Autophagic degradation of leaf starch in plants

Abstract

Autophagy is an evolutionarily conserved process in eukaryotic cells that functions to degrade cytoplasmic components in the vacuole or lysosome. Previous research indicates that the core molecular machinery of autophagosome formation works well in plants, and plant autophagy plays roles in diverse biological processes such as nutrient recycling, development, immunity and responses to a variety of abiotic stresses. Recently, we reported that autophagy contributed to leaf starch degradation, which had been thought to be a process confined to chloroplasts. This finding demonstrated a previously unidentified pathway of leaf starch depletion and a new role of basal autophagy in plants.

Starch is the major storage polysaccharide in plants. There are two types of starch: reserve starch in sink organs, and transitory starch in leaves (the latter is also referred to as leaf starch). Leaf starch is synthesized in chloroplasts during the day and degraded during the night to produce neutral sugars such as maltose and glucose. Using biochemical and microscopy approaches, we monitored the nocturnal changes of leaf starch in Nicotiana benthamiana. As the night deepened, both the size and average number of visible starch granules per chloroplast diminishes. Accordingly, the leaf starch content decreases and starch reserves are almost exhausted at dawn. It seems that the first half of the night is a critical stage of starch degradation because most of the starch mobilized (about 70%) during the night is depleted within the first 4 h of darkness.

To investigate the involvement of autophagy in leaf starch degradation, we detected nocturnal changes in expression levels of autophagy-related (ATG) genes and autophagic activities in leaves. In the first 2 or 4 h of darkness, several ATG genes including ATG6, ATG7, ATG9 and PI3K are upregulated. Concurrently, autophagic activities measured by diverse approaches (monodansylcadaverine staining, CFP-ATG8f labeling method and transmission electron microscopy) exhibit a 3- to 4-fold increase. This temporal coincidence between autophagy upregulation and starch degradation implies the involvement of autophagy in nocturnal starch metabolism. Furthermore, disruption of autophagy by 3-methyladenine treatment or silencing of ATG genes reduces leaf starch degradation and results in excessive starch accumulation at the end of the night. These data validate the contribution of autophagy in the leaf starch depletion process.

It was long thought that mobilization of leaf starch occurs inside the chloroplasts. Our findings raise an interesting question about how autophagy, a cytoplasmic process, contributes to chloroplast-localized leaf starch degradation. Observations of starch components outside the chloroplast make it easier to be understood. Ultrastructural analysis of mesophyll cells exposed to darkness show a type of small starch granule-like structure (SSGL) in the cytoplasm and vacuole. The SSGL has a diameter of less than 0.5 μm, which is much smaller than that of starch granules (1 to 2 μm) synthesized under light. Consistently, we observed SSGLs labeled by YFP-tagged granule-bound starch synthase I (GBSSI-YFP), a starch granule marker, ouside chloroplasts. Using the periodic acid-thiocarbohydrazide-silver proteinate (PA-TCH-SP) staining method, we confirmed the starchy nature of SSGLs in TEM sections. These data reveal the objective existence of starch outside the chloroplasts at night.

Next, we investigated whether autophagy contributes to the delivery of SSGLs to the vacuole. In the ultrastructural analysis of SSGLs, we observed SSGLs wrapped in a single- or double-membrane vesicle in the vacuole, which resembles an autophagic body. SSGLs labeled by GBSSI-YFP could also be sequestered by CFP-ATG8f-labeled autophagosomes in the cytoplasm and transported to the central vacuole. Furthermore, blocking autophagy by silencing ATG6 reduces the number of vacuole-localized SSGLs. These results indicate that the cytoplasm-to-vacuole traffic of SSGLs is dependent on autophagosome vehicles. Additionally, we observed SSGLs that had almost been degraded in the vacuole, suggesting that the fate of vacuole-localized SSGLs would be degradation by functional enzymes. Other researchers detected considerable β-amylase activities in vacuolar fractions of several plant species two decades ago. Here, we identified SSGLs in the vacuole, which suggest the possible substrates for the vacuolar amylase activities. However, it is still unknown whether these vacuolar amylase activities, or which specific enzyme, account for the degradation of SSGLs.

Another interesting question raised by the appearance of SSGLs outside the chloroplasts is how the chloroplastic starch components are exported. In our study, we observed increased numbers of stromules (stroma-filled tubules) at the surface of plastids after exposure to darkness. These stromules are labeled by the released GBSSI-YFP in stroma and appear usually together with SSGLs exhibiting concentrated fluorescence of GBSSI-YFP. They have diameters ranging from 0.4 μm to 1.2 μm, which are large enough to accommodate SSGLs. Furthermore, we identified SSGLs in stromules or their derivative structures, chloroplast protrusions. These data suggest that stromules may function as the exit channel of SSGLs.

In addition, we showed that the autophagic pathway and classic chloroplast pathway contribute to the breakdown of leaf starch independently and the chloroplast pathway makes a larger contribution.

Taken together, SSGLs exported from chloroplasts can be engulfed by autophagosomes and then transported to the vacuole for breakdown. This finding reveals a new, nonplastid pathway of leaf starch degradation and a new role of autophagy in plants. In the future, there are still many questions that remain to be answered. For example, is the autophagic starch degradation a selective or nonselective process? Assuming that it is selective, does a cargo receptor exist, and, if so, what is it? How is the nocturnal autophagic activity regulated? Is the activity light- or circadian-regulated in similar ways as leaf starch degradation per se? For plants, what is the biological significance of keeping two pathways of leaf starch degradation? Further efforts to answer these questions will greatly improve our understanding of the autophagic pathway in leaf starch degradation.

Wang Y, Yu B, Zhao J, Guo J, Li Y, Han S, et al. Autophagy contributes to leaf starch degradation. Plant Cell. 2013;25:1383–99. doi: 10.1105/tpc.112.108993.

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.