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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

Starch overaccumulation in TUB8-silenced leaves triggers chloroplast malformation and SEX chlorophagy. (A) Ultrastructural analysis shows varying degrees of starch accumulation in mesophyll cells of TUB8-silenced plants at different stages of silencing. Blue arrows refer to the vacuole-localized SEX chloroplast. (B) Ultrastructure of misshapen chloroplasts in chlorotic leaves of TUB8-silenced plants at 5 wk post-agroinfiltration (wpi). Red arrowheads indicate vesicles adjacent to starchy chloroplast or chloroplastic structures. Yellow and cyan arrows indicate smaller chloroplastic structures with or without starch granules, which were probably generated by a budding-like process (yellow arrowhead) from the whole, large chloroplasts. Occasionally, remnants of disrupted chloroplast, like starch granule (red asterisk) and segments of thylakoids (black arrows), were observed in the vacuole. (C) Ultrastructural analysis of starch accumulation and SEX chlorophagy in mesophyll cells of TUB8-silenced and APS1- and TUB8-cosilenced plants at 3 wk post-agroinfiltration (wpi). (D) Quantification of vacuole-localized SEX chloroplast count per cell in TEM images of TUB8-silenced, as well as APS1- and TUB8-cosilenced leaves. Values are means ± SE from more than 50 cells. Two asterisks indicate a highly significant difference (P < 0.01; the Student t test). All the leaf samples used for TEM sectioning were taken from plants that had just finished nocturnal metabolism at the indicated weeks post-agroinfiltration (wpi) for VIGS. S, starch; V, vacuole; Cp, chloroplast; CW, cell wall. Black scale bars: 5 μm; white scale bars: 2 μm.
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f0006: Starch overaccumulation in TUB8-silenced leaves triggers chloroplast malformation and SEX chlorophagy. (A) Ultrastructural analysis shows varying degrees of starch accumulation in mesophyll cells of TUB8-silenced plants at different stages of silencing. Blue arrows refer to the vacuole-localized SEX chloroplast. (B) Ultrastructure of misshapen chloroplasts in chlorotic leaves of TUB8-silenced plants at 5 wk post-agroinfiltration (wpi). Red arrowheads indicate vesicles adjacent to starchy chloroplast or chloroplastic structures. Yellow and cyan arrows indicate smaller chloroplastic structures with or without starch granules, which were probably generated by a budding-like process (yellow arrowhead) from the whole, large chloroplasts. Occasionally, remnants of disrupted chloroplast, like starch granule (red asterisk) and segments of thylakoids (black arrows), were observed in the vacuole. (C) Ultrastructural analysis of starch accumulation and SEX chlorophagy in mesophyll cells of TUB8-silenced and APS1- and TUB8-cosilenced plants at 3 wk post-agroinfiltration (wpi). (D) Quantification of vacuole-localized SEX chloroplast count per cell in TEM images of TUB8-silenced, as well as APS1- and TUB8-cosilenced leaves. Values are means ± SE from more than 50 cells. Two asterisks indicate a highly significant difference (P < 0.01; the Student t test). All the leaf samples used for TEM sectioning were taken from plants that had just finished nocturnal metabolism at the indicated weeks post-agroinfiltration (wpi) for VIGS. S, starch; V, vacuole; Cp, chloroplast; CW, cell wall. Black scale bars: 5 μm; white scale bars: 2 μm.

