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 ATG5, an autophagy-related gene, in Physcomitrella, and confirmed that atg5 mutants are deficient in the process of autophagy. On carbon or nitrogen starvation medium, atg5 colonies turned yellow earlier than the wild-type (WT) colonies, showing that Physcomitrella atg5 mutants, like yeast and Arabidopsis, 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 atg5 colonies than WT colonies. The sugar content was almost the same between WT and atg5 colonies, indicating that the early-senescence phenotype of atg5 is not explained by sugar deficiency. However, the levels of 7 amino acids showed significantly different alteration between atg5 and WT in the dark: 6 amino acids, particularly arginine and alanine, were much more deficient in the atg5 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 atg5 mutants is partly explained by amino acid imbalance because of the lack of cytoplasmic degradation by autophagy in Physcomitrella.

细胞自噬（autophagy）是细胞通过液泡或溶酶体降解自身部分细胞质的生物学通路。为探明细胞自噬在植物中的生理功能，我们在小立碗藓（Physcomitrella）中敲除了自噬相关基因ATG5，并证实atg5突变体存在细胞自噬功能缺陷。在碳源或氮源饥饿培养基上，atg5突变体菌落的黄化进程早于野生型（WT）菌落，表明小立碗藓atg5突变体与酵母菌、拟南芥一致，对营养饥饿敏感。
在黑暗环境下，即便处于营养充足的培养基中，突变体菌落也会发生黄化，伴随叶绿素与核酮糖-1,5-二磷酸羧化酶/加氧酶（Rubisco）的净降解，同时多个衰老相关基因（senescence-associated genes）的表达量上调。该黄化过程可被蛋白质合成抑制剂环己酰亚胺（cycloheximide）抑制，提示小立碗藓原丝体菌落与高等植物绿叶一样，会经历黑暗诱导的衰老，且atg5突变体菌落的黑暗诱导衰老进程早于野生型。
野生型与atg5突变体菌落的糖含量基本一致，表明atg5突变体的早衰表型并非由糖缺乏所致。但在黑暗环境下，二者的7种氨基酸水平呈现显著差异：6种氨基酸（尤其是精氨酸与丙氨酸）在atg5突变体中的含量大幅降低，即便此时Rubisco蛋白已发生早期降解。
在添加酪蛋白氨基酸（casamino acids）的营养充足培养基上，atg5突变体的早衰表型得到一定程度缓解。我们据此提出：小立碗藓atg5突变体因缺失细胞自噬介导的细胞质降解过程，其早衰表型可部分归因于氨基酸失衡。