Diversification and conservation of DNA binding specificities of SPL family of transcription factors

O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=89 SRC="FIGDIR/small/612952v2_ufig1.gif" ALT="Figure 1"> View larger version (24K): org.highwire.dtl.DTLVardef@1154c42org.highwire.dtl.DTLVardef@1419f5borg.highwire.dtl.DTLVardef@1454a7aorg.highwire.dtl.DTLVardef@1cbd5f_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOGRAPHICAL ABSTRACTC_FLOATNO C_FIG SQUAMOSA Promoter-Binding Protein-Like (SPL) transcription factors are key regulators of plant development and stress responses. Here, we present a comprehensive DNA affinity purification sequencing (DAP-seq) analysis of 14 of the 16 SPL members in Arabidopsis thaliana, revealing two major classes based on distinct DNA-binding motifs. Gene Ontology enrichment of target genes indicated both shared and specialized functions among AtSPLs, including roles in hormone signaling, metal ion homeostasis, and developmental transitions. Comparative analysis of closely related paralogs, AtSPL9 and AtSPL15, uncovered divergence in their genomic binding locations and target gene regulation, particularly in auxin and abscisic acid pathways. These differences were supported by motif analysis and protoplast reporter assays. Extending our study across species, we analyzed DAP-seq data from maize (Zea mays) and wheat (Triticum aestivum) SPL homologs, identifying conserved target genes but species-specific DNA motifs. Notably, wheat SPLs preferentially bind to longer, palindromic motifs distinct from the GTAC core motif in Arabidopsis. Using AlphaFold3 structure modeling, we show that these motif differences may arise from enhanced protein-protein interactions that stabilize dimeric binding. Our results highlight how SPL family members diversify through DNA-binding evolution and suggest that transcription factor dimerization contributes to motif complexity and regulatory specialization across plant genomes.