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First published online November 26, 2003; 10.1104/pp.103.031963 Plant Physiology 133:2048-2060 (2003) © 2003 American Society of Plant Biologists Lipid Storage Metabolism Is Limited by the Prevailing Low Oxygen Concentrations within Developing Seeds of Oilseed Rape1Max Planck Institute of Molecular Plant Physiology, Am Mühlenberg 1, 14476 Golm, Germany
The aim of this study was to investigate whether endogenous restrictions in oxygen supply are limiting for storage metabolism in developing oilseed rape (Brassica napus) seeds. Siliques were studied 30 d after flowering, when rapid lipid accumulation is occurring in the seeds. (a) By using microsensors, oxygen concentrations were measured within seeds and in the silique space between seeds. At ambient external oxygen (21% [v/v]) in the light, oxygen fell to 17% (v/v) between and 0.8% (v/v) within seeds. A step-wise reduction of the external oxygen concentration led within 2 h to a further decrease of internal oxygen concentrations, and a step-wise increase of the external oxygen concentration up to 60% (v/v) resulted in an increase in internal oxygen that rose to 30% (v/v) between and 8% (v/v) within seeds. (b) The increase in oxygen levels in the seeds was accompanied by a progressive increase in the levels of ATP, UTP, and the ATP to ADP and UTP to UDP ratios over the entire range from 0% to 60% (v/v) external oxygen. (c) To investigate metabolic fluxes in planta, 14C-sucrose was injected into seeds, which remained otherwise intact within their siliques. The increase in oxygen in the seeds was accompanied by a progressive increase in the rate of lipid (including triacylglycerol), protein and cell wall synthesis, and an increase in glycolytic flux over a range from sub- to superambient oxygen concentrations. In contrast to lipid synthesis, starch synthesis was not significantly increased at superambient oxygen levels. The levels of fermentation products such as lactate and glycerol-3P increased only at very low (0%-4% [v/v]) external oxygen concentrations. (d) When 14C-acetate or 14C-acetyl-coenzyme A (CoA) was injected into seeds, label incorporation into triacylglycerol progressively increased over the whole range of external oxygen concentrations from 0% to 60% (v/v). (e) Stimulation of lipid synthesis was accompanied by an increase in sugar levels and a decrease in the levels of hexose-phosphates and acetyl-CoA, indicating sucrose unloading and the use of acetyl-CoA as possible regulatory sites. (f) Increased lipid synthesis was also accompanied by an increase in the maximal activities of invertase and diacylglycerol acyltransferase. (g) The developmental shift from starch to lipid storage between 15 and 45 d after flowering was accompanied by an increase in the seed energy state. (h) The results show that at ambient oxygen levels, the oxygen supply is strongly limiting for energy metabolism and biosynthetic fluxes in growing rape seeds, affecting lipid synthesis more strongly than starch synthesis. The underlying mechanisms and implications for strategies to increase yield and storage product composition in oilseed crops are discussed.
Plants synthesize reduced carbon compounds from inorganic matter during photosynthesis in leaves, which are then exported to reproductive organs to be used for storage and growth. In developing seeds, Suc is converted to oil, protein, and starch, which are laid down as storage reserves. The percentage of reduced carbon stored as oil (triacylglycerol [TAG]) in seeds varies between species from 1% to 60% of total dry weight (Ohlrogge and Browse, 1995  ). Although the understanding of the pathways involved in Suc to TAG conversion in oil-storing seeds has improved in recent years, little is known about how the flux of carbon through these pathways is controlled or how the partitioning of carbon between oil and other storage products is determined (Rawsthorne, 2002).
