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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 the immunogold localization of α-amylase in attached cotyledons cells (A–E), detached cotyledons cells (F), or control for immunogold labeling (G). (A) Anti–α-amylase antibody immunogold-stained PSV, but not SG. (B and C) Golgi complex and PSVs were both immunogold-labeled with anti– α-amylase antibody. (D) Immunogold localization of α-amylase (15-nm particles) and SH-EP (10-nm particles). α-Amylase and SH-EP were localized in PSVs and KVs, respectively. (E) Gold particles from anti–α-amylase antibody were detected in LVs as well as PSVs. (F) PSVs in detached cotyledons were immunogold labeled with anti– α-amylase antibody. (G) Immunogold staining of detached cotyledon cells without first antibody (anti–α-amylase antibody). No gold particles were observed in the cell. CW, cell wall; G, Golgi complex; KV, KDEL-tailed cysteine proteinase-accumulating vesicle; LV, lytic vacuole; Mt, mitochondrion; PSV, protein storage vacuole; SG, starch granule. Bars, 200 nm.
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fig3: Electron micrographs showing the immunogold localization of α-amylase in attached cotyledons cells (A–E), detached cotyledons cells (F), or control for immunogold labeling (G). (A) Anti–α-amylase antibody immunogold-stained PSV, but not SG. (B and C) Golgi complex and PSVs were both immunogold-labeled with anti– α-amylase antibody. (D) Immunogold localization of α-amylase (15-nm particles) and SH-EP (10-nm particles). α-Amylase and SH-EP were localized in PSVs and KVs, respectively. (E) Gold particles from anti–α-amylase antibody were detected in LVs as well as PSVs. (F) PSVs in detached cotyledons were immunogold labeled with anti– α-amylase antibody. (G) Immunogold staining of detached cotyledon cells without first antibody (anti–α-amylase antibody). No gold particles were observed in the cell. CW, cell wall; G, Golgi complex; KV, KDEL-tailed cysteine proteinase-accumulating vesicle; LV, lytic vacuole; Mt, mitochondrion; PSV, protein storage vacuole; SG, starch granule. Bars, 200 nm.

Mentions: Cotyledon cells of day 3 dark-grown V. mungo seedlings were fractionated into microsome and PSV fractions, and both fractions were separated by SDS-PAGE. In the PSV fraction, polypeptides around 50 kD that were derived from storage globulins (Okamoto and Minamikawa, 1998) were enriched (Fig. 2 A). When the proteins in the gel were analyzed by immunoblotting with anti–SH-EP antibody, proSH-EP of 43-kD (ER and/or KDEL-tailed cysteine proteinase–accumulating vesicle [KV] form) and 33-kD mature SH-EP (vacuolar form) were detected in microsome and PSV fractions, respectively (Fig. 2 B; Okamoto et al., 1994). This suggests that fractionations of the microsome and PSV from cotyledon cells were successfully conducted. Probing the blots with anti–α-amylase antibody resulted in detection of α-amylase with molecular mass of 44 kD in both the PSV and microsomal fractions (Fig. 2 C), suggesting that the enzyme is localized in PSV without proteolytic processing of the enzyme in PSV. By immunocytochemical analysis of the cotyledon cells with anti–α-amylase antibody, it was revealed that α-amylase is localized in both PSVs and LVs (Fig. 3 , A–F), which is possibly converted from the PSV by fusion of KV (Toyooka et al., 2000). Fig. 3 A shows that α-amylase is localized in the PSV but not in the SG. Immunogold staining of the Golgi complex with anti–α-amylase antibody indicates that α-amylase is transported to the PSV through a Golgi complex–dependent pathway (Fig. 3, B and C). When cotyledon cells were double immunogold–stained with anti–α-amylase polyclonal antibody and anti–SH-EP monoclonal antibody, α-amylase and SH-EP were localized in the PSV and KV, respectively (Fig. 3 D), supporting the sorting of α-amylase is Golgi complex dependent but not KV dependent. In addition to the PSV, the LV was densely immunogold labeled (Fig. 3 E). This suggests that α-amylase is transported to both the PSV and LV or sorted to the PSV before the conversion of PSVs to LVs occurs. When the detached cotyledons were incubated for 3 days and the cotyledon cells were analyzed with the same procedures, it was indicated that α-amylase is transported to the PSV also in the detached cotyledons (Fig. 3, F and G).


