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. 2004 Sep;136(1):2676–2686. doi: 10.1104/pp.104.046854

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 Rape1

Helene Vigeolas 1,2, Torsten Möhlmann 1,2, Norbert Martini 1, H Ekkehard Neuhaus 1, Peter Geigenberger 1,*
PMCID: PMC523332  PMID: 15333758

Abstract

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; Focks and Benning, 1998). Recent research has clarified the route of starch synthesis in developing rape seed embryos (Rawsthorne, 2002). Incoming Suc is degraded via two different pathways involving invertase or Suc synthase (SuSy) to produce hexose-phosphates (Da Silva et al., 1997). The hexose-phosphates then enter the plastid via a Glc6P/Pi-translocator (Da Silva et al., 1997). Within the plastid, Glc6P is metabolized to Glc1P by phosphoglucomutase (Caspar et al., 1985; Periappuram et al., 2000) followed by the ATP-dependent conversion of Glc1P to ADPGlc by ADPGlc pyrophosphorylase (AGPase) and the subsequent incorporation of the Glc moiety into the starch granule by starch synthases (Kang and Rawsthorne, 1994; 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). Alternatively, starch could serve as an important carbon source required to sustain the high rates of lipid synthesis during rapid oil deposition (Norton and Harris, 1975) or sugar synthesis to achieve desiccation tolerance (Leprince et al., 1990) in later stages of seed development. Finally, Da Silva et al. (1997) suggested that the early development of a capacity for starch synthesis may contribute to the establishment of the embryo as a sink organ prior to the onset of lipid synthesis. To address this question, a plastidial phosphoglucomutase mutant of Arabidopsis, which lacks starch, was studied (Caspar et al., 1985; Periappuram et al., 2000). Interestingly, seeds of the mutant accumulated 40% less oil compared to the wild type, indicating that starch is in some way positively linked to oil accumulation in seeds (Periappuram et al., 2000). However, interpretation of these results is limited by the lack of information about changes in seed metabolism and possible pleiotropic effects due to reduction of phosphoglucomutase in leaves and other tissues of the mutant.

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.

RESULTS

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), essentially as described by de Block et al. (1989). The cDNA was expressed under the control of a thioesterase promoter from Cuphea lancelota, ClFatb4, identified to be embryo-specific in transgenic oilseed rape (G. Hemmann, J. Bautor, and N. Martini, unpublished data). After regeneration of plants, transformation was verified in several lines via PCR and resistance against glufosinate-ammonium. Lines were selected showing a 10% to 50% decrease in AGPase activity in developing embryos due to antisense and cosuppression, and this was accompanied by a 40% to 70% decrease in starch content (Table I).

Table I.

AGPase activity and starch content in embryos of transgenic rape seeds (T3-generation) with decreased expression of AGPase due to antisense or cosuppression (AGPase-sense)

Line Construct AGPase Activity Starch Content
nmol min−1 embryo−1 nmol C-6-units embryo−1
Wild type – 0.80 ± 0.20 600 ± 90
No. 25 AGPase-sense 0.78 ± 0.06 370 ± 15*
No. 88 AGPase-sense 0.60 ± 0.21 310 ± 62*
No. 97 AGPase-sense 0.48 ± 0.09 130 ± 79*
No. 43 AGPase-antisense 0.59 ± 0.14 320 ± 20*
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Results are means ± se (n = 3–5) and refer to a developmental stage of 25 to 30 DAF. Significant changes from the wild type are marked with an asterisk (P < 0.05 using the Student's t test). –, Not applicable.

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).

Figure 1.

Figure 1.

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Decreased expression of AGPase in rape seed embryos leads to decreased AGPase activity and starch content. At 25, 30, and 55 DAF, whole seeds from the lines number 43 (dark-gray bars) and number 97 (light-gray bars) and from wild type (black bars) were analyzed for AGPase activity (A) and starch content (B). In a separate experiment, seeds at 25 DAF were dissected to analyze AGPase activity (C) and starch content (D) in embryo and testa, separately. The dissected testa also included the endosperm. Small subunit AGPS1-mRNA levels of whole seeds were also analyzed by northern-blot analysis. The relative signals for AGPS1 transcript were analyzed using a phospho-imager and were 79% ± 2% and 59% ± 5% of wild-type level in lines number 43 and number 97, respectively. Results are means ± se (n = 6). Significant changes from the wild type are marked with an asterisk (P < 0.05 using the Student's t test). MS indicates mature seeds and refers to 55 DAF.

