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

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.     

Analysis of subcellular metabolite levels of potato tubers (Solanum tuberosum) displaying alterations in cellular or extracellular sucrose metabolism

The expression of a heterologous invertase in potato tubers (Solanum tuberosum) in either the cytosol or apoplast leads to a decrease in total sucrose content and to an increase in glucose. Depending on the targeting of the enzyme different changes in phenotype and metabolism of the tubers occur: the cytosolic invertase expressing tubers show an increase in the glycolytic flux, accumulation of amino acids and organic acids, and the appearance of novel disaccharides; however, these changes are not observed when the enzyme is expressed in the apoplast [Roessner et al. (2001). Plant Cell, 13, 11-29]. The analysis of these lines raised several questions concerning the regulation of compartmentation of metabolites in potato tubers. In the current study we addressed these questions by performing comparative subcellular metabolite profiling. We demonstrate that: (i) hexoses accumulate in the vacuole independently of their site of production, but that the cytosolic invertase expression led to a strong increase in the cytosolic glucose concentration and decrease in cytosolic sucrose, whereas these effects were more moderate in the apoplastic expressors; (ii) three out of four of the novel compounds found in the cytosolic overexpressors accumulate in the same compartment; (iii) despite changes in absolute cellular content the subcellular distribution of amino acids was invariant in the invertase overexpressing tubers. These results are discussed in the context of current models of the compartmentation of primary metabolism in heterotrophic plant tissues. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Subcellular Volumes and Metabolite Concentrations in Potato (Solanum tuberosum cv. Désirée) Leaves1

Cellular and subcellular volumes in mature leaves of potato (Solanum tuberosum cv. Désirée) were determined stereologically from light and electron micrographs. Leaves of ten-week-old plants with a total leaf volume of 623 μL per mg chlorophyll (Chl) were found to be composed of 12 % epidermis, 68 % mesophyll, 5 % vascular tissue, 3 % apoplast and 16 % gas space. In the epidermal cells 97 % of the volume was occupied by the vacuole. The mesophyll cells consisted (as expressed per mg Chl) of 323 μL (76 %) vacuole, 35 μL (8 %) chloroplast stroma, 22 μL (5 %) cytosol plus nucleus and peroxisomes, and 4μL, (1 %) mitochondria. A comparison of these values with subcellular volumes previously determined for spinach and barley leaves, shows that the relative sizes of the subcellular volumes are strikingly similar. Subcellular concentrations of carbohydrates, of the phosphorylated intermediates of carbohydrate metabolism, of malate, and of amino acids have been evaluated from measurements of the corresponding subcellular metabolite contents determined using the non-aqueous fractionation technique. Malate, glucose and fructose were found to accumulate in the vacuole, whereas the concentration of sucrose and amino acids in the cytosol were much higher than in the vacuole. The amino acid concentration in the chloroplast stroma is similar to that in the cytosol. Phosphorylated intermediates of carbohydrate metabolism are confined to the chloroplast stroma and the cytosol, confirming the validity of the fractionation method. Whereas triose phosphates and fructose-l,6-bisphosphate are concentrated in the stroma, the concentrations of hexose monophosphates were highest in the cytosol. Since the subcellular metabolite distribution in potato leaves reported here is very similar to that previously described for spinach and barley leaves, we conclude that it may be characteristic for mesophyll cells in general.     

Subcellular pyrophosphate metabolism in developing tubers of potato (Solanum tuberosum)

PPi has previously been implicated specifically in the co-ordination of the sucrose–starch transition and in the broader context of its role as co-factor in heterotrophic plant metabolism. In order to assess the compartmentation of pyrophosphate (PPi) metabolism in the potato tuber we analysed the effect of expressing a bacterial pyrophosphatase in the amyloplast of wild type tubers or in the cytosol or amyloplast of invertase-expressing tubers. The second and third approaches were adopted since we have previously characterized the invertase expressing lines to both exhibit highly altered sucrose metabolism and to contain elevated levels of PPi (Farré et al. (2000a) Plant Physiol 123:681) and therefore this background rendered questions concerning the level of communication between the plastidic and cytosolic pyrophosphate pools relatively facile. In this study we observed that the increase in PPi in the invertase expressing lines was mainly confined to the cytosol. Accordingly, the expression of a bacterial pyrophosphatase in the plastid of either wild type or invertase-expressing tubers did not lead to a decrease in total PPi content. However, the expression of the heterologous pyrophosphatase in␣the cytosol of cytosolic invertase-expressing tubers led to strong metabolic changes. These results are discussed both with respect to our previous hypotheses and to current models of the compartmentation of potato tuber metabolism.     