The metabolism of Suc to storage lipids in developing oilseed rape (Brassica napus) involves the breakdown of the incoming Suc to glycolytic intermediates (hexose-P, phosphoenolpyruvate, pyruvate, and malate), which are subsequently imported into the plastid by specific transport proteins and used to synthesize acetyl-CoA via the pyruvate dehydrogenase complex (Rawsthorne, 2002
Most of the information on the regulation of lipid biosynthesis derives from studies performed in leaves. Here, it could be shown that changes in intermediates of fatty acid synthesis during light-dark transitions are consistent with control being exerted at the level of ACCase (Post-Beittenmiller et al., 1991
In heterotrophic seeds, ATP is produced by respiration and imported into plastids via an ATP/ADP exchanger or produced by glycolysis directly within the plastid (Rawsthorne, 2002
Previous reports document an unexpected inhibition of seed production when plants are grown at low external oxygen, with a critical oxygen pressure of around 5% (v/v) external oxygen (compared with 21% [v/v] in ambient conditions; Quebedeaux and Hardy, 1975 The following study investigates whether internal oxygen concentration are limiting for storage metabolism in oilseed rape. Rape seeds were chosen; since being larger, they are more accessible than Arabidopsis seeds and are an economically important oil crop. (a) Optical microsensors were inserted into siliques and seeds to analyze in situ oxygen concentrations in the silique airspace and inside the seeds. This was performed at ambient external oxygen (21% [v/v]), after decreasing external oxygen to 12% and 0% (v/v) and after increasing external oxygen to 30% and 60% (v/v). (b) To analyze the influence of changes in the oxygen concentration on cellular energy state, the levels of ATP, ADP, UTP, and UDP were analyzed in seeds in parallel. (c) To analyze the influence of changes in oxygen on metabolic fluxes to TAG, starch, structural elements, and glycolysis, 14C-Suc was injected into seeds and the metabolism of radiolabel analyzed after 2 h. In addition to this, more immediate precursors of fatty acid biosynthesis (14C-acetate and 14C-acetyl-CoA) were injected, and label incorporation into TAG was investigated. (d) Metabolite levels and enzyme activities were measured in the seeds to elucidate possible regulatory sites and mechanisms. The results show that ambient oxygen concentrations are very low within growing rape seeds (approximately 0.8% [v/v]) and can be markedly increased to more than 8% (v/v) by elevating external oxygen supply. Increased oxygen is accompanied by an increase in the energy state within seeds, increased metabolic fluxes to storage TAG biosynthesis (but not starch), and an increase in DAGAT and invertase activity. We conclude that oilseed metabolism is restricted by the prevailing low oxygen concentrations, providing an adaptive mechanism that allows oxygen consumption to be decreased to prevent internal anoxia. These results have obvious implications for strategies to increase yield and storage product composition in oilseed crops.
Influence of Changes in the External Oxygen Concentration on Internal Oxygen Concentrations in Siliques and Seeds of Rape
Experiments were performed with growing rape plants in the middle of the light period using siliques at 30 d after flowering (DAF). At this stage, developing seeds contained substantial amounts of lipids, starch, and protein and were characterized by high rates of lipid accumulation (Fig. 1). To analyze internal oxygen concentrations within the silique space between and inside growing seeds, an oxygen sensor (30-µm-diameter tip) was impaled into growing siliques. At ambient external oxygen (21% [v/v]) in the light, the internal oxygen concentration was 17% (v/v) in the silique airspace between seeds (Fig. 2A) and 0.8% (v/v) inside the seeds (Fig. 2B). The value for the silique airspace was similar to the value reported by Porterfield et al. (1999
The increase in oxygen levels in the seeds was accompanied by a progressive increase in the levels of ATP (Fig. 3A), UTP (Fig. 3D), and the ratios of ATP to ADP (Fig. 3C) and UTP to UDP (Fig. 3F) over the entire range from 0% to 60% (v/v) external oxygen. These results imply that at ambient oxygen levels, the oxygen supply is strongly limiting for energy metabolism in rape seeds.
