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First published online November 21, 2008; 10.1104/pp.108.130518 Plant Physiology 149:653-669 (2009) © 2009 American Society of Plant Biologists OPEN ACCESS ARTICLE
Decreased Mitochondrial Activities of Malate Dehydrogenase and Fumarase in Tomato Lead to Altered Root Growth and Architecture via Diverse Mechanisms1,[W],[OA]Max Planck Institute of Molecular Plant Physiology, D–14476 Potsdam-Golm, Germany (M.J.v.d.M., S.O., A.N.-N., A.R.F.); and Umeå Plant Science Center, Department of Forest Genetics and Plant Physiology, Swedish University of Agricultural Sciences, SE–90183 Umeå, Sweden (T.M.)
Transgenic tomato (Solanum lycopersicum) plants in which either mitochondrial malate dehydrogenase or fumarase was antisense inhibited have previously been characterized to exhibit altered photosynthetic metabolism. Here, we demonstrate that these manipulations also resulted in differences in root growth, with both transgenics being characterized by a dramatic reduction of root dry matter deposition and respiratory activity but opposite changes with respect to root area. A range of physiological, molecular, and biochemical experiments were carried out in order to determine whether changes in root morphology were due to altered metabolism within the root itself, alterations in the nature of the transformants' root exudation, consequences of alteration in the efficiency of photoassimilate delivery to the root, or a combination of these factors. Grafting experiments in which the transformants were reciprocally grafted to wild-type controls suggested that root length and area were determined by the aerial part of the plant but that biomass was not. Despite the transgenic roots displaying alteration in the expression of phytohormone-associated genes, evaluation of the levels of the hormones themselves revealed that, with the exception of gibberellins, they were largely unaltered. When taken together, these combined experiments suggest that root biomass and growth are retarded by root-specific alterations in metabolism and gibberellin contents. These data are discussed in the context of current models of root growth and biomass partitioning.
The structure of the plant tricarboxylic acid (TCA) cycle has been established for decades (Beevers, 1961  ), and in vitro studies have established regulatory properties of many of its component enzymes (Budde and Randall, 1990; Millar and Leaver, 2000; Studart-Guimarães et al., 2005). That said, relatively little is known, as yet, regarding how this important pathway is regulated in vivo (Fernie et al., 2004a; Sweetlove et al., 2007). Indeed, even fundamental questions concerning the degree to which this pathway operates in illuminated leaves (Tcherkez et al., 2005; Nunes-Nesi et al., 2007a) and the influence it has on organic acid levels in fruits (Burger et al., 2003) remain contentious. Furthermore, in contrast to many other pathways of primary metabolism, the TCA cycle has been subjected to relatively few molecular physiological studies. To date, the functions of pyruvate dehydrogenase, citrate synthase, aconitase, isocitrate dehydrogenase, succinyl-CoA ligase, fumarase, and malate dehydrogenase have been studied via this approach (Landschütze et al., 1995; Carrari et al., 2003; Yui et al., 2003; Nunes-Nesi et al., 2005, 2007a; Lemaitre et al., 2007; Studart-Guimarães et al., 2007); however, several of these studies were relatively cursory. Despite this fact, they generally corroborate one another, with at least two studies providing clear evidence for an important role of the TCA cycle in flower development (Landschütze et al., 1995; Yui et al., 2003) or in the coordination of photosynthetic and respiratory metabolisms of the illuminated leaf (Carrari et al., 2003; Nunes-Nesi et al., 2005, 2007a).
