Journal of Experimental Botany, Vol. 54, No. 381, pp. 259-270,
January 2, 2003
© 2003 Oxford University Press
Biochemical and physiological studies of Arabidopsis thaliana transgenic lines with repressed expression of the mitochondrial pyruvate dehydrogenase kinase1
Received 22 April 2002; Accepted 30 August 2002
2 National Research Council of Canada, Plant Biotechnology Institute, 110 Gymnasium Place, Saskatoon, Saskatchewan S7N 0W9, Canada
3 The University of Guelph, Department of Plant Agriculture, Guelph, Ontario N1G 2W1, Canada
1 GenBank/EMBL Accession Number: AJ007312.
4 To whom correspondence should be addressed. Fax: +1 306 975 4839. E-mail: david.taylor{at}nrc.ca
Abbreviations: DAG, diacylglycerol; FFA, free fatty acid; GC, gas chromatography; PA, phosphatidic acid; PC, phosphatidylcholine; PL, polar lipid; SE, standard error; TLE, total lipid extract; TLC, thin layer chromatography; TAG, triacylglycerol.
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Abstract |
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Pyruvate dehydrogenase kinase (PDHK), a negative regulator of the mitochondrial pyruvate dehydrogenase complex (mtPDC), plays a pivotal role in controlling mtPDC activity, and hence, the TCA cycle and cell respiration. Previously, the cloning of a PDHK cDNA from Arabidopsis thaliana and the effects of constitutively down-regulating its expression on plant growth and development has been reported. The first detailed analyses of the biochemical and physiological effects of partial silencing of the mtPDHK in A. thaliana using antisense constructs driven by both constitutive and seed-specific promoters are reported here. The studies revealed an increased level of respiration in leaves of the constitutive antisense PDHK transgenics; an increase in respiration was also found in developing seeds of the seed-specific antisense transgenics. Both constitutive and seed-specific partial silencing of the mtPDHK resulted in increased seed oil content and seed weight at maturity. Feeding 3-14C pyruvate to bolted stems containing siliques (constitutive transgenics), or to isolated siliques or immature seeds (seed-specific transgenics) confirmed a higher rate of incorporation of radiolabel into all seed lipid species, particularly triacylglycerols. Neither constitutive nor seed-specific partial silencing of PDHK negatively affected overall silique and seed development. Instead, oil and seed yield, and overall plant productivity were improved. These findings suggest that a partial reduction of the repression of the mtPDC by antisense PDHK expression can alter carbon flux and, in particular, the contribution of carbon moieties from pyruvate to fatty acid biosynthesis and storage lipid accumulation in developing seeds, implicating a role for mtPDC in fatty acid biosynthesis in seeds.
Key words: Arabidopsis thaliana, early flowering, mitochondrial pyruvate dehydrogenase complex, plant development, pyruvate dehydrogenase kinase (EC 2.7.1.99), respiration, seed oil content, seed weight.
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Introduction |
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The pyruvate dehydrogenase complex (PDC) is a large multienzyme system catalysing the oxidative decarboxylation of pyruvate with the concomitant release of acetyl-CoA and reduction of NAD+ through the following reaction sequence: pyruvate+NAD++CoA-SH
acetyl-CoA+NADH+CO2. The complex contains three primary components: pyruvate dehydrogenase (PDH; E1, EC 1.2.4.1); dihydrolipoamide transacetylase (E2, EC 2.3.1.12); and dihydrolipoamide dehydrogenase (E3, EC 1.8.1.4). Plants are unique in having PDCs in two isoforms, one located in the mitochondrial matrix as in other eukaryotic cells, and the other located in the plastid stroma (Lernmark and Gardeström, 1994). Although both plastidic and mitochondrial PDC (mtPDC) isoforms are very sensitive to product feedback regulation (by NADH and acetyl-CoA), only the mtPDC is regulated through inactivation/reactivation by phosphorylation/dephosphorylation (Randall et al., 1981; Budde et al., 1988). Current knowledge about the molecular structure of plant mtPDCs is largely based on studies of its mammalian counterparts. As in mammalian systems, plant mtPDC contains the regulatory proteins, PDH kinase (PDHK, EC 2.7.1.99) and the PDH phosphatase (PDHP, EC 3.1.3.43) (Budde and Randall, 1990). PDHK is the negative regulator of the PDC. PDH has an 
2ß2 structure and it is the E1 component which is phosphorylated by PDHK, resulting in the inactivation of PDH. PDHP reactivates PDH through dephosphorylation. The in situ steady-state level of phosphorylation of the E1 subunit can be regulated by pyruvate, ATP, and the particular substrates being oxidized by the mitochondria (Budde et al., 1988). Light also exerts an indirect effect on the inhibition of mtPDC activity, likely associated with photosynthesis and/or photorespiration (Budde and Randall, 1990). Maximum PDC activity appears to vary developmentally, with the highest catalytic activity observed during seed germination and early seedling development (Hill et al., 1992; Grof et al., 1995). MtPDC acts as a link between glycolytic carbon metabolism and the tricarboxylic acid (TCA) cycle. Because of the irreversible nature of the reaction, PDC is a particularly important site for metabolic regulation. The importance of the TCA cycle in plant development, especially for the initiation of the reproductive phase of plant growth has been demonstrated (Landschütze et al., 1995). In that study, antisense repression of the mitochondrial citrate synthase gene resulted in the delayed flowering phenotype and a specific disintegration of the ovary tissues of flowers. The reaction catalysed by plastidial PDC provides acetyl-CoA for fatty acid biosynthesis both in vegetative and reproductive tissues, including seeds, but a role for mtPDC in fatty acid biosynthesis in seeds has not been conclusively demonstrated.
