An engineered phosphoenolpyruvate carboxylase redirects carbon and nitrogen flow in transgenic potato plants

Authors


* For correspondence (fax +49 241 802 2637; e-mail cp@bio1.rwth-aachen.de)

Summary

Phosphoenolpyruvate carboxylase (PEPC) plays a central role in the anaplerotic provision of carbon skeletons for amino acid biosynthesis in leaves of C3 plants. Furthermore, in both C4 and CAM plants photosynthetic isoforms are pivotal for the fixation of atmospheric CO2. Potato PEPC was mutated either by modifications of the N-terminal phosphorylation site or by an exchange of an internal cDNA segment for the homologous sequence of PEPC from the C4 plant Flaveria trinervia. Both modifications resulted in enzymes with lowered sensitivity to malate inhibition and an increased affinity for PEP. These effects were enhanced by a combination of both mutated sequences and pulse labelling with 14CO2in vivo revealed clearly increased fixation into malate for this genotype. Activity levels correlated well with protein levels of the mutated PEPC. Constitutive overexpression of PEPC carrying both N-terminal and internal modifications strongly diminished plant growth and tuber yield. Metabolite analysis showed that carbon flow was re-directed from soluble sugars and starch to organic acids (malate) and amino acids, which increased four-fold compared with the wild type. The effects on leaf metabolism indicate that the engineered enzyme provides an optimised starting point for the installation of a C4-like photosynthetic pathway in C3 plants.

Introduction

Phosphoenolpyruvate carboxylase (PEPC, EC 4.1.1.31) catalyses the synthesis of oxaloacetate (OAA) from phosphoenolpyruvate (PEP) and bicarbonate. The reaction product is rapidly converted to malate by malate dehydrogenase or transaminated to aspartate by aminotransferases. PEPC acts as a homotetramer of approximately 400 kDa in the cytoplasm of plant cells and serves multiple functions (Latzko and Kelly, 1983). In leaves of C3 plants, PEPC is involved in the anaplerotic replenishment of citric acid cycle intermediates, by providing precursors for several biosynthetic pathways including biosynthesis of amino acids and phenolic compounds (Andrews, 1986; Melzer and O'Leary, 1987). Furthermore, in concert with malic enzyme (Davies, 1986) it is involved in the fine regulation of the cytoplasmic pH and compensates for the alkalinisation of the cytoplasm during nitrate reduction (Manh et al., 1993). Such isoforms are also characterised as C3 or root isoforms.

C4 and CAM plants with their specialised photosynthetic features apply PEPC for the initial fixation of atmospheric CO2. The specific isoforms evolved from the anaplerotic (C3) enzyme, but show different expression levels and tissue specificities (Ku et al., 1996; Nimmo, 2000; Sheen, 1999). Also, they differ in their catalytic properties concerning substrate affinity and allosteric regulation by metabolic intermediates. As a general tendency, photosynthetic isoforms display lower affinities for PEP compared with the anaplerotic enzyme, but they are also less sensitive to product inhibition by malate (e.g. Svensson et al., 1997).

PEPC activity is also regulated by phosphorylation of a N-terminal serine residue, which reduces the sensitivity of the enzyme for allosteric inhibitors, especially malate. In C4 plants, this specific phosphorylation of PEPC occurs in the light leading to a lower sensitivity towards feedback inhibition by malate. The importance of the phosphorylation site for protein activity has been proven in vitro for the photosynthetic isoforms from Sorghum and maize. In order to mimic constitutive phosphorylation, exchanges of crucial residues for negatively charged amino acids clearly reduced the sensitivity of the enzyme for malate inhibition (Ueno et al., 1997; Wang and Chollet, 1993). In CAM plants, the corresponding regulatory phosphorylation occurs during the night, when CO2 is fixed into organic acids. Recently, a PEPC kinase has been cloned from the CAM plant Kalanchoë fedtschenkoi. The kinase is negatively regulated by light on the transcriptional level providing first insights into the molecular regulation of PEPC phosphorylation (Nimmo, 2000). Likewise phosphorylation of the N-terminal residue has also been shown for the C3 isoforms, but the degree of phosphorylation is again dependent on specific metabolic states and on the cytosolic pH (Duff and Chollet, 1995).

In this study, the impact of constitutive overexpression of an engineered physiologically active PEPC on plant metabolism was investigated. The data obtained indicate that plant performance is drastically deteriorated by constitutive overexpression of the recombinant enzyme and that carbon flow is redirected from sugars to organic acids and amino acids. The latter underlines that the mutated enzyme might become an optimised starting point for establishing a fully operational single cell C4-like cycle in C3 plants (Häusler et al., 2002).

Results

Engineered PEPC proteins showed altered catalytic properties in vitro

We were aiming to overexpress the endogenous PEPC in potato plants as a basis for the establishment of a single cell C4 cycle. As the sensitivity for malate inhibition of C3 PEPC is high, the potato ppc coding sequence was modified to attenuate this – for our approach – undesirable effect. First, the N-terminal serine was replaced by aspartate to mimic constitutive phosphorylation. As listed in Table 1, this modification already reduced the Km for PEP by 20% and increased the I50 for malate approximately twofold. The additional deletion of an adjacent Lys residue with known importance for allosteric inhibition (Ueno et al., 1997) did not affect the substrate affinity, but further decreased the sensitivity to malate inhibition. In a parallel approach, a part of the coding sequence of the potato PEPC was replaced for the homologous region from the C4 plant Flaveria trinervia. By this, similar alterations in the catalytic properties of the enzyme were obtained as for the modification of the N-terminal region. A combination of both manipulations of the amino acid sequence of the enzyme led to more than additive effects on the catalytic properties. Substrate affinity was doubled compared with the wild-type and I50 values for malate increased by more than one order of magnitude. The attenuation of product inhibition was reproducibly apparent at PEP concentrations of between 50 µm and 1 mm (data not shown). The best performing constructs according to the in vitro analysis (stppdC4 and stppcS9D-C4) were chosen for further physiological and biochemical characterisation of the transgenic potato plants.

Table 1.  PEPC constructs and enzymatic properties
 stppcstppcS11DstppcS9DstppcC4stppcS9D-C4
  1. a at 0,25 mm PEP; baccording to GenBank gi 1168761.

