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

Abstract

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, Pi 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.