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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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Changes with time in amounts of SH-EP, α-amylase, and VmPE-1 in cotyledons of V. mungo seedlings (Attached cotyledons) or embryo axis–removed cotyledons of V. mungo seeds (Detached cotyledons). Attached or detached cotyledons were prepared as described in Materials and methods. 10 pairs of cotyledons were homogenized with 3 ml of 50 mM Tris-Cl (pH 7.4), and the homogenates were centrifuged at 15,000 g for 10 min. Supernatants (10 μl each) were analyzed by SDS-PAGE immunoblotting with anti–SH-EP, α-amylase, or VmPE-1 antibody.
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fig1: Changes with time in amounts of SH-EP, α-amylase, and VmPE-1 in cotyledons of V. mungo seedlings (Attached cotyledons) or embryo axis–removed cotyledons of V. mungo seeds (Detached cotyledons). Attached or detached cotyledons were prepared as described in Materials and methods. 10 pairs of cotyledons were homogenized with 3 ml of 50 mM Tris-Cl (pH 7.4), and the homogenates were centrifuged at 15,000 g for 10 min. Supernatants (10 μl each) were analyzed by SDS-PAGE immunoblotting with anti–SH-EP, α-amylase, or VmPE-1 antibody.

Mentions: We have previously reported that removal of the embryonic axes from V. mungo seeds had effects on expression of proteinase activity and degradation of storage reserves in cotyledons (Minamikawa, 1979; Yamauchi et al., 1994). When V. mungo seeds were allowed to germinate at 27°C in darkness, proteinase and amylase activities in the cotyledons (attached cotyledons) increased and reached a maximum level at 3 d after imbibition, and the amounts of storage proteins and starch gradually decreased during seed germination and subsequent seedling growth. When detached cotyledons (embryonic axes were removed from dry seeds) were prepared and incubated under the same conditions as above, mobilization of storage proteins and starch was not observed, and the proteinase activity remained at a low level. However, amylase activity appeared even in the detached cotyledons at an equivalent level to that in the attached cotyledons (Yamauchi et al., 1994). In this study, changes with time in amounts of a papain-type proteinase (SH-EP), α-amylase and an asparaginyl endopeptidase (VmPE-1; Okamoto et al., 1994) in the attached or detached cotyledons were analyzed by SDS-PAGE and immunoblotting with anti–SH-EP, α-amylase, or VmPE-1 antibody. In the attached cotyledons, these three hydrolases were synthesized de novo, and the amounts of these enzymes increased in V. mungo seeds within 4 d after imbibition (Fig. 1) . In the detached cotyledons, expressions of SH-EP and VmPE-1 were markedly suppressed, but the expression level of α-amylase was almost the same as that in the attached cotyledons (Fig. 1). These results indicate that α-amylase is normally expressed even in detached cotyledon cells in which starch stored in SGs is intact (Minamikawa 1979; Yamauchi et al., 1994), and that α-amylase is not directly transported to SGs but to other cell compartment(s) in the cotyledon cells. Thus, to determine the intracellular localization of the enzyme, subcellular fractionation and immunocytochemical assays of cotyledon cells were conducted.


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)

Changes with time in amounts of SH-EP, α-amylase, and VmPE-1 in cotyledons of V. mungo seedlings (Attached cotyledons) or embryo axis–removed cotyledons of V. mungo seeds (Detached cotyledons). Attached or detached cotyledons were prepared as described in Materials and methods. 10 pairs of cotyledons were homogenized with 3 ml of 50 mM Tris-Cl (pH 7.4), and the homogenates were centrifuged at 15,000 g for 10 min. Supernatants (10 μl each) were analyzed by SDS-PAGE immunoblotting with anti–SH-EP, α-amylase, or VmPE-1 antibody.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2196185&req=5

fig1: Changes with time in amounts of SH-EP, α-amylase, and VmPE-1 in cotyledons of V. mungo seedlings (Attached cotyledons) or embryo axis–removed cotyledons of V. mungo seeds (Detached cotyledons). Attached or detached cotyledons were prepared as described in Materials and methods. 10 pairs of cotyledons were homogenized with 3 ml of 50 mM Tris-Cl (pH 7.4), and the homogenates were centrifuged at 15,000 g for 10 min. Supernatants (10 μl each) were analyzed by SDS-PAGE immunoblotting with anti–SH-EP, α-amylase, or VmPE-1 antibody.
Mentions: We have previously reported that removal of the embryonic axes from V. mungo seeds had effects on expression of proteinase activity and degradation of storage reserves in cotyledons (Minamikawa, 1979; Yamauchi et al., 1994). When V. mungo seeds were allowed to germinate at 27°C in darkness, proteinase and amylase activities in the cotyledons (attached cotyledons) increased and reached a maximum level at 3 d after imbibition, and the amounts of storage proteins and starch gradually decreased during seed germination and subsequent seedling growth. When detached cotyledons (embryonic axes were removed from dry seeds) were prepared and incubated under the same conditions as above, mobilization of storage proteins and starch was not observed, and the proteinase activity remained at a low level. However, amylase activity appeared even in the detached cotyledons at an equivalent level to that in the attached cotyledons (Yamauchi et al., 1994). In this study, changes with time in amounts of a papain-type proteinase (SH-EP), α-amylase and an asparaginyl endopeptidase (VmPE-1; Okamoto et al., 1994) in the attached or detached cotyledons were analyzed by SDS-PAGE and immunoblotting with anti–SH-EP, α-amylase, or VmPE-1 antibody. In the attached cotyledons, these three hydrolases were synthesized de novo, and the amounts of these enzymes increased in V. mungo seeds within 4 d after imbibition (Fig. 1) . In the detached cotyledons, expressions of SH-EP and VmPE-1 were markedly suppressed, but the expression level of α-amylase was almost the same as that in the attached cotyledons (Fig. 1). These results indicate that α-amylase is normally expressed even in detached cotyledon cells in which starch stored in SGs is intact (Minamikawa 1979; Yamauchi et al., 1994), and that α-amylase is not directly transported to SGs but to other cell compartment(s) in the cotyledon cells. Thus, to determine the intracellular localization of the enzyme, subcellular fractionation and immunocytochemical assays of cotyledon cells were conducted.

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