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The carnitine biosynthetic pathway in Arabidopsis thaliana shares similar features with the pathway of mammals and fungi
Plant Physiology and Biochemistry 60 (2012) 109e114
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Short communication
The carnitine biosynthetic pathway in Arabidopsis thaliana shares similar features with the pathway of mammals and fungi Sonia Rippa, Yingjuan Zhao, Franck Merlier, Aurélie Charrier 1, Yolande Perrin* Génie Enzymatique et Cellulaire (GEC), UMR 6022 CNRS, Université de Technologie de Compiègne, Centre de Recherche Royallieu, BP 20529, 60205 Compiègne Cedex, France
a r t i c l e i n f o
a b s t r a c t
Article history: Received 27 June 2012 Accepted 2 August 2012 Available online 11 August 2012
Carnitine is an essential quaternary ammonium amino acid that occurs in the microbial, plant and animal kingdoms. The role and synthesis of this compound are very well documented in bacteria, fungi and mammals. On the contrary, although the presence of carnitine in plant tissue has been reported four decades ago and information about its biological implication are available, nothing is known about its synthesis in plants. We designed experiments to determine if the carnitine biosynthetic pathway in Arabidopsis thaliana is similar to the pathway in mammals and in the fungi Neurospora crassa and Candida albicans. We first checked for the presence of trimetyllysine (TML) and g-butyrobetaine (g-BB), two precursors of carnitine in fungi and in mammals, using liquid chromatography-electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS). Both compounds were shown to be present in plant extracts at concentrations in the picomole range per mg of dry weight. We next synthesized deuterium-labeled TML and transferred A. thaliana seedlings on growth medium supplemented with 1 mM of the deuterated precursor. LC-ESI-MS/MS analysis of plant extracts clearly highlighted the synthesis of deuterium labeled g-BB and labeled carnitine in deuterated-TML fed plants. The similarities between plant, fungal and mammalian pathways provide very useful information to search homologies between genomes. As a matter of fact the analysis of A. thaliana protein database provides homology for several enzymes responsible for carnitine synthesis in fungi and mammals. The study of mutants affected in the corresponding genes would be very useful to elucidate the plant carnitine biosynthetic pathway and to investigate further the role of carnitine in plant physiology. Ó 2012 Elsevier Masson SAS. All rights reserved.
Keywords: Arabidopsis thaliana Carnitine biosynthesis Lipid metabolism Mass spectrometry
1. Introduction Carnitine is a ubiquitous compound found in the animal, plant and microbial kingdoms and it plays several essential metabolic and physiological roles. In mammals, carnitine is known to be responsible for importing activated fatty acids (FA) into the mitochondrion, as acylcarnitines, to feed the b-oxidation process [1]. Carnitine also allows the export of activated medium chain FA from
Abbreviations: ALDH, aldehyde dehydrogenase; g-BB, g-butyrobetaine; BBD, gbutyrobetaine dioxygenase; FA, fatty acid; HTML, 3-hydroxy-6-N-trimethyllysine; HTMLA, HTML aldolase; MRM, multiple reaction monitoring; MSM, Murashige and Skoog medium; PLP, pyridoxal 50 -phosphate; RT, retention time; SHMT, serine hydroxymethyltransferase; THA, threonine aldolase; THF, tetrahydrofolate; THP, 3(2,2,2-trimethylhydrazine)propionate; TMABA, 4-N-trimethylaminobutyraldehyde; TMABADH, 4-N-trimethylaminobutyraldehyde dehydrogenase; TML, trimethyllysine; TMLD, TML dioxygenase. * Corresponding author. Tel.: þ33 344234416; fax: þ33 344234423. E-mail address: [email protected] (Y. Perrin). 1 Present address: Université d’Angers, UMR 1191 Physiologie Moléculaire des Semences IFR 149 QUASAV, 2 Bd Lavoisier, 49045 Angers Cedex, France. 0981-9428/$ e see front matter Ó 2012 Elsevier Masson SAS. All rights reserved. http://dx.doi.org/10.1016/j.plaphy.2012.08.001
peroxisomes in the case of peroxisomal b-oxidation of very long chain FA [2]. The involvement of carnitine in the energy metabolism of mammals also covers the buffering of the mitochondrial acetyl-CoA pool in situation of excess or inadequate acetyl-CoA provision [1,3]. In fungi, carnitine can be recruited for the transfer of acetyl-CoA from the glyoxysome to the mitochondrion when the glyoxylate cycle is impaired [4]. Besides its basic metabolic function, carnitine has been assigned additional roles in recent years, all connected with handling inadequate cellular environments. In bacteria carnitine can accumulate at high levels within the cell without altering cytoplasmic functions and acts as an osmolyte by helping cells to deal with chilling induced [5] or salt induced [6] osmotic stress. It is also suggested that carnitine could take part in the FA intracellular transport essential to the process of membrane lipid adjustment in the case of chill stress [5]. In mammals, linked to its involvement in FA metabolism, carnitine plays a detoxifying role by facilitating tissue excretion, as carnitine esters, of drug-derived FA that cannot be metabolised by the cell [7]. In addition carnitine has been assigned antioxidant properties in both fungi [8] and mammals [9,10].
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The discovery of carnitine (acetyl-, octanoyl- or palmitoyl-) transferase activities in preparations of mitochondria and chloroplasts from pea or mung-bean seedlings [11e16] and the quantification of acylcarnitines alongside free carnitine in several plant species (Arabidopsis thaliana, tobacco, rapeseed and flax) [17] demonstrate the involvement of carnitine in plant lipid metabolism. This result is coherent with the probable existence of carnitine transporters in plant cells such as the A. thaliana plasma membrane transporter OCT1 [18]. The link between carnitine and FA appears to be partly inherent to a mitochondrial b-oxidation process in plant cells [19,20] connected to some specific metabolic requirements notably for chloroplast maturation during greening of the emerging plumules of pea seedlings [21e24]. Finally as observed in animals, fungi and bacteria we recently showed that carnitine confers tolerance to abiotic stress in plants, enhancing the development and recovery of A. thaliana seedlings in conditions of salt and oxidative stress [25]. In animal species carnitine is mainly provided by food but can also be synthesized in the liver, kidney and brain (for review see Ref. [26]). The precursor of carnitine is the 6-N-trimethyllysine (TML) derived from lysosomal or proteasomal protein degradation, leading to carnitine in a four-step pathway (Fig. 1). This compound is first hydroxylated by TML dioxygenase (TMLD; EC 1.14.11.8) and the resulting 3-hydroxy-6-N-trimethyllysine (HTML) is cleaved by HTML aldolase (HTMLA; EC 4.1.2.X) into 4-N-trimethylaminobutyraldehyde (TMABA) and glycine. TMABA dehydrogenase (TMABADH; EC 1.2.1.47) transforms next TMABA into 4-Ntrimethylaminobutyrate (g-butyrobetaine, g-BB). Finally g-BB is hydroxylated by g-BB dioxygenase (BBD; EC 1.14.11.1) to produce carnitine. The fungi Neurospora crassa and Candida albicans do produce carnitine using a similar biosynthetic pathway to mammals [27,28]. To date there is no available datum on the
synthesis of carnitine in plants. The presence of endogenous carnitine in axenic cultures shows that plants synthesize carnitine, not excluding that on soil, carnitine can come partly from rhizosphere interactions. Elucidation of the carnitine biosynthetic pathway is crucial for establishing its biological significance in plants, by giving us clues about its regulation and by allowing approaches of reverse genetic. 2. Results 2.1. Detection and quantification of likely precursors of carnitine in A. thaliana We first checked by LC-ESI-MS/MS for the presence of the two known carnitine precursors TML and g-BB for which commercial standards are available. We found that both compounds are present in extracts from 7-day-old A. thaliana seedlings alongside carnitine, as shown on Fig. 2A. A clear signal is obtained for the MRM transitions selected according to the fragmentation patterns for TML (189 > 84), for g-BB (146 > 87) and for carnitine (162 > 85 and 162 > 103). Quantifications by adding internal standards show that the three metabolites are present in similar amounts, in the picomole range per mg of dry weight, and with a higher content for TML (Table 1). 2.2. Monitoring the incorporation of deuterium labelled TML Based on this initial result, we checked if carnitine synthesis truly starts from TML in A. thaliana as it does in mammal and fungal cells. We synthesized [2H9]-TML and seeds were sown on MSM supplemented with 1 mM of the deuterated compound. We checked for deuterium incorporation in g-BB and carnitine by
NH O
N
OH
NH O
N OH
OH
O
N
O
N
OH
O
N OH
OH
Fig. 1. Carnitine biosynthesis pathway in mammals. From Ref. [26]. TML is released from lysosomal or proteosomal protein degradation and converted into carnitine in a four-step pathway. PLP: pyridoxal 50 -phosphate; TMLD: TML dioxygenase; HTMLA: HTML aldolase; TMABADH: TMABA dehydrogenase; BBD: g-BB dioxygenase.
