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

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 Km 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 Km 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.

Quantitative Multilevel Analysis of Central Metabolism in Developing Oilseeds of Oilseed Rape during in Vitro Culture

“…These were then compared with their respective K eq (the ratio of product and reactant concentrations at which forward and reverse reaction rates are equal) to reveal how far each reaction was displaced from its chemical equilibrium (Table IV). Following Tiessen et al (2012), we considered a reaction as irreversible when the mass-action ratio is displaced from its K eq by a factor of more than 10. The outcomes indicated that both Suc cleavage mediated by Suc synthase and hexose mobilization mediated by glucokinases/fructokinases were essentially irreversible in vivo in the oilseed rape embryo (Table IV).…”

“…7) of metabolic control of lipid/ starch partitioning in oilseeds has been established with only limited consideration of cellular compartmentation. Application of nonaqueous fractionation protocols would be needed to experimentally resolve metabolite compartmentation (Tiessen et al, 2012), but this technically challenging approach was not applicable. While we could not determine the distribution of metabolites among different subcellular compartments, for the interpretation of metabolite data in our study, it is of interest to know which of the measured metabolites are likely to be found predominantly in the metabolically highly active compartments (i.e.…”

Functions of Multiple Genes Encoding ADP-Glucose Pyrophosphorylase Subunits in Maize Endosperm, Embryo, and Leaf

“…In contrast to chloroplasts, which produce ATP in the stroma, nonphotosynthetic plastids in endosperm import ATP from the cytosol, where its concentration is approximately 6-to 7-fold higher than in the organelle (Tiessen et al, 2012). Despite these considerations, cereals appear to have maintained selection not only for the cytosolic AGPase but also for the plastidial form(s) of the enzyme.…”

“…the decrease in the starch deposition rate in maize endosperm at approximately 20 DAP; Prioul et al, 1994). The concentrations of the AGPase products and substrates in barley amyloplasts at mid development are such that the plastidial enzyme operates near equilibrium (Tiessen et al, 2012). Thus, plastidial AGPase potentially could respond to the metabolic environment so that, in some conditions, it catalyzes the reverse reaction and generates Glc-1-P from imported ADPGlc.…”

Modulation of Allosteric Regulation by E38K and G101N Mutations in the Potato Tuber ADP-glucose Pyrophosphorylase

“…31,32) During active photosynthesis, the levels of 3-PGA, GAP, G6P, R5P, and F6P are elevated in the plastid, while in the developing cereal endosperm, these metabolites together with F16BP, F26BP, and PEP, are expected to be present at significant levels due to the pronounced glycolysis that occurs in this tissue. [33][34][35][36] Thus the introduction of PLS-E38K, PLS-G101N, and PLS-E38K/G101N, which possess a broader range of activators, should result in enhanced starch production over that predicted based on increased sensitivity to 3-PGA alone.…”