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).

Influence of Decreased Expression of AGPase on Metabolism of 14C-Suc Injected into Seeds

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). In parallel experiments, we checked that this manipulation did not significantly alter subsequent growth and lipid accumulation of the seeds when compared to untreated controls (data not shown). In the wild type, more than 50% of the injected 14C-Suc was metabolized (Fig. 2, A and B), and most of the label was converted to lipids (18%; Fig. 2G), starch (15%; Fig. 2H), and structural elements (protein and cell wall; 6%; Fig. 2F). Most of the label in lipids was recovered in triacylglycerol (TAG; 10%; Fig. 2I) and to a lesser extent in diacylglycerol (DAG; 2.5%; Fig. 2J). In lines number 43 and number 97 with reduced AGPase activity, less of the injected label was metabolized and converted to lipids (including TAG and DAG) or starch (Fig. 2, G–J), whereas percentage incorporation of label into amino acids, organic acids, and structural elements (protein and cell walls) was not substantially changed (Fig. 2, C, D, and F). More label was retained in the sugar pool (Fig. 2A), which is consistent with an inhibition of Suc degradation.

Figure 2.

Figure 2.

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Decreased expression of AGPase affects metabolism of [U-14C]Suc injected directly into seeds. To determine the influence of decreased AGPase on metabolic fluxes, 0.5 μL of a buffered solution containing [U-14C]Suc was injected into seeds (25 DAF), which remained otherwise intact within their siliques. After 28 h, siliques were frozen in liquid N2 and seeds separated from the silique wall and extracted to determine distribution of label into different fractions. A, Label remaining in sugars. B, Percentage of injected 14C-Suc that was metabolized. C to J, Percentage of injected label metabolized to amino acids (C), organic acids (D), phosphoesters (E), structural components (protein plus cell wall; F), total lipids (G), starch (H), TAG (I), and DAG (J). The results are means ± se (n = 4–5 separate siliques from different plants). Significant changes from the wild type are marked with an asterisk (P < 0.05 using the Student's t test).

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.

Figure 3.

Figure 3.

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Decreased expression of AGPase affects metabolic fluxes in developing seeds at 25 DAF. The specific activity of the hexose-phosphate pool (see below) and label incorporation into the relevant fractions (see Fig. 2) was used to calculate absolute rates for metabolic fluxes in seeds. The specific activity of the hexose-phosphate pool was calculated by dividing the label retained in phosphoesters (Fig. 2E) by the total carbon of the hexose phosphate pool (see Fig. 4). The values were 77 ± 14, 115 ± 7, and 135 ± 24 dpm nmol−1 for wild type, line 43 and line 97, respectively. A, Glycolytic flux (the sum of the flux to the organic acids and amino acids). B, Flux into structural components (cell wall plus protein). C, The rate of starch synthesis. D, Rate of total lipid synthesis. E, Rate of TAG synthesis. F, Rate of DAG synthesis. The results are means ± se (n = 4–5 separate siliques from different plants). Significant changes from the wild type are marked with an asterisk (P < 0.05 using the Student's t test).

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.

Influence of Decreased AGPase on Metabolite Levels in Seeds

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.

Figure 4.

Figure 4.

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Influence of decreased expression of AGPase on metabolite levels and energy state in developing seeds at 25 DAF. Siliques were rapidly frozen in liquid nitrogen and the seeds separated from the silique wall under liquid N2 to analyze metabolite levels. A, Suc. B, Gly3P. C, Glc6P. D, Glc1P. E, ADPGlc. F, Acetyl-CoA. G, ATP. H, ADP levels. The ratio of ATP to ADP (I) is also shown. The results are means ± se (n = 4–6 separate siliques from different plants). Significant changes from the wild type are marked with an asterisk (P < 0.05 using the Student's t test).