To investigate the effect of changes in external oxygen around siliques on metabolic fluxes in seeds, 14C-Suc was injected into seeds, which remained otherwise intact within their siliques. Seeds were harvested 2 h later to investigate the fate of the label (Fig. 4). This in planta labeling method provides a minimally invasive technique by which to study the metabolism of labeled precursors within developing seeds. In parallel experiments, we checked that this manipulation did not significantly alter subsequent growth and lipid accumulation of the seeds when compared with untreated controls (data not shown). During the short labeling period of 2 h, only a small portion of the labeled Suc was metabolized (Fig. 4A). The percentage label found in hexose-phosphates (Fig. 4B), amino acids (Fig. 4B), and organic acids (Fig. 4C) was low and showed no marked changes over the whole range of external oxygen concentrations, except at 0% (v/v) oxygen, where labeling of phosphoester and organic acids increased and of amino acids decreased (Fig. 4B). The increase in organic acids was especially marked (approximately 10-fold), reflecting the onset of lactic fermentation. Most of the metabolized label was converted to structural components (Fig. 4D), starch (Fig. 4E), and lipids (Fig. 4F). The proportion of the injected label metabolized to lipids (Fig. 4F), and structural components (proteins plus cell walls, Fig. 4D) progressively increased when the external oxygen was increased over the entire range from 0% to 60% (v/v). Most of the label in lipids was recovered in TAG (Fig. 4G) and to a lesser extent in DAG (Fig. 4H). In contrast to this, the percentage labeling of starch did not change substantially over the whole range of external oxygen concentrations, except at 4% and 0% (v/v) oxygen, when labeling of starch decreased (Fig. 4E). Interestingly, there was incorporation of label into starch despite overall levels of starch decreasing in seeds at this stage of development (Fig. 1B), indicating simultaneous synthesis and degradation of starch in the seeds. This is in confirmation with previous studies documenting turnover of starch in developing rape seed embryos (Eastmond and Rawsthorne, 2000
The 14C-Suc taken up into the cells will mix with internal unlabeled pools, so movement of the label will not necessarily reflect fluxes into the various pools. This is especially true in the case of Suc because Suc represents a very large internal pool in these seeds (approximately 200 µmol g fresh weight-1, see below). Label incorporated into the phosphoester fraction at the end of the 2-h incubation (see Fig. 4B) was divided by the total carbon found in phosphoesters (see below) to calculate the specific activity of the hexose-phosphate pool at the end of the labeling interval. To calculate the mean specific activity during the 2-h incubation, values were divided by a factor of 2 (for data on mean specific activities, see legend of Fig. 5; for a discussion of the assumptions involved in these calculations, see Geigenberger et al., 1997
Influence of the Oxygen Concentration on the Metabolism of 14C-Acetate to TAG The influence of the external oxygen concentration on TAG biosynthesis was also investigated by supplying 14C-acetate to the seeds, which is a more immediate precursor of fatty acid synthesis. Figure 6 shows the incorporation of label from 14C-acetate into TAG as percentage of the total label injected. Due to the short incubation time (2 h), only a small percentage of the injected label was metabolized (data not shown). Label incorporation into TAG increased over the whole range of external oxygen concentrations from 0% to 60% (Fig. 6). Similar results were obtained when 14C-acetyl-CoA was injected into seeds (data not shown). However, in this case, we cannot exclude that 14C-acetyl-CoA was converted to 14C-acetate prior to uptake into the plastid. The data indicate that at least one of the regulatory sites leading to changes in TAG biosynthesis in response to oxygen is upstream of acetyl-CoA.
Metabolite levels in seeds are summarized in Figure 7. The increase in oxygen levels in seeds was accompanied by a slight but progressive increase in the level of Suc (Fig. 7A) and a strong almost linear increase in the levels of Glc (Fig. 7B) and Fru (Fig. 7C) over the whole range of external oxygen concentrations from 0% to 60% (v/v). Interestingly, sugars increased even though at the same time metabolic fluxes were increasing (see Fig. 5). This implies that Suc import has been stimulated.
