Singlet oxygen leads to structural changes to chloroplasts during degradation in the Arabidopsis thaliana plastid ferrochelatase two mutant

During photosynthesis, chloroplasts can produce large amounts of reactive oxygen species (ROS), particularly under stressful conditions. Along with other nutrients, chloroplasts also contain 80% of a leaf's nitrogen supply. For these reasons, chloroplasts are prime targets for cellular degradation to protect cells from photo-oxidative damage and to redistribute nutrients to sink tissues. Multiple chloroplast degradation pathways have been described and are induced by photo-oxidative stress and nutrient starvation. However, the mechanisms by which damaged or senescing chloroplasts are identified, transported to the central vacuole, and ultimately degraded are not well characterized. Here, we investigated the subcellular structures involved with degrading chloroplasts induced by the ROS singlet oxygen (1O2) in the Arabidopsis thaliana plastid ferrochelatase two (fc2) mutant. Using a three-dimensional serial-block face electron microscopy analysis, we show up to 35% of degrading chloroplasts in fc2 mutants protrude into the central vacuole. While the location of a chloroplast within a cell had no effect on the likelihood of its degradation, chloroplasts in spongy mesophyll cells were degraded at a higher rate than those in palisade mesophyll cells. To determine if degrading chloroplasts have unique structural characteristics allowing them to be distinguished from healthy chloroplasts, we analyzed fc2 seedlings grown under different levels of photo-oxidative stress. A clear correlation was observed between chloroplast swelling, 1O2-signaling, and the state of degradation. Finally, plastoglobule enzymes involved in chloroplast disassembly were shown to be upregulated while plastoglobules increased their association with the thylakoid grana, implicating an interaction between 1O2-induced chloroplast degradation and senescence pathways.

The core autophagy machinery is not required for chloroplast singlet oxygen-mediated cell death in the Arabidopsis thaliana plastid ferrochelatase two mutant

Background Chloroplasts respond to stress and changes in the environment by producing reactive oxygen species (ROS) that have specific signaling abilities. The ROS singlet oxygen (1O2) is unique in that it can signal to initiate cellular degradation including the selective degradation of damaged chloroplasts. This chloroplast quality control pathway can be monitored in the Arabidopsisthaliana mutant plastid ferrochelatase two (fc2) that conditionally accumulates chloroplast 1O2 under diurnal light cycling conditions leading to rapid chloroplast degradation and eventual cell death. The cellular machinery involved in such degradation, however, remains unknown. Recently, it was demonstrated that whole damaged chloroplasts can be transported to the central vacuole via a process requiring autophagosomes and core components of the autophagy machinery. The relationship between this process, referred to as chlorophagy, and the degradation of 1O2-stressed chloroplasts and cells has remained unexplored. Results To further understand 1O2-induced cellular degradation and determine what role autophagy may play, the expression of autophagy-related genes was monitored in 1O2-stressed fc2 seedlings and found to be induced. Although autophagosomes were present in fc2 cells, they did not associate with chloroplasts during 1O2 stress. Mutations affecting the core autophagy machinery (atg5, atg7, and atg10) were unable to suppress 1O2-induced cell death or chloroplast protrusion into the central vacuole, suggesting autophagosome formation is dispensable for such 1O2–mediated cellular degradation. However, both atg5 and atg7 led to specific defects in chloroplast ultrastructure and photosynthetic efficiencies, suggesting core autophagy machinery is involved in protecting chloroplasts from photo-oxidative damage. Finally, genes predicted to be involved in microautophagy were shown to be induced in stressed fc2 seedlings, indicating a possible role for an alternate form of autophagy in the dismantling of 1O2-damaged chloroplasts. Conclusions Our results support the hypothesis that 1O2-dependent cell death is independent from autophagosome formation, canonical autophagy, and chlorophagy. Furthermore, autophagosome-independent microautophagy may be involved in degrading 1O2-damaged chloroplasts. At the same time, canonical autophagy may still play a role in protecting chloroplasts from 1O2-induced photo-oxidative stress. Together, this suggests chloroplast function and degradation is a complex process utilizing multiple autophagy and degradation machineries, possibly depending on the type of stress or damage incurred.

