Salt-Sensitive Signaling Networks in the Mediation of K+/Na+ Homeostasis Gene Expression in Glycyrrhiza uralensis Roots

Tao Lang, Shurong Deng, Nan Zhao, Chen Deng, Yinan Zhang, Yanli Zhang, Huilong Zhang, Gang Sa, Jun Yao, Caiwu Wu, Yanhong Wu, Qun Deng, Shanzhi Lin, Jianxin Xia, Shaoliang Chen

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 Ca(2+), H2O2, 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 Ca(2+), H2O2, NO, and eATP. The salt-adaptive effects of these molecules (Na(+) extrusion and K(+) maintenance) were reduced by pharmacological agents, including LaCl3 (a PM Ca(2+) 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, Ca(2+), H2O2, NO, and eATP promoted transcription of GuSOS3 (salt overly sensitive 3) and/or GuCIPK (CBL-interacting protein kinase) to activate the predominant Ca(2+)-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 H2O2 in NaCl-stressed G. uralensis roots. Our results indicated that eATP triggered downstream components and interacted with Ca(2+), H2O2, 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.