Autophagy contributes to leaf starch degradation

Abstract

Transitory starch, a major photosynthetic product in the leaves of land plants, accumulates in chloroplasts during the day and is hydrolyzed to maltose and Glc at night to support respiration and metabolism. Previous studies in Arabidopsis thaliana indicated that the degradation of transitory starch only occurs in the chloroplasts. Here, we report that autophagy, a nonplastidial process, participates in leaf starch degradation. Excessive starch accumulation was observed in Nicotiana benthamiana seedlings treated with an autophagy inhibitor and in autophagy-related (ATG) gene-silenced N. benthamiana and in Arabidopsis atg mutants. Autophagic activity in the leaves responded to the dynamic starch contents during the night. Microscopy showed that a type of small starch granule-like structure (SSGL) was localized outside the chloroplast and was sequestered by autophagic bodies. Moreover, an increased number of SSGLs was observed during starch depletion, and disruption of autophagy reduced the number of vacuole-localized SSGLs. These data suggest that autophagy contributes to transitory starch degradation by sequestering SSGLs to the vacuole for their subsequent breakdown.

Figure 1.

3-MA Treatment Results in a Starch-Excess Phenotype. (A) Iodine staining of 3-MA–treated seedlings. Two-week-old N. benthamiana seedlings germinated on MS plates containing or not containing 5 mM 3-MA were harvested at the end of the day and night for determination of starch content. (B) Quantification of starch in 3-MA–treated seedlings. Approximately 30 seedlings were harvested and regarded as one sample for the starch quantification test. Values are means ± se of four replicate samples. Student’s t test was applied to determine statistically significant differences (*P <0.05; **P <0.01).

Figure 2.

Transcript Pattern of Autophagy-Related Genes during the Night. Real-time RT-PCR analysis was performed using total RNA isolated from leaves at the indicated time points. Expression data relative to 0 h were normalized to that of actin. Values are means ± se from two independent experiments.

Figure 3.

Visualization of Autophagy in Leaves in Night Conditions by Confocal Microscopy. (A) to (C) Dynamic autophagic activity as revealed by MDC staining. (A) MDC-stained autophagosomes in the leaves after various periods in darkness. MDC-labeled structures are in green and the chloroplasts are in red. Bars = 10 μm. (B) Magnification of the mesophyll cells surrounded by a dashed line in (A). MDC-stained autophagosomes are indicated by arrowheads. The white and magenta arrowheads indicate the individual and aggregated autophagic structures in the vacuole, respectively. The blue arrowheads refer to the autophagosomes in the cytoplasm. Bars = 10 μm. (C) Relative autophagic activity normalized to the activity at the beginning of night. Quantification of the MDC-stained autophagosomes in leaves at each time point was performed to calculate the autophagic activity relative to that at time 0 h, which was set to 1. Over 300 mesophyll cells for each time point were used for the quantification. Values represent means ± se from two independent experiments. Student’s t test was applied to determine significant differences (*P < 0.05; **P < 0.01). (D) to (F) Dynamic autophagic activity showed by autophagy marker CFP-ATG8f. (D) Autophagosomes labeled by CFP-ATG8f in leaves at different time points after dark treatment. CFP-ATG8f fusion proteins are in cyan, and chloroplasts are in red. Bars = 10 μm. (E) Magnification of the mesophyll cells surrounded by a dashed line in (D). Autophagosomes labeled by CFP-ATG8f are indicated by arrows. Yellow arrows indicate autophagosomes in the cytoplasm. White arrows indicate autophagosomes in the vacuole. Bars = 10 μm. (F) Relative autophagic activity normalized to the activity at the beginning of night. Quantification of the CFP-ATG8–labeled autophagosomes in leaves at each time point was performed to calculate the autophagic activity relative to that at time 0 h, which was set to 1. More than 100 mesophyll cells for each time point were used for the quantification. Values represent means ± se from two independent experiments. Student’s t test was used to determine significant differences (**P < 0.01).

Figure 4.

