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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.


Related in: MedlinePlus

Silencing of TUB8 affects plant development and disorganizes microtubule arrays. (A) Leaf curling and crinkling phenotype in TUB8-silenced plants. Photos were taken at 2 wk post-agroinfiltration (wpi) for VIGS. (B) Realtime RT-PCR shows relative mRNA levels of TUB8 in silenced or nonsilenced plants. ACT7 was used as the internal control. Values are means ± SE of 5 replicate samples. The Student t test was applied to determine statistically significant differences (**P <0.01). (C and D) Developmental defects in TUB8-silenced plants at later stages of silencing, including defective flower development in (C) and leaf chlorosis in (D). Photos were taken at 7 wpi for VIGS. (E) Disordered cortical microtubule arrays in TUB8-silenced leaves. Various aberrations of GFP-MBD-labeled microtubules, including segmentation (magenta dashed oval), bending (white dashed square) and parallel distribution (yellow dashed rectangle) are shown in the pavement cells of TUB8-silenced plants. Scale bars: 10 μm.
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f0003: Silencing of TUB8 affects plant development and disorganizes microtubule arrays. (A) Leaf curling and crinkling phenotype in TUB8-silenced plants. Photos were taken at 2 wk post-agroinfiltration (wpi) for VIGS. (B) Realtime RT-PCR shows relative mRNA levels of TUB8 in silenced or nonsilenced plants. ACT7 was used as the internal control. Values are means ± SE of 5 replicate samples. The Student t test was applied to determine statistically significant differences (**P <0.01). (C and D) Developmental defects in TUB8-silenced plants at later stages of silencing, including defective flower development in (C) and leaf chlorosis in (D). Photos were taken at 7 wpi for VIGS. (E) Disordered cortical microtubule arrays in TUB8-silenced leaves. Various aberrations of GFP-MBD-labeled microtubules, including segmentation (magenta dashed oval), bending (white dashed square) and parallel distribution (yellow dashed rectangle) are shown in the pavement cells of TUB8-silenced plants. Scale bars: 10 μm.

Mentions: To investigate the function of tubulin genes, we used a Tobacco rattle virus (TRV)-based virus-induced gene silencing (VIGS) system40 to silence tubulin genes. TUB8-silenced plants developed a leaf curling and crinkling phenotype at 2 wk post-agroinfiltration (Fig. 3A) and showed more severe developmental aberrations including stunted top and root growth, leaf chlorosis and flower defects at later stages of silencing (Fig. 3C and D; Fig. S6A and B). Similar phenotypes were also observed in TUA6/α-tubulin 6 (TUBULIN ALPHA-6)-silenced plants (Fig. S6C). Real-time RT-PCR showed that silencing of TUB8 or TUA6 in N. benthamiana greatly reduces the mRNA level of its respective target gene but has no significant effect on mRNA abundance of Actin (ACT7) and other nontargeted tubulin genes such as TUB1 or TUA5 (Fig. 3Band Fig. S7).Figure 3.


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 affects plant development and disorganizes microtubule arrays. (A) Leaf curling and crinkling phenotype in TUB8-silenced plants. Photos were taken at 2 wk post-agroinfiltration (wpi) for VIGS. (B) Realtime RT-PCR shows relative mRNA levels of TUB8 in silenced or nonsilenced plants. ACT7 was used as the internal control. Values are means ± SE of 5 replicate samples. The Student t test was applied to determine statistically significant differences (**P <0.01). (C and D) Developmental defects in TUB8-silenced plants at later stages of silencing, including defective flower development in (C) and leaf chlorosis in (D). Photos were taken at 7 wpi for VIGS. (E) Disordered cortical microtubule arrays in TUB8-silenced leaves. Various aberrations of GFP-MBD-labeled microtubules, including segmentation (magenta dashed oval), bending (white dashed square) and parallel distribution (yellow dashed rectangle) are shown in the pavement cells of TUB8-silenced plants. Scale bars: 10 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f0003: Silencing of TUB8 affects plant development and disorganizes microtubule arrays. (A) Leaf curling and crinkling phenotype in TUB8-silenced plants. Photos were taken at 2 wk post-agroinfiltration (wpi) for VIGS. (B) Realtime RT-PCR shows relative mRNA levels of TUB8 in silenced or nonsilenced plants. ACT7 was used as the internal control. Values are means ± SE of 5 replicate samples. The Student t test was applied to determine statistically significant differences (**P <0.01). (C and D) Developmental defects in TUB8-silenced plants at later stages of silencing, including defective flower development in (C) and leaf chlorosis in (D). Photos were taken at 7 wpi for VIGS. (E) Disordered cortical microtubule arrays in TUB8-silenced leaves. Various aberrations of GFP-MBD-labeled microtubules, including segmentation (magenta dashed oval), bending (white dashed square) and parallel distribution (yellow dashed rectangle) are shown in the pavement cells of TUB8-silenced plants. Scale bars: 10 μm.
Mentions: To investigate the function of tubulin genes, we used a Tobacco rattle virus (TRV)-based virus-induced gene silencing (VIGS) system40 to silence tubulin genes. TUB8-silenced plants developed a leaf curling and crinkling phenotype at 2 wk post-agroinfiltration (Fig. 3A) and showed more severe developmental aberrations including stunted top and root growth, leaf chlorosis and flower defects at later stages of silencing (Fig. 3C and D; Fig. S6A and B). Similar phenotypes were also observed in TUA6/α-tubulin 6 (TUBULIN ALPHA-6)-silenced plants (Fig. S6C). Real-time RT-PCR showed that silencing of TUB8 or TUA6 in N. benthamiana greatly reduces the mRNA level of its respective target gene but has no significant effect on mRNA abundance of Actin (ACT7) and other nontargeted tubulin genes such as TUB1 or TUA5 (Fig. 3Band Fig. S7).Figure 3.

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.


Related in: MedlinePlus