N-terminalMATRNLDMATRNLDMATRNLSMATRNLDMATRNLS
sequencebKLASIDAKLADIDAADIDAKLASIDAADIDA
ReplacementAA 384(→)420
(F. trinervia)
AA 384(→)420
(F. trinervia)
Km (PEP) [µM]57 ± 6,043 ± 1,244 ± 1,941 ± 1,823 ± 1,7
I50 (malate)a29 ± 1,553 ± 0,9163 ± 14149 ± 1,51123 ± 20
[µM]

Expression levels of mutated PEPC proteins correlate well with in vitro activities

The modified coding sequences of the Solanum tuberosum PEPC were transformed into potato and expressed under constitutive control of the CaMV 35SS promoter (for details see Experimental procedures). Extracts from 34 independent regenerants were tested for PEPC activity in vitro and recombinant proteins were detected on Western blots and quantified densitometrically. In both populations, the average PEPC activity was clearly higher than in the wild-type. However, individual lines showed both lowered and increased activities. As shown in Table 2, PEPC activities correlated well with the amount of protein. Levels of overexpression up to approximately threefold and enhanced activities up to fivefold that of the wild-type were observed. When PEPC activities in the transformants were lower than in the wild-type (presumably due to co-suppression), this was also reflected in the amount of PEPC protein. Thus, in vitro PEPC activities in the transgenic lines correlate well with the amount of PEPC protein extracted from leaves.

Table 2.  Relative PEPC amounts and enzymatic activities in transgenic potato lines
 LinePEPC proteinaEnzymatic activityb
  1. a total PEPC amount (x fold compared with wild-type); b ×fold compared to wild-type.

stppcC4213,44,9
1512,83,8
2512,52,7
stppcS9D-C4212,94,7
412,42,7
722,73,5
912,62,6
1810,40,27
2410,30,32

Plant growth is impaired by constitutive overexpression of the mutated PEPC

Growth and development of the transgenic potato lines was monitored. A clear retardation in growth was observed for lines expressing the stppcS9D-C4 construct with modified phosphorylation site (Figures 1 and 2a). Again, the degree of retardation correlated well with the PEPC expression level. Differences in the final heights of the shoots were based on a reduced internode length because the number of nodes was even increased in the plants with decreased growth (Figure 2b). Additionally, leaves appeared to be darker and more brittle. The fresh weight increased to approximately 150% of the wild-type in the highest expressing transgenic line. As the dry weight was identical, this was due to a higher water content per leaf area (data not shown). Due to the formation of basal lateral shoots, the overexpressors had a bushy appearance (Figure 1). Transgenic lines expressing the construct without modified phosphorylation site (i.e. stppcC4) lacked any effects on growth and appearance and were hence undistinguishable from the wild-type.

Figure 1.

Phenotype of stppcS9D-C4 lines.

The photograph shows representative cuttings from transgenic plants 6 weeks after potting. Plants were sorted for increasing PEPC activity levels from the left to the right, as indicated below the plants, in multiples compared with the wild-type.

Figure 2.

Growth retardation of transgenic potato lines.

Columns indicate the mean height (a) or internode length (b) of five 10-week-old-cuttings from each transgenic line. Genotypes are as given in the figure. Vertical bars indicate standard errors. An* denotes deviations from the wild-type with P < 0.05. Numbers below the columns indicate the multiples of PEPC activity in transgenic lines compared with the wild-type.

The tubers are the main sink for carbon in potato plants. As shown in Figure 3, tuber yield was drastically reduced to 20% of the wild-type in the stppcS9D-C4 lines. Again, these effects were absent in transformants expressing the stppcC4 construct.

Figure 3.

Yield of tubers from transgenic lines.

The figure shows the total tuber fresh weight from 10-week-old cuttings. Columns show the mean values from five cuttings for each transgenic line. Vertical bars indicate standard errors. An* denotes deviations from the wild-type with P < 0.05. Numbers below the columns indicate the multiples of PEPC activity in transgenic lines compared to the wild-type.

Transgenic lines are not compromised in photosynthetic performance

In order to assess whether an increased flux through PEPC affects photosynthetic performance and is thereby responsible for the stunted growth of the transgenic lines, gas exchange and modulated chlorophyll fluorescence characteristics determined in air at a saturating photon flux density of 500 µmol m−2 s−1 (approximately twice the PFD the plants experienced during growth) of the lines stppcS9D-C4 72 and 21 were compared with the wild-type. As shown in Table 3, overexpression of the engineered PEPC had no major effects on photosynthetic parameters. Rates of CO2 assimilation (A), leaf intercellular CO2 concentrations (Ci), stomatal conductances (g) and transpiration rates (E) in the transgenic lines were similar to the wild-type. However, there was a slight increase in electron transport rates (ETR) in the highest expressing line stppcS9D-C4 21, which resulted in a marginal enhancement of the electron requirement for CO2 assimilation (e : A ratio). These changes were less apparent when CO2 assimilation rates were corrected for the rates of dark CO2 release as listed in Table 4 (see also below) indicating that electron requirement for CO2 assimilation in the transgenic plants is similar to the wild-type. Also, there was no consistent effect on photosynthetic parameters determined under non-photorespiratory conditions, that is in an atmosphere containing 2% O2, or at elevated temperature, that is at 35°C (data not shown).

Table 3.  Gas exchange and Chl a fluorescence characteristics of wild-type and transgenic potato lines in the light.
 Wild-typestppcS9D-C4 72stppcS9D-C4 21
  1. * The rate of CO2 assimilation was corrected for dark respiration rates (Table 4).

A (µmol m−2 s−1)12.75 ± 0.7510.5 ± 1.5412.54 ± 0.22
Ci (µl l−1)154 ± 15152 ± 13154 ± 29
E (mmol m−2 s−1)3.25 ± 0.372.79 ± 0.613.29 ± 0.30
g (mol m−2 s−1)0.316 ± 0.0920.217 ± 0.0840.32 ± 0.12
Fv : Fm0.767 ± 0.00750.797 ± 0.00570.809 ± 0.006
ETR (µmol m−2 s−1)125.5 ± 7.5122.9 ± 15.8138.4 ± 2.6
qP0.883 ± 0.0390.907 ± 0.0320.884 ± 0.010
QAred (%)11.7 ± 3.910.0 ± 2.311.6 ± 1.0
qN0.344 ± 0.0650.375 ± 0.0410.427 ± 0.061
e : A9.85 ± 0.4811.73 ± 1.7211.04 ± 0.33
e : A*8.85 ± 0.4310.10 ± 1.688.92 ± 0.30
E : A0.25 ± 0.0150.26 ± 0.020.26 ± 0.02
Experimentsn = 5n = 3n = 3
Table 4.  Gas exchange characteristics of wild-type and transgenic potato lines kept in the dark for 20 min (A) or 5 min after illumination (B)
  Wild-typestppcS9D-C4 72stppcS9D-C4 21
R (µmol·m−2·s−1)A1.43 ± 0.341.66 ± 0.202.98 ± 0.26-
B1.54 ± 0.381.55 ± 0.12.44 ± 0.31
Ci (µl·l−1)A439 ± 99674 ± 65396 ± 73
B317 ± 49434 ± 88345 ± 50
g (mol·m−2·s−1)A0.017 ± 0.0150.001 ± 0.0010.09 ± 0.01
B0.157 ± 0.0900.045 ± 0.0250.14 ± 0.09
Experiments n = 5n = 3n = 3

There were no large effects on the Fv : Fm ratio, which provides information on the intactness of photosystem II (PS II), or on photochemical quenching (qp), which correlates with the redox state of QA, the first quinone electron acceptor in PSII (i.e. inline image (5) = (1-qP)*100). However, there was a trend of an increase in non-photochemical quenching (qN), which was most apparent in the line 21 suggesting changes in the stromal redox state and/or the proton gradient across the thylakoid membrane.

