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Seed-specific expression of a bacterial phosphoenolpyruvate carboxylase in Vicia narbonensis increases protein content and improves carbon economy

Seed-specific expression of a bacterial phosphoenolpyruvate carboxylase in Vicia narbonensis... <h1>Introduction</h1> Legume seeds are an important source of plant-derived protein with economic importance in the worldwide feed and food industry. Increasing the storage protein content of crop seeds is difficult to achieve because conventional breeding always favours yield in general. Biotechnological approaches to manipulate seed protein have introduced foreign or modified endogenous storage protein genes to increase sink demand ( Gueguen and Popineau, 1998 ; Hofman et al ., 1988 ; Saalbach et al ., 1995 ), but with limited success. Consequently, novel approaches are necessary, based on the understanding of the mechanisms that regulate storage protein accumulation. Thus, to elevate seed nitrogen levels, it could be advantageous to focus on assimilate partitioning into the different storage products ( Golombek et al ., 2001 ). Storage proteins are synthesized during the maturation phase of seed development. Amino acid precursors are unloaded from the phloem, and subsequently taken up by the symplasmically isolated embryo ( Miranda et al ., 2001 ). Storage protein synthesis is regulated at different levels. The availability and partitioning of assimilates and nitrogen compounds are of primary importance ( Golombek et al ., 2001 ). Interestingly, much of the control is exerted by the embryo itself, rather than by the maternal plant ( Hayati et al ., 1993 ). Glutamine and/or asparagine are preferentially imported into legume cotyledons ( Miflin and Lea, 1977 ). The biosynthesis of other amino acids requires the provision of carbon skeletons from glycolytic and citric acid cycle products. Some carbon derived from sucrose must therefore be diverted into storage proteins, a process controlled by the anaplerotic reaction of phosphoenolpyruvate carboxylase (PEPC, EC 4.1.1.31) ( Turpin and Weger, 1990 ). PEPC has been investigated in seeds of pea, soybean, wheat and Vicia ( Flinn, 1985 ; Golombek et al ., 1999 ; Gonzalez et al ., 1998 ; Hedley et al ., 1975 ; Smith et al ., 1989 ). The enzyme re-fixes HCO 3 − liberated by respiration and, together with PEP, yields oxaloacetate that can be converted to aspartate, malate or other intermediates of the citric acid cycle. Thus, PEPC controls the anaplerotic carbon flow and can potentially improve seed carbon economy. Correlative evidence points to a rate-limiting role of PEPC in the anaplerotic carbon flow during storage protein synthesis in V. faba ( Golombek et al ., 2001 ). In soybean cultivars differing in seed protein content, PEPC correlates with seed protein content ( Smith et al ., 1989 ; Sugimoto et al ., 1989 ). Thus, storage protein synthesis is partly controlled by the PEPC-catalysed anaplerotic reaction. PEPC is a ubiquitous enzyme in plants which is highly regulated. The allosteric properties of PEPC are subject to opposite and antagonistic effects of metabolites, malate and glucose-6-phosphate. Phosphorylation modulates the metabolic regulation with respect to feedback inhibition by malate ( Jeanneau et al ., 2002 ). Biotechnological approaches using PEPC primarily have aimed to improve CO 2 assimilation by suppressing photorespiration in leaves ( Jeanneau et al ., 2002 ; Ku et al ., 1999 ). Ectopic expression of PEPC should introduce a part of a C 4 -like CO 2 concentrating mechanism in leaf cells. Potato plants were transformed with PEPC from Corynebacterium glutamicum ( Gehlen et al ., 1996 ) or with a genetically engineered potato PEPC with decreased feedback inhibition ( Rademacher et al ., 2002 ) driven by the constitutive 35S promoter. Over-expressers showed a moderate increase in malate and amino acids demonstrating increased anaplerotic fluxes ( Gehlen et al ., 1996 ; Häusler et al ., 1999 ). However, plants were severely disturbed in shoot and tuber growth ( Rademacher et al ., 2002 ). In PEPC over-expressing leaves, the CO 2 assimilation rates and the CO 2 compensation point remained unchanged, partly due to increased CO 2 release from PEP carboxylation products initiated by endogenous cytosolic NAD(P)-malic enzyme (NAD, nicotinamide adenine dinucleotide) ( Häusler et al ., 1999 ). Here, we report on the ectopic expression of C. glutamicum PEPC targeted into maturing seeds of the bean V. narbonensis . A seed storage protein promoter restricted expression to maturing cotyledons where large amounts of storage products were synthesized and probably a high demand of anaplerotic flux was required. The engineered embryos showed a clear stimulation of [ 14 C]-CO 2 uptake and the label was specifically incorporated into proteins, thus indicating a shift in metabolic fluxes from sugars/starch into amino acids. Dry seeds from transgenic lines had a significantly higher content of crude protein (by up to 20% per gram) and a higher seed dry weight. Combining both effects, it was revealed that the protein content per individual seed increased by 40%−50%. It can be concluded that PEPC in seeds is a promising target for plant breeding and biotechnology. <h1>Results</h1> <h2>Seeds expressing Corynebacterium PEPC are larger and contain more protein</h2> A full-length cDNA of C. glutamicum phosphoenolpyruvate carboxylase (CgPEPC, Eikmanns et al ., 1989 ) was cloned under the control of the Legumin B4 promoter ( Bäumlein et al ., 1992 ), which confers seed-specific expression. The cDNA construct was cloned into pGA 482 ( An et al ., 1987 ) and introduced into V. narbonensis using an Agrobacterium -mediated protocol ( Pickardt et al ., 1991 ). A total of 23 F 0 lines were regenerated. In order to obtain stable lines, the plants were allowed to self-pollinate and polymerase chain reaction (PCR)-positive plants were further propagated. Seeds from three independent transgenic lines (PPC-9, PPC-10, PPC-12) of the F 4 to F 6 generation were chosen for further analysis. The expression of the CgPEPC was shown by reverse transcriptase