Influence of Decreased AGPase on Energy State in Seeds

Starch and fatty acid synthesis are both dependent upon provision of ATP from the cytosol (Rawsthorne, 2002). An inhibition of starch synthesis might therefore be expected to lead to an increase in the availability of ATP for lipid synthesis. The levels of ATP and ADP in seeds of the transgenic lines at 25 DAF are shown in Figure 4, G and H. Decreased expression of AGPase led to an unexpected decrease in the level of ATP (Fig. 4G), whereas ADP (Fig. 4H) was not significantly changed. Thus the ATP to ADP ratio decreased 2-fold in seeds with reduced AGPase activity (Fig. 4I) and could have led to a restriction of acetyl-CoA carboxylase (ACCase) and other ATP-dependent enzymes in vivo at this developmental stage. Decreased expression of AGPase led to a similar decrease in ATP level in seeds at 30 DAF, whereas no significant changes were observed when mature seeds (55 DAF) were compared (Fig. 5A). The levels of Glc6P (Fig. 5B) and Suc (Fig. 5C) are shown for comparison. Irrespective of the developmental stage investigated, neither Glc6P nor Suc showed any significant changes compared to wild type. This indicates that AGPase is unlikely to be involved to determine the sugar levels in maturated seeds.

Figure 5.

Figure 5.

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Influence of decreased expression of AGPase on ATP, Glc6P, and Suc levels during seed development. At 25, 30, and 55 DAF (the latter representing mature seeds), siliques were harvested and seeds separated as described in Figure 4 to prepare extracts for measurement of ATP (A), Glc6P (B), and Suc (C) levels in wild type (black bars), line number 43 (dark-gray bars), and line number 97 (light-gray bars). The results are means ± se (n = 4–5 separate siliques from different plants). Significant changes from the wild type are marked with an asterisk (P < 0.05 using the Student's t test).

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; Focks and Benning, 1998), different developmental stages of the seeds were investigated (Fig. 6). At 25 DAF, reduction in AGPase led to a 40% increase in alkaline (Fig. 6A) and acid invertase activity (data not shown), whereas UGPase activity remained unchanged (Fig. 6B) and SuSy activity decreased by 40% in the strongest repressed line, number 97 (data not shown). Interestingly, glycolytic enzyme activities such as hexokinase (Fig. 6C) and PPi-dependent PFK (Fig. 6D) were significantly decreased compared to wild type, which is consistent with the decrease in the estimated rate of glycolytic flux (see Fig. 3A). In contrast, the maximal activity of ACCase (Fig. 6E), representing the key enzyme of fatty acid synthesis using acetyl-CoA as a direct substrate, was not significantly changed compared to wild type. Similar results were observed at 30 DAF, whereas in mature seeds (55 DAF) no significant changes in the investigated enzyme activities were observed between wild-type and the transgenic lines.

Figure 6.

Figure 6.

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Influence of decreased expression of AGPase on enzyme activities in developing seeds. Siliques were harvested at 25, 30, and 55 DAF (the latter is equivalent to maturated seeds) and seeds separated as described in Figure 4 to prepare extracts for measurement of enzyme activities in wild type (black bars), line number 43 (dark-gray bars), and line number 97 (light-gray bars). A, Alkaline invertase. B, UGPase. C, Hexokinase. D, PPi-dependent PFK. E, ACCase. The results are means ± se (n = 4–5 separate siliques from different plants). Significant changes from the wild type are marked with an asterisk (P < 0.05 using the Student's t test).

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 (α-linolenic acid), whereas the relative amount of 18:1 (oleic acid), which is an intermediate of desaturation, was slightly decreased in the transformants.

Figure 7.

Figure 7.

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Decreased expression of AGPase affects oil accumulation during seed development. At different time points between 25 DAF and maturity (55 DAF), seeds were harvested and extracted to analyze the total fatty acid content in wild type (black bars), line number 43 (dark-gray bars), and line number 97 (light-gray bars). The results are means ± se (n = 4 separate siliques from different plants). Significant changes from the wild type are marked with an asterisk (P < 0.05 using the Student's t test).

Table II.