In our own studies on tomato (Solanum lycopersicum), we have observed that modulation of fumarase and mitochondrial malate dehydrogenase activities leads to contrasting shoot phenotypes, with the former displaying stunted growth while the later exhibited an enhanced photosynthetic performance (Nunes-Nesi et al., 2005
Establishment of the Experimental System
Given the previous observations that plants exhibiting reduced activities of several enzymes of the mitochondrial TCA cycle display a restricted root biomass (Carrari et al., 2003
Having established the experimental system with which to address our questions, we next turned our attention to the preliminary characterization of root metabolite exudation. As a first experiment, we analyzed the levels of both endogenous root tip (the entire 100-mm region proximal to the apex) metabolites and exuded metabolites during a 24-h cycle. Very few clear trends were apparent in the data from the endogenous metabolites. All amino acids analyzed remained stable throughout the time course (Supplemental Fig. S1A), and this was also true for the majority of the organic acid and sugar compounds (Supplemental Fig. S1, B and C). Tomato root systems subjected to an additional 2 d of darkness displayed a sharp decrease in both organic acid and sugar (mainly Suc and Fru) contents (Supplemental Fig. S1, E and F). By contrast, the amino acid levels seemed either to remain unaltered by this experimental treatment (e.g. Ser, Met, Ala, and Asp) or peaked during defined times in the extended night period (e.g. Ile and Glu; Supplemental Fig. S1D). In the case of the exometabolites, a more pronounced circadian-like rhythm could be observed (Supplemental Fig. S2). The pattern of organic acid displayed a peak toward the end of the light period, with the lowest reported values for organic acids and sugars in the morning and during midday (Supplemental Fig. S2, B and C). As was observed for the endometabolites, there were few trends in the levels of amino acids, although Lys and Cys both peaked toward the end of the light period (Supplemental Fig. S2A). Most exometabolites displayed similar rhythmic fluctuations on the extension of the night. However, the levels of Glu, isocitrate, citrate, Suc, Glc, and Fru were dramatically decreased on extended darkness, and their patterns displayed a dramatic decrease in amplitude (Supplemental Fig. S2, D–F; Supplemental Table S1).
As a first experiment, the activities of malate dehydrogenase and fumarase were detected in root extracts of the previously characterized transgenic lines (Nunes-Nesi et al., 2005
Root Morphology and Physiology in the Mitochondrial Malate Dehydrogenase and Fumarase Transformants When grown in vermiculite, the transgenic lines displayed dramatic alterations in root area that were markedly reduced in the mMDH lines and significantly enhanced in two of the three FL lines (Fig. 1A ). A similar picture was apparent in root length, although in this instance the increase was only significant in one of the FL lines (line FL41; Fig. 1B). Measuring the cell length of equivalent cells from the root tip revealed that both lines FL11 and FL41 were significantly longer (up to 2-fold as long) but that the other lines were invariant in this parameter (Fig. 1C). Perhaps surprisingly, the FL lines exhibited an invariant cell width whereas lines mMDH7 and mMDH8 were characterized as having reduced cell widths (Fig. 1D). When the exudates of the transgenics were harvested (as described in "Materials and Methods") and their pH quantified, it became apparent that the exudates from all three mMDH lines were considerably more acidic than those of the wild type (Fig. 1E). Intriguingly, there was only a minor, and nonsignificant, decrease in total root fresh weight in both sets of transformants (Fig. 1F) but relatively dramatic reductions in root dry mass (down to 25% of that observed in the wild type; Fig. 1G). Direct analysis of the rate of root respiration, however, confirmed that this was impaired in both sets of transgenics, being reduced to levels corresponding to approximately 20% of those observed for the wild type (Fig. 1H). The root hair density was largely unaltered in the transgenic lines, being increased in line mMDH8 but invariant in all other lines (Fig. 1I). Similarly, mMDH7 and mMDH8 displayed an increased vascular diameter, but all other lines were invariant from the wild type (Fig. 1J). Furthermore, both the number of secondary roots and the number of cell layers were invariant in all transgenic lines in comparison with the wild type (data not shown). Thus, despite displaying similar reductions in root respiration and dry mass, the transformants displayed markedly different root phenotypes, with the mitochondrial malate dehydrogenase plants exhibiting stunted root growth and reduced root area while roots of the FL lines were, if anything, longer and had a greater surface area than those of the wild type.
