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First published online August 27, 2004; 10.1104/pp.104.046854 Plant Physiology 136:2676-2686 (2004) © 2004 American Society of Plant Biologists Embryo-Specific Reduction of ADP-Glc Pyrophosphorylase Leads to an Inhibition of Starch Synthesis and a Delay in Oil Accumulation in Developing Seeds of Oilseed Rape1Max Planck Institute of Molecular Plant Physiology, Am Mühlenberg 1, 14476 Golm, Germany (H.V., P.G.); University of Kaiserslautern, 67653 Kaiserslautern, Germany (T.M., H.E.N.); and Max Planck Institute for Breeding Research, 50829 Cologne, Germany (N.M.)
In oil-storing Brassica napus (rape) seeds, starch deposition occurs only transiently in the early stages of development, and starch is absent from mature seeds. This work investigates the influence of a reduction of ADP-Glc pyrophosphorylase (AGPase) on storage metabolism in these seeds. To manipulate the activity of AGPase in a seed-specific manner, a cDNA encoding the small subunit of AGPase was expressed in the sense or antisense orientation under the control of an embryo-specific thioesterase promoter. Lines were selected showing an embryo-specific decrease in AGPase due to antisense and cosuppression at different stages of development. At early developmental stages (25 days after flowering), a 50% decrease in AGPase activity was accompanied by similar decreases in starch content and the rate of starch synthesis measured by injecting 14C-Suc into seeds in planta. In parallel to inhibition of starch synthesis, the level of ADP-Glc decreased, whereas Glc 1-phosphate levels increased, providing biochemical evidence that inhibition of starch synthesis was due to repression of AGPase. At 25 days after flowering, repression of starch synthesis also led to a decrease in the rate of 14C-Suc degradation and its further metabolism via other metabolic pathways. This was not accompanied by an increase in the levels of soluble sugars, indicating that Suc import was inhibited in parallel. Flux through glycolysis, the activities of hexokinase, and inorganic pyrophosphate-dependent phosphofructokinase, and the adenylate energy state (ATP to ADP ratio) of the transgenic seeds decreased, indicating inhibition of glycolysis and respiration compared to wild type. This was accompanied by a marked decrease in the rate of storage lipid (triacylglycerol) synthesis and in the fatty acid content of seeds. In mature seeds, glycolytic enzyme activities, metabolite levels, and ATP levels remained unchanged, and the fatty acid content was only marginally lower compared to wild type, indicating that the influence of AGPase on carbon metabolism and oil accumulation was largely compensated for in the later stages of seed development. Results indicate that AGPase exerts high control over starch synthesis at early stages of seed development where it is involved in establishing the sink activity of the embryo and the onset of oil accumulation.
Starch is the major storage carbohydrate in most plants, with many important functions. Starch accumulates in leaves during the day and is remobilized at night to support continued Suc synthesis, export, and respiration (Caspar et al., 1985  ; Geiger and Servaites, 1994; Geiger et al., 1995). Starch accumulates in plants when they grow slowly, for example due to nutrient deficiency or low temperature, and can be remobilized when conditions become more favorable or the plant enters the reproductive phase (Schulze et al., 1991; Stitt and Schulze, 1994). Large amounts of starch are accumulated in the stems and roots of overwintering biennials and perennials, in vegetative storage organs such as potato (Solanum tuberosum) tubers, and in seeds.
