JXB Advance Access originally published online on June 18, 2004
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Journal of Experimental Botany, Vol. 55, No. 402, pp. 1455-1462, July 2004
Journal of Experimental Botany, Vol. 55, No. 402, © Society for Experimental Biology 2004; all rights reserved
RESEARCH PAPER |
The import of phosphoenolpyruvate by plastids from developing embryos of oilseed rape, Brassica napus (L.), and its potential as a substrate for fatty acid synthesis
Department of Metabolic Biology, John Innes Centre, Norwich Research Park, Colney, Norwich NR4 7UH, UK
To whom correspondence should be addressed. Fax: +44 (0)1603 450014. E-mail: steve.rawsthorne{at}bbsrc.ac.uk
Received 7 November 2003; Accepted 18 March 2004
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Abstract |
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The plastidial phosphoenolpyruvate (PEP)/phosphate translocator (PPT) is expressed in the developing embryos of oilseed rape (Brassica napus L.). PEP can be imported by plastids isolated from embryos and used for fatty acid synthesis at rates that are sufficient to account for one-third of the rate of fatty acid synthesis in vivo. This provides the first experimental evidence for uptake of PEP and incorporation of carbon from it into fatty acids by plastids. PEP metabolism in isolated plastids is able to provide some of the ATP required for fatty acid synthesis. Expression of the PPT and related glucose 6-phosphate (Glc-6-P) translocator (GPT) is high in early embryo and leaf development and then declines. The marked decline in the abundance of PPT and GPT transcripts between the pre- and mid-oil accumulating stages of embryo development in B. napus does not correlate with the corresponding translocator activities, which both increase over the same period. This means that transcript abundance cannot be used to infer the activity of the translocators.
Key words: Brassica napus, embryo, fatty acid synthesis, phosphoenolpyruvate translocator, plastid
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Introduction |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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Fatty acid synthesis in the plastids of heterotrophic organs is dependent on the supply of carbon substrates from the cytosol. Previous studies have shown that a range of metabolites is used for fatty acid synthesis by plastids from heterotrophic tissues of different plant species including glucose 6-phosphate (Glc-6-P), triose phosphate, malate, and pyruvate (Smith et al., 1992
; Kang and Rawsthorne, 1994; Qi et al., 1995; Eastmond and Rawsthorne, 2000) (Fig. 1). The rate of uptake of each of these metabolites varies. For example, pyruvate uptake by plastids from castor endosperm is weak compared with the transporter-mediated import of malate for fatty acid synthesis (Eastmond et al., 1997). The relative rate of uptake, and the extent to which different metabolites contribute to fatty acid synthesis, depend upon the species, the organ, and the developmental stage of the organ. For example, in Brassica napus (L.) embryos, variation during development in the activity of the pyruvate transporter is reflected in the different rates of incorporation of exogenous pyruvate into fatty acids by plastids isolated from embryos of different ages (Eastmond and Rawsthorne, 2000). Transport rates depend upon the amounts of specific translocator proteins in the plastid envelope, but could also vary due to regulation of transporter activity. For example, the Glc-6-P translocator (GPT) is inhibited by acyl-Coenzyme As, the end-products of plastidial fatty acid synthesis and acyl chain export (Fox et al., 2000; Johnson et al., 2000, 2002).
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In addition to the transporters and metabolites mentioned above, PEP import could also contribute to fatty acid synthesis. Recent studies based on 13C-isotope feeding of cultured B. napus embryos (Schwender and Ohlrogge, 2002
; Schwender et al., 2003) have revealed that the plastidial PEP pool is a major source of carbon for fatty acid synthesis (J Schwender, personal communication). While this does not provide direct evidence for PEP import, it does reveal the importance of understanding the relative extents to which PEP can be imported into plastids or synthesized within them.