Root Carbohydrate, Adenylate, and Redox Contents of the Transformants
We next decided to evaluate the content of carbohydrates in the roots of material harvested at the end of the light period from 5-week-old vermiculite-grown plants. Soluble sugars and starch were extracted and measured using established spectrophotometric methods (Müller-Röber et al., 1992
Redox profiling of the transgenic lines revealed a significant decreases in reduced glutathione in the FL lines, while oxidized glutathione levels were reduced to approximately 60% of that found in the wild type in both the FL and mMDH lines (Fig. 2 ). In addition, a tendency of increase in total ascorbate was observed in both the mMDH and FL lines, although in both sets of plants ascorbate levels were only significantly elevated in a single line (mMDH21 and FL11, respectively). Furthermore, the deduced ratio of total ascorbate to ascorbate was largely unaltered in the mMDH lines but dramatically lower in the FL lines. Despite these changes, the levels of reducing equivalents were essentially invariant in the transgenic lines, with only a single significant difference being observed (the mild decrease in NAD content in line FL41). In contrast, the levels of adenylates appeared to be somewhat reduced in the lines, significantly so in the case of line mMDH8 (ADP) and line FL11 (AMP and ATP), but the ATP-ADP ratios and the adenylate energy charges of the transformants were invariant from the wild type (Fig. 2).
Analysis of Endometabolites in the Transformants
To further characterize the metabolic changes in the roots of these lines, an established gas chromatography-mass spectrometry (GC-MS)-based metabolite-profiling method (Fernie et al., 2004b
Analysis of Exometabolites in the Transformants We next utilized GC-MS to determine the relative levels of metabolites excreted from the roots of transgenic and wild-type plants in order to assess the potential influence of exudation on the root phenotype. For this purpose, exudates were collected at the end of the light period as described above from all lines. As noted previously for the metabolite levels within the transgenic roots, the number and magnitude of changes observed was much greater in the FL lines than in the mMDH lines (Supplemental Table S3). The malate dehydrogenase transformants were characterized by only minor differences in their exudation profiles, namely, decreased exudation of trehalose (significant in line mMDH7) and of Glc, Gal, and Man (significant in line mMDH8). By contrast, there was a large increase in the relative levels of metabolites exuded by the roots of the FL lines (despite a relatively high level of biological variance in these samples). Assessment of the sugars and sugar derivatives revealed an increase in isomaltose, maltitol, trehalose, rhamnose, Ara, Xyl, and Man (however, this was only significant in line FL11) as well as in Glc (significant in line FL63), Gal (significant in lines FL11 and FL63), glycerol 1-phosphate (significant in line FL63), glycerol, and Rib (significant in lines FL11 and FL63). Changes were also apparent in the organic acids, with increased exudation in shikimate (significant in all lines), quinate, fumarate, and glycerate (significant in lines FL11 and FL63), and citrate, succinate, oxalate, benzoate, glutarate, malate, and saccharate (significant in line FL11). Furthermore, the levels of several amino acids were elevated in the FL line exudates, with the levels of Gly (significant in all lines), β-Ala and Asp (significant in lines FL11 and FL63), and Glu, hydoxy-Pro, Asn, Met, Thr, Glu, and Ile (significant in line FL11) all increasing. Gln levels also increased 10-fold in line FL41.
In order to evaluate the exudation data in context with that of the endometabolites, we ran a series of calibration curves of authentic standards alongside our experimental extracts to allow us to perform absolute quantification of the metabolite levels (these data can all be viewed in Supplemental Tables S2 and S3). Several interesting features arise from this data transformation. Consistent with previous reports (Carrari et al., 2005
In order to gain further insight into the root metabolism of the transgenic lines, we next performed feeding experiments in which we incubated root material of both transformants in [13C]Glc for a period between 3 and 5 h and determined the redistribution of heavy label using a modified version of the GC-MS metabolite profiling method described above (Roessner-Tunali et al., 2004
Evaluation of Phytohormone Content and the Expression of Phytohormone-Associated Genes in Roots of the Transgenics
Given the observed alterations in root architecture, we thought it prudent to evaluate the levels of the phytohormones abscisic acid (ABA), auxin (IAA), and gibberellins (GAs), which have all been strongly implicated to have great importance in root development (Yaxley et al., 2001
Despite the relative paucity of changes in the levels of the hormones themselves, we next decided to evaluate the expression of genes associated with hormone perception or signal transduction relay. In order to achieve this, we isolated RNA from the same material used for the phytohormone measurements, with the exception that only one representative transgenic line was considered, and evaluated gene expression using the TOM1 microarray (Alba et al., 2004 ). The pertinent data from this transcript profiling study is presented in Figure 4
, with transcripts being grouped on the basis of their MapMan ontologies and detailed description of their identities being provided in Supplemental Table S4. While this presentation revealed a large number of changes, these generally did not correspond to the observed similarities or differences in phenotype across the lines. Given that the phenotypes displayed by both mMDH and fumarase were relatively dramatic and were either conserved or opposite, we had postulated that we may see a similar behavior at the transcript level of key genes. Studying Figure 4, however, revealed only a few putative homologous genes displaying antagonistic expression in the transgenics, for example, auxin response factor 3 (EC 4.2.9.21), ABA-responsive protein (EC 5.2.19.15), and MADS box transcription, and a few that displayed similar changes in gene expression, for example, GA 3-oxidase 1 (EC 6.4.15.16) GA 2-oxidase (EC 4.4.17.20), and the response regulator protein (EC 4.4.4.17).
