Comparative Genomics and Co-Expression Profiling of MADS-Box Genes Reveal Conserved Candidate Regulators of Secondary Cell Wall Formation in Lignified Endocarp and Seed Coat Across Four Angiosperm Species

Fruit endocarp and seed coat are essential protective structures that influence key agronomic and mechanical traits in species with lignified protective tissues, yet their regulatory mechanisms remain incompletely understood. Here, we conducted a comprehensive genome-wide analysis of the MADS-box gene family in four angiosperm species: Juglans sigillata, Carya illinoinensis, Macadamia integrifolia, and Ricinus communis. A total of 58, 55, 57, and 57 MADS-box genes were identified, respectively, and systematically characterized through phylogenetic, structural, and evolutionary analyses. Comparative results revealed that MIKCc-type genes are highly conserved and primarily expanded via segmental duplication under strong purifying selection. Co-expression network analysis identified MADS-box genes as high-connectivity hub candidates that are strongly associated with genes involved in tissue specification, hormone signaling, and secondary cell wall biosynthesis. Promoters analysis indicated that these genes contain diverse cis-regulatory elements; however, these results are based on sequence prediction and do not demonstrate functional regulatory interactions. Across species, MADS-box genes exhibited analogous temporal expression dynamics during lignified endocarp and seed coat development, consistent with a potentially conserved transcriptional framework. Collectively, this study provides new insights into the evolutionary diversification and putative functions of MADS-box genes, and proposes a putative hierarchical regulatory framework for lignified endocarp and seed coat development. These findings supply valuable candidate target genes for future molecular breeding aimed at improving shell thickness, hardness, and related agronomic traits in woody nut and oilseed species.