The levels of glucose-6-phosphate (Fig. 7D), glucose-1-phosphate (Fig. 7E), and acetyl-CoA (Fig. 7F) decreased when external oxygen was increased from 12% to 30% (v/v), reflecting the stimulation of respiratory and biosynthetic fluxes (see Fig. 5). It is noteworthy that in this range of external oxygen concentrations, the stimulation of lipid synthesis was accompanied by a decrease of acetyl-CoA. This provides additional evidence that one of the sites at which flux is stimulated lies downstream of acetyl-CoA. The levels of glycerol-3P (Fig. 7G) and lactate (Fig. 7H), which are both regarded as fermentation products in plants, showed no substantial changes when external oxygen was increased from 12% to 60% (v/v) but increased progressively at 4% and 0% (v/v) external oxygen, showing that fermentation was induced at these very low external oxygen concentrations.
The data so far document a progressive increase in metabolic fluxes to respiratory and biosynthetic pathways in response to increasing oxygen levels within seeds. To investigate whether this can be correlated with changes in key enzymes of Suc degradation, fatty acid synthesis, or TAG biosynthesis, we measured the maximal activities of Suc synthase (SuSy; Fig. 8A), invertase (Fig. 8B), ACCase (Fig. 8C), G3PAT (Fig. 8D), and DAGAT (Fig. 8E) in the seeds. Under ambient conditions (21% [v/v] oxygen), the activities of SuSy, ACCase, G3PAT, and DAGAT were in the same range as the values published previously for developing rape seeds (Weselake et al., 1993
The activity of ACCase, which catalyzes the first step of fatty acid synthesis, increased when external oxygen was increased from 0% to 12% (v/v) but decreased at higher external oxygen concentrations (Fig. 8C). Although G3PAT, which catalyzes the first acylating step of TAG assembly and membrane lipid synthesis, showed no significant changes in activity over the whole range of external oxygen concentrations, there was a progressive increase in the activity of DAGAT especially between 0% and 30% (v/v) external oxygen. DAGAT catalyzes the final acylation of DAG to TAG and is the only step that is unique to TAG biosynthesis.
Oxygen Falls to Low Levels within Developing Rape Seeds in Planta
By using microsensors inserted into the tissue, we have shown that oxygen falls to low concentrations (0.8% [v/v]) within oil-storing rape seeds, growing at ambient external oxygen (21% [v/v]) in the light (Fig. 2B). A similar fall in internal oxygen concentrations has previously been found in other heterotrophic tissues like starch-storing pea (Pisum sativum) and broad bean (Vicia faba) seeds (Rolletschek et al., 2002
The results of the present paper show that there is a decrease in metabolic activity in response to the prevailing low oxygen concentrations within seeds. When oxygen concentrations within seeds were decreased below 0.8% (v/v), the concentration found with ambient external levels, ATP to ADP and UTP to UDP ratios decreased, indicating a restriction in respiration (Fig. 3), and there was a general decrease in various biosynthetic fluxes to lipids (including TAG), starch, protein, and cell walls (Fig. 5). Conversely, when oxygen concentrations within seeds were increased above 0.8% (v/v), there was an increase in the adenylate and uridinylate energy states, an increase in glycolytic flux, and an increase in the rate of lipid, protein, and cell wall synthesis. Also, flux to TAG increased, reflecting the increase in total lipid synthesis, whereas flux to starch was not significantly changed at elevated oxygen. These results show that rapeseed metabolism is restricted by the prevailing low internal oxygen levels and that this restriction can be relieved by elevating the external oxygen supply. Crucially, these metabolic changes occurred at internal oxygen levels between 0.8% and 8% (v/v) and could clearly be separated from fermentation that occurred only at lower seed oxygen concentrations (below 0.7%), see Figures 5A and 7, G and H.