Analysis of the Influence of Modified Shoot Metabolism on Root Function in the Transgenics Given that the majority of the experiments described thus far have focused directly on the roots (or root system), we decided to additionally evaluate basic aspects of root morphology and metabolism from the whole plant perspective. For this purpose, reciprocal grafting experiments between transgenic lines (mMDH8 and FL41) and wild-type controls were performed and root morphological parameters of the grafted material were determined. Plants were grown on vermiculite for a period of 4 weeks, after which graftings were carried out between wild-type stock and transgenic scions and vice versa. The resultant grafts were then left for another 2 weeks prior to the determination of morphological attributes. The grafting of wild-type scions onto transgenic stock was able to revert the transgenic root-length and root-area phenotypes to resemble those of the wild type (i.e. they resulted in a relative reduction in the length of roots in the FL line and a relative increase in the mMDH line; Fig. 5, A and B ). By contrast, the grafts were not able to alter the root biomass phenotype of either line, indicating that this parameter was independent of changes in the phenotype of the source organs (Fig. 5C). For all morphological parameters under consideration, wild type-to-wild type grafts were not significantly different from the untreated wild type, allowing us to exclude that the effects we observed were a direct consequence of the grafting process per se.
In a second experiment, we repeated the grafting but only with the most extreme transgenic line, and this time we harvested root tips from the rootstock of the grafted plants and determined their metabolite content by GC-MS (Table V ). As would perhaps be expected, the metabolite levels of the untreated transgenic lines were highly consistent with those reported in Supplemental Table S2, while the reciprocal grafts contained far fewer metabolic changes than observed in the ungrafted transgenic plants (irrespective of the direction of the graft). Evaluating the entire metabolite data sets by hierarchical cluster analysis (Saeed et al., 2003 ) revealed that the ungrafted FL metabolome is the most divergent from that of the ungrafted wild type. However, close inspection of the data in Table V reveals specific patterns of metabolic change that can be discerned that may contribute to the phenotypic restoration. Using this approach, we were able to identify that the levels of β-Ala and Pro were significantly different in the ungrafted roots of both transgenic lines but returned to wild-type levels following grafting of a wild-type scion. Using this approach, other subsets of metabolites could be identified that responded to the morphological restoration associated with grafting in one or other of the transgenic lines. Analysis of these data clearly revealed that levels of the metabolites glycerol and glycerol-3 phosphate were altered by the grafting process itself.
