High-throughput screening and structure-guided design of small molecules enable modulation of SAL-PAP stress signaling.

Chloroplasts sense environmental stress and activate chloroplast-to-nucleus retrograde signalling, reprogramming nuclear gene expression for plant acclimation. One such pathway is regulated by the phosphatase, SAL, which hydrolyses the nucleotide signal 3'-phosphoadenosine 5'-phosphate (PAP), a by-product of secondary sulfur metabolism. In Arabidopsis thaliana, genetic loss of AtSAL1 elevates PAP and enhances stress tolerance but causes pleiotropic growth defects. To uncouple stress signalling from genetic pleiotropy, we conducted a high-throughput in vitro screen of 13 000 small molecules and identified V20, a competitive inhibitor of AtSAL1 with 3-fold greater potency than the known Li+ inhibitor. Structural analoguing of V20 and biochemical assays defined key pharmacophore features required for inhibition. Accelerated molecular dynamics simulations revealed two previously uncharacterized V20-binding pockets adjacent to the catalytic site. V20 binding induces conformational changes which restrict substrate access to the catalytic site. Exogenous application of V20 to Arabidopsis led to increased PAP accumulation, activated PAP-responsive gene expression, and enhanced oxidative tolerance. Our findings reveal new insights into the regulatory domains of SAL enzymatic activity for control of PAP-mediated signalling and establish a proof-of-concept for targeted chemical modulation of SAL activity, which offers novel strategies to selectively manipulate oxidative stress signalling and sulfur metabolism in plants.