The transport of phosphoenolpyruvate (PEP) across the plastid envelope by a specific phosphate exchange translocator has been described previously (Fischer et al., 1997). In Arabidopsis, two genes encode PEP translocators (PPT) and the expression patterns for PPT1 (At5g33320) and PPT2 (At3g01550) have been defined using RT-PCR and gene promoter–GUS reporter fusions (Knappe et al., 2003a, b). The expression of PPT1 was mostly associated with vascular tissues, but GUS staining also appeared in the mesophyll of expanded leaves. While the PPT2 promoter expressed more widely, it was not active in seeds where PPT1 expression was clearly seen. Comparative, quantitative measurements of the expression of the two genes have not been reported.
In Arabidopsis, the importance of PPT1 in providing PEP for plastidial fatty acid synthesis has been questioned. Support for the role of the PPT1 is taken from expressed sequence tag (EST) analysis of developing seed of Arabidopsis. This revealed that, amongst the phosphate exchange translocator family for which function had been assigned, the relative abundance of the respective ESTs was PPT1=GPT>TPT (White et al., 2000). No PPT2 ESTs were detected in the seed analysis consistent with lack of PPT2::GUS reporter expression in developing seeds (White et al., 2000; Knappe et al., 2003b). However, analysis of the cue1 mutant of Arabidopsis, originally identified by Li et al. (1995), has led to the conclusion that PPT1 is not required for the maintenance of normal levels of lipids in Arabidopsis (Streatfield et al., 1999). The cue1 mutant lacks a functional PPT1 gene, but the mutation has no effect on lipid content in the seeds or leaves. Interpretation of any effect of cue1 on lipids is complex in leaves due to the differential expression patterns of PPT1 and PPT2, but in seeds only PPT1 expression was reported (Knappe et al., 2003b).
Both of the conclusions drawn above about the importance of PPT1 in lipid synthesis in Arabidospis seeds can be criticized. First, EST abundance is not a reliable measure of the activity of the protein encoded. Second, lack of an absolute requirement for PPT1 does not rule out its involvement in fatty acid synthesis in the wild type. There are many plastid transporters potentially supplying intermediates for fatty acid synthesis (Fig. 1), and lack of an individual transporter such as PPT1 in the cue1 mutant may be compensated for either by (a) an increase in the rate of import via one or more of the other transporters, or (b) an increase in the duration of oil synthesis such that the final oil content in the mutant is similar to that in the wild type.
To shed further light on the importance of PPT for fatty acid synthesis, its activity has been studied during embryo development in B. napus and the incorporation of carbon from PEP into fatty acids by isolated plastids has been measured. To enable comparison with previous studies using isolated oilseed rape plastids (Kang and Rawsthorne, 1996; Eastmond and Rawsthorne, 2000), the activity of the related GPT was determined in parallel. The developmental and organ-specific patterns of transcript abundances have also been determined for the PPT, GPT, and TPT in B. napus.
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Materials and methods |
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Reagents, radiochemicals and synthesis of [2-14C]PEP
Isotopes were purchased from NEN (Dupont, Herts, UK): [1-14C]Glc-6-P, [1-14C] and [2-14C]pyruvate and from Amersham Pharmacia Biotech (Little Chalfont, UK): [1-14C]PEP. Coupling enzymes were from Roche Diagnostics Ltd (Lewes, UK). All other reagents were of analytical grade or higher.
