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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.


The occurrence of SEX chlorophagy Is independent of ATG6, ATG5 and ATG7. (A) Real-time RT-PCR suggests silencing of the corresponding target genes in either individually silenced or cosilenced plants. ACT7 was used as the internal control. Values are means ± SE of 2 replicate samples. (B) Quantification of the number of vacuole-localized SEX chloroplast per cell in TEM images of TUB8-silenced, as well as ATG and TUB8-cosilenced plants. Values are means ± SE from more than 30 cells. (C) Ultrastructural analysis shows occurrence of SEX chlorophagy in ATG and TUB8-cosilenced plants. Leaf samples used for TEM sectioning were taken from plants that had just finished nocturnal metabolism at 25 d post-agroinfiltration (3 or 4 wpi) for VIGS. Blue arrows refer to the vacuole-localized SEX chloroplasts. Scale bars: 5 μm.
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f0007: The occurrence of SEX chlorophagy Is independent of ATG6, ATG5 and ATG7. (A) Real-time RT-PCR suggests silencing of the corresponding target genes in either individually silenced or cosilenced plants. ACT7 was used as the internal control. Values are means ± SE of 2 replicate samples. (B) Quantification of the number of vacuole-localized SEX chloroplast per cell in TEM images of TUB8-silenced, as well as ATG and TUB8-cosilenced plants. Values are means ± SE from more than 30 cells. (C) Ultrastructural analysis shows occurrence of SEX chlorophagy in ATG and TUB8-cosilenced plants. Leaf samples used for TEM sectioning were taken from plants that had just finished nocturnal metabolism at 25 d post-agroinfiltration (3 or 4 wpi) for VIGS. Blue arrows refer to the vacuole-localized SEX chloroplasts. Scale bars: 5 μm.

Mentions: ATG-dependent autophagic pathway plays a role in the degradation of whole or partial chloroplast during senescence in individually darkened leaves.47 The partially suppressed autophagosome-mediated autophagic activities in TUB8-silenced plants indicate that the degradation of SEX chloroplast may be independent of macroautophagy. To further test this hypothesis, we cosilenced TUB8 together with several key ATG genes, including ATG6, ATG5 and ATG7 (Fig. 7A), and investigated the involvement of these ATG genes in SEX chlorophagy. TEM observations showed that the aberrant phenotypes of starch overaccumulation and chloroplast degradation in vacuole were still developed when TUB8 was cosilenced together with ATG6, ATG5 or ATG7 (Fig. 7C). Quantitative analysis of vacuole-localized SEX chloroplasts demonstrated that no obvious difference existed in the level of SEX chlorophagy between the TUB8 individually silenced and ATG/TUB8 cosilenced plants (Fig. 7B). These results suggest that SEX chlorophagy is ATG6-,ATG5- and ATG7-independent.Figure 7.


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)

The occurrence of SEX chlorophagy Is independent of ATG6, ATG5 and ATG7. (A) Real-time RT-PCR suggests silencing of the corresponding target genes in either individually silenced or cosilenced plants. ACT7 was used as the internal control. Values are means ± SE of 2 replicate samples. (B) Quantification of the number of vacuole-localized SEX chloroplast per cell in TEM images of TUB8-silenced, as well as ATG and TUB8-cosilenced plants. Values are means ± SE from more than 30 cells. (C) Ultrastructural analysis shows occurrence of SEX chlorophagy in ATG and TUB8-cosilenced plants. Leaf samples used for TEM sectioning were taken from plants that had just finished nocturnal metabolism at 25 d post-agroinfiltration (3 or 4 wpi) for VIGS. Blue arrows refer to the vacuole-localized SEX chloroplasts. Scale bars: 5 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4835195&req=5

f0007: The occurrence of SEX chlorophagy Is independent of ATG6, ATG5 and ATG7. (A) Real-time RT-PCR suggests silencing of the corresponding target genes in either individually silenced or cosilenced plants. ACT7 was used as the internal control. Values are means ± SE of 2 replicate samples. (B) Quantification of the number of vacuole-localized SEX chloroplast per cell in TEM images of TUB8-silenced, as well as ATG and TUB8-cosilenced plants. Values are means ± SE from more than 30 cells. (C) Ultrastructural analysis shows occurrence of SEX chlorophagy in ATG and TUB8-cosilenced plants. Leaf samples used for TEM sectioning were taken from plants that had just finished nocturnal metabolism at 25 d post-agroinfiltration (3 or 4 wpi) for VIGS. Blue arrows refer to the vacuole-localized SEX chloroplasts. Scale bars: 5 μm.
Mentions: ATG-dependent autophagic pathway plays a role in the degradation of whole or partial chloroplast during senescence in individually darkened leaves.47 The partially suppressed autophagosome-mediated autophagic activities in TUB8-silenced plants indicate that the degradation of SEX chloroplast may be independent of macroautophagy. To further test this hypothesis, we cosilenced TUB8 together with several key ATG genes, including ATG6, ATG5 and ATG7 (Fig. 7A), and investigated the involvement of these ATG genes in SEX chlorophagy. TEM observations showed that the aberrant phenotypes of starch overaccumulation and chloroplast degradation in vacuole were still developed when TUB8 was cosilenced together with ATG6, ATG5 or ATG7 (Fig. 7C). Quantitative analysis of vacuole-localized SEX chloroplasts demonstrated that no obvious difference existed in the level of SEX chlorophagy between the TUB8 individually silenced and ATG/TUB8 cosilenced plants (Fig. 7B). These results suggest that SEX chlorophagy is ATG6-,ATG5- and ATG7-independent.Figure 7.

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.