Based on the sequence information from the molecular characterization of mammalian mtPDCs, the cloning of a number of plant cDNAs encoding mtPDC components has been reported (Luethy et al., 1994, 1995; Grof et al., 1995; Guan et al., 1995; Thelen et al., 1998a, b, 1999; Zou and Taylor, 1998; Zou et al., 1999). Rather than typical Ser/Thr kinase domains for phosphorylation, PDHKs from plants contain five conserved domains more characteristic of prokaryotic two-component histidine kinases (Thelen et al., 1998a, 2000; Zou et al., 1999). Prokaryotic histidine kinases typically autophosphorylate a His residue, followed by phosphotransfer to Asp or Glu residues of their response regulator protein, thereby inducing signal transduction (Stock et al., 1989). Recently, through the use of histidine modifying agents, it has been shown that in the A. thaliana PDCK, His residues are critical for both the autophosphorylation and the phosphotransfer mechanisms associated with PDCK, and thus confirmed the A. thaliana PDHK to be a His kinase (Mooney et al., 2000). Recombinant A. thaliana PDCK was shown to inactivate kinase-depleted corn mtPDC, by phosphorylating Ser residues. When using site-directed mutagenesis on a conserved His-121 residue (changed to alanine or glutamine), both the autophosphorylation and transphosphorylation functions of the PDCK were reduced by about 50%, implicating the His-121 residue as at least one residue critical for PDCK function. By covalent alkylation of both His residues 121 and 168, serine phosphorylation and kinase function were reduced to 0–12%, thereby implicating both His 121 and His 168 (the only two invariant His residues amongst the cloned PDHKs) as essential for complete PDCK function (Thelen et al., 2000).
In a previous study, using a transgenic approach to characterize mtPDHK, it was demonstrated that the activity of the A. thaliana PDC could be manipulated by modulating the expression level of its regulator, the PDCK gene. The effects of partial repression of PDHK expression on plant growth and development were described for transgenic plants expressing an antisense construct encoding an A. thaliana PDHK (AtPDHK) cDNA using a CaMV 35S promoter for constitutive expression in the plant. Constitutive antisense expression of the AtPDHK cDNA in transgenic plants resulted in reduced PDHK expression and elevated PDC activity based on measurements of in vitro enzyme activity. These transgenic plants also had an altered growth phenotype with reduced accumulation of vegetative mass, early flower initiation, and a shortened generation time (Zou et al., 1999). Immunoblot analyses of control and transgenic antisense PDHK mitochondrial protein preparations were probed with a monoclonal antibody to the E1 subunit of maize mitochondrial PDH. The maize antibody had shown a strong cross-reactivity with the Arabidopsis 43 kDa PDH E1 subunit (Thelen et al., 1998b). However, as reported previously, a comparison of non-transformed wild-type (n-WT), pBI121 plasmid-only transgenic control and antisense PDHK transgenic line 9-5 mitochondrial proteins showed that there was no evidence that the amount of the E1 subunit was increased in the antisense PDHK transgenic line. Similar results were obtained in western analyses of the other antisense PDHK transgenic lines. Therefore the increase in mtPDC activity was not associated with an increase in the amount of PDH protein, but in the level of activated enzyme (Zou et al., 1999). This previous work did not examine whether the metabolic flux through the reaction catalysed by PDC was actually increased in the transgenics.
Studies of the A. thaliana antisense PDHK transgenic plants that contain the AtPDHK cDNA under the control of either a constitutive or seed-specific promoter are reported here. Particular emphasis is placed on the physiological and biochemical changes and alterations of carbon flux resulting from altered mtPDHK activity and it is shown that these changes can lead to altered phenotypes exhibiting increased harvest index, seed storage lipids and seed weight, thereby improving plant productivity. Further more, the data also support the involvement of mtPDC in fatty acid biosynthesis in developing seeds.
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Plant material
Arabidopsis thaliana plants, ecotype Columbia, both wild type and transgenics, were grown at the same time on Terra-lite Redi-earth (WR Grace & Co., Canada Ltd., Ajax, ON) in a growth chamber set at 22 °C with a diurnal photoperiod of 16 h light (200 µE m–2 s–1) and 8 h dark, unless otherwise stated. Under these growth conditions, one generation of wild-type Columbia plants took 75–77 d (Zou et al., 1999).