[2-14C]PEP was prepared enzymaticaly from [2-14C]pyruvate using pyruvate phosphate dikinase (PPDK). Recombinantly expressed PPDK (20 µg; a gift of Dr P Geigenberger and A Teissen, MPI, Golm, Germany; Gibon et al., 2002) was added to a reaction buffer containing 50 mM HEPES/KOH pH 8.0, 4.5 mM MgCl2, 0.5 mM ATP, 2.0 mM NaH2PO4, 2.0 mM NH4Cl, 1.0 mM PEP, 0.5 units inorganic pyrophosphatase, and 185 kBq [2-14C] pyruvate (30 µl total reaction volume) and the mixture incubated at 25 °C for 60 min. The reaction was stopped by cooling on ice. The cooled reaction was transferred to a microcrystalline cellulose TLC plate (Polygram Cel 400, Machery-Nagel GmbH & Co. KG, Düren, Germany). Standards of [1-14C]PEP and [2-14C]pyruvate (0.5 kBq) were also loaded. The plate was run for 80 min in a solvent tank containing 160 ml diethyl ether, formic acid, water (5:2:1, by vol.), removed, and dried. The TLC plate was exposed to a Fuji imaging plate (BAS-IP MS 2040; Raytrek Scientific Ltd, Sheffield, UK) overnight and visualized using a Fujix bio-imaging analysis system 1000 (Fuji UK Ltd, London). Radioactive bands containing PEP were scraped off the plate and the PEP eluted with several washes of water followed by centrifugation to pellet the cellulose (final volume 0.75 ml). The yield of PEP was approximately 50% based upon quantification of the image of the pyruvate and PEP containing bands on the plate.
The PEP-containing solution was checked for authenticity and purity using Dowex columns and enzyme degradation. This was based upon interconversion of PEP to lactate and the differential elution patterns of PEP, lactate, and pyruvate from the Dowex column verified using [1-14C]PEP and [2-14C]pyruvate as standards. Enzymatic interconversion was accomplished by incubating 100 µl of eluted PEP solution with lactate dehydrogenase (1 unit: added alone to form lactate) and pyruvate kinase (2 units: added in addition to form pyruvate) in 150 µl of reaction medium (50 mM HEPES/KOH pH 7.0, 5 mM MgCl2, 0.3 mM NADH, and 0.7 mM ADP). Untreated or enzyme-treated samples were loaded onto 0.7 ml Dowex 1x8 100–200 (Supelco, Bellefonte, USA) columns that were sequentially eluted with 6x500 µl 100 mM succinate/NaOH pH 5.5, 2x500 µl 100 mM acetate/NaOH pH 5.5, and finally 5x500 µl 2 M HCl. The purity of the [2-14C]PEP was 98% based on the elution profile of radioactivity from the Dowex column compared with the elution profiles of [1-14C]PEP and [2-14C]pyruvate.
Growth of plants and preparation of plastids
Growth of plants and preparation of plastids from developing embryos of oilseed rape (Brassica napus L. cv. Topas) was as described previously (Kang and Rawsthorne, 1994) with the following modification. Peeled embryos (500–550) were homogenized with two single-edged razor blades five times for 1 min in 2 ml plastid isolation medium. After each homogenization, the extract was filtered through two layers of Miracloth (Calbiochem Ltd, Beeston, UK).
Uptake and utilization of metabolites
Short-term uptake experiments were performed using the silicone oil centrifugation method (Heldt and Sauer, 1971). The silicone oil consisted of a 1:2 (w/w) mix of AP100:AR200 oils (Wacker Chemicals Ltd., Walton-on-Thames, UK). Centrifuge tubes (400 µl, M102 polyethylene; Thermo Life Sciences, Basingstoke, UK) were filled with 60 µl oil mix. Incubations were carried out in plastid incubation media according to Eastmond and Rawsthorne (1998) except that they were started by mixing 170 µl of washed plastids with 30 µl uptake mix containing [1-14C]PEP or [1-14C]Glc-6-P at the given concentration (respective specific activities of 57–75 GBq mol–1 and 74–84 GBq mol–1). From this mixture, 170 µl was transferred rapidly into the 400 µl tubes containing oil and incubations were stopped by centrifugation at 12 000 g for 10 s. Rates of uptake were expressed per unit GAPDH (NADP-dependent glyceraldehyde-3-phosphate dehydrogenase) recovered in the post-centrifugation pellet in control experiments without added isotope. There was a 16.6±2.7% recovery of NADP-GAPDH activity with no difference between the A and C stages (mean ±SE of seven independent preparations). Rates were linear for up to 120 s and final measurements were made over the first minute. For competition experiments the given amount of unlabelled second substrate was included in the 30 µl uptake mix. Expression of uptake on the basis of NADP-GAPDH activity recovered in the pellet leads to measured rates of PEP and Glc-6-P uptake that are between 5- and 10-fold greater than those reported previously, depending on substrate and stage of embryo development (Eastmond and Rawsthorne, 2000; Kubis and Rawsthorne, 2000). This difference largely arises because the earlier data were expressed on the basis of total NADP-GAPDH activity added to each incubation prior to centrifugation, thereby underestimating the rate for the plastids recovered in the pellet.