PCR in embryos of the lines PPC-9, PPC-10 and PPC-12 at 25 days after pollination (DAP) ( Figure 1A ). For all lines, vegetative growth and flowering were phenotypically normal. In order to analyse whether the transgenic plants had an altered seed composition, we analysed dry embryos for the content of storage proteins and starch. Compared with wild-type seeds, those from the CgPEPC-expressing lines contained 15%−25% more protein on a per gram basis ( Figure 1B ). The starch content was not significantly changed, although the mean values were slightly lower ( Figure 1C ). Storage protein composition was also analysed after extraction of embryo powder with aqueous buffers. Total globulins, containing the major 7s and 11s storage proteins, were significantly increased by approximately 20% in the transgenic lines compared with the wild-type ( Figure 1D ). Similarly, the albumins were also increased by about 20% ( Figure 1E ). This class includes the sum of water-soluble storage and non-storage proteins. Remarkably, the seeds from all lines were characterized by increased individual dry weight by 20%−30% ( Figure 2A ). To analyse whether higher seed weights were translated into a greater yield per plant, we analysed, from the same batch of growth chamber plants, the total seed number and the total seed weight per plant, as well as the seed number per pod. The seed number per plant was not significantly different for the transgenics, although the mean values were slightly lower ( Figure 2B ). The total seed weight per plant also did not change significantly, but the mean values were slightly higher ( Figure 2C ). However, there was a significant decrease in seed number per pod for the CgPEPC plants by 20%−30% ( Figure 2D ). We believe that the results must be interpreted cautiously with respect to yield because such parameters need to be analysed using field trials. Therefore, at present, it is not clear whether an improved yield can also be expected from CgPEPC expression. In summary, the expression of CgPEPC leads to characteristic changes in seed composition, i.e. an increase in both protein content on a per gram basis and individual seed dry weight. By adding both effects, there is a clear and drastic increase in the crude protein content on an individual seed basis by as much as 40%−50%. <h2>Seeds expressing Corynebacterium PEPC fix more [ 14 C]-CO 2 into proteins</h2> We localized the CgPEPC protein directly in tissue sections by immunohistochemistry. Cotyledons were fixed, embedded in paraplast and sectioned. Immunofluorescence measurements revealed a clear green signal all over the cytoplasm of the cotyledonary parenchyma cells of the transgenic line ( Figure 3A ), whereas no signal was present in the corresponding sections of wild-type cotyledons ( Figure 3B ). This clearly indicates that the bacterial PEPC protein is present in the cotyledonary parenchyma cells of transgenic lines. The detection of CgPEPC protein was not possible by conventional immunoblot analysis using crude extracts, polyclonal antiserum against CgPEPC and a cocktail of proteinase inhibitors within the extraction buffer (data not shown), probably due to rapid degradation of the bacterial protein during extraction. In order to analyse the in vivo PEPC activity, we measured the CO 2 dark fixation. Towards this aim, embryos of wild-type and line PPC-12 at 28–30 DAP were pulse-labelled with [ 14 C]-HCO 3 − . In Figure 3(C) , it is shown that cotyledons of line PPC-12 took up approximately 50% more [ 14 C]-HCO 3 − compared with the wild-type, indicating that the engineered CgPEPC increased the CO 2 dark fixation rate of growing embryos. To further assess the fate of the 14 C label, we analysed the partitioning into the starch and protein fractions, which represent the two major storage products. Remarkably, transgenic embryos partitioned threefold more label into the protein fraction compared with the wild-type ( Figure 3D ). Only a very low amount of the 14 C label appeared in the starch fraction, with no significant difference between the wild-type and PPC-12. The in vitro enzyme assay only revealed a minor increase in PEPC activity in the transgenic plants ( Figure 3E ), probably due to rapid degradation of the bacterial protein during extraction. Our data suggest that the CgPEPC protein is present and physiologically active in maturing embryos and most probably increases the dark fixation rate of [ 14 C]-CO 2 . Furthermore, the higher amount of CO 2 taken up is specifically channelled into the protein fraction of the seeds. <h2>Changed metabolite profiles indicate re-directed carbon flow into organic and amino acids</h2> PEPC activity represents a side-branch of the glycolytic pathway, and may affect carbon flux towards the synthesis of proteins. PEPC carboxylates PEP to oxaloacetate, which can either be converted to aspartate by aspartate aminotransferase or to malate by malate dehydrogenase or, together with pyruvate, can be fed into the citric acid cycle to produce 2-oxo-glutarate (see the general metabolic pathway in Figure 4 ). The latter can serve as a carbon acceptor for amino acid biosynthesis. In order to analyse the changes in metabolic pathways within CgPEPC-expressing seeds, steady state levels were measured for sucrose, free amino acids, intermediates of glycolysis and the citric acid cycle, as well as for adenosine triphosphate and diphosphate ( Table 1 ). There was a general trend towards lower sucrose levels and lower pool sizes of phosphorylated sugars (glucose-6-phosphate, glucose-1-phosphate, fructose-1,6-bisphosphate), but increases in the pool of free amino acids. Levels of nucleotide sugars (uridine diphosphate glucose, adenosine diphosphate glucose) did not change. The acetyl-CoA content was higher in all three lines. There was an overall trend towards higher levels of intermediates of the citric acid cycle. Thus, CgPEPC expression in V. narbonensis seeds leads to changes in the pattern of seed metabolites, indicating a shift of metabolic fluxes from sugars/starch into amino acids. In addition, phosphorylated intermediates from the glycolytic pathway were