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

C4 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 C4 grasses by investigating candidate genes and their upstream regions from Zea mays by looking for genes with adaptive evolution in solely C4 orthologs and motifs present uniquely in C4 plants. Three genes out of 70 genes analyzed, namely myb89, spb21, and ereb80 were observed from Maize with adaptive evolution uniquely present in multiple C4 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 C4 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. HIGHLIGHTIn this paper, we analyze the putative regulator sets of Kranz anatomy to discover important regulators and novel cis-regulatory elements that possibly regulate Kranz anatomy using evolutionary approaches.