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First published online October 9, 2003; 10.1104/pp.103.026716 Plant Physiology 133:1322-1335 (2003) © 2003 American Society of Plant Biologists Reduced Expression of Aconitase Results in an Enhanced Rate of Photosynthesis and Marked Shifts in Carbon Partitioning in Illuminated Leaves of Wild Species Tomato1Department Willmitzer, Max-Planck-Institut für Molekulare Pflanzenphysiologie, Am Mühlenberg 1, 14476 Golm, Germany (F.C., A.N.-N., Y.G., A.L., A.R.F.); and Departamento de Biologia Vegetal, Federal University of Vicosa, 36571–000 Viçosa-MG, Brazil (M.E.L.)
Wild species tomato (Lycopersicon pennellii) plants bearing a genetic lesion in the gene encoding aconitase (Aco-1; aconitate hydratase EC 4.2.1.3) were characterized at molecular and biochemical levels. The genetic basis of this lesion was revealed by cloning the wild-type and mutant alleles. The mutation resulted in lowered expression of the Aco-1 transcript and lowered levels of both cytosolic and mitochondrial aconitase protein and activity. After in silico analysis, we concluded that in the absence of a recognizable target sequence, the best explanation for the dual location of this protein is inefficient targeting. Biochemical analysis of leaves of the Aco-1 accession suggested that they exhibited a restricted flux through the Krebs cycle and reduced levels of Krebs cycle intermediates but were characterized by elevated adenylate levels and an enhanced rate of CO2 assimilation. Furthermore, the analysis of both steady-state metabolite levels and metabolic fluxes revealed that this accession also exhibited elevated rates of photosynthetic Suc synthesis and a corresponding increase in fruit yield. Therefore, we conclude that the Krebs cycle normally competes with the Suc synthetic pathway for carbon but is not essential for the supply of energy to fuel the operation of this pathway.
The mitochondrial metabolism of plants, like that of all eukaryotes, is dominated by the synthesis of ATP, a fundamental component of this process being the Krebs cycle, which links the pathway of glycolysis to that of the electron transport chain. Despite the fact that the operation and location of the complete Krebs cycle was demonstrated in plant cells decades ago (Beevers, 1961  ), the function of this important pathway in plants is still far from clear (Hill, 1997; Siedow and Day, 2000). Even fundamental questions such as whether the Krebs cycle operates in illuminated photosynthetic tissue and if it contributes to the energy requirements of photosynthetic Suc synthesis remain controversial (Graham, 1980; Krömer, 1995; Padmasree et al., 2002). In addition to its role in energy production, a second essential feature of the Krebs cycle in plants is meeting the demand for carbon skeletons that is imposed by anabolic processes such as porphyrin and amino acid synthesis (Douce and Neuberger, 1989; Mackenzie and McIntosh, 1999).
The concerted action of citrate synthase, aconitase, and isocitrate dehydrogenase transform acetyl CoA into
Here, we describe the molecular and genetic analysis of Aco-1, a tomato (Lycopersicon pennellii) accession that was isolated as deficient in aconitase activity—a trait that was subsequently mapped to chromosome 12 (Bernatzky and Tanksley, 1986 The results of these analyses will be discussed in the context of current models of the role of the Krebs cycle in photosynthetic metabolism.
Genetic Analysis of Aconitase within Tomato Full-length cDNAs encoding aconitase were isolated from leaves of the tomato accessions LP1940 (Lp) and 2901 (Aco-1) by a PCR-based approach as described in "Materials and Methods." Using this strategy, we isolated two 2.8-kb clones, the sequences of which have been deposited in GenBank (accession nos. bk524167 and bk524171 for the Lp and Aco-1 genotypes, respectively). Sequence analysis of the clones from both genotypes indicated that the whole coding region had been amplified and revealed open reading frames encoding proteins of 898 amino acids in both instances. The predicted proteins of the two accessions differ in 12 amino acid residues with only four of these producing changes in polarity of the protein (data not shown). Comparison at the nucleotide level revealed that both alleles show between 79 and 97% identity to all plant aconitases in the databases (potato [Solanum tuberosum], X97012; tobacco [Nicotiana tabacum], AF194945; pumpkin [Cucurbita maxima], D29629; Arabidopsis, AY136414; lemon [Citrus limon], AF073507; and melon [Cucumis melo], X82840). In addition to the high identity with other plant aconitases, the two alleles share high identity (50%–60%) with mammalian cytosolic aconitases but not with mammalian mitochondrial isoforms or the bacterial aconitase A (Fig. 1A).