Incorporation of carbon from [2-14C]PEP (6–12 GBq mol–1) or [1-14C]Glc-6-P (46–75 GBq mol–1) into fatty acids was carried out using 1 h incubations according to Kang and Rawsthorne (1994). The end-products of incorporation of carbon were verified to be free fatty acids as described in Fox et al. (2000). Rates of incorporation of carbon from PEP or Glc-6-P were expressed per unit GAPDH. To estimate ‘intactness’ of the plastid preparations used in the fatty acid synthesis experiments, the latency of NADP-GAPDH was measured in each plastid preparation (Kang and Rawsthorne, 1994).
Determination of PEP content in embryos
The in vivo PEP content was determined following TCA–ether extraction (Jelitto et al., 1992) of 60 embryos (average 3.1 mg FW embryo–1). Extracts were analysed enzymatically by measuring the change in A340 following sequential addition of lactate dehydrogenase (to give pyruvate) and then pyruvate kinase (to give PEP), as described above for the analysis of [1-14C]PEP.
Cloning, total nucleic acid extraction, and northern hybridization
RT-PCR was performed to amplify B. napus PPT and TPT gene fragments using primers designed from published sequences (PPT: U13632
[GenBank]
.1 from B. oleracea and U66321
[GenBank]
from A. thaliana (At5g33320), 5'-AGATCTCCCACGATGCAGAGC, 3'-TGCTTAGGCAGTCTTAGGCTTTG; TPT: U13630
[GenBank]
.1 from B. oleracea (Fischer et al., 1994, 1997), 5'-TCGAGATGGAGTCACGCG, 3'-TCTACGCCGTCTTACCTTGC). DNaseI-treated total RNA (using TRIZOL-reagent, Invitrogen Ltd, Paisley, UK) prepared from embryos in early–mid stages of oil deposition was used as a template. RT-PCR products were cloned into pUC18 and sequenced (Big Dye Terminator Sequencing Kit, Applied Biosystems, Warrington, UK) to verify that they represented the B. napus orthologues of the respective published gene sequences. A clone representing part of the B. napus GPT cDNA was kindly provided by Dr M Hills (JIC, Norwich).
Total RNA was isolated from 100 mg of freshly harvested plant material using the RNeasy plant mini kit from Qiagen (Crawley, UK) according to the manufacturer's instructions. Total RNA was fractionated and blotted to nylon membranes (Hybond N+, Amersham) as previously described (Hobbs et al., 1999). Membranes were probed with 32P-labelled gene-specific DNA fragments described above (Oligolabelling Kit, Amersham Pharmacia Biotech, Little Chalfont, UK) in a 0.3 M sodium-phosphate buffer pH 7.2 containing 7% (w/v) SDS, 1 mM EDTA pH 8.0, and 2% (w/v) BSA overnight at 65 °C. Filters were washed at 65 °C twice in 2x SSC/0.1% (w/v) SDS for 20 min, and once each in 1x SSC/0.1% (w/v) SDS for 10 min and 0.5x SSC/0.1% (w/v) SDS for 10 min. Cross-hybridization between the different Pi translocator probes (sequence homologies at the nucleotide level range from 60% to 70%) can be excluded, since the wash-stringency only allowed probe–target combinations with more than 80% homology to remain hybridized. Where a single membrane was reprobed it was first washed by boiling it in 1% (w/v) SDS for 30 min. Consistent with previous observations (Streatfield et al., 1999) exposure times for PPT and GPT on blots were up to 20-fold longer than those for TPT. Scanning densitometry of blots was carried out using a BioRad GS-710 Imaging Densitometer with Bio-Rad Quantity One software (Bio-Rad, Hemel Hempstead, UK).
