrated transcriptomic and metabolomic characterization of knockout mutants (Stgame9) revealed extensive reprogramming of gene expression and metabolism, affecting not only SGA and sterol pathways but also a broader range of metabolic processes, with stress-related metabolic responses being attenuated in Stgame9 tubers. Despite these changes, Stgame9 plants displayed a normal growth phenotype under both greenhouse and field conditions. Our findings substantiate a pivotal role of GAME9 in potato for the regulation of basal and induced SGA biosynthesis. The results further indicate that StGAME9 is involved in the regulation of a broader, complex, and interconnected network along biosynthetic pathways, where potato metabolism exhibits substantial robustness and compensatory capacity to buffer the loss of StGAME9.