Isolation of dimorphic chloroplasts from the single-cell C 4 species Bienertia sinuspersici
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PLANT METHODS
METHODOLOGY
Open Access
Isolation of dimorphic chloroplasts from the single-cell C4 species Bienertia sinuspersici Shiu-Cheung Lung, Makoto Yanagisawa and Simon DX Chuong*
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. Keywords: Bienertia sinuspersici, Chloroplast isolation, Dimorphic chloroplasts, Osmotic swelling, Photosynthesis, protoplast, Single-cell C4, Vacuole isolation
Background 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]. The physiological relevance of the Kranz anatomy in relation to the C4 photosynthetic pathways, however, had not been elucidated until the successful separation of the two types of chlorenchyma cells and their respective dimorphic chloroplasts. With the development of various mechanical and enzymatic methods for separating the mesophyll and bundle sheath cells, the biochemistry of C4 cycles has been intensively studied over the past few decades focusing explicitly on characterizing the enzymatic properties and determining their precise subcellular locations in these cell types (for review, see [4]), leading to the current C4 models. In the C4 model, atmospheric CO 2 is initially converted into C 4 acids by * Correspondence: [email protected] Department of Biology, University of Waterloo, 200 University Avenue West, Waterloo, Ontario N2L 3G1, Canada
phosphoenolpyruvate carboxylase (PEPC) in mesophyll cells. The C4 acids are broken down by a C4 subtype-specific decarboxylation enzyme in bundle sheath cells, and the liberated CO2 is subsequently re-fixed by ribulose1,5-bisphosphate carboxylase/oxygenase (Rubisco). The C4 pathway concentrates CO2 at the site of Rubisco and minimizes the photorespiration process, an unfavorable oxygenase activity of Rubisco with O2. The indispensable relationship between the Kranz anatomy and C4 photosynthesis has been an
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