In oil seeds such as rape (Brassica napus) and Arabidopsis, starch deposition occurs only transiently in the early stages of embryo development, accounting for approximately 8% to 10% of the dry weight, and is absent in mature seeds (Da Silva et al., 1997
Despite these advances in the characterization of the route of Suc-starch conversion, we know little about the metabolic regulation of this pathway in oil seeds. Furthermore, the role of transient starch accumulation in these seeds and the factors leading to the developmental shift from starch to lipid storage during seed filling has not yet been defined. It has been proposed that starch synthesis competes with lipid synthesis and therefore restricts oil synthesis in seeds (Bettey and Smith, 1990 This study investigates the effect of a reduction in AGPase on storage metabolism in developing rape seeds. To manipulate the activity of AGPase in a seed-specific manner, a cDNA encoding the small subunit of AGPase was expressed in the sense or antisense orientation under the control of an embryo-specific thioesterase promoter. This promoter confers strong expression in developing embryos (N. Martini, unpublished data). Lines were selected showing an embryo-specific decrease in AGPase activity due to antisense or cosuppression. To investigate the influence of a reduction in AGPase activity on seed metabolism, we analyzed (1) the starch content of the seeds; (2) the metabolism of 14C-Suc injected into the seeds; (3) the levels of metabolites involved in starch, lipid, and energy metabolism; and (4) the rate of lipid accumulation during seed development. Our results show that AGPase exerts a high degree of control over starch and lipid synthesis in developing rape seeds mainly during early developmental stages.
Generation of Plants with Decreased Expression of AGPase
Hypocotyl segments from oilseed rape plants (cv Drakkar) were transformed with either an antisense or a sense construct containing a sequence encoding the small subunit of AGPase isolated from an embryo-specific oilseed rape cDNA library (Elborough et al., 1994)
Decreased Expression of AGPase and Decreased Starch Are Both Restricted to the Embryo The influence of decreased expression of AGPase on starch synthesis was investigated in more detail in two representative lines, line 43 (antisense) and line 97 (cosuppression), which showed the strongest decrease in AGPase activity. In these lines, overall AGPase mRNA levels (see legend to Fig. 1), AGPase activity (Fig. 1A), and starch content (Fig. 1B) of the seeds was reduced down to 50% of the wild-type level at early developmental stages (25 and 30 days after flowering [DAF]). The data of Figure 1 are expressed per seed. Similar changes were observed when the data were expressed on a fresh weight basis: At 25 DAF, AGPase activities (in nmol gFW–1 min–1) were 292 ± 33, 199 ± 11, and 156 ± 18 and starch content (in µmol gFW–1) 129 ± 2, 65 ± 2, and 60 ± 4 in wild type, line 43 and line 97, respectively. At 30 DAF, AGPase activities (in nmol gFW–1 min–1) were 200 ± 29, 101 ± 19, and 107 ± 33 and starch content (in µmol gFW–1) 73 ± 3, 55 ± 1, and 34 ± 1 in wild type, line 43 and line 97, respectively. During seed maturation, AGPase activity decreased markedly in the wild type, and there was a further decrease in the transgenic lines (Fig. 1A). In this late developmental stage (55 DAF), starch decreased to extremely low levels and was barely detectable in seeds of the wild-type and the transgenic lines (Fig. 1B).
At 25 DAF, most of the AGPase activity was found in the embryo (more than 90%), and only marginal activity was found in the testa and endosperm (Fig. 1C). Compared to wild type, AGPase activity in the transgenic lines was reduced to 50% in the embryo, whereas no significant change was observed in testa and endosperm (Fig. 1C). Similar results were observed when the data were expressed on a fresh weight basis (nmol gFW–1 min–1), with AGPase activities in the embryo being 622 ± 88, 418 ± 76, and 325 ± 39, and in the testa (including endosperm) being 51 ± 21, 62 ± 11, and 90 ± 58 in wild type, line 43 and line 97, respectively. This shows that the reduction in AGPase expression was indeed embryo-specific. Furthermore, starch was mainly recovered in the embryo, and significant decreases in starch content due to reduction of AGPase were only seen in the embryo and not in the testa or endosperm (Fig. 1D).