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Results |
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Measurement of PEP transporter activity in isolated plastids
Uptake of [1-14C]PEP by plastids isolated from B. napus embryos at the pre-oil accumulation stage (i.e. early cotyledon filling; approximately 1.5 mg FW embryo–1) and at mid-oil accumulation (i.e. during mid–late cotyledon filling; approximately 3.5 mg FW embryo–1) was measured using the silicone oil centrifugation method (Heldt and Sauer, 1971
). These two stages correspond, respectively, to stages A and C as defined by Eastmond and Rawsthorne (2000). To carry out uptake and feeding experiments with isolated plastids under conditions representative of those in vivo, the PEP concentration were estimated first in vivo. The PEP content determined for mid-oil-stage embryos was 0.57±0.01 nmol embryo–1 (mean ±SE of three separate extractions). Assuming a uniform distribution of PEP in the aqueous compartments and a water content of 45% (w:w) for the embryos (da Silva, 1993), this figure equates to a minimum concentration of 0.4 mM. This value is well in excess of the apparent Km for PPT (0.1 mM) (Kubis and Rawsthorne, 2000). The rate of uptake of PEP (at 0.4 mM) by plastids isolated from mid-oil-stage embryos was 2.0–3.8-fold greater than that for plastids from younger embryos at the pre-oil stage (Table 1). This was true whether the rate of uptake was expressed relative to the activity of the plastid marker enzyme, NADP-glyceraldehyde 3-phosphate dehydrogenase (NADP-GAPDH), or on a per embryo basis. By contrast, using the same plastid preparations, the rate of Glc-6-P uptake changed less than 2.0-fold between the pre- and mid-oil stages (Table 1). At all stages, and with all bases of expression, the rate of uptake of Glc-6-P was greater than that of PEP, although the differences between the uptake rates for the two metabolites were smaller at the mid-oil stage.
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To determine whether the PEP transport activity of the embryo plastids was attributable to the activity of a specific PPT or to transport via the GPT and/or TPT which have some affinity for PEP (Flügge, 1999
), the uptake was measured of 0.4 mM PEP in the presence of 2.0 mM Glc-6-P or 1.0 mM 3-PGA by plastids isolated from mid-oil-stage embryos. Under these conditions, Glc-6-P and 3-PGA inhibited PEP uptake by 10% and 27%, respectively (data not shown). Such weak inhibition of PEP uptake by these metabolites, combined with a Km for PEP of 0.1 mM, strongly suggests that the uptake of PEP measured here is facilitated by a specific PPT.
PEP can be used as a source of both carbon and ATP for fatty acid synthesis by isolated plastids
The PEP that is imported into the plastids of B. napus by the PPT is a potential source of carbon for fatty acid synthesis by the plastids, in addition to that from imported Glc-6-P and pyruvate. To investigate whether carbon from imported PEP could be used for fatty acid synthesis, isolated plastids were incubated with 14C-PEP and the rate of incorporation of 14C into fatty acids was measured. The C-1 carbon of PEP is lost during its conversion to acetyl-CoA, the immediate substrate for fatty acid synthesis, in the plastidial pyruvate dehydrogenase step. Therefore, [2-14C]PEP was used in these experiments. A method for the synthesis of [2-14C]PEP was devised in which [2-14C]pyruvate was incubated, under optimized conditions, with recombinantly-expressed pyruvate, Pi dikinase and the PEP produced was purified using TLC (Fig. 2). The yield of [2-14C]PEP from [2-14C]pyruvate was approximately 50% with a purity of 98%.