generally decreased, whereas non-phosphorylated compounds from the citric acid cycle were increased. Such a shift in the ratio of phosphorylated to non-phosphorylated products can be expected because PEPC catalyses the cleavage of inorganic phosphate (Pi) from PEP. Adenosine triphosphate levels were lower, probably due to the higher energy demand by increased protein synthesis. Taken together, increased PEPC in seeds causes characteristic changes in the metabolite pattern, which are consistent with a re-directed carbon flow from the starch synthesis pathway towards the protein synthesis pathway. In addition, we measured nearby enzymes upstream or downstream of PEPC in growing embryos of all three transgenic lines and the wild-type (28–32 DAP) ( Table 2 ). Aspartate aminotransferase was about 30% higher in cotyledons of line PPC-12, but not significantly different from the wild-type in the other lines. NAD-malic enzyme, NAD-malate dehydrogenase and pyruvate kinase were not different. <h1>Discussion</h1> Conventional plant breeding mainly aims to increase yield. However, selection for yield favours a relative increase in the ‘low-energy product’ starch at the expense of protein. Quality parameters, such as the protein content, are very important for human food and animal feedstuffs. Recent studies on seed physiology have pointed to PEPC as a regulator of partitioning of photoassimilates into proteins ( Golombek et al ., 1999, 2001 ). In growing seeds, PEPC re-fixes carbon released by respiration. PEPC catalyses the anaplerotic flux, thereby satisfying the carbon acceptor demands for amino acid biosyntheses (see Figure 4 ). The labelling pattern obtained after short-term feeding of [ 14 C]-CO 2 is consistent with CO 2 dark fixation by PEPC, initially into oxaloacetate, and then into malate and aspartate ( Flinn, 1985 ). In order to increase PEPC in vivo , it is essential to circumvent the down-regulation by endogenous mechanisms, e.g. malate feedback inhibition. This can be achieved either by using an enzyme of non-plant origin, which cannot be regulated by the plant's own mechanisms ( Häusler et al ., 1999 ), or by using an engineered plant isoform, which lacks the regulatory phosphorylation site ( Rademacher et al ., 2002 ). To achieve positive effects by PEPC over-expression, it is also important to consider gene-dosage effects and the location of the enzyme. Severe negative effects on growth and development have been observed in potato plants that constitutively over-express PEPC controlled by the 35S promoter ( Rademacher et al ., 2002 ). In this work, we used a bacterial PEPC that is not prone to malate feedback regulation ( Gehlen et al ., 1996 ; Häusler et al ., 1999 ) for seed-specific expression. On the one hand, our transgenic seeds showed a dramatically changed seed composition. On the other hand, we found high lethality during transgene invasion. In 70% of the F 0 plants, the seeds aborted at mid-development and viable seeds could not be obtained. This indicates that plant seeds tolerate only moderately low levels of CgPEPC expression. Lethality, due to an over-dosage of PEPC, is not surprising given the fact that endogenous PEPC is tightly regulated on different levels in order to prevent uncontrolled increase ( Chollet et al ., 1996 ). Thus, the analysed lines expressed relatively low levels of RNA and enzyme, probably explaining the fact that changes in metabolite levels were often at the border of significance. Nevertheless, we showed that the low-level expression of CgPEPC was sufficient to considerably increase CO 2 dark fixation ( Figure 3C ). In addition, characteristic changes in metabolic profiles occurred: (i) a shift in metabolic fluxes from sugars/starch into amino acids; (ii) a decrease in the level of phosphorylated compounds away from the glycolytic pathway; and (iii) an increase in non-phosphorylated intermediates of the citric acid cycle (shown schematically in Figure 4 ). These changes are consistent with an increased anaplerotic flux and implicate a re-directed carbon flow from sugars/starch towards organic acids and amino acids. During recent years, attempts have been undertaken to introduce a C 4 -like photosynthesis using the over-expression of unregulated PEP isoforms ( Jeanneau et al ., 2002 ). Although this aim has not been achieved at present, a clear induction of the anaplerotic pathway with changes in the metabolite profiles in leaves has been described ( Rademacher et al ., 2002 ), very similar to that observed in our CgPEPC seeds. With respect to the PEPC-catalysed reaction, the bean cotyledons differ from leaves in two aspects. Firstly, cotyledons are highly respiring organs with a low photosynthetic CO 2 fixation capacity. Therefore, PEPC-catalysed CO 2 dark fixation is desirable. Secondly, cotyledons are organs specialized for protein synthesis and storage, and thus have a high demand for carbon acceptors derived from the anaplerotic pathway. These two aspects explain our observation that the altered metabolic fluxes introduced by the seed-specific expression of CgPEPC resulted in a 15%−25% higher seed protein content. In addition, we measured a considerable increase in seed dry weight of 20%−30%, apparently without a reduction in seed number per plant. The increased dry weight can be attributed to an increased import of assimilates, especially nitrogenous compounds, into the seeds, and/or an improved carbon economy due to higher dark fixation rates and reduced carbon loss. The results also showed that CO 2 release by respiratory activity within the seed is not a wasteful process. CO 2 dark fixation by PEPC is essential to channel carbon specifically into the synthesis of organic acids and thereby into proteins. We showed that a high flux through this pathway is an important determinant for seed protein content. It is unclear at present whether CgPEPC expression increases yield as well as improving protein content. This can only be checked by field experiments. Our results also raise another interesting aspect. CgPEPC expression causes the diversion of carbon flow into organic acids, probably improves carbon economy and