The vacuole is a unique plant organelle that plays an important role in maintaining cellular homeostasis under various environmental stress conditions. However, the effects of biotic stress on vacuole structure has not been examined using three-dimensional (3D) visualization. Here, we performed 3D electron tomography to compare the ultrastructural changes in the vacuole during infection with different viruses. The 3D models revealed that vacuoles are remodeled in cells infected with cucumber mosaic virus (CMV) or tobacco necrosis virus A Chinese isolate (TNV-A C), resulting in the formation of spherules at the periphery of the vacuole. These spherules contain neck-like channels that connect their interior with the cytosol. Confocal microscopy of CMV replication proteins 1a and 2a and TNV-A C auxiliary replication protein p23 showed that all of these proteins localize to the tonoplast. Electron microscopy revealed that the expression of these replication proteins alone is sufficient to induce spherule formation on the tonoplast, suggesting that these proteins play prominent roles in inducing vacuolar membrane remodeling. This is the first report of the 3D structures of viral replication factories built on the tonoplasts. These findings contribute to our understanding of vacuole biogenesis under normal conditions and during assembly of plant (+) RNA virus replication complexes.

Electron tomography is a powerful tool for visualizing subcellular organelles. With the advances in cryo-fixation techniques, it is now possible to reconstruct complex structures in cells preserved close to their native states in three-dimension (3D) using electron tomography. In order to better visualize these objects, 3D models are made from outlines of organelles in individual tomographic slices, which can be used to display morphological features and quantify structural parameters. While outlines of simple organelles can be drawn by hand fairly quickly, it is possible to accelerate 3D modeling of more complex organelles by means of semiautomatic segmentation. In this chapter, we use the example of reconstructing Golgi cisternae of a plant cell into 3D models using the semiautomatic protocol.

Bienertia sinuspersici is a single-cell C4 plant species of which chlorenchyma cells have two distinct groups of chloroplasts spatially segregated in the cytoplasm. The central vacuole encloses most chloroplasts at the cell center and confines the rest of the chloroplasts near the plasma membrane. Young chlorenchyma cells, however, do not have large vacuoles and their chloroplasts are homogenous. Therefore, maturing Bienertia chlorenchyma cells provide a unique opportunity to investigate chloroplast proliferation in the central cluster and the remodeling of chloroplasts that have been displaced by the vacuole to the cell periphery. Chloroplast numbers and sizes increased, more notably, during later stages of maturation than the early stages. Electron tomography analyses indicated that chloroplast enlargement is sustained by thylakoid growth and that invaginations from the inner envelope membrane contributed to thylakoid assembly. Grana stacks acquired more layers, differentiating them from stroma thylakoids as central chloroplasts matured. In peripheral chloroplasts, however, grana stacks stretched out to a degree that the distinction between grana stacks and stroma thylakoids was obscured. In central chloroplasts undergoing division, thylakoids inside the cleavage furrow were kinked and severed. Grana stacks in the division zone were disrupted, and large complexes in their membranes were dislocated, suggesting the existence of a thylakoid fission machinery.

Chloroplast development is a complex process that is critical to the growth and development of plants. However, the detailed mechanism of chloroplast development in woody plants remains unclear. In this study, we showed that chloroplasts with elaborate thylakoids could develop from proplastids in the cells of calli derived from leaf tissues of Populus tomentosa upon exposure to light. Chloroplast development was confirmed at the molecular and cellular levels. Transcriptome analysis revealed that genes related to photoreceptors and photosynthesis were significantly up-regulated during chloroplast development in a time-dependent manner. In light-induced chloroplast development, a key process was the removal of hydrogen peroxide, in which thylakoid-localized PtotAPX played a major role; light-induced chloroplast development was enhanced in PtotAPX-overexpressing transgenic P. tomentosa callus with lower levels of hydrogen peroxide, but was suppressed in PtotAPX antisense transgenic callus with higher levels of hydrogen peroxide. Moreover, the suppression of light-induced chloroplast development in PtotAPX antisense transgenic callus was relieved by the exogenous reactive oxygen species scavenging agent N,N′-dimethylthiourea (DMTU). Based on these results, we propose that PtotAPX-mediated removal of reactive oxygen species plays a key role in chloroplast development from proplastids upon exposure to light in P. tomentosa.

C4 plants enhance photosynthesis efficiency by concentrating CO 2 to the site of Rubisco action. Chloroplasts in C4 plants exhibit structural dimorphism because thylakoid architectures vary depending on energy requirements. Advances in electron microscopy imaging capacity and sample preparation technologies allowed characterization of thylakoid structures and their macromolecular arrangements with unprecedented precision mostly in C3 plants. The thylakoid is assembled during chloroplast biogenesis through collaboration between the plastid and nuclear genomes. Recently, the membrane dynamics involved in the assembly process has been investigated with 3D electron microscopy, and molecular factors required for thylakoid construction have been characterized. The two classes of chloroplasts in C4 plants arise from common precursors, but little is known about how a single type of chloroplasts grow, divide, and differentiate to mature into distinct chloroplasts. Here, we outline the thylakoid structure and its assembly processes in C3 plants to discuss ultrastructural analyses of dimorphic chloroplast biogenesis in C4 plant species. Future directions for electron microscopy research of C4 photosynthetic systems are also proposed.