AtAUGs Suppress the Expression of PP2C Genes to Redundantly Regulate ABA Responses in Arabidopsis

The modulation of plant responses to abscisic acid (ABA) and/or abiotic stresses can be manipulated by the expression of ABA-responsive genes, which is affected by phytohormone ABA. While some ABA-responsive genes have been shown to regulate plant responses to ABA and/or abiotic stresses, the functions of numerous ABA-responsive genes remain unknown. Therefore, characterizing these unstudied genes would provide a practical way to identify novel regulators of plant adaptations to ABA and/or abiotic stresses. Here, we characterized four closely related unstudied ABA-responsive genes in Arabidopsis thaliana, named Arabidopsis thaliana ABA-up regulated genes (AtAUGs). We found that ABA treatment induces AtAUGs expression level, and our results in transfected protoplasts show that AtAUGs exhibit nucleus localization and downregulate the co-transfected reporter expression level. The results of ABA sensitivity assays, including seed germination, cotyledon greening, and root extension assay show that transgenic plants overexpressing AtAUGs had increased sensitivity, but ataugs mutants generated by isolating T-DNA insertion lines or through CRISPR/Cas9 gene-editing of AtAUGs had decreased sensitivity; in addition, the greatest decrease in ABA sensitivity was observed in the ataug1 ataug2 ataug3 ataug4 (ataug1234) quadruple mutants. The qRT-PCR results show that the expression levels of several Type 2C Protein Phosphatase (PP2C) genes, the key negative regulator genes of ABA signaling including PP2CA, Hypersensitive to ABA 1 (HAB1), HAB2, Highly ABA-Induced PP2C protein 3 (HAI3), ABA-Hypersensitive Germination 1 (AHG1), and ABA Insensitive 2 (ABI2) decreased in 35S:AtAUGs transgenic plants, but increased in the ataug1234 quadruple mutants. Taken together, these results suggest that AtAUGs are ABA-responsive genes, and AtAUGs positively regulate ABA responses in a redundant manner, by downregulating the expression of crucial negative regulator genes in ABA signaling.