An Engineered Abscisic Acid Receptor Enhances ABA Signaling and Improves Abiotic Stress Tolerance in Rice

Abscisic acid (ABA) is a key phytohormone that orchestrates adaptive responses in plants exposed to abiotic stress. The ABA signaling cascade is triggered by ABA-mediated binding of PYRABACTIN RESISTANCE1-LIKE (PYL) receptors to clade A Type 2 C protein phosphatases (PP2CAs). In Arabidopsis thaliana, several constitutively active variants of ABA receptors have been described, offering valuable tools for improving plant tolerance to environmental stresses. To identify amino acid residues in the rice ABA receptor OsPYL5 that are involved in ABA-independent interactions, we implemented a random mutagenesis strategy followed by yeast two-hybrid (Y2H) screening. We identified residues L-93 and N-102 as key residues that influence the ABA independent interaction of OsPYL5 with OsPP2CA51. Substituting these residues with T or Y significantly enhanced the activity of an ABA-responsive reporter in rice protoplasts, even in the absence of ABA treatment. We therefore engineered a double-point mutant, OsPYL5^{L93W N102Y}, which demonstrated a strong ABA-independent interaction with OsPP2CA51 in Y2H assays, elevated activation of ABA-responsive reporter in rice protoplasts, and suppression of PP2CA phosphatase activity in vitro in the absence of ABA. Transgenic rice lines overexpressing OsPYL5^{L93W N102Y} showed delayed germination and growth retardation in the absence of ABA treatment. They also exhibited increased sensitivity to ABA during germination and in early seedling growth assays compared to an OsPYL5-overexpressing transgenic rice line (OsPYL5-OX). Moreover, compared to OsPYL5-OX, they showed dramatic upregulation of ABA-responsive genes both without ABA and with low concentrations of ABA. These transgenic lines also showed enhanced tolerance to drought and salt stress compared to both the control cultivar and OsPYL5-OX. Taken together, our findings not only identify key residues of OsPYL5 that enable ABA-independent receptor function, but also highlight the feasibility of engineering ABA receptors to improve abiotic stress tolerance.