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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.


Silencing of TUB8 in N. benthamiana blocks leaf starch degradation. (A) Iodine staining of leaf discs taken from plants subjected to prolonged dark treatment. The plants were kept in darkness for up to 120 h of treatment until all the samples of each time point were collected. At each time point, 5 to 6 leaf discs were punched from the plants and incubated in ethanol to remove leaf pigments. Iodine staining was carried out when all the samples were harvested. Representative results are presented. (B to D) The SEX phenotype in TUB8-silenced leaves is caused by a blockage of starch degradation. (B) Iodine staining of leaves detached from silenced and nonsilenced plants at the indicated time. These results were reproduced in 2 experiments using more than 3 leaves in each experiment. Representative results are presented. (C) Real-time RT-PCR shows successful silencing of target genes in either individually silenced or cosilenced plants. ACT7 was used as the internal control. Values are means ± SE of 2 replicate samples. (D) Quantitative analysis of leaf starch content in silenced and nonsilenced plants at the indicated time. Values are means ± SE of 2 or 3 replicate samples.
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f0005: Silencing of TUB8 in N. benthamiana blocks leaf starch degradation. (A) Iodine staining of leaf discs taken from plants subjected to prolonged dark treatment. The plants were kept in darkness for up to 120 h of treatment until all the samples of each time point were collected. At each time point, 5 to 6 leaf discs were punched from the plants and incubated in ethanol to remove leaf pigments. Iodine staining was carried out when all the samples were harvested. Representative results are presented. (B to D) The SEX phenotype in TUB8-silenced leaves is caused by a blockage of starch degradation. (B) Iodine staining of leaves detached from silenced and nonsilenced plants at the indicated time. These results were reproduced in 2 experiments using more than 3 leaves in each experiment. Representative results are presented. (C) Real-time RT-PCR shows successful silencing of target genes in either individually silenced or cosilenced plants. ACT7 was used as the internal control. Values are means ± SE of 2 replicate samples. (D) Quantitative analysis of leaf starch content in silenced and nonsilenced plants at the indicated time. Values are means ± SE of 2 or 3 replicate samples.

Mentions: Since macroautophagy contributes to leaf starch degradation44 and silencing of TUB8 blocks the upregulation of nocturnal autophagy, we reasoned that TUB8 silencing could affect the breakdown of transitory starch in leaves. Indeed, we detected large amounts of starch reserves in TUB8-silenced leaves at the end of night using iodine staining (Fig. 5). Strikingly, these reserves were conspicuously higher than those found in ATG6-silenced plants (Fig. S10) and could not be consumed efficiently even when the time of dark treatment was prolonged to 120 h (Fig. 5A). This extremely strong starch-excess (SEX) phenotype in TUB8-silenced plants implies that leaf starch degradation is largely blocked when microtubule cytoskeletons are disorganized.Figure 5.


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)

Silencing of TUB8 in N. benthamiana blocks leaf starch degradation. (A) Iodine staining of leaf discs taken from plants subjected to prolonged dark treatment. The plants were kept in darkness for up to 120 h of treatment until all the samples of each time point were collected. At each time point, 5 to 6 leaf discs were punched from the plants and incubated in ethanol to remove leaf pigments. Iodine staining was carried out when all the samples were harvested. Representative results are presented. (B to D) The SEX phenotype in TUB8-silenced leaves is caused by a blockage of starch degradation. (B) Iodine staining of leaves detached from silenced and nonsilenced plants at the indicated time. These results were reproduced in 2 experiments using more than 3 leaves in each experiment. Representative results are presented. (C) Real-time RT-PCR shows successful silencing of target genes in either individually silenced or cosilenced plants. ACT7 was used as the internal control. Values are means ± SE of 2 replicate samples. (D) Quantitative analysis of leaf starch content in silenced and nonsilenced plants at the indicated time. Values are means ± SE of 2 or 3 replicate samples.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f0005: Silencing of TUB8 in N. benthamiana blocks leaf starch degradation. (A) Iodine staining of leaf discs taken from plants subjected to prolonged dark treatment. The plants were kept in darkness for up to 120 h of treatment until all the samples of each time point were collected. At each time point, 5 to 6 leaf discs were punched from the plants and incubated in ethanol to remove leaf pigments. Iodine staining was carried out when all the samples were harvested. Representative results are presented. (B to D) The SEX phenotype in TUB8-silenced leaves is caused by a blockage of starch degradation. (B) Iodine staining of leaves detached from silenced and nonsilenced plants at the indicated time. These results were reproduced in 2 experiments using more than 3 leaves in each experiment. Representative results are presented. (C) Real-time RT-PCR shows successful silencing of target genes in either individually silenced or cosilenced plants. ACT7 was used as the internal control. Values are means ± SE of 2 replicate samples. (D) Quantitative analysis of leaf starch content in silenced and nonsilenced plants at the indicated time. Values are means ± SE of 2 or 3 replicate samples.
Mentions: Since macroautophagy contributes to leaf starch degradation44 and silencing of TUB8 blocks the upregulation of nocturnal autophagy, we reasoned that TUB8 silencing could affect the breakdown of transitory starch in leaves. Indeed, we detected large amounts of starch reserves in TUB8-silenced leaves at the end of night using iodine staining (Fig. 5). Strikingly, these reserves were conspicuously higher than those found in ATG6-silenced plants (Fig. S10) and could not be consumed efficiently even when the time of dark treatment was prolonged to 120 h (Fig. 5A). This extremely strong starch-excess (SEX) phenotype in TUB8-silenced plants implies that leaf starch degradation is largely blocked when microtubule cytoskeletons are disorganized.Figure 5.

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.