The aim of this work was to determine the importance of enzymes of malate metabolism for root morphology and function. Initial observations previously suggested that root biomass was decreased in transgenic tomato, exhibiting decreased expression either of the mitochondrial malate dehydrogenase or the predominantly expressed isoform of fumarase. On evaluation of root metabolism, in plants prior to flowering, we confirmed the reduction in root biomass in both sets of lines. In addition, we observed that the mMDH lines displayed decreased root length and a decreased exudate pH (whereas the FL lines displayed slightly increased root length). We also documented that, as would be expected, the rate of respiration in the roots was more severely compromised than in the leaves of these lines. This finding is interesting as it suggests that there is less flexibility in respiration in heterotrophic organs; however, currently we can only speculate about the reasons underlying it. It is highly likely that this observation merely reflects the relative importance of the TCA cycle in the various organs; however, we cannot formally exclude that alternative respiratory substrates are more efficiently used in the leaf. In keeping with the first hypothesis are the facts that the respiration rates documented here in the roots of the wild type (Fig. 1I) are far in excess of those reported previously for wild-type tomato leaves (Nunes-Nesi et al., 2005 , 2007a) and that the rate of the TCA cycle in the illuminated leaf is, at least to some extent, restricted (Tcherkez et al., 2005; Nunes-Nesi et al., 2007b). In addition, the results of the 13C-labeling experiments (Table IV) reinforce this, since the results from tomato root are suggestive of higher fluxes than those previously reported for tomato leaves (Studart-Guimarães et al., 2007). Furthermore, a severe inhibition of respiration is also evidenced by the reduced levels of adenylates observed in the transgenic lines (Fig. 2). Moreover, there is no current evidence to suggest that TCA cycle bypasses do not function as effectively in roots as in leaves (Ishizaki et al., 2005; Nunes-Nesi et al., 2005). Indeed, the facts that the levels of reductant are not dramatically reduced and that the level of ascorbate is even enhanced in the transgenic lines (Fig. 2) are highly suggestive that these bypasses are indeed engaged in the transformants. That said, irrespective of the exact reason behind the observed differences, it is clear that the TCA cycle plays a less redundant role in root metabolism than in leaf metabolism.
A major goal of this work was to improve our understanding of the influence of malate metabolism on root function in the tomato. Particular attention was paid to dissecting whether the restriction of root biomass observed here was the consequence of altered source function, altered root exudation, or merely an effect of altering metabolism in the root. In order to address this question, we took a range of approaches, including metabolite profiling of both cellular (endo-) and exuded (exo-) metabolites of the root system as well as analyzing the levels of phytohormones and the expression of genes associated with their perception and/or signal relay. For this purpose, we adopted an established GC-MS protocol to evaluate endometabolite and exometabolite contents of the transgenic root systems as well as the endometabolite contents in the root tips of grafted plants and used targeted approaches to measure phytohormones, carbohydrates, adenylates, and redox compounds in the root tip. GC-MS analysis of the endometabolites revealed very few changes in the mMDH lines, while the majority of endometabolites decreased in the FL lines. Comparison of the changes in exometabolite contents in the FL and mMDH lines at the end of the light period revealed that exudation was also markedly different between the lines. The levels of a wide range of exometabolites were elevated in the FL lines, while there was little change in the mMDH lines. These results provide compelling evidence that the reduced root biomass phenotype is not a consequence of a reduced organic acid exudation in the transgenics. This observation is somewhat at odds with previous studies showing that manipulations of TCA cycle metabolism in the roots can greatly improve fitness via a root exudate-mediated mechanism (Koyama et al., 2000
The fact that one of the sets of transgenics was previously demonstrated to display an enhanced rate of photosynthesis and the other displayed a decreased rate (Nunes-Nesi et al., 2005
What the results from these studies do clearly demonstrate, however, is that the reduced root biomass is due to a root-specific effect. One possible cause for this is the dramatic reduction in respiration in this organ. This reduction can be seen in both the direct oxygen consumption measurements and the results obtained from the 13C isotope-labeling experiments, which imply a slower general metabolism in the transformants. This trend, however, is not apparent in the steady-state levels of endometabolites, presumably due to the different exudation rates of the different transgenics. That the rate of respiration and the reduced root biomass correlate, taken alongside the previously observed links between energy metabolism, biosynthesis, and growth (Regierer et al., 2002
From our current study, we can state that the steady-state levels of these compounds in the root per se do not influence root branching and other morphological effects observed in the transgenics. We cannot, however, formally exclude that control of root development could be mediated by the influx of phytohormones from aerial parts of the plant, since several of these phenotypes were rescued by the grafting of a wild-type scion onto the transgenic rootstock. While we were able to demonstrate that this was able, at least partially, to complement the metabolic phenotype of the transgenics, we did not determine if this resulted in an altered exchange rate of phytohormones between the aerial and subterranean parts of the plant. Our results, however, do allow us to conclude that, despite the presence of compelling evidence for roles for IAA and ABA in mediating root development (Grieneisen et al., 2007
Plant Material
Previously characterized tomato (Solanum lycopersicum ‘Moneymaker’) seeds, exhibiting reduced expression of mitochondrial malate dehydrogenase and fumarase (Nunes-Nesi et al., 2005
Unless stated otherwise, all chemicals, cofactors, and enzymes were purchased from either Sigma-Aldrich or Merck.
