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

Leaves of flowering plants are characterized 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 analyze the spatiotemporal localization 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 localization 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 prepatterned 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 polarized as differentiation proceeds. Collectively, our data suggest that both lineage- and position-based mechanisms may underpin vein patterning in maize leaves.

The bHLH transcription factor OsPRI1 activates the Setaria viridis PEPC1 promoter in rice.

Photosynthetic efficiency is reduced by the dual role of Rubisco, which acts either as a carboxylase or as an oxygenase, the latter leading to photorespiration. C4 photosynthesis evolved as a carbon-concentrating mechanism to reduce photorespiration. To engineer C4 into a C3 plant, it is essential to understand how C4 genes, such as phosphoenolpyruvate carboxylase (PEPC1), are regulated to be expressed at high levels and in a cell-specific manner. Yeast one-hybrid screening was used to show that OsPRI1, a rice bHLH transcription factor involved in iron homeostasis, binds to the Setaria viridis PEPC1 promoter. This promoter drives mesophyll-specific gene expression in rice. The role of OsPRI1 in planta was characterized using a rice line harbouring SvPEPC1pro ::GUS. We show that OsPRI1 activates the S. viridis PEPC1 promoter by binding to an N-box in the proximal promoter, and that GUS activity is highly reduced in SvPEPC1pro ::GUS lines when OsPRI1 is mutated. Cross-species comparisons showed that the SvPRI1 homolog binds to the SvPEPC1 promoter but the maize ZmPRI1 does not bind to the ZmPEPC1 promoter. Our results suggest that elements of the iron homeostasis pathway were co-opted to regulate PEPC1 gene expression during the evolution of some but not all C4 species.

The WIP6 transcription factor TOO MANY LATERALS specifies vein type in C4 and C3 grass leaves.

Grass leaves are invariantly strap shaped with an elongated distal blade and a proximal sheath that wraps around the stem. Underpinning this shape is a scaffold of leaf veins, most of which extend in parallel along the proximo-distal leaf axis. Differences between species are apparent both in the vein types that develop and in the distance between veins across the medio-lateral leaf axis. A prominent engineering goal is to increase vein density in leaves of C3 photosynthesizing species to facilitate the introduction of the more efficient C4 pathway. Here, we discover that the WIP6 transcription factor TOO MANY LATERALS (TML) specifies vein rank in both maize (C4) and rice (C3). Loss-of-function tml mutations cause large lateral veins to develop in positions normally occupied by smaller intermediate veins, and TML transcript localization in wild-type leaves is consistent with a role in suppressing lateral vein development in procambial cells that form intermediate veins. Attempts to manipulate TML function in rice were unsuccessful because transgene expression was silenced, suggesting that precise TML expression is essential for shoot viability. This finding may reflect the need to prevent the inappropriate activation of downstream targets or, given that transcriptome analysis revealed altered cytokinin and auxin signaling profiles in maize tml mutants, the need to prevent local or general hormonal imbalances. Importantly, rice tml mutants display an increased occupancy of veins in the leaf, providing a step toward an anatomical chassis for C4 engineering. Collectively, a conserved mechanism of vein rank specification in grass leaves has been revealed.