In vitro grown potato microtubers are a suitable system for the study of primary carbohydrate metabolism

The aim of this work was to determine the suitability of tissue culture microtubers for the study of primary carbohydrate metabolism in potato plants (Solanum tuberosum L. cv. Desirée). We have determined the levels of key enzymes and intermediates in the pathways of sucrose metabolism, starch metabolism and glycolysis in wild type microtubers. Comparison of the metabolite levels in microtubers with a range of published studies on soil-grown developing tubers showed that the two systems were similar both in the absolute levels and in the ratios between metabolites, despite some differences in the maximum catalytic activities of some glycolytic enzymes. We conclude that in vitro grown microtubers are an adequate model system for studying primary carbohydrate metabolism in developing potato tubers.     

Induction of the activity of glycolytic enzymes correlates with enhanced hydrolysis of sucrose in the cytosol of transgenic potato tubers

The aim of this work was to define the metabolic factors which regulate the respiratory pathways in trangenic potato tubers. We previously found that respiration is enhanced in transgenic tubers which express a yeast invertase and a glucokinase from Zymomonas mobilis . In this study we investigated glycolysis in three further transgenic potato lines with profound changes in the mobilization of sucrose. We studied antisense ADPglucose pyrophosphorylase lines which are characterized by a reduction in starch accumulation and a significant build up of sucrose and related metabolic intermediates. We also report the generation of two novel double transgenic lines where the yeast invertase is expressed specifically in tubers of the ADPglucose pyrophosphorylase antisense line, targeted to either the cytosol or apopolast. We evaluated whether the localization of sucrose cleavage had an impact on the glycolytic induction, and assessed if invertase expression in the high-sucrose background had any further effects on glycolysis. We found that induction of the glycolytic enzymes only occurs when the invertase is targeted to the cytosol, and that the extent of this induction was comparable in the wild type and antisenseADPglucose pyrophosphorylase backgrounds. We conclude that the signal regulating glycolysis is directly linked to cytosolic sucrose hydrolysis.     

Accumulation of hexoses in leaf vacuoles: Studies with transgenic tobacco plants expressing yeast-derived invertase in the cytosol,vacuole or apoplasm

The subcellular distribution of hexoses, sucrose and amino acids among the stromal, cytosolic and vacuolar compartments was analysed by a nonaqueous fractionation technique in leaves of tobacco (Nicotiana tabaccum L.) wild-type and transgenic plants expressing a yeast-derived invertase in the cytosolic, vacuolar or apoplasmic compartment. In the wild-type plants the amino acids were found to be located in the stroma and in the cytosol, sucrose mainly in the cytosol and up to 98% of the hexoses in the vacuole. In the leaves of the various transformants, where the contents of hexoses were greater than in wild-type plants, again 97–98% of these hexoses were found in the vacuoles. It is concluded that leaf vacuoles contain transporters for the active uptake of glucose and fructose against a high concentration gradient. A comparison of estimated metabolite concentrations in the subcellular compartments of wild-type and transformant plants indicated that the decreased photosynthetic capacity of the transformants is not due to an osmotic effect on photosynthesis, as was shown earlier to be the case in transformed potato leaves, but is the result of a long-term dedifferentiation of tobacco leaf cells to heterotrophic cells.Abbreviations apo-inv tobacco plant with yeast invertase in the apoplasm - Chl chlorophyll - cy-inv tobacco plant with yeast invertase in the cytosol - vac-inv tobacco plant with yeast invertase in the vacuole - WT wild-type tobacco plant The authors thank A. Großpietsch for her able technical assistance. This work has been supported by the Bundesminister für Forschung und Technologie.     