Interestingly, starch and lipid biosynthesis were differentially affected by elevated oxygen supply. Although storage lipid synthesis increased more than 2-fold when oxygen was increased above ambient levels, there was no further increase in starch synthesis above ambient oxygen concentrations (compare Fig. 5, B with D). This indicates that starch synthesis is already at its maximal rate at ambient internal oxygen levels in seeds (0.8% [v/v]), whereas the rate of lipid biosynthesis is clearly restricted under these conditions and has an optimum at superambient oxygen concentrations (8% [v/v] internal oxygen or higher). An optimum for starch synthesis at relatively low oxygen concentrations (between 1% and 12% [v/v]) has also been reported for discs of starch-storing potato tubers (Geigenberger et al., 2000
The differential effect of oxygen on starch and lipid synthesis could also be linked to the different energy requirements of the two biosynthetic processes in seeds. Based on theoretical considerations of the stoichiometry of the reaction pathways, addition of a six-carbon unit would cost one ATP in the case of starch and three ATP in the case of lipid synthesis. Experiments on isolated plastids from developing embryos of oilseed rape showed that lipid synthesis requires more ATP than starch synthesis (Neuhaus and Emes, 2000
Changes in metabolite levels (Fig. 7) indicate that the stimulation of lipid synthesis in response to elevated oxygen occurs at two regulatory sites. The increase in the sugar levels in the face of increased metabolic fluxes provides evidence that Suc unloading has been stimulated by increased oxygen supply. This is consistent with recent studies showing an inhibition of metabolic activity paralleled by an inhibition of Suc unloading (using C-11 isotope labeling techniques) in developing wheat and rice (Oryza sativa) seeds when external oxygen was decreased from 21% to 10% (v/v; J.T. van Dongen, G. Roeb, A. Fröhlich, and P. Geigenberger, unpublished data). It is also consistent with studies documenting low oxygen concentrations within the vascular bundles of stems, restricting phloem energy metabolism and Suc transport rates (van Dongen et al., 2003 The stimulation of lipid synthesis at elevated oxygen was also accompanied by a decrease in the levels of hexose-phosphates and acetyl-CoA (Fig. 7, D-F), indicating that one or more reactions using acetyl-CoA have been stimulated. The increased incorporation of 14C-acetate into TAG (Fig. 6) provides additional evidence that the use of acetyl-CoA for lipid synthesis has been stimulated. This could be due to increased levels of ATP (Fig. 3A), leading to a stimulation of ACCase activity in planta. However, this is not reflected by changes in the maximal ACCase activity assayed under optimal conditions in vitro (Fig. 8C), indicating the importance of metabolic fine control of ACCase for the regulation of lipid synthesis. The factors leading to a decrease in the maximal ACCase activity at elevated oxygen are not known, but it is interesting to note that changes in overall ACCase activity obviously correlate with changes in the in planta concentration of acetyl-CoA (compare Figs. 8C and 7F). It must be noted, however, that the reported metabolite levels are overall levels and may not reflect the concentrations in the plastid. Direct measurements of subcellular metabolite levels will be needed to confirm our interpretation.
The increase in overall DAGAT activity (Fig. 8E) could provide a further explanation for the stimulation of oil biosynthesis. DAGAT catalyzes the final acylating step of TAG biosynthesis. Maximal DAGAT activity increased at elevated oxygen and correlated with increased TAG biosynthesis. The reason(s) leading to increased DAGAT activity at elevated oxygen in the 2-h time frame are unclear and could involve transcriptional and/or posttranscriptional regulation. Previous studies document that increased expression of DAGAT leads to increased oil accumulation in seeds of transgenic Arabidopsis plants (Jako et al., 2001
Lipid synthesis also requires NADPH and NADH for each C-2 addition to a growing acyl chain (Rawsthorne, 2002
Plants, unlike animals, lack specialized circulation systems for efficient oxygen transport to internal tissues, and oxygen can fall to quite low concentrations within bulky, dense, or metabolically active plant tissues (Geigenberger, 2003