S. Rippa et al. / Plant Physiology and Biochemistry 60 (2012) 109e114
A
MRM of 6 Channels ES+ 189.18 > 83.96 1.58e4
6.17
100
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B 2
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19
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MRM of 6 Channels ES+ 146.1 > 87 1.35e4
5.27
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MRM of 6 Channels ES+ 155.1 > 87 6.87e4 2
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MRM of 6 Channels ES+ 162.2 > 85.1 6.49e3
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MRM of 6 Channels ES+ 171.2 > 85.1 4.93e3
5.18
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[ H9]-carnitine
carnitine %
%
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Time
MRM of 6 Channels ES+ 162.2 > 103.1 8.64e3
5.15
100
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MRM of 6 Channels ES+ 171.2 > 103.1 5.99e3
5.31
100
2
[ H9]-carnitine
carnitine %
33
10.00
[ H9]-γ-BB
%
44
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5.18
100
γ-BB
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MRM of 6 Channels ES+ 198.18 > 83.96 3.85e6
6.12
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%
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50
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Fig. 2. LC-ESI-MS/MS chromatograms of an extract obtained from 7-day-old A. thaliana seedlings grown on normal MSM (A) and on 1 mM [2H9]-TML supplemented MSM (B). MRM transitions of TML (189 > 84), [2H9]-TML (198 > 84), g-BB (146 > 87), [2H9]-g-BB (155 > 87), carnitine (162 > 85 and 162 > 103) and [2H9]-carnitine (171 > 85 and 171 > 103) are shown. Retention times are given on top of every specific signal.
LC-ESI-MS/MS on extracts obtained from [2H9]-TML-treated 7-dayold seedlings and from 7-day-old control seedlings grown on normal MSM. Results show that [2H9]-TML is efficiently taken-up by the A. thaliana seedlings during their development on [2H9]-TMLsupplemented medium (Fig. 2B). The corresponding extract contains a compound that we identify as [2H9]-TML since it presents a mass increment of 9 Da as compared to non-deuterated TML Table 1 Content of TML, g-BB and carnitine in 7-day-old A. thaliana seedlings grown on MSM. Compound
Content (pmol mg DW1)
TML g-BB Carnitine
74.5 13.1 18.5 2.5 1.72 0.6
Each value is given as mean standard deviation of results obtained from three independent experiments.
(198 Da versus 189 Da), fragments in the same way (fragment of 84 Da) and shows an identical retention time (RT) of 6 min in our HPLC conditions. The quantification has confirmed an endogenous concentration of 4890 pmol DW1 of [2H9]-TML. It appears that part of [2H9]-TML is metabolised into [2H9]-g-BB and [2H9]-carnitine since both molecules are present in the [2H9]-TML-treated plant extract as shown on Fig. 2B. The compound that we identify as [2H9]-g-BB presents an identical RT as the non-deuterated g-BB, and fragments into a 87 Da derivative that gives a signal at transition 155 > 87 similarly to the non-deuterated g-BB fragmentation (146 > 87). More interestingly [2H9]-carnitine is observable at two transitions 171 > 85 and 171 > 103 corresponding to the same fragmentation as non-deuterated carnitine, but with a mass increment of 9 Da and an identical retention time of 5 min. The presence of [2H9]-carnitine was further verified by performing its positive ESI-MS m/z (mode daughter) spectrum (Fig. 3). The mass pattern reveals daughter ions of m/z ¼ 69 corresponding to the typical
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A
8.41e6
102.8
100
H3C
84.9 59.9
%
CH3
OH
+
N
CH3
42.8
OH
O
162.1
56.8
101.6
0
B
103.0
100
69.0
3.15e3
2
2
H3C
3. Discussion
CH3
OH
+
N
2
CH3
OH
O 171.3
%
84.9
0 40
60
80
100
120
content was of 0.9 pmol mg DW1 and the carnitine content of 6.1 pmol mg DW1 (the ratio [2H9]-carnitine/carnitine was 1/7). In the plant sample grown on 0.5 mM [2H9]-TML and 0.5 mM TML the [2H9]-carnitine fell to 0.3 pmol mg DW1 whereas the unlabeled carnitine content was of 22.8 pmol mg DW1 (the ratio [2H9]carnitine/carnitine was 1/76). To note that the plant extract from seedlings grown on MSM contained 7.2 pmol mg DW1 of carnitine and no [2H9]-carnitine. In parallel, g-BB was supplied at 0.5 mM at the same time as [2H9]-TML at 1 mM. In this plant extract [2H9]-carnitine was undetectable (Table 2). However this last result must be considered more precautiously due to a highly deleterious effect on seedling development of exogenous g-BB at this concentration, similar but more pronounced than the TML effect.
