Evolutionary approaches predicted changes in transcription factors and cis-elements that associate with Kranz anatomy development in maize
C4 plants exhibit higher photosynthetic efficiency under high temperatures and low water availability due to their unique anatomical and biochemical adaptations, but the evolutionary mechanisms and regulatory components underlying their Kranz development remain partly known. If C4 traits evolved through adaptive changes, its developmental regulators should show detectable evolutionary signatures in C4 lineages. We investigated the adaptive evolutionary changes in both protein sequences and cis-motifs upstream of candidate genes differentially expressed during Kranz anatomy establishment in C4 grasses. We identified signatures of convergent evolution in candidate genes and their upstream regions by comparing C4 orthologs with their C3 counterparts, and enriched non-syntenic motifs present upstream of C4 orthologs through phylogeny-aware analysis. Out of 191 candidate genes analyzed, ten orthogroups containing maize orthologs of EREB160, DOF11, bHLH116, MADS9, bHLH33/bHLH105, SCRO1/SCR2, CADTFR3, THX8, C3H28, and SHR1/SHR2 showed positive selection pressure specific to C4 orthologs. Among them, DOF11, SCRO1/SCR2, and SHR1/SHR2 have previously been implicated in Kranz regulation by previous studies. Additionally, we identified a conserved position mutation present in bHLH116 across C4 lineages, which may influence its DNA-binding domain. Moreover, we identified 39 annotated non-syntenic/C4-shifted DNA motifs, upstream of 28 gene orthologs, enriched in C4 species. Notably, genes with these motifs did not intersect with the genes under positive selection, suggesting these pathways evolved separately without much crosstalk, nevertheless, a few putative regulatory-network-motifs were observed. These motifs are potential additions to the limited Kranz-specific motifs and can aid in predicting new putative regulators after experimental validation. Significance statementC4 plants evolved as an adaptive response through anatomical and biochemical changes in their C3 relatives. Unlike the biochemical changes, the regulators/drivers of anatomical changes and the nature of underlying changes remains only partially understood. Using signatures of adaptive evolution, the current study screened candidate regulatory factors and sequences, including a few previously known and many novel candidates. These findings complement the existing experimental knowledge and advance the discovery of the gene regulatory network underlying the anatomical changes associated with C4 evolution.