Kinase SnRK1.1 Regulates SLAH3 Engaged In Nitrate-Dependent Alleviation Of Ammonium Toxicity In Arabidopsis

Nitrate(NO₃^-)and ammonium(NH₄⁺)are major inorganic nitrogen(N)nutrient for plants.Compared with NO₃^-,when NH₄⁺serves as the sole or dominant N source,it ussualy causes growth inhibition in many plants,known as ammonium toxicity.Elevating p H in the medium can significantly alleviate ammonium toxicity,indicating that medium acidification is one of the main causes of ammonium toxicity.Small amount of NO₃^-can significantly mitigate ammonium toxicity,which is known as nitrate-depedent alleviation of ammonium toxicity.Our previous study revealed that anion channel SLAC1 homologue 3(SLAH3)is involved in this process.However,the detailed mechanism how SLAH3 regulates this process is still unknown.In this study,a yeast two-hybrid screen was used to identify proteins that interact with SLAH3.Sn RK1.1 was identified as a SLAH3-interactor in Arabidopsis(Arabidopsis thaliana).As the homolog of AMPK in mammals and SNF1 in yeast,Sn RK1 in plants is involved in regulating energy balance.These kinases are highly conserved in the structure and function.AMPK/SNF1/Sn RK1 family protein kinases are signaling regulators involved in energy homeostasis and various stress responses.They are involved in cellular responses to energy stress and interact with molecules of other signaling components to regulate growth,stress,and nutrient balance.First,yeast two-hybrid(Y2H)assay was used to confirm that Sn RK1.1 interacts with SLAH3 and that loss of Sn RK1.1 kinase activity does not affect its interaction with SLAH3.Second,in order to understand the biological significance of Sn RK1.1 in regulating SLAH3,GUS(?-glucuronidase)reporter gene expression driven by the own promoters of SLAH3 and Sn RK1.1 was analyzed.The results showed that both Sn RK1.1 and SLAH3are highly expressed in roots.Meanwhile,protein interaction experiments confirmed the interaction between Sn RK1.1 and SLAH3 in vivo.According to the phenotypes of Sn RK1.1 overexpression plants,this study demonstrated that Sn RK1.1 is involved in regulating the response to high-NH₄⁺/low-p H stress,which is influenced by sugar signaling.The genetic results indicated Sn RK1.1 is a negative regulator of SLAH3.Kinase assays further showed Sn RK1.1 strongly phosphorylates S601 at SLAH3 C-terminal.Most of the reported regulatory regions of SLAH3 and its homologous protein SLAC1 are currently in the intracellular N-terminal,which is important for their positive regulation.However,this study found Sn RK1.1 can phosphorylate S601 at SLAH3 C-terminal.It was demonstrated that SLAH3 S601 plays an important role under high-NH₄⁺/low-p H conditions.Finally,Sn RK1.1 migrates from cytoplasm to nucleus under high-NH₄⁺/low-p H conditions,releasing the inhibition of SLAH3 activity.In summary,this study concluded that the translocation of Sn RK1.1 from cytoplasm to nucleus under high-ammonium stress releases the inhibition on SLAH3,which allows SLAH3-mediated NO₃^-efflux leading to alleviation of high-NH₄⁺/low-p H stress in the rhizosphere.This study revealed that the C-terminal phosphorylation also plays important role in SLAH3 regulation and provided a new insight into the molecular mechanisms of nitrate-dependent mitigation of ammonium toxicity in plants.