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Selective scattering spectra of granal and agranal chloroplasts were measured in the red spectral region and compared with calculations based on the Mie theory. The spectra were influenced considerably by the intactness and ultrastructural pattern of the chloroplasts. It was demonstrated that the spectra consist of two components: one attributable to grana and the other, to single lamellae. The dependence of the selective scattering spectra on the ultrastructural characteristics offers a convenient method for monitoring the quality of chloroplast preparations by a procedure much faster than electron microscopy. Selective scattering spectra of algal cells such as Chlorella and Anacystis, as well as of spinach chloroplasts and fragments, have been reported in the literature (1)(2)(3)(4)(5)(6)(7)(8). In these studies the photosynthetic lamellar system was considered as a whole with no regard to the ultrastructural pattern characteristic of the chloroplasts. As shown under the electron microscope, chloroplasts consist of two types of lamellae: single lamellae that appear never to fuse with each other, and stacked lamellae that build up the highly ordered piles called "grana." The individual contributions of single and stacked lamellae in the selective scattering spectra have not been distinguished in previous reports.The aim of the present work was to study the selective scattering spectra of chloroplasts containing various amounts of grana and to compare intact class A and class B chloroplasts (9) in order to obtain information on the relationship between ultrastructure and selective scattering. Selective spectra of chloroplasts were shown to be dependent on their intactness and ultrastructure. This phenomenon offers a convenient method for checking the quality of chloroplast preparations. MATERIALS AND METHODSChloroplast preparations with high and low granum contents were obtained from maize leaves (Zea mays L. var. MV SC 660). The plants were grown in a greenhouse for 9-l1 days.Class A chloroplasts were isolated from a suspension of mesophyll protoplasts (10) or bundle sheath cells. Mesophyll protoplasts were prepared in a digestion medium containing 0.6 M d-sorbitol, 0.1 M potassium phosphate, 0.5% (wt/vol) Macerozyme R-10, 2.0% (wt/vol) cellulase Onozuka R-10, and 0.5% (wt/vol) potassium dextran sulfate, pH 5.8. Bundle sheath cells were separated in a maceration medium containing 0.4 M d-sorbitol, 0.1 M potassium phosphate, 0.5% Macerozyme R-10, 1.0% cellulase Onozuka R-10, and 1.0% Helicase, pH 5.8. Macerozyme and cellulase preparations were purchased from Kinki Yakult MFG Co. Ltd., Nishinomiya, Japan; the Helicase was obtained from Industrie Biologique Frangaise, Gennevilliers, France; and potassium dextran sulfate was from Meito Sangyo Co. Ltd., Nagoya, Japan. The incubation was for 90 min at 37°. Protoplasts and cells were washed three times in 0.6 M d-sorbitol and transferred to the medium of Anderson and Boardman (11) containing 0.3 M sucrose, 0.05 M potassium phosphate, and 0.01 M potassium chlor...

A procedure was developed to isolate mesophyll and bundle sheath chloroplasts of a high degree of intactness and low cross-contamination. Light-induced (14)CO2 fixation of isolated chloroplasts was similar to that of protoplasts and cells in that it was low and was stimulated by the addition of exogenous substrates. O2 evolution was absent in both bundle sheath chloroplasts and cells. The flash-induced 515 nm absorbance change of intact mesophyll chloroplasts showed a biphasic rise, previously known to be a characteristic only of intact algae. With bundle sheath chloroplasts or cells, no 515 nm signal could be detected. In the presence of 10 μmol l(-1) phenazine methosulphate, bundle sheath chloroplasts exhibited a flash-induced 515 nm signal with a monophasic rise and amplitude comparable to that of the mesophyll chloroplasts. A similar signal was obtained with bundle sheath chloroplasts suspended in an extract prepared from the mesophyll tissue. Both the substrate stimulation of the CO2 fixation and the reconstitution of the 515 nm signal in bundle sheath chloroplasts by the mesophyll extract indicate the requirement of cooperation between the mesophyll and bundle sheath cells of maize leaves.

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