The strong dependence of Suc to lipid conversion on oxygen is remarkable. Lipid biosynthesis is clearly restricted by the prevailing oxygen concentrations within seeds and can be increased by increasing oxygen supply. From radiolabeling studies, fluxes from Suc to lipids were calculated to be 12 nmol Suc seed-1 h-1 at ambient (21% [v/v]) external oxygen and 23 nmol Suc seed-1 h-1 at elevated (60% [v/v]) external oxygen (Fig. 5D). If we assume that only the 12 CH2 groups of a Suc molecule (molecular weight = 168 g mol-1) are incorporated into lipids, the rate of lipid synthesis would correspond to 48.4 and 92.7 µg lipid seed-1 d-1 at 21% and 60% (v/v) external oxygen, respectively. The value for the rate of lipid synthesis in ambient conditions (48 µg lipid seed-1 d-1) is well in the range of the maximum lipid accumulation rate derived from measurements of total lipid contents during seed development (approximately 35 µg seed-1 d-1, calculated from Fig. 1A). Similar rates of lipid accumulation have been reported in previous studies in developing rape seeds (Turnham and Northcote, 1983
Plant Material Spring rapeseed (Brassica napus cv Drakkar) plants were grown in a phytotron (25°C/day and 20°C/night) with a 16-h photoperiod at a light 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 30 DAF, when the seed diameter was about 3 mm, and lipid content was approximately 200 µg seed-1 (see Fig. 1). 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 lipids of developing seeds between 15 and 45 DAF were extracted according to the method of Bligh and Dyer (1959
Protein content was measured as by Eastmond and Rawsthorne (2000
To determine the influence of changing oxygen concentrations on carbon flux and metabolism within the seed, a side branch bearing several siliques was enclosed in a transparent plastic bag through which air with various oxygen tensions was passed. The premixed gases (Messer Griesheim GmbH, Magdeburg, Germany) used for this treatment contained 350 ppm CO2, different oxygen concentrations as indicated in the text, and N2. Using a 5-µL Hamilton syringe (470-µm needle diameter, neolab, Heidelberg, Germany), 0.5 µL of a solution containing 115 µM [U-14C] Suc, 2.8 mM [U-14C] acetate, or 110 µM [1-14C] acetyl-CoA (Amersham-Buchler, Freiburg, Germany; specific activities 22.8 MBq µmol-1, 2.11 MBq µmol-1, or 2.11 MBq µmol-1, respectively) in 20 mM MES-buffer (pH 5.7) was injected directly into seeds. After 2 h, siliques were harvested and immediately frozen in liquid N2. During the whole experiment, seeds remained otherwise intact within their siliques.
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 by Bligh and Dyer (1959
To determine the influence of changing oxygen concentrations on metabolite levels, nucleotide levels, and enzyme activities in seeds, side branches containing several siliques were enclosed in a transparent plastic bag through which air with various oxygen tensions was passed. The premixed gases (Messer Griesheim GmbH) used for this treatment contained 350 ppm CO2, different oxygen concentrations as indicated in the text, and N2. After 2 h, siliques were rapidly frozen in liquid nitrogen, and seeds separated from silique walls under liquid nitrogen. Metabolite and nucleotide levels were analyzed in trichloroacetic acid extracts as described by Jelitto et al. (1992
In situ oxygen tensions were measured in the light, using an oxygen microsensor with a tip diameter of approximately 30 µm, and connected to a fiber optic oxygen meter (MicroxTX2, Presens, Regensburg, Germany) as by van Dongen et al. (2003
We wish to thank Mark Stitt for his support and interest in this work, stimulating discussions, and helpful comments on the manuscript. We are grateful to Peter Dörmann for providing the gas chromatography facilities, to John E. Lunn and Alisdair R. Fernie for critical readings of the manuscript, and to Britta Hausmann and Karin Koehl for taking care of the plants. Received August 19, 2003; returned for revision September 8, 2003; accepted September 19, 2003.
1 This work was supported by the Deutsche Forschungsgemeinschaft (grant no. Ge 878/1-3).  Article, publication date, and citation information can be found at www.plantphysiol.org/cgi/doi/10.1104/pp.103.031963. * Corresponding author; e-mail geigenberger{at}mpimp-golm.mpg.de; fax 49-331-567-8408.
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Zeng Y, Wu Y, Avigne WT, Koch KE (1999) Rapid repression of maize invertases by low oxygen: invertase/sucrose synthase balance, sugar signaling potential, and seedling survival. Plant Physiol 121: 599-608 Related articles in Plant Physiol.:
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