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First published online October 1, 2008; 10.1104/pp.108.126516 Plant Physiology 148:1640-1654 (2008) © 2008 American Society of Plant Biologists OPEN ACCESS ARTICLE
Decreased Expression of Cytosolic Pyruvate Kinase in Potato Tubers Leads to a Decline in Pyruvate Resulting in an in Vivo Repression of the Alternative Oxidase1,[W],[OA]Max-Planck Institute of Molecular Plant Physiology, 14476 Potsdam-Golm, Germany (S.N.O., J.E.L., E.U.W., A.L., J.T.v.D., B.F., A.R.F., P.G.); Leibniz-Institute of Vegetable and Ornamental Crops, 14979 Grossbeeren, Germany (P.G.); and University of Potsdam, Institute of Chemistry and Interdisciplinary Center for Photonics, 14476 Potsdam-Golm, Germany (E.S.)
The aim of this work was to investigate the effect of decreased cytosolic pyruvate kinase (PKc) on potato (Solanum tuberosum) tuber metabolism. Transgenic potato plants with strongly reduced levels of PKc were generated by RNA interference gene silencing under the control of a tuber-specific promoter. Metabolite profiling showed that decreased PKc activity led to a decrease in the levels of pyruvate and some other organic acids involved in the tricarboxylic acid cycle. Flux analysis showed that this was accompanied by changes in carbon partitioning, with carbon flux being diverted from glycolysis toward starch synthesis. However, this metabolic shift was relatively small and hence did not result in enhanced starch levels in the tubers. Although total respiration rates and the ATP to ADP ratio were largely unchanged, transgenic tubers showed a strong decrease in the levels of alternative oxidase (AOX) protein and a corresponding decrease in the capacity of the alternative pathway of respiration. External feeding of pyruvate to tuber tissue or isolated mitochondria resulted in activation of the AOX pathway, both in the wild type and the PKc transgenic lines, providing direct evidence for the regulation of AOX by changes in pyruvate levels. Overall, these results provide evidence for a crucial role of PKc in the regulation of pyruvate levels as well as the level of the AOX in heterotrophic plant tissue, and furthermore reveal that these parameters are interlinked in vivo.
Pyruvate kinase (PK; EC 2.7.1.40) catalyzes the final reaction of the glycolytic pathway, converting ADP and phosphoenolpyruvate (PEP) to ATP and pyruvate, which is subsequently imported into mitochondria and used as a substrate for respiration. PK catalyzes an irreversible reaction in vivo in castor bean (Ricinus communis; Geigenberger et al., 1993  ) and is probably a primary regulation site of glycolysis and respiration, as indicated by various biochemical studies in mammalian and bacterial systems (Pilkis and Granner, 1992; Teusink et al., 2000) and plants (Thomas et al., 1997; Geigenberger et al., 1998; Schwender et al., 2004; Plaxton and Podesta, 2006). In plants, PK is believed to provide bottom-up control of plant glycolytic flux from hexose-phosphates to pyruvate owing to PEP's pronounced feedback allosteric inhibition of ATP- and inorganic pyrophosphate-dependent phosphofructokinases (Dennis and Greyson, 1987). PK in plants has been found to be subject to a number of regulatory mechanisms, including allosteric regulation by Glu and Asp (Turner et al., 2005), activation by changes in cytosolic pH (Podesta and Plaxton 1991, 1992, 1994), inhibition by ATP (Podesta and Plaxton, 1991, 1992, 1993), and phosphorylation and ubiquitination (Tang et al., 2003). While the role of PK in the regulation of glycolysis in response to hormone signals has been intensively studied in mammalian and bacterial systems (Pilkis and Granner, 1992; Teusink et al., 2000), much less is known about the signals regulating PK in plants.