140
160
m/z 180
Fig. 3. Positive ESI-MS m/z (mode daughter) spectra of carnitine and [2H9]-carnitine extracted from 7-day-old A. thaliana seedlings grown on 1 mM [2H9]-TML supplemented MSM. (A) Carnitine ESI-MS spectrum: parent ion m/z 162.1 and daughter ion m/z 59.9 corresponding to the trimethylammonium fragment are surrounded in black; (B) ESI-MS spectrum of [2H9]-carnitine: parent ion m/z 171.3 and daughter ion m/z 69 corresponding to the trimethylammonium fragment, with a mass increment of 9 Da as compared to (A), are circled in green. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
unlabeled carnitine daughter ion of m/z ¼ 60 generated by the trimethylammonium fragment with a mass increment of 9 Da due to the deuteriums. As expected the deuterium labeled compounds were not detected in the control extract (data not shown). An additional experiment was carried out in order to strengthen the above findings. [2H9]-TML was supplied at the same time as unlabeled TML (0.5 mM each) and compared, in terms of [2H9]carnitine synthesis, to a 1 mM supply of [2H9]-TML alone. This experiment was performed during the first 3 days of seedling development and not on 7-day-old seedlings as for the previous experiment, in order to optimize our results since we measured a higher carnitine content in 3-day-old seedlings. Moreover, after 3 days of development a negative effect of TML was observed through a slowdown of shoot and root growth, and through the visible accumulation of anthocyanin in leaves, reflecting the stress suffered by the seedlings. As expected the [2H9]-TML dilution with TML led to a decrease of [2H9]-carnitine content in the seedlings as compared to seedlings receiving [2H9]-TML only (Table 2). In the plant sample grown on 1 mM [2H9]-TML, the [2H9]-carnitine
Table 2 Content of carnitine and [2H9]-carnitine in 3-day-old A. thaliana seedlings grown on different growth mediums for isotope dilution experiments. Growth medium
[2H9]-carnitine (pmol mg DW1)
Carnitine (pmol mg DW1)
MSM 1 mM [2H9]-TML 0.5 mM [2H9]-TML/0.5 mM TML 1 mM [2H9]-TML /0.5 mM g-BB
Undetectable 0.9 0.3 Undetectable
7.2 6.1 22.8 10.5
The presence of TML and g-BB in A. thaliana has been a first indication that both compounds could be precursors for carnitine synthesis in plants, as in mammals and fungi. Moreover, our quantifications reveal that in A. thaliana, TML and g-BB are present in higher amounts than carnitine, a situation very different than in mammals. In mouse for instance, in organs that synthesize carnitine such as liver, kidney and brain, the carnitine content can be 25e70 fold higher than TML, and 8e40 fold higher than g-BB, depending on the organ [29]. This indicates that the regulation and/or the flux through plant carnitine biosynthesis is very different than in mammal. The results also reveal that an exogenous supply of 1 mM TML does not lead to any measurable increase of endogenous carnitine. Therefore, exogenous TML is not a good substrate for carnitine synthesis in A. thaliana. Considering that our work is carried out on a whole organism, it is possible that most part of exogenous TML does not reach the site of carnitine synthesis at the scale of the tissue or of the organelle. This explains the low amounts of [2H9]-carnitine synthesized in the seedlings despite a highly concentrated supply of [2H9]-TML. On this point, if TML was efficiently metabolized into carnitine, we probably would not observe a deleterious effect on seedling development, considering that carnitine is a compatible solute that can accumulate in the plant at very high concentrations without affecting plant development [25]. At this stage of our knowledge, we cannot explain the toxicity of TML and g-BB that are compounds with a quaternary ammonium moiety like compatible solutes such as glycinebetaine and carnitine. This signifies that despite a chemical proximity, all betaines are not trivial to cellular functions, probably depending on the nature and the strength of their interaction with metabolism and/or signalling pathways. The detection of [2H9]-g-BB and [2H9]-carnitine alongside endogenous [2H9]-TML clearly proves that as in animal and fungal cells, trimethyllysine is a precursor of carnitine in A. thaliana cells. Regarding the dilution experiment, the absence of linearity between the two-fold dilution of [2H9]-TML and the resultant tenfold reduction of [2H9]-carnitine content, could reflect the activity of a non-MichaeliseMenten enzyme for which substrate affinity would increase non linearly with substrate concentration. Moreover, the lowering effect of the supply of unlabelled g-BB on the synthesis of [2H9]-carnitine from [2H9]-TML tends to confirm that g-BB is an intermediate in the carnitine biosynthesis pathway from TML. Through those results, it can be expected that similar enzymatic activities, performed by homologous enzymes, could control carnitine synthesis in fungi, mammals and plants. The identification of the enzymes belonging to the carnitine biosynthetic pathway will allow reverse genetic approaches to investigate further the physiological roles of carnitine in plants. We have
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already performed BLASTP analyses using the human or fungal protein sequences to identify the probable enzymes implicated in A. thaliana carnitine synthesis and have found good candidates for step 2 and 3 (Fig. 1). It is noteworthy that we did not find any significant sequence homology between the mammalian or fungal enzymes implicated in steps 1 and 4 and the proteins found in the A. thaliana database. This result suggests that the two hydroxylation steps might be performed by different enzymes in plants and in fungi and mammals. However, feeding of A. thaliana seedlings with THP (3-(2,2,2-trimethylhydrazine)propionate), a well characterized inhibitor of the mammalian g-BB dioxygenase [30], has led to an average 50% decrease of the endogenous carnitine, for exogenous supplies ranging from 500 mM to 1 mM THP (data not shown). This result suggests that despite the absence of sequence homology between plants and mammals, the active sites of the mammalian g-BB dioxygenase and of the corresponding plant enzyme might share similar conformations. Concerning step 2, the human HTML aldolase (HTMLA) might be identical to the cytosolic serine hydroxymethyltransferase (SHMT; EC 2.1.2.1) but this remains to be established [26,31]. SHMT are PLP-dependent enzymes that catalyse the reversible conversion of serine and tetrahydrofolate (THF) to glycine and 5,10-methylene THF [32]. A. thaliana genome encodes 7 SHMTs that display high sequence similarity with the human cytosolic SHMT1 (identity of 58% and similarity of 74% regarding the A. thaliana SHMT1 for instance). Although this A. thaliana protein (also called SHM1) has been implicated in the photorespiratory pathway [33], we do not exclude the possible role of an A. thaliana SHMT in converting HTML into TMABA when considering the homology between A. thaliana and human SHMT proteins. Recently the complete carnitine biosynthesis pathway has been identified and characterized in C. albicans and a threonine aldolase has been proposed to be the main HTMLA in this organism [28]. Threonine aldolases (EC 4.1.2.5) are PLPdependent enzymes that convert threonine to acetaldehyde and glycine. A BLASTP search with the fungal protein sequence has identified the A. thaliana threonine aldolase 1 and 2 (THA1 and THA2) as the most likely homologues (sequence identity of 36% and 37% respectively). These two proteins are implicated in plant amino acid metabolism and particularly in glycine and acetaldehyde formation [34]. The third step in human carnitine formation is realized by an aldehyde dehydrogenase (ALDH) named TMABADH. In A. thaliana the ALDH gene superfamily contains 14 sequences distributed in 9 families, implicated in cytoplasmic male sterility, plant defence and abiotic stress tolerance [35]. Among them, family 2 and 10 are closely homologous to the human TMABADH (identity of approximately 37e39%). Interestingly it has been shown recently that in Pisum sativum two aminoaldehyde dehydrogenases belonging to the large ALDH superfamily can oxidize N,N,N-trimethyl-4-aminobutyraldehyde (TMABA) into g-BB [36]. A. thaliana ALDH10A8 and 10A9, belonging to the family 10 of the ALDH superfamily, are quite similar to these pea proteins (more than 70% of identity). Recent results show that in A. thaliana, the peroxisomal ALDH10A9 protein possesses betaine aldehyde, 4Aminobutyraldehyde (ABAL) and 3-Aminopropionalehyde (APAL) dehydrogenase in vitro activities [37]. The A. thaliana ALDH10 proteins could therefore be also able to convert TMABA into g-BB. The availability of knockout mutants for some of the candidate enzymes and the possibility of finding more KO mutants from the A. thaliana T-DNA insertion libraries will allow us to investigate further the involvement of the candidate enzymes in the plant carnitine biosynthetic pathway. In the present paper we report data showing that plants employ similar precursors to mammals and fungi to synthesize carnitine, bringing the first evidence that the carnitine biosynthetic pathways in those eukaryotic organisms share similar features, and most
