Anatomy and ultrastructure of embryonic leaves of the C-4 species Setaria viridis

Background and Aims Setaria viridis is being promoted as a model C-4 photosynthetic plant because it has a small genome (similar to 515 Mb), a short life cycle (60 d) and it can be transformed. Unlike other C-4 grasses such as maize, however, there is very little information about how C-4 leaf anatomy (Kranz anatomy) develops in S. viridis. As a foundation for future developmental genetic studies, we provide an anatomical and ultrastructural framework of early shoot development in S. viridis, focusing on the initiation of Kranz anatomy in seed leaves. Methods Setaria viridis seeds were germinated and divided into five stages covering development from the dry seed (stage S0) to 36 h after germination (stage S4). Material at each of these stages was examined using conventional light, scanning and transmission electron microscopy. Key Results Dry seeds contained three embryonic leaf primordia at different developmental stages (plastochron 1-3 primordia). The oldest (P3) leaf primordium possessed several procambial centres whereas P2 displayed only ground meristem. At the tip of P3 primordia at stage S4, C-4 leaf anatomy typical of the malate dehydrogenase-dependent nicotinamide dinucleotide phosphate (NADP-ME) subtype was evident in that vascular bundles lacked a mestome layer and were surrounded by a single layer of bundle sheath cells that contained large, centrifugally located chloroplasts. Two to three mesophyll cells separated adjacent vascular bundles and one mesophyll cell layer on each of the abaxial and adaxial sides delimited vascular bundles from the epidermis. Conclusions The morphological trajectory reported here provides a foundation for studies of gene regulation during early leaf development in S. viridis and a framework for comparative analyses with other C-4 grasses.