Molecular cloning of the AtPDCK gene and production of transgenic plants
General molecular techniques such as plasmid DNA isolation, restriction digestion, modification and ligation of DNA, polymerase chain reaction, agarose gel electrophoresis, Northern blots, transformation and culture of E. coli were carried out according to standards protocols (Sambrook et al., 1989).
An Arabidopsis mtPDHK clone (YA5) previously isolated and characterized, was first cloned in an antisense orientation behind the double 35S promoter and used to transform Arabidopsis plants with Agrobacterium as described (Zou et al., 1999).
For seed-specific down-regulation, the same fragment (975 bp) of the PDHK YA5 clone was inserted in an antisense orientation behind the napin promoter of the pDH 1 vector (a customized vector derived from pUC19) at the XbaI and BamHI sites. The orientation of the PDCK fragment in the pDH 1 vector was verified by restriction digestion and DNA sequencing. A HindIII/EcoRI digestion excised the expression cassette which was then ligated to the corresponding sites of the transforming vector pRD400 (Datla et al., 1992; courtesy of Dr R Datla, PBI/NRC). Agrobacterium transformation (strain GV3101 pMP10) and plant vacuum infiltration were the same as described previously (Zou et al., 1999), with the plasmid-only control being pRD400.
Selection of transgenic plants, propagation and the convention for designating the transgenic generation (T1, T2, T3, etc) are as described by Katavic et al. (1994).
For this study, the analyses were performed on progeny of the T3 generation. The analysis of the transgenic plants, pyruvate dehydrogenase and citrate synthase enzyme assays were conducted as described by Zou et al. (1999). Seed oil content and lipid species analyses were performed as cited previously (Katavic et al., 1995; Zou et al., 1997).
Substrate incorporation studies
Feeding experiments were conducted on either developing siliques, as in the case of the constitutive antisense transgenic line 9-5-A, and its corresponding plasmid-only control (pBI121), or on both developing siliques and individual immature seeds from the seed-specific antisense transgenic line 5-6-E, and its corresponding plasmid-only control (pRD400). All experiments were carried out twice.
For silique feedings, bolted stems were harvested 2 weeks after the first flower and fed at room temperature with 1 µCi 3-14C sodium pyruvate (American Radiolabelled Chemicals) in 100 µl water, followed by a 500 µl ‘chase’ of distilled water, and incubated for 48 h in a growth chamber as described above. For each line, siliques were harvested and pooled within the same stage of development, numbered from one to eight as described previously ((Zou et al., 1996) for preparation of a total lipid extract (TLE). The different families of lipids in the TLE were resolved by thin layer chromatography (TLC). The silica gel areas corresponding to triacylglycerols (TAGs), diacylglycerols (DAGs), free fatty acids (FFAs), and polar lipids (PLs; primarily phosphatidylcholines (PCs) and phosphatidic acids (PAs)) were scraped and placed in scintillation vials with 7 ml of Omni-Solv (DuPont) to assess the incorporation of radioactivity using a scintillation counter (LSC 1219 Rackbeta, LKB Wallac) as described previously (Taylor et al., 1992).
In the case of immature seed feedings, two weeks after the first flower about 10 developing seeds were excised from three to four siliques of the primary stem, and pooled within the same developmental stage. Each pool of seeds was incubated in 2 µCi 3-14C sodium pyruvate or 5 µCi U-14C sucrose (American Radiolabeled Chemicals) in 500 µl HEPES pH 7.4, for 20 h in the light, at 25 °C, with shaking at 100 rpm, and washed three times with distilled water before preparing the TLE as described by Taylor et al. (1992). A portion of the TLE was transmethylated using 3 N methanolic HCl and the resulting fatty acid methyl esters (FAMEs) were redissolved in acetonitrile and separated using reverse-phase radio-HPLC analysis as described previously (Taylor et al., 1992).
Respiration measurements
Transgenic lines 3-1-J, 9-5-A, 10-4-C (constitutive expression) and the pBI121 plasmid-only control, and transgenic lines 1-1-B, 5-6-E, 10-2-K (seed specific expression) and pRD400 plasmid-only control were grown under environmental conditions favouring the full development of the leaves, i.e. short days (12 h light), in order to facilitate gas exchange measurements. Individual leaves or 25–30 fully immature siliques containing embryos at mid-development (developmental stages 5 to 6 as described previously (Zou et al., 1996) were placed in a cuvette linked to a Li-Cor (Lincoln, NE 68504) model LI-6400 portable gas exchange system (open gas exchange system) for respiration and photosynthetic measurements. It was necessary to humidify the air going into the leaf cuvette for the Arabidopsis measurements to maintain a vapour pressure deficit (VPD) between the leaf and air of 1–1.2 kPa (at the temperature used for the measurements, this is equivalent to around 60% RH). The humidity was maintained by using a system involving two water baths where the incoming air from the growth chamber was first passed through a flask containing distilled water at 25 °C to add water to the air, and the air was then passed through a coil in a second water bath set at a lower temperature to give the appropriate water vapour pressure in the air. (The Arabidopsis leaves do not transpire enough to depend only on that water vapour source in the chamber.)