Gas exchange characteristics of plants, which were dark adapted or darkened shortly after illumination were compared (Table 4). There was a clear increase in dark CO2 release (R) in line 21 suggesting higher rates of decarboxylation. For line 72, a potentially higher rate in dark respiration appeared to be masked by an extremely low stomatal conductance in the dark. This assumption is reinforced by a substantial increase in the Ci values both in the dark adapted state or shortly after illumination, probably as a consequence of increased dark CO2 release at almost completely closed stomata.

The above data indicate that the retardation in growth observed for the PEPC overexpressors is not likely to be caused by a limitation in photosynthesis on a leaf area basis. However, whole plant carbon assimilation is determined by the total leaf area, which is reduced in the transgenic lines due to the stunted growth.

PEPC overexpressors fix more CO2 into malate

In order to gain further insights into the contribution of engineered PEPC to carbon fixation, short-term incorporation of 14CO2 into malate was assessed (Figure 4). Interestingly, wild-type potato already incorporated approximately 15% of newly fixed 14CO2 into malate after a 10-sec pulse. All transgenic genotypes differed significantly from the wild-type. As expected, the co-suppressed lines showed reduced malate labelling, whereas overexpressing lines showed enhanced labelling. In the stppcS9D-C4 lines 72 and 21 labelling of malate was more than doubled. When these plants were compared with plants of the stppcC4 genotype with similar PEPC activities, the mutants with modified phosphorylation site showed always increased malate labelling (P < 0.1). The above data support the idea that an overexpressed physiologically active PEPC is capable of redirecting carbon metabolism. However, the flux into PEP carboxylation products was too low to cause any significant changes in gross CO2 assimilation or in the electron requirement for CO2 assimilation under ambient conditions.

Figure 4.

Percentage of 14CO2 incorporation into malate in wild-type and transgenic potato lines.

Total radioactivity of extracts was measured by scintillation counting and the relative amount of radioactive malate was determined by densitometry of thin layer plates after one-dimensional separation. Each data point is the mean of at least three independent experiments. Vertical lines indicate standard errors. All transgenic lines were different from wild-type with P < 0.025. Numbers below the columns indicate the multiples of PEPC activity in transgenic lines compared with the wild-type.

Carbon and nitrogen metabolism is redirected in PEPC overexpressors

PEP is a central intermediate of glycolysis and amino acid biosynthesis. PEP also serves as a precursor for the shikimate pathway, which is entirely localised within the plastids (Schmid and Amrhein, 1995). In green tissues, PEP is generated from triose phosphates exported from the chloroplast in the light or by the degradation of starch and/or sucrose in the dark. Thus, the overexpression of a physiologically active PEPC in potato plants might perturb fluxes into organic acids, amino acids and carbohydrates as well as secondary metabolites (the latter has already been published in part for the lines under investigation in Häusler et al. (2001)). Overexpression of the stppcS9D-C4 construct resulted in a gene dose dependent increase in the contents of malate and amino acids (Figure 5). It is likely that the additional flux of carbon occurs at the expense of starch, glucose and fructose as the contents of these carbohydrates decreased inversely proportionally to the increase in malate and amino acids. Interestingly, contents of sucrose were not altered in the transgenic lines compared with the wild-type. For the lines carrying the stppcC4 construct, changes in carbon partitioning were absent and contents of starch, soluble sugars, organic acids and amino acids were comparable with the wild-type (data not shown).

Figure 5.

Impact of PEPC activity on the leaf content of sugars and free amino acids in stppcS9D-C4 plants.Leaf sections were cut from the third leaf from the top of 6-week-old cuttings after 8 h of illumination and metabolites were determined. Columns show the mean values from six cuttings for each transgenic line. Vertical bars indicate standard errors. An* denotes deviations from the wild-type with P < 0.05. Numbers below the columns indicate the multiples of PEPC activity in transgenic lines compared with the wild-type.

HPLC analysis of the amount of free amino acids in the transgenic lines and control plants in the light revealed that the sum of glutamine and glutamate (Glx) as well as the sum of asparagine and aspartate (Asx) increased up to five-fold (Figure 6). More detailed enzymatic assays showed that these alterations were mainly due to changes in the concentration of glutamate and aspartate, whilst the corresponding amides were apparently unaffected.

Figure 6.

Impact of PEPC activity on the content of free amino acids in leaves of stppcS9D-C4 plants.

Leaf sections were cut from the third leaf from the top of 6-week-old cuttings after 8 h of illumination and free amino acids (a) and nitrate (b) contents were determined. Columns show the mean values from four cuttings for each transgenic line. Vertical bars indicate standard errors. An* denotes deviations from the wild-type with P < 0.05. For wild-type and overexpressing lines, columns for Asx and Glx are split into Asp and Asn or Glu and Gln, respectively. n.d = not determined.

In the dark, glutamine contents were close to the detection limit in the wild-type, but increased substantially in the PEPC overexpressors, whereas glutamate contents were only doubled in the transgenics (data not shown). The contents of apartate and asparagine were not significantly affected after 8 h in the dark (data not shown). In the light, there was also an increase in the contents of threonine, alanine, and glycine as well as valine that correlated with the expression level of PEPC.

Overexpression of PEPC decreases the contents of phosphorylated metabolic intermediates

The carboxylation of PEP is accompanied by the release of Pi as one of the reaction products. The second reaction product, oxaloacetate (OAA) is readily converted into non-phosphorylated compounds such as malate or aspartate or it can be fed into the citric acid cycle in concert with pyruvate to produce 2-OG. The latter can be used for amino acid biosynthesis via the GS/GOGAT cycle (Goodwin and Mercer, 1983). Hence, overexpression of an unregulated PEPC may cause a substantial accumulation of non-phosphorylated compounds as has been observed for malate and total amino acids (Figure 5). In order to gain further insights into the impact of the overexpression of PEPC on steady state levels of metabolic intermediates that undergo a high turnover, a number of phosphorylated metabolites and a selection of non-phosphorylated intermediates were determined in the light and the dark (Table 5). At the end of the dark period, the amounts of esterified phosphate in metabolic intermediates were severely decreased (by almost 50%) in leaves of the PEPC overexpressing lines stppcS9D-C4 72 and 21. Contents of phosphorylated metabolites were also lower in the light. In contrast, Pi contents in the transformants were increased at the end of the dark period. This is consistent with the substantial allocation from phosphorylated intermediates into non-phosphorylated compounds (i.e. organic acids, amino acids, or sugars) triggered by PEPC. However, Pi contents were not severely affected in the transformants in the light.

