Abstract
Mitogen-activated protein kinase kinase kinases (MAPKKKs) are central components of the MAPK cascade and play crucial roles in plant growth, development, and stress responses. Nevertheless, the evolutionary dynamics and functional diversification of this gene family within the Asteraceae lineage remain poorly characterized. In this study, we conducted a comprehensive comparative genomic analysis of the MAPKKK family across ten representative Asteraceae species. A total of 1,009 MAPKKK genes were identified and phylogenetically classified into three subfamilies-RAF, MEKK, and ZIK-which were further subdivided into 14 distinct clusters. Structural analysis revealed considerable variation in intron length and gene architecture among subfamilies and species. All Asteraceae MAPKKK genes were grouped into 64 orthologous gene groups (OGGs), including 16 conserved, 28 variable, and 30 rare OGGs. Conserved OGGs comprised 296 genes, accounting for 29.34% of the total MAPKKK genes, and were subject to significantly stronger purifying selection compared with dispensable genes. Chromosomal localization and synteny analyses indicated that whole-genome duplication (WGD) events were the primary drivers of MAPKKK family expansion, with conserved genes retained preferentially under stronger selective constraints than those arising from small-scale duplication (SSD) events. Collinearity analysis further showed that conserved genes constituted over 47.91% of syntenic gene pairs, underscoring their high evolutionary conservation. Promoter analysis identified stress-responsive cis-elements as the most abundant category, representing 41.6% of all detected elements, highlighting the potential role of MAPKKKs in environmental adaptation. Transcriptomic and qRT-PCR assays in sunflower under drought, salt, and alkaline stresses revealed several MAPKKK genes with distinct and stress-specific expression profiles, such as the conserved genes Hann_MAPKKK16 and Hann_MAPKKK135. Together, these results provide important insights into the evolutionary mechanisms governing MAPKKK family expansion and functional specialization, advancing our understanding of stress adaptation in Asteraceae species.