Gas exchange measurements were performed after the stabilization of the CO2 exchange rate, i.e. 10–15 min after the leaf or siliques were placed in the cuvette. Respiration rates were determined in two ways. First, during the daylight cycle, the CO2 exchange rate was measured with the plant material in the cuvette exposed to normal light, and then again with the cuvette covered to prevent any exposure to light. Second, during the dark period, the plant material was previously in the dark for at least 2 h prior to measurements. In the latter case, the whole plant was already acclimated to dark conditions, whereas in the first case, only the measured leaf was placed in the dark. All gas exchange measurements were performed on eight sets of leaves and on four to six sets of siliques for each line studied. To calculate CO2 exchange rates, the leaf area in the cuvette was determined by tracing the leaf, and for inflorescences the portion of inflorescence measured was cut, dried at 60 °C, and the weight determined. Dry weights of leaves, inflorescences and roots were determined by drying the tissue at 60 °C for 3 d and then weighing the dried tissue.
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Studies of the tandem 35S:antisense AtPDHK transgenic lines
In the present study, three of the constitutively repressed PDHK lines (3-1-J, 9-5-A and 10-4-C) were selected, as well as the non-transformed wild type (n-WT) and the plasmid only control (pBI121), and the respiration rates of leaves and siliques were measured using a Li-Cor gas measuring system. As shown in Fig. 1A, there was a significant increase (up to 1.6-fold) in dark respiration of the leaves of the constitutively-repressed antisense PDHK transgenic lines compared to the non-transformed wild type (n-WT) and to the plasmid-only (pBI121) transgenic control lines. There was also some indication that leaf photosynthetic rates were lower in these PDHK transgenic lines, but the differences were not statistically significant.
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Figure 1B shows that constitutively-repressed antisense PDHK plants exhibited a significantly reduced leaf dry weight (40–60% of that of the controls). Root and inflorescence dry weights were reduced in the PDHK transgenics compared to both controls, most notably in transgenic line 9-5-A .
It is important to note that the leaf measurements were performed on plants grown with 16 h light and 8 h dark. Thus the rates of photosynthesis can automatically be doubled to normalize the rates of photosynthesis versus respiration over time. In addition, the rates of respiration were made on plants in the first half of the dark period, and thus it is unlikely that the respiration rates measured occurred over the whole night period, since starch in the leaf can be depleted relatively quickly at night. Certainly the size of the leaves was greatly reduced in the constitutive antisense transgenics, indicating that the net difference between the average absolute values of photosynthesis and respiration in the two controls versus the three transgenics was slightly lower in the transgenics, as the data indicate.
The increased dark respiration in leaves of the antisense PDHK transgenics was directly correlated with increased relative activities of mitochondrial PDC and of citrate synthase in the TCA cycle (Fig. 2). Since mtPDC represents the primary entry point of carbohydrates into the TCA cycle, it also plays an important role in modulating this respiratory process. In contrast to leaves, the difference in the rate of dark respiration in developing siliques (seeds) of the constitutively-repressed antisense PDHK transgenic lines was not significantly different compared to the non-transformed control or pBI121 plasmid-only transgenic control lines (Fig. 3).
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When the mature seeds from these constitutively repressed PDHK lines were examined, two consistent trends were found (1) a significant increase in the accumulation of seed storage lipids on a per-100-seed basis (Fig. 4) and (2) an increase in the average 1000-seed weight (Fig. 5).
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In order to study the link between respiratory flux, lipid deposition and sink size in more detail, and to facilitate the interpretation of data, antisense line 9-5-A was chosen because it had a single insert and was homozygous for the antisense trait. Transgenic line 9-5-A transformed with the constitutively-expressed antisense PDHK construct exhibited increases of 13% and 20% in seed oil content and seed weight, respectively, in the T3 generation, compared with n-WT and pBI121 control transformants. Furthermore, the average number of siliques per 15 cm segment of bolted stem (30±3) and the average number of seeds per silique (50±7) were not significantly different from the n-WT and pBI121 control transgenics. Though it flowered earlier (Fig. 6A) and reached maturity sooner (68–70 d versus 75–77 d for pBI121 controls) (Zou et al., 1999), the inflorescence of the antisense PDHK line 9-5-A was as robust at maturity as the pBI121 and non-transformed control plants (Fig. 6B). This indicated that seed yield was not adversely affected in the antisense PDHK transformants. In fact, the harvest index is increased in the constitutively-repressed PDHK transgenics despite the fact that leaf and root dry weights are significantly reduced (Fig. 1A). The reduction in leaf dry weight is consistent with the fewer and smaller rosette leaves, as reported previously (Zou et al., 1999).
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To study carbon flux into seed storage lipids, bolted stems from antisense PDHK line 9-5-A and pBI121 control transgenics weighing 85–90 mg FW and containing 24–27 developing siliques were fed with 3-14C pyruvate. The results clearly show that there was a general increase in radiolabel found in all lipid classes, but in particular, 3- and 4-fold increases in the accumulation of radiolabelled polar lipids and TAGs, respectively (Fig. 7).