To assess the complexity of the gene encoding the aconitase enzyme of tomato, Southern hybridization of genomic DNA from both accessions was performed using the full open reading frame of the cloned aconitase cDNA from tomato (Fig. 1B). Using four different tomato expressed sequence tags (ESTs) with high homology with other aconitase genes as probes (for details, see "Materials and Methods"), identical bands were revealed (data not shown).
On comparison of the digestion of the genomic DNA with EcoRV and DraI allelic differences between the genotypes became apparent (Fig. 1B). Despite the fact that the above experiments hinted to the presence of only a single gene when the chromosomal map location of the cloned cDNA was determined by using a set of tomato introgression lines (Eshed and Zamir, 1994 Analysis of mRNA northern blots using the Lp aconitase cDNA as a probe suggests constitutive expression of this gene, the transcript being present in leaves, flowers, fruits, and roots (Fig. 1C). Comparison of the relative mRNA levels between the genotypes suggests a considerably lower expression level of aconitase mRNA in leaves, flowers, and fruits of the Aco-1 genotype but an elevated expression in the roots.
Having demonstrated that leaves of the Aco-1 plants exhibited lower expression of the aconitase gene, we next turned our attention to determining the effect this had on the protein amount and activity. Given the considerable difficulty of measuring aconitase activity in crude extracts, we attempted activity elution using the method of Slaughter et al. (1977
The aconitase protein level in the pooled fractions was determined by western blot of cytoplasmic and mitochondrial proteins with antiserum raised against pumpkin aconitase (Hayashi et al., 1995
Having confirmed that accession 2901 (Aco-1) that we obtained from the University of California (Davis) Stock Center was deficient in aconitase activity (with respect to the control accession LP1940 [Lp]), we grew both alongside one another in 2.5-L pots in the greenhouse and determined morphological parameters of the genotypes. The genotypes were distinguishable from one another at a very early stage. Up to 3 weeks after transfer to the greenhouse, the Aco-1 plants displayed stunted growth (Fig. 3A). Over the course of development, however, this growth phenotype became less pronounced and by the time of fruit set was no longer visibly apparent. Leaves from the Aco-1 plants were also characterized by a deeper pigmentation color (Fig. 3A). To confirm this, we next determined the chlorophyll and carotenoid content of leaf samples harvested from 6-week-old plants. These determinations revealed significantly higher levels of chlorophyll a and b and carotenoids in leaves from the Aco-1 plants (Fig. 3B). As a further experiment, we recorded the growth, flowering, and biomass of the different organs of the plants. Despite the fact that the Aco-1 plants initially showed a stunted phenotype after 10 weeks of growth, they were marginally taller than the wild-type accession.
After 20 weeks of growth, we determined the biomass of separate organs of the plants. Although there were no significant difference between the mass of stem or leaf across the genotypes, the Aco-1 plants produced slightly less root matter but considerably more fruit matter per plant (Table I). Interestingly, the time of flowering was also approximately 1 week later in the Aco-1 plants, and the number of flowers was significantly lower at this time point; however, the total number of flowers was eventually the same across the genotypes.
