Shifts in leaf vein density through accelerated vein formation in C4 Flaveria (Asteraceae)

Background and Aims Leaf venation in many C-4 species is characterized by high vein density, essential in facilitating rapid intercellular diffusion of C-4 photosynthetic metabolites between different tissues (mesophyll, bundle sheath). Greater vein density has been hypothesized to be an early step in C-4 photosynthesis evolution. Development of C-4 vein patterning is thought to occur from either accelerated or prolonged procambium formation, relative to ground tissue development. Methods Cleared and sectioned tissues of phylogenetically basal C-3 Flaveria robusta and more derived C-4 Flaveria bidentis were compared for vein pattern in mature leaves and vein pattern formation in developing leaves. Key Results In mature leaves, major vein density did not differ between C-3 and C-4 Flaveria species, whereas minor veins were denser in C-4 species than in C-3 species. The developmental study showed that both major and minor vein patterning in leaves of C-3 and C-4 species were initiated at comparable stages (based on leaf length). An additional vein order in the C-4 species was observed during initiation of the higher order minor veins compared with the C-3 species. In the two species, expansion of bundle sheath and mesophyll cells occurred after vein pattern was complete and xylem differentiation was continuous in minor veins. In addition, mesophyll cells ceased dividing sooner and enlarged less in C-4 species than in C-3 species. Conclusions Leaf vein pattern characteristic to C-4 Flaveria was achieved primarily through accelerated and earlier offset of higher order vein formation, rather than other modifications in the timing of vein pattern formation, as compared with C-3 species. Earlier cessation of mesophyll cell division and reduced expansion also contributed to greater vein density in the C-4 species. The relatively late expansion of bundle sheath and mesophyll cells shows that vein patterning precedes ground tissue development in C-4 species.