Relationships between histological and functional differentiation in C_4 plants

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1.Immunocytochemical methods for observation of higher plant leaves were investigated. An effective method for light microscopy was to treat de-waxd paraffin sections with primary antiserum and apply R-phycoerythrin-conjugated secondary antibody. Observation was made under a fluorescence microscope equipped with an exciter filter unit B.For electron microscopy tissues were fixed with 3% glutaraldehyde for 2h and embedded in LR White resin.Sections were treated with primary antiserum and then with colloidal gold-conjugated secondary antibody.2.Chloroplast structure and localization of Rubisco in various cell types of Portulaca grandiflora were examined by immunocytochemical electron microscopy. Accumulation of Rubisco and suppression of granal development were confirmed in the chloroplasts of typical bundle sheath cells around peripheral vascular bundles. However, characteristics as a bundle sheath chloroplast were also detected in the chloroplasts of water storage cells adjacent to the central vascular bundle although the Kranz anatomy was not developed around the central vascular bundle.3.Chloroplast structure and localization of Rubisco were examined in green callus tissues and regenerating callus tissues of P.grandiflora. Chloroplasts possessed grana even in the cells around vascular bundles of callus. Suppression of granal development was observed in chloroplasts of cells around vascular bundles in regeneration leaf tissues. Labeling of Rubisco was detected in chloroplasts of most of the green tissues in both subculturing and regenerating calli. Labeling of Rubisco was also detected even in mesophyll chloroplasts of intact plants grown heterotrophically under low light intensity.4.It was assumed that vascular bundles play important roles in structural and functional differentiation of C_4 plant leaves but this regulatory mechanism was not function in callus tissues and plants grown heterotrophically.