Autophagy Promotes Cell Death Induced by Hydrogen Peroxide in Physcomitrium patens.

The autophagy-defective mutants (atg5 and atg7) of Physcomitrium patens exhibit strong desiccation tolerance. Here, we examined the effects of H2O2 on wild-type (WT) and autophagy-defective mutants of P. patens, considering that desiccation induces reactive oxygen species (ROS). We found that atg mutants can survive a 30-min treatment with 100 mM H2O2, whereas WT cannot, implying that autophagy promotes cell death induced by H2O2. Concomitant with cell death, vacuole collapse occurred. Intracellular H2O2 levels in both WT and atg5 increased immediately after H2O2 treatment and subsequently reached plateaus, which were higher in WT than in atg5. The ROS scavenger N-acetylcysteine lowered the plateau levels in WT and blocked cell death, suggesting that higher H2O2 plateau caused cell death. The uncoupler of electron transport chain (ETC) carbonyl cyanide m-chlorophenylhydrazone also lowered the H2O2 plateaus, showing that ROS produced in the ETC in mitochondria and/or chloroplasts elevated the H2O2 plateau. The autophagy inhibitor 3-methyladenine lowered the H2O2 plateau and the cell death rate in WT, suggesting that autophagy occurring after H2O2 treatment is involved in the production of ROS. Conversely, the addition of bovine serum albumin, which is endocytosed and supplies amino acids instead of autophagy, elevated the H2O2 plateau in atg5 cells, suggesting that amino acids produced through autophagy promote H2O2 generation. These results clearly show that autophagy causes cell death under certain stress conditions. We propose that autophagy-derived amino acids are catabolized using ETCs in mitochondria and/or chloroplasts and produce H2O2, which in turn promotes the cell death accompanying vacuole collapse.

Hydrogen Peroxide Mediates Premature Senescence Caused by Darkness and Inorganic Nitrogen Starvation in Physcomitrium patens.

Leaf senescence accompanied by yellowing and Rubisco degradation occurs prematurely in response to various stresses. However, signaling pathways between stress perception and senescence responses are not understood fully, although previous studies suggest the involvement of reactive oxygen species (ROS). While investigating the physiological functions of autophagy in Physcomitrium patens using wild-type (WT) and autophagy-deficient atg5 strains, we found that Physcomitrium colonies senesce prematurely under dark or nitrogen-deficient conditions, with atg5 senescing earlier than WT. In the present study, we measured cellular H2O2, and examined whether H2O2 mediates premature senescence in Physcomitrium colonies. Methyl viologen, an ROS generator, increased cellular H2O2 levels and caused senescence-like symptoms. H2O2 levels were also elevated to the same plateau levels in WT and atg5 under dark or nitrogen-deficient conditions. The ROS scavenger N-acetylcysteine and the ROS source inhibitor carbonyl cyanide m-chlorophenylhydrazone inhibited the increase in H2O2 levels as well as senescence. Upon transfer to a nitrogen-deficient medium, H2O2 levels increased earlier in atg5 than in WT by ~18 h, whereas atg5 yellowed earlier by >2 days. We conclude that the increased H2O2 levels under dark or nitrogen-deficient conditions mediate premature senescence in Physcomitrium but do not explain the different senescence responses of WT and atg5 cells.

Amino Acids Supplied through the Autophagy/Endocytosis Pathway Promote Starch Synthesis in Physcomitrella Protonemal Cells.

The physiological implications of autophagy in plant cells have not been fully elucidated. Therefore, we investigated the consequences of autophagy in the moss Physcomitrella by measuring biochemical parameters (fresh and dry weights; starch, amino acid, carbohydrate, and NH3 content) in wild-type (WT) and autophagy-deficient atg5 Physcomitrella cells. We found higher starch levels and a higher net starch synthesis rate in WT cells than in atg5 cells cultured in a glucose-containing culture medium, whereas net starch degradation was similar in the two strains cultured in a glucose-deficient culture medium. Additionally, the treatment of cells with the autophagy inhibitor 3-methyladenine suppressed starch synthesis. Loading bovine serum albumin into atg5 cells through endocytosis, i.e., supplying proteins to vacuoles in the same way as through autophagy, accelerated starch synthesis, whereas loading glutamine through the plasma membrane had no such effect, suggesting that Physcomitrella cells distinguish between different amino acid supply pathways. After net starch synthesis, NH3 levels increased in WT cells, although the change in total amino acid content did not differ between WT and atg5 cells, indicating that autophagy-produced amino acids are oxidized rapidly. We conclude that autophagy promotes starch synthesis in Physcomitrella by supplying the energy obtained by oxidizing autophagy-produced amino acids.