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 the immunogold localization of α-amylase in attached cotyledons cells (A–E), detached cotyledons cells (F), or control for immunogold labeling (G). (A) Anti–α-amylase antibody immunogold-stained PSV, but not SG. (B and C) Golgi complex and PSVs were both immunogold-labeled with anti– α-amylase antibody. (D) Immunogold localization of α-amylase (15-nm particles) and SH-EP (10-nm particles). α-Amylase and SH-EP were localized in PSVs and KVs, respectively. (E) Gold particles from anti–α-amylase antibody were detected in LVs as well as PSVs. (F) PSVs in detached cotyledons were immunogold labeled with anti– α-amylase antibody. (G) Immunogold staining of detached cotyledon cells without first antibody (anti–α-amylase antibody). No gold particles were observed in the cell. CW, cell wall; G, Golgi complex; KV, KDEL-tailed cysteine proteinase-accumulating vesicle; LV, lytic vacuole; Mt, mitochondrion; PSV, protein storage vacuole; SG, starch granule. Bars, 200 nm.
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Related In: Results  -  Collection

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fig3: Electron micrographs showing the immunogold localization of α-amylase in attached cotyledons cells (A–E), detached cotyledons cells (F), or control for immunogold labeling (G). (A) Anti–α-amylase antibody immunogold-stained PSV, but not SG. (B and C) Golgi complex and PSVs were both immunogold-labeled with anti– α-amylase antibody. (D) Immunogold localization of α-amylase (15-nm particles) and SH-EP (10-nm particles). α-Amylase and SH-EP were localized in PSVs and KVs, respectively. (E) Gold particles from anti–α-amylase antibody were detected in LVs as well as PSVs. (F) PSVs in detached cotyledons were immunogold labeled with anti– α-amylase antibody. (G) Immunogold staining of detached cotyledon cells without first antibody (anti–α-amylase antibody). No gold particles were observed in the cell. CW, cell wall; G, Golgi complex; KV, KDEL-tailed cysteine proteinase-accumulating vesicle; LV, lytic vacuole; Mt, mitochondrion; PSV, protein storage vacuole; SG, starch granule. Bars, 200 nm.
Mentions: Cotyledon cells of day 3 dark-grown V. mungo seedlings were fractionated into microsome and PSV fractions, and both fractions were separated by SDS-PAGE. In the PSV fraction, polypeptides around 50 kD that were derived from storage globulins (Okamoto and Minamikawa, 1998) were enriched (Fig. 2 A). When the proteins in the gel were analyzed by immunoblotting with anti–SH-EP antibody, proSH-EP of 43-kD (ER and/or KDEL-tailed cysteine proteinase–accumulating vesicle [KV] form) and 33-kD mature SH-EP (vacuolar form) were detected in microsome and PSV fractions, respectively (Fig. 2 B; Okamoto et al., 1994). This suggests that fractionations of the microsome and PSV from cotyledon cells were successfully conducted. Probing the blots with anti–α-amylase antibody resulted in detection of α-amylase with molecular mass of 44 kD in both the PSV and microsomal fractions (Fig. 2 C), suggesting that the enzyme is localized in PSV without proteolytic processing of the enzyme in PSV. By immunocytochemical analysis of the cotyledon cells with anti–α-amylase antibody, it was revealed that α-amylase is localized in both PSVs and LVs (Fig. 3 , A–F), which is possibly converted from the PSV by fusion of KV (Toyooka et al., 2000). Fig. 3 A shows that α-amylase is localized in the PSV but not in the SG. Immunogold staining of the Golgi complex with anti–α-amylase antibody indicates that α-amylase is transported to the PSV through a Golgi complex–dependent pathway (Fig. 3, B and C). When cotyledon cells were double immunogold–stained with anti–α-amylase polyclonal antibody and anti–SH-EP monoclonal antibody, α-amylase and SH-EP were localized in the PSV and KV, respectively (Fig. 3 D), supporting the sorting of α-amylase is Golgi complex dependent but not KV dependent. In addition to the PSV, the LV was densely immunogold labeled (Fig. 3 E). This suggests that α-amylase is transported to both the PSV and LV or sorted to the PSV before the conversion of PSVs to LVs occurs. When the detached cotyledons were incubated for 3 days and the cotyledon cells were analyzed with the same procedures, it was indicated that α-amylase is transported to the PSV also in the detached cotyledons (Fig. 3, F and G).

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.

Show MeSH
Related in: MedlinePlus