Title : AtAIRP2, an Arabidopsis RING E3 Ubiquitin Ligase, Positively Regulates ABA- and High Salinity-mediated Seed Germination by Stimulating ATP1/SDIRIP1 Turn-over
Abstract:
A systemic network of proteolytic regulation via the ubiquitin-26S proteasome system plays significant roles in environmental adaptation and hormone signal transduction in plants. The abscisic acid (ABA) signaling is an essential pathway plants to survive against the abiotic stress, especially drought and osmotic stresses. AtAIRP2 is a cytosolic RING-type E3 ubiquitin (Ub) ligase that positively regulates an ABA response in Arabidopsis. Yeast two-hybrid screening using AtAIRP2 as bait identified ATP1 (AtAIRP2 Target Protein 1) as a substrate of AtAIRP2. ATP1, previously known as SDIRIP1 of which SDIR1 substrate protein, was identified as an interacting partner of AtAIRP2, a positive regulator of ABA-mediated drought response. ATP1 was accordingly renamed ATP1/SDIRIP1. A specific interaction between AtAIRP2 and ATP1/SDIRIP1 and ubiquitination of ATP1/SDIRIP1 by AtAIRP2 were demonstrated in vitro and in planta. The turn-over of ATP1/SDIRIP1 was regulated by AtAIRP2 in cell-free degradation and protoplast co-transfection assays. The ABA-mediated germination assay of 35S:ATP1/SDIRIP1-RNAi/atairp2 double-mutant progeny revealed that ATP1/SDIRIP1 acts downstream of AtAIRP2. AtAIRP2 and SDIR1 reciprocally complemented the ABA- and salt-insensitive germination phenotypes of sdir1 and atairp2 mutants, respectively, indicating their combinatory roles in seed germination. Subcellular localization and BiFC experiments in the presence of MG132, a 26S proteasome inhibitor, showed that AtAIRP2 and ATP1/SDIRIP1 were co-localized to the cytosolic spherical body, which lies in close proximity to the nucleus, in tobacco leaf cells. The 26S proteasome subunits RPN12a and RPT1 and molecular chaperones HSP70 and HSP104 were co-localized to these discrete punctae-like structures. These results raised the possibility that AtAIRP2 and ATP1/SDIRIP1 interact in the cytosolic spherical compartment. Collectively, our data suggest that down-regulation of ATP1/SDIRIP1 by the combinatory actions of AtAIRP2 and SDIR1 RING E3 ligases is critical for ABA and high salinity responses during germination in Arabidopsis.
