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Cotyledon cells of Vigna mungo seedlings use at least two distinct autophagic machineries for degradation of starch granules and cellular components.

Toyooka K, Okamoto T, Minamikawa T - J. Cell Biol. (2001)

Bottom Line: The results revealed that SG is inserted into LV through autophagic function of LV and subsequently degraded by vacuolar alpha-amylase.When the embryo axes were removed from seeds and the detached cotyledons were incubated, the autophagosome-mediated autophagy was observed, but the autophagic process for the degradation of SG was not detected, suggesting that these two autophagic processes were mediated by different cellular mechanisms.The two distinct autophagic processes were thought to be involved in the breakdown of SG and cell components in the cells of germinated cotyledon.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Sciences, Tokyo Metropolitan University, Tokyo, 192-0397 Japan.

ABSTRACT
alpha-Amylase is expressed in cotyledons of germinated Vigna mungo seeds and is responsible for the degradation of starch that is stored in the starch granule (SG). Immunocytochemical analysis of the cotyledon cells with anti-alpha-amylase antibody showed that alpha-amylase is transported to protein storage vacuole (PSV) and lytic vacuole (LV), which is converted from PSV by hydrolysis of storage proteins. To observe the insertion/degradation processes of SG into/in the inside of vacuoles, ultrastructural analyses of the cotyledon cells were conducted. The results revealed that SG is inserted into LV through autophagic function of LV and subsequently degraded by vacuolar alpha-amylase. The autophagy for SG was structurally similar to micropexophagy detected in yeast cells. In addition to the autophagic process for SG, autophagosome-mediated autophagy for cytoplasm and mitochondria was detected in the cotyledon cells. When the embryo axes were removed from seeds and the detached cotyledons were incubated, the autophagosome-mediated autophagy was observed, but the autophagic process for the degradation of SG was not detected, suggesting that these two autophagic processes were mediated by different cellular mechanisms. The two distinct autophagic processes were thought to be involved in the breakdown of SG and cell components in the cells of germinated cotyledon.

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Electron micrographs showing ultrastructures of cotyledon cells of germinated V. mungo seeds (A–G), immunogold localization of α-amylase in cotyledon cells (H), and ultrastructure of cells of detached cotyledons (I). Toluidine blue (TB) staining of sections from cotyledons of day 3 V. mungo seedlings. Conversion from TB-stained cells (region I) to TB-stainless cells (region III) was accompanied with that of the PSV to LV. (B) SGs and PSVs in TB-stained cells (A, region I). The PSV was filled with storage proteins. Arrowheads indicate border between SGs and the cytoplasm. (C) SGs and PSVs in cotyledons at region II in A. Electron density of PSVs became low. SGs were surrounded with membranous structure (arrows). LED areas were found between SGs and the cytoplasm. (D) SG and LV in TB-stainless cells (region III in A). The PSV was converted to the LV in the cells. The areas around SG with LED were enlarged. (E) Ultrastructure of TB- stainless cells. SGs wrapped with a LED area contacted with LVs (arrowheads). Vesicles with similar density to LVs were observed. (F) LED membranes around the SGs fused with the LV membranes (arrow). (G) SGs were observed in LVs. The shape of SGs were largely different from those in B–E. (H) An Immunogold image representing degradation of SGs by α-amylase localized in LVs. Gold particles from anti–α-amylase antibody were densely detected in the peripheral region of SGs, which is inserted into the inside of the LV. (I) PSVs were not converted to LVs, and SGs were not taken up into PSVs in the cells of detached cotyledons. Neither low density areas around SGs nor vesicles with similar density to LV was observed in the cells. LV, lytic vacuole; Mt, mitochondrion; PSV, protein storage vacuole; SG, starch granule. Bars: (A) 50 μm; (E and G) 2 μm; (D, F, and I) 1 μm; (B, C, and H) 200 nm.
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fig4: Electron micrographs showing ultrastructures of cotyledon cells of germinated V. mungo seeds (A–G), immunogold localization of α-amylase in cotyledon cells (H), and ultrastructure of cells of detached cotyledons (I). Toluidine blue (TB) staining of sections from cotyledons of day 3 V. mungo seedlings. Conversion from TB-stained cells (region I) to TB-stainless cells (region III) was accompanied with that of the PSV to LV. (B) SGs and PSVs in TB-stained cells (A, region I). The PSV was filled with storage proteins. Arrowheads indicate border between SGs and the cytoplasm. (C) SGs and PSVs in cotyledons at region II in A. Electron density of PSVs became low. SGs were surrounded with membranous structure (arrows). LED areas were found between SGs and the cytoplasm. (D) SG and LV in TB-stainless cells (region III in A). The PSV was converted to the LV in the cells. The areas around SG with LED were enlarged. (E) Ultrastructure of TB- stainless cells. SGs wrapped with a LED area contacted with LVs (arrowheads). Vesicles with similar density to LVs were observed. (F) LED membranes around the SGs fused with the LV membranes (arrow). (G) SGs were observed in LVs. The shape of SGs were largely different from those in B–E. (H) An Immunogold image representing degradation of SGs by α-amylase localized in LVs. Gold particles from anti–α-amylase antibody were densely detected in the peripheral region of SGs, which is inserted into the inside of the LV. (I) PSVs were not converted to LVs, and SGs were not taken up into PSVs in the cells of detached cotyledons. Neither low density areas around SGs nor vesicles with similar density to LV was observed in the cells. LV, lytic vacuole; Mt, mitochondrion; PSV, protein storage vacuole; SG, starch granule. Bars: (A) 50 μm; (E and G) 2 μm; (D, F, and I) 1 μm; (B, C, and H) 200 nm.