As a first experiment, we analyzed the carbohydrate content of leaves from 6-week-old plants during a diurnal cycle (Fig. 3, C and D). There was a significant increase both in the starch content and the rate of starch accumulation in the Aco-1 plants when compared with the Lp plants; however, in both cases, the amount of starch accumulated by the end of the day was approximately twice that at the beginning of the light period. A similar if less dramatic pattern was observed in the Suc content of Aco-1 and Lp plants with a minor, yet significantly higher Suc content in the Aco-1 plants throughout the majority of the light period. These changes were accompanied by an elevated Glc content (data not shown) and a small reduction in the total levels of amino acids (especially prominent at the middle of both light and dark periods; Fig. 3E). Despite the changes observed in amino acid content, there were no differences in total protein content between the accessions (data not shown).
Considering that both Suc and starch levels increased within the Aco-1 plants, we decided to investigate whether they exhibited an altered photosynthetic rate. First, fluorescence emission was measured in vivo using a pulse amplitude modulation (PAM) fluorometer, and relative electron transport rates (ETRs) were calculated. When exposed to higher irradiance (photon flux density [PFD] of 700 µmol m–2 s–1), the Aco-1 plants exhibited a significantly elevated rate of electron transport (Fig. 4A). Second, gas exchange was measured in the two genotypes under PFDs that ranged from 100 to 1,000 µmol m–2 s–1 (Fig. 4, B and C). Aco-1 plants exhibited assimilation rates that were significantly higher than the wild type under all conditions except the lowest irradiance (Fig. 4B). Analysis of other parameters of gas exchange revealed that the Aco-1 genotype also exhibited a higher transpiration rate (Fig. 4C) at all PFDs, and there were minor changes in the internal CO2 concentration (data not shown), suggesting that the enhanced photosynthesis may, in part, be due to a more efficient stomatal conductance.
In a complementary approach, we determined the effects of reduced aconitase activity on photosynthetic fluxes by studying the metabolism of 14CO2 by excised leaf discs illuminated for 30 min. In keeping with the data presented above, the assimilation rate was markedly increased in plants of the Aco-1 genotype. Furthermore, this increased carbon fixation was coupled with an increase in the accumulation of both [14C]Suc and [14C]starch (Table II).
When these data are expressed as a percentage of the total 14CO2 metabolized, decreased aconitase activity led to decreased partitioning into organic and amino acids. These data imply a reduced respiration rate in these plants. Further studies also revealed that dark respiration was also reduced in the lines (–0.36 ± 0.03 and –0.86 ± 0.28 µmol m–2 s–1 for Aco-1 and Lp plants, respectively; values presented as mean ± SE, n = 6).
Although the results of the above experiments indicate an enhanced rate of photosynthesis, we decided to study this in more detail by examining the levels of metabolic intermediates of the major pathways of plant photosynthetic metabolism. We began by evaluating the consequence of altered aconitase activity on the levels of Krebs cycle intermediates utilizing a recently established gas chromatography (GC)-mass spectrometry (MS) protocol. Several of these intermediates (namely fumarate, succinate, and
Organic acids that are not associated with the Krebs cycle display no discernable trend; for example, saccharic acid is 2-fold higher in Aco-1, whereas threonic acid is only about 30% of the level determined in Lp plants (data not shown). Although it is technically unfeasible to obtain the concentration of metabolites within the mitochondria, at least one metabolite, succinate, reflects the mitochondrial situation because in plant cells it is present exclusively within the mitochondria (Hill, 1997
Next, we determined the levels of adenylates and uridinylates by HPLC in tricarboxylic acid extracts from the two accessions (Table III). Perhaps it is surprising, given that the Aco-1 plants are deficient in the activity of an enzyme of the Krebs cycle, that they display increased levels of total adenylates containing a minor increase in the level of ATP but almost a 2-fold higher ADP content than the Lp accession, with a resultant significant decrease in the deduced ATP to ADP ratio. Notably, however, the ATP to UTP ratio remains the same across the genotypes. Interestingly, the content of UDP is markedly lower in the Aco-1 lines, resulting in a large change in the deduced UTP to UDP ratio. Also, these plants were further characterized by large accumulation of UDP-Glc. In addition to the nucleotide measurements, we determined the levels of the key photosynthetic metabolites Glc 6-phosphate, Glc 1-phosphate, Fru 6-phosphate, and 3-phosphoglycerate using sensitive cycling assays that have been established recently (Gibon et al., 2002
To better understand the enhanced photosynthesis observed in Aco-1, we determined the activities of a broad range of enzyme activities important in photosynthesis, starch and Suc synthesis, and tricarboxylic acid cycle function (Table IV). There were no significant differences in the maximal catalytic activities of UDP-Glc pyrophosphorylase, Suc phosphate synthase, Rubisco, NADP+-dependent glyceraldehyde phosphate dehydrogenase, transaldolase, transketolase, phosphoribulokinase, phosphoglycerate kinase, both pyrophosphate-dependent and ATP-dependent PFPs, or pyruvate kinase. Likewise, the initial Rubisco activities and the deduced activation state of Rubisco and the maximal catalytic activities of AGPase and phosphoglucomutase were unaltered.
