Abstract
Three terrestrial plants are known to perform C4 photosynthesis without the dual-cell system by partitioning two distinct types of chloroplasts in separate cytoplasmic compartments. We report herein a protocol for isolating the dimorphic chloroplasts from Bienertia sinuspersici. Hypo-osmotically lysed protoplasts under our defined conditions released intact compartments containing the central chloroplasts and intact vacuoles with adhering peripheral chloroplasts. Following Percoll step gradient purification both chloroplast preparations demonstrated high homogeneities as evaluated from the relative abundance of respective protein markers. This protocol will open novel research directions toward understanding the mechanism of single-cell C4 photosynthesis.
Highlights
The majority of terrestrial plants house chloroplasts primarily in one major cell type of leaves, and perform C3 photosynthesis to assimilate atmospheric CO2 into a 3-carbon product, 3-phosphoglyceric acid
Rationale of the isolation procedures in relation to cell anatomy For a better clarification of the each isolation step as described in the subsequent sections, we first summarize the major changes in the complex subcellular organization of chlorenchyma cells throughout the process as illustrated with schematic diagrams (Figure 1)
In a mature chlorenchyma cell, a large central vacuole separates the cytoplasm into the peripheral (PCC) and the central (CCC) cytoplasmic compartments, which are interconnected by cytoplasmic channels traversing the vacuole (Figure 1A)
Summary
The majority of terrestrial plants house chloroplasts primarily in one major cell type of leaves (i.e. mesophyll cells), and perform C3 photosynthesis to assimilate atmospheric CO2 into a 3-carbon product, 3-phosphoglyceric acid. In C4 species, on the other hand, a Kranz-type leaf anatomy featuring the second type of chlorenchyma cells surrounding the vascular bundles (i.e. bundle sheath cells) was reported as early as in the late 1800’s [1]. In these species, the initial carbon fixation into 4-carbon acids was first documented in the 1960’s [2,3]. In the C4 model, atmospheric CO2 is initially converted into C4 acids by
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