Table 5.  Leaf contents of metabolites in wild-type potato plants and transgenic lines overexpressing PEPC. Samples were taken either after 8 h in the light or in the dark (at the end of the dark period), respectively. Significance levels were determined according to the Welsch-test
  Wild-type stppcS9D-C4 72 stppcS9D-C4 21 
  1. P-values are: 1 = P < 0.005,2 = P < 0.01,3 = P < 0.02, 4 = P < 0.05, and 5 = P < 0.1.

   matom·m−2 (% of total phosphate)    
Total esterified phosphateLight0.34 ± 0.07(10.2)0.24 ± 0.05(7.1)0.27 ± 0.06(9.3)
Dark0.24 ± 0.08(11.5)0.14 ± 0.04(3.4)0.14 ± 0.03(4.1)
   matom·m−2    
Inorganic phosphateLight2.99 ± 0.783.12 ± 0.902.62 ± 0.26
Dark1.84 ± 0.583.97 ± 1.313.42 ± 0.24
(a) Light µmol·m−2 (% of total esterified phosphate)    
RubP18.9 ± 2.1(11.2)14.5 ± 4.2(12.2)8.4 ± 0.84(6.2)
3-PGA63.4 ± 15.4(18.8)25.6 ± 2.33(10.8)45.6 ± 4.55(16.8)
2-PGA2.9 ± 0.2(0.9)11.5 ± 1.61(4.8)6.9 ± 1.53(2.6)
PEP17.5 ± 3.3(5.2)10.1 ± 2.03(4.2)11.1 ± 4.34(4.1)
DHAP14.2 ± 0.7(4.2)8.0 ± 2.01(3.4)13.0 ± 4.2(4.8)
GAP2.5 ± 0.2(0.8)3.7 ± 0.95(1.6)2.9 ± 4.0(1.1)
Fru1,6P216.1 ± 3.4(9.6)12.1 ± 3.5(10.3)14.4 ± 4.9(10.6)
Glc6P40.0 ± 12.9(11.8)21.6 ± 3.05(9.2)29.7 ± 6.9(10.9)
Glc1P3.3 ± 1.5(1.0)1.8 ± 0.8(0.8)3.6 ± 2.0(1.3)
Fru6P15.2 ± 2.5(4.5)7.8 ± 2.92(3.3)12.5 ± 3.8(4.6)
UDPG13.5 ± 2.5(7.1)8.1 ± 0.93(6.9)13.1 ± 1.8(9.7)
ATP17.2 ± 1.5(15.3)14.8 ± 3.3(18.8)16.6 ± 1.8(18.4)
ADP6.8 ± 2.5(4.0)8.9 ± 3.5(7.5)8.7 ± 2.6(6.4)
AMP11.5 ± 2.4(3.3)7.8 ± 4.0(3.3)7.4 ± 0. 2.45(2.7)
Pyruvate25.6 ± 9.929.7 ± 5.027.5 ± 6.74
2-OG44.8 ± 21.660.3 ± 19.063.1 ± 14.6
OAA2.8 ± 0.93.6 ± 1.23.1 ± 0.4
(b) Dark µmol·m−2 (% of total esterified phosphate)    
RubPn.d.n.d.n.d.
3-PGA66.5 ± 9.3(27.7)18.2 ± 3.61(12.6)21.8 ± 4.61(15.2)
2-PGA7.7 ± 1.9(3.2)10.1 ± 2.2(9.3)5.9 ± 2.4(4.1)
PEP13.6 ± 1.8(5.6)8.2 ± 1.82(5.7)8.1 ± 2.02(5.6)
DHAP1.1 ± 2.2(0.5)6.8 ± 2.52(4.7)6.5 ± 0.42(4.5)
GAPn.d.1.9 ± 1.7(1.3)2.3 ± 0.5(1.6)
Fru1,6P21.1 ± 2.2(0.9)1.3 ± 1.1(1.2)2.4 ± 0.24(3.4)
Glc6P37.1 ± 11.3(15.4)13.9 ± 2.74(9.7)13.8 ± 2.74(9.6)
Glc1P4.0 ± 2.4(1.7)3.2 ± 1.0(2.2)3.5 ± 2.0(2.4)
Fru6P7.1 ± 1.8(2.9)3.7 ± 2.45(2.6)4.3 ± 0.35(3.0)
UDPG8.6 ± 1.3(7.2)7.8 ± 2.9(10.8)7.3 ± 0.9(10.2)
ATP14.5 ± 4.0(18.1)12.2 ± 4.5(25.4)10.4 ± 4.0(21.8)
ADP15.8 ± 1.8(13.1)7.0 ± 1.01(9.7)8.4 ± 2.31(11.7)
AMP9.1 ± 3.5(3.8)9.4 ± 3.4(6.5)8.9 ± 2.15(6.2)
Pyruvate18.2 ± 1.917.1 ± 6.513.6 ± 2.14
2-OG26.0 ± 7.922.4 ± 6.315.1 ± 7.3
OAA4.2 ± 3.73.0 ± 1.22.3 ± 0.6

There were interesting changes in the absolute contents of individual phosphorylated intermediates as well as in their proportion relative to the total content of esterified phosphate. Most prominently, the contents of 3-phosphoglycerate (3-PGA) decreased in leaves kept both in the light and dark. This decrease was accompanied by a decline in PEP contents and an increase in 2-PGA in the light. However, when expressed relative to summarised phosphate esters, PEP and 3-PGA contents were not significantly altered indicating that the decrease in the absolute contents followed the overall trend of a decline in phosphorylated intermediates. Besides 3-PGA and PEP, ribulose-1,5-bisphosphate (RuBP), hexose phosphates (apart from Glu-1P) and AMP contents were decreased in the light, whereas the contents of triose phosphates, fructose-1,6-bisphosphate (Fru1,6P2), UDP-glucose (the precursor for sucrose biosynthesis) and ATP were not severely altered in the transformants relative to the wild-type. ADP levels were slightly increased in the light. For the 2-oxoacids, there was an increase in 2-oxoglutarate (2-OG), which acts as a precursor for glutamate synthesis, and a trend of an increase in oxaloacetate (OAA), the product of PEPC, whereas pyruvate contents remained unaffected.