increases the supply of carbon acceptors for amino acid synthesis. Obviously, the increase in the carbon acceptor pool within the embryo may cause a higher uptake of amino nitrogen into the seed. Because CgPEPC expression is restricted to the seed, a kind of seed-specific signal has to be assumed which couples the higher demand of nitrogen within the embryo to an increased supply from the vegetative organs. How this can be fulfilled is not clear at present, but will be investigated in the future. Possible routes include increased nitrogen translocation, uptake from the soil and/or nodule fixation. In summary, the data presented here clearly demonstrate that, in protein-storing seeds, the anaplerotic flow catalysed by PEPC represents a bottleneck, and thus is a most promising target for future efforts in plant breeding and biotechnology. <h1>Experimental procedures</h1> <h2>Plant transformation</h2> A full-length cDNA of C. glutamicum PEPC was kindly provided by B. J. Eikmanns, University of Ulm, Germany ( GenBank accession no. X14234 , Eikmanns et al ., 1989 ) and cloned under the control of the Legumin B4 promoter, as described in Weber et al . (1998 ), into the binary vector pGA482 ( An et al ., 1987 ). Transformation of V. narbonensis via Agrobacterium -mediated gene transfer was performed according to Pickardt et al . (1991 ). Plants were grown in growth chambers under a light/dark regime of 16 h light and 8 h dark at 20 °C and 18 °C, respectively. <h2>Biochemical analysis</h2> Plant material was harvested and extracted as described in Heim et al . (1993 ) and Rolletschek et al . (2002 ). Free amino acids, sugars, starch, proteins, total carbon and nitrogen, nucleotides and nucleotide sugars were extracted and measured as described in Rolletschek et al . (2002 ). Total crude protein was calculated from the total nitrogen content according to Schroeder (1982 ). PEPC activity in embryos was determined spectrophotometrically according to Golombek et al . (1999 ). Freshly harvested cotyledons were ground in three volumes (w/v) of 50 m m N -2-hydroxyethylpiperazine- N -2-ethanesulphonic acid (HEPES), pH 7.4, 5 m m MgCl 2 , 5 m m dithiothreitol (DTT), 1 m m ethylenediaminetetraacetic acid (EDTA), 1 m m ethleneglycoltetraacetic acid (EGTA) and 10% glycerine. The assay was performed in 25 m m Tris, pH 8, 5 m m MgCl 2 , 1 m m KHCO 3 , 0.2 m m NADH, 2 U malate dehydrogenase and 30 µL extract in a total volume of 0.5 mL and started with 5 m m PEP. Other enzymes were measured according to Dey and Harborne (1990 ). <h2>Metabolite analysis</h2> Plant material was extracted as in Rolletschek et al . (2002 ). Metabolites were measured by ion chromatography coupled to mass spectrometry (IC-MS). This allowed separation according to retention times and molecular masses, and enabled parallel quantitative determinations to be made with low detection limits. Chromatography was performed using a DX-600 ion chromatography system (Dionex, USA). Separation was carried out on a Dionex AS11-HC column (2 × 250 mm) and a guard column (AG11-HC) at 25 °C. A binary gradient at a constant flow rate of 0.5 mL/min was applied using distilled water (eluent A) and 100 m m sodium hydroxide (eluent B). The gradient was produced by the following linear concentration changes: 8 min 4% B, 20 min 30% B, 10 min 60% B, 10 min 100% B, hold 100% B for 3 min, return to 4% B in 3 min and equilibrate for 10 min. Column effluents were directed to an ASRS-ULTRA (2-mm) anion self-regenerating suppressor (Dionex) working in the external water mode (2 mL/min) at 100 mA. After passing the conductivity cell, the effluent was directed into the mass spectrometer without further splitting via the electrospray interface. MS analysis was performed using a single quadrupole (MSQ, Dionex) with enhanced low mass option. The following parameters were employed: probe temperature, 400 °C; sheath gas, nitrogen; capillary voltage, 3.5 kV; detection in negative ion mode using a cone voltage of 50 V and a dwell time of 1 s. Up to 20 metabolites were measured in parallel using the single ion monitoring (SIM) mode. Deprotonated ions [M − H] − were monitored with a span of 1 amu. Single SIMs were performed in small time windows of about 5 min during the total run time (64 min), i.e. up to five SIMs were run in parallel at a maximum. This allowed parallel monitoring to be minimized and sensitivity to be enhanced. By comparing the results of IC-MS analyses with those from enzymatic assays, we were able to validate the concentration determined by IC-MS. To establish the efficiency of the whole extraction and measurement procedure, the recovery of metabolites was checked by the addition of standards to tissue samples (about threefold excess) before extraction. Recoveries were between 77% and 98%. <h2>Immunocytochemistry</h2> Procedures were performed as described in Borisjuk et al . (1995 ) and were modified as follows. To block nonspecific antibody binding, the tissue sections were treated with 50 m m phosphate-buffered saline (PBS) (pH 7) containing 0.1% bovine serum albumin (BSA), 0.2 m methyl-α- d -galactopyranoside, 0.2 m methyl-ॆ- d -galactopyranoside and 0.1% Tween 20. To increase the sensitivity and specificity of immunostaining, Alexa Fluor 488-conjugated secondary antibody (1 : 100) was applied instead of streptavidine-alkaline phosphatase assay for the detection of primary antibody and confocal laser scanning microscopy for the localization of the fluorescent signal within the tissues. Polyclonal antiserum against Corynebacterium PEPC was kindly donated by B. J. Eikmanns, University of Ulm, Germany. <h2>[ 14 C]-CO 2 partitioning</h2> Cotyledons (28–30 DAP) were prepared and processed as in Rolletschek et al . (2002 ). Pulse labelling was performed for 8 h with [U- 14 C]-NaHCO 3 (3.7 MBq/mmol, Amersham-Buchler, Braunschweig, Germany) in a buffer containing 20 m m HCO 3 − . http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Plant Biotechnology Journal Wiley

Seed-specific expression of a bacterial phosphoenolpyruvate carboxylase in Vicia narbonensis increases protein content and improves carbon economy

