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Subcellular analysis of starch metabolism in developing barley seeds using a non-aqueous fractionation method.

Tiessen A, Nerlich A, Faix B, Hümmer C, Fox S, Trafford K, Weber H, Weschke W, Geigenberger P - J. Exp. Bot. (2011)

Bottom Line: This reflects the different subcellular distribution of ADPGlc pyrophosphorylase (AGPase) in these tissues. (iv) Cytosolic concentrations of ADPGlc were found to be close to the published K(m) values of AGPase and the ADPGlc/ADP transporter at the plastid envelope.Also the concentrations of the reaction partners glucose-1-phosphate, ATP, and inorganic pyrophosphate were close to the respective K(m) values of AGPase. (v) Knock-out of cytosolic AGPase in Riso16 mutants led to a strong decrease in ADPGlc level, in both the cytosol and plastid, whereas knock-down of the ADPGlc/ADP transporter led to a large shift in the intracellular distribution of ADPGlc. (v) The thermodynamic structure of the pathway of sucrose to starch was determined by calculating the mass-action ratios of all the steps in the pathway.The reversibility of AGPase in the plastid has important implications for the regulation of carbon partitioning between different biosynthetic pathways.

View Article: PubMed Central - PubMed

Affiliation: Departamento de Ingeniería Genética, CINVESTAV, Campus Guanajuato, Irapuato, México.

ABSTRACT
Compartmentation of metabolism in developing seeds is poorly understood due to the lack of data on metabolite distributions at the subcellular level. In this report, a non-aqueous fractionation method is described that allows subcellular concentrations of metabolites in developing barley endosperm to be calculated. (i) Analysis of subcellular volumes in developing endosperm using micrographs shows that plastids and cytosol occupy 50.5% and 49.9% of the total cell volume, respectively, while vacuoles and mitochondria can be neglected. (ii) By using non-aqueous fractionation, subcellular distribution between the cytosol and plastid of the levels of metabolites involved in sucrose degradation, starch synthesis, and respiration were determined. With the exception of ADP and AMP which were mainly located in the plastid, most other metabolites of carbon and energy metabolism were mainly located outside the plastid in the cytosolic compartment. (iii) In developing barley endosperm, the ultimate precursor of starch, ADPglucose (ADPGlc), was mainly located in the cytosol (80-90%), which was opposite to the situation in growing potato tubers where ADPGlc was almost exclusively located in the plastid (98%). This reflects the different subcellular distribution of ADPGlc pyrophosphorylase (AGPase) in these tissues. (iv) Cytosolic concentrations of ADPGlc were found to be close to the published K(m) values of AGPase and the ADPGlc/ADP transporter at the plastid envelope. Also the concentrations of the reaction partners glucose-1-phosphate, ATP, and inorganic pyrophosphate were close to the respective K(m) values of AGPase. (v) Knock-out of cytosolic AGPase in Riso16 mutants led to a strong decrease in ADPGlc level, in both the cytosol and plastid, whereas knock-down of the ADPGlc/ADP transporter led to a large shift in the intracellular distribution of ADPGlc. (v) The thermodynamic structure of the pathway of sucrose to starch was determined by calculating the mass-action ratios of all the steps in the pathway. The data show that AGPase is close to equilibrium, in both the cytosol and plastid, whereas the ADPGlc/ADP transporter is strongly displaced from equilibrium in vivo. This is in contrast to most other tissues, including leaves and potato tubers. (vi) Results indicate transport rather than synthesis of ADPGlc to be the major regulatory site of starch synthesis in barley endosperm. The reversibility of AGPase in the plastid has important implications for the regulation of carbon partitioning between different biosynthetic pathways.

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Comparison of the thermodynamic structure of the pathway of sucrose to starch in growing potato tubers (A) and developing barley endosperm (B). For each step in the pathway, thermodynamic properties are indicated using a false-colour code. False-colour symbols represent the ratio T/Keq showing how far each reaction is displaced from equilibrium, with T being the ratio between the in vivo subcellular concentrations of the products and the substrates of each reaction. Data are taken from Table 3 (see also Geigenberger et al., 2004 for potato tubers). For designation of the different steps, see Fig. 1. In general, a reaction is regarded as irreversible when the mass–action ratio is displaced from its Keq by a factor >10.
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fig5: Comparison of the thermodynamic structure of the pathway of sucrose to starch in growing potato tubers (A) and developing barley endosperm (B). For each step in the pathway, thermodynamic properties are indicated using a false-colour code. False-colour symbols represent the ratio T/Keq showing how far each reaction is displaced from equilibrium, with T being the ratio between the in vivo subcellular concentrations of the products and the substrates of each reaction. Data are taken from Table 3 (see also Geigenberger et al., 2004 for potato tubers). For designation of the different steps, see Fig. 1. In general, a reaction is regarded as irreversible when the mass–action ratio is displaced from its Keq by a factor >10.