In darkened leaves, most of the changes in metabolite contents were similar or even more pronounced. There was a significant decline in 3-PGA (but not in 2-PGA), PEP, hexose phosphates, ATP and ADP. In contrast, triose phosphates and Fru1,6P2, which were close to the detection limit in the wild-type, exhibited a substantial increase in the dark. AMP contents remained unaffected. The contents of the 2-oxoacids pyruvate, 2-OG, and OAA, showed a trend of decline. For 2-OG, the decline was inversely correlated with the increase in glutamine (data not shown) suggesting a substantial flux into Gln biosynthesis in the dark.

The data indicate a drain of phosphorylated intermediates by overexpression of the engineered PEPC.

Discussion

Modification of the catalytic properties of potato PEPC

Five attempts towards overexpression of ppc genes in plants have been described so far. Three groups expressed the maize PEPC cDNA or gene under control of different promoters (Hudspeth et al., 1992; Kogami et al., 1994; Ku et al., 1999) and increases in enzyme activity up to 100-fold were detected with the complete intact maize gene in rice (Ku et al., 1999). Recently, the overexpression of PEPC and malate dehydrogenase from Sorghum in potato has been described, but in this case expression levels were low and no modification in photosynthetic parameters was observed (Beaujean et al., 2001). To avoid any possible influence of improper post-translational modification, we have previously used a bacterial ppc cDNA under control of a modified 35S promoter in potato (Gehlen et al., 1996). This resulted in a clear increase in PEPC activity (Häusler et al., 2001) and slowed down growth in axenic culture with some minor impact on photosynthetic and metabolic parameters (Gehlen et al., 1996). Opposite effects were observed in plants with antisense stppc constructs and diminished PEPC activities (Häusler et al., 1999). In order to further increase the activity of PEPC in vivo under physiological conditions we introduced selected modifications into the coding sequence of the potato ppc cDNA. The exchange of an internal part of the sequence for the homologous stretch from the C4 plant Flaveria trinervia was initially thought to allow discrimination of the transgene and the endogenous protein by specific antibodies in Western analyses, but surprisingly resulted in a significant shift of the enzymatic properties in vitro. Blasing et al. (2000) applied domain shuffling to analyse regions responsible for C4-specific characteristics of Flaveria PEPC proteins. A region between aa 296 and 437 and a single serine residue at the carboxy-terminus were shown to control these characteristics. The domain exchanged in our study from the Flaveria trinervia to the potato PEPC is part of the identified region. Beside a decrease in the Km we observed an increase of the I50 for malate, a change towards values observed for C4 isoforms. Interestingly, the reciprocal exchange from the C3 enzyme to the C4 enzyme decreased the I50 for malate (Thomas Rademacher, unpublished data) supporting the importance of this region for the enzymatic properties of PEPC proteins. Blasing et al. (2000) used kinetic parameters and enzyme activation by Glc6P as indicative parameters instead. It will be interesting to test whether these parameters are also affected in the engineered potato protein.

Mutations at the N-terminal phosphorylation site of the engineered potato sequence also increased the affinity for PEP and decreased product inhibition of the enzyme. These alterations have previously been shown to reduce the malate sensitivity of C4 PEPC enzymes from Sorghum and maize (see also introduction). We made similar observations with a photosynthetic PEPC from Flaveria trinervia (unpublished data). Furthermore, the importance of this region for the activity of C3 PEPC enzymes was shown by in vitro and in vivo phosphorylation experiments (Duff and Chollet, 1995; Schuller and Werner, 1993; Zhang et al., 1995).

We obtained best effects with the potato enzyme carrying both the N-terminal and internal modifications. Allosteric inhibition of the engineered construct was in the range of values normally observed for photosynthetic isoforms and in parallel the affinity for PEP was further increased beyond the levels normally observed for anaplerotic isoforms. We expected this combination to be optimally suited for overexpression of PEPC with high and stable activities in vivo. This was tested by correlation of expression rates and the percentage of 14CO2 fixed into malate. Overexpression of PEPC clearly increased pulse labelling of malate. When the enzyme with both N-terminal and internal modifications was used, effects were clearly enhanced compared with the proteins carrying only the internal modification. The maximum rate of carbon fixation into malate in the transgenic lines was similar to the levels detected for C3-C4-intermediate species like Flaveria ramosissima where C3 and C4 cycles compete for carbon fixation (Rumpho et al., 1984). Thus, the degree of malate inhibition in vitro and the amount of malate labelled in vivo correlate well suggesting that product inhibition might be one limiting step for the enzymatic activity inside the cell.

However, the net rate of CO2 fixation into malate could additionally be dependent on reaction sequences metabolising malate. We have recently reported that the introduction of either bacterial PEPC or the modified potato PEPC described here induced an endogenous cytosolic NADP malic enzyme and increased the activity of mitochondrial NAD malic enzyme in potato leaves (Häusler et al., 2001). The decarboxylation reaction catalysed by these enzymes might liberate labelled carbon that had already been fixed and by this reduce the gross incorporation. This assumption is also supported by the increase in dark CO2 release observed in the PEPC overexpressing lines.

Our data indicate that the introduced modifications are necessary to induce enhanced in vivo PEPC activity in the light. This is in agreement with additional experiments where neither overexpression of the Corynebacterium glutamicum nor of the intact Flaveria trinervia PEPC in potato induced comparable rates of CO2 fixation into malate (Thomas Rademacher, unpublished data). However, similar observations concerning enhanced labelling of C4 acids have been made by Suzuki et al. (2000) following overexpression of phosphoenolpyruvate carboxykinase (PCK) in rice. The authors assume that these effects are due to an enhanced activity of the endogenous PEPC in response to increased PEP levels produced by the overexpressed PCK.

Leaf metabolism is perturbed by overexpression of PEPC

Striking differences were observed for plant growth and metabolite levels comparing plants overexpressing PEPC with and without modified phosphorylation site. Plants overexpressing the constructs with a modified phosphorylation site were stunted in growth and tuber yield was severely impaired. Growth retardation appeared to be correlated with changes in metabolite content suggesting a redirection in metabolic fluxes. The most striking effect was the increase in malate and total amino acids. Both changes fit very well into the proposed role of PEPC in C3 plants. Moreover, the increase in malate and amino acids was inversely proportional to the decline in starch and soluble sugars in the light. The loss of carbon in starch and soluble sugars of about 34 matom C·m−2 in line stppcS9D-C4 21 was compensated by a gain of 23 matom C·m−2 in malate, 12 matom C·m−2 in Glu/Gln and 1 matom C·m−2 in Asp/Asn. This underlines the redirection of carbon flow from starch and soluble sugars into organic acids. The decrease in starch content and the increases in soluble, osmotically active compounds could also account for the higher leaf water content.