Mentions: Ultrastructural analyses of the cotyledon cells of normally germinated V. mungo seeds were conducted to observe how SG interacts with vacuoles to be degraded by α-amylase. A cotyledon of a day 3 seedling is composed of heterologous cells with respect to the amount of reserves in the cells. Cells positioned near a vascular bundle (VB) were filled with storage materials (Fig. 4 A, region I), the reserves were degraded mostly in the cells far from the VB (Fig. 4 A, region III), and middle phase cells between filled cells and empty cells were observed (Fig. 4 A, region II). In the cells filled with reserves (filled cells), the border between the SG and cytoplasm was clear, and the electron density of the PSV was high since the storage proteins were intact (Fig. 4 B). The electron density of the PSV became low when the degradation of proteins started (Fig. 4 C). In the same cells, a membranous structure surrounding SGs was observed, and some regions with low electron density (LED) were found (Fig. 4 C). The LED area around the SG was enlarged in the empty cells in which most reserves were degraded and the PSV was converted to an LV (Fig. 4 D). In addition, vesicles with similar density to the LV and LED regions were observed in the empty cells (Fig. 4 E). These vesicles would be formed/synthesized de novo, since such vesicles were not found in the filled cells (Fig. 4 B). An SG surrounded with the LED region interacted with the LV (Fig. 4 E), and membrane fusion between the LED area around the SG and LV was observed (Fig. 4 F). The SG was found to be in the inside of the LV, and the shape of the SG was not round (Fig. 4 G), suggesting that degradation of SGs in LVs results in a change of shape of the SG. Fig. 4 H represents the immunogold image of degradation of an SG by α-amylase in an LV. A major part of α-amylase was found at the peripheral region of the SG, which was inserted into the LV (Fig. 4 H), suggesting the degradation of starch occurs at the peripheral area of SGs. In contrast to cells of attached cotyledons, conversion of PSVs to LVs was not observed, and SGs were not taken up into a PSV in the cells of detached cotyledons, which were incubated for 3 d (Fig. 4 I). In addition, neither the LED region around the SG nor the possibly de novo–synthesized vesicle with similar density to the LV was observed in the cells (Fig. 4 I). The degradation of the SG in LVs (Fig. 4, E–G) will indicate that the conversion of PSVs to LVs must occur before starch breakdown. By biochemical analyses for changes with time in amount of proteins and starch in the cotyledons of germinated V. mungo seeds, it has been revealed that the amount of proteins decreases in the cotyledons before starch breakdown (Minamikawa, 1979). The observation of degradation of the SG in LVs supports the previous report, since degradation of storage proteins in PSVs results in PSV conversion to LVs. Next, SGs, cells containing SGs, and thick sections were prepared from the cotyledons of day 3 seedlings and subsequently stained with LysoTracker red, an acidic organelle-selective probe. Small foci stained with the probe were found around the SG in broken cotyledon cells (Fig. 5 , A–D) and in cells of cotyledon sections (Fig. 5, E–H). In addition, SGs were found to be wrapped with the acidic cell compartment (Fig. 5, I and J) and to be in the inside of a putative vacuole (Fig. 5, K and L). These LysoTracker red–stained small foci and cell compartments wrapping SGs explain the character of de novo–synthesized vesicles and the LED region around the SG, which were observed in the ultrastructual analyses of the cotyledon cells. The de novo–synthesized vesicles shown in Fig. 4 E may correspond to the small foci (Fig. 5, B, D, F, and H); and the LED region (Fig. 4, D and E), to the LysoTracker red–stained cell compartment wrapping SGs (Fig. 5 J). This also suggests the possibilities that vesicles or small cell compartments with acidic pH fuse with SGs and that LED regions around SGs are built up by the fusion of such acidic vesicles/small cell compartments. When sections were prepared from detected cotyledons, which were incubated for 3 d and subsequently stained with the probe, no small foci were observed in the cells (Fig. 5, M–P). This is consistent with the result of ultrastructural observation, showing that neither de novo vesicle or LED region around SGs was detected in the cells of detached cotyledons (Fig. 4 I).


