Salt-sensitive signaling networks in the mediation of K⁺/Na⁺ homeostasis gene expression in Glycyrrhiza uralensis roots

We investigated the effects of salt-sensitive signaling molecules on ionic fluxes and gene expression related to K⁺/Na⁺ homeostasis in a perennial herb, Glycyrrhiza uralensis, during short-term NaCl stress (100 mM, 24 h). Salt treatment caused more pronounced Na⁺ accumulation in root cells than in leaf cells. Na⁺ ions were mostly compartmentalized in vacuoles. Roots exposed to NaCl showed increased levels of extracellular ATP (eATP), cytosolic Ca²⁺, H₂ O₂, and NO. Steady-state flux recordings revealed that these salt-sensitive signaling molecules enhanced NaCl-responsive Na⁺ efflux, due to the activated Na⁺/H⁺ antiport system in the plasma membrane (PM). Moreover, salt-elicited K⁺ efflux, which was mediated by depolarization-activated cation channels, was reduced with the addition of Ca²⁺, H₂ O₂, NO, and eATP. The salt-adaptive effects of these molecules (Na⁺ extrusion and K⁺ maintenance) were reduced by pharmacological agents, including LaCl₃ (a PM Ca²⁺ channel inhibitor), DMTU (a reactive oxygen species scavenger), cPTIO (an NO scavenger), or PPADS (an antagonist of animal PM purine P2 receptors). RT-qPCR data showed that the activation of the PM Na⁺/H⁺ antiport system in salinized roots most likely resulted from the upregulation of two genes, GuSOS1 and GuAHA, which encoded the PM Na⁺/H⁺ antiporter, salt overly sensitive 1 (SOS1), and H⁺ -ATPase, respectively. Clear interactions occurred between these salt-sensitive agonists to accelerate transcription of salt-responsive signaling pathway genes in G. uralensis roots. For example, Ca²⁺, H₂ O₂, NO, and eATP promoted transcription of GuSOS3 (salt overly sensitive 3) and/or GuCIPK (CBL-interacting protein kinase) to activate the predominant Ca²⁺ -SOS signaling pathway in salinized liquorice roots. eATP, a novel player in the salt response of G. uralensis, increased the transcription of GuSOS3, GuCIPK, GuRbohD (respiratory burst oxidase homolog protein D), GuNIR (nitrate reductase), GuMAPK3, and GuMAPK6 (the mitogen-activated protein kinases 3 and 6). Moreover, GuMAPK3 and GuMAPK6 expression levels were enhanced by H₂ O₂ in NaCl-stressed G. uralensis roots. Our results indicated that eATP triggered downstream components and interacted with Ca²⁺, H₂ O₂, and NO signaling to maintain K⁺/Na⁺ homeostasis. We propose that a multiple signaling network regulated K⁺/Na⁺ homeostasis in NaCl-stressed G. uralensis roots.