Mentions: For comparison, ADPGlc levels were also analysed in the different fractions obtained after non-aqueous density fractionation of material from growing potato tubers (Fig. 5D). The overall level of ADPGlc in growing potato tubers was 20-fold lower than in wild-type barley endosperm and 4-fold lower than in the Riso16 mutant lacking cytosolic AGPase (Table 2). In contrast to barley endosperm, almost all of the ADPGlc in growing potato tubers (>97%) was located in the plastid, whereas there was only a very minor amount in the cytosol (<3%). The very low amount of ADPGlc found in the cytosol of growing tubers (∼2% of the total, or 0.4 μM) is possibly due to a small percentage of ADPGlc leaking out of the plastid. A comparison between ADPGlc (0.4 μM) and UDPGlc (571 μM; see Tiessen et al., 2002) concentrations in the cytosol of growing tubers suggests that sucrose synthase is almost exclusively involved in UDPGlc synthesis in vivo, and that ADPGlc is at its best only a very minor by-product of sucrose synthase activity under these conditions. In addition to this, the estimated concentration of ADPGlc in the cytosol of growing potato tubers (∼0.4 μM) was 500 times lower than that in the cytosol of barley endosperm (∼236–268 μM), which is consistent with a very minor role, if any, for ADPGlc in the cytosol of growing tubers (Tables 1, 2). This is in agreement with the classical pathway of starch synthesis, involving ADPGlc synthesis in the plastid rather than the cytosol (Fig 1A). It also shows that the recently proposed new pathway of starch synthesis, involving synthesis of ADPGlc in the cytosol by sucrose synthase (Baroja-Fernandez et al., 2004), is unlikely to operate in growing potato tubers.


Subcellular analysis of starch metabolism in developing barley seeds using a non-aqueous fractionation method.

Tiessen A, Nerlich A, Faix B, Hümmer C, Fox S, Trafford K, Weber H, Weschke W, Geigenberger P - J. Exp. Bot. (2011)

Comparison of the thermodynamic structure of the pathway of sucrose to starch in growing potato tubers (A) and developing barley endosperm (B). For each step in the pathway, thermodynamic properties are indicated using a false-colour code. False-colour symbols represent the ratio T/Keq showing how far each reaction is displaced from equilibrium, with T being the ratio between the in vivo subcellular concentrations of the products and the substrates of each reaction. Data are taken from Table 3 (see also Geigenberger et al., 2004 for potato tubers). For designation of the different steps, see Fig. 1. In general, a reaction is regarded as irreversible when the mass–action ratio is displaced from its Keq by a factor >10.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC3295393&req=5

fig5: Comparison of the thermodynamic structure of the pathway of sucrose to starch in growing potato tubers (A) and developing barley endosperm (B). For each step in the pathway, thermodynamic properties are indicated using a false-colour code. False-colour symbols represent the ratio T/Keq showing how far each reaction is displaced from equilibrium, with T being the ratio between the in vivo subcellular concentrations of the products and the substrates of each reaction. Data are taken from Table 3 (see also Geigenberger et al., 2004 for potato tubers). For designation of the different steps, see Fig. 1. In general, a reaction is regarded as irreversible when the mass–action ratio is displaced from its Keq by a factor >10.
Mentions: For comparison, ADPGlc levels were also analysed in the different fractions obtained after non-aqueous density fractionation of material from growing potato tubers (Fig. 5D). The overall level of ADPGlc in growing potato tubers was 20-fold lower than in wild-type barley endosperm and 4-fold lower than in the Riso16 mutant lacking cytosolic AGPase (Table 2). In contrast to barley endosperm, almost all of the ADPGlc in growing potato tubers (>97%) was located in the plastid, whereas there was only a very minor amount in the cytosol (<3%). The very low amount of ADPGlc found in the cytosol of growing tubers (∼2% of the total, or 0.4 μM) is possibly due to a small percentage of ADPGlc leaking out of the plastid. A comparison between ADPGlc (0.4 μM) and UDPGlc (571 μM; see Tiessen et al., 2002) concentrations in the cytosol of growing tubers suggests that sucrose synthase is almost exclusively involved in UDPGlc synthesis in vivo, and that ADPGlc is at its best only a very minor by-product of sucrose synthase activity under these conditions. In addition to this, the estimated concentration of ADPGlc in the cytosol of growing potato tubers (∼0.4 μM) was 500 times lower than that in the cytosol of barley endosperm (∼236–268 μM), which is consistent with a very minor role, if any, for ADPGlc in the cytosol of growing tubers (Tables 1, 2). This is in agreement with the classical pathway of starch synthesis, involving ADPGlc synthesis in the plastid rather than the cytosol (Fig 1A). It also shows that the recently proposed new pathway of starch synthesis, involving synthesis of ADPGlc in the cytosol by sucrose synthase (Baroja-Fernandez et al., 2004), is unlikely to operate in growing potato tubers.