Cotyledon cells of Vigna mungo seedlings use at least two distinct autophagic machineries for degradation of starch granules and cellular components.

Toyooka K, Okamoto T, Minamikawa T - J. Cell Biol. (2001)

Electron micrographs showing ultrastructures of cotyledon cells of germinated V. mungo seeds (A–G), immunogold localization of α-amylase in cotyledon cells (H), and ultrastructure of cells of detached cotyledons (I). Toluidine blue (TB) staining of sections from cotyledons of day 3 V. mungo seedlings. Conversion from TB-stained cells (region I) to TB-stainless cells (region III) was accompanied with that of the PSV to LV. (B) SGs and PSVs in TB-stained cells (A, region I). The PSV was filled with storage proteins. Arrowheads indicate border between SGs and the cytoplasm. (C) SGs and PSVs in cotyledons at region II in A. Electron density of PSVs became low. SGs were surrounded with membranous structure (arrows). LED areas were found between SGs and the cytoplasm. (D) SG and LV in TB-stainless cells (region III in A). The PSV was converted to the LV in the cells. The areas around SG with LED were enlarged. (E) Ultrastructure of TB- stainless cells. SGs wrapped with a LED area contacted with LVs (arrowheads). Vesicles with similar density to LVs were observed. (F) LED membranes around the SGs fused with the LV membranes (arrow). (G) SGs were observed in LVs. The shape of SGs were largely different from those in B–E. (H) An Immunogold image representing degradation of SGs by α-amylase localized in LVs. Gold particles from anti–α-amylase antibody were densely detected in the peripheral region of SGs, which is inserted into the inside of the LV. (I) PSVs were not converted to LVs, and SGs were not taken up into PSVs in the cells of detached cotyledons. Neither low density areas around SGs nor vesicles with similar density to LV was observed in the cells. LV, lytic vacuole; Mt, mitochondrion; PSV, protein storage vacuole; SG, starch granule. Bars: (A) 50 μm; (E and G) 2 μm; (D, F, and I) 1 μm; (B, C, and H) 200 nm.
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fig4: Electron micrographs showing ultrastructures of cotyledon cells of germinated V. mungo seeds (A–G), immunogold localization of α-amylase in cotyledon cells (H), and ultrastructure of cells of detached cotyledons (I). Toluidine blue (TB) staining of sections from cotyledons of day 3 V. mungo seedlings. Conversion from TB-stained cells (region I) to TB-stainless cells (region III) was accompanied with that of the PSV to LV. (B) SGs and PSVs in TB-stained cells (A, region I). The PSV was filled with storage proteins. Arrowheads indicate border between SGs and the cytoplasm. (C) SGs and PSVs in cotyledons at region II in A. Electron density of PSVs became low. SGs were surrounded with membranous structure (arrows). LED areas were found between SGs and the cytoplasm. (D) SG and LV in TB-stainless cells (region III in A). The PSV was converted to the LV in the cells. The areas around SG with LED were enlarged. (E) Ultrastructure of TB- stainless cells. SGs wrapped with a LED area contacted with LVs (arrowheads). Vesicles with similar density to LVs were observed. (F) LED membranes around the SGs fused with the LV membranes (arrow). (G) SGs were observed in LVs. The shape of SGs were largely different from those in B–E. (H) An Immunogold image representing degradation of SGs by α-amylase localized in LVs. Gold particles from anti–α-amylase antibody were densely detected in the peripheral region of SGs, which is inserted into the inside of the LV. (I) PSVs were not converted to LVs, and SGs were not taken up into PSVs in the cells of detached cotyledons. Neither low density areas around SGs nor vesicles with similar density to LV was observed in the cells. LV, lytic vacuole; Mt, mitochondrion; PSV, protein storage vacuole; SG, starch granule. Bars: (A) 50 μm; (E and G) 2 μm; (D, F, and I) 1 μm; (B, C, and H) 200 nm.
