Immunogold localization of photosynthetic enzymes in leaves of Aristida latifolia, a unique C4 grass with a double chlorenchymatous bundle sheath

C₄ species of the genus Aristida (Poaceae) have 3 distinct types of photosynthetic cells in their leaves, the mesophyll (M) cells, the outer bundle sheath (BS) cells, and the inner BS cells, and exhibit a unique Kranz-type leaf anatomy. The cellular localization of C₃ and C₄ photosynthetic enzymes was investigated in leaves of Aristida latifolia Domin by the protein A-gold immunocytochemical technique. The outer BS cells contained centripetally located small chloroplasts, which were structurally similar to those of the M cells. The inner BS cells contained centrifugally located large chloroplasts, which lacked well-developed grana and exhibited rudimentary grana for the most part. The leaves contained high levels of NADP-malic enzyme (EC 1.1.1.40) activity, and the plant was classified as being of the NADP-malic enzyme type. The immunocytochemical study revealed that labeling of ribulose 1,5-bisphos-phate carboxylase/oxygenase (EC 4.1.1.39) was present in the chloroplasts of the outer and inner BS cells, but was undetectable in the M cells. Labeling of phoshoen-olpyruvate carboxylase (EC 4.1.1.31) was observed in the cytosol of M cells, but not in that of BS cells. By contrast, labeling of pyruvate, P_i dikinase (PPDK, EC 2.7.9.1) was evident not only in the chloroplasts of M cells but also in those of outer BS cells, but was absent from the inner BS cells. The density of labeling in the chloroplasts of M cells was higher than that in chloroplasts of outer BS cells. These results indicate that the two carboxylating enzymes are differentially distributed between the M cells and the two types of BS cells, whereas PPDK shows a more complex distribution pattern. The locations of these enzymes are discussed in relation to C₄ photosynthesis.