Isolation of Autolysosomes from Tobacco BY-2 Cells.

Autolysosomes are organelles that sequester and degrade a portion of the cytoplasm during autophagy. Although autophagosomes are short lived compared to other organelles such as mitochondria, plastids, and peroxisomes, many autolysosomes accumulate in tobacco BY-2 cells cultured under sucrose starvation conditions in the presence of a cysteine protease inhibitor. We here describe our methodology for isolating autolysosomes from BY-2 cells by conventional cell fractionation using a Percoll gradient. The autolysosome fraction separates clearly from fractions containing mitochondria and peroxisomes. It contains acid phosphatase, vacuolar H+-ATPase, and protease activity. Electron micrographs show that the fraction contains partially degraded cytoplasm seen in autolysosomes before isolation although an autolysosome structure is only partially preserved.

Plant Cell Wall-Penetrable, Redox-Responsive Silica Nanoprobe for the Imaging of Starvation-Induced Vesicle Trafficking.

Autophagy is a self-protection process against reactive oxygen species (ROS). The intracellular level of ROS increased when cells were cultured under nutrient starvation. Antioxidants such as glutathione and ascorbic acid play an important role in ROS removal. However, the cellular redox state in the autophagic pathway is still unclear. Herein, we developed a new redox-sensitive probe with a disulfide-linked silica scaffold to enable the sensing of the reduction environment in cell organelles. This redox-responsive silica nanoprobe (ReSiN) could penetrate the plant cell wall and release fluorescent molecules in response to redox states. By applying the ReSiN to tobacco BY-2 cells and tracing the distribution of fluorescence, we found a higher reducing potential in the central vacuole than in the autolysosomes. Upon cysteine protease inhibitor (E64-c) treatment in sucrose-free medium, the disulfide-silica structures of the ReSiNs were broken down in the vacuoles but were not degraded and were accumulated in the autolysosomes. These results reveal the feasibility of our nanoprobe for monitoring the endocytic and macroautophagic pathways. These pathways merge upstream of the central vacuole, which is the final destination of both pathways. In addition, different redox potentials were observed in the autophagic pathway. Finally, the expression of the autophagy-related protein (Atg8) fused with green fluorescence protein confirmed that the ReSiN treatment itself did not induce the autophagic pathway under normal physiological conditions, indicating the versatility of this nanoprobe in studying stimuli-triggered autophagy-related trafficking.

Atg29 phosphorylation regulates coordination of the Atg17-Atg31-Atg29 complex with the Atg11 scaffold during autophagy initiation.

Macroautophagy (hereafter autophagy) functions in the nonselective clearance of cytoplasm. This process participates in many aspects of cell physiology, and is conserved in all eukaryotes. Autophagy begins with the organization of the phagophore assembly site (PAS), where most of the AuTophaGy-related (Atg) proteins are at least transiently localized. Autophagy occurs at a basal level and can be induced by various types of stress; the process must be tightly regulated because insufficient or excessive autophagy can be deleterious. A complex composed of Atg17-Atg31-Atg29 is vital for PAS organization and autophagy induction, implying a significant role in autophagy regulation. In this study, we demonstrate that Atg29 is a phosphorylated protein and that this modification is critical to its function; alanine substitution at the phosphorylation sites blocks its interaction with the scaffold protein Atg11 and its ability to facilitate assembly of the PAS. Atg29 has the characteristics of an intrinsically disordered protein, suggesting that it undergoes dynamic conformational changes on interaction with a binding partner(s). Finally, single-particle electron microscopy analysis of the Atg17-Atg31-Atg29 complex reveals an elongated structure with Atg29 located at the opposing ends.