Isolation of dimorphic chloroplasts from the single-cell C4 species Bienertia sinuspersici

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 CO2 is initially converted into C4 acids by 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 ribulose-1,5-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 accepted feature until the discovery of three terrestrial single-cell C4 species, Suaeda aralocaspica (formerly called Borszczowia aralocaspica) [5], Bienertia cycloptera [6,7], and B. sinuspersici [8] in the Chenopodia