The MAPK regulatory module TaMPKKK18 ‐ 3B / TaMPKK1 .2‐ 6B / TaMPK2 ‐ 1D / TaNAC2 ‐ 5B regulates low‐P stress response in Triticum aestivum

The mitogen-activated protein kinase (MAPK) signaling cascades play crucial roles in mediating abiotic stress responses. This study characterized TaMPKKK18-3B, a member of the MAPK kinase kinase family, in mediating low-P stress response in Triticum aestivum. TaMPKKK18-3B contains conserved motifs shared by MAPKKK that target the nucleus. TaMPKKK18-3B transcripts were significantly upregulated under low-P stress conditions, an upregulation that was associated with the cis-acting element referred to as phosphate induction binding site (PIBS) situated in its promoter. Y-2H, BiFC and Co-IP assays indicated the interactions between TaMPKKK18-3B and the MAPKK member TaMPKK1.2-6B, which interacted with the MAPK member TaMPK2-1D. In turn, TaMPK2-1D interacted with the NAC transcription factor TaNAC2-5B via distinct conserved domains. These results suggested the formation of a MAPK regulatory module TaMPKKK18-3B/TaMPKK1.2-6B/TaMPK2-1D/TaNAC2-5B. Transgenic analysis on TaMPKKK18-3B module genes indicated their positive roles in modulating low-P adaptation by regulating phosphate (Pi) acquisition, acid phosphatase (AP) activity, and root system architecture (RSA) establishment. Dual luciferase, Y-1H, and EMSA assays suggested that TaNAC2-5B binds to the promoters of phosphate transporter gene TaPT2, AP gene TaAP1, and RSA-associated gene TaPIN3 and activates their transcription. Moreover, transgenic analyses validated the functions of stress-responsive genes in regulating Pi uptake, AP activity, and RSA behavior under deficient-P conditions. Strong positive correlations were observed between the transcript levels of TaMPKKK18-3B module genes and wheat yield under P-depleted field environments, with haplotype TaMPKKK18-3B-Hap 1 conferring wheat cultivars improved low-P stress tolerance. This study offers valuable insights into plant low-P response underlying the MAPK signaling pathway and provides markers for breeding the high-P-use-efficient cultivars in T. aestivum.