Identification of salt-responsive mitogen-activated protein kinase cascade members in the halophyte N. sibirica

Soil salinization severely limits global agricultural productivity. Halophytes such as Nitraria sibirica (N. sibirica) serve as valuable model systems for unraveling the sophisticated mechanisms underlying salt adaptation. Although mitogen-activated protein kinase (MAPK) cascades function as central signaling modules in plant stress responses, their specific architecture and functional organization remain largely unexplored in extremophytes .

Through a genome-wide analysis of N. sibirica, we systematically identified 79 MAPK cascade components, including 14 MAPKs, 7 MAPKKs, and 58 MAPKKKs. All identified proteins harbor canonical structural features, including conserved T(E/D)Y motifs in MAPKs, active site S/T-X₅-S/T motifs in MAPKKs, and subfamily-specific signatures in MAPKKKs. Evolutionary analysis classified these components into highly conserved subfamilies aligned with the established Arabidopsis framework. Transcriptomic profiling, coupled with qRT-PCR validation, uncovered multiple salt-responsive genes that exhibited diverse subcellular localization patterns. Furthermore, by integrating AlphaFold-based structural modeling with yeast two-hybrid assays, we identified a linearly organized MAPK cascade module: NsMAPKKK (NISI07G2543) - NsMAPKK (NISI03G1309) - NsMAPK (NISI09G1183). This signaling module was consistently upregulated under salinity stress, and its physical interactions were demonstrated in vitro.

This study presents a genome-wide identification of the MAPK cascade family in N. sibirica. By delineating a candidate salt-responsive module, our findings provide insights into stress signal transduction in extremophytes and offer potential target candidates for engineering salt tolerance in crops.