The abscisic acid (ABA) signaling pathway is crucial for regulating downstream ABA-responsive genes, which influence plant responses to ABA and abiotic stresses. However, many ABA-responsive genes remain poorly characterized. This study reports on the identification and characterization of ABA up-regulated EAR motif-containing proteins (AtAUEs), a novel family of EAR motif-containing proteins in Arabidopsis thaliana. From a previous transcriptome dataset, AtAUEs were identified as a family of unknown-function ABA-response genes with only five members, and the up-regulation of AtAUEs by ABA was further confirmed by quantitative RT-PCR (qRT PCR). All AtAUEs contain at least one LxLxL EAR motif and can repress reporter gene expression in Arabidopsis protoplasts. We generated CRISPR/Cas9 gene-edited ataue1, ataue2 and ataue3 single, ataue1 ataue2 (ataue12) double, and ataue1 ataue2 ataue3 (ataue123) triple mutants, as well as transgenic plants overexpressing AtAUE1, and examined their ABA sensitivity. We found that the single and double mutants displayed wild-type responses to ABA treatment, while the ataue123 triple mutants showed increased sensitivity in seed germination and cotyledon greening assays but decreased sensitivity to ABA treatment in root elongation assays. Conversely, the 35S:AtAUE1 showed decreased sensitivity in seed germination and cotyledon greening assays but increased sensitivity to ABA treatment in root elongation assays. The qRT PCR results show that the expression level of ABI5 was increased in the ataue123 mutants and decreased in the 35S:AtAUE1 plants. These findings suggest that AtAUEs function redundantly to regulate ABA responses in Arabidopsis, likely by modulating the expression of key regulatory genes in ABA-signaling pathway.
Keywords: Arabidopsis; AtAUEs; CRISPR/Cas9; EAR proteins; abscisic acid; transcription repressor.