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Incorporation of carbon from PEP into fatty acids occurred at a rate equal to or greater than that from Glc-6-P at both stages of embryo development examined, whether expressed per unit plastid marker enzyme or per embryo (Table 2). The rates of incorporation of carbon from PEP and Glc-6-P by isolated plastids at the mid-oil stage were between 25 and 30 nmol acetate embryo–1 h–1. This rate is of the same order as the estimated rate of fatty acid synthesis in vivo (78 nmol acetate embryo–1 h–1; Eastmond and Rawsthorne, 2000
). At the pre-oil stage of development, the rate of incorporation of carbon from PEP into fatty acids was, on a per embryo basis, similar to that at mid-oil synthesis (Table 2). By contrast, the rate of incorporation of carbon from Glc-6-P into fatty acids at the pre-oil stage was much less than that at the mid-oil stage (Table 2). While marked, this change in Glc-6-P utilization should be interpreted cautiously because [1-14C]Glc-6-P was used to determine the rate of fatty acid synthesis in these experiments. It is possible that the activity of the oxidative pentose phosphate pathway in these plastids (Kang and Rawsthorne, 1996) could lead to a loss of 14C label, leading to a potential dilution of the specific activity of the acetyl-CoA used in fatty acid synthesis and, therefore, an underestimation of the true rate. Consistent with this, differences were observed in the rate of fatty acid synthesis from [1-14C]Glc-6-P and [U-14C]Glc-6-P (D Hutchings, M Emes, S Rawsthorne, unpublished results). The uniform rate of fatty acid synthesis from PEP did not reflect the changing activity of PPT (Table 1). The capacity for PEP import was comparable to the in vitro rate of fatty acid synthesis in plastids isolated from embryos at the pre-oil stage, but it exceeded the rate of synthesis at the mid-oil stage.
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In addition to acting as a carbon source, PEP metabolism via the plastidial pyruvate kinase reaction could also provide ATP for fatty acid synthesis in isolated plastids (Boyle et al., 1990
; Kleinig and Liedvogel, 1980; Qi et al., 1994). To test whether this occurred in B. napus embryos, isolated plastids from mid-oil-stage embryos were incubated with saturating [2-14C]PEP in the presence or absence of a saturating ATP concentration (4.0 mM; Kang and Rawsthorne, 1994). In the presence of ATP, the rate of fatty acid synthesis was 0.65±0.04 µmol unit–1 GAPDH h–1 (data are the mean ±SE of values from three separate incubations for each treatment and are similar to the values for replicate plastid preparations in Table 2). When ATP was excluded from incubations, the rate of synthesis was almost 60% lower at 0.27±0.07 µmol unit–1 GAPDH h–1, but it was not abolished.
The expression of plastidial phosphate exchange translocators
The relationship between translocator activity and the expression of the PPT gene(s) throughout development of B. napus embryos was analysed. Using northern blots, the pattern of expression of PPT was compared with those of GPT and TPT. Five different stages of development were chosen, ranging from pre-oil synthesis to mature embryos. A full-length B. napus PPT probe that is an orthologue of the PPT1 genes of Arabidopsis and B. oleracea (Fischer et al., 1997) was used. The PPT was most strongly expressed in the pre- and early-oil stages (Fig. 3). Expression then declined markedly although transcripts were still clearly detectable at the mid-oil, desiccating, and mature embryo stages (Fig. 3). The patterns of mRNA abundance for GPT and TPT were similar to that of PPT, with the strongest expression in pre-oil embryos, declining mRNA abundance at the early-oil stage, and then very weak to no detectable expression (even when blots were overexposed; data not shown) thereafter (Fig. 3).