To investigate the effect of decreased AGPase activity on metabolic fluxes in seeds at 25 DAF, 14C-Suc was injected into seeds, which otherwise remained intact within their siliques. Seeds were harvested 28 h later to investigate the fate of the label (Fig. 2). After this time, approximately 90% of the injected Suc was recovered in the embryo where it was mainly incorporated into storage products (data not shown). This in planta labeling method provides a minimally invasive technique to study the metabolism of labeled precursors within developing seeds (Vigeolas et al., 2003
The 14C-Suc taken up into the cells will mix with internal unlabeled pools of Suc, so movement of the label will not necessarily reflect fluxes into the various pools. This is especially true in the case of Suc, since Suc represents a very large internal pool in these seeds (approximately 120 µmol gFW–1, see below). To correct for this, the specific activity of the hexose-phosphate pool was calculated by dividing the amount of label recovered in phosphoesters (Fig. 2E) by the total carbon found in the hexose-phosphate pool (see Geigenberger et al., 1997 , for a discussion of the assumptions involved in these calculations). The specific activity of the hexose-phosphate pool was higher in the transgenic lines than in the wild type, with values of 77 ± 14, 115 ± 8, and 135 ± 24 dpm per nmol for wild type, line number 43, and line number 97, respectively (data are means ± SE; n = 4). This indicates that reduction of AGPase leads to less dilution of the label by internal unlabeled pools and implies that starch or sugar degradation might have been decreased in the transgenic lines. To estimate the absolute rate of starch synthesis, the amount of label in the starch fraction was divided by the specific activity of the phosphoester pool. Reduction of AGPase activity led to a significant decrease in the estimated rate of starch synthesis, being 2-fold lower in line 43 and 3-fold lower in line 97 (Fig. 3C). There was a similar decrease in the estimated rate of lipid synthesis (Fig. 3D), which was reflected by a decreased flux to TAG (Fig. 3E) and DAG (Fig. 3F), and a smaller decrease in the estimated flux to organic and amino acids (Fig. 3A), which was calculated as the sum of label in these fractions divided by the specific activity of the phosphoester pool. Although the rate of CO2 evolution has not been measured, labeling of organic and amino acids provides a rough estimate of the flux into glycolytic pathways. In contrast, the estimated flux to structural components (cell wall plus protein; Fig. 3B) was not significantly changed.
Calculation of the sum of the estimated fluxes to organic and amino acids, starch, lipids, and structural elements (protein and cell wall) using the data in Figure 3 resulted in values of 7.09 ± 0.74, 4.03 ± 0.36, and 2.86 ± 0.41 for wild type, line number 43, and line number 97, respectively (in nmol Suc seed–1 h–1; values are means ± SE, n = 4). As these are the major fluxes in seeds, their sum provides an estimate of the net rate of Suc breakdown. The combined flux reflecting net Suc degradation is severely decreased in response to reduction in AGPase.
To investigate why starch and lipid synthesis were inhibited, metabolite levels were measured in the seeds at 25 DAF (Fig. 4). Suc (Fig. 4A) was the main soluble sugar in these seeds, with hexoses being only a negligible component of the total sugar pool (Glc as well as Fru represented only 1.6% of the total sugar pool, data not shown). Decreased expression of AGPase did not lead to significant changes in the level of Suc in seeds (Fig. 4A). Interestingly, sugars stayed the same even though the rate of Suc degradation and metabolic fluxes to starch and lipids decreased (see Figs. 2 and 3). This implies that Suc import had been inhibited. Inhibition of starch synthesis was accompanied by an increase in the level of Glc1P (Fig. 4D) and a decrease in the level of ADPGlc (Fig. 4E), which are the immediate substrate and the immediate product of AGPase, respectively. This provides independent biochemical evidence that AGPase was indeed the step at which starch synthesis was inhibited. Inhibition of lipid synthesis was accompanied by an increase in the level of acetyl-CoA (Fig. 4F), which is the direct precursor for fatty acid synthesis in the plastid, whereas glycerol3-P (Fig. 4B), which is a precursor for TAG synthesis did not change significantly. This would suggest that the inhibition of lipid synthesis is not due to a general restriction in carbon-precursor supply and indicates that one or more of the steps using hexose-phosphates or acetyl-CoA for fatty acid synthesis had been inhibited.
