Disruption of microtubules in plants suppresses macroautophagy and triggers starch excess-associated chloroplast autophagy

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.

Keywords: ATG6; chloroplast autophagy; leaf starch degradation; macroautophagy; microtubules; plants.

Figure 1.

ATG6 interacts with TUB8 in yeast and N. benthamiana. (A) Interaction of NtATG6 with NtTUB8 in yeast. Yeast cells harboring both BD-ATG6 and AD-TUB8 were able to turn blue on X-Gal-containing plate and grow on Leu-deficient medium in the presence of galactose and raffinose. Yeast harboring other control constructs showed no growth or color-changes, on corresponding induction plates. (B) NtTUB8 coimmunoprecipitates with NtATG6. Immunoprecipitation (IP) by anti-HA antibody was performed on total protein extracts from N. benthamiana leaves transiently expressing HA-TUB8 or other control groups. Precipitates were then analyzed by immunoblotting (IB) using anti-MYC (top panel) or anti-HA (middle panel) antibodies. Expression of MYC-tagged proteins was checked with anti-MYC (bottom panel). (C) Firefly luciferase complementation imaging (LCI) assay shows the interaction of ATG6 with TUB8. Coexpression of cLUC-TUB8 with ATG6-nLUC, but not other negative controls, reconstituted LUC activities and generated luminescence in the presence of luciferin. Infiltration areas of various combinations of constructs are indicated by the dashed circle.

Figure 2.

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.

Figure 3.

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.

Figure 4.

Disruption of microtubules suppresses upregulation of nocturnal autophagy. (A and B) Suppressed nocturnal autophagy in TUB8-silenced leaves at midnight. (A) Representative images of CFP-ATG8f-labeled autophagic structures in leaves. CFP-ATG8f is in cyan, and chloroplasts are in red. White arrows indicate CFP-ATG8f-labeled autophagic structures. Scale bars: 20 μm. (B) Relative autophagic activity in TUB8-silenced plants. The autophagic activity in nonsilenced plants was set to 1. This experiment was repeated 6 times. Values represent means ± SE. The Student t test was used to determine significant differences (**, P < 0.01). (C and D) Suppressed nocturnal autophagy in microtubule inhibitor-treated leaves at midnight. (C) Relative autophagic activity in microtubule inhibitor-treated leaves. The autophagic activity in leaves infiltrated with 0.1% dimethyl sulfoxide (Mock) was set to 1. This experiment was repeated 10 times. Values represent means ± SE. The Student t test was used to determine significant differences (**, P < 0.01). (D) Representative images of autophagic structures and cortical microtubule arrays in leaves after 4 h of treatment with microtubule inhibitors. Magenta arrowheads indicate short segments of depolymerized microtubules. Scale bars: 20 μm.

Figure 5.

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.

Figure 6.

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.

Figure 7.

The occurrence of SEX chlorophagy Is independent of ATG6, ATG5 and ATG7. (A) Real-time RT-PCR suggests silencing of the corresponding target genes in either individually silenced or cosilenced plants. ACT7 was used as the internal control. Values are means ± SE of 2 replicate samples. (B) Quantification of the number of vacuole-localized SEX chloroplast per cell in TEM images of TUB8-silenced, as well as ATG and TUB8-cosilenced plants. Values are means ± SE from more than 30 cells. (C) Ultrastructural analysis shows occurrence of SEX chlorophagy in ATG and TUB8-cosilenced plants. Leaf samples used for TEM sectioning were taken from plants that had just finished nocturnal metabolism at 25 d post-agroinfiltration (3 or 4 wpi) for VIGS. Blue arrows refer to the vacuole-localized SEX chloroplasts. Scale bars: 5 μm.

Figure 8.

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.