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

Abstract

BackgroundIn 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.ResultsConfocal 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.ConclusionsBoth CCC RLSB localization and the differential chloroplast redox state are suggested to have a role in post-transcriptional rbcL expression.

The unique structural and biochemical development of single cell C4photosynthesis along longitudinal leaf gradients inBienertia sinuspersiciandSuaeda aralocaspica(Chenopodiaceae)

“…The selective localization of Rubisco (consisting of the nuclear-encoded rbcS and chloroplast-encoded rbcL) for function within the C 4 -D domain is one of the most definitive biochemical features of SC-C 4 . Transient expression studies using constructs expressing a GFP fusion with the B. sinuspersici rbcS plastid transit peptide in B. sinuspersici SC-C 4 mesophyll protoplasts provided no evidence for selective import within these cells ( Rosnow et al , 2014 ). In the current study, chloroplast-encoded Rubisco rbcL was used as an indicator of changes in gene expression that occurs during the differentiation of dimorphic chloroplast along the longitudinal gradient of SC-C 4 development.…”

“…Recently it was shown that the rbc L-specific mRNA binding protein RLSB has a role in post-transcriptional rbc L expression and possibly selective plastid-specific accumulation of Rubisco in several C 4 species, including SC-C 4 B. sinuspersici as well as the C 4 dicots Suaeda taxifolia and Flaveria bidentis , and the C 4 monocots Zea mays and Setaria viridis ( Bowman et al , 2013 ; Rosnow et al , 2014 ). In B. sinuspersici , RLSB is co-localized with Rubisco specifically within the central compartment chloroplasts ( Rosnow et al , 2014 ), while in Kranz-type species it co-localizes with Rubisco only in BS chloroplasts. It has been proposed that RLSB and the differential redox status of the dimorphic chloroplasts might work together to selectively control synthesis of Rubisco in the C 4 -D domain in B. sinuspersici ( Rosnow et al , 2014 ).…”

“…In B. sinuspersici , RLSB is co-localized with Rubisco specifically within the central compartment chloroplasts ( Rosnow et al , 2014 ), while in Kranz-type species it co-localizes with Rubisco only in BS chloroplasts. It has been proposed that RLSB and the differential redox status of the dimorphic chloroplasts might work together to selectively control synthesis of Rubisco in the C 4 -D domain in B. sinuspersici ( Rosnow et al , 2014 ). The established changes in rbc L mRNA and protein at different stages of leaf development demonstrated in this current study will serve as a framework for analyses currently in progress to determine how RLSB and other regulatory factors determine plastid-specific Rubisco accumulation in SC-C 4 plant species.…”

Regulation of Rubisco gene expression in C4 plants

“…For this laser scanning confocal image, Rubisco is observed as a gold color, while the rest of the cell is observed by differential interfering contrast (DIC) imaging. Reaction of the B. sinuspersic sections with primary and secondary antisera was performed as described for Figure 1 top panel, and [13 ]. Scale bar = 50 mM.…”

“…In mature C 4 leaves, specificity to BSC occurs at the levels of mRNA and protein accumulation (Graphical abstract, and Figure 1). In the mature leaves of plants that utilize the less common single cell C 4 system (SC-C 4 ), Rubisco is compartmentalized within only one type of dimorphic chloroplast that is localized to a specific region of the leaf chlorenchyma cell ( Figure 2) [5,12,13 ].…”