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probably involve similar enzymatic activities. This result makes it now possible to identify the genes involved in carnitine biosynthesis in plants. Protein homology searches will allow notably reverse genetic approaches that will facilitate studies of the roles that carnitine plays in plants, and new insights into comparative physiology between plant and animal kingdoms. 4. Methods 4.1. [2H9]-TML synthesis We synthesized deuterium labeled 6-N-trimethyllysine according to the method described in Ref. [38], from L-lysine HCl (Merck, ref. K15007900) and using [Me-2H6]dimethyl sulfate (Sigma, ref. 164526) as methyl donor. The [2H9]-TML was purified twice on a 4-ml Dowex 50WX8 (Serva) column, washed with 20 volumes of milli-Q grade water and eluted with 120 ml of 2 mol/L NH4OH. Ammonia was evaporated under a flux of nitrogen at room temperature. Then the effluent was concentrated by lyophilisation. A sterile one molar [2H9]-TML stock solution was prepared in 70% ethanol. The absence of lysine in the solution was checked by LCESI-MS/MS. 4.2. Plant culture and treatment A. thaliana (L.) Heynh, ecotype Columbia (Col-0), was used in this study. For aseptic growth, seeds were first surface-sterilized and sown under sterile conditions on Murashige and Skoog medium (MSM) containing 8% agar [39]. They were placed at 4 C for 3 days for stratification. Seedlings were subsequently grown at 24 C under long day conditions (16-h-light cycle at 150 mE m2 s1 illumination) for three or seven days before being frozen in liquid N2 for subsequent extraction. One molar stocks of TML, g-BB (Sigma ref. T1660 and 403245 respectively) and [2H9]-TML prepared in 70% ethanol or filter sterilized were directly added in the MSM to desired concentrations for plant treatments. The pH of the medium was adjusted to 5.8 when necessary. 4.3. Analysis of TML, g-BB and carnitine by tandem mass spectrometry The extractions of carnitine and its precursors were performed according to Ref. [17] with some modifications. Standards for TML, g-BB and L-carnitine were purchased from Sigma (ref. T1660, 403245 and C0158 respectively). The equipment consists in a Famos autosampler and an Ultimate 3000 LC Packing capillarity HPLC (Dionex) combined to a Quattro Micro triple quadruple mass spectrometer (Waters). Approximately 200 mg of seedlings were used for extractions performed in 700 ml of acetonitrile:water (8:2, v:v). The HPLC separation was carried out on a 5 mm Luna C8(2) 150 mm 0.5 mm capillarity column (Phenomenex) maintained at 35 C. The elution gradient was performed according to Ref. [40] using water (A) and methanol (B) as mobile phases. Both A and B contained ammonium acetate (10 mM) and heptafluorobutyric acid (10 mM). The gradient started with 10% B, raised to 100% in 20 min and lowered to 10% from 20 to 25 min. The flow was set at 8 ml/min and the injected sample volume was 1 ml. Collision energy of 20 eV was set using standards. Standards were used to assess transitions 189 > 84 for TML, 146 > 87 for g-BB, 162 > 85 and 162 > 103 for carnitine. Plant extracts were diluted 5 times in methanol:water (80:20) when analysing TML and [2H9]-TML due to the high content of TML in plant tissues. Quantification of TML, g-BB and carnitine was performed by measuring the area under each specific peak using MassLynx 4.0 (Waters). Samples were first run without an internal standard, secondly with a standard at known
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concentration and finally with a standard at a double concentration. A linear regression permitted quantification of the concentration of samples. Acknowledgments We gratefully thank Professor Béla Melegh for his useful information on the synthesis and purification of deuterium labeled TML. We are grateful to Professor Stephan Krähenbühl for having offered us the THP compound. We also would like to thank Mr Michael Jones for careful reading of the manuscript. References [1] J. Kerner, C. Hoppel, Fatty acid import into mitochondria, BBA Mol. Cell Biol. Lipids 1486 (2000) 1e17. [2] F. Fraser, V.A. Zammit, Submitochondrial and subcellular distributions of the carnitine-acylcarnitine carrier, FEBS Lett. 445 (1999) 41e44. [3] F.B. Stephens, D. Constantin-Teodosiu, P.L. Greenhaff, New insights concerning the role of carnitine in the regulation of fuel metabolism in skeletal muscle, J. Physiol. 581 (2007) 431e444. [4] C.W.T. Van Roermund, E.H. Hettema, M. Van Den Berg, H.F. Tabak, R.J.A. Wanders, Molecular characterization of carnitine-dependent transport of acetyl-CoA from peroxisomes to mitochondria in Saccharomyces cerevisiae and identification of a plasma membrane carnitine transporter, Agp2p, EMBO J. 18 (1999) 5843e5852. [5] A.S. Angelidis, G.M. Smith, Role of the glycine betaine and carnitine transporters in adaptation of Listeria monocytogenes to chill stress in defined medium, Appl. Environ. Microbiol. 69 (2003) 7492e7498. [6] M. Canovas, V. Bernal, A. Sevilla, J.L. Iborra, Salt stress effects on the central and carnitine metabolisms of Escherichia coli, Biotechnol. Bioeng. 96 (2007) 722e737. [7] E. Arrigoni-Martelli, V. Caso, Carnitine protects mitochondria and removes toxic acyls from xenobiotics, Drugs Exp. Clin. Res. 27 (2001) 27e49. [8] J. Franken, S. Kroppenstedt, J.H. Swiegers, F.F. Bauer, Carnitine and carnitine acetyltransferases in the yeast Saccharomyces cerevisiae, a role for carnitine in stress protection, Curr. Genet. 53 (2008) 347e360. lu, H. Satiroglu, I. Alican, A. Kaçmaz, A. Sakarcan, L[9] G. Sener, K. Paskalog carnitine ameliorates oxidative damage due to chronic renal failure in rats, J. Cardiovasc. Pharmacol. 43 (2004) 698e705. [10] D. Silva-Adaya, V. Pérez-De La Cruz, M.N. Herrera-Mundo, K. Mendoza-Macedo, J. Villeda-Hernández, Z. Binienda, S.F. Ali, A. Santamaría, Excitotoxic damage, disrupted energy metabolism, and oxidative stress in the rat brain: antioxidant and neuroprotective effects of L-carnitine, J. Neurochem. 105 (2008) 677e689. [11] C. Wood, M. Noh Hj Jalil, A. Ariffin, B.C.S. Yong, D.R. Thomas, Carnitine shortchain acyltransferase in pea mitochondria, Planta 158 (1983) 175e178. [12] C. Wood, M. Noh Hj Jalil, I. McLaren, B.C.S. Yong, A. Ariffin, P.H. McNeil, N. Burgess, D.R. Thomas, Carnitine long-chain acyltransferase and oxidation of palmitate, palmitoyl coenzyme A and palmitoylcarnitine by pea mitochondria preparations, Planta 161 (1984) 255e260. [13] H. Gerbling, B. Gerhardt, Carnitine-acyltransferase activity of mitochondria from mung-bean hypocotyls, Planta 174 (1988) 90e93. [14] H. Schwabedissen-Gerbling, B. Gerhardt, Purification and characterization of carnitine acyltransferase from higher plant mitochondria, Phytochemistry 39 (1995) 39e44. [15] C. Masterson, C. Wood, Pea chloroplast carnitine acetyltransferase, Proc. Biol. Sci. 267 (2000) 1e6. [16] C. Masterson, C. Wood, Carnitine palmitoyltransferases in pea leaf chloroplasts: partial purification, location, and properties, Can. J. Bot. 78 (2000) 328e335. [17] B. Bourdin, H. Adenier, Y. Perrin, Carnitine is associated with fatty acid metabolism in plants, Plant Physiol. Biochem. 45 (2007) 926e931.
