Genome-Wide Identification Analysis of the MAPKKK Gene Family in Cotton and Its Role in Development and Stress Response

Mitogen-activated protein kinase kinase kinases (MAPKKKs) are pivotal upstream regulators of MAPK cascades, integrating signals that coordinate plant development and stress responses. However, the specific functions of MAPKKKs, particularly within the MEKK subfamily, in mediating cotton resistance to Verticillium wilt and Fusarium wilt remain poorly characterized. To address this, we conducted a systematic, cross-species analysis of the MAPKKK family in four key cotton species: <i>Gossypium arboreum</i>, <i>Gossypium barbadense</i>, <i>Gossypium hirsutum</i>, and <i>Gossypium raimondii</i>. Genome-wide identification and phylogenetic analysis revealed 660 MAPKKK genes, classifying them into the MEKK, Raf, and ZIK subfamilies. Evolutionary analysis indicated that Whole-Genome Duplication (WGD) events were the primary driver of family expansion. Promoter <i>cis</i>-element and Gene Ontology (GO) enrichment analyses implicated these genes in hormone signaling and stress adaptation. Expression profiling demonstrated functional modularity, with distinct members responding specifically to cold stress or cooperatively to drought and salt stresses. Upon pathogen infection, members diverged into regulatory modules associated with immune homeostasis, tissue-specific defense, and core signaling potentially governing systemic acquired resistance (SAR). The temporal expression patterns of core candidate genes were validated by qRT-PCR. This study provides, for the first time, a comprehensive evolutionary and functional framework for the MEKK subfamily within the cotton MAPKKK family. It reveals the conserved and divergent roles of this subfamily in stress adaptation and identifies key candidate genes for breeding disease-resistant cotton varieties.