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Disruption of microtubules in plants suppresses macroautophagy and triggers starch excess-associated chloroplast autophagy.

Wang Y, Zheng X, Yu B, Han S, Guo J, Tang H, Yu AY, Deng H, Hong Y, Liu Y - Autophagy (2015)

Bottom Line: Here, we found that ATG6 interacts with TUB8/β-tubulin 8 and colocalizes with microtubules in Nicotiana benthamiana.Disruption of microtubules by either silencing of tubulin genes or treatment with microtubule-depolymerizing agents in N. benthamiana reduces autophagosome formation during upregulation of nocturnal or oxidation-induced macroautophagy.Furthermore, a blockage of leaf starch degradation occurred in microtubule-disrupted cells and triggered a distinct ATG6-, ATG5- and ATG7-independent autophagic pathway termed starch excess-associated chloroplast autophagy (SEX chlorophagy) for clearance of dysfunctional chloroplasts.

View Article: PubMed Central - PubMed

Affiliation: a Center for Plant Biology ; Beijing , China.

ABSTRACT
Microtubules, the major components of cytoskeleton, are involved in various fundamental biological processes in plants. Recent studies in mammalian cells have revealed the importance of microtubule cytoskeleton in autophagy. However, little is known about the roles of microtubules in plant autophagy. Here, we found that ATG6 interacts with TUB8/β-tubulin 8 and colocalizes with microtubules in Nicotiana benthamiana. Disruption of microtubules by either silencing of tubulin genes or treatment with microtubule-depolymerizing agents in N. benthamiana reduces autophagosome formation during upregulation of nocturnal or oxidation-induced macroautophagy. Furthermore, a blockage of leaf starch degradation occurred in microtubule-disrupted cells and triggered a distinct ATG6-, ATG5- and ATG7-independent autophagic pathway termed starch excess-associated chloroplast autophagy (SEX chlorophagy) for clearance of dysfunctional chloroplasts. Our findings reveal that an intact microtubule network is important for efficient macroautophagy and leaf starch degradation.

No MeSH data available.


Disruption of microtubules suppresses upregulation of nocturnal autophagy. (A and B) Suppressed nocturnal autophagy in TUB8-silenced leaves at midnight. (A) Representative images of CFP-ATG8f-labeled autophagic structures in leaves. CFP-ATG8f is in cyan, and chloroplasts are in red. White arrows indicate CFP-ATG8f-labeled autophagic structures. Scale bars: 20 μm. (B) Relative autophagic activity in TUB8-silenced plants. The autophagic activity in nonsilenced plants was set to 1. This experiment was repeated 6 times. Values represent means ± SE. The Student t test was used to determine significant differences (**, P < 0.01). (C and D) Suppressed nocturnal autophagy in microtubule inhibitor-treated leaves at midnight. (C) Relative autophagic activity in microtubule inhibitor-treated leaves. The autophagic activity in leaves infiltrated with 0.1% dimethyl sulfoxide (Mock) was set to 1. This experiment was repeated 10 times. Values represent means ± SE. The Student t test was used to determine significant differences (**, P < 0.01). (D) Representative images of autophagic structures and cortical microtubule arrays in leaves after 4 h of treatment with microtubule inhibitors. Magenta arrowheads indicate short segments of depolymerized microtubules. Scale bars: 20 μm.
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f0004: Disruption of microtubules suppresses upregulation of nocturnal autophagy. (A and B) Suppressed nocturnal autophagy in TUB8-silenced leaves at midnight. (A) Representative images of CFP-ATG8f-labeled autophagic structures in leaves. CFP-ATG8f is in cyan, and chloroplasts are in red. White arrows indicate CFP-ATG8f-labeled autophagic structures. Scale bars: 20 μm. (B) Relative autophagic activity in TUB8-silenced plants. The autophagic activity in nonsilenced plants was set to 1. This experiment was repeated 6 times. Values represent means ± SE. The Student t test was used to determine significant differences (**, P < 0.01). (C and D) Suppressed nocturnal autophagy in microtubule inhibitor-treated leaves at midnight. (C) Relative autophagic activity in microtubule inhibitor-treated leaves. The autophagic activity in leaves infiltrated with 0.1% dimethyl sulfoxide (Mock) was set to 1. This experiment was repeated 10 times. Values represent means ± SE. The Student t test was used to determine significant differences (**, P < 0.01). (D) Representative images of autophagic structures and cortical microtubule arrays in leaves after 4 h of treatment with microtubule inhibitors. Magenta arrowheads indicate short segments of depolymerized microtubules. Scale bars: 20 μm.