Subcellular compartmentation of pyrophosphate and alkaline pyrophosphatase in leaves

The subcellular localisation of pyrophosphate and alkaline pyrophosphatase in leaves has been studied using non-aqueous density gradient centrifugation of spinach leaves, and membrane filtration of wheat mesophyll protoplasts. The pyrophosphate was measured in extracts prepared in trichloroacetic acid, and could be quantitatively recovered from the leaf material. It was located predominantly in the cytosol, with a concentration of 0.2–0.3 mM. In contrast, the alkaline pyrophosphatase was largely, if not, exclusively, located in the chloroplast. By comparing the pyrophosphate levels in the cytosol with previously published data on the cytosolic levels of phosphate and metabolic intermediates, it is shown that the reactions catalysed by pyrophosphate: fructose-6-phosphate phosphotransferase and UDP-glucose pyrophosphorylase are close to the thermodynamic equilibrium and, thus, freely reversible in vivo. Comparison of the pyrophosphate levels with the reported electrical and pH gradient across the tonoplast membrane shows the free energy released during pyrophosphate hydrolysis is similar to that required to move a proton across the tonoplast membrane. It is suggested that pyrophosphate could operate as a secondary energy donor in the cytosol of plant cells.     

Overexpression of pyrophosphatase leads to increased sucrose degradation and starch synthesis, increased activities of enzymes for sucrose-starch interconversions, and increased levels of nucleotides in growing potato tubers

Overexpression of inorganic pyrophosphatase (PPase) from Escherichia coli in the cytosol of plants (ppa1 plants) leads to a decrease of inorganic pyrophosphate (PPi; U. Sonnewald, 1992, Plant J 2: 571–581). The consequences for sucrose-starch interconversions have now been studied in growing potato (Solanum tuberosum L. cv. Desirée) tubers. Sucrose is degraded via sucrose synthase and UDP-glucose pyrophosphorylase in growing tubers, and it was expected that the low PPi in the ppa1 transformants would restrict the mobilisation of sucrose and conversion to starch. Over-expression of PPase resulted in an accumulation of sucrose and UDP-glucose, and decreased concentrations of hexose phosphates and glycerate-3-phosphate in growing ppa1 tubers. Unexpectedly, the rate of degradation of [¹⁴C] sucrose was increased by up to 30%, the rate of starch synthesis was increased, and the starch content was increased by 20–30% in ppa1 tubers compared to wild-type tubers. Reasons for this unexpectedly efficient conversion of sucrose to starch in the ppa1 tubers were investigated. (i) The transformed tubers contained increased activities of several enzymes required for sucrose-starch interconversions including two- to threefold more sucrose synthase and 60% more ADP-glucose pyrophosphorylase. They also contained 30–100% increased activities of several glycolytic enzymes and amylase, increased protein, and unaltered or slightly decreased starch phosphorylase, acid invertase and mannosidase. (ii) The transformants contained higher pools of uridine nucleotides. As a result, although the UDP-glucose pool is increased two- to threefold, this does not lead to a decrease of UTP or UDP. (iii) The transformants contained twofold larger pools of ATP and ADP, and ADP-glucose was increased by up to threefold. In stored ppa1 tubers, there were no changes in the activities of glycolytic enzymes, and nucleotides did not increase. It is concluded that in growing tubers PPi has a wider significance than just being an energy donor for specific reactions in the cytosol. Increased rates of PPi hydrolysis also affect general aspects of cell activity including the levels of nucleotides and protein. Possible ways in which PPi hydrolysis could affect these processes are discussed. Received: 9 July 1997 / Accepted: 3 November 1997     

Metabolomic analysis of the potato tuber life cycle

An analysis of the potato (Solanum tuberosum L.) tuber life cycle has been completed using a range of mass-spectrometry (MS) based approaches. Six stages have been examined which included developing and mature tubers, sprouting mature tubers and mature tubers stored at 5 or 10°C. The impact of excising developing tubers from the mother plant (source-sink manipulation) was also determined. Data was subjected to Principal Components Analysis, Analysis of Variance and Hierarchical Cluster Analysis to assess the potential for separating the life cycle stages, to define the major profiles for metabolite changes during the life cycle stages examined, and to inform on which metabolites underpinned these profiles. We have shown that it is possible to separate all of the stages using combined analytical approaches and that five major profiles can be used to describe the changes in metabolite levels. Data also indicate that, within a relatively short timeframe, manipulation of source-sink relations has a significant impact on metabolite pools beyond what is currently known for sugar–starch metabolism. We have also demonstrated that the metabolomics data can be mined to provide answers to specific questions––in this case to identify temporal changes in metabolites related to acrylamide-forming potential.     

