Developmental and Subcellular Organization of Single-Cell C₄ Photosynthesis in Bienertia sinuspersici Determined by Large-Scale Proteomics and cDNA Assembly from 454 DNA Sequencing

Abstract

Kranz C4 species strictly depend on separation of primary and secondary carbon fixation reactions in different cell types. In contrast, the single-cell C4 (SCC4) species Bienertia sinuspersici utilizes intracellular compartmentation including two physiologically and biochemically different chloroplast types; however, information on identity, localization, and induction of proteins required for this SCC4 system is currently very limited. In this study, we determined the distribution of photosynthesis-related proteins and the induction of the C4 system during development by label-free proteomics of subcellular fractions and leaves of different developmental stages. This was enabled by inferring a protein sequence database from 454 sequencing of Bienertia cDNAs. Large-scale proteome rearrangements were observed as C4 photosynthesis developed during leaf maturation. The proteomes of the two chloroplasts are different with differential accumulation of linear and cyclic electron transport components, primary and secondary carbon fixation reactions, and a triose-phosphate shuttle that is shared between the two chloroplast types. This differential protein distribution pattern suggests the presence of a mRNA or protein-sorting mechanism for nuclear-encoded, chloroplast-targeted proteins in SCC4 species. The combined information was used to provide a comprehensive model for NAD-ME type carbon fixation in SCC4 species.

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

“…Their preferential accumulation in the P- and C-chloroplasts has been demonstrated previously by in situ localization studies815 as well as western blot analysis17 and proteomics16. Thus, we used these two markers as a starting point to analyze the mechanism behind selective protein accumulation in Bienertia.…”

“…After conversion by TPI to dihydroxyacetone phosphate (DHAP), triose-phosphates can then shuttle back to the bundle sheath (in case of Kranz C4) or the C-chloroplasts (in case of Bienertia) as part of the triose-phosphate shuttle (also called “open Calvin cycle”). In Bienertia, the most abundant isoform of TPI was shown to be strongly enriched in the P-chloroplasts16 and its GFP fusion showed a localization pattern similar to that of PPDK and AK (Fig. 1D).…”

“…This can be concluded, since deactivation of these elements leads to import into both chloroplast types and not to a complete loss of import which would be expected if the elements are required for specific uptake into the P-chloroplasts. This mechanism might seem surprising, but it makes sense in the greater context: The general proteome of the two chloroplast types is similar in Bienertia16 and accordingly, the vast majority of nuclear encoded plastid targeted proteins has to be imported into both chloroplast types. Therefore, information for both P- and C-targeting would be required for all those proteins to ensure equal distribution.…”

“…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.…”