[18] C. Lelandais-Brière, M. Jovanovic, G.A. Torres, Y. Perrin, R. Lemoine, F. CorreMenguy, C. Hartmann, Disruption of AtOCT1, an organic cation transporter gene, affects root development and carnitine-related responses in Arabidopsis, Plant J. 51 (2007) 154e164. [19] R.A. Panter, J.B. Mudd, Some aspects of carnitine metabolism in avocado (Persea americana), Biochem. J. 134 (1973) 655e658. [20] C. Masterson, C. Wood, Mitochondrial b-oxidation of fatty acids in higher plants, Physiol. Plantarum 109 (2000) 217e224. [21] D.R. Thomas, A. Ariffin, M. Noh Hj Jalil, B.C.S. Yong, R.J. Cooke, C. Wood, Effect of carnitine on greening barley leaves, Phytochemistry 20 (1981) 1241e1244. [22] C. Wood, C. Masterson, D.R. Thomas, The role of carnitine in plant cell metabolism, in: A.K. Tobin (Ed.), Plant Organelles: Compartmentation of Metabolism in Photosynthetic Cells, S.E.B. Seminar Series, vol. 50, Cambridge University Press, Cambridge UK, 1992, pp. 229e263. [23] C. Wood, C. Masterson, D.R. Thomas, Carnitine-dependent fatty acyl transport in plant cells, in: H. Lambers, L.H.W. van der Plas (Eds.), Molecular, Biochemical and Physiological Aspects of Plant Respiration, SPB Publishing, The Hague, Holland, 1992, pp. 195e207. [24] C. Masterson, C. Wood, Influence of mitochondrial b-oxidation on early pea seedling development, New Phytol. 181 (2009) 832e842. [25] A. Charrier, S. Rippa, A. Yu, P.J. Nguyen, J.P. Renou, Y. Perrin, The effect of carnitine on Arabidopsis development and recovery in salt stress conditions, Planta 235 (2012) 123e135. [26] F.M. Vaz, R.J. Wanders, Carnitine biosynthesis in mammals, Biochem. J. 361 (2002) 417e429. [27] R.A. Kaufman, H.P. Broquist, Biosynthesis of carnitine in Neurospora crassa, J. Biol. Chem. 252 (1977) 7437e7439. [28] K. Strijbis, C.W. van Roermund, G.P. Hardy, J. van den Burg, K. Bloem, J. de Haan, N. van Vlies, R.J. Wanders, F.M. Vaz, B. Distel, Identification and characterization of a complete carnitine biosynthesis pathway in Candida albicans, FASEB J. 23 (2009) 2349e2359. [29] N. van Vlies, L. Tian, H. Overmars, A.H. Bootsma, W. Kulik, R.J.A. Wanders, P.A. Wood, F.M. Vaz, Characterization of carnitine and fatty acid metabolism in the long-chain acyl-CoA dehydrogenase-deficient mouse, Biochem. J. 387 (2005) 185e193. [30] M. Spaniol, H. Brooks, L. Auer, A. Zimmermann, M. Solioz, B. Stieger, S. Krähenbühl, Development and characterization of an animal model of carnitine deficiency, Eur. J. Biochem. 268 (2001) 876e1887. [31] K. Strijbis, F.M. Vaz, B. Distel, Enzymology of the carnitine biosynthesis pathway, IUBMB Life 62 (2010) 357e362. [32] L. Schirch, Serine hydroxymethyltransferase, Adv. Enzymol. 53 (1982) 83e112. [33] L.M. Voll, A. Jamai, P. Renné, H. Voll, C.R. McClung, A.P. Weber, The photorespiratory Arabidopsis shm1 mutant is deficient in SHM1, Plant Physiol. 140 (2006) 59e66. [34] V. Joshi, K.M. Laubengayer, N. Schauer, A.R. Fernie, G. Jander, Two Arabidopsis threonine aldolases are nonredundant and compete with threonine deaminase for a common substrate pool, Plant Cell 18 (2006) 3564e3575. [35] H.H. Kirch, D. Bartels, Y. Wei, P.S. Schnable, A.J. Wood, The ALDH gene superfamily of Arabidopsis, Trends Plant Sci. 9 (2004) 371e377. [36] M. Tylichová, D. Kopecný, S. Moréra, P. Briozzo, R. Lenobel, J. Snégaroff, M. Sebela, Structural and functional characterization of plant aminoaldehyde dehydrogenase from Pisum sativum with a broad specificity for natural and synthetic aminoaldehydes, J. Mol. Biol. 396 (2010) 870e882. [37] T.D. Missihoun, J. Schmitz, R. Klug, H.H. Kirch, D. Bartels, Betaine aldehyde dehydrogenase genes from Arabidopsis with different sub-cellular localization affect stress responses, Planta 233 (2011) 369e382. [38] F.M. Vaz, B. Melegh, J. Bene, D. Cuebas, D.A. Gage, A. Bootsma, P. Vreken, A.H. van Gennip, L.L. Bieber, R.J. Wanders, Analysis of carnitine biosynthesis metabolites in urine by HPLC-electrospray tandem mass spectrometry, Clin. Chem. 48 (2002) 826e834 (Erratum in: Clin. Chem. 48 (2002) 1633). [39] T. Murashige, F. Skoog, A revised medium for rapid growth and bioassays with tobacco tissue cultures, Physiol. Plant 15 (1962) 473e497. [40] L. Vernez, G. Hopfgartner, M. Wenk, S. Krähenbühl, Determination of carnitine and acylcarnitines in urine by high-performance liquid chromatographyelectrospray ionization ion trap tandem mass spectrometry, J. Chromatogr. A 984 (2003) 203e213.
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Short communication
The carnitine biosynthetic pathway in Arabidopsis thaliana shares similar features with the pathway of mammals and fungi Sonia Rippa, Yingjuan Zhao, Franck Merlier, Aurélie Charrier 1, Yolande Perrin* Génie Enzymatique et Cellulaire (GEC), UMR 6022 CNRS, Université de Technologie de Compiègne, Centre de Recherche Royallieu, BP 20529, 60205 Compiègne Cedex, France
a r t i c l e i n f o
a b s t r a c t
Article history: Received 27 June 2012 Accepted 2 August 2012 Available online 11 August 2012
Carnitine is an essential quaternary ammonium amino acid that occurs in the microbial, plant and animal kingdoms. The role and synthesis of this compound are very well documented in bacteria, fungi and mammals. On the contrary, although the presence of carnitine in plant tissue has been reported four decades ago and information about its biological implication are available, nothing is known about its synthesis in plants. We designed experiments to determine if the carnitine biosynthetic pathway in Arabidopsis thaliana is similar to the pathway in mammals and in the fungi Neurospora crassa and Candida albicans. We first checked for the presence of trimetyllysine (TML) and g-butyrobetaine (g-BB), two precursors of carnitine in fungi and in mammals, using liquid chromatography-electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS). Both compounds were shown to be present in plant extracts at concentrations in the picomole range per mg of dry weight. We next synthesized deuterium-labeled TML and transferred A. thaliana seedlings on growth medium supplemented with 1 mM of the deuterated precursor. LC-ESI-MS/MS analysis of plant extracts clearly highlighted the synthesis of deuterium labeled g-BB and labeled carnitine in deuterated-TML fed plants. The similarities between plant, fungal and mammalian pathways provide very useful information to search homologies between genomes. As a matter of fact the analysis of A. thaliana protein database provides homology for several enzymes responsible for carnitine synthesis in fungi and mammals. The study of mutants affected in the corresponding genes would be very useful to elucidate the plant carnitine biosynthetic pathway and to investigate further the role of carnitine in plant physiology. Ó 2012 Elsevier Masson SAS. All rights reserved.
Keywords: Arabidopsis thaliana Carnitine biosynthesis Lipid metabolism Mass spectrometry
1. Introduction Carnitine is a ubiquitous compound found in the animal, plant and microbial kingdoms and it plays several essential metabolic and physiological roles. In mammals, carnitine is known to be responsible for importing activated fatty acids (FA) into the mitochondrion, as acylcarnitines, to feed the b-oxidation process [1]. Carnitine also allows the export of activated medium chain FA from
Abbreviations: ALDH, aldehyde dehydrogenase; g-BB, g-butyrobetaine; BBD, gbutyrobetaine dioxygenase; FA, fatty acid; HTML, 3-hydroxy-6-N-trimethyllysine; HTMLA, HTML aldolase; MRM, multiple reaction monitoring; MSM, Murashige and Skoog medium; PLP, pyridoxal 50 -phosphate; RT, retention time; SHMT, serine hydroxymethyltransferase; THA, threonine aldolase; THF, tetrahydrofolate; THP, 3(2,2,2-trimethylhydrazine)propionate; TMABA, 4-N-trimethylaminobutyraldehyde; TMABADH, 4-N-trimethylaminobutyraldehyde dehydrogenase; TML, trimethyllysine; TMLD, TML dioxygenase. * Corresponding author. Tel.: þ33 344234416; fax: þ33 344234423. E-mail address: [email protected] (Y. Perrin). 1 Present address: Université d’Angers, UMR 1191 Physiologie Moléculaire des Semences IFR 149 QUASAV, 2 Bd Lavoisier, 49045 Angers Cedex, France. 0981-9428/$ e see front matter Ó 2012 Elsevier Masson SAS. All rights reserved. http://dx.doi.org/10.1016/j.plaphy.2012.08.001
peroxisomes in the case of peroxisomal b-oxidation of very long chain FA [2]. The involvement of carnitine in the energy metabolism of mammals also covers the buffering of the mitochondrial acetyl-CoA pool in situation of excess or inadequate acetyl-CoA provision [1,3]. In fungi, carnitine can be recruited for the transfer of acetyl-CoA from the glyoxysome to the mitochondrion when the glyoxylate cycle is impaired [4]. Besides its basic metabolic function, carnitine has been assigned additional roles in recent years, all connected with handling inadequate cellular environments. In bacteria carnitine can accumulate at high levels within the cell without altering cytoplasmic functions and acts as an osmolyte by helping cells to deal with chilling induced [5] or salt induced [6] osmotic stress. It is also suggested that carnitine could take part in the FA intracellular transport essential to the process of membrane lipid adjustment in the case of chill stress [5]. In mammals, linked to its involvement in FA metabolism, carnitine plays a detoxifying role by facilitating tissue excretion, as carnitine esters, of drug-derived FA that cannot be metabolised by the cell [7]. In addition carnitine has been assigned antioxidant properties in both fungi [8] and mammals [9,10].