Isolation and Characterization of a cDNA Encoding the Tomato and Aco-1 Alleles of Aconitase and Molecular Characterization of the Aco-1 Accession
Full-length 2.8-kb clones were obtained from both Lp and Aco-1 accessions of tomato utilizing a PCR-based approach using primers based on a consensus of the publicly available plant aconitase sequences. These cDNAs both encode proteins of molecular masses of 98 kD and exhibit high homology to all sequenced aconitases but particularly to bacterial and mammalian cytoplasmic aconitases (Peyret et al., 1995
Searching a tomato EST collection for clones with significant homology to other reported plant aconitases resulted in the detection of a total of 45 clones within two highly homologous tentative consensus sequences, suggesting the possibility of a unique gene. However, further map-based localization studies using the clone described here were in close agreement with the previous annotations based on isozyme analysis (Tanksley and Rick, 1980
When taken together with the demonstration that the Aco-1 accession is deficient in aconitase protein and activity in both the cytoplasm and mitochondria, these data suggest that the both cytosolic and mitochondrial aconitases from tomato are encoded by the gene cloned here. A range of observations, some of them purely circumstantial, indicate that this is probably also the case in a range of other plant species. First, cytoplasmic and mitochondrial isoforms of aconitase have been separated both from Acer pseudoplatanus cell suspension cultures (Brouquisse et al., 1987
Reduction of aconitase activity led to a stunted phenotype at early stages of development; however, after a period of 4 to 5 weeks, the Aco-1 plants were equivalent in height to the control accession. However, leaves from the Aco-1 plants did display a darker pigmentation characterized by elevated levels of chlorophylls and carotenoids and exhibited higher rates of assimilation, transpiration, and electron transport through the PSs. In keeping with elevated rates of both carbon assimilation and photosynthesis, the Aco-1 plants were characterized by elevated accumulation of Suc and starch during the light period and an increased fruit yield. Although it seems likely that the increased fruit yield results from increases in leaf photoassimilate supply, the data presented in the current study do not preclude the possibility that the increased fruit yield is, at least in part, a direct effect of modulating the aconitase activity in the fruit. The enhancement of photosynthesis is somewhat surprising, particularly given the limited success in achieving this result through direct modification of the Calvin cycle (for review, see Raines, 2003
Although the reduction in aconitase activity clearly results in an elevated rate of photosynthesis in the Aco-1 plants, the exact mechanism by which this comes about remains unresolved. However, a broad screen of other key enzymes of photosynthetic carbohydrate metabolism revealed that only the activity of aconitase was altered in the Aco-1 accession. A number of other possible explanations for the increased rate of photosynthesis exist—the most likely of these is that it is a consequence of a reduction of partitioning of assimilate toward the Krebs cycle. Results from the 14CO2 feeding experiment demonstrate a clear reduction in labeling of organic and amino acids. Although some of these acids may well be formed by fixation of carbon dioxide by phosphoenolpyruvate carboxylase, and the data do not constitute a direct measurement of flux, we nevertheless believe that these results imply that the reduction in aconitase activity results in a reduced operation the Krebs cycle. Furthermore, the data from these feeding experiments are in accordance with the metabolite levels determined in this study—with the Aco-1 plants displaying decreased levels of fumarate, succinate, and
