Multiplexed in situ hybridization reveals distinct lineage identities for major and minor vein initiation during maize leaf development

ABSTRACT Leaves of flowering plants are characterised by diverse venation patterns. Patterning begins with the selection of vein-forming procambial initial cells from within the ground meristem of a developing leaf, a process which is considered to be auxin-dependent, and continues until veins are anatomically differentiated with functional xylem and phloem. At present, the mechanisms responsible for leaf venation patterning are primarily characterized in the model eudicot Arabidopsis thaliana which displays a reticulate venation network. However, evidence suggests that vein development may proceed via a different mechanism in monocot leaves where venation patterning is parallel. Here, we employed Molecular Cartography, a multiplexed in situ hybridization technique, to analyse the spatiotemporal localisation of a subset of auxin related genes and candidate regulators of vein patterning in maize leaves. We show how different combinations of auxin influx and efflux transporters are recruited during leaf and vein specification, and how major and minor vein ranks develop with distinct identities. The localisation of the procambial marker PIN1a and the spatial arrangement of procambial initial cells that give rise to major and minor vein ranks further suggests that vein spacing is pre-patterned across the medio-lateral leaf axis prior to accumulation of the PIN1a auxin transporter. In contrast, patterning in the adaxial-abaxial axis occurs progressively, with markers of xylem and phloem gradually becoming polarised as differentiation proceeds. Collectively our data suggest that both lineage- and position-based mechanisms may underpin vein patterning in maize leaves. SIGNIFICANCE STATEMENT During the development of multicellular organisms specialized cell-types differentiate from pluripotent stem cells, with cell identity acquired via lineage- or position-based mechanisms. In plants, most organs develop post-embryogenesis and as such developmental processes are influenced by the external environment. To adapt to different environmental contexts and yet still form recognizable structures, position-based differentiation mechanisms are deployed in which cells adopt a certain fate depending on the activity of neighbouring cells. Such is the prevalence of position-based mechanisms in plant development that a role for lineage is rarely contemplated. Here we show that stem cells which give rise to different vein types in maize leaves are transcriptionally distinct, possibly reflecting a role for lineage-based mechanisms in the differentiation of leaf veins.