The ZxCNIH4-ZxHKT1;1 module responding to external NaCl orchestrates Na⁺ partitioning in a xerophyte

Na⁺-accumulating xerophytes adapt to arid environments by sequestering large amounts of Na⁺ in their leaves as an osmolyte. Zygophyllum xanthoxylum is one such species, exhibiting growth promotion at 50 mM NaCl but inhibition at 150 mM NaCl, reflecting a finely tuned salt-adaptation strategy. The plasma membrane-localized ZxHKT1;1 mediates Na⁺ retrieval from the root xylem sap and plays a pivotal role in this process, governing Na⁺ distribution within Z. xanthoxylum under varying NaCl conditions. However, the intracellular mechanisms guiding ZxHKT1;1 trafficking, especially the modulation of ZxHKT1;1 targeting under different NaCl concentrations, remain unclear. Here, we identified five ZxCNIHs, all of which interact with ZxHKT1;1. Yeast complementation assays demonstrated that ZxCNIH4 functions as the specific cargo receptor for ZxHKT1;1, mediating its correct targeting to the plasma membrane. Under 50 mM NaCl, ZxCNIH4 shows only weak induction, and the resulting limited plasma-membrane targeting of ZxHKT1;1 favors leaf Na⁺ accumulation for osmotic adjustment. Whereas under 150 mM NaCl, sharp upregulation of ZxCNIH4 enhances ZxHKT1;1 trafficking and plasma membrane targeting, thereby promoting Na⁺ retrieval from the xylem sap. Conversely, silencing ZxCNIH4 leads to cytosolic retention of ZxHKT1;1, thereby reducing net Na⁺ flux into root xylem parenchyma cells and consequently disrupting leaf Na⁺ balance and reducing tolerance of Z. xanthoxylum to high NaCl. Our findings uncover a previously uncharacterized NaCl concentration-dependent mechanism whereby ZxCNIH4-mediated sorting directs ZxHKT1;1 trafficking, linking external NaCl to the dynamic orchestration of Na⁺ partitioning between roots and shoots, and advances our understanding of salt adaptation in a Na⁺-accumulating xerophyte.