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.