Mentions: To better understand the extreme SEX phenotype of TUB8-silenced plants, transmission electron microscopy (TEM) was used to analyze the cellular ultrastructure of TUB8-silenced mesophyll cells when the nocturnal metabolism was finished. Large oval-shaped granules accumulated in the chloroplasts of TUB8-silenced plants, whereas no visible starch granules remained in the nonsilenced plants at the end of night (Fig. 6A), consistent with the results of starch assays (Fig. 5; Fig. S10). Additionally, we visualized cells with variant degrees of starch accumulation in TUB8-silenced leaves at different stages of silencing (Fig. 6A). At 3 wk post-agroinfiltration, some chloroplasts of TUB8-silenced leaves accumulated starch at levels of illuminated wild-type cells (Fig. 6A, panel 2), whereas more chloroplasts accumulated large granules, which severely exceeded the capacity of stroma (Fig. 6A, panel 3). The former contained well-organized thylakoid membranes and grana stacks, had normal shapes and were located in an orderly fashion in the thin layer of cytoplasm (Fig. 6A, panel 2), while the latter contained fewer visible thylakoid membrane systems, became swollen, enlarged and globular in shape and sometimes were separated from the cytosol and taken into the vacuole (Fig. 6A, panel 3). Measurements of ultrastructural characteristics of chloroplast (the length/width ratio and cross-sectional area) further supported the changes in chloroplast morphology of TUB8-silenced leaves (Fig. S11). The misshapen phenotypes of chloroplasts were more evident in chlorotic leaves of TUB8-silenced plants at about 5 wk post-agroinfiltration (Fig. 6A panels 4 and 5, and Fig. 6B). Nearly all the chloroplasts were fully filled with massive starch granules, appearing as if specific storage organelles of starch, and an increasing number of chloroplasts swarmed into vacuoles (Fig. 6A panels 4 and 5). In addition to whole chloroplasts with several granules, we sometimes observed vacuolar localization of smaller chloroplastic structures with one starch granule bounded by extremely thin thylakoid membrane (Fig. 6B, yellow arrows) or small spherical structures with only aberrant thylakoids but no granules (Fig. 6B, cyan arrows). These smaller chloroplastic structures are likely products of a budding process from whole, large chloroplasts (Fig. 6B, yellow arrowhead). Vesicles were occasionally found to be close to, but never engulf, starchy chloroplast or chloroplastic structures (Fig. 6B, red arrowhead). In a few cases, starch granules were released from the extremely disrupted chloroplasts into vacuoles (Fig. 6B, red asterisk). Other remnants of chloroplasts, like fragmented thylakoids (Fig. 6B, black arrows), were observed in vacuoles. In chlorotic leaves, collapsed cells were occasionally observed, in which massive starchy chloroplasts and other organelles were taken into the central vacuole (Fig. 6B panel 5). We called the above self-eating process for clearing disorganized starchy chloroplast ‘starch excess-associated chloroplast autophagy’ (hereafter short for SEX chlorophagy). Remarkably, the ultrastructural characteristics of aberrant chloroplasts were well matched with some special findings in chloroplast morphologies of TUB8-silenced leaves under CLSM, including lower or hollow chlorophyll fluorescence (Fig. S12) and vacuolar localization of chloroplast or its derivative structures (Movies S2 to S4).Figure 6.


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 overaccumulation in TUB8-silenced leaves triggers chloroplast malformation and SEX chlorophagy. (A) Ultrastructural analysis shows varying degrees of starch accumulation in mesophyll cells of TUB8-silenced plants at different stages of silencing. Blue arrows refer to the vacuole-localized SEX chloroplast. (B) Ultrastructure of misshapen chloroplasts in chlorotic leaves of TUB8-silenced plants at 5 wk post-agroinfiltration (wpi). Red arrowheads indicate vesicles adjacent to starchy chloroplast or chloroplastic structures. Yellow and cyan arrows indicate smaller chloroplastic structures with or without starch granules, which were probably generated by a budding-like process (yellow arrowhead) from the whole, large chloroplasts. Occasionally, remnants of disrupted chloroplast, like starch granule (red asterisk) and segments of thylakoids (black arrows), were observed in the vacuole. (C) Ultrastructural analysis of starch accumulation and SEX chlorophagy in mesophyll cells of TUB8-silenced and APS1- and TUB8-cosilenced plants at 3 wk post-agroinfiltration (wpi). (D) Quantification of vacuole-localized SEX chloroplast count per cell in TEM images of TUB8-silenced, as well as APS1- and TUB8-cosilenced leaves. Values are means ± SE from more than 50 cells. Two asterisks indicate a highly significant difference (P < 0.01; the Student t test). All the leaf samples used for TEM sectioning were taken from plants that had just finished nocturnal metabolism at the indicated weeks post-agroinfiltration (wpi) for VIGS. S, starch; V, vacuole; Cp, chloroplast; CW, cell wall. Black scale bars: 5 μm; white scale bars: 2 μm.
