Recently, Molecular Plant published online a research article from Professor Yu Sun’s team at Hebei Normal University, titled “Two E-clade Protein Phosphatase 2Cs enhance ABA signaling by dephosphorylating ABI1 in Arabidopsis” (Impact Factor: 24.1). This study revealed that the E-clade PP2C phosphatases ADEP1 and ADEP2 enhance protein stability of ABI1 through dephosphorylation, thereby enabling a rapid and precise regulation of the plant abscisic acid (ABA) response.
In the experiments, Arabidopsis plants older than three weeks were transferred to the JIUPO artificial climate chamber and cultivated in soil under a 22 °C photoperiod regime and 55% relative humidity.
A phosphatase yeast library screen in Arabidopsis was used to identify protein phosphatases capable of dephosphorylating ABI1. The results identified two E-clade PP2C phosphatases, DEP1 and ADEP2, which showed strong interaction with both ABI1 and ABI2 (Figure 1A). Subsequent experiments including luciferase complementation imaging (LCI) (Figure 1B), co-immunoprecipitation (Co-IP) (Figures 1C–D), pull-down assays (Figure 1E), and gel overlay assays (Figure 1F) confirmed the physical interaction between ADEP1/2 and ABI1/2. (Note: MBP-tagged ADEP1/2 proteins were able to directly bind GST-tagged ABI1/2 immobilized on nitrocellulose membranes, further validating the physical association.)
Figure 1. Interaction of ADEP1 and ADEP2 with ABI1 and ABI2
To investigate the functional roles of ADEP1 and ADEP2 in ABA responses, adep1, adep2 single mutants and adep1/adep2 double mutants were generated. These mutants exhibited significantly reduced sensitivity to ABA, as demonstrated by higher seed germination rates, longer primary root lengths, and reduced stomatal closure upon ABA treatment. The double mutant showed even lower sensitivity than either single mutant. Complementation with ADEP1/2 restored the phenotypes related to seed germination, root growth, and stomatal behavior (Figures 2A–F), indicating that ADEP1/2 act as positive regulators of the ABA response and function redundantly.
Because ABA signaling induces accumulation of the ABI5 protein, its levels were examined in the adep1/2 mutants and complemented lines. The results showed reduced ABI5 protein levels in the mutants, while the complemented lines restored levels comparable to wild type (Figures 2G–H), indicating that the adep1/2 mutant phenotypes are associated with ABA signaling, and that ADEP1/2 act as positive components in the ABA signaling pathway.
Figure 2. Model of ADEP1/2 precisely regulating ABA response by dephosphorylating ABI1
Furthermore, transcriptomic (RNA-seq) analysis was conducted to study the role of ADEP1/2 in ABA signaling. The results showed that the number and magnitude of ABA-induced differentially expressed genes (DEGs) were markedly higher in wild-type Col-0 plants compared to the adep1/2 mutants (Figures 3A–C). These findings demonstrate that loss of ADEP1/2 weakens the transcriptional response to ABA.
Figure 3. RNA-seq analysis supports ADEP1/2 as positive regulators of ABA signaling
This study represents another important advancement by Professor Yu Sun’s group at Hebei Normal University in understanding how E-clade PP2Cs regulate the ABA signaling pathway. Previously, the team discovered that EGR1/2, members of the PP2C E subfamily, positively regulate ABA signaling by dephosphorylating Ser31 of SnRK2.2, thereby reducing its kinase activity. The current study reveals a novel mechanism in which the homologous proteins ADEP1/2 modulate ABA signaling by dephosphorylating ABI1/2, offering new theoretical insight into how plants achieve a precise balance between stress response and developmental growth.