Root exometabolites were collected at the end of the light period (unless stated otherwise) by gently excavating the root system from the vermiculite growth medium, washing, and incubating in double distilled water (pH 6.8) under gentle agitation (100 rpm) for 1 h.
The presence of colony-forming units was determined in order to monitor microbial activity exactly as defined previously (Weisskopf et al., 2005
Enzymes were extracted as described previously (Tauberger et al., 2000
Protein-blot analysis of fumarase protein was carried out on 33 µg of crude protein extract according to Nunes-Nesi et al. (2007a)
Cellular metabolite levels were analyzed as outlined by Roessner et al. (2001)
The levels of starch, Suc, Fru, and Glc in the leaf tissue were determined exactly as described previously (Müller-Röber et al., 1992
Isotopic feeding of root material was done following the protocol outlined by Roessner-Tunali et al. (2004)
Total RNA extraction for 5-week-old root material was done using Trizol according to the manufacturer's specifications. Following this, first-strand cDNA synthesis was carried out using the SuperScript indirect cDNA labeling kit (Invitrogen). Typically, 15 to 20 µg of total RNA (DNase I treated) was incubated at 70°C for 10 min with 5 µg of oligo(dT)20 primer in a total volume of 18 µL and placed on ice for 5 min. Subsequently, a master mix consisting of 5x first-strand buffer, 0.15 µmol of dithiothreitol, 15 mmol of deoxynucleoside triphosphate mix, RNAseOUT, and SuperScript III reverse transcriptase was added and the tube was incubated at 46°C. The mixture was hydrolyzed with 1 N NaOH, thoroughly mixed, and incubated at 70°C for 10 min, after which it was neutralized with 1 N HCl. Sodium acetate (3 M, pH 5.2) was added before proceeding to first-strand cDNA purification, following the kit instructions. Labeling and hybridization of the TOM1 array were followed directly as described previously (Alba et al., 2004
Root tips (25 mg) were cut with a sharp blade, washed, and incubated in 10 mM MES-KOH (pH 6.5) in a Clark-type oxygen electrode. The rate of oxygen consumption was calculated as described by Geigenberger et al. (2000)
Root phenotypes were digitally obtained and analyzed with Rootedge software (version 2.3; Iowa State University Research Foundation) as described by Kaspar and Ewing (1997)
Determination of abscisic acid was performed essentially as described by Peng et al. (1999)
Unless otherwise specified, statistical analyses were performed using the t test embedded in the Microsoft Excel software. Only the return of P < 0.05 was designated significant.
The following materials are available in the online version of this article.
We thank Drs. Matthew Hannah and Anna Lytovchenko (Max Planck Institute of Molecular Plant Physiology) for advice on grafting and proofreading, respectively. We thank Ingabritt Carlsson for help with IAA and GA analysis. We are also indebted to Dr. Karin Köhl and Helga Kulka (Max Planck Institute of Molecular Plant Physiology) for taking excellent care of the plants. Received September 29, 2008; accepted November 19, 2008; published November 21, 2008.
1 This work was supported by the Max-Planck-Gesellschaft (M.J.v.d.M., A.N.-N., A.R.F.), the South African National Research Foundation and the Deutscher Akademischer Austausch Dienst (M.J.v.d.M.), the Deutsch Forschungsgemeinschaft (S.O.), and the Swedish Research Council (T.M.).  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: Alisdair R. Fernie (fernie{at}mpimp-golm.mpg.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.108.130518 * Corresponding author; e-mail fernie{at}mpimp-golm.mpg.de.
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subunit in anther tapetum causes male sterility. Plant J 34: 57–66
