Resolving the Compartmentation and Function of C4 Photosynthesis in the Single-Cell C4 Species Bienertia sinuspersici

Bienertia sinuspersici is a land plant known to perform C-4 photosynthesis through the location of dimorphic chloroplasts in separate cytoplasmic domains within a single photosynthetic cell. A protocol was developed with isolated protoplasts to obtain peripheral chloroplasts (P-CP), a central compartment (CC), and chloroplasts from the CC (C-CP) to study the subcellular localization of photosynthetic functions. Analyses of these preparations established intracellular compartmentation of processes to support a NAD-malic enzyme (ME)-type C-4 cycle. Western-blot analyses indicated that the CC has Rubisco from the C-3 cycle, the C-4 decarboxylase NAD-ME, a mitochondrial isoform of aspartate aminotransferase, and photo-respiratory markers, while the C-CP and P-CP have high levels of Rubisco and pyruvate, Pidikinase, respectively. Other enzymes for supporting a NAD-ME cycle via an aspartate-alanine shuttle, carbonic anhydrase, phosophoenolpyruvate carboxylase, alanine, and an isoform of aspartate aminotransferase are localized in the cytosol. Functional characterization by photosynthetic oxygen evolution revealed that only the C-CP have a fully operational C-3 cycle, while both chloroplast types have the capacity to photoreduce 3-phosphoglycerate. The P-CP were enriched in a putative pyruvate transporter and showed light-dependent conversion of pyruvate to phosphoenolpyruvate. There is a larger investment in chloroplasts in the central domain than in the peripheral domain (6-fold more chloroplasts and 4-fold more chlorophyll). The implications of this uneven distribution for the energetics of the C-4 and C-3 cycles are discussed. The results indicate that peripheral and central compartment chloroplasts in the single-cell C-4 species B. sinuspersici function analogous to mesophyll and bundle sheath chloroplasts of Kranz-type C-4 species.