Fatty acid composition of mature wild-type and transgenic seeds with decreased expression of AGPase

Fatty Acid Composition
Wild Type No. 43 No. 97
mol %
16:0 4.3 ± 0.2 4.9 ± 0.1 4.9 ± 0.1
18:0 n.d. n.d. n.d.
18:1 69.4 ± 0.8 67.6 ± 0.5 67 ± 0.5
18:2 12.2 ± 0.4 13.2 ± 0.4 13 ± 0.3
18:3 5.4 ± 0.3 5.6 ± 0.2 6.9 ± 0.2*
20:0 0.6 ± 0.04 0.67 ± 0.01 0.5 ± 0.04
20:1 1 ± 0.2 1.2 ± 0.02 1 ± 0.09
22:0 0.35 ± 0.03 0.36 ± 0.008 0.3 ± 0.01
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Results are means ± se (n = 4). Significant changes from the wild type are marked with an asterisk (P < 0.05 using the Student's t test). n.d., Not detectable.

DISCUSSION

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) or Vicia narbonensis (Rolletschek et al., 2002), a large decrease in AGPase only lead to a minor reduction in the amount of starch in the embryo, indicating flux-control coefficients below 0.1. This shows that the control exerted by AGPase over starch synthesis differs between seeds of different species, being much higher in oil seeds than in legume seeds. The data can be compared to results obtained on Arabidopsis leaves (Neuhaus and Stitt, 1990) or potato tubers (Müller-Röber et al., 1992; Geigenberger et al., 1999), where flux control coefficients between 0.3 to 0.6 have been reported.

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) and potato tubers (Tiessen et al., 2002; Geigenberger, 2003), AGPase is subject to posttranslational redox modification leading to dramatic changes in the allosteric properties of the enzyme. This posttranslational mechanism leads to a compensatory stimulation of the remaining AGPase activity in transgenic tubers with decreased AGPase expression, resulting in an underestimation of the real extent of flux control (Tiessen et al., 2002).

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). Previous studies demonstrate that there is a simultaneous synthesis and degradation of starch in developing rape seeds at early developmental stages (Da Silva et al., 1997; Eastmond and Rawsthorne, 2000; Vigeolas et al., 2003). This might explain why the decrease in AGPase activity led to a more marked decrease in the overall starch content of the seeds in some of the lines at 25 DAF. Whether there is a similar starch cycle in mature seeds remains to be determined. In mature seeds, starch was virtually absent in all lines indicating that the net rate of starch synthesis was zero (Fig. 1B). This could be due to the rate of unidirectional starch synthesis being lower than that of unidirectional starch degradation or to a complete inhibition of unidirectional starch synthesis. The first interpretation is more likely since there was still some AGPase activity remaining in mature seeds (Fig. 1A). However, these seeds had relatively low levels of hexose-phosphates (Fig. 5B) and ATP (Fig. 5A) representing the substrates of AGPase, and therefore the remaining AGPase activity might have been inhibited in vivo.

An Embryo-Specific Decrease in AGPase Is Accompanied by an Inhibition of Glycolysis and of Storage Lipid Biosynthesis in Young But Not in Mature Seeds of Oilseed Rape

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), whereas invertase activities decrease (King et al., 1997). In this context, it is possible that the lower PPi-dependent PFK activity and the higher invertase activities in the transformants at 25 and 30 DAF (see Fig. 6) were due to a delay in seed filling. The absence of any changes in fully matured seeds is consistent with this notion.

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). Inhibition of glycolysis will lead to a decrease in the supply of glycolytic intermediates and ATP for fatty acid synthesis in the plastid and hence to an inhibition of oil synthesis. There was a strong decrease in the level of ATP and in the ATP to ADP ratio in the transgenic seeds at early developmental stages (Fig. 4, G and H), which could have led to an inhibition of ACCase activity in vivo. The accumulation of acetyl-CoA—the substrate of ACCase—in the transgenic seeds (Fig. 4F) is in keeping with this notion. However, direct measurements of subcellular metabolite levels will be needed to confirm this interpretation.