Potato plants are often limited in nitrogen supply during growth. Thus, an increase in the amino acid content in the transgenic lines might be interpreted as an indirect consequence of slower growth rates attenuating the plants from nitrogen limitation. However, nitrate contents in the transgenic lines were considerably increased both in the light and dark ruling out nitrogen limitation at the site of nitrate supply. Contents of Glu and Gln as well as Asp increased in the PEPC overexpressors during illumination. Interestingly, in the dark Gln contents increased considerably from a very low value (2 µmol m-2) in the wild type to 200 µmol m-2 in the line stppcS9D-C4 21, whereas Glu contents remained unchanged (data not shown). Kaiser and Brendle-Behnisch (1995) have shown that nitrate reductase activity is enhanced in leaf discs kept in the dark up to levels normally observed in the light when the pH is changed into the acidic range. In the PEPC overexpressors, such an acidification might be brought about by the increased accumulation of malate and thus nitrate reductase activity might be enhanced compared with the wild-type providing additional reduced nitrogen for amino acid synthesis. It is likely that 2-oxoglutarate (2-OG) deriving from the anaplerotic action of PEPC is utilised as the corresponding carbon source for amino acid biosynthesis in the dark. This idea is supported by the observed decline in 2-OG contents with an increase in PEPC activity. The increase in Gln combined with unaltered Glu contents suggests a limitation at the site of plastidial Fd-GOGAT, which requires reducing equivalents supplied by photosynthetic electron transport in the light.

Besides Glu and Asp, the contents of Thr, Ala, Gly and Val were clearly increased in the PEPC overexpressors compared with the wild-type. Thr is derived from Asp, Gly can be synthesized from Glu, Val from pyruvate, and Ala either from aspartate or by transamination from pyruvate (Goodwin and Mercer, 1983). Increased contents of these amino acids suggest higher fluxes starting from the respective precursors. Similar changes in the levels of individual amino acids were observed in plants, which were shifted from low to high nitrate nutrition. The most pronounced increase was obtained for Asp/Asn, Glu/Gln, Ser, and Gly (Champigny, 1995). Moreover, if plants depleted in nitrate or mutated in genes necessary for nitrogen metabolism are exposed to a nitrate pulse, carbon partitioning is redirected towards organic acids (Duff and Chollet, 1995; Scheible et al., 1997; Stitt et al., 2002). Interestingly, this is accompanied by an increase in PEPC kinase activity protecting PEPC from product inhibition (Champigny, 1995). The mutations introduced into the phosphorylation site mimic exactly this physiological state supporting the idea that a diminished malate sensitivity is the decisive step for the increase of in vivo PEPC activities. The data are consistent with a relief of nitrogen limitation in the transgenic lines caused by the slower growth and deviations in the relative ratios of amino acids due to indirect impacts of PEPC overexpression on the amino acid metabolism.

There were clear changes in the contents of intermediates that undergo high metabolic fluxes, such as phosphorylated metabolites and 2-oxoacids both in the light and the dark. Most strikingly, contents of RubP, 3-PGA, PEP, Glc6P, Fru6P and AMP declined in the light, whereas 2-PGA, ADP, 2-OG and OAA increased and trioseP, Fru1,6P2, Glc1P, ATP and pyruvate remained unaffected. As in the light the majority of 3-PGA is localised in the stroma (Leidreiter et al., 1995; Wirtz et al., 1980), a decrease in 3-PGA would be consistent with a limitation of starch biosynthesis at the site of ADP-glucose phosphorylase (AGPase) because this enzyme strongly responds to the stromal 3-PGA : Pi ratio in that a decrease in this ratio limits the flux of carbon into starch (Preiss, 1982). In fact, starch levels were significantly diminished in the transgenic lines. Moreover, AGPase activity also responds negatively to nitrate levels that were enhanced in transgenic leaves and high nitrate levels correlate with reduced levels of 3-PGA and PEP (Scheible et al., 1997; Stitt et al., 2002) consistent with the situation observed in the PEPC overexpressors.

RubP levels have been shown to respond to the leaf intercellular CO2 concentration (Ci) and the photon flux density (PFD), in that the contents decline with an increase in Ci above ambient and increase with an increase in PFD (Badger et al., 1984). Thus, CO2 assimilation rates can switch from RubP saturating rates to RubP limiting rates and vice versa. However, despite the decline in RubP in stppcS9D-C4 21 by more than 50% of the wild-type, CO2 assimilation was not severely impaired suggesting that RubP content in the transgenic lines was still sufficient for the maintenance of CO2 assimilation rates comparable with the wild-type. Interestingly, 3-PGA, which has been shown to be inversely correlated to RubP over a wide range of Ci-values and PFDs (Badger et al., 1984), declined as well suggesting that the Calvin cycle intermediates are drained from the stroma, most likely by excessive Pi liberation from PEP in the cytosol. Furthermore, changes in the metabolite ratios of the reactions catalysed by phosphoglyceromutase (3-PGA : 2-PGA) and enolase (PEP : 2-PGA) in the transgenic plants showed clear deviations from the wild-type. As chloroplasts usually lack a full glycolytic pathway (because they are deficient in phosphoglyceromutase and enolase), these metabolite ratios should be indicative of processes taking place in the cytosol. Assuming that approximately 20% of the 3-PGA measured in the light is localised in the cytosol (Wirtz et al., 1980), the 3-PGA : 2-PGA ratio in wild-type plants is 4.4 and thus near to the reported Keq of 5.0. In the transgenic lines the distribution of 3-PGA between the stroma and the cytosol is unknown. The overall ratios of 3-PGA : 2-PGA were diminished from 22.0 in the wild-type to 2.2 and 6.6 in the lines stppcS9D-C4 72 and 21, respectively. A similar scenario holds true for the PEP:2-PGA ratio. Again, the ratio of 6.1 in the wild-type is near to the reported constant equilibrium (Keq = 6.7) whereas ratios of 0.88 (line 72) and 1.3 (line 21) suggest that the reaction catalysed by enolase is far away from equilibrium in the transgenic lines. Enolase activity appears to become flux limiting as PEP : 2-PGA ratios declined. However, it is not quite clear why 3-PGA : 2-PGA ratios decline in concert. It is conceivable that 3-PGA in the cytosol falls far below the Km value of phosphoglyceromutase for 3-PGA.

For transgenic rice plants overexpressing the complete maize PEPC gene phosphate limitation of photosynthesis has been proposed (Fukayama et al., 2001; Matsuoka et al., 2001) and was recently supported experimentally (Agarie et al., 2002). From our metabolite data, phosphate limitation of photosynthesis (Sharkey, 1985) appears to be unlikely in the potato system.