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The discovery of carnitine (acetyl-, octanoyl- or palmitoyl-) transferase activities in preparations of mitochondria and chloroplasts from pea or mung-bean seedlings [11e16] and the quantification of acylcarnitines alongside free carnitine in several plant species (Arabidopsis thaliana, tobacco, rapeseed and flax) [17] demonstrate the involvement of carnitine in plant lipid metabolism. This result is coherent with the probable existence of carnitine transporters in plant cells such as the A. thaliana plasma membrane transporter OCT1 [18]. The link between carnitine and FA appears to be partly inherent to a mitochondrial b-oxidation process in plant cells [19,20] connected to some specific metabolic requirements notably for chloroplast maturation during greening of the emerging plumules of pea seedlings [21e24]. Finally as observed in animals, fungi and bacteria we recently showed that carnitine confers tolerance to abiotic stress in plants, enhancing the development and recovery of A. thaliana seedlings in conditions of salt and oxidative stress [25]. In animal species carnitine is mainly provided by food but can also be synthesized in the liver, kidney and brain (for review see Ref. [26]). The precursor of carnitine is the 6-N-trimethyllysine (TML) derived from lysosomal or proteasomal protein degradation, leading to carnitine in a four-step pathway (Fig. 1). This compound is first hydroxylated by TML dioxygenase (TMLD; EC 1.14.11.8) and the resulting 3-hydroxy-6-N-trimethyllysine (HTML) is cleaved by HTML aldolase (HTMLA; EC 4.1.2.X) into 4-N-trimethylaminobutyraldehyde (TMABA) and glycine. TMABA dehydrogenase (TMABADH; EC 1.2.1.47) transforms next TMABA into 4-Ntrimethylaminobutyrate (g-butyrobetaine, g-BB). Finally g-BB is hydroxylated by g-BB dioxygenase (BBD; EC 1.14.11.1) to produce carnitine. The fungi Neurospora crassa and Candida albicans do produce carnitine using a similar biosynthetic pathway to mammals [27,28]. To date there is no available datum on the
synthesis of carnitine in plants. The presence of endogenous carnitine in axenic cultures shows that plants synthesize carnitine, not excluding that on soil, carnitine can come partly from rhizosphere interactions. Elucidation of the carnitine biosynthetic pathway is crucial for establishing its biological significance in plants, by giving us clues about its regulation and by allowing approaches of reverse genetic. 2. Results 2.1. Detection and quantification of likely precursors of carnitine in A. thaliana We first checked by LC-ESI-MS/MS for the presence of the two known carnitine precursors TML and g-BB for which commercial standards are available. We found that both compounds are present in extracts from 7-day-old A. thaliana seedlings alongside carnitine, as shown on Fig. 2A. A clear signal is obtained for the MRM transitions selected according to the fragmentation patterns for TML (189 > 84), for g-BB (146 > 87) and for carnitine (162 > 85 and 162 > 103). Quantifications by adding internal standards show that the three metabolites are present in similar amounts, in the picomole range per mg of dry weight, and with a higher content for TML (Table 1). 2.2. Monitoring the incorporation of deuterium labelled TML Based on this initial result, we checked if carnitine synthesis truly starts from TML in A. thaliana as it does in mammal and fungal cells. We synthesized [2H9]-TML and seeds were sown on MSM supplemented with 1 mM of the deuterated compound. We checked for deuterium incorporation in g-BB and carnitine by
NH O
N
OH
NH O
N OH
OH
O
N
O
N
OH
O
N OH
OH
Fig. 1. Carnitine biosynthesis pathway in mammals. From Ref. [26]. TML is released from lysosomal or proteosomal protein degradation and converted into carnitine in a four-step pathway. PLP: pyridoxal 50 -phosphate; TMLD: TML dioxygenase; HTMLA: HTML aldolase; TMABADH: TMABA dehydrogenase; BBD: g-BB dioxygenase.
S. Rippa et al. / Plant Physiology and Biochemistry 60 (2012) 109e114
A
MRM of 6 Channels ES+ 189.18 > 83.96 1.58e4
6.17
100
111
B 2
[ H9]-TML
TML %
%
19
2.00
4.00
6.00
8.00
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Time
MRM of 6 Channels ES+ 146.1 > 87 1.35e4
5.27
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0
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Time
MRM of 6 Channels ES+ 155.1 > 87 6.87e4 2
%
2.00
4.00
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MRM of 6 Channels ES+ 162.2 > 85.1 6.49e3
5.11
100
4
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4.00
6.00
8.00
10.00
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Time
MRM of 6 Channels ES+ 171.2 > 85.1 4.93e3
5.18
100
2
[ H9]-carnitine
carnitine %
%
2.00
4.00
6.00
8.00
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Time
MRM of 6 Channels ES+ 162.2 > 103.1 8.64e3
5.15
100
61
2.00
4.00
6.00
8.00
10.00
12.00
Time
MRM of 6 Channels ES+ 171.2 > 103.1 5.99e3
5.31
100
2
[ H9]-carnitine
carnitine %
33
10.00
[ H9]-γ-BB
%
44
8.00
5.18
100
γ-BB
22
MRM of 6 Channels ES+ 198.18 > 83.96 3.85e6
6.12
100
%
2.00
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6.00
8.00
10.00
12.00
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50
2.00
4.00
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8.00
10.00
12.00
Time
Fig. 2. LC-ESI-MS/MS chromatograms of an extract obtained from 7-day-old A. thaliana seedlings grown on normal MSM (A) and on 1 mM [2H9]-TML supplemented MSM (B). MRM transitions of TML (189 > 84), [2H9]-TML (198 > 84), g-BB (146 > 87), [2H9]-g-BB (155 > 87), carnitine (162 > 85 and 162 > 103) and [2H9]-carnitine (171 > 85 and 171 > 103) are shown. Retention times are given on top of every specific signal.
LC-ESI-MS/MS on extracts obtained from [2H9]-TML-treated 7-dayold seedlings and from 7-day-old control seedlings grown on normal MSM. Results show that [2H9]-TML is efficiently taken-up by the A. thaliana seedlings during their development on [2H9]-TMLsupplemented medium (Fig. 2B). The corresponding extract contains a compound that we identify as [2H9]-TML since it presents a mass increment of 9 Da as compared to non-deuterated TML Table 1 Content of TML, g-BB and carnitine in 7-day-old A. thaliana seedlings grown on MSM. Compound
Content (pmol mg DW1)
TML g-BB Carnitine
74.5 13.1 18.5 2.5 1.72 0.6
Each value is given as mean standard deviation of results obtained from three independent experiments.