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Publisher
Wiley
Copyright
© 2004 Blackwell Publishing Ltd
ISSN
1467-7644
eISSN
1467-7652
DOI
10.1111/j.1467-7652.2004.00064.x
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17147612
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Abstract

<h1>Introduction</h1> Legume seeds are an important source of plant-derived protein with economic importance in the worldwide feed and food industry. Increasing the storage protein content of crop seeds is difficult to achieve because conventional breeding always favours yield in general. Biotechnological approaches to manipulate seed protein have introduced foreign or modified endogenous storage protein genes to increase sink demand ( Gueguen and Popineau, 1998 ; Hofman et al ., 1988 ; Saalbach et al ., 1995 ), but with limited success. Consequently, novel approaches are necessary, based on the understanding of the mechanisms that regulate storage protein accumulation. Thus, to elevate seed nitrogen levels, it could be advantageous to focus on assimilate partitioning into the different storage products ( Golombek et al ., 2001 ). Storage proteins are synthesized during the maturation phase of seed development. Amino acid precursors are unloaded from the phloem, and subsequently taken up by the symplasmically isolated embryo ( Miranda et al ., 2001 ). Storage protein synthesis is regulated at different levels. The availability and partitioning of assimilates and nitrogen compounds are of primary importance ( Golombek et al ., 2001 ). Interestingly, much of the control is exerted by the embryo itself, rather than by the maternal plant ( Hayati et al ., 1993 ). Glutamine and/or asparagine are preferentially imported into legume cotyledons ( Miflin and Lea, 1977 ). The biosynthesis of other amino acids requires the provision of carbon skeletons from glycolytic and citric acid cycle products. Some carbon derived from sucrose must therefore be diverted into storage proteins, a process controlled by the anaplerotic reaction of phosphoenolpyruvate carboxylase (PEPC, EC 4.1.1.31) ( Turpin and Weger, 1990 ). PEPC has been investigated in seeds of pea, soybean, wheat and Vicia ( Flinn, 1985 ; Golombek et al ., 1999 ; Gonzalez et al ., 1998 ; Hedley et al ., 1975 ; Smith et al ., 1989 ). The enzyme re-fixes HCO 3 − liberated by respiration and, together with PEP, yields oxaloacetate that can be converted to aspartate, malate or other intermediates of the citric acid cycle. Thus, PEPC controls the anaplerotic carbon flow and can potentially improve seed carbon economy. Correlative evidence points to a rate-limiting role of PEPC in the anaplerotic carbon flow during storage protein synthesis in V. faba ( Golombek et al ., 2001 ). In soybean cultivars differing in seed protein content, PEPC correlates with seed protein content ( Smith et al ., 1989 ; Sugimoto et al ., 1989 ). Thus, storage protein synthesis is partly controlled by the PEPC-catalysed anaplerotic reaction. PEPC is a ubiquitous enzyme in plants which is highly regulated. The allosteric properties of PEPC are subject to opposite and antagonistic effects of metabolites, malate and glucose-6-phosphate. Phosphorylation modulates the metabolic regulation with respect to feedback inhibition by malate ( Jeanneau et al ., 2002 ). Biotechnological approaches using PEPC primarily have aimed to improve CO 2 assimilation by suppressing photorespiration in leaves ( Jeanneau et al ., 2002 ; Ku et al ., 1999 ). Ectopic expression of PEPC should introduce a part of a C 4 -like CO 2 concentrating mechanism in leaf cells. Potato plants were transformed with PEPC from Corynebacterium glutamicum ( Gehlen et al ., 1996 ) or with a genetically engineered potato PEPC with decreased feedback inhibition ( Rademacher et al ., 2002 ) driven by the constitutive 35S promoter. Over-expressers showed a moderate increase in malate and amino acids demonstrating increased anaplerotic fluxes ( Gehlen et al ., 1996 ; Häusler et al ., 1999 ). However, plants were severely disturbed in shoot and tuber growth ( Rademacher et al ., 2002 ). In PEPC over-expressing leaves, the CO 2 assimilation rates and the CO 2 compensation point remained unchanged, partly due to increased CO 2 release from PEP carboxylation products initiated by endogenous cytosolic NAD(P)-malic enzyme (NAD, nicotinamide adenine dinucleotide) ( Häusler et al ., 1999 ). Here, we report on the ectopic expression of C. glutamicum PEPC targeted into maturing seeds of the bean V. narbonensis . A seed storage protein promoter restricted expression to maturing cotyledons where large amounts of storage products were synthesized and probably a high demand of anaplerotic flux was required. The engineered embryos showed a clear stimulation of [ 14 C]-CO 2 uptake and the label was specifically incorporated into proteins, thus indicating a shift in metabolic fluxes from sugars/starch into amino acids. Dry seeds from transgenic lines had a significantly higher content of crude protein (by up to 20% per gram) and a higher seed dry weight. Combining both effects, it was revealed that the protein content per individual seed increased by 40%−50%. It can be concluded that PEPC in seeds is a promising target for plant breeding and biotechnology. <h1>Results</h1> <h2>Seeds expressing Corynebacterium PEPC are larger and contain more protein</h2> A full-length cDNA of C. glutamicum phosphoenolpyruvate carboxylase (CgPEPC, Eikmanns et al ., 1989 ) was cloned under the control of the Legumin B4 promoter ( Bäumlein et al ., 1992 ), which confers seed-specific expression. The cDNA construct was cloned into pGA 482 ( An et al ., 1987 ) and introduced into V. narbonensis using an Agrobacterium -mediated protocol ( Pickardt et al ., 1991 ). A total of 23 F 0 lines were regenerated. In order to obtain stable lines, the plants were allowed to self-pollinate and polymerase chain reaction (PCR)-positive plants were further propagated. Seeds from three independent transgenic lines (PPC-9, PPC-10, PPC-12) of the F 4 to F 6 generation were chosen for further analysis. The expression of the CgPEPC was shown by reverse transcriptase PCR in