SLAH3-like anion channels mediate nitrate-dependent alleviation of ammonium toxicity in rice

Nitrate (NO₃⁻) and ammonium (NH₄⁺) are major inorganic nitrogen (N) sources for plants. However, when supplied as the sole N source, NH₄⁺ often inhibits growth in many species, a phenomenon known as ammonium toxicity. NO₃⁻ can alleviate this toxicity, a process referred to as nitrate-dependent alleviation of ammonium toxicity. In Arabidopsis thaliana , the SLOW ANION CHANNEL HOMOLOG 3 (AtSLAH3), which mediates NO₃⁻ efflux, plays a key role in this process. However, the roles of its homologs in rice ( Oryza sativa ) have remained unknown. In this study, we identified nine OsSLAC/SLAH genes in the rice genome, comprising OsSLAC1 and OsSLAH1 – OsSLAH8 . Functional complementation assays in the atslah3-4 mutant showed that expression of OsSLAC1 , OsSLAH3 , OsSLAH4 , OsSLAH5 , or OsSLAH8 rescued the high-ammonium hypersensitivity phenotype, restoring chlorophyll content, biomass, and root growth to wild-type levels. Non-invasive Micro-test Technology (NMT) further confirmed that these complementation lines regained NO₃⁻ efflux capacity under ammonium stress. Phylogenetic and structural analyses identified the root-expressed OsSLAH3 , OsSLAH4 , and OsSLAH5 proteins as functional counterparts of AtSLAH3 , supporting their specific roles in nitrate-dependent alleviation of ammonium toxicity. We further demonstrated that excessive NH₄⁺ causes ammonium toxicity in rice primarily through rhizosphere acidification. Loss-of-function mutations in OsSLAH3 , OsSLAH4 , or OsSLAH5 impaired this nitrate-dependent alleviation. Notably, OsSLAH3/4/5 physically interact with NITRATE TRANSPORTER 1.1B (OsNRT1.1B), a key regulator of ammonium tolerance. Collectively, our findings reveal that OsSLAH3/4/5 mediate NO₃⁻ efflux to maintain rhizosphere pH homeostasis, thereby mitigating NH₄⁺ stress in rice. This study establishes functional conservation of the SLAC/SLAH family in ammonium detoxification between dicot Arabidopsis and monocot rice and reveals a functional coupling mechanism involving OsNRT1.1B.