Supplementary material from "Identification of F-Box Proteins in ABA- and GA-regulated Seed Germination: Interaction of GASA1 signaling peptide and ABA-Induced Ubiquitination"

Large gene families and the frequent overlapping functions of homologous genes remain a major challenge for functional forward genetic screens in plants. The recent development of homology-based gene silencing using computationally generated artificial microRNAs (amiRNAs) has been demonstrated to be a promising tool for unbiased functional genomics in plants by circumventing redundancies and lethality. In this study, through a forward genetics screen, we isolated an abscisic acid-insensitive amiRNA line targeting five previously uncharacterized F-box Insensitive to ABA (FIA) genes. Notably, a triple mutant in the identified FIA genes FIA1, FIA3 and FIA4 (f1/f3/f4), that are expressed in germinating seeds, exhibited insensitivity in abscisic acid (ABA) inhibition of seed germination. In contrast, this ABA insensitivity was not observed in a double mutant of two FIA genes FIA1 and FIA4. Further investigation of the FIA1 interactome using F-box decoy lines revealed the gibberellin (GA)-responsive GASA1 protein that has been reported to encode a small stress signaling peptide as an interacting partner. We found that ABA promoted the ubiquitination of GASA1 in Arabidopsis, leading to its degradation via the 26S proteasome pathway. Together, our study reveals that ABA represses seed germination through FIA proteins and regulates the FIA interactor, GA-responsive GASA1.