Starch and fatty acid synthesis are both dependent upon provision of ATP from the cytosol (Rawsthorne, 2002
Influence of Decreased AGPase on Enzyme Activities in Seeds
To investigate whether the inhibition of Suc degradation and lipid synthesis in seeds with decreased expression of AGPase is attributable to an inhibition of enzymes involved in Suc degradation and fatty acid synthesis, we measured the maximal activities of invertase, SuSy, UDP-Glc pyrophosphorylase (UGPase), hexokinase, inorganic pyrophosphate (PPi)-dependent phosphofructokinase (PFK), and ACCase. Since these activities can show marked changes during development (King et al., 1997
Influence of Decreased AGPase on Lipid Accumulation and Fatty Acid Composition in Seeds
The total lipid content of developing seeds at different developmental stages was investigated by gas chromatography analysis of fatty-acid methyl esters (Fig. 7). In the wild type, lipid content of seeds increased from 150 µg seed–1 at 25 DAF to 480 µg seeds–1 at 35 DAF and plateaued at 790 µg seed–1 in mature seeds (55 DAF). Reduction of AGPase led to a decreased lipid content of the seeds, the decrease being more marked (down to 50% of wild-type level) at 25 and 30 DAF than at later stages of development. In mature seeds, the lipid content was only slightly lower in lines with decreased AGPase than in wild type. There were no large changes in fatty acid composition between mature wild-type and transgenic seeds (Table II). There was a slight increase in the relative amounts of the end products of desaturation such as 18:3 (
An Embryo-Specific Decrease in AGPase Leads to a Strong Inhibition of Starch Biosynthesis in Developing Seeds of Oilseed Rape
To investigate the role of AGPase in developing oil seeds, transgenic oilseed rape plants were generated with decreased expression of AGPase using an embryo-specific promoter. Several independent lines were established and propagated up to the T3 generation showing an embryo-specific decrease in AGPase activity. In seeds at 25 DAF, a 50% decrease in AGPase activity led to a 50% to 60% decrease in the rate of starch synthesis analyzed by injecting 14C-Suc into seeds in planta and an approximately 50% to 70% decrease in starch content. Inhibition of starch synthesis was accompanied by a decrease in the level of ADPGlc, the immediate product of AGPase and the direct precursor of starch, whereas the immediate substrate of AGPase, Glc1P, increased. This metabolic crossover provides unequivocal biochemical evidence that the inhibition of starch synthesis was due to inhibition of AGPase. These data show that AGPase exerts high control over the rate of starch synthesis in developing oilseed rape embryos, with flux-control coefficients estimated to be close to 1.0. Interestingly, in legume seeds such as pea (Pisum sativum; Denyer et al., 1995
The reason for the different levels of control of flux exerted by AGPase in different tissues remains to be determined. It could be related to variations in overall AGPase expression or AGPase regulation. In leaves (Hendriks et al., 2003
Determinations of flux control coefficients are often based on the analysis of overall starch levels, neglecting the possibility that starch degradation and synthesis could operate in parallel (Geigenberger et al., 2004
Our results show that the repression of starch synthesis also affected other metabolic processes in the seeds at 25 and 30 DAF but not in mature seeds. Unexpectedly, the estimated flux through glycolysis (Fig. 3A), glycolytic enzyme activities (Fig. 6), and ATP levels (Fig. 4) decreased in seeds with decreased expression of AGPase, indicating that glycolysis and respiration were inhibited compared to wild type. Inhibition of glycolysis was not due to a restriction in overall carbon precursor supply since hexose-phosphate levels were high in the transformants (Fig. 4). It is more likely that one or more steps utilizing hexose-phosphates for glycolysis and respiration have been inhibited. The lower maximal activity of PPi-dependent PFK in the transformants could provide a possible explanation for the inhibition of glycolysis. It is known from earlier studies in wild type that the activities of glycolytic enzymes, specifically PPi-dependent PFK, increase during seed filling (Focks and Benning, 1998
Interestingly, the inhibition of glycolysis was accompanied by a decrease in the rate of storage lipid synthesis in the transformants at early stages of seed development (Fig. 3). This is consistent with previous studies on a low-seed-oil Arabidopsis mutant where decreased lipid accumulation was attributed to a decrease in glycolytic enzyme activities (mainly hexokinase and PPi-dependent PFK) in the seeds (Focks and Benning, 1998
Previous studies of Da Silva et al. (1997)
The results of this paper provide evidence that AGPase is specifically important in the early stages of oil seed development where the enzyme has a high control coefficient for starch synthesis and is involved in establishing the sink activity of the embryo and the onset of storage lipid accumulation. There is no substantial influence on carbon metabolism and lipid content in mature seeds, which makes it unlikely that starch serves as an important carbon source for lipid or Suc synthesis during the embryo maturation process.