Interestingly, despite a probable reduction in the flux through the Krebs cycle, the Aco-1 plants are characterized by enhanced adenylate levels but a decrease in the cellular ATP to ADP ratio. There are two possibilities that can be proposed to explain the enhanced levels of adenylates. First, it is highly likely that a large proportion of the increase in adenylate pool size is met by the increased rate of photosynthesis discussed above. However, it is also conceivable that the increased size of the adenylate pools is the consequence of a compensatory induction of photorespiration in this accession. In keeping with the second explanation, the levels of the photorespiratory intermediates Gly and to a lesser extent Ser accumulate in the Aco-1 accession, and experimental evidence has been provided that the mitochondrial electron transport chain oxidizes not only NADH produced by the Krebs cycle but also that produced by photorespiration (Krömer, 1995
Although the above mechanism is the one we favor to explain the increase in photosynthesis, other plausible explanations exists. First, although the increase in pigment contents cannot be explained directly by the metabolic changes we describe here, it is conceivable the reduction in aconitase protein levels could result in a surplus of cellular iron, which could in turn explain the increase pigment contents in the Aco-1 plants. A second plausible explanation, albeit purely speculative, is that the reduction in the aconitase activity disrupts a cellular signaling pathway. Such a role for aconitase is not without precedence because it has been demonstrated to be a key component of cellular iron homoeostasis in animal systems (for review, see Cairo and Pietrangelo, 2000
The pattern of change in the amino acid pool sizes in the tomato accessions between the day and night was similar to those previously reported in tobacco (Scheible et al., 2000
In this paper, we have shown that repression of both mitochondrial and cytosolic aconitase activities in wild tomato has dramatic effects on the photosynthetic metabolism and fruit yield of the plant. This repression of activity resulted in a reduction in the levels of  -ketoglutarate and, hence, in Gln, but despite an apparent reduction in flux through the Krebs cycle, it did not result in a repression but rather an increase in the rate of photosynthetic Suc synthesis. When taken together, these results suggest that the Krebs cycle normally competes with the Suc synthetic pathway for carbon but is not essential for the supply of energy to fuel the operation of this pathway. That said, these changes are observed during a point at which the plants have achieved developmental equivalence, and further experimentation is required to elucidate factors involved in the dramatic phenotype observed during early development.
Plant Material and Growth of Plants
Tomato (Lycopersicon pennellii) seeds of accession numbers LP1940 (Lp) and LP2901 (Aco-1) were obtained from the true-breeding monogenic stocks maintained by the Tomato Genetics Stock Center (University of California, Davis). The seeds were germinated on Murashige and Skoog media (Murashige and Skoog, 1962
A pair of primers was designed based on the consensus sequence of all cloned plant aconitase genes (potato [Solanum tuberosum], tobacco (Nicotiana tabacum), pumpkin [Cucurbita maxima], Arabidopsis, and lemon [Citrus limon]), and these were named ACO-15 (5' CCATGGCTGCAGAGAACCC 3') and ACO-13 (5' ATGTGGATTCTCATTGCTG 3'), respectively. Total RNA (3 µg) was obtained from LP1940 and Aco-1 leaves with Trizol (Gibco BRL, Karlsruhe, Germany), followed by DNAse treatment, and converted into cDNA with SuperScript II reverse transcriptase (Gibco BRL). The resultant cDNAs were then used as template for PCR amplification using 1 unit of Pfu DNA polymerase (Stratagene, Amsterdam) in 50 µL of the recommended buffer (0.1 mM dNTPs and 0.25 µM of each primer). Conditions were: initial denaturation at 94°C for 2 min and 30 cycles, each consisting of denaturation at 94°C for 45 s, annealing at 48°C for 1 min, and elongation at 72°C for 2 min. The last round of elongation was for 10 min at 72°C. PCR products were separated by 1% (w/v) agarose gel electrophoresis and purified by using the NucleoSpin Extract kit (Macherey-Nagel, Düren, Germany) and subjected to DNA sequence analysis at AGOWA GmbH (Berlin). Two independent PCR products from each genotype were sequenced and verified by comparison with sequences present in GenBank.
