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Abstract. A method is described for separating mesophyll and bundle sheath chloroplasts from the leaves of C4 plants. The agranal bundle sheath chloroplasts are inactive in the Hill reaction, whereas granal bundle sheath and granal mesophyll chloroplasts exhibit normal photosystem II activity. The agranal bundle sheath chloroplasts are deficient in photosystem II; they lack cytochrome b-559 and the fluorescence bands associated with photosystem II. All the chloroplasts exhibit photosystem I activity.Leaves of plants with the C4-dicarboxylic acid pathway of photosynthesis1 contain two distinctive layers of chlorophyll-containing cells: the outer mesophyll layer and the inner bundle sheath layer surrounding the vascular bundles.2 Chloroplasts of the mesophyll cells contain grana, but those of the bundle sheath exhibit varying degrees of grana development depending on the species. Earlier studies' in which leaf sections were treated with the Hill oxidant, tetranitro blue tetrazolium chloride (TNBT), showed that noncyclic electron flow from water was restricted to chloroplasts contaiuing grana. The agranal bundle sheath chloroplasts of Sorghum were incapable of TNBT reduction unless artificial electron donors were provided to photosystem I. This paper describes a technique for separating mesophyll and bundle sheath chloroplasts from the leaves of (X plants. The method is based on the differential resistance of the bundle sheath and mesophyll cells to breakage.4 The species used in this study were selected to provide a suitable range of grana development in the bundle sheath chioroplasts.We have examined the photochemical and fluorescence properties and cytochrome contents of the isolated mesophyll and bundle sheath chloroplasts. The results indicate that the bundle sheath chloroplasts lacking grana are deficient in photosystem II but they contain an active photosystem I. Granacontaining chloroplasts, both mesophyll and bundle sheath, have a functional photosystem II.

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