Plant Material Spring rape seed plants (Brassica napus cv Drakkar) were grown in a phytotron (25°C day and 20°C night) with a 16-h photoperiod at an irradiance of 300 µmol photons m–2 s–1. Emerging flowers were tagged, and seed age was expressed in DAF. If no developmental stage is indicated in the text, then experiments were performed with seeds at the age of 25 DAF when the seed diameter was about 3 mm and the lipid content was approximately 150 µg/seed (see Fig. 7). All of these experiments were done in the middle of the light period.
Unless stated otherwise, chemicals were obtained from Sigma (Taufkirchen, Germany) or Merck (Darmstadt, Germany).
Total RNA was extracted from seeds by using the RNA plant reagent of Invitrogen GmbH (Karlsruhe, Germany), and AGPase mRNA levels were analyzed according to Kossmann et al. (1999)
Total lipids of developing seeds at different developmental stages were extracted according to the method of Bligh and Dyer (1959)
Starch content was measured as described in Geigenberger et al. (1998)
Using a 5-µL Hamilton syringe (needle diameter 470 µm), 0.5 µL of a solution containing 115 µM [U-14C]Suc (Amersham-Buchler, Freiburg, Germany; specific activity 22.8 MBq µmol–1) in 20 mM MES buffer (pH 5.7) was injected directly into seeds as described in Vigeolas et al. (2003)
Seeds were manually separated from the silique wall under liquid N2. For each replicate, five seeds were pooled and ground to a fine powder in liquid nitrogen using a ball mill (Retsch Schwingmühle M200, Haan, Germany). Using the extraction method described in Bligh and Dyer (1959)
Siliques were rapidly frozen in liquid nitrogen and seeds separated from silique walls under liquid nitrogen. Metabolite and nucleotide levels were analyzed in TCA extracts as described in Jelitto et al. (1992) Sequence data from this article have been deposited with the EMBL/GenBank data libraries under accession number AJ271162.
We gratefully acknowledge Jaqueline Bautor (MPIZ, Cologne) for her skillful assistance in rapeseed transformation, Britta Hausmann and Karin Köhl (MPIMP, Golm) for greenhouse work, Anja Fröhlich (MPIMP, Golm) for excellent technical assistance, Peter Dörmann (MPIMP, Golm) for providing the GC facilities, and John E. Lunn (MPIMP, Golm) for critical reading of the manuscript. Received May 24, 2004; returned for revision June 21, 2004; accepted June 21, 2004.
1 This work was supported by the Deutsche Forschungsgemeinschaft (grant no. Ge 878/1–3 to P.G.) and by the Bundesministerium für Ernährung, Landwirtschaft u. Forsten through the Fachagentur Nachwachsende Rohstoffe (to H.E.N., T.M.). 
2 These authors contributed equally to the paper. Article, publication date, and citation information can be found at www.plantphysiol.org/cgi/doi/10.1104/pp.104.046854. * Corresponding author; e-mail geigenberger{at}mpimp-golm.mpg.de; fax 49–331–567–8408.
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Vigeolas H, van Dongen JT, Waldeck P, Hühn D, Geigenberger P (2003) Lipid metabolism is limited by the prevailing low oxygen concentrations within developing seeds of oilseed rape. Plant Physiol 133: 2048–2060 This article has been cited by other articles:
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-linolenic acid), whereas the relative amount of 18:1 (oleic acid), which is an intermediate of desaturation, was slightly decreased in the transformants. 
