Identification of F-box proteins in ABA- and GA-regulated seed germination: interaction of GASA1 signalling 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 (ABA)-insensitive amiRNA line targeting five previously uncharacterized <i>F-box Insensitive to ABA</i> (<i>FIA</i>) genes. Notably, a triple mutant in the identified <i>FIA</i> genes <i>FIA1</i>, <i>FIA3</i> and <i>FIA4</i> (<i>f1/f3/f4</i>), that are expressed in germinating seeds, exhibited insensitivity in ABA inhibition of seed germination. In contrast, this ABA insensitivity was not observed in a double mutant of two <i>FIA</i> genes <i>FIA1</i> and <i>FIA4</i>. 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 signalling peptide as an interacting partner. We found that ABA promoted the ubiquitination of GASA1 in <i>Arabidopsis</i>, 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.This article is part of the theme issue 'Crops under stress: can we mitigate the impacts of climate change on agriculture and launch the 'Resilience Revolution'?'.