Mentions: We have previously shown that nocturnal autophagy in N. benthamiana leaves undergoes an upregulation before midnight and a downregulation by dawn.21 Using this as a model system, we tested the role of microtubule in macroautophagy. CFP-tagged ATG8f (CFP-ATG8f) was used to label autophagic structures in mesophyll cells as described previously.21 As expected, a lot of punctate structures labeled by CFP-ATG8f appeared in mesophyll cells of nonsilenced plants after 4 h of exposure to darkness (Fig. 4A, upper panel). However, many fewer autophagic structures were detected in the TUB8-silenced leaves (Fig. 4A, lower panel). Quantitative analysis of autophagic structures showed that a 66% reduction of autophagic activities occurred in TUB8-silenced leaves at midnight (Fig. 4B). Similar reduction of autophagic activity was observed in TUA6-silenced leaves (Fig. S8C and D). We also tested the basal autophagic activity occurring in plants exposed to 0 h or 8 h of darkness. No obvious differences, however, were detected between TUB8-silenced and nonsilenced plants (Fig. S8A and B). These results indicate that microtubules may not be required but are still important for efficient autophagosome biogenesis during upregulation of nocturnal autophagy.Figure 4.


Disruption of microtubules in plants suppresses macroautophagy and triggers starch excess-associated chloroplast autophagy.

Wang Y, Zheng X, Yu B, Han S, Guo J, Tang H, Yu AY, Deng H, Hong Y, Liu Y - Autophagy (2015)

Disruption of microtubules suppresses upregulation of nocturnal autophagy. (A and B) Suppressed nocturnal autophagy in TUB8-silenced leaves at midnight. (A) Representative images of CFP-ATG8f-labeled autophagic structures in leaves. CFP-ATG8f is in cyan, and chloroplasts are in red. White arrows indicate CFP-ATG8f-labeled autophagic structures. Scale bars: 20 μm. (B) Relative autophagic activity in TUB8-silenced plants. The autophagic activity in nonsilenced plants was set to 1. This experiment was repeated 6 times. Values represent means ± SE. The Student t test was used to determine significant differences (**, P < 0.01). (C and D) Suppressed nocturnal autophagy in microtubule inhibitor-treated leaves at midnight. (C) Relative autophagic activity in microtubule inhibitor-treated leaves. The autophagic activity in leaves infiltrated with 0.1% dimethyl sulfoxide (Mock) was set to 1. This experiment was repeated 10 times. Values represent means ± SE. The Student t test was used to determine significant differences (**, P < 0.01). (D) Representative images of autophagic structures and cortical microtubule arrays in leaves after 4 h of treatment with microtubule inhibitors. Magenta arrowheads indicate short segments of depolymerized microtubules. Scale bars: 20 μm.
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Related In: Results  -  Collection