Subcellular Localization and Vesicular Structures of Anthocyanin Pigmentation by Fluorescence Imaging of Black Rice (Oryza sativa L.) Stigma Protoplast

Anthocyanins belong to the group of flavonoid compounds broadly distributed in plant species responsible for attractive colors. In black rice (Oryza sativa L.), they are present in the stems, leaves, stigmas, and caryopsis. However, there is still no scientific evidence supporting the existence of compartmentalization and trafficking of anthocyanin inside the cells. In the current study, we took advantage of autofluorescence with anthocyanin’s unique excitation/emission properties to elucidate the subcellular localization of anthocyanin and report on the in planta characterization of anthocyanin prevacuolar vesicles (APV) and anthocyanic vacuolar inclusion (AVI) structure. Protoplasts were isolated from the stigma of black and brown rice and imaging using a confocal microscope. Our result showed the fluorescence displaying magenta color in purple stigma and no fluorescence in white stigma when excitation was provided by a helium–neon 552 nm and emission long pass 610–670 nm laser. The fluorescence was distributed throughout the cell, mainly in the central vacuole. Fluorescent images revealed two pools of anthocyanin inside the cells. The diffuse pools were largely found inside the vacuole lumen, while the body structures could be observed mostly inside the cytoplasm (APV) and slightly inside the vacuole (AVI) with different shapes, sizes, and color intensity. Based on their sizes, AVI could be grouped into small (Ф < 0.5 um), middle (Ф between 0.5 and 1 um), and large size (Ф > 1 um). Together, these results provided evidence about the sequestration and trafficking of anthocyanin from the cytoplasm to the central vacuole and the existence of different transport mechanisms of anthocyanin. Our results suggest that stigma cells are an excellent system for in vivo studying of anthocyanin in rice and provide a good foundation for understanding anthocyanin metabolism in plants, sequestration, and trafficking in black rice.

Electron Tomography Analysis of Thylakoid Assembly and Fission in Chloroplasts of a Single-Cell C4 plant, Bienertia sinuspersici

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.

Polyploid monolayer Ishikawa endometrial cells form unicellular hollow spheroids capable of migration

The results in this paper demonstrate that Ishikawa endometrial monolayer cells become multinucleated by a process of nuclear “donation” from neighboring cells. As the resulting polyploid cell detaches from the colony in which it was formed, it is possible to detect mitonucleon(s) in the center of the cell. The mitonucleon is a transient mitochondrial superstructure surrounding aggregated chromatin (Fleming et al. 1998) with characteristics of the family of mitochondrial superstructures that are sometimes called spheroids or cup-shaped mitochondria (Fleming, 2016a). As was recently demonstrated gas vacuoles form within mitonucleons (Fleming, 2018). In the free-floating single cell, the retained gas creates a central vacuole, and the cell becomes a spheroid that floats above the monolayer. It resembles a “signet ring cell” in being characterized by a central vacuole and chromatin compressed against the vacuole membrane. The resulting structure is a spheroids that is hollow and unicellular, albeit polyploid. But whereas signet ring cells are assumed to be undergoing apoptosis, that is not the case for unicellular spheroids. Complete spheres with chromatin and cytosolic cell contents compressed against the cell membrane can be found floating independently above Ishikawa monolayers. When an isolated sphere settles back onto the surface of the petri dish, it is possible to observe dissipating gas bubbles within the now flattened sphere for a short period of time. When the gas is discharged the resulting cell looks like a typical giant polyploid cell.

Polyploid monolayer Ishikawa endometrial cells form unicellular hollow spheroids capable of migration

The results in this paper demonstrate that Ishikawa endometrial monolayer cells become multinucleated by a process of nuclear “donation” from neighboring cells. As the resulting polyploid cell detaches from the colony in which it was formed, it is possible to detect mitonucleon(s) in the center of the cell. The mitonucleon is a transient mitochondrial superstructure surrounding aggregated chromatin (Fleming et al. 1998) with characteristics of the family of mitochondrial superstructures that are sometimes called spheroids or cup-shaped mitochondria (Fleming, 2016a). As was recently demonstrated gas vacuoles form within mitonucleons (Fleming, 2018). In the free-floating single cell, the retained gas creates a central vacuole, and the cell becomes a spheroid that floats above the monolayer. It resembles a “signet ring cell” in being characterized by a central vacuole and chromatin compressed against the vacuole membrane. The resulting structure is a spheroids that is hollow and unicellular, albeit polyploid. But whereas signet ring cells are assumed to be undergoing apoptosis, that is not the case for unicellular spheroids. Complete spheres with chromatin and cytosolic cell contents compressed against the cell membrane can be found floating independently above Ishikawa monolayers. When an isolated sphere settles back onto the surface of the petri dish, it is possible to observe dissipating gas bubbles within the now flattened sphere for a short period of time. When the gas is discharged the resulting cell looks like a typical giant polyploid cell.