The Anion Channel SLAH3 Interacts With Potassium Channels To Regulate Nitrogen-potassium Homeostasis And The Membrane Potential In Arabidopsis

Nitrogen(N)and potassium(K)are essential macronutrients for plants.Sufficient N and K uptake from the environment is required for successful growth and development.However,how N and K influence each other at the molecular level in plants is largely unknown.In this study,we found loss-of-function mutation in SLAH3(SLAC1 HOMOLOGUE 3),encoding a NO3-efflux channel in Arabidopsis thaliana,enhanced tolerance to high KNO3 concentrations.Unexpectedly,K+ and NO3-medium proved that slah3 mutants show less sensitivity to high K+alone but not NO3-alone.Ion exclusion experiments showed that the tolerance of slah3 to high concentrations of K+,independent of Na+,Cl-or osmotic stress.Arabidopsis seedling transfer experiments showed that SLAH3 functions mainly in roots in response to high K+stress.SLAH3 belongs to the SLAC/SLAH family,SLAH3 is the only SLAC/SLAH gene that plays a dominant role in the high K+response.Increasing NO3-concentration in high K+medium resulted in reduced phenotypic differences between wild-type(WT)and slah3 mutant plants,indicating that SLAH3mediated high-K+tolerance was closely related to NO3-concentrations and appropriate NO3-can alleviate high K+stress.Under high K+conditions,NO3-efflux was reduced and K+efflux was enhanced in slah3 mutants,demonstrating that SLAH3-mediated NO3-efflux and SLAH3-affected K+flux are important in the response to high K+.SLAH3 is involved in NO3--K+ homeostasis.The complementation lines in slah3 background,SLAH3WT can restore the slah3 phenotype to WT-like one under high K+condition.SLAH3F517L with partial channel activity can partially restore slah3 phenotype,while SLAH3G264D without channel activity failed to alter slah3 phenotype.Genetic experiments have demonstrated that the channel activity of SLAH3 is critical in plant response to high K+stress.Under high K+conditions,the K+content of Col-0 and slah3 increased significantly,and the NO3-content decreased significantly.High K+leads to an imbalance in N-K homeostasis in plants.But there are no significant differences in K+and NO3-contents between Col-0 and slah3 under high K+conditions.The phenotypic difference between Col-0 and slah3 is not due to excess K+or deprivation of NO3-in the plants.NO3-and K+are the abundant anions and cations in plant cells.In addition to being nitrogen and potassium nutrients,NO3-and K+also play an important role in charge balance and membrane potential homeostasis.In WT,prompt and severe membrane potential depolarization was induced when treated with high concentration of KCl,while the application of high concentration of KNO3 led to a lower level of depolarization.By contrast,KCl caused less membrane potential depolarization in slah3.The membrane potential results support the observed phenotype that slah3 is less sensitive to high concentrations of K+.Exogenously applied NO3-promoted membrane repolarization.We hypothesize that NO3-is an important anion in the regulation of plant membrane potential,and more importantly,that SLAH3 plays an important role in regulating membrane potential by mediating NO3-efflux.The potassium efflux channels GORK and SKOR can be activated by depolarized membrane potential.The gork and skor mutants show more sensitive under high K+conditions,and the double mutants gork slah3 and skor slah3 can tolerate high potassium stress with a phenotype consistent with slah3.After high K+treatment,compared with WT,less depolarization is observed in slah3 mutants and enhanced depolarization occurs in gork/skor mutants and the double mutants gork slah3 and skor slah3 showed a similar degree of depolarization to slah3 single mutant.The enhancement of high K+induced membrane potentials depolarization of gork and skor relies on SLAH3.SLAH3 mediated NO3-efflux and GORK/SKOR mediated K+efflux are involved in membrane potential regulation in response to high K+conditions.The genetic results indicated that the potassium ion efflux channels GORK and SKOR are involved in the plant response to high K+stress,and SLAH3 acts upstream of GORK and SKOR.SLAH3 was expressed in root hairs and root stellar cells.SLAH3 had similar expression patterns to GORK and SKOR in roots.BiFC and Co-IP experiments showed that the two potassium efflux channels GORK and SKOR interacted with SLAH3.Electrophysiological results showed that SLAH3 inhibited the activity of GORK and SKOR in Xenopus laevis oocytes.This inhibition is independent of the channel activity of SLAH3.The NO3-channel activity of SLAH3 is not affected by GORK and SKOR.The results of electrophysiological experiments were further verified in plants by NMT experiments.In this study,a two-layer regulation mechanism of SLAH3 on the K+efflux channel was proposed for the first time:(1)SLAH3 regulated the voltage-dependent K+efflux channels GORK/SKOR through the changes in the free running membrane potential induced by the electrogenic transport processes.(2)SLAH3 is also presented as an interaction partner of GORK and SKOR,inhibiting their activities.This study reveals that NO3-channel SLAH3 forms functional units with K+channels GORK and SKOR to modulate membrane potential by coordinating nitrogen-potassium balance.In agricultural production,balanced application of N and K fertilizers is critical.This study clarified the molecular mechanism of the anion channel SLAH3 forms complexes with potassium channels to modulate membrane potential via regulating nitrogen-potassium homeostasis,providing a theoretical basis for improving plant nitrogen and potassium utilization,increasing crop yield and reducing environment pollution.The elucidation of the molecular mechanisms of SLAH3-GORK/SKOR regulation is potentially beneficial for the cultivation of new crop varieties that are more adaptable to N-K imbalance environments in agricultural practice.