Ontogenetic and time-course transcriptomic analyses of maize leaf primordia reveal regulators of transverse vein development

Leaf vein patterning varies greatly among land plants and is linked to leaf structure and function. C4 plants, such as maize, have a denser arrangement of veins than C3 plants, which is hypothesized to be a pre-adaptive trait for C4 photosynthesis. Therefore, understanding how leaf veins are patterned has implications for maximizing photosynthetic efficiency. Major maize leaf veins, called longitudinal veins, are arranged in parallel and are interconnected by transverse veins. Recent work has established molecular factors controlling longitudinal vein development; however, it is unknown how transverse veins are patterned. Here, we investigated how transverse veins establish connections within the maize vascular network. We performed anatomical analyses of transverse veins and found that despite asynchronous development, transverse veins fully integrate into the longitudinal vein network. Tracing the ontogeny of transverse vein development using the marker PIN1a-YFP, we found that two sets of two initial cells on opposing sides of adjacent longitudinal veins recruit intervening ground cells to form the provascular strand. Treatment with auxin inhibitors did not abolish transverse vein initiation, suggesting an auxin-independent pre-pattern for transverse vein initiation. Through time-course RNAseq of leaf primordia, we identified a transcriptional signature that precedes transverse vein development. Two genes that belong to this gene cluster, EREB41 and EREB114, are necessary for transverse vein formation. Together, our studies establish the ontogeny of maize transverse vein development and reveal how new vascular connections are specified, assembled, and integrated into an existing vascular network.