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To compare expression of the PPT, GPT, and TPT translocators in embryos with that in other organs and to discover whether the pattern of expression in other organs also changed during development, the expression of PPT, GPT, and TPT was studied in leaves of B. napus (Fig. 4). The transcript abundance in leaves of a range of developmental stages was compared with that in pre-oil-stage embryos. The PPT was most strongly expressed in pre-oil-stage embryos and immature leaves on seedlings and mature plants. The weakest expression was seen in cotyledon leaves and the older primary leaves. Expression of the GPT followed a similar pattern, although in this case expression in the immature primary leaves was twice that in other tissues (Fig. 4). The abundance of the TPT transcript was much lower in pre-oil embryos, 1 cm long primary leaves and immature leaves, than in cotyledon leaves, and the older primary and secondary leaves (Fig. 4).
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Discussion |
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The activity of the PPT, incorporation of carbon from PEP into fatty acids by isolated plastids, and the availability of PEP in vivo at concentrations that would nearly saturate the PPT all suggest that PEP could act as a substrate for oil synthesis in the developing embryos of B. napus. The in vitro rate of PEP-dependent fatty acid synthesis by plastids isolated from mid-oil-stage embryos was at least equal to that from Glc-6-P (Table 1) and was sufficient to account for 33% of the highest rate of lipid accumulation that was observed in the embryo in vivo (78 nmol acetate equivalents per embryo–1 h–1; Eastmond and Rawsthorne, 2000
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Phosphoenolpyruvate can therefore be considered, along with pyruvate and Glc-6-P (Eastmond and Rawsthorne, 2000), as a metabolite of potential importance for the supply of carbon skeletons for the synthesis of fatty acids in B. napus embryos (Fig. 1). It has been discussed previously that additivity of substrate utilization for driving high rates of fatty acid synthesis by isolated plastids implies that the simultaneous use of more than one substrate is highly likely, and may even be a requirement in vivo (Kang and Rawsthorne, 1996; Eastmond and Rawsthorne, 2000). Assessing the relative contributions of these translocators to fatty acid synthesis in vivo will require in vivo manipulation of the activities of the relevant translocators specifically in the embryo. Such an approach would prevent effects on embryo development and nutrition that might arise from the detrimental effects of knocking out activity in the whole plant, as seen in the cue1 mutant (Streatfield et al., 1999).
In addition to providing carbon skeletons for plastidial metabolism, PEP could also provide, through the pyruvate kinase reaction, a proportion of the ATP requirement for fatty acid synthesis in isolated embryo plastids. The present study confirms earlier reports for a number of species and tissues (Kleinig and Liedvogel, 1980; Boyle et al., 1990; Qi et al., 1994) that PEP can act as an ATP source for fatty acid synthesis in isolated plastids. However, in these earlier studies, the incorporation of carbon from PEP itself into fatty acids was not measured which means that the effectiveness of PEP as an ATP source was not fully addressed. As metabolism of Glc-6-P to acetyl-CoA would also provide ATP through the pyruvate kinase reaction, it is puzzling that fatty acid synthesis from Glc-6-P is almost entirely dependent on exogenously supplied ATP (Eastmond and Rawsthorne, 1998), while that from PEP is not. There is not a simple explanation for these two contrasting observations. Further experimentation would be required to determine, more completely, the fates of Glc-6-P and PEP in order to resolve this.
The pattern of expression of the PPT and GPT during B. napus embryo development that is reported here is broadly comparable to that reported previously for developing Arabidopsis seeds. In B. napus, it was found that the expression of these transporters was high in early embryo development and then declined during later stages of development. In Arabidopsis, PPT1 is expressed uniformly between 5 DAF and 11 DAF (microarray analysis; Ruuska et al., 2002), but declines by 50% between 9 DAF and 13 DAF (supplementary quantitative data provided by Ruuska et al. (2002) including Real Time-PCR (at: http://www.bpp.msu.edu/Seed/SeedArray.htm or http://www.bpp.msu.edu/Seed/sari/fig1.pdf)). Microarray analysis also showed that the expression of GPT declined considerably between 8 DAF and 13 DAF (Ruuska et al., 2002). Based on the published profiles for oil accumulation in Arabidopsis seeds (Baud et al., 2002; Ruuska et al., 2002), 8 DAF and 13 DAF corresponds to the pre- to early-oil stages defined for B. napus embryo development.
