Structural changes in the vacuole and cytoskeleton are key to development of the two cytoplasmic domains supporting single-cell C(4) photosynthesis in Bienertia sinuspersici.

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.