embryos of the lines PPC-9, PPC-10 and PPC-12 at 25 days after pollination (DAP) ( Figure 1A ). For all lines, vegetative growth and flowering were phenotypically normal. In order to analyse whether the transgenic plants had an altered seed composition, we analysed dry embryos for the content of storage proteins and starch. Compared with wild-type seeds, those from the CgPEPC-expressing lines contained 15%−25% more protein on a per gram basis ( Figure 1B ). The starch content was not significantly changed, although the mean values were slightly lower ( Figure 1C ). Storage protein composition was also analysed after extraction of embryo powder with aqueous buffers. Total globulins, containing the major 7s and 11s storage proteins, were significantly increased by approximately 20% in the transgenic lines compared with the wild-type ( Figure 1D ). Similarly, the albumins were also increased by about 20% ( Figure 1E ). This class includes the sum of water-soluble storage and non-storage proteins. Remarkably, the seeds from all lines were characterized by increased individual dry weight by 20%−30% ( Figure 2A ). To analyse whether higher seed weights were translated into a greater yield per plant, we analysed, from the same batch of growth chamber plants, the total seed number and the total seed weight per plant, as well as the seed number per pod. The seed number per plant was not significantly different for the transgenics, although the mean values were slightly lower ( Figure 2B ). The total seed weight per plant also did not change significantly, but the mean values were slightly higher ( Figure 2C ). However, there was a significant decrease in seed number per pod for the CgPEPC plants by 20%−30% ( Figure 2D ). We believe that the results must be interpreted cautiously with respect to yield because such parameters need to be analysed using field trials. Therefore, at present, it is not clear whether an improved yield can also be expected from CgPEPC expression. In summary, the expression of CgPEPC leads to characteristic changes in seed composition, i.e. an increase in both protein content on a per gram basis and individual seed dry weight. By adding both effects, there is a clear and drastic increase in the crude protein content on an individual seed basis by as much as 40%−50%. <h2>Seeds expressing Corynebacterium PEPC fix more [ 14 C]-CO 2 into proteins</h2> We localized the CgPEPC protein directly in tissue sections by immunohistochemistry. Cotyledons were fixed, embedded in paraplast and sectioned. Immunofluorescence measurements revealed a clear green signal all over the cytoplasm of the cotyledonary parenchyma cells of the transgenic line ( Figure 3A ), whereas no signal was present in the corresponding sections of wild-type cotyledons ( Figure 3B ). This clearly indicates that the bacterial PEPC protein is present in the cotyledonary parenchyma cells of transgenic lines. The detection of CgPEPC protein was not possible by conventional immunoblot analysis using crude extracts, polyclonal antiserum against CgPEPC and a cocktail of proteinase inhibitors within the extraction buffer (data not shown), probably due to rapid degradation of the bacterial protein during extraction. In order to analyse the in vivo PEPC activity, we measured the CO 2 dark fixation. Towards this aim, embryos of wild-type and line PPC-12 at 28–30 DAP were pulse-labelled with [ 14 C]-HCO 3 − . In Figure 3(C) , it is shown that cotyledons of line PPC-12 took up approximately 50% more [ 14 C]-HCO 3 − compared with the wild-type, indicating that the engineered CgPEPC increased the CO 2 dark fixation rate of growing embryos. To further assess the fate of the 14 C label, we analysed the partitioning into the starch and protein fractions, which represent the two major storage products. Remarkably, transgenic embryos partitioned threefold more label into the protein fraction compared with the wild-type ( Figure 3D ). Only a very low amount of the 14 C label appeared in the starch fraction, with no significant difference between the wild-type and PPC-12. The in vitro enzyme assay only revealed a minor increase in PEPC activity in the transgenic plants ( Figure 3E ), probably due to rapid degradation of the bacterial protein during extraction. Our data suggest that the CgPEPC protein is present and physiologically active in maturing embryos and most probably increases the dark fixation rate of [ 14 C]-CO 2 . Furthermore, the higher amount of CO 2 taken up is specifically channelled into the protein fraction of the seeds. <h2>Changed metabolite profiles indicate re-directed carbon flow into organic and amino acids</h2> PEPC activity represents a side-branch of the glycolytic pathway, and may affect carbon flux towards the synthesis of proteins. PEPC carboxylates PEP to oxaloacetate, which can either be converted to aspartate by aspartate aminotransferase or to malate by malate dehydrogenase or, together with pyruvate, can be fed into the citric acid cycle to produce 2-oxo-glutarate (see the general metabolic pathway in Figure 4 ). The latter can serve as a carbon acceptor for amino acid biosynthesis. In order to analyse the changes in metabolic pathways within CgPEPC-expressing seeds, steady state levels were measured for sucrose, free amino acids, intermediates of glycolysis and the citric acid cycle, as well as for adenosine triphosphate and diphosphate ( Table 1 ). There was a general trend towards lower sucrose levels and lower pool sizes of phosphorylated sugars (glucose-6-phosphate, glucose-1-phosphate, fructose-1,6-bisphosphate), but increases in the pool of free amino acids. Levels of nucleotide sugars (uridine diphosphate glucose, adenosine diphosphate glucose) did not change. The acetyl-CoA content was higher in all three lines. There was an overall trend towards higher levels of intermediates of the citric acid cycle. Thus, CgPEPC expression in V. narbonensis seeds leads to changes in the pattern of seed metabolites, indicating a shift of metabolic fluxes from sugars/starch into amino acids. In addition, phosphorylated intermediates from the glycolytic pathway were generally