TEM of Dynamic Autophagic Activity in Leaves during the Night. (A) Representative TEM images of autophagic structures at different time points after dark treatment. Lots of autophagic bodies (blue arrows) appeared in the central vacuole of mesophyll cells after 4 h of exposure to darkness, but fewer were visible at other time points. The red asterisk indicates a mitochondrion that has entered the vacuole. Cp, chloroplast; M, mitochondrion; S, starch; V, vacuole. Bars = 2.5 μm. (B) Representative ultrastructure of autophagosomes observed in the cytoplasm of mesophyll cells. In addition to the classic double-membrane autophagosomes (black arrows), an isolation membrane (autophagosome precursor, indicated by black arrowhead) was observed. CW, cell wall. Bars = 500 nm. (C) Relative autophagic activity normalized to the activity at the beginning of the night. Approximately 20 cells were used to quantify autophagic structures. Values represent means ± se from two independent experiments. Student’s t test was applied to indicate significant differences (***P < 0.001).

Figure 5.

Starch Content Declined during the Night. (A) Leaf discs stained with iodine solution reveal the decreased starch content during the night. Four time points (0, 2, 4, and 8 h after dark treatment) were selected for monitoring the starch content. Bars = 1 mm. (B) Ultrastructural analysis of mesophyll cells indicates changes of starch granules during the night. The percentage of chloroplasts with visible starch granules (black arrowheads) in a mesophyll cell declined (top panel). The size and number of visible starch granules per chloroplast also diminished (bottom panel). Cp, chloroplast; S, starch; V, vacuole. Bars = 5 μm. (C) Quantification of leaf starch contents during the night (left panel) and determination of the percentage of starch degraded in different time periods (right panel). Three replicate leaf samples for each time point were used in the starch quantification. Values are means ± se. The percentage of starch degraded in the initial and last 4 h was determined according to the starch content measured at each time point (left panel). [See online article for color version of this figure.]

Figure 6.

Starch-Excess Phenotype in ATG6-Silenced Nicotiana benthamiana. (A) Iodine staining of leaves from ATG6-silenced and nonsilenced plants. Leaves were harvested at the end of the night for detection of starch content. Starch was almost exhausted in control leaves but was readily detected in the ATG6-silenced leaves. These results were reproduced in 10 independent experiments using two to three leaves in each experiment. Representative results are presented. (B) Quantification of leaf starch content in ATG6-silenced and nonsilenced plants. Values are means ± se from three replicate leaf samples. Two asterisks indicate a significant difference (P < 0.01; Student’s t test). (C) Ultrastructural analysis of starch accumulation in mesophyll cells. At the end of the night, almost no starch granules were visible in the chloroplasts of control leaves, whereas starch granules were still present in the chloroplasts (arrowheads) of ATG6-silenced leaves. Cp, chloroplast; CW, cell wall; M, mitochondrion; S, starch; V, vacuole. Bars = 5 μm. [See online article for color version of this figure.]

Figure 7.

Silencing of Other ATG Genes Leads to Starch Accumulation at the End of the Night. (A) Iodine staining of leaves from other ATG gene-silenced and nonsilenced plants. Results were reproduced in more than three independent experiments using two to three leaves per experiment. Representative results are presented. (B) Quantification of leaf starch in other ATG gene-silenced and nonsilenced plants. Values are means ± se from at least two replicate samples. [See online article for color version of this figure.]

Figure 8.

Confocal Microscopy of SSGLs Labeled by the Starch Granule Marker GBSSI-YFP. (A) to (C) GBSSI-YFP could be used as a starch granule marker. Bars = 10 μm. (A) GBSSI-YFP transiently expressed in leaves of N. benthamiana plants at different time points during the night. At the beginning of the night (0 h), GBSSI-YFP localized mainly on the starch granules in the chloroplasts. As the night progressed, the restricted fluorescence of GBSSI-YFP on starch granules diminished, while diffuse fluorescence appeared in the stroma and stromules (2 to 4 h, magenta arrowheads). Sometimes, SSGLs (white arrows) were observed in leaves exposed to darkness for 2 and 4 h. Yellow, GBSSI-YFP; red, chloroplasts. (B) Enlarged area enclosed by a dashed line in (A) at 0 h. (C) Enlarged area enclosed by a dashed line in (A) at 4 h. (D) SSGLs appeared concurrently with stromules in leaves exposed to darkness for 2 or 4 h. Arrowheads indicate the stromules, while arrows refer to the SSGLs. One SSGL (cyan arrow) was in the stromule (cyan arrowhead). Bars = 10 μm. (E) and (F) SSGLs could be sequestered in autophagosomes. (E) SSGLs labeled by GBSSI-YFP colocalized with autophagosomes labeled by CFP-ATG8f. Before dark treatment, SSGLs and autophagosomes were rarely observed in mesophyll cells (top panel). After a 4-h dark treatment, both SSGLs (white arrows) and autophagosomes (magenta arrows) appeared and some colocalized (magenta arrowheads; bottom panel). Bars = 10 μm. (F) Time course of an autophagosome engulfing the SSGL and delivering it to the vacuole. The white arrowhead tracks the location of the SSGL within the autophagosome as it travels from the cytoplasm to the vacuole. Bars = 2 μm.