Genomic DNA (10 µg) from LP1940 and Aco-1 was isolated and Southern blotted as described by Hoisington et al. (1994
Total RNA was isolated using the commercially available Trizol kit (Gibco BRL) according to the manufacturer's suggestions for extraction from plant material. The RNA (15 µg) was then size fractionated on a 1% (w/v) agarose MOPS-formaldehyde gel before transfer to a nylon membrane filter. This filter was subsequently probed using the same clone as was used in the Southern experiment described above. Hybridization was carried out using standard protocols (Sambrook et al., 1989
Western analysis of aconitase protein was carried out either on crude protein extract or on mitochondrial protein extract, exactly as described by Hayashi et al. (1995
With the exceptions of the enzymes mentioned below, all enzymes were determined following the extraction and assay procedures detailed by Tauberger et al. (2000
Fluorescence emission was measured in vivo using a PAM fluorometer (Walz, Effeltrich, Germany) on 6-week-old plants maintained at fixed irradiance (200 and 700 µmol photons m–2 s–1) for 30 min previous to measure chlorophyll fluorescence yield, and relative ETR was calculated using the WinControl software package (Walz). Gas-exchange measurements were performed in a special custom-designed open system (Walz; for detailed description, see Muschak et al., 1997
The 14C-labeling pattern of Suc, starch, and other cellular constituents was performed by illuminating leaf discs (10-mm diameter) in a leaf disc oxygen electrode (Hansatech, Kings Lynn, Norfolk, UK) at 250 µmol photosynthetically active radiation m–2 s–1 at 20°C for 30 min. The carbon dioxide was supplied from 400 µL of 1 M NaH14CO3 (specific activity 1.96GBq mmol–1 [pH 9.3]) and placed on a felt mat at the base of the oxygen electrode chamber. After this experiment, the leaf discs were frozen in liquid nitrogen until further analysis. Frozen leaf tissue was fractionated to allow the determination of the metabolic fate of the assimilated 14CO2 exactly as detailed by Lytovchenko et al. (2002
Tissue samples were rapidly frozen in liquid nitrogen. Subsequently, the samples were extracted in either ethanol (for determination of carbohydrate and amino acid content) or in trichloroacetic acid (for the determination of triose and hexose phosphates, phosphate, 3PGA, and nucleotides) as detailed by Fernie et al. (2001a
Student's t tests were performed using the algorithm embedded into Microsoft Excel (Microsoft Corporation, Seattle). The term significant is used in the text only when the change in question has been confirmed to be significant (P < 0.05) with the Student's t test.
Discussions and support of Prof. Lothar Willmitzer throughout this work are most gratefully acknowledged. We are very grateful to Dani Zamir for carrying out the mapping analysis described in this paper. We are also thankful to Prof. Mark Stitt for provision of technical facilities, to Dr. John Lunn for many helpful comments during the writing of this manuscript, and to Dr. Joachim Fisahn for help in organization of gas-exchange measurements and discussion of the results. We are also thankful to Helga Kulka for excellent care of the plants. Received May 11, 2003; returned for revision May 27, 2003; accepted July 17, 2003.
Article, publication date, and citation information can be found at www.plantphysiol.org/cgi/doi/10.1104/pp.103.026716.
1 This work was supported by Max-Planck-Gesellschaft (to F.C. and A.N.-N.), by Conselho Nacional de Desenvolvimento Científìco e Technológico (Brazil; to A.N.-N. and A.R.F.), and by DIP (to A.N.-N. and A.R.F.). * Corresponding author; e-mail fernie{at}mpimp-golm.mpg.de; fax 49–0–331–5678408.
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