Previous studies in vascular plants revealed that PK exists as tissue-specific isozymes that show pronounced differences in their respective physical and kinetic properties (Plaxton and Podesta, 2006
Given the likely importance of PKc for generating pyruvate for entry into the mitochondrial tricarboxylic acid (TCA) cycle, we focused our studies on understanding the effect of altered PKc activity on carbon metabolism, particularly on respiratory processes and the coordinated regulation of glycolysis, the TCA cycle, and the mitochondrial electron transport chain. It is becoming apparent that a coordinated regulation of these respiratory processes exists (Fernie et al., 2004 In this study, we have generated and analyzed transgenic potato tubers with a decrease in PKc levels using an RNA interference (RNAi) approach. Decreased PKc levels led to decreased levels of pyruvate and several TCA cycle intermediates, which were accompanied by changes in carbon partitioning between glycolysis and starch and by a decrease in AOX protein level and respiratory capacity. The decrease in AOX-dependent respiratory capacity could be reverted by external feeding of pyruvate to tuber tissue. These results thus provide evidence for an important role of PKc in regulating pyruvate levels and the alternative pathway of respiration in heterotrophic potato tubers.
Generation of Potato Tubers with Decreased PKc Expression and Activity
Plants contain multiple isozymes of PK (e.g. Arabidopsis contains 14 isozymes; Arabidopsis Genome Initiative 2000
To generate potato tubers with decreased PKc expression, we targeted the PKCYT1 gene in an RNAi gene silencing approach. The PKCYT1 gene was chosen because our database searches indicated that transcripts of PKCYT1 are highly abundant in tubers and it is therefore likely to encode a major tuber isozyme. An RNAi construct was created, containing a PCR-amplified, 650-bp cDNA fragment homologous to the PKCYT1 gene in a hairpin orientation between the tuber-specific B33 promoter and the OCS terminator (Fig. 2A ), and transformed into potato. The resultant transgenic lines were screened by measuring total PK activity in 10-week-old tubers using an enzyme assay at pH 6.9. Five lines were selected for further analysis: PKC-25, -6, and -15 had significantly reduced PK activities down to 40%, 37%, and 29% of wild-type level, respectively, PKC-26 had significantly reduced activity to 64% of wild-type activity, while activity in PKC-11 was not significantly different from wild type (Fig. 2B).
PKc protein levels in the transgenic tubers were determined from western blots using a PKc-specific antibody raised against PKc from Brassica napus (Smith et al., 2000 ; Fig. 2C). This antibody has previously been shown to recognize only PKc proteins and not PKp proteins in different plant species (Smith et al., 2000; Plaxton et al., 2002; Rivoal et al., 2002). Analysis of wild-type potato showed that tubers contain two strongly immunoreactive proteins of approximately 60 and 55 kD (proteins 1 and 2; Fig. 2C) and one less immunoreactive protein (approximately 50 kD, protein 3; Fig. 2C). In the PKc transgenic lines, levels of the two major PKc immunoreactive polypeptides (proteins 1 and 2) are strongly decreased in the strongly silenced lines (PKC-25, -6, and -15) but not in the weakly silenced lines (PKC-26 and -11; Fig. 2C). Interestingly, the minor immunoreactive polypeptide (protein 3) appears to be induced in PKC-25, -6, and -15. It is difficult to assign the different immunoreactive bands to particular genes because the predicted molecular masses of the PKc polypeptides are similar (54–57 kD); nevertheless, it is likely that protein 2 corresponds to the PKCYT1 protein, based on the predicted molecular mass (55 kD) and the reduced levels of this protein in the transgenic tubers. Protein 3 appears to be the only PKc polypeptide abundant in potato leaves (data not shown) and could be a posttranslationally cleaved product of one of the PKc proteins, similar to that described in soybean (Glycine max; Tang et al., 2003).
