Evolutionary approaches predicted changes in transcription factors and cis -elements that associate with Kranz anatomy development in maize

C ₄ plants are more efficient in high temperatures and less water due to unique anatomy and biochemical modifications, but critical regulator genes controlling Kranz anatomy remain undiscovered. This study aims to investigate the genes and cis -motifs that may be critical regulators in establishing and maintaining Kranz anatomy features in leaves of C ₄ grasses by investigating candidate genes and their upstream regions from Zea mays by looking for genes with adaptive evolution in solely C ₄ orthologs and motifs present uniquely in C ₄ plants. Three genes out of 70 genes analyzed, namely myb89 , spb21, and ereb80 were observed from Maize with adaptive evolution uniquely present in multiple C ₄ species, with the first two supported by expression data from different studies to be possibly involved in Kranz regulation, with further experimental validation required to investigate their exact roles in Kranz development. 12 motifs were found upstream of gene orthologs specific to or enriched in C ₄ species. Three motifs upstream of IDP8537 and gras19 were found in a subset of their coexpression cluster, further suggesting their potential involvement in Kranz anatomy regulation. These novel motifs are potential additions to the limited Kranz-specific motifs and can aid in predicting new regulators after experimental validation.