In the dark, the contents of 3-PGA, PEP, hexoseP, ATP, ADP, and all three 2-oxoacids decreased in the transformants compared with the wild-type, whereas trioseP and Fru1,6P increased significantly and UDPG and AMP remained unchanged. A possible scenario for the delivery of precursors for PEP carboxylation could be the phosphorolytic degradation of starch that will lead to triose phosphates as end products of the oxidative pentose phosphate pathway. This is consistent with the depletion in Glc6P and concomitant with an increase in relative Glc1P levels and the increase in trioseP, which are the major exchange components linking the pentose phosphate pathway to glycolysis.

Interestingly, sucrose biosynthesis seemed not to be affected severely. The key metabolite UDP-glucose (UDPG) exhibited neither altered absolute levels nor clear changes in the relative abundance compared to the total esterified phosphates. This is in agreement with sucrose levels in wild-type and transgenic lines, which were not severely affected by PEPC overexpression (Figure 5). As sucrose is the main carbohydrate transported in the plant, export of this compound from leaves might be reduced. This would again match the reduced deposition of carbohydrates in tubers.

Taken together, the data indicate significant changes in carbohydrate metabolism and a depletion of phosphorylated intermediates due to the overexpression of PEPC in the transgenic lines.

Growth retardation in the transgenic lines overexpressing functional PEPC is a yet unexplained phenomenon

Internodial growth was severely impaired in the transgenic lines overexpressing a functional PEPC in a gene dose dependent manner. Stunted growth was accompanied by a diminished total leaf area and lowered rates of CO2 assimilation per plant, particularly if it is considered that photosynthetically active leaves were at a larger distance to the light source since plants were smaller. The lower carbon fixation on the whole plant level might be in part responsible for the decreased tuber yield. For transgenic potato plants overexpressing the PEPC from C. glutamicum a moderate growth retardation was only observed in axenic culture (Gehlen et al., 1996) and there was no reported effect on growth parameters in rice plants overexpressing the complete maize PEPC gene under the control of its own promoter (Ku et al., 1999). Moreover, even if the increase in nocturnal CO2 release observed in the potato plants carrying the mutated potato PEPC is considered, this would not result in the growth retardation observed, particularly if it is taken into account that nocturnal carbon loss was diminished in the line stppcS9D-C4 72 by almost complete stomatal closure. Likewise N-limitation as an explanation for growth retardation appears to be unlikely as nitrate contents are even increased in the transgenic lines. However, at this stage there is no information available on the impact an increase in malate could have on cytosolic pH or regulatory properties of enzymes involved in nitrogen metabolism. It also appears likely that the growth retardation observed here is an indirect consequence of an imbalance of growth factors by the constitutive overexpression of functional PEPC in all tissues and cell types. In order to tackle this problem it is intended to overexpress the same PEPC construct under the control of a leaf specific promoter.

Our approaches towards overexpression of a physiologically active PEPC are finally aimed to install a C4-like pathway in C3 plants. In this publication, we were able to show that our engineered enzyme induces massive accumulation of malate inside the cell. Still, this does not lead to a reduction of the CO2 compensation point (Γ) characteristic for C4 carbon fixation (data not shown). We have previously reported that combination of PEPC with a decarboxylating activity inside the chloroplast is capable of modulating photosynthetic parameters in potato (Lipka et al., 1999). Plants expressing additional activities for PEP regeneration are currently under investigation in our laboratory. We are convinced that integration of the novel protein into such a C4-like pathway will avoid the observed site effects of increased PEPC activity in the cytoplasm and lead to the establishment of an effective HCO3–/CO2 pump in C3 plants.

Experimental procedures

Plant transformation and growth

Leaf discs of Solanum tuberosum cv. Désirée were transformed by infection with Agrobacterium tumefaciens strain GV3101 (Dietze et al., 1995). Transgenic lines were selected on kanamycin according to (Koncz and Schell, 1986). Plants used for physiological experiments were cultivated in growth chambers at a temperature of 22°C in the light for 16 h and 20°C in the dark for 8 h. They were illuminated with Osram Superstar HQI-T 400 W DH−1 lamps (Osram, München, Germany). The photon flux density was between 200 and 300 µmol m−2 s−1. Prior to physiological experiments individual transgenic lines were propagated by cuttings. If not stated otherwise, all plant samples were taken between 6 and 8 h after onset of illumination.

DNA manipulation, plasmids, oligonucleotides

DNA manipulations were performed according to standard methods. All modified ppc genes were first cloned into pTrc99A vectors (Amersham Bioscience, Freiburg, Germany) to examine activity by complementation of E. coli strain PCR1 defective in endogenous PEPC (Sabe et al., 1984). Mutations in the N-terminal region of stppc were induced by PCR-based techniques. In all constructs, the second amino acid was exchanged from threonine to alanine to generate a NcoI site for cloning. For the S11D mutant, S11 was replaced by asparagine. For the S9D mutant, amino acids D7, K8, and L9 were additionally deleted and replaced by serine. An internal fragment from aa384 to aa420 (with respect to the potato sequence) was exchanged in between the potato and the Flaveria PEPC using the bordering BstEII and PmaCI restriction sites of the cDNA. The particular genotypes are listed in Table 1. For plant transformation, the gene constructs were inserted into the binary plant expression vector pSPAM, a derivative of pPAM (GenBank: AY027531). The expression cassette was flanked by the scaffold attachment region of the tobacco RB7 gene (GenBank: U67919). The nptII cassette of pPCV002 (Koncz and Schell, 1986) was used for selection of transgenic plants. Expression of ppc genes is under constitutive control of the transcriptionally enhanced CaMV 35SS promoter (Reichel et al., 1996). A schematic representation of the T-DNA is given in Figure 7.

Figure 7.

Schematic representation of the T-DNA construct transferred to potato plants.

Details on the different elements are given in experimental procedures. Abbreviations are: LB, RB = left and right border, Pnos = nopalin synthase promoter, nptII = neomycin phosphotransferase, pAocs = octopin synthase polyadenylation signal, SAR = scaffold attachment region, P35SS/pA35S = transcriptionally enhanced promoter and polyadenylation signal of the 35S gene from cauliflower mosaic virus, TL = tobacco etch virus 5′untranslated region.

PEPC activity assays

Enzymatic properties of modified PEPC proteins expressed in bacteria were determined in a coupled spectrophotometrical assay. Desalted extracts were prepared in the presence of 10 µg ml-1 chymostatin. Substrate affinity and sensitivity to malate inhibition were determined at a suboptimal pH as suggested before (Li et al., 1996). Reaction conditions were: 50 mm HEPES-NaOH (pH 7.3), 5 mm MgCl2, 1 mm NaHCO3, 20% (w/v) glycerol, 0.2 mm NADH, 2.5 mm PEP, 6 U ml−1 MDH in a volume of 1 ml. One unit of enzyme activity was defined as 1 µmol of NADH oxidation per min at 30°C. Controls were without PEP. PEPC activity of transgenic potato plants was determined at an optimal pH (pH 8.0) from non-desalted leaf extracts.