To assess the transcript levels of PK genes in the transgenic lines, quantitative real-time reverse transcription (RT)-PCR was performed with cDNA derived from tuber RNA using primers specific for the potato PK transcripts. The results showed that the targeted PKCYT1 transcript was strongly reduced to 3%, 2%, and 1% of wild-type levels in the lines with low PK activity (PKC-25, -6, and -15, respectively), reduced to 56% in PKC-26, and not reduced in PKC-11 (Fig. 2D). The expression of two other PKCYT genes, PKCYT2 and PKCYT3, was also measured, with both showing minor but nonsignificant decreases in transcript level (data not shown). Two PKP genes, PKP
Measurement of maximal catalytic activities of the glycolytic enzymes hexokinase, fructokinase, phosphoglucomutase, and phosphofructokinase showed no significant difference in the PKc transgenics compared to wild type (Table I
). The maximal activities of enzymes that bypass PKc to produce pyruvate via malate (PEP carboxylase [PEPC], malate dehydrogenase, and NAD malic enzyme) were also not significantly different from wild type, nor was the activity of PEP phosphatase, which is a vacuolar acid phosphatase that can convert PEP to pyruvate without requiring ATP (Plaxton, 1996
Effect of Decreased PKc Expression on Growth and Metabolite Levels Wild-type and PKc transgenic plants were grown under greenhouse conditions for metabolic analysis of tubers from 10-week-old plants. Total tuber mass per plant was slightly elevated in all transgenic lines (significant for PKC-25; Fig. 3A ), while tuber number per plant was significantly higher in all transgenic lines compared to wild type (Fig. 3B). The reason for the increase in tuber number upon reduction of PKc is not revealed by our data. However, the average tuber mass was not significantly different from wild type (Fig. 3C). Starch content in the transgenics was not different to wild type but protein content was reduced (significant for PKC-26, -25, and -15; Fig. 3, D and E). Respiration was measured as the rate of oxygen consumption by tuber discs cut from freshly harvested tubers. While PKC-25 showed a significant reduction in respiration, all other lines were not significantly different from wild type (Fig. 3F).
The levels of hexose-phosphates (sum of Glc-1-P, Fru-6-P, and Glc-6-P) and PEP were similar in all lines (Fig. 3, R and L), while pyruvate was significantly decreased in PKC-6 and -15 (Fig. 3M), resulting in a significant increase in the PEP to pyruvate ratio in these lines (Fig. 3N), which is consistent with the decrease in PK activity. Measurement of sugars revealed that PKC-6 had significantly higher Glc and Suc, while PKC-11 had significantly higher Glc (Fig. 3, O–Q). Measurement of adenine nucleotides showed that ATP levels were similar in all lines, while ADP levels showed a trend of being slightly higher than wild type, and the ATP to ADP ratio a trend of being slightly decreased in PKC-25, -6, and -15 (Fig. 3, G–I). These changes were, however, not significant. UTP and UDP levels were similar in all lines (Fig. 3, J and K).
Metabolite profiling using an established gas chromatography-mass spectrometry (GC-MS) method (Roessner et al., 2001
Effect of Decreased PKc Expression on Metabolic Fluxes The effect of reduced PKc on carbon fluxes was determined by incubating tuber discs from wild type and lines PKC-11, -25, and -15 with [U-14C]Glc followed by fractionation of the labeled material to determine the label distribution into different pathways (Fig. 4 ). The transgenic tubers showed no difference from wild type in total label uptake (Fig. 4A), amount metabolized (Fig. 4B), or in the percentage of metabolized label incorporated into carbon dioxide (Fig. 4C), cell wall (Fig. 4F), or amino acids (Fig. 4H). However, there was a tendency of increased label incorporation into starch (Fig. 4D), significant reduction of label incorporated into protein (Fig. 4E, significant for lines PKC-25 and -15), and a tendency of reduction of label into organic acids (Fig. 4G). PKC-11 showed a significant increase of label into phosphate esters and Suc, while PKC-15 had reduced label incorporation into Suc (Fig. 4, I and J). The total sum of label incorporation into glycolysis was calculated as the sum of label in carbon dioxide, protein, organic acids, and amino acids (Fig. 4K). The ratio of label incorporation into starch versus glycolysis was significantly increased in PKC-25 and -15 (Fig. 4L).
Metabolic fluxes were calculated according to Geigenberger et al. (1997) by determining the specific activity of the hexose phosphate pool, which was similar in all lines (wild type = 1.11 ± 0.06, PKC-11 = 1.46 ± 0.14, PKC-25 = 0.91 ± 0.21, PKC-15 = 1.17 ± 0.25 Bq nmol–1). There was an increased flux of carbon into starch in transgenic lines PKC-25 and -15 compared to wild type (significant for line PKC-15; Fig. 4M). Fluxes into protein and glycolysis were not significantly different from wild type, but the ratio of flux into starch versus glycolysis was significantly higher than wild type in PKC-25 and -15 (Fig. 4, N, O, and R). Flux into Suc was significantly higher than wild type in PKC-11 but lower in PKC-15, while flux into cell wall was not significantly different from wild type (Fig. 4, P and Q). These data show that reduced PKc causes an altered redistribution of carbon fluxes, with slightly less flux through glycolysis and greater flux into starch synthesis.