A possible role for pyrophosphate in the coordination of cytosolic and plastidial carbon metabolism within the potato tuber

The early stages of tuber development are characterized by cell division, high metabolic activity, and the predominance of invertase as the sucrose (Suc) cleaving activity. However, during the subsequent phase of starch accumulation the cleavage of Suc occurs primarily by the action of Suc synthase. The mechanism that is responsible for this switch in Suc cleaving activities is currently unknown. One striking difference between the invertase and Suc synthase mediated cleavage of Suc is the direct involvement of inorganic pyrophosphate (PPi) in the latter case. There is presently no convincing explanation of how the PPi required to support this process is generated in potato (Solanum tuberosum) tubers. The major site of PPi production in a maturing potato tubers is likely to be the reaction catalyzed by ADP-glucose pyrophosphorylase, the first committed step of starch biosynthesis in amyloplasts. We present data based on the analysis of the PPi levels in various transgenic plants altered in starch and Suc metabolism that support the hypothesis that PPi produced in the plastid is used to support cytosolic Suc breakdown and that PPi is an important coordinator of cytosolic and plastidial metabolism in potato tubers.     

Photosynthetic assimilation of 14C into amino acids in potato (Solanum tuberosum) and asparagine in the tubers

Asparagine is the predominant free amino acid in potato tubers and the present study aimed to establish whether it is imported from the leaves or synthesised in situ. Free amino acid concentrations are important quality determinants for potato tubers because they react with reducing sugars at high temperatures in the Maillard reaction. This reaction produces melanoidin pigments and a host of aroma and flavour volatiles, but if free asparagine participates in the final stages, it results in the production of acrylamide, an undesirable contaminant. ¹⁴CO₂ was supplied to a leaf or leaves of potato plants (cv. Saturna) in the light and radioactivity incorporated into amino acids was determined in the leaves, stems, stolons and tubers. Radioactivity was found in free amino acids, including asparagine, in all tissues, but the amount incorporated in asparagine transported to the tubers and stolons was much less than that in glutamate, glutamine, serine and alanine. The study showed that free asparagine does not play an important role in the transport of nitrogen from leaf to tuber in potato, and that the high concentrations of free asparagine that accumulate in potato tubers arise from synthesis in situ. This indicates that genetic interventions to reduce free asparagine concentration in potato tubers will have to target asparagine metabolism in the tuber.     

Kinetics of labelling of organic and amino acids in potato tubers by gas chromatography-mass spectrometry following incubation in (13)C labelled isotopes

Metabolic pathways of primary metabolism of discs isolated from potato tubers were evaluated by the use of a gas chromatography-mass spectrometry (GC-MS) method generated specifically for this purpose. After testing several possible methods including chemical ionization, it was decided for reasons of sensitivity, reproducibility and speed to use electron impact ionization-based GC-MS analysis. The specific labelling and label accumulation of over 30 metabolites including a broad number of sugars, organic and amino acids was analysed following the incubation of tuber discs in [U-(13)C]glucose. The reproducibility of this method was similar to that found for other GC-MS-based analyses and comparison of flux estimates from this method with those obtained from parallel, yet less comprehensive, radiolabel experiments revealed close agreement. Therefore, the novel method allows quantitatively evaluation of a broad range of metabolic pathways without the need for laborious (and potentially inaccurate), chemical fractionation procedures commonly used in the estimation of fluxes following incubation in radiolabelled substrates. As a first experiment the GC-MS method has been applied to compare the metabolism of wild type and well-characterized transgenic potato tubers exhibiting an enhanced sucrose mobilization. The fact that this method is able to rapidly yield further comprehensive information into primary metabolism illustrates its power as a further phenotyping tool for the analysis of plant metabolism.     

