Establishment of Monitoring Methods for Autophagy in Rice Reveals Autophagic Recycling of Chloroplasts and Root Plastids during Energy Limitation

Abstract

Autophagy is an intracellular process leading to vacuolar or lysosomal degradation of cytoplasmic components in eukaryotes. Establishment of proper methods to monitor autophagy was a key step in uncovering its role in organisms, such as yeast (Saccharomyces cerevisiae), mammals, and Arabidopsis (Arabidopsis thaliana), in which chloroplastic proteins were found to be recycled by autophagy. Chloroplast recycling has been predicted to function in nutrient remobilization for growing organs or grain filling in cereal crops. Here, to develop our understanding of autophagy in cereals, we established monitoring methods for chloroplast autophagy in rice (Oryza sativa). We generated transgenic rice-expressing fluorescent protein (FP) OsAuTophaGy8 (OsATG8) fusions as autophagy markers. FP-ATG8 signals were delivered into the vacuolar lumen in living cells of roots and leaves mainly as vesicles corresponding to autophagic bodies. This phenomenon was not observed upon the addition of wortmannin, an inhibitor of autophagy, or in an ATG7 knockout mutant. Markers for the chloroplast stroma, stromal FP, and FP-labeled Rubisco were delivered by a type of autophagic body called the Rubisco-containing body (RCB) in the same manner. RCB production in excised leaves was suppressed by supply of external sucrose or light. The release of free FP caused by autophagy-dependent breakdown of FP-labeled Rubisco was induced during accelerated senescence in individually darkened leaves. In roots, nongreen plastids underwent both RCB-mediated and entire organelle types of autophagy. Therefore, our newly developed methods to monitor autophagy directly showed autophagic degradation of leaf chloroplasts and root plastids in rice plants and its induction during energy limitation.

ATG9 regulates autophagosome progression from the endoplasmic reticulum in Arabidopsis

“…The phosphoinositide 3-kinase (PI3K) inhibitor wortmannin has been used to block this process, in which the formation of autophagosome-related structures [labeled by ATG5, ATG8, or SH3 domain-containing protein (SH3P2)] were suppressed upon wortmannin treatment in plant (10,27,28). Interestingly, we found that the wortmannin treatment abrogates the formation of YFP-ATG8e-labeled abnormal structures in the atg9-3 background during autophagy (Fig.…”

Entire Photodamaged Chloroplasts Are Transported to the Central Vacuole by Autophagy

“…Our previous studies showed that nutrient recycling during senescence or starvation is achieved mainly by piecemeal autophagy of chloroplasts via the RCB pathway (Ishida et al, 2008;Ono et al, 2013;Izumi et al, 2015). RCB pathways are specifically activated during energy limitation due to impaired photosynthetic conditions such as darkness (Izumi et al, 2010.…”

“…Immunoblotting was performed as previously described (Ishida et al, 2008;Izumi et al, 2015), with slight modification. Leaves were homogenized in HEPES-NaOH (pH 7.5) containing 14 mM 2-mercaptoethanol, 10% (v/v) glycerol, 2% (w/v) SDS, and protease inhibitor cocktail (Roche) and then centrifuged at 10,000g for 5 min.…”

Stromules: Probing Formation and Function

“…Small bodies containing stroma-localized fluorescent protein can be visualized in the vacuoles, provided their lytic activity is inhibited. The accumulation of these bodies in the vacuole occurs during nutrient stress, and is dependent on the autophagy system, as the stroma-containing bodies are not seen in mutants lacking ATG7 (Ishida et al, 2008;Izumi et al, 2010Izumi et al, , 2015. Whether stromules are the actual source of these bodies through tip-shedding is not known.…”