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Studies of the napin:antisense AtPDHK transgenic lines
Once again, from a group of transgenics, three napin:antisense PDHK lines (1-1-B, 5-6-E and 10-2-K) were chosen together with the corresponding non-transformed wild-type control (n-WT) and plasmid only transgenic control line pRD400, for more detailed studies. Physiologically, the antisense PDHK transgenic lines, where the construct was driven by a seed-specific promoter (napin), were indistinguishable from the pRD400 and non-transformed controls with respect to plant development: rosette leaf size and number, flowering, and generation time were unaffected (data not shown). While leaf respiration was not significantly different than the controls, respiration in the developing siliques of the napin:antisense PDHK transgenic lines was consistently higher (Fig. 8). In particular, lines 5-6-E and 10-2-K were statistically different at a 95% confidence level. Based on the absolute values for n-WT, the three antisense PDHK lines 1-1B, 5-6-E and 10-2-K showed rates of respiration of 0.010, 0.011 and 0.013 µmol CO2 s–1 g–1 DW greater than the n-WT control.
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The connection to seed lipid deposition was able to be studied more directly by using transgenic lines wherein the repressed PDHK expression was seed-specific. Once again, a significant increase was observed in the accumulation of seed storage lipids on a per 100-seed basis (cf. Fig. 4) and an increase in the average 1000-seed weight (cf. Fig. 5).
Single insert homozygous line 5-6-E was chosen to be studied in more detail, having exhibited 50% and 25% increments in seed oil content and seed weight, respectively, in the T3 generation, compared with n-WT controls and the pRD400 control transformants. Line 5-6-E developed identically to wild type and the pRD400 controls, with no visible vegetative or reproductive phenotype. Like the controls, line 5-6-E matured at about 77 d after planting; the number of siliques per plant and number of seeds per silique were also indistinguishable from those of n-WT and pRD400 plants.
Green siliques were harvested at specific developmental stages (as described by Zou et al., 1996) and fed with 3-14C pyruvate. The results indicated that as the feedings were conducted on the less mature (stages 3 and 4) to the mid-developing (stages 5 and 6) siliques, there was a progressive increase in the 14C label found in total lipid extracts of the transgenic line compared to the pRD400 control. At stages 3 and 4, the increase was the strongest in the PL and DAG fractions; by stages 5 and 6, the relative increase of radiolabel was most obvious in the TAG fraction of Nap-5-6 (Fig. 9). These gradual changes in the size of the labelled pools of each lipid class generally followed a progressive movement of label through the known Kennedy pathway intermediates and into TAGs, as the developing seeds matured.
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To measure the flux of carbon from pyruvate directly into developing embryos, without a contribution or effect of the maternal silique tissues, embryos were harvested from the various stages of developing siliques and the embryos incubated directly, with shaking in the presence of 3-14C pyruvate for 20 h. Previous time-course studies on the incorporation of labelled pyruvate and sucrose into components of immature seeds showed that over a period of 18 h, the incorporation rate was linear (Focks and Benning, 1998). Once again, the antisense napin:PDHK transgenic line exhibited a dramatic increase in label incorporation into seed lipids (radio-specific activity of the lipid pool) at the mid-development stages (5 and 6) compared to the corresponding lipid pool found in the pRD400 control embryos (Fig. 0
). By stage 7, there continued to be a very strong increase in the size of the endogenous fatty acid pools. Line 5-6-E at stage 7 exhibited a 45% increase compared to stage 6, while the pRD400 line showed a smaller, but significant corresponding increase from stage 6 to stage 7 (30%). Thus, even though the activity in line 5-6-E is still about 2.5–3-fold higher than in pRD400 seeds, the specific activity on a µmol basis is strongly reduced in both lines. A similar trend of increased movement of carbon into seed storage lipids was observed when the transgenic line 5-6-E was fed with radiolabelled sucrose, except that the accumulation pattern was typically maximized one to two stages behind that observed during direct pyruvate feeding (data not shown). This is not unexpected given that sucrose must first be broken down by glycolysis before providing pyruvate moieties for fatty acid biosynthesis and other biosynthetic processes.
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While the flux of acyl moieties into seed lipids was enhanced in the napin:antisense PDHK lines, the acyl composition of TAGs and other seed lipids in mature seeds was unchanged. For example, the proportions of saturates, mono-unsaturates, polyunsaturates and very long chain fatty acids (C20 or >) were, repectively, 13.3±0.4, 34.2±0.7, 52.5±0.9, and 23.1±0.8 in the pRD400 controls versus 13.5±0.4, 35.8±1.0, 50.7±1.0, and 23.2±0.4 in the napin:antisense PDHK line 5-6-E.
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Discussion |
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Biochemically, respiration can be said to involve glycolysis, the oxidative pentose phosphate pathway, the TCA cycle, and the mitochondrial electron transport system. In addition to generating energy and reducing equivalents, the respiration process also provides an array of intermediates (carbon skeletons) as the building blocks for many essential biosynthetic processes. Mitochondrial PDC acts as a link between glycolytic carbon metabolism and the TCA cycle. Because of the irreversible nature of the reaction, PDC is a particularly important site for metabolic regulation. Acetyl-CoA, the product of PDC, is the starting point for the synthesis of fatty acids, isoprenoids and a number of secondary products.