In these northern blot experiments, it was not known whether the full-length B. napus PPT1 probe that was used hybridized to PPT1 alone or to all PPT transcripts in this species. To date, only one PPT gene (PPT1) has been identified in brassica species (Fischer et al., 1997). However, Arabidopsis also has a PPT2 gene and, due to the close phylogenetic relationship between Arabidopsis and B. napus, PPT2 gene orthologues would also be expected to be present in B. napus. If conservation of gene function between the two species occurs, then it is likely that the expression of PPT in B. napus embryos is determined by PPT1 orthologues based on histochemical staining for activity of the PPT promoters in whole Arabidopsis plants (Knappe et al., 2003b).
In these studies the expression of the PPT, GPT, and TPT in B. napus and Arabidopsis varied between organs and with the developmental stage of the organ. In both leaves and developing embryos the expression of PPT and GPT was strongest in sink tissues, such as leaves in early expansion and in pre-oil embryos. By contrast, the expression of TPT was strongest, not surprisingly, in photosynthetically active source tissues such as mature and cotyledon leaves. The expression of PTP and GPT declined as the leaves developed, but they did not entirely disappear in source leaves. The variation in expression pattern of PPT that is shown here contrasts with previous reports that PPT is expressed at low and uniform levels in different tissues of Arabidopsis and maize (Streatfield et al., 1999; Kammerer et al., 1998). These data are more like those of Fischer et al. (1997) and Knappe et al. (2003b) who have reported both tissue-specific and developmental variation in PPT gene expression.
In B. napus embryos, the marked decline in the abundance of PPT and GPT transcripts between pre- and mid-oil stages (Fig. 3) contrasts with (i) the increase in the activity of these two translocators, and (ii) maintained utilization of PEP for fatty acid synthesis on a per embryo basis over the same time period (Table 1). These disparities between the translocator activity and transcript abundance imply that the translocator proteins are stable in the plastid envelope and/or that there may be changes in translational efficiency. The separation in developmental time of mRNA abundance from a later peak of protein abundance/activity in developing B. napus embryos has been reported previously for enzymes involved in fatty acid metabolism such as enoyl-ACP reductase, a component of the fatty acid synthetase complex (Fawcett et al., 1994), and acyl-CoA synthetase (Pongdontri and Hills, 2001). Importantly, this study shows that measurement of the activity of a transporter is more likely to provide information relevant to interpretation of its role in metabolism during development than can be drawn from measurements of transcript abundance.
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Acknowledgements |
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We thank Professor Alison Smith and Dr Kay Denyer for their constructive comments on the manuscript. Dr Christoph Benning (Michigan State University) is thanked for alerting the authors to the results of the EST analysis on developing Arabidopsis seed prior to its publication. We also thank Dr Joerg Schwender and Professor John Ohlrogge (Michigan State University) for sharing unpublished data from 13C-isotope experiments using isolated B. napus embryos. Dr Steve Bowra provided assistance in isolating the PCR-derived clones of the B. napus phosphate exchange translocators. Dr Peter Geigenberger and Axel Tiessen (MPI, Golm, Germany) are thanked for the kind gift of recombinantly-expressed PPDK. Ian Hagon and his team produced the plant material used in these experiments and the seed of B. napus cv. Topas was a kind gift of Dalgety Agriculture (Essex, UK). This work was supported by the Biotechnology and Biological Sciences Research Council through its Core Strategic Grant to the John Innes Centre and Research Grant 208/D09342 to SR. Additional support came from the European Union Framework V program via research grant QLK3 CT2000 00349, and from Renessen LLC.
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Footnotes |
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* These two authors contributed equally to this study.
Present address: Department of Biology, University of Leicester, Leicester LE1 7RH, UK.
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References |
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