Mentions: Ultrastructural analyses of the cotyledon cells of normally germinated V. mungo seeds were conducted to observe how SG interacts with vacuoles to be degraded by α-amylase. A cotyledon of a day 3 seedling is composed of heterologous cells with respect to the amount of reserves in the cells. Cells positioned near a vascular bundle (VB) were filled with storage materials (Fig. 4 A, region I), the reserves were degraded mostly in the cells far from the VB (Fig. 4 A, region III), and middle phase cells between filled cells and empty cells were observed (Fig. 4 A, region II). In the cells filled with reserves (filled cells), the border between the SG and cytoplasm was clear, and the electron density of the PSV was high since the storage proteins were intact (Fig. 4 B). The electron density of the PSV became low when the degradation of proteins started (Fig. 4 C). In the same cells, a membranous structure surrounding SGs was observed, and some regions with low electron density (LED) were found (Fig. 4 C). The LED area around the SG was enlarged in the empty cells in which most reserves were degraded and the PSV was converted to an LV (Fig. 4 D). In addition, vesicles with similar density to the LV and LED regions were observed in the empty cells (Fig. 4 E). These vesicles would be formed/synthesized de novo, since such vesicles were not found in the filled cells (Fig. 4 B). An SG surrounded with the LED region interacted with the LV (Fig. 4 E), and membrane fusion between the LED area around the SG and LV was observed (Fig. 4 F). The SG was found to be in the inside of the LV, and the shape of the SG was not round (Fig. 4 G), suggesting that degradation of SGs in LVs results in a change of shape of the SG. Fig. 4 H represents the immunogold image of degradation of an SG by α-amylase in an LV. A major part of α-amylase was found at the peripheral region of the SG, which was inserted into the LV (Fig. 4 H), suggesting the degradation of starch occurs at the peripheral area of SGs. In contrast to cells of attached cotyledons, conversion of PSVs to LVs was not observed, and SGs were not taken up into a PSV in the cells of detached cotyledons, which were incubated for 3 d (Fig. 4 I). In addition, neither the LED region around the SG nor the possibly de novo–synthesized vesicle with similar density to the LV was observed in the cells (Fig. 4 I). The degradation of the SG in LVs (Fig. 4, E–G) will indicate that the conversion of PSVs to LVs must occur before starch breakdown. By biochemical analyses for changes with time in amount of proteins and starch in the cotyledons of germinated V. mungo seeds, it has been revealed that the amount of proteins decreases in the cotyledons before starch breakdown (Minamikawa, 1979). The observation of degradation of the SG in LVs supports the previous report, since degradation of storage proteins in PSVs results in PSV conversion to LVs. Next, SGs, cells containing SGs, and thick sections were prepared from the cotyledons of day 3 seedlings and subsequently stained with LysoTracker red, an acidic organelle-selective probe. Small foci stained with the probe were found around the SG in broken cotyledon cells (Fig. 5 , A–D) and in cells of cotyledon sections (Fig. 5, E–H). In addition, SGs were found to be wrapped with the acidic cell compartment (Fig. 5, I and J) and to be in the inside of a putative vacuole (Fig. 5, K and L). These LysoTracker red–stained small foci and cell compartments wrapping SGs explain the character of de novo–synthesized vesicles and the LED region around the SG, which were observed in the ultrastructual analyses of the cotyledon cells. The de novo–synthesized vesicles shown in Fig. 4 E may correspond to the small foci (Fig. 5, B, D, F, and H); and the LED region (Fig. 4, D and E), to the LysoTracker red–stained cell compartment wrapping SGs (Fig. 5 J). This also suggests the possibilities that vesicles or small cell compartments with acidic pH fuse with SGs and that LED regions around SGs are built up by the fusion of such acidic vesicles/small cell compartments. When sections were prepared from detected cotyledons, which were incubated for 3 d and subsequently stained with the probe, no small foci were observed in the cells (Fig. 5, M–P). This is consistent with the result of ultrastructural observation, showing that neither de novo vesicle or LED region around SGs was detected in the cells of detached cotyledons (Fig. 4 I).

