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

Abstract

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 photorespiratory 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.Rubisco is the central CO 2 fixation enzyme in plants. It is a bifunctional enzyme, catalyzing the carboxylation as well as the oxygenation of ribulose-bisphosphate (RuBP) to 3-phosphoglycerate (3-PGA) and phosphoglycolate, respectively. While 3-PGA is subsequently utilized in the C 3 cycle to produce triose-phosphates, phosphoglycolate has no known metabolic use (Ogren, 1984) and needs to be detoxified by the plant. This process is known as photorespiration, which is not only energy consuming but also results in a net loss of previously fixed carbon and nitrogen. The rate of carboxylation versus oxygenation through Rubisco is directly related to the availability of CO 2 and the concentration of oxygen competing for the catalytic site of the enzyme. Since Rubisco is thought to have evolved under much higher levels of CO 2 than current atmospheric concentrations, it is believed that photorespiration was insignificant earlier in photosynthetic organisms. Under current atmospheric CO 2 levels, however, significant amounts (e.g. up to 25%) of previously fixed carbon may be lost through RuBP oxygenase activity under conditions that favor photorespiration, like high temperatures or water stres...

Single cell C4 photosynthesis in aquatic and terrestrial plants: A gas exchange perspective

“…the central compartment to study the subcellular compartmentalization of enzymes and transporters in B. sinuspersici (Offermann et al, 2011). This study provided functional evidence of NAD-ME type biochemistry in B. sinuspersici.…”

“…Also curious was the large investment in chloroplast and chlorophyll to the central compartment not dissimilar to the investment of chlorophyll and nitrogen to the BS compartment seen in terrestrial NAD-ME species (Ghannoum et al, 2005). The study by Offermann et al (2011) also identified a pyruvate transporter in the peripheral chloroplasts for import of this substrate for PPDK, which supports another requirement for C 4 photosynthesis. The third requirement of symplastic connection between the two cytosolic compartments is probably more easily met; however, how the necessary metabolite gradients are established between the two compartments is intriguing.…”

C4 Cycles: Past, Present, and Future Research on C4 Photosynthesis

“…Leaf development in these species is quite remarkable in that chlorenchyma cells are organized into two distinct cytoplasmic compartments that are maintained by an organized network of actin filaments and microtubules (Chuong et al, 2006). In Binertia, there is a centrally located compartment surrounded by a more peripheral compartment (Voznesenskaya et al, 2002;Offermann et al, 2011), whereas in Suaeda, the two compartments are distal (toward the outside of the leaf) and proximal in the cell (Voznesenskaya et al, 2001). In each of the two compartments, chloroplasts accumulate a distinct complement of photosynthetic enzymes with the peripheral/distal chloroplasts analogous to M cell chloroplasts of the Kranz system and the central/proximal chloroplasts analogous to the BS cell chloroplasts.…”

Transit peptide elements mediate selective protein targeting to two different types of chloroplasts in the single-cell C4 species Bienertia sinuspersici

“…CO 2 released from mitochondrial decarboxylation can then be re-fixed by the adjacent C-chloroplasts. In summary, protein localization studies, gas exchange properties and physiological experiments indicated that the P- and C-chloroplasts of SCC4 species basically resemble the biochemistry and function of MC and BSC chloroplasts of Kranz C4 species16172021.…”

“…Numerous studies have demonstrated that the two chloroplast types accumulate different sets of nuclear encoded proteins according to their specific functions in the C4 and C3 cycle respectively8915161718. For example, nuclear encoded pyruvate, Pi-dikinase (PPDK), the key enzyme needed for generation of the primary CO 2 acceptor phospho enol pyruvate (PEP) is found specifically in the P-chloroplasts only.…”