The present study is the first to demonstrate that partial silencing of PDHK can increase the metabolic flux through the reaction catalysed by mtPDC, based on measured increases in respiratory CO2 evolution and fatty acid production, in the anti-sense PDHK transgenics. In a previous study (Zou et al., 1999), it was noted that flowering time could be hastened by reducing PDHK expression, and it was hypothesized that this was due to enhanced availability of mitochondrial acetyl-CoA via increased pyruvate oxidation, and correlated with increased TCA cycle activity. These data supported earlier findings (Landschütze et al., 1995) suggesting that flowering time could be delayed by reduced TCA cycle activity and reduced respiration. This is in general agreement with the observation that an increase in the number of mitochondria and respiratory activity in the shoot apex upon induction of flowering appears to be causally linked to flower formation (Havelange, 1980). However, respiration rates had not been directly measured in the transgenic lines, and such measurements would be necessary to test the hypothesis.
Collectively, the results in this and a previous study (Zou et al., 1999) where partial silencing of PDHK was achieved by an antisense approach, indicated that the antisense transgenics exhibited higher PDC activity, higher respiration rates, higher TCA enzyme activities and, in the case of the constitutively expressed antisense transgenics (e.g. 35S35S:antisense line 9-5-A), earlier flowering and shorter maturity times. Therefore, these data support the earlier conclusion by Landschütze et al. (1995), that the TCA cycle is of major importance during the transition from the vegetative to the generative phase and that the regulation of PDC activity directly affects mitochondrial respiration. As far as is known, the current results achieved by down-regulating the expression of a regulator of mitochondrial respiration, rather than focusing on a specific catabolic enzyme in the respiratory pathway, are the first to support this hypothesis.
The increase in respiratory CO2 evolution measured for the intact siliques of the seed-specific anti-sense PDHK trangenics can be explained by the theoretical respiratory CO2 evolution associated with increased seed oil production in these transformants. Using an average increase of 300 µg total fatty acids per 100 seeds as a basis for the calculation (cf. Fig. 4), and assuming that each triacylglycerol has an average chemical formula of C57H110O6 (MW=890; i.e. 3 C18 fatty acids attached to a glycerol backbone), it can be calculated that there is an increase of 336 nmol of triacylglycerol per 100 seeds or 1000 nmol of C18 fatty acids per 100 seeds. Since there is 1 pyruvate required per 2 carbons in the fatty acid skeleton, there are 9 mol of pyruvate required per C18 fatty acid, or approximately 9 µmol (1000 nmolx9) of additional pyruvate for the extra 300 µg of fatty acids. Since there is one CO2 released per pyruvate oxidized by the PDC, this is equivalent to an extra 9 µmol CO2 evolved per 300 µg fatty acids. In addition, the extra respiratory CO2 evolution expected in response to the NADPH and ATP requirements for this additional fatty acid synthesis can be calculated. For each C18 fatty acid synthesized, there are 16 NADPH required and approximately 19 ATP (1 ATP per conversion of acetyl-CoA to malonyl Co-A (8ATP per C18 fatty acid), 1 ATP per acetyl-CoA shunted out of the mitochondria (9 ATP per C18), and 2 ATP per ester linkage between fatty acids and glycerol. To generate 16 NADPH equivalents and 19 ATP, considering glycolysis and then either the TCA cycle or the oxidative pentose phosphate pathway as sources of reducing equivalents and energy, would require approximately another 9 mol of CO2 released per mol C18 fatty acid formed (or another 9 µmol CO2 evolved per 300 µg fatty acids). Therefore, a total of 18 µmol CO2 would be evolved per 300 µg fatty acids formed. Since an average A. thaliana seed weighs 20 µg, 100 seeds is equivalent to 2 mg. Also, assuming that oil deposition in a seed occurs over a 4 week period (2.4 million seconds), the average rate of CO2 evolution would be 18 µmol CO2 per 2 mg per 2.4 million seconds or 0.004 µmol CO2 g–1 dry weight s–1. From Fig. 8, it can be calculated that the average increase in silique respiration rates for the seed-specific PDHK transgenics compared to the nt-WT control is 0.011 µmol CO2 s–1 g–1 dry weight. This calculation demonstrates that the increased respiratory rate can more than account for that required to support the increased oil accumulation in the developing seeds. Thus, greater respiration is occurring overall in the antisense PDHK transgenic seeds both for oil production and general respiration.