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f0004: Disruption of microtubules suppresses upregulation of nocturnal autophagy. (A and B) Suppressed nocturnal autophagy in TUB8-silenced leaves at midnight. (A) Representative images of CFP-ATG8f-labeled autophagic structures in leaves. CFP-ATG8f is in cyan, and chloroplasts are in red. White arrows indicate CFP-ATG8f-labeled autophagic structures. Scale bars: 20 μm. (B) Relative autophagic activity in TUB8-silenced plants. The autophagic activity in nonsilenced plants was set to 1. This experiment was repeated 6 times. Values represent means ± SE. The Student t test was used to determine significant differences (**, P < 0.01). (C and D) Suppressed nocturnal autophagy in microtubule inhibitor-treated leaves at midnight. (C) Relative autophagic activity in microtubule inhibitor-treated leaves. The autophagic activity in leaves infiltrated with 0.1% dimethyl sulfoxide (Mock) was set to 1. This experiment was repeated 10 times. Values represent means ± SE. The Student t test was used to determine significant differences (**, P < 0.01). (D) Representative images of autophagic structures and cortical microtubule arrays in leaves after 4 h of treatment with microtubule inhibitors. Magenta arrowheads indicate short segments of depolymerized microtubules. Scale bars: 20 μm.
Mentions: We have previously shown that nocturnal autophagy in N. benthamiana leaves undergoes an upregulation before midnight and a downregulation by dawn.21 Using this as a model system, we tested the role of microtubule in macroautophagy. CFP-tagged ATG8f (CFP-ATG8f) was used to label autophagic structures in mesophyll cells as described previously.21 As expected, a lot of punctate structures labeled by CFP-ATG8f appeared in mesophyll cells of nonsilenced plants after 4 h of exposure to darkness (Fig. 4A, upper panel). However, many fewer autophagic structures were detected in the TUB8-silenced leaves (Fig. 4A, lower panel). Quantitative analysis of autophagic structures showed that a 66% reduction of autophagic activities occurred in TUB8-silenced leaves at midnight (Fig. 4B). Similar reduction of autophagic activity was observed in TUA6-silenced leaves (Fig. S8C and D). We also tested the basal autophagic activity occurring in plants exposed to 0 h or 8 h of darkness. No obvious differences, however, were detected between TUB8-silenced and nonsilenced plants (Fig. S8A and B). These results indicate that microtubules may not be required but are still important for efficient autophagosome biogenesis during upregulation of nocturnal autophagy.Figure 4.

Bottom Line: Here, we found that ATG6 interacts with TUB8/β-tubulin 8 and colocalizes with microtubules in Nicotiana benthamiana.Disruption of microtubules by either silencing of tubulin genes or treatment with microtubule-depolymerizing agents in N. benthamiana reduces autophagosome formation during upregulation of nocturnal or oxidation-induced macroautophagy.Furthermore, a blockage of leaf starch degradation occurred in microtubule-disrupted cells and triggered a distinct ATG6-, ATG5- and ATG7-independent autophagic pathway termed starch excess-associated chloroplast autophagy (SEX chlorophagy) for clearance of dysfunctional chloroplasts.

View Article: PubMed Central - PubMed

Affiliation: a Center for Plant Biology ; Beijing , China.

ABSTRACT
Microtubules, the major components of cytoskeleton, are involved in various fundamental biological processes in plants. Recent studies in mammalian cells have revealed the importance of microtubule cytoskeleton in autophagy. However, little is known about the roles of microtubules in plant autophagy. Here, we found that ATG6 interacts with TUB8/β-tubulin 8 and colocalizes with microtubules in Nicotiana benthamiana. Disruption of microtubules by either silencing of tubulin genes or treatment with microtubule-depolymerizing agents in N. benthamiana reduces autophagosome formation during upregulation of nocturnal or oxidation-induced macroautophagy. Furthermore, a blockage of leaf starch degradation occurred in microtubule-disrupted cells and triggered a distinct ATG6-, ATG5- and ATG7-independent autophagic pathway termed starch excess-associated chloroplast autophagy (SEX chlorophagy) for clearance of dysfunctional chloroplasts. Our findings reveal that an intact microtubule network is important for efficient macroautophagy and leaf starch degradation.

No MeSH data available.