Deciphering Microprotein–TF Regulatory Crosstalk: An Integrative Transcriptomic and Evolutionary Analysis of MIF1–ZHD Interaction in Brassica napus Seed Development

Seed development in Brassica napus is tightly regulated at many stages. Microproteins (miPs), small regulatory peptides derived from larger ancestral proteins, have emerged as key post-translational modulators in plants; however, their role in seed development remains poorly understood. Here, a genome-wide screen for putative miPs in B. napus was performed, identifying 32 candidates. Among these, MIF1 (BnaC06g35530D), with domain similarity to ZF-HD transcription factors (TFs), showed robust expression dynamics. The integration of RNA-seq profiling, qRT-PCR, co-expression network modeling, and protein–protein interaction revealed a strong regulatory association between MIF1 and its predicted TF partner, i.e., ZHD (BnaC01g16510D), particularly at the zygote and bending cotyledon stages. Promoter analysis identified shared cis-elements among co-expressed genes, while structural modeling supported a stable MIF1–ZHD interaction interface. Evolutionary analysis demonstrated the conservation of zinc finger motifs, syntenic neighborhoods, and purifying selection on MIF1 across Brassicaceae species. Collectively, our findings suggest that MIF1 potentially functions as a conserved post-translational repressor of ZHD during seed development, contributing to fine-tuned transcriptional regulation in polyploid oilseed crops. This study provides a testable hypothesis about the interaction between MIF1 and ZHD and also paves the way for the future functional characterization of miP-TF modules in plant development.