Photosynthetic enzyme accumulation during leaf development of Arundinella hirta, a C4 grass having Kranz cells not associated with veins

The leaf of the NADP-malic enzyme type C(4) grass, Arundinella hirta, has not only mesophyll cells (MCs) and bundle sheath cells (BSCs, usual Kranz cells) but also another type of Kranz cells (distinctive cells; DCs) that are not associated with vascular bundles. We investigated photosynthetic enzyme accumulation along the base-to-tip maturation gradient of developing leaves by immunogold electron microscopy. In mature leaves, phosphoenolpyruvate carboxylase (PEPC) and ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco) were detected in the MC cytosol and in the BSC and DC chloroplasts, respectively. Pyruvate, P(i) dikinase (PPDK) was present in the chloroplasts of all photosynthetic cells but with higher levels in the MCs. Rubisco was first detected in the basal region of emerging leaf blades where the BSCs and DCs became discernable. Subsequently, the accumulation of PEPC and PPDK was initiated in the region where the granal proliferation in the chloroplasts was conspicuous; and, suberized lamellae were formed in the cell walls of the Kranz cells. There was no difference in the patterns of cellular development and enzyme accumulation between the BSCs and DCs or between the MCs adjacent to each type of Kranz cells. These results demonstrate that, although the DCs are not associated with veins, they behaved like BSCs with respect to enzyme induction and cellular differentiation.