(198 Da versus 189 Da), fragments in the same way (fragment of 84 Da) and shows an identical retention time (RT) of 6 min in our HPLC conditions. The quantification has confirmed an endogenous concentration of 4890 pmol DW1 of [2H9]-TML. It appears that part of [2H9]-TML is metabolised into [2H9]-g-BB and [2H9]-carnitine since both molecules are present in the [2H9]-TML-treated plant extract as shown on Fig. 2B. The compound that we identify as [2H9]-g-BB presents an identical RT as the non-deuterated g-BB, and fragments into a 87 Da derivative that gives a signal at transition 155 > 87 similarly to the non-deuterated g-BB fragmentation (146 > 87). More interestingly [2H9]-carnitine is observable at two transitions 171 > 85 and 171 > 103 corresponding to the same fragmentation as non-deuterated carnitine, but with a mass increment of 9 Da and an identical retention time of 5 min. The presence of [2H9]-carnitine was further verified by performing its positive ESI-MS m/z (mode daughter) spectrum (Fig. 3). The mass pattern reveals daughter ions of m/z ¼ 69 corresponding to the typical
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S. Rippa et al. / Plant Physiology and Biochemistry 60 (2012) 109e114
A
8.41e6
102.8
100
H3C
84.9 59.9
%
CH3
OH
+
N
CH3
42.8
OH
O
162.1
56.8
101.6
0
B
103.0
100
69.0
3.15e3
2
2
H3C
3. Discussion
CH3
OH
+
N
2
CH3
OH
O 171.3
%
84.9
0 40
60
80
100
120
content was of 0.9 pmol mg DW1 and the carnitine content of 6.1 pmol mg DW1 (the ratio [2H9]-carnitine/carnitine was 1/7). In the plant sample grown on 0.5 mM [2H9]-TML and 0.5 mM TML the [2H9]-carnitine fell to 0.3 pmol mg DW1 whereas the unlabeled carnitine content was of 22.8 pmol mg DW1 (the ratio [2H9]carnitine/carnitine was 1/76). To note that the plant extract from seedlings grown on MSM contained 7.2 pmol mg DW1 of carnitine and no [2H9]-carnitine. In parallel, g-BB was supplied at 0.5 mM at the same time as [2H9]-TML at 1 mM. In this plant extract [2H9]-carnitine was undetectable (Table 2). However this last result must be considered more precautiously due to a highly deleterious effect on seedling development of exogenous g-BB at this concentration, similar but more pronounced than the TML effect.
140
160
m/z 180
Fig. 3. Positive ESI-MS m/z (mode daughter) spectra of carnitine and [2H9]-carnitine extracted from 7-day-old A. thaliana seedlings grown on 1 mM [2H9]-TML supplemented MSM. (A) Carnitine ESI-MS spectrum: parent ion m/z 162.1 and daughter ion m/z 59.9 corresponding to the trimethylammonium fragment are surrounded in black; (B) ESI-MS spectrum of [2H9]-carnitine: parent ion m/z 171.3 and daughter ion m/z 69 corresponding to the trimethylammonium fragment, with a mass increment of 9 Da as compared to (A), are circled in green. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
unlabeled carnitine daughter ion of m/z ¼ 60 generated by the trimethylammonium fragment with a mass increment of 9 Da due to the deuteriums. As expected the deuterium labeled compounds were not detected in the control extract (data not shown). An additional experiment was carried out in order to strengthen the above findings. [2H9]-TML was supplied at the same time as unlabeled TML (0.5 mM each) and compared, in terms of [2H9]carnitine synthesis, to a 1 mM supply of [2H9]-TML alone. This experiment was performed during the first 3 days of seedling development and not on 7-day-old seedlings as for the previous experiment, in order to optimize our results since we measured a higher carnitine content in 3-day-old seedlings. Moreover, after 3 days of development a negative effect of TML was observed through a slowdown of shoot and root growth, and through the visible accumulation of anthocyanin in leaves, reflecting the stress suffered by the seedlings. As expected the [2H9]-TML dilution with TML led to a decrease of [2H9]-carnitine content in the seedlings as compared to seedlings receiving [2H9]-TML only (Table 2). In the plant sample grown on 1 mM [2H9]-TML, the [2H9]-carnitine
Table 2 Content of carnitine and [2H9]-carnitine in 3-day-old A. thaliana seedlings grown on different growth mediums for isotope dilution experiments. Growth medium
[2H9]-carnitine (pmol mg DW1)
Carnitine (pmol mg DW1)
MSM 1 mM [2H9]-TML 0.5 mM [2H9]-TML/0.5 mM TML 1 mM [2H9]-TML /0.5 mM g-BB
Undetectable 0.9 0.3 Undetectable
7.2 6.1 22.8 10.5
The presence of TML and g-BB in A. thaliana has been a first indication that both compounds could be precursors for carnitine synthesis in plants, as in mammals and fungi. Moreover, our quantifications reveal that in A. thaliana, TML and g-BB are present in higher amounts than carnitine, a situation very different than in mammals. In mouse for instance, in organs that synthesize carnitine such as liver, kidney and brain, the carnitine content can be 25e70 fold higher than TML, and 8e40 fold higher than g-BB, depending on the organ [29]. This indicates that the regulation and/or the flux through plant carnitine biosynthesis is very different than in mammal. The results also reveal that an exogenous supply of 1 mM TML does not lead to any measurable increase of endogenous carnitine. Therefore, exogenous TML is not a good substrate for carnitine synthesis in A. thaliana. Considering that our work is carried out on a whole organism, it is possible that most part of exogenous TML does not reach the site of carnitine synthesis at the scale of the tissue or of the organelle. This explains the low amounts of [2H9]-carnitine synthesized in the seedlings despite a highly concentrated supply of [2H9]-TML. On this point, if TML was efficiently metabolized into carnitine, we probably would not observe a deleterious effect on seedling development, considering that carnitine is a compatible solute that can accumulate in the plant at very high concentrations without affecting plant development [25]. At this stage of our knowledge, we cannot explain the toxicity of TML and g-BB that are compounds with a quaternary ammonium moiety like compatible solutes such as glycinebetaine and carnitine. This signifies that despite a chemical proximity, all betaines are not trivial to cellular functions, probably depending on the nature and the strength of their interaction with metabolism and/or signalling pathways. The detection of [2H9]-g-BB and [2H9]-carnitine alongside endogenous [2H9]-TML clearly proves that as in animal and fungal cells, trimethyllysine is a precursor of carnitine in A. thaliana cells. Regarding the dilution experiment, the absence of linearity between the two-fold dilution of [2H9]-TML and the resultant tenfold reduction of [2H9]-carnitine content, could reflect the activity of a non-MichaeliseMenten enzyme for which substrate affinity would increase non linearly with substrate concentration. Moreover, the lowering effect of the supply of unlabelled g-BB on the synthesis of [2H9]-carnitine from [2H9]-TML tends to confirm that g-BB is an intermediate in the carnitine biosynthesis pathway from TML. Through those results, it can be expected that similar enzymatic activities, performed by homologous enzymes, could control carnitine synthesis in fungi, mammals and plants. The identification of the enzymes belonging to the carnitine biosynthetic pathway will allow reverse genetic approaches to investigate further the physiological roles of carnitine in plants. We have