decreased, whereas non-phosphorylated compounds from the citric acid cycle were increased. Such a shift in the ratio of phosphorylated to non-phosphorylated products can be expected because PEPC catalyses the cleavage of inorganic phosphate (Pi) from PEP. Adenosine triphosphate levels were lower, probably due to the higher energy demand by increased protein synthesis. Taken together, increased PEPC in seeds causes characteristic changes in the metabolite pattern, which are consistent with a re-directed carbon flow from the starch synthesis pathway towards the protein synthesis pathway. In addition, we measured nearby enzymes upstream or downstream of PEPC in growing embryos of all three transgenic lines and the wild-type (28–32 DAP) ( Table 2 ). Aspartate aminotransferase was about 30% higher in cotyledons of line PPC-12, but not significantly different from the wild-type in the other lines. NAD-malic enzyme, NAD-malate dehydrogenase and pyruvate kinase were not different. <h1>Discussion</h1> Conventional plant breeding mainly aims to increase yield. However, selection for yield favours a relative increase in the ‘low-energy product’ starch at the expense of protein. Quality parameters, such as the protein content, are very important for human food and animal feedstuffs. Recent studies on seed physiology have pointed to PEPC as a regulator of partitioning of photoassimilates into proteins ( Golombek et al ., 1999, 2001 ). In growing seeds, PEPC re-fixes carbon released by respiration. PEPC catalyses the anaplerotic flux, thereby satisfying the carbon acceptor demands for amino acid biosyntheses (see Figure 4 ). The labelling pattern obtained after short-term feeding of [ 14 C]-CO 2 is consistent with CO 2 dark fixation by PEPC, initially into oxaloacetate, and then into malate and aspartate ( Flinn, 1985 ). In order to increase PEPC in vivo , it is essential to circumvent the down-regulation by endogenous mechanisms, e.g. malate feedback inhibition. This can be achieved either by using an enzyme of non-plant origin, which cannot be regulated by the plant's own mechanisms ( Häusler et al ., 1999 ), or by using an engineered plant isoform, which lacks the regulatory phosphorylation site ( Rademacher et al ., 2002 ). To achieve positive effects by PEPC over-expression, it is also important to consider gene-dosage effects and the location of the enzyme. Severe negative effects on growth and development have been observed in potato plants that constitutively over-express PEPC controlled by the 35S promoter ( Rademacher et al ., 2002 ). In this work, we used a bacterial PEPC that is not prone to malate feedback regulation ( Gehlen et al ., 1996 ; Häusler et al ., 1999 ) for seed-specific expression. On the one hand, our transgenic seeds showed a dramatically changed seed composition. On the other hand, we found high lethality during transgene invasion. In 70% of the F 0 plants, the seeds aborted at mid-development and viable seeds could not be obtained. This indicates that plant seeds tolerate only moderately low levels of CgPEPC expression. Lethality, due to an over-dosage of PEPC, is not surprising given the fact that endogenous PEPC is tightly regulated on different levels in order to prevent uncontrolled increase ( Chollet et al ., 1996 ). Thus, the analysed lines expressed relatively low levels of RNA and enzyme, probably explaining the fact that changes in metabolite levels were often at the border of significance. Nevertheless, we showed that the low-level expression of CgPEPC was sufficient to considerably increase CO 2 dark fixation ( Figure 3C ). In addition, characteristic changes in metabolic profiles occurred: (i) a shift in metabolic fluxes from sugars/starch into amino acids; (ii) a decrease in the level of phosphorylated compounds away from the glycolytic pathway; and (iii) an increase in non-phosphorylated intermediates of the citric acid cycle (shown schematically in Figure 4 ). These changes are consistent with an increased anaplerotic flux and implicate a re-directed carbon flow from sugars/starch towards organic acids and amino acids. During recent years, attempts have been undertaken to introduce a C 4 -like photosynthesis using the over-expression of unregulated PEP isoforms ( Jeanneau et al ., 2002 ). Although this aim has not been achieved at present, a clear induction of the anaplerotic pathway with changes in the metabolite profiles in leaves has been described ( Rademacher et al ., 2002 ), very similar to that observed in our CgPEPC seeds. With respect to the PEPC-catalysed reaction, the bean cotyledons differ from leaves in two aspects. Firstly, cotyledons are highly respiring organs with a low photosynthetic CO 2 fixation capacity. Therefore, PEPC-catalysed CO 2 dark fixation is desirable. Secondly, cotyledons are organs specialized for protein synthesis and storage, and thus have a high demand for carbon acceptors derived from the anaplerotic pathway. These two aspects explain our observation that the altered metabolic fluxes introduced by the seed-specific expression of CgPEPC resulted in a 15%−25% higher seed protein content. In addition, we measured a considerable increase in seed dry weight of 20%−30%, apparently without a reduction in seed number per plant. The increased dry weight can be attributed to an increased import of assimilates, especially nitrogenous compounds, into the seeds, and/or an improved carbon economy due to higher dark fixation rates and reduced carbon loss. The results also showed that CO 2 release by respiratory activity within the seed is not a wasteful process. CO 2 dark fixation by PEPC is essential to channel carbon specifically into the synthesis of organic acids and thereby into proteins. We showed that a high flux through this pathway is an important determinant for seed protein content. It is unclear at present whether CgPEPC expression increases yield as well as improving protein content. This can only be checked by field experiments. Our results also raise another interesting aspect. CgPEPC expression causes the diversion of carbon flow into organic acids, probably improves carbon economy and increases the