Structural changes in the vacuole and cytoskeleton are key to development of the two cytoplasmic domains supporting single-cell C4 photosynthesis in Bienertia sinuspersici

Abstract

Bienertia sinuspersici Akhani has an unusual mechanism of C4 photosynthesis which occurs within individual chlorenchyma cells. To perform C4, the mature cells have two cytoplasmic compartments consisting of a central (CCC) and a peripheral (PCC) domain containing dimorphic chloroplasts which are interconnected by cytoplasmic channels. Based on leaf development studies, young chlorenchyma cells have not developed the two cytoplasmic compartments and dimorphic chloroplasts. Fluorescent dyes which are targeted to membranes or to specific organelles were used to follow changes in cell structure and organelle distribution during formation of C4-type chlorenchyma. Chlorenchyma cell development was divided into four stages: 1-the nucleus and chloroplasts occupy much of the cytoplasmic space and only small vacuoles are formed; 2-development of larger vacuoles, formation of a pre-CCC with some scattered chloroplasts; 3-the vacuole expands, cells have directional growth; 4-mature stage, cells have become elongated, with a distinctive CCC and PCC joined by interconnecting cytoplasmic channels. By staining vacuoles with a fluorescent dye and constructing 3D images of chloroplasts, and by microinjecting a fluorescence dye into the vacuole of living cells, it was demonstrated that the mature cell has only one vacuole, which is traversed by cytoplasmic channels connecting the CCC with the PCC. Immunofluorescent studies on isolated chlorenchyma cells treated with cytoskeleton disrupting drugs suspended in different levels of osmoticum showed that both microtubules and actin filaments are important in maintaining the cytoplasmic domains. With prolonged exposure of plants to dim light, the cytoskeleton undergoes changes and there is a dramatic shift of the CCC from the center toward the distal end of the cell.

Isolation of dimorphic chloroplasts from the single-cell C4 species Bienertia sinuspersici

“…The hypo-osmotic shock causes the plasma membrane to stretch beyond its extension limit that eventually breaks, releasing the intact CCC and vacuole with attached peripheral chloroplasts (P-Chls; Figure 1D). This is in agreement with the previous observation showing that the two cytoplasmic compartments are separated by a single vacuole [9,11]. Eventually, the central vacuole carrying P-Chls on the external surface (Figure 1E) and the isolated CCC containing central chloroplasts (C-Chls; Figure 1F) can be separated into two discrete entities.…”

“…Interested readers should refer to our previous report for detailed technical considerations on plant growth conditions and chlorenchyma protoplast isolation [12]. Previously, progressive developmental variation has been identified at different stages of B. sinuspersici leaves in terms of the unique subcellular compartmentation and photosynthetic gene expression [11,13]. Similar developmental gradients were also observed across the base-to-tip dimension of leaves [13].…”

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

“…Nevertheless, it was speculated that in vivo, the tonoplast membrane (mediated by the pressure of the vacuole) might be partially responsible for maintaining the structure of the CC Park et al, 2009). Our study shows that, even after disrupting the cell and thereby disrupting the vacuole, the CC remains undamaged and is very resistant to mechanical force.…”

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

“…It has been demonstrated that the unique SCC4 related subcellular compartmentalization and the morphological and biochemical specialization of the two chloroplast types develop gradually from very young towards more mature cells13141543 (Fig. 8B).…”

“…Most information on single-cell C4 photosynthesis comes from the model species Bienertia sinuspersici (hereafter referred to as Bienertia). In Bienertia, it has been shown that the two chloroplast types are separated by a single large vacuole13, which presumably serves as a diffusion barrier between the two reaction compartments. The peripheral and the central compartments are connected by numerous cytoplasmic channels for metabolite exchange814.…”