One of the most remarkable examples of convergent evolution is the transition from C to C photosynthesis, an event that occurred on over 60 independent occasions. The evolution of C is particularly noteworthy because of the complexity of the developmental and metabolic changes that took place. In most cases, compartmentalized metabolic reactions were facilitated by the development of a distinct leaf anatomy known as Kranz. C Kranz anatomy differs from ancestral C anatomy with respect to vein spacing patterns across the leaf, cell-type specification around veins, and cell-specific organelle function. Here we review our current understanding of how Kranz anatomy evolved and how it develops, with a focus on studies that are dissecting the underlying genetic mechanisms. This research field has gained prominence in recent years because understanding the genetic regulation of Kranz may enable the C-to-C transition to be engineered, an endeavor that would significantly enhance crop productivity.

ORCID IDs: 0000-0003-4287-6927 (M.L.S.); 0000-0002-7609-0378 (O.V.S.); 0000-0001-5932-6468 (B.J.W.); 0000-0002-4621-1490 (P.W.); 0000-0001-7648-3924 (J.A.L.).The coordinated positioning of veins, mesophyll cells, and stomata across a leaf is crucial for efficient gas exchange and transpiration and, therefore, for overall function. In monocot leaves, stomatal cell files are positioned at the flanks of underlying longitudinal leaf veins, rather than directly above or below. This pattern suggests either that stomatal formation is inhibited in epidermal cells directly in contact with the vein or that specification is induced in cell files beyond the vein. The SHORTROOT pathway specifies distinct cell types around the vasculature in subepidermal layers of both root and shoots, with cell type identity determined by distance from the vein. To test whether the pathway has the potential to similarly pattern epidermal cell types, we expanded the expression domain of the rice (Oryza sativa ssp japonica) OsSHR2 gene, which we show is restricted to developing leaf veins, to include bundle sheath cells encircling the vein. In transgenic lines, which were generated using the orthologous ZmSHR1 gene to avoid potential silencing of OsSHR2, stomatal cell files were observed both in the normal position and in more distant positions from the vein. Contrary to theoretical predictions, and to phenotypes observed in eudicot leaves, the increase in stomatal density did not enhance photosynthetic capacity or increase mesophyll cell density. Collectively, these results suggest that the SHORTROOT pathway may coordinate the positioning of veins and stomata in monocot leaves and that distinct mechanisms may operate in monocot and eudicot leaves to coordinate stomatal patterning with the development of underlying mesophyll cells.The coordinated differentiation of cell types within an organ is a crucial component of morphogenesis, necessary to ensure that the final form is appropriate for function. In this regard, photosynthetic function in plant leaves requires that chloroplast-containing cells in the middle leaf layers are interspersed with veins (to supply water and to redistribute metabolites) and are overlaid with stomatal pores through which carbon dioxide can enter the leaf. In grass leaves, cellular arrangements are defined by parallel longitudinal veins that extend from the base of the leaf sheath to the tip of the leaf blade, with short transverse veins interconnecting the longitudinal network (Sharman, 1942;Esau, 1943;Nelson and Dengler, 1997). This vascular framework underpins linear files of stomata in the epidermis, with each vein being flanked by one to three rows of stomata on both the medial and lateral sides (Stebbins and Shah, 1960). The genetic mechanisms that ensure optimal photosynthetic capacity in grass leaves, by coordinating the development of veins, photosynthetic cell types, and stomata, are not known.Grass leaves develop basipetally such that cellular differentiation proceeds from the tip of the leaf to the base, ...

16 17 Summary statement: Two duplicated maize SCARECROW genes control the development of the 18 endodermis in roots and the mesophyll in leaves 19 2 ABSTRACT 20 The highly efficient C4 photosynthetic pathway is facilitated by 'Kranz' leaf anatomy. In Kranz leaves, 21 closely spaced veins are encircled by concentric layers of photosynthetic bundle sheath (inner) and 22 65 recruited in the leaf rather than the root in maize, but the subtle phenotype reported in leaves of 66 Zmscr1 mutants precludes an understanding of the precise role played during Kranz development. 67 68 Both gene and whole genome duplication events are highly prevalent throughout the plant phylogeny 69 (Adams and Wendel, 2005; Blanc and Wolfe, 2004) and if retained in the genome, duplicated genes 70 are free to sub-or neo-functionalize (Moore and Purugganan, 2005; Ohno, 1970). Perhaps more 71 553 Adams, K. L. and Wendel, J. F. (2005). Polyploidy and genome evolution in plants. Curr. Opin.

The highly efficient C 4 photosynthetic pathway is facilitated by ‘Kranz’ leaf anatomy. In Kranz leaves, closely spaced veins are encircled by concentric layers of photosynthetic bundle sheath (inner) and mesophyll (outer) cells. Here, we demonstrate that, in the C 4 monocot maize, Kranz patterning is regulated by redundant function of SCARECROW 1 (ZmSCR1) and a previously uncharacterized homeologue: ZmSCR1h. ZmSCR1 and ZmSCR1h transcripts accumulate in ground meristem cells of developing leaf primordia and in Zmscr1;Zmscr1h mutant leaves, most veins are separated by one rather than two mesophyll cells; many veins have sclerenchyma above and/or below instead of mesophyll cells; and supernumerary bundle sheath cells develop. The mutant defects are unified by compromised mesophyll cell development. In addition to Kranz defects, Zmscr1;Zmscr1h mutants fail to form an organized endodermal layer in the root. Collectively, these data indicate that ZmSCR1 and ZmSCR1h redundantly regulate cell-type patterning in both the leaves and roots of maize. Leaf and root pathways are distinguished, however, by the cell layer in which they operate – mesophyll at a two-cell distance from leaf veins versus endodermis immediately adjacent to root vasculature.

All grass leaves are strap-shaped with a series of parallel veins running from base to tip, but the distance between each pair of veins, and the cell-types that develop between them, differs depending on whether the plant performs C3 or C4 photosynthesis. As part of a multinational effort to introduce C4 traits into rice to boost crop yield, candidate regulators of C4 leaf anatomy were previously identified through an analysis of maize leaf transcriptomes. Here we tested the potential of 60 of those candidate genes to alter leaf anatomy in rice. In each case, transgenic rice lines were generated in which the maize gene was constitutively expressed. Lines grouped into three phenotypic classes: (1) indistinguishable from wild-type; (2) aberrant shoot and/or root growth indicating possible perturbations to hormone homeostasis; and (3) altered secondary cell wall formation. One of the genes in class 3 defines a novel monocot-specific family. None of the genes were individually sufficient to induce C4-like vein patterning or cell-type differentiation in rice. A better understanding of gene function in C4 plants is now needed to inform more sophisticated engineering attempts to alter leaf anatomy in C3 plants.