Using isolated intact plastids, it has been shown that acetate is the preferred substrate for fatty acid synthesis, and there is evidence that a multienzyme system including acetyl-CoA synthetase and acetyl-CoA carboxylase, exists in plastids, which channels acetate into lipids (Roughan and Ohlrogge, 1996). It is almost certain that at least some of the acetyl-CoA in plastids is formed by plastidic pyruvate dehydrogenase, using pyruvate imported from the cytosol or produced locally by plastidial glycolysis. A further possibility, especially in non-photosynthetic tissues (e.g. roots and developing embryos), is that acetyl-CoA, generated in the mitochondria, is an alternative means to provide acetate moieties for fatty acid synthesis (Ohlrogge and Browse, 1995). Mitochondrially-generated acetyl-CoA could be hydrolysed to yield free acetate, which could move into the plastid for conversion to acetyl-CoA via plastidial acetyl-CoA synthetase, an enzyme with 5-15-fold higher activity than the in vivo rate of fatty acid synthesis (Roughan and Ohlrogge, 1994). Liedvogel (1986) demonstrated the presence of a short-chain acyl-CoA hydrolase localized specifically in the matrix space of spinach mitochondria. It is inferred, therefore, that acetyl-CoA in the mitochondria is hydrolysed by this enzyme to yield free acetate. Free acetate may move to the plastid for conversion to acetyl-CoA by a plastidic acetyl-CoA synthase, thereby contributing, in part, to the acetyl-CoA pool required for fatty acid biosynthesis. Alternatively, the mitochondrial acetyl-CoA could be converted to acetylcarnitine and transported directly into the plastid. Hence, in theory, the mitochondrial pyruvate dehydrogenase complex has an important role to play in fatty acid biosynthesis.
One of our major interests in pursuing the characterization of the AtPDHK gene and the results of its partial silencing, has been to determine whether or not mtPDH plays a role in converting pyruvate to acetate moieties which may contribute to fatty acid biosynthesis in the primary lipid sink of oilseeds (developing embryos). Results in the present study support the hypothesis that mtPDH plays an important role in fatty acid biosynthesis in the developing embryos of oilseeds.
As shown in the case of the constitutively-silenced PDCK transgenics, silique respiration was not significantly increased, yet these lines still displayed a significant increase in lipid accumulation in the seeds. It is believed that this is because these lines have a much reduced leaf area and overall rate of photosynthesis; thus carbon supply to the silique is probably reduced in these plants. Therefore, the increased lipid level cannot be attributed to the increased level of NADH or ATP, resulting from an increased TCA activity and respiration rate (a secondary effect of the elevated PDC activity). Rather, the radiolabelling data support the hypothesis that the observed increase in seed lipid biosynthesis results from an increased supply of acetyl-CoA from the mitochondria due to enhanced pyruvate decarboxylation.
Given that there is some evidence that plants can sense and alter gene expression in response to the size of metabolic pools (e.g. organic acids; see Landschütze et al., 1995), it is possible that some of the effects observed in the present study might be secondary consequences of perturbed metabolite (e.g. organic acid) contents. Further studies are required to examine this possibility.
In any case, these data provide evidence that alterations in PDC activity in the mitochondria can act as a signal to affect lipid biosynthesis in seeds by a mechanism not directly related to increased respiratory activity.
To summarize, the collective data from the present studies conducted with transgenic lines where PDHK was partially silenced in both a constitutive and a seed-specific manner support the hypothesis that enhanced mitochondrial PDC activity can result in an increased availability of acetyl moities from mitochondrial pyruvate turnover which are utilized in the synthesis of fatty acids and storage lipids in the developing seed. The proof of this hypothesis has been hindered until now. This question has been addressed directly using a transgenic approach. The antisense PDHK transgenics exhibit an increase in fatty acid biosynthesis and accumulation as well as a significant increase in the average seed weight of the progeny.
When looking at the constitutive antisense PDHK transgenics, it is indeed interesting that, despite an altered vegetative phenotype (smaller leaf size and number) and a significant increase in leaf (but not silique) respiration, there was no penalty with respect to seed yield and oil deposition. On the contrary, both traits showed increases in the antisense PDHK transgenics. The same can be said with respect to the seed-specific antisense PDHK transgenics, wherein fatty acid biosynthesis, lipid deposition and sink size (seed weight) were all increased over that observed in the seed of plasmid-only transgenic controls (silique respiration was increased, although respiration in the vegetative tissues was not affected).
These findings contrast with those observed in plants where sink tissues accumulate starch (e.g. potato tubers). Starch biosynthesis takes place exclusively in plastids that are the sole location for starch synthases and starch branching enzymes (Preiss, 1997). While current understanding of how sucrose to starch conversion is regulated in potato tubers is limited, some transgenic studies have shed some light on this process. Starch deposition has been altered by genetic engineering. By expression of a mutant E. coli glgC16 gene encoding an ADP glucose pyrophosphorylase in potato tubers, an increase in starch accumulation was observed (Stark et al., 1992). More recently, studies of potato tubers in which the expression of the plastidic adenylate transporter was down-regulated, revealed that tubers contained drastically reduced starch content, starch grains of only about 50% normal size, and an increased level of soluble sugars, all of which was accompanied by a 2-fold increase in the respiration rate observed in tuber slices and a large reduction in tuber volumetric weight (Tjaden et al., 1998; Geigenberger et al., 2001). In another study, it was shown that combined over-expression of a yeast invertase and a glucokinase from Zhymomonas mobilis, in potato tubers resulted in a large increase in glycolytic metabolites, 2–3-fold increases in the activities of key enzymes of respiratory pathways and 3–5-fold increases in carbon dioxide production. This increased respiration resulted in a dramatic decrease in starch content. The double transgenic lines showed starch levels that were as little as 35% of that found in wild-type tubers (Trethewey et al., 1998).