Pyrophosphorylases in Solanum tuberosum (IV. Purification,Tissue Localization,and Physicochemical Properties of UDP-Glucose Pyrophosphorylase)

The enzyme UDP-glucose pyrophosphorylase (UGPase) from potato (Solanum tuberosum L. cv Norchip) tubers was purified 177-fold to near homogeneity and to a specific activity of 1099 international units/mg of protein. The molecular mass of the purified enzyme was 53 kD as determined by SDS-PAGE and gel filtration. Immunological and activity assays detected UGPase at similar levels in potato stems, stolons, and tubers. Leaves and roots contained lower levels of UGPase activity and protein. Lineweaver-Burk plots for substrates inorganic pyrophosphate and UDP-glucose were linear in the pyrophosphorolytic direction, yielding Km values of 0.13 and 0.14 mM, respectively. However, Lineweaver-Burk plots for the substrates glucose-1-P and UTP were biphasic in nature when UGPase was assayed in the direction of UDP-glucose synthesis. At physiological substrate concentrations (i.e. from 0.05-0.20 mM), Km values of 0.08 mM (glucose-1-P) and 0.12mM (UTP) were obtained. When substrate concentrations increased above 0.20 mM, Km values increased to 0.68 mM (glucose-1-P) and 0.53 mM (UTP). These kinetic patterns of potato UGPase suggest a "negative cooperative effect" (A. Conway, D.E. Koshland, Jr. [1968] Biochemistry 7: 4011-4022) with respect to the substrates glucose-1-P and UTP. The biphasic substrate saturation curves were similar to the kinetics of the dimeric form of UGPase purified from Salmonella typhimurium (T. Nakae [1971] J Biol Chem 246: 4404-4411). The in vivo significance of the enzyme's "negative cooperativity" in the direction of UDP-glucose synthesis and potato sweetening is discussed.     

Effect of elicitors on accumulation of protease inhibitors in injured potato tubers

The time course of accumulation and the composition of proteinase-inhibiting proteins in diffusates from potato tubers treated with elicitors such as salicylic, jasmonic, and arachidonic acids were studied. The 40-kDa reserve protein patatin and the chymotrypsin inhibitors, among which proteins of 24.6, 22.0, and 16.0 kDa were prevalent, accumulated in diffusates from potato tubers. Jasmonic and arachidonic acids activated the accumulation of the chymotrypsin inhibitors in tubers in response to the injury stress, whereas salicylic acid inhibited this process. The effects of jasmonic and arachidonic acids increased when their concentrations decreased to 10(-6) M. The data suggest an important role of the lipoxygenase metabolism in signal transduction of the anti-injury defense system in the dormant potato tubers.     

Metabolite profiling and assessment of metabolome compartmentation of soybean leaves using non-aqueous fractionation and GC-MS analysis

In the present study, non-aqueous fractionation (NAQF) and GC-MS were used to obtain a spatially resolved view of metabolism in mature leaves of soybean (Glycine max Merr.). NAQF of lyophilized soybean leaves was performed using CCl₄-n-heptane and ultracentrifugation that yielded a gradient comprised of six fractions. Chlorophyll content, and marker enzyme activities, phosphoenolpyruvate carboxylase (PEPC) and α-mannosidase, were utilized as stroma, cytosol and vacuole markers, respectively. GC-MS analyses of each fraction resulted in the identification of around 100 different metabolites. The distribution of these identified compounds showed a decreasing order from the vacuole to cytosol to chloroplast stroma. In other words, a greater number of identified compounds were found in the vacuole when compared to the cytosol or stroma. Levels of sugars, organic acids and fatty acids showed greater relative abundances in the vacuole with 50, 55, and 50% of the respective pools. A greater relative abundance of amino acids was observed in the cytosol where 45% of the total of amino acids content was recorded. The relatively large pool of sugars and phenolic acids in the vacuole compartment implies high levels of starch metabolism and phenylpropanoid biosynthesis. The low amino acids pool, on the other hand, suggests low nitrogen accumulation in the leaves of soybean. Hierarchical cluster analysis on the most abundant metabolites revealed three clusters containing 10, 20, and 2 of the 32 selected metabolites. The data were discussed in term of NAQF and GC-MS analysis of soybean mature leaves, and also in term of distribution and compartmentation of metabolites at subcellular levels.     