Bottom Line: This reflects the different subcellular distribution of ADPGlc pyrophosphorylase (AGPase) in these tissues. (iv) Cytosolic concentrations of ADPGlc were found to be close to the published K(m) values of AGPase and the ADPGlc/ADP transporter at the plastid envelope.Also the concentrations of the reaction partners glucose-1-phosphate, ATP, and inorganic pyrophosphate were close to the respective K(m) values of AGPase. (v) Knock-out of cytosolic AGPase in Riso16 mutants led to a strong decrease in ADPGlc level, in both the cytosol and plastid, whereas knock-down of the ADPGlc/ADP transporter led to a large shift in the intracellular distribution of ADPGlc. (v) The thermodynamic structure of the pathway of sucrose to starch was determined by calculating the mass-action ratios of all the steps in the pathway.The reversibility of AGPase in the plastid has important implications for the regulation of carbon partitioning between different biosynthetic pathways.

View Article: PubMed Central - PubMed

Affiliation: Departamento de Ingeniería Genética, CINVESTAV, Campus Guanajuato, Irapuato, México.

ABSTRACT
Compartmentation of metabolism in developing seeds is poorly understood due to the lack of data on metabolite distributions at the subcellular level. In this report, a non-aqueous fractionation method is described that allows subcellular concentrations of metabolites in developing barley endosperm to be calculated. (i) Analysis of subcellular volumes in developing endosperm using micrographs shows that plastids and cytosol occupy 50.5% and 49.9% of the total cell volume, respectively, while vacuoles and mitochondria can be neglected. (ii) By using non-aqueous fractionation, subcellular distribution between the cytosol and plastid of the levels of metabolites involved in sucrose degradation, starch synthesis, and respiration were determined. With the exception of ADP and AMP which were mainly located in the plastid, most other metabolites of carbon and energy metabolism were mainly located outside the plastid in the cytosolic compartment. (iii) In developing barley endosperm, the ultimate precursor of starch, ADPglucose (ADPGlc), was mainly located in the cytosol (80-90%), which was opposite to the situation in growing potato tubers where ADPGlc was almost exclusively located in the plastid (98%). This reflects the different subcellular distribution of ADPGlc pyrophosphorylase (AGPase) in these tissues. (iv) Cytosolic concentrations of ADPGlc were found to be close to the published K(m) values of AGPase and the ADPGlc/ADP transporter at the plastid envelope. Also the concentrations of the reaction partners glucose-1-phosphate, ATP, and inorganic pyrophosphate were close to the respective K(m) values of AGPase. (v) Knock-out of cytosolic AGPase in Riso16 mutants led to a strong decrease in ADPGlc level, in both the cytosol and plastid, whereas knock-down of the ADPGlc/ADP transporter led to a large shift in the intracellular distribution of ADPGlc. (v) The thermodynamic structure of the pathway of sucrose to starch was determined by calculating the mass-action ratios of all the steps in the pathway. The data show that AGPase is close to equilibrium, in both the cytosol and plastid, whereas the ADPGlc/ADP transporter is strongly displaced from equilibrium in vivo. This is in contrast to most other tissues, including leaves and potato tubers. (vi) Results indicate transport rather than synthesis of ADPGlc to be the major regulatory site of starch synthesis in barley endosperm. The reversibility of AGPase in the plastid has important implications for the regulation of carbon partitioning between different biosynthetic pathways.

Show MeSH