Bottom Line: The results revealed that SG is inserted into LV through autophagic function of LV and subsequently degraded by vacuolar alpha-amylase.When the embryo axes were removed from seeds and the detached cotyledons were incubated, the autophagosome-mediated autophagy was observed, but the autophagic process for the degradation of SG was not detected, suggesting that these two autophagic processes were mediated by different cellular mechanisms.The two distinct autophagic processes were thought to be involved in the breakdown of SG and cell components in the cells of germinated cotyledon.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Sciences, Tokyo Metropolitan University, Tokyo, 192-0397 Japan.

ABSTRACT
alpha-Amylase is expressed in cotyledons of germinated Vigna mungo seeds and is responsible for the degradation of starch that is stored in the starch granule (SG). Immunocytochemical analysis of the cotyledon cells with anti-alpha-amylase antibody showed that alpha-amylase is transported to protein storage vacuole (PSV) and lytic vacuole (LV), which is converted from PSV by hydrolysis of storage proteins. To observe the insertion/degradation processes of SG into/in the inside of vacuoles, ultrastructural analyses of the cotyledon cells were conducted. The results revealed that SG is inserted into LV through autophagic function of LV and subsequently degraded by vacuolar alpha-amylase. The autophagy for SG was structurally similar to micropexophagy detected in yeast cells. In addition to the autophagic process for SG, autophagosome-mediated autophagy for cytoplasm and mitochondria was detected in the cotyledon cells. When the embryo axes were removed from seeds and the detached cotyledons were incubated, the autophagosome-mediated autophagy was observed, but the autophagic process for the degradation of SG was not detected, suggesting that these two autophagic processes were mediated by different cellular mechanisms. The two distinct autophagic processes were thought to be involved in the breakdown of SG and cell components in the cells of germinated cotyledon.

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Related in: MedlinePlus