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f0006: Starch overaccumulation in TUB8-silenced leaves triggers chloroplast malformation and SEX chlorophagy. (A) Ultrastructural analysis shows varying degrees of starch accumulation in mesophyll cells of TUB8-silenced plants at different stages of silencing. Blue arrows refer to the vacuole-localized SEX chloroplast. (B) Ultrastructure of misshapen chloroplasts in chlorotic leaves of TUB8-silenced plants at 5 wk post-agroinfiltration (wpi). Red arrowheads indicate vesicles adjacent to starchy chloroplast or chloroplastic structures. Yellow and cyan arrows indicate smaller chloroplastic structures with or without starch granules, which were probably generated by a budding-like process (yellow arrowhead) from the whole, large chloroplasts. Occasionally, remnants of disrupted chloroplast, like starch granule (red asterisk) and segments of thylakoids (black arrows), were observed in the vacuole. (C) Ultrastructural analysis of starch accumulation and SEX chlorophagy in mesophyll cells of TUB8-silenced and APS1- and TUB8-cosilenced plants at 3 wk post-agroinfiltration (wpi). (D) Quantification of vacuole-localized SEX chloroplast count per cell in TEM images of TUB8-silenced, as well as APS1- and TUB8-cosilenced leaves. Values are means ± SE from more than 50 cells. Two asterisks indicate a highly significant difference (P < 0.01; the Student t test). All the leaf samples used for TEM sectioning were taken from plants that had just finished nocturnal metabolism at the indicated weeks post-agroinfiltration (wpi) for VIGS. S, starch; V, vacuole; Cp, chloroplast; CW, cell wall. Black scale bars: 5 μm; white scale bars: 2 μm.
Mentions: To better understand the extreme SEX phenotype of TUB8-silenced plants, transmission electron microscopy (TEM) was used to analyze the cellular ultrastructure of TUB8-silenced mesophyll cells when the nocturnal metabolism was finished. Large oval-shaped granules accumulated in the chloroplasts of TUB8-silenced plants, whereas no visible starch granules remained in the nonsilenced plants at the end of night (Fig. 6A), consistent with the results of starch assays (Fig. 5; Fig. S10). Additionally, we visualized cells with variant degrees of starch accumulation in TUB8-silenced leaves at different stages of silencing (Fig. 6A). At 3 wk post-agroinfiltration, some chloroplasts of TUB8-silenced leaves accumulated starch at levels of illuminated wild-type cells (Fig. 6A, panel 2), whereas more chloroplasts accumulated large granules, which severely exceeded the capacity of stroma (Fig. 6A, panel 3). The former contained well-organized thylakoid membranes and grana stacks, had normal shapes and were located in an orderly fashion in the thin layer of cytoplasm (Fig. 6A, panel 2), while the latter contained fewer visible thylakoid membrane systems, became swollen, enlarged and globular in shape and sometimes were separated from the cytosol and taken into the vacuole (Fig. 6A, panel 3). Measurements of ultrastructural characteristics of chloroplast (the length/width ratio and cross-sectional area) further supported the changes in chloroplast morphology of TUB8-silenced leaves (Fig. S11). The misshapen phenotypes of chloroplasts were more evident in chlorotic leaves of TUB8-silenced plants at about 5 wk post-agroinfiltration (Fig. 6A panels 4 and 5, and Fig. 6B). Nearly all the chloroplasts were fully filled with massive starch granules, appearing as if specific storage organelles of starch, and an increasing number of chloroplasts swarmed into vacuoles (Fig. 6A panels 4 and 5). In addition to whole chloroplasts with several granules, we sometimes observed vacuolar localization of smaller chloroplastic structures with one starch granule bounded by extremely thin thylakoid membrane (Fig. 6B, yellow arrows) or small spherical structures with only aberrant thylakoids but no granules (Fig. 6B, cyan arrows). These smaller chloroplastic structures are likely products of a budding process from whole, large chloroplasts (Fig. 6B, yellow arrowhead). Vesicles were occasionally found to be close to, but never engulf, starchy chloroplast or chloroplastic structures (Fig. 6B, red arrowhead). In a few cases, starch granules were released from the extremely disrupted chloroplasts into vacuoles (Fig. 6B, red asterisk). Other remnants of chloroplasts, like fragmented thylakoids (Fig. 6B, black arrows), were observed in vacuoles. In chlorotic leaves, collapsed cells were occasionally observed, in which massive starchy chloroplasts and other organelles were taken into the central vacuole (Fig. 6B panel 5). We called the above self-eating process for clearing disorganized starchy chloroplast ‘starch excess-associated chloroplast autophagy’ (hereafter short for SEX chlorophagy). Remarkably, the ultrastructural characteristics of aberrant chloroplasts were well matched with some special findings in chloroplast morphologies of TUB8-silenced leaves under CLSM, including lower or hollow chlorophyll fluorescence (Fig. S12) and vacuolar localization of chloroplast or its derivative structures (Movies S2 to S4).Figure 6.

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