Adaptive evolutionary changes in regulatory genes and cis -elements associated with Kranz development in monocots

Abstract C 4 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 C 4 traits evolved through adaptive changes, its developmental regulators should show detectable evolutionary signatures in C 4 lineages. In this study, we investigated the adaptive evolutionary changes in both protein sequences and cis -motifs upstream of genes associated with Kranz anatomy in C 4 grasses. We identified signatures of convergent evolution in candidate genes and their upstream regions by comparing C 4 orthologs with their C 3 counterparts and identifying enriched non-syntenic motifs present upstream of C 4 orthologs through phylogeny-aware analysis. Out of 191 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 C 4 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 C 4 lineages, which may influence its DNA-binding domain. Moreover, we identified 39 annotated non-syntenic/C 4 -shifted DNA motifs, upstream of 28 gene orthologs, enriched in C 4 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 regulatory-network-motifs were observed. These motifs are potential additions to the limited Kranz-specific motifs and can aid in predicting new regulators after experimental validation. Significance statement C 4 plants are highly efficient under heat and water stress, yet the genetic mechanism behind their establishment is poorly understood. Adaptive changes in the coding region of regulatory genes were fewer in number and largely convergent in nature, whereas, the non-coding regulatory DNA elements, predicted in genes associated with early leaf development in C 4 grasses, showed their acquisition exclusively to C 4 plants, very unlikely and supported previous reports of acquisition from their ancestral C 3 plants. These findings shed new light on evolutionary trends associated with Kranz evolution and new candidates for improving crop efficiency through future research.