The fundamental difference in productivity observed between the A. thaliana antisense PDHK transgenics (this study) and transgenically engineered potatoes may lie in the nature of the storage product accumulated in developing seed embryos and tubers, the respective sink organs, and also in the fact that the result of partial silencing of PDHK is an increase in acetate moieties, the building blocks for fatty acid synthesis. In the case of organs storing starch, there is a requirement for sucrose moieties for conversion to starch in the plastids. Any process which causes an unexpected turnover in fixed carbon to CO2 (e.g. higher respiration in tuber tissue) will detrimentally affect the proportion of sucrose destined for starch biosynthesis, and the size or volumetric weight of the sink (tuber).
In contrast, A. thaliana, like other members of the Brassicaceae, accumulates oil (triacylglycerol) as the primary storage component in its seeds. It has been shown that in rapeseed embryos, there is an import of sucrose for the biosynthesis of starch which occurs very early in embryo development (at the ‘milky’ stage); however, this starch is quickly broken down to provide fixed carbon as acetate, for fatty acid and storage lipid bioassembly as the embryo reaches mid-maturity (Kang and Rawsthorne, 1994). Indeed, mature Brassica seeds contain little or no detectable starch. Yet this initial import and metabolism of carbohydrate is essential for the eventual switchover to fatty acid biosynthesis and storage lipid deposition: Focks and Benning (1998) have shown that an A. thaliana mutant defective in the seed-specific regulation of carbohydrate metabolism develop wrinkled seeds with drastically reduced oil content. In another study, overexpressing ADP glucose pyrophosphorylase in canola prolonged the period of starch accumulation at the expense of oil biosynthesis (Boddupalli et al., 1995).
These studies, in which the mtPDHK is partially silenced, strongly support the hypothesis that if mitochondrial PDC activity is increased, the resulting enhanced conversion of pyruvate to acetate results in a net increment in the size of the mitochondrially-generated acetate pool available for lipid biosynthesis. Thus the antisense PDHK transgenics exhibit both an increase in lipid deposition and sink size (seed weight).
Besides the increased productivity observed as a result of PDHK silencing, there are other potentially important applications of this concept for crop improvement. Given that the generation time in Arabidopsis control plants is about 76 d under the growth conditions used in these studies, a 7 d earlier flowering and earlier maturing phenotype in the antisense PDHK plants represents a shortening of generation time by about 10%. Similar modification of flowering time to extend the geographical range of cultivation is an important goal for Brassica crops (Lagercrantz et al., 1996). In related Brassicaceae (e.g. canola), this would provide the advantage of an earlier harvest and permit more northerly cultivation (Murphy and Scarth, 1994). Late season frost damage in temperate climates (e.g. on the Canadian Prairies) could be avoided with cultivars that mature earlier, and this could also significantly alleviate problems associated with late-season clearing of chlorophyll from the maturing seeds (which can lead to ‘green oil’ during processing and necessitate expensive bleaching steps).
Yet another application of silencing the negative regulation of mitochondrial PDC may be to increase respiration in sink tissues (e.g. potato tubers transformed with an antisense PDHK construct under control of the strong tissue-specific promoter, patatin B33; Trethewey et al., 1998), thereby reducing starch deposition while providing additional acetyl-CoA moieties for the biosynthesis and deposition of plant-produced bioplymers, including bioplastics (e.g. based on potatoes transformed with bacterial gene(s) allowing acetate to be re-channelled into polyhydroxyalkanoates; (Padgette et al., 1997).
There is still an important question that remains to be answered: during the partial silencing of the negative regulator of mtPDC, can an increased synthesis of fatty acids occur directly within the mitochondria and, if so, can these fatty acids be exported to the ER for triacylglycerol bioassembly? An answer to this question is important in the light of reports by other researchers (Wada et al., 1997; Gueguen et al., 2000), regarding the presence of fatty acid synthase machinery within the mitochondrial matrix, and the capacity for this organelle to make 16:0-ACP and 18:0-ACP, for example (Gueguen et al., 2000). However, given the logistics of extracting significant numbers of highly purified mitochondria to address this question, these experiments must await the production of antisense PDHK transgenic lines in a higher Brassica species, like B. napus canola, a project currently underway in this laboratory.
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The authors gratefully acknowledge M Giblin, D Barton, D Reed, and Dr V Katavic for technical assistance, and D Schwab, B Panchuk and Dr L Pelcher of the PBI DNA Technology Group for primer synthesis and DNA sequencing. We thank Dr WA Keller for his critical review of this manuscript. This is National Research Council of Canada Publication No. 43919.
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