ATG5-knockout mutants of Physcomitrella provide a platform for analyzing the involvement of autophagy in senescence processes in plant cells

Autophagy is a pathway in which a cell degrades part of its cytoplasm in vacuoles or lysosomes. To identify the physiological functions of autophagy in plants, we disrupted <i>ATG5</i>, an autophagy-related gene, in <i>Physcomitrella</i>, and confirmed that <i>atg5</i> mutants are deficient in the process of autophagy. On carbon or nitrogen starvation medium, <i>atg5</i> colonies turned yellow earlier than the wild-type (WT) colonies, showing that <i>Physcomitrella atg5</i> mutants, like yeast and <i>Arabidopsis</i>, are sensitive to nutrient starvation. In the dark, even under nutrient-sufficient conditions, colonies turned yellow and the net degradation of chlorophyll and Rubisco protein occurred together with the upregulation of several senescence-associated genes. Yellowing reactions were inhibited by the protein synthesis inhibitor cycloheximide, suggesting that protonemal colonies undergo dark-induced senescence like the green leaves of higher plants. Such senescence responses in the dark occurred earlier in <i>atg5</i> colonies than WT colonies. The sugar content was almost the same between WT and <i>atg5</i> colonies, indicating that the early-senescence phenotype of <i>atg5</i> is not explained by sugar deficiency. However, the levels of 7 amino acids showed significantly different alteration between <i>atg5</i> and WT in the dark: 6 amino acids, particularly arginine and alanine, were much more deficient in the <i>atg5</i> mutants, irrespective of the early degradation of Rubisco protein. On nutrient-sufficient medium supplemented with casamino acids, the early-senescence phenotype was slightly moderated. We propose that the early-senescence phenotype in <i>atg5</i> mutants is partly explained by amino acid imbalance because of the lack of cytoplasmic degradation by autophagy in <i>Physcomitrella</i>.