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already performed BLASTP analyses using the human or fungal protein sequences to identify the probable enzymes implicated in A. thaliana carnitine synthesis and have found good candidates for step 2 and 3 (Fig. 1). It is noteworthy that we did not find any significant sequence homology between the mammalian or fungal enzymes implicated in steps 1 and 4 and the proteins found in the A. thaliana database. This result suggests that the two hydroxylation steps might be performed by different enzymes in plants and in fungi and mammals. However, feeding of A. thaliana seedlings with THP (3-(2,2,2-trimethylhydrazine)propionate), a well characterized inhibitor of the mammalian g-BB dioxygenase [30], has led to an average 50% decrease of the endogenous carnitine, for exogenous supplies ranging from 500 mM to 1 mM THP (data not shown). This result suggests that despite the absence of sequence homology between plants and mammals, the active sites of the mammalian g-BB dioxygenase and of the corresponding plant enzyme might share similar conformations. Concerning step 2, the human HTML aldolase (HTMLA) might be identical to the cytosolic serine hydroxymethyltransferase (SHMT; EC 2.1.2.1) but this remains to be established [26,31]. SHMT are PLP-dependent enzymes that catalyse the reversible conversion of serine and tetrahydrofolate (THF) to glycine and 5,10-methylene THF [32]. A. thaliana genome encodes 7 SHMTs that display high sequence similarity with the human cytosolic SHMT1 (identity of 58% and similarity of 74% regarding the A. thaliana SHMT1 for instance). Although this A. thaliana protein (also called SHM1) has been implicated in the photorespiratory pathway [33], we do not exclude the possible role of an A. thaliana SHMT in converting HTML into TMABA when considering the homology between A. thaliana and human SHMT proteins. Recently the complete carnitine biosynthesis pathway has been identified and characterized in C. albicans and a threonine aldolase has been proposed to be the main HTMLA in this organism [28]. Threonine aldolases (EC 4.1.2.5) are PLPdependent enzymes that convert threonine to acetaldehyde and glycine. A BLASTP search with the fungal protein sequence has identified the A. thaliana threonine aldolase 1 and 2 (THA1 and THA2) as the most likely homologues (sequence identity of 36% and 37% respectively). These two proteins are implicated in plant amino acid metabolism and particularly in glycine and acetaldehyde formation [34]. The third step in human carnitine formation is realized by an aldehyde dehydrogenase (ALDH) named TMABADH. In A. thaliana the ALDH gene superfamily contains 14 sequences distributed in 9 families, implicated in cytoplasmic male sterility, plant defence and abiotic stress tolerance [35]. Among them, family 2 and 10 are closely homologous to the human TMABADH (identity of approximately 37e39%). Interestingly it has been shown recently that in Pisum sativum two aminoaldehyde dehydrogenases belonging to the large ALDH superfamily can oxidize N,N,N-trimethyl-4-aminobutyraldehyde (TMABA) into g-BB [36]. A. thaliana ALDH10A8 and 10A9, belonging to the family 10 of the ALDH superfamily, are quite similar to these pea proteins (more than 70% of identity). Recent results show that in A. thaliana, the peroxisomal ALDH10A9 protein possesses betaine aldehyde, 4Aminobutyraldehyde (ABAL) and 3-Aminopropionalehyde (APAL) dehydrogenase in vitro activities [37]. The A. thaliana ALDH10 proteins could therefore be also able to convert TMABA into g-BB. The availability of knockout mutants for some of the candidate enzymes and the possibility of finding more KO mutants from the A. thaliana T-DNA insertion libraries will allow us to investigate further the involvement of the candidate enzymes in the plant carnitine biosynthetic pathway. In the present paper we report data showing that plants employ similar precursors to mammals and fungi to synthesize carnitine, bringing the first evidence that the carnitine biosynthetic pathways in those eukaryotic organisms share similar features, and most
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probably involve similar enzymatic activities. This result makes it now possible to identify the genes involved in carnitine biosynthesis in plants. Protein homology searches will allow notably reverse genetic approaches that will facilitate studies of the roles that carnitine plays in plants, and new insights into comparative physiology between plant and animal kingdoms. 4. Methods 4.1. [2H9]-TML synthesis We synthesized deuterium labeled 6-N-trimethyllysine according to the method described in Ref. [38], from L-lysine HCl (Merck, ref. K15007900) and using [Me-2H6]dimethyl sulfate (Sigma, ref. 164526) as methyl donor. The [2H9]-TML was purified twice on a 4-ml Dowex 50WX8 (Serva) column, washed with 20 volumes of milli-Q grade water and eluted with 120 ml of 2 mol/L NH4OH. Ammonia was evaporated under a flux of nitrogen at room temperature. Then the effluent was concentrated by lyophilisation. A sterile one molar [2H9]-TML stock solution was prepared in 70% ethanol. The absence of lysine in the solution was checked by LCESI-MS/MS. 4.2. Plant culture and treatment A. thaliana (L.) Heynh, ecotype Columbia (Col-0), was used in this study. For aseptic growth, seeds were first surface-sterilized and sown under sterile conditions on Murashige and Skoog medium (MSM) containing 8% agar [39]. They were placed at 4 C for 3 days for stratification. Seedlings were subsequently grown at 24 C under long day conditions (16-h-light cycle at 150 mE m2 s1 illumination) for three or seven days before being frozen in liquid N2 for subsequent extraction. One molar stocks of TML, g-BB (Sigma ref. T1660 and 403245 respectively) and [2H9]-TML prepared in 70% ethanol or filter sterilized were directly added in the MSM to desired concentrations for plant treatments. The pH of the medium was adjusted to 5.8 when necessary. 4.3. Analysis of TML, g-BB and carnitine by tandem mass spectrometry The extractions of carnitine and its precursors were performed according to Ref. [17] with some modifications. Standards for TML, g-BB and L-carnitine were purchased from Sigma (ref. T1660, 403245 and C0158 respectively). The equipment consists in a Famos autosampler and an Ultimate 3000 LC Packing capillarity HPLC (Dionex) combined to a Quattro Micro triple quadruple mass spectrometer (Waters). Approximately 200 mg of seedlings were used for extractions performed in 700 ml of acetonitrile:water (8:2, v:v). The HPLC separation was carried out on a 5 mm Luna C8(2) 150 mm 0.5 mm capillarity column (Phenomenex) maintained at 35 C. The elution gradient was performed according to Ref. [40] using water (A) and methanol (B) as mobile phases. Both A and B contained ammonium acetate (10 mM) and heptafluorobutyric acid (10 mM). The gradient started with 10% B, raised to 100% in 20 min and lowered to 10% from 20 to 25 min. The flow was set at 8 ml/min and the injected sample volume was 1 ml. Collision energy of 20 eV was set using standards. Standards were used to assess transitions 189 > 84 for TML, 146 > 87 for g-BB, 162 > 85 and 162 > 103 for carnitine. Plant extracts were diluted 5 times in methanol:water (80:20) when analysing TML and [2H9]-TML due to the high content of TML in plant tissues. Quantification of TML, g-BB and carnitine was performed by measuring the area under each specific peak using MassLynx 4.0 (Waters). Samples were first run without an internal standard, secondly with a standard at known
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concentration and finally with a standard at a double concentration. A linear regression permitted quantification of the concentration of samples. Acknowledgments We gratefully thank Professor Béla Melegh for his useful information on the synthesis and purification of deuterium labeled TML. We are grateful to Professor Stephan Krähenbühl for having offered us the THP compound. We also would like to thank Mr Michael Jones for careful reading of the manuscript. References [1] J. Kerner, C. Hoppel, Fatty acid import into mitochondria, BBA Mol. Cell Biol. Lipids 1486 (2000) 1e17. [2] F. Fraser, V.A. Zammit, Submitochondrial and subcellular distributions of the carnitine-acylcarnitine carrier, FEBS Lett. 445 (1999) 41e44. [3] F.B. Stephens, D. Constantin-Teodosiu, P.L. Greenhaff, New insights concerning the role of carnitine in the regulation of fuel metabolism in skeletal muscle, J. Physiol. 581 (2007) 431e444. [4] C.W.T. Van Roermund, E.H. Hettema, M. Van Den Berg, H.F. Tabak, R.J.A. Wanders, Molecular characterization of carnitine-dependent transport of acetyl-CoA from peroxisomes to mitochondria in Saccharomyces cerevisiae and identification of a plasma membrane carnitine transporter, Agp2p, EMBO J. 18 (1999) 5843e5852. [5] A.S. Angelidis, G.M. Smith, Role of the glycine betaine and carnitine transporters in adaptation of Listeria monocytogenes to chill stress in defined medium, