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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.


ATG6 colocalizes with microtubules in vivo. Images were taken when CFP-ATG6 and the microtubule reporter, GFP-MBD, were transiently coexpressed in N. benthamiana leaves for 48 h. GFP-MBD-labeled microtubules appear green and CFP-ATG6-labeled punctate structures are pseudocolored red. Scale bars: 20 μm.
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f0002: ATG6 colocalizes with microtubules in vivo. Images were taken when CFP-ATG6 and the microtubule reporter, GFP-MBD, were transiently coexpressed in N. benthamiana leaves for 48 h. GFP-MBD-labeled microtubules appear green and CFP-ATG6-labeled punctate structures are pseudocolored red. Scale bars: 20 μm.

Mentions: Since ATG6 interacts with tubulins, the major components of microtubules, we hypothesize that a putative interaction exists between ATG6 and microtubules. To test this, we performed a colocalization assay in plant cells. MAP4 is one of the well-studied microtubule-associated proteins (MAPs) in mammalian cells and GFP-tagged MAP4 or its microtubule binding domain (MBD) has been used as a microtubule reporter in mammals and plants.38,39 Here, we constructed a GFP-tagged MBD domain of HsMAP4, GFP-MBD, to label the cortical microtubule networks in leaf cells of N. benthamiana. Cyan fluorescent protein (CFP)-tagged ATG6 (CFP-ATG6) was transiently coexpressed with GFP-MBD in leaves for 48 h, followed by microscopic observation under confocal laser scanning microscopy (CLSM). Interestingly, CFP-ATG6 decorated a number of punctate structures and all of them displayed localizations on or adjacent to cortical microtubules (Fig. 2). Detailed observation of movement of CFP-ATG6 by time-lapse microscopy showed that some dots of CFP-ATG6 could move along microtubules for short distances (Fig. S5B and C; Movie S1). We also captured one CFP-ATG6-decorated structure showing synchronous movement with the shrinking microtubule (Fig. S5C; Movie S1). Similar colocalization of ATG6 with microtubules was observed when CFP-ATG6 was coexpressed with YFP-MBD (Fig. S5A). Taken together, these data suggest that ATG6 binds to microtubules in vivo.Figure 2.


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)

ATG6 colocalizes with microtubules in vivo. Images were taken when CFP-ATG6 and the microtubule reporter, GFP-MBD, were transiently coexpressed in N. benthamiana leaves for 48 h. GFP-MBD-labeled microtubules appear green and CFP-ATG6-labeled punctate structures are pseudocolored red. Scale bars: 20 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f0002: ATG6 colocalizes with microtubules in vivo. Images were taken when CFP-ATG6 and the microtubule reporter, GFP-MBD, were transiently coexpressed in N. benthamiana leaves for 48 h. GFP-MBD-labeled microtubules appear green and CFP-ATG6-labeled punctate structures are pseudocolored red. Scale bars: 20 μm.
Mentions: Since ATG6 interacts with tubulins, the major components of microtubules, we hypothesize that a putative interaction exists between ATG6 and microtubules. To test this, we performed a colocalization assay in plant cells. MAP4 is one of the well-studied microtubule-associated proteins (MAPs) in mammalian cells and GFP-tagged MAP4 or its microtubule binding domain (MBD) has been used as a microtubule reporter in mammals and plants.38,39 Here, we constructed a GFP-tagged MBD domain of HsMAP4, GFP-MBD, to label the cortical microtubule networks in leaf cells of N. benthamiana. Cyan fluorescent protein (CFP)-tagged ATG6 (CFP-ATG6) was transiently coexpressed with GFP-MBD in leaves for 48 h, followed by microscopic observation under confocal laser scanning microscopy (CLSM). Interestingly, CFP-ATG6 decorated a number of punctate structures and all of them displayed localizations on or adjacent to cortical microtubules (Fig. 2). Detailed observation of movement of CFP-ATG6 by time-lapse microscopy showed that some dots of CFP-ATG6 could move along microtubules for short distances (Fig. S5B and C; Movie S1). We also captured one CFP-ATG6-decorated structure showing synchronous movement with the shrinking microtubule (Fig. S5C; Movie S1). Similar colocalization of ATG6 with microtubules was observed when CFP-ATG6 was coexpressed with YFP-MBD (Fig. S5A). Taken together, these data suggest that ATG6 binds to microtubules in vivo.Figure 2.

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.