Amino Acid and sucrose content determined in the cytosolic, chloroplastic, and vacuolar compartments and in the Phloem sap of spinach leaves

Amino acid and sucrose contents were analyzed in the chloroplastic, cytosolic, and vacuolar compartments and in the phloem sap of illuminated spinach leaves (Spinacia oleracea L.). The determination of subcellular metabolite distribution was carried out by nonaqueous fractionation of frozen and lyophilized leaf material using a novel three-compartment calculation method. The phloem sap was collected by aphid stylets which had been severed by a laser beam. Subcellular analysis revealed that the amino acids found in leaves are located mainly in the chloroplast stroma and in the cytosol, the sum of their concentrations amounting to 151 and 121 millimolar, respectively, whereas the amino acid concentrations in the vacuole are one order of magnitude lower. The amino acid concentrations in the phloem sap are found to be not very different from the cytosolic concentrations, whereas the sieve tube concentration of sucrose is found to be one order of magnitude higher than in the cytosol. It is concluded that the phloem loading results in a preferential extraction of sucrose from the source cells.     

Metabolic and developmental adaptations of growing potato tubers in response to specific manipulations of the adenylate energy status

Heterotrophic carbon metabolism has been demonstrated to be limited by oxygen availability in a variety of plant tissues, which in turn inevitably affects the adenylate status. To study the effect of altering adenylate energy metabolism, without changing the oxygen supply, we expressed a plastidially targeted ATP/ADP hydrolyzing phosphatase (apyrase) in tubers of growing potato (Solanum tuberosum) plants under the control of either inducible or constitutive promoters. Inducible apyrase expression in potato tubers, for a period of 24 h, resulted in a decrease in the ATP-content and the ATP-ADP ratio in the tubers. As revealed by metabolic profiling, this was accompanied by a decrease in the intermediates of sucrose to starch conversion and several plastidially synthesized amino acids, indicating a general depression of tuber metabolism. Constitutive tuber-specific apyrase expression did not lead to a reduction of ATP, but rather a decrease in ADP and an increase in AMP levels. Starch accumulation was strongly inhibited and shifted to the production of amylopectin instead of amylose in these tubers. Furthermore, the levels of almost all amino acids were decreased, although soluble sugars and hexose-Ps were highly abundant. Respiration was elevated in the constitutively expressing lines indicating a compensation for the dramatic increase in ATP hydrolysis. The increase in respiration did not affect the internal oxygen tensions in the tubers. However, the tubers developed a ginger-like phenotype having an elevated surface-volume ratio and a reduced mass per tuber. Decreased posttranslational redox activation of ADP-glucose pyrophosphorylase and a shift in the ratio of soluble starch synthase activity to granule-bound starch synthase activity were found to be partially responsible for the alterations in starch structure and abundance. The activity of alcohol dehydrogenase was decreased and pyruvate decarboxylase was induced, but this was neither reflected by an increase in fermentation products nor in the cellular redox state, indicating that fermentation was not yet induced in the transgenic lines. When taken together the combined results of these studies allow the identification of both short- and long-term adaptation of plant metabolism and development to direct changes in the adenylate status.     

Isotope labelling of Rubisco subunits provides in vivo information on subcellular biosynthesis and exchange of amino acids between compartments

The architecture of plant metabolism includes substantial duplication of metabolite pools and enzyme catalyzed reactions in different subcellular compartments. This poses challenges for understanding the regulation of metabolism particularly in primary metabolism and amino acid biosynthesis. To explore the extent to which amino acids are made in single compartments and to gain insight into the metabolic precursors from which they derive, we used steady state (13) C labelling and analysed labelling in protein amino acids from plastid and cytosol. Ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco) is a major component of green tissues and its large and small subunits are synthesized from different pools of amino acids in the plastid and cytosol, respectively. Developing Brassica napus embryos were cultured in the presence of [U-(13) C]-sucrose, [U-(13) C]-glucose, [U-(13) C]-glutamine or [U-(13) C]-alanine to generate proteins. The large subunits (LSU) and small subunits (SSU) of Rubisco were isolated and the labelling in their constituent amino acids was analysed by gas chromatography-mass spectrometry. Amino acids including alanine, glycine and serine exhibited different (13) C enrichment in the LSU and SSU, demonstrating that these pools have different metabolic origins and are not isotopically equilibrated between the plastid and cytosol on the time scale of cellular growth. Potential extensions of this novel approach to other macromolecules, organelles and cell types of eukaryotes are discussed.