supply of carbon acceptors for amino acid synthesis. Obviously, the increase in the carbon acceptor pool within the embryo may cause a higher uptake of amino nitrogen into the seed. Because CgPEPC expression is restricted to the seed, a kind of seed-specific signal has to be assumed which couples the higher demand of nitrogen within the embryo to an increased supply from the vegetative organs. How this can be fulfilled is not clear at present, but will be investigated in the future. Possible routes include increased nitrogen translocation, uptake from the soil and/or nodule fixation. In summary, the data presented here clearly demonstrate that, in protein-storing seeds, the anaplerotic flow catalysed by PEPC represents a bottleneck, and thus is a most promising target for future efforts in plant breeding and biotechnology. <h1>Experimental procedures</h1> <h2>Plant transformation</h2> A full-length cDNA of C. glutamicum PEPC was kindly provided by B. J. Eikmanns, University of Ulm, Germany ( GenBank accession no. X14234 , Eikmanns et al ., 1989 ) and cloned under the control of the Legumin B4 promoter, as described in Weber et al . (1998 ), into the binary vector pGA482 ( An et al ., 1987 ). Transformation of V. narbonensis via Agrobacterium -mediated gene transfer was performed according to Pickardt et al . (1991 ). Plants were grown in growth chambers under a light/dark regime of 16 h light and 8 h dark at 20 °C and 18 °C, respectively. <h2>Biochemical analysis</h2> Plant material was harvested and extracted as described in Heim et al . (1993 ) and Rolletschek et al . (2002 ). Free amino acids, sugars, starch, proteins, total carbon and nitrogen, nucleotides and nucleotide sugars were extracted and measured as described in Rolletschek et al . (2002 ). Total crude protein was calculated from the total nitrogen content according to Schroeder (1982 ). PEPC activity in embryos was determined spectrophotometrically according to Golombek et al . (1999 ). Freshly harvested cotyledons were ground in three volumes (w/v) of 50 m m N -2-hydroxyethylpiperazine- N -2-ethanesulphonic acid (HEPES), pH 7.4, 5 m m MgCl 2 , 5 m m dithiothreitol (DTT), 1 m m ethylenediaminetetraacetic acid (EDTA), 1 m m ethleneglycoltetraacetic acid (EGTA) and 10% glycerine. The assay was performed in 25 m m Tris, pH 8, 5 m m MgCl 2 , 1 m m KHCO 3 , 0.2 m m NADH, 2 U malate dehydrogenase and 30 µL extract in a total volume of 0.5 mL and started with 5 m m PEP. Other enzymes were measured according to Dey and Harborne (1990 ). <h2>Metabolite analysis</h2> Plant material was extracted as in Rolletschek et al . (2002 ). Metabolites were measured by ion chromatography coupled to mass spectrometry (IC-MS). This allowed separation according to retention times and molecular masses, and enabled parallel quantitative determinations to be made with low detection limits. Chromatography was performed using a DX-600 ion chromatography system (Dionex, USA). Separation was carried out on a Dionex AS11-HC column (2 × 250 mm) and a guard column (AG11-HC) at 25 °C. A binary gradient at a constant flow rate of 0.5 mL/min was applied using distilled water (eluent A) and 100 m m sodium hydroxide (eluent B). The gradient was produced by the following linear concentration changes: 8 min 4% B, 20 min 30% B, 10 min 60% B, 10 min 100% B, hold 100% B for 3 min, return to 4% B in 3 min and equilibrate for 10 min. Column effluents were directed to an ASRS-ULTRA (2-mm) anion self-regenerating suppressor (Dionex) working in the external water mode (2 mL/min) at 100 mA. After passing the conductivity cell, the effluent was directed into the mass spectrometer without further splitting via the electrospray interface. MS analysis was performed using a single quadrupole (MSQ, Dionex) with enhanced low mass option. The following parameters were employed: probe temperature, 400 °C; sheath gas, nitrogen; capillary voltage, 3.5 kV; detection in negative ion mode using a cone voltage of 50 V and a dwell time of 1 s. Up to 20 metabolites were measured in parallel using the single ion monitoring (SIM) mode. Deprotonated ions [M − H] − were monitored with a span of 1 amu. Single SIMs were performed in small time windows of about 5 min during the total run time (64 min), i.e. up to five SIMs were run in parallel at a maximum. This allowed parallel monitoring to be minimized and sensitivity to be enhanced. By comparing the results of IC-MS analyses with those from enzymatic assays, we were able to validate the concentration determined by IC-MS. To establish the efficiency of the whole extraction and measurement procedure, the recovery of metabolites was checked by the addition of standards to tissue samples (about threefold excess) before extraction. Recoveries were between 77% and 98%. <h2>Immunocytochemistry</h2> Procedures were performed as described in Borisjuk et al . (1995 ) and were modified as follows. To block nonspecific antibody binding, the tissue sections were treated with 50 m m phosphate-buffered saline (PBS) (pH 7) containing 0.1% bovine serum albumin (BSA), 0.2 m methyl-α- d -galactopyranoside, 0.2 m methyl-ॆ- d -galactopyranoside and 0.1% Tween 20. To increase the sensitivity and specificity of immunostaining, Alexa Fluor 488-conjugated secondary antibody (1 : 100) was applied instead of streptavidine-alkaline phosphatase assay for the detection of primary antibody and confocal laser scanning microscopy for the localization of the fluorescent signal within the tissues. Polyclonal antiserum against Corynebacterium PEPC was kindly donated by B. J. Eikmanns, University of Ulm, Germany. <h2>[ 14 C]-CO 2 partitioning</h2> Cotyledons (28–30 DAP) were prepared and processed as in Rolletschek et al . (2002 ). Pulse labelling was performed for 8 h with [U- 14 C]-NaHCO 3 (3.7 MBq/mmol, Amersham-Buchler, Braunschweig, Germany) in a buffer containing 20 m m HCO 3 − .

Journal

Plant Biotechnology Journal – Wiley

Published: May 1, 2004

Keywords: anaplerotic pathway; assimilate partitioning; phosphoenolpyruvate carboxylase; seed development; seed storage protein; transformation

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