14CO2 labelling experiments and extraction of metabolites

Labelling experiments were performed in a plexiglass chamber of 200 ml volume (custom made at the Institute for Biology I, RWTH-Aachen). 14CO2 was released from NaH14CO3 (53 µCi µ mol−1) in air, which resulted in an increase of the CO2 concentration from 380 µl l-1 to 410 µl l-1. Leaf discs (2,5 cm2) were taken from the third uppermost leaf 8 h after onset of illumination, and were preincubated for 30 min in Petridishes on moistured filter paper at a PFD of 100 µmol m−2 s−1. After preincubation, the discs were inserted into and removed from the plexiglass chamber by means of a slide plate. After incorporation of 14CO2 in the light, leaf discs were submersed instantaneously in 1 ml of 80% (v/v) boiling ethanol. Hot ethanol extraction was repeated twice. The ethanolic fractions were pooled, evaporated to dryness and the residues dissolved in 100 µl 40% (v/v) ethanol. Total radioactivity incorporated in the soluble fraction was determined in aliquots of extracts. Soluble compounds in the ethanolic extracts were separated two-dimensionally on cellulose thin-layer plates (Merck, Darmstadt, Germany) as described by Schürmann (1969). Quantitatively dominant compounds were identified by cochromatography of authentic substances. The chromatograms were scanned using a Phosphoimager (FUJI FLA 3000; Raytest, Straubenhardt, Germany) and the intensity of the signals in the individual spots was quantified with the AIDA 3.1 software supplied with the device.

Determination of metabolites

Sugars and amino acids were extracted from leaf discs as described above for 14C labelled metabolites. Dried pellets from the ethanol extraction were resuspended in water and insoluble material removed by centrifugation (5 min, 14 900 g). Starch was extracted from bleached leaf discs that were homogenised in 500 µl 2 m KOH. The solution was incubated at 95°C for 45 min. Insoluble material was removed by centrifugation (see above). 900 µl 1 m acetic acid was added to adjust the pH to 4.8. Samples were stored at − 20°C. Starch was hydrolysed enzymatically in the presence of α-amylase and amyloglucosidase. The glucose released from starch and contents of soluble sugars in the ethanolic extracts were determined enzymatically according to Stitt et al. (1989).

A coupled assay was used for the determination of malate. Reaction conditions were as follows: 250 mm glycylglycin, 50 mm l-glutamate, 4 mm NAD, 0.75 U glutamate oxaloacetate transaminase ml-1, pH 10. Malate concentration was calculated from the extinction difference at A340 after 24 U of malate dehydrogenase were added and incubated (30°C) until extinction was constant.

Total amino acid concentration was determined with ninhydrin as described in Häusler et al. (1999). The concentration of individual amino acids as listed in Figure 7 was determined with an amino acid analyser (Biotronic LC 5001; Maintal, Germany) after derivatisation of amino acids with o-phtalaldehyde. Contents of Glu, Gln, Asp and Asn were determined enzymatically according to Bergmeyer (1970). Gln contents were estimated from the difference in Glu contents in the presence or absence of glutaminase in an assay coupled to glutamate dehydrogenase. The reaction product 2-oxogluarate (2-OG) was converted to its hydrazone form in the presence of hydrazine. Asp and Asn were quantified in an assay coupled to oxaloacetate (OAA) reduction catalysed by NAD-MDH. OAA formation from Asp was initiated by the addition of aspartate:oxoglutarate aminotransferase in the presence of an excess of 2-OG and Asn was subsequently converted to Asp by the addition of asparaginase.

All other low abundant metabolites were determined in neutralised perchloric acid extracts from potato leaf discs (10 cm2) enzymatically according to Häusler et al. (2000) with a microtiter plate reader (Tecan, Crailsheim, Germany) in the fluorescence mode (excitation: λ = 340 nm, emission: λ = 465 nm). The basic protocols for enzymatic metabolite determinations were modified from Bergmeyer (1970) and Stitt et al. (1989). As potato leaf extracts contain substantial amounts of phenolic compounds, which are capable of quenching NAD(P)H fluorescence care was taken with the calibration of the assay. The quenching of extracts obtained with PEPC overexpressors was far less pronounced compared with the wild-type suggesting lower levels of phenolics in the transformants. High rates of non-enzymatic NADH oxidation in the presence of the extracts were accounted for by preincubation of the extracts in the presence of 10 µm NADH. Only when the rate of NADH oxidation was negligible the assay was initiated by the addition of coupling enzymes.

Nitrate was converted to nitrite according to Bergmeyer (1970) in the presence of nitrate reductase. The reaction product nitrite was quantified colorimetrically according to Rao et al. (1981) in the presence of N-naphtylethylendiamoniumchloride and sulfonylamide at a wavelength of 540 nm. Standard curves were constructed with known concentrations of KNO3 after conversion to NO2.

Inorganic phosphate was determined following Wirtz et al. (1980) by phosphorylytic cleavage of oyster glycogen in the presence of rabbit muscle phosphorylase. The reaction product glucose-1-phosphate (Glc1P) was converted to glucose-6-phosphate (Glc6P) in the presence of phosphoglucomutase and this was quantified coupled to Glc6P dehydrogenase.

Determination of photosynthetic parameters

Gas exchange and modulated Chlorophyll a fluorescence measurements were essentially performed as described in Häusler et al. (1999). Shortly, steady state photosynthesis was determined 30–40 min after illumination at a constant temperature of 25°C and a PFD of 500 µmol m−2 s−1. The relative humidity was kept at approximately 30% water vapour saturation. For the determination of the Fv : Fm ratio, plants were darkened for 20 min prior to the application of a saturated pulse.

Statistical evaluation of experimental data

The data provided are all mean values ± standard deviations (SD) or ± standard errors (SE) of the indicated number of independent data acquisitions using leaves from different plants of individual transgenic or control lines. Significance of differences between wild-type plants and transformants were assayed using the Welsch-test based on SD values (Lorenz, 1984), which allows for unequal relative errors between two groups of measurements assuming that a Gauss distribution is applicable (the authors are aware that this is an idealisation for some of the data sets).

Footnotes

  • Current address: Fraunhofer Institute for Molecular Biology and Applied Ecology, 52074 Aachen, Germany

  • Current address: Max-Planck Institute for Plant Breeding Research, 50829 Koeln, Germany

Acknowledgements

This work was supported in part by a grant from the Deutsche Forschungsgemeinschaft (DFG). Thanks to the Institute for Biochemistry, University Clinic Aachen, for help with analysis of individual amino acids.

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