An Embryo-Specific Decrease in AGPase Leads to Decreased Suc Metabolism and Delayed Lipid Accumulation in Developing Seeds of Oilseed Rape

Previous studies of Da Silva et al. (1997) showed that most of the carbon that is used for starch synthesis in developing rape seeds comes from imported Suc and proposed that the early development of a capacity of starch synthesis may contribute to the establishment of the embryo as a sink. Our in planta labeling studies using seeds at 25 DAF showed that decreased expression of AGPase led to a 2-fold decrease in the rate of Suc degradation (Fig. 2B and data in text above), indicating a severe decrease in the sink activity of the embryo at this stage. There was no substantial increase in the levels of soluble sugars, providing evidence that inhibition of Suc metabolism was accompanied by a decrease in the rate of Suc import (Fig. 4A). Delayed seed filling and sink development is also documented by the delayed increase in total lipid content during seed development (Fig. 7). The results provide direct evidence that starch synthesis is required to establish the growing embryo as a sink at this early developmental stage, which is consistent to the initial proposal of Da Silva et al. (1997). Interestingly, there were no significant changes in final seed size or dry weight (data not shown) or in total lipid content (Fig. 7) in mature seeds. This indicates that the influence of AGPase on Suc import and seed filling is largely compensated for in later stages of development, which are characterized by high rates of oil accumulation. This contrasts with potato tubers, which accumulate starch throughout their development where reduced expression of AGPase leads to a strong decrease in final starch and dry-weight accumulation and tuber size (Müller-Röber et al., 1992). Further studies are required to define the factors leading to the developmental shift from starch to lipid storage and to assess their role in controlling overall sink activity in developing oil seeds.

CONCLUSION

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.

MATERIALS AND METHODS

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.

Chemicals

Unless stated otherwise, chemicals were obtained from Sigma (Taufkirchen, Germany) or Merck (Darmstadt, Germany).

Northern-Blot Analysis

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) using the cDNA of the small subunit of AGPase (AGPS1) from oilseed rape as hybridization probe.

Analysis of Total Lipid Content of Seeds

Total lipids of developing seeds at different developmental stages were extracted according to the method of Bligh and Dyer (1959), and the lipid content was measured by gas chromatography of fatty acid methyl esters using pentadecanoic acid as internal standard (Benning and Somerville, 1992).

Determination of Starch Content of Seeds

Starch content was measured as described in Geigenberger et al. (1998).

In Planta Labeling Experiments

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). After 28 h, siliques were harvested and immediately frozen in liquid N2. During the whole experiment, seeds remained otherwise intact within their siliques and attached to the plant.

Extraction and Fractionation of Radiolabeled Seeds

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), the material was separated into a chloroform phase (lipid fraction), a water/methanol phase (water-soluble fraction), and an insoluble pellet containing starch, protein, and cell wall material. Lipids were fractionated by thin-layer chromatography as described in Stobart et al. (1997). The radioactive TAG and DAG spots were scraped from the plate and the radioactivity measured in a liquid scintillation counter. The methanol/water fraction was dried under an airstream at 45°C. The pellet was taken up in 1 mL of water, and neutral, anionic, and cationic compounds were separated on cationic (AG 50W-X8 Superfine, NH4+ form) and anionic (AG 1-X8 Superfine, OH− form) columns according to the manufacturer's instructions (Bio-Rad Laboratories, Munich). The label in phosphate esters was measured according to Geigenberger et al. (1997). The insoluble fraction was separated into starch, protein, and cell wall as in Merlo et al. (1993).

Metabolite, Nucleotide, and Enzyme Analysis

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). Glc6P, Glc1P, glycerol-3P, and acetyl-CoA were measured as in Gibon et al. (2002). Glc, Fru, and Suc were measured as in Geigenberger et al. (1998). ATP and ADP were quantified by high-pressure liquid chromatography using a Partisil-SAX10 anion-exchange column (Kontron Instruments, Eiching, Germany) as given in Geigenberger et al. (1997). SuSy, invertases, UGPase, hexokinase, and PPi-dependent PFK were extracted according to Geigenberger and Stitt (1993) and measured as in Merlo et al. (1993). ACCase was extracted with 0.5 m sorbitol, HEPES-NaOH (pH 7.4), 10 mm KCl, 1 mm MgCl2, 1 mm EDTA, 10% (v/v) ethanediol, 5 mm dithiothreitol, and 1% (w/v) bovine serum albumin. The activity was determined by measuring the incorporation of 14C from NaH14CO3 into malonyl-CoA (Kang et al., 1994).

Sequence data from this article have been deposited with the EMBL/GenBank data libraries under accession number AJ271162.

Acknowledgments

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.

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.).

Article, publication date, and citation information can be found at www.plantphysiol.org/cgi/doi/10.1104/pp.104.046854.

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