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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.


Starch accumulation and SEX chlorophagy in microtubule depolymerizing agent-treated plants. (A) Iodine staining of leaves indicates massive starch reserves in the microtubule depolymerizing agent-treated plants from transplanting assay. Leaves used for determination of starch content were harvested from the plants treated with 10 μM APM or oryzalin at the end of night. These results were reproduced in 2 experiments using 6 to 8 leaves in each experiment. Representative results are presented. Scale bars: 0.5 cm. (B) Quantitative analysis of leaf starch content. Values are means ± SE of 3 replicate samples. (C) Ultrastructural analysis shows occurrence of starchy chloroplast autophagy in microtubule depolymerizing agent-treated plants from the transplanting assay. Blue arrows refer to the vacuole-localized starchy chloroplasts. Scale bars: 5 μm. Leaf samples used here for starch assay and TEM sectioning were all collected at the end of night.
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f0008: Starch accumulation and SEX chlorophagy in microtubule depolymerizing agent-treated plants. (A) Iodine staining of leaves indicates massive starch reserves in the microtubule depolymerizing agent-treated plants from transplanting assay. Leaves used for determination of starch content were harvested from the plants treated with 10 μM APM or oryzalin at the end of night. These results were reproduced in 2 experiments using 6 to 8 leaves in each experiment. Representative results are presented. Scale bars: 0.5 cm. (B) Quantitative analysis of leaf starch content. Values are means ± SE of 3 replicate samples. (C) Ultrastructural analysis shows occurrence of starchy chloroplast autophagy in microtubule depolymerizing agent-treated plants from the transplanting assay. Blue arrows refer to the vacuole-localized starchy chloroplasts. Scale bars: 5 μm. Leaf samples used here for starch assay and TEM sectioning were all collected at the end of night.

Mentions: To further confirm whether the aberrant phenotypes of TUB8-silenced plants, including developmental defects, blocked starch depletion and concomitant chloroplast degradation, were caused by a dysfunctional microtubule cytoskeleton, we tested effects of microtubule-depolymerizing drugs, APM and oryzalin, on these biological processes. Two approaches were adopted for the application of microtubule inhibitors: one was germination of N. benthamiana seeds directly on drug-containing MS plates for 2 to 3-weeks growth (hereafter referred to as germination assay); the other one was transplanting seedlings to the drug-containing MS plates for a 4-wk treatment (hereafter referred to as transplanting assay). Normally, N. benthamiana seedlings grown on MS media for 3 wk were 15 to 20 mm in height and had 4 to 7 leaves and elongated hypocotyls and roots. However, seedlings germinated on MS plates containing either 10 μM APM or 10 μM oryzalin stopped growing after cotyledon opening and displayed defects in hypocotyl elongation and root development (Fig. S13A). Similar growth retardation was observed in N. benthamiana plants from the transplanting assay (Fig. S13D). Additionally, chlorosis phenotype was evident in the transplanted plants treated with either 10 μM APM, 50 μM APM or 50 μM oryzalin (Fig. S13D). Iodine staining and quantitative measurement of starch contents revealed that large amounts of starch remained in drug treated-seedlings or leaves that had finished nocturnal metabolism (Fig. 8Aand B; Fig. S13B and C). Furthermore, TEM observations shows that SEX chlorophagy occurred in mesophyll cells of transplanted plants treated with APM or oryzalin (Fig. 8C). Taken together, these results suggest that functional microtubules are required for leaf starch degradation and normal plant development.Figure 8.


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)

Starch accumulation and SEX chlorophagy in microtubule depolymerizing agent-treated plants. (A) Iodine staining of leaves indicates massive starch reserves in the microtubule depolymerizing agent-treated plants from transplanting assay. Leaves used for determination of starch content were harvested from the plants treated with 10 μM APM or oryzalin at the end of night. These results were reproduced in 2 experiments using 6 to 8 leaves in each experiment. Representative results are presented. Scale bars: 0.5 cm. (B) Quantitative analysis of leaf starch content. Values are means ± SE of 3 replicate samples. (C) Ultrastructural analysis shows occurrence of starchy chloroplast autophagy in microtubule depolymerizing agent-treated plants from the transplanting assay. Blue arrows refer to the vacuole-localized starchy chloroplasts. Scale bars: 5 μm. Leaf samples used here for starch assay and TEM sectioning were all collected at the end of night.
© Copyright Policy - open-access
Related In: Results  -  Collection

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f0008: Starch accumulation and SEX chlorophagy in microtubule depolymerizing agent-treated plants. (A) Iodine staining of leaves indicates massive starch reserves in the microtubule depolymerizing agent-treated plants from transplanting assay. Leaves used for determination of starch content were harvested from the plants treated with 10 μM APM or oryzalin at the end of night. These results were reproduced in 2 experiments using 6 to 8 leaves in each experiment. Representative results are presented. Scale bars: 0.5 cm. (B) Quantitative analysis of leaf starch content. Values are means ± SE of 3 replicate samples. (C) Ultrastructural analysis shows occurrence of starchy chloroplast autophagy in microtubule depolymerizing agent-treated plants from the transplanting assay. Blue arrows refer to the vacuole-localized starchy chloroplasts. Scale bars: 5 μm. Leaf samples used here for starch assay and TEM sectioning were all collected at the end of night.
Mentions: To further confirm whether the aberrant phenotypes of TUB8-silenced plants, including developmental defects, blocked starch depletion and concomitant chloroplast degradation, were caused by a dysfunctional microtubule cytoskeleton, we tested effects of microtubule-depolymerizing drugs, APM and oryzalin, on these biological processes. Two approaches were adopted for the application of microtubule inhibitors: one was germination of N. benthamiana seeds directly on drug-containing MS plates for 2 to 3-weeks growth (hereafter referred to as germination assay); the other one was transplanting seedlings to the drug-containing MS plates for a 4-wk treatment (hereafter referred to as transplanting assay). Normally, N. benthamiana seedlings grown on MS media for 3 wk were 15 to 20 mm in height and had 4 to 7 leaves and elongated hypocotyls and roots. However, seedlings germinated on MS plates containing either 10 μM APM or 10 μM oryzalin stopped growing after cotyledon opening and displayed defects in hypocotyl elongation and root development (Fig. S13A). Similar growth retardation was observed in N. benthamiana plants from the transplanting assay (Fig. S13D). Additionally, chlorosis phenotype was evident in the transplanted plants treated with either 10 μM APM, 50 μM APM or 50 μM oryzalin (Fig. S13D). Iodine staining and quantitative measurement of starch contents revealed that large amounts of starch remained in drug treated-seedlings or leaves that had finished nocturnal metabolism (Fig. 8Aand B; Fig. S13B and C). Furthermore, TEM observations shows that SEX chlorophagy occurred in mesophyll cells of transplanted plants treated with APM or oryzalin (Fig. 8C). Taken together, these results suggest that functional microtubules are required for leaf starch degradation and normal plant development.Figure 8.

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.