Exploring mechanisms linked to differentiation and function of dimorphic chloroplasts in the single cell C₄ species Bienertia sinuspersici

Background: In the model single-cell C₄ plant Bienertia sinuspersici, chloroplast- and nuclear-encoded photosynthetic enzymes, characteristically confined to either bundle sheath or mesophyll cells in Kranz-type C₄ leaves, all occur together within individual leaf chlorenchyma cells. Intracellular separation of dimorphic chloroplasts and key enzymes within central and peripheral compartments allow for C₄ carbon fixation analogous to NAD-malic enzyme (NAD-ME) Kranz type species. Several methods were used to investigate dimorphic chloroplast differentiation in B. sinuspersici.Results: Confocal analysis revealed that Rubisco-containing chloroplasts in the central compartment chloroplasts (CCC) contained more photosystem II proteins than the peripheral compartment chloroplasts (PCC) which contain pyruvate,Pi dikinase (PPDK), a pattern analogous to the cell type-specific chloroplasts of many Kranz type NAD-ME species. Transient expression analysis using GFP fusion constructs containing various lengths of a B. sinuspersici Rubisco small subunit (RbcS) gene and the transit peptide of PPDK revealed that their import was not specific to either chloroplast type. Immunolocalization showed the rbcL-specific mRNA binding protein RLSB to be selectively localized to the CCC in B. sinuspersici, and to Rubisco-containing BS chloroplasts in the closely related Kranz species Suaeda taxifolia. Comparative fluorescence analyses were made using redox-sensitive and insensitive GFP forms, as well comparative staining using the peroxidase indicator 3,3-diaminobenzidine (DAB), which demonstrated differences in stromal redox potential, with the CCC having a more negative potential than the PCC.Conclusions: Both CCC RLSB localization and the differential chloroplast redox state are suggested to have a role in post-transcriptional rbcL expression.