Figure 9.

Ultrastructural Observation of SSGL Structures in the Cytoplasm and Vacuole. (A) SSGLs in the cytoplasm. In leaves of plants exposed to darkness for 2 and 4 h, some SSGLs (cyan arrowheads) were observed in the cytoplasm in addition to the regular starch granules (S) located in the chloroplast. Note the boundary between the chloroplast and cytoplasm. (B) to (D) SSGLs in the vacuole. In the same samples as mentioned above, SSGLs also appeared in the central vacuole. Three types of SSGLs were observed. The red arrows refer to SSGLs that were sequestered in single- or double-membrane vesicles (B). The yellow arrows refer to SSGLs located directly in the vacuole (C). The blue arrows refer to SSLGs that had almost completely degraded (D). Cp, chloroplast; CW, cell wall; ER, endoplasmic reticulum; M, mitochondrion; S, starch; V, vacuole. Bars = 500 nm.

Figure 10.

Ultrastructural Confirmation of the Starch Components in SSGLs by the Silver Proteinate Staining. (A) Photographs of starch granules in the chloroplast stained by SP. (B) Ultrastructural observation of SP-stained SSGLs in the cytoplasm (cyan arrowhead) and vacuole (arrows). The red, yellow, and blue arrows refer to SSGLs engulfed by a single-membrane vesicle, SSGLs located directly in the vacuole, and SSLGs that had almost completely degraded, respectively. In addition, some diffuse silver depositions with less intensity (representative areas enclosed by the magenta dashed lines) were often observed around the SSGLs. (C) Image of SP-stained SSGLs (white arrowheads) deposited in a chloroplast protrusion that lacks thylakoid membranes. (D) Quantification of the number of vacuole-localized SSGLs per cell in TEM images of ATG6-silenced and nonsilenced leaves exposed to darkness for 2 h. Values are means ± se from 30 cells. Two asterisks indicate a significant difference (P < 0.01; Student’s t test). Cp, chloroplast; CW, cell wall; M, mitochondrion; S, starch; V, vacuole. Bars = 500 nm.

Figure 11.

The Autophagic Pathway Contributes Independently to Leaf Starch Degradation. (A) Iodine staining of the leaves from gene-silenced plants at the end of the night. Excess starch was detected in both ATG6-silenced and SEX-silenced leaves. Furthermore, more starch accumulated in the cosilenced leaves than in the individually silenced plants. The results were reproduced in three independent experiments using three leaves in each experiment. Representative results are presented. (B) Relative gene expression in the gene-silenced plants. Real-time RT-PCR analysis showed no significant difference in the reduced expression levels between the individually silenced plants and cosilenced ones. Strikingly, increased expression of SEX1 was detected in the ATG6-silenced leaves. Student’s t test was applied to determine statistically significant differences (**P < 0.01). (C) Quantification of leaf starch accumulated in the gene-silenced plants at the end of night. This assay showed similar results as those shown in (A). Values are means ± se from more than four replicate leaf samples. (D) Iodine staining of leaf discs taken from a gene-silenced plant exposed to prolonged periods of darkness. The plants were kept in a dark room for as long as 120 h. More than three leaf discs were punched from the plants at a time point and incubated in ethanol to remove the leaf pigments. When all of the samples were harvested, they were stained with Lugol’s solution. Representative results are presented. [See online article for color version of this figure.]