The relative flux of carbon through the TCA cycle was measured by incubating tuber discs in positionally labeled Glc isotopomers ([1-14C]Glc, [3,4-14C]Glc, and [6-14C]Glc) and measuring the label released as CO2. Carbon from the C3,4 positions can only be released as CO2 via the mitochondrial TCA cycle, whereas carbon from the C6 position can be released as CO2 from both mitochondrial respiration and the oxidative pentose phosphate (OPP) pathway. The relative TCA cycle flux can be compared among the plant lines from the ratio of label evolution from the C3,4 to C1 incubations, while the C6 to C1 ratio is a measurement of flux through the OPP pathway relative to the other pathways of carbon oxidation (ap Rees and Beevers, 1960
Respiration was measured as the overall rate of oxygen consumption by tuber discs cut from freshly harvested tubers. While PKC-25 showed a significant reduction in respiration, all other lines were not significantly different from wild type (Fig. 3F).
We also measured internal oxygen concentrations in intact tubers. Because changes in respiration rates will lead to inverse changes in internal oxygen concentrations (see Bologa et al., 2003
The AOX capacity was calculated as the rate of KCN-insensitive, salicylhydroxamic acid (SHAM)-sensitive oxygen consumption in tuber slices, and the level of stimulation by pyruvate was also determined. Without exogenous pyruvate addition, AOX capacity was significantly lower than wild type in the strongest transgenic lines (PKC-25, -6, and -15) but no different than wild type in the nonsilenced line PKC-11 (Fig. 6A
). Upon addition of pyruvate, respiration was significantly increased in all genotypes but remained significantly lower than wild type in PKC-25, -6, and -15 (Fig. 6A). As reported previously (Millar et al., 1993
In isolated tuber mitochondria, similar results were obtained, with a significantly lower AOX capacity in PKC-25, -6, and -15 compared to wild type and PKC-11 (Fig. 6B). However, the difference in AOX capacity between the transgenics and wild type was not as marked as in intact tuber tissue. Mitochondria in intact cells are subject to other influences, such as redox poise, that are known to influence AOX activity (Umbach and Siedow, 1993 ; Day et al., 1995; Mackenzie and McIntosh, 1999; Vanlerberghe et al., 1999).
The results presented above indicate that decreased PKc led to a decrease in the capacity of the alternative pathway of respiration. To confirm these results, AOX protein levels were measured in isolated mitochondria by western blots of proteins separated under reducing conditions, using an AOX-specific antibody (Elthon and McIntosh, 1987
The role of PKc in plants has not yet been fully resolved, despite a number of physiological and biochemical approaches published in the past. In this article, we have studied the effect of decreased PKc levels in potato tubers on tuber metabolism using an RNAi silencing approach. Silencing the PKCYT1 gene in tubers resulted in a large decrease in total PK activity and decreased levels of the two major immunoreactive PKc polypeptides present in wild-type tubers (of sizes approximately 55 and 60 kD; see Fig. 1). The large reduction of PKCYT1 transcript levels to 1% to 3% of wild type levels in the PKc transgenic tubers probably accounts for the strongly decreased level of the 55-kD PKc immunoreactive band. However, the strong reduction of the 60-kD protein does not appear to be solely due to reduced transcription of the corresponding gene, because there was only weak cross-silencing of other PKC genes. It has previously been demonstrated that PKc in tobacco leaves and castor oil leaves is a heterotetramer consisting of two subunits of 57 kD and 56 kD (Plaxton, 1989 ; Knowles et al., 1998) and our results indicate PKc in potato tubers may also be composed of two different subunits. While this remains to be investigated, it is possible that the presence of the 55-kD protein is necessary to maintain levels of the 60-kD protein, as has been described for the heterotetrameric plant AGPase enzyme, in which the presence of the 49-kD subunit is required to maintain stability of the 52-kD subunit (Wang et al., 1998). Furthermore, analysis of tobacco plants lacking PKc activity and protein levels in leaves showed that the 56-kD and 57-kD PKc polypeptides were either both present or both absent (Gottlob et al., 1992). In the PKc transgenic plants analyzed here, the third PKc immunoreactive PKc polypeptide detected in tubers (approximately 50 kD) was slightly induced in the transgenics, but this also does not appear to be due to increased transcription of the PKC genes measured. This protein band may represent either another isozyme or a posttranslationally cleaved product of one of the larger isozymes, similar to that described by Tang et al. (2003).
Decreased PKc expression led to an increased PEP to pyruvate ratio in growing tubers, indicating that PEP to pyruvate conversion was indeed inhibited in the transgenic lines (Fig. 3N). This was mainly due to a decrease in pyruvate levels, while PEP levels remained rather unchanged. The fact that the levels of PEP and 3-phosphoglycerate were not substantially increased could be due to a coordinated inhibition of additional regulation sites upstream in glycolysis and Suc degradation. The decrease in pyruvate level was accompanied by a decrease in the levels of some other organic acids involved in the TCA cycle and decreases in the level of Asp (Table II). There was also a decrease in the level of total protein in the tubers. Labeling experiments using [14C]Glc showed a slight decrease in the partitioning of carbon toward protein and organic acid synthesis upon reduction of PKc levels. These results indicate that PKc plays an important role in the regulation of the levels of organic acids in tubers, as well as preferentially partitioning carbon toward the TCA cycle. This contention is strongly supported by the finding that PKc is part of a functional association of glycolytic enzymes with the mitochondrial membrane in potato as well as Arabidopsis and that cyanide treatment reduces the association of these enzymes while increasing the demand on the alternative pathway of respiration (Graham et al., 2007
Surprisingly, the strong decrease in PKc levels did not lead to substantial changes in overall respiration rates and TCA cycle flux in tubers (Figs. 3F and 5A), confirming previous studies with transgenic tobacco plants lacking PKc in leaves (Grodzinski et al., 1999
While a decrease in PKc did not affect the overall rate of oxygen consumption (Fig. 3F), there was a clear effect on the capacity of the AOX-mediated alternative pathway of mitochondrial electron transport (Fig. 6). AOX capacity was measured as the level of KCN-insensitive, SHAM-sensitive respiration, which has previously been used to assess AOX capacity in Arabidopsis and potato (Hiser et al., 1996
Our results are consistent with previous studies showing that AOX activity is increased after addition of pyruvate or other organic acids in vitro (Millar et al., 1993
The results presented provide evidence for a role of PKc to regulate both the level and the subsequent use of pyruvate via the AOX-mediated alternative pathway of respiration. Moderate in vivo changes in pyruvate levels were shown to have a strong impact on the alternative pathway of respiration, while the COX-dependent pathway was hardly affected. Obviously, plants have to regulate cytosolic pyruvate levels very carefully to keep alternative respiration to a minimum. The role of PKc thus clearly contrasts with that of the plastidial PK isozyme, which is crucial for provision of pyruvate for fatty acid synthesis in the plastid (Andre and Benning, 2007 In conclusion, the study presented here provides new insights into the specific role of a major PKc isozyme in plants and complements previous detailed analyses of the plastidial isozymes in Arabidopsis. However, further studies will be required to fully understand the function of the other plant PK isozymes revealed by our phylogenetic analysis.
Database Searching and Phylogenetic Analysis
PK cDNA or EST sequences were obtained from publicly available data from the Joint Genome Institute (http://www.phytozome.net) and the National Center for Biotechnology Information (http://www.ncbi.nml.nih.gov). A basic alignment of the deduced protein sequences was created using the ClustalW program (Chenna et al., 2003
Potato (Solanum tuberosum) cv Desiree was obtained from Saatzucht Lange AG. Plants were maintained in tissue culture and then transferred to greenhouse conditions as described by Fernie et al. (2002)
A 650-bp fragment of the tomato (Solanum lycopersicum) cDNA clone (TC172280) showing 97% nucleotide identity to the potato PKCYT1 gene was amplified by PCR (5' primer, CACCCGTGCTGAGGCTAC; 3' primer, CAGGTGTGGGGGAGTTCA) and cloned via the GATEWAY method into a modified GATEWAY binary vector (based on pK7GWIWG2(I)0; Karimi et al., 2002
Proteins were extracted and desalted according to Trethewey et al. (1998)
For measurement of PEP, pyruvate, nucleotides, and hexose phosphates, 200 mg tuber material was extracted with trichloroacetic acid according to Trethewey et al. (1998)
Tuber discs were prepared from growing plants and [U-14C]Glc labeling experiments were performed as described by Geigenberger et al. (1997)
TCA cycle flux analysis was performed based on Nunes-Nesi et al. (2005)
Tuber discs (8 mm x 2 mm) were prepared from freshly harvested, 10-week-old tubers and briefly rinsed in buffer (10 mM MES-KOH, pH 6.5) to remove broken cells, then two discs were immediately transferred to the temperature-controlled chamber of a Clark-type electrode (Hansatech) containing 1 mL buffer at 25°C. AOX capacity was calculated as the KCN-insensitive, SHAM-sensitive respiration rates according to Umbach et al. (2005)
To investigate whether there is a possible wound-induced respiration during the time course of our tuber-disc experiments (<3 h; see above), we measured the rates of oxygen consumption at different time points after slicing the tubers (Supplemental Fig. S2). The data show that there is no increase in respiration rates within the first 3 h of incubation. Previous studies on respiration rates in tuber slices are consistent with this (Hajirezaei and Stitt, 1991
Mitochondria were isolated from freshly-harvested, 10-week-old tubers according to Jenner et al. (2001)
Pots were removed from plants and tubers were freed from soil and immediately fixed in a micromanipulator (Saur Laborbedarf) while remaining attached to the plant without previous washing with water. Without delay, a 230-µm diameter, minimally invasive oxygen micro-optode (Löhmannsröben et al., 2006
Total RNA was isolated from 100 mg ground tuber material using the Qiagen RNeasy Plant Minikit (Qiagen). RNA quality was assessed by agarose gel electrophoresis prior to DNaseI digestion (Promega) and cDNA synthesis using the Superscript III RT-PCR kit (Invitrogen). Quantitative real-time RT-PCR was performed as described by Czechowski et al. (2004)
A total of 25 µg of protein from mitochondria isolated from tubers (for AOX and COX) or from tuber tissue (for PKc) in Laemmli buffer (Laemmli, 1970
Data were analyzed using the Student's t test and deemed significant if P < 0.05.
Sequence data from this article can be found in the GenBank/EMBL or TIGR transcript contig data libraries under accession numbers S53332 (potato PKCYT1), TC134535 (potato PKCYT2), TC141201 (potato PKCYT3), TC118783 (potato PKCYT4), TC135950 (potato PKCYT5), TC135043 (potato PKP
The following materials are available in the on-line version of this article.
We gratefully acknowledge Anett Doering for excellent technical assistance, Helga Kulka for plant cultivation, Romy Baran for plant transformation, and Prof. Bill Plaxton (Queen's University, Ontario, Canada) for kindly providing the anti-PKc antibody, and Dr. Thomas Elthon (University of Nebraska, Lincoln, NE) for kindly providing the anti-AOX antibody. Received July 16, 2008; accepted September 24, 2008; published October 1, 2008.
1 This work was supported by the Deutsche Forschungsgemeinschaft and by the Max Planck Society. 
2 Present address: CSIRO Plant Industry, Clunies Ross St., Canberra, ACT 2601, Australia.
3 Present address: Samuel Roberts Noble Foundation, 2510 Sam Noble Pkwy., Ardmore, OK 73401. The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantphysiol.org) is: Peter Geigenberger (geigenberger{at}igzev.de).
[W] The online version of this article contains Web-only data.
[OA] Open Access articles can be viewed online without a subscription. www.plantphysiol.org/cgi/doi/10.1104/pp.108.126516 * Corresponding author; e-mail geigenberger{at}igzev.de.
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ABSTRACT
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-keto acids to isolated mitochondrial preparations under conditions where the cytochrome c pathway is inhibited (e.g. in the presence of antimycin a or KCN) stimulates the activity of the mitochondrial alternative oxidase (AOX; Millar et al., 1993
-amino-n-caproic acid, 0.5 mM phenylmethylsulfonyl fluoride, 0.1% (w/v) fatty-acid-free bovine serum albumin, 10% (v/v) glycerol, and 0.1% (v/v) Triton X-100, as in Geigenberger and Stitt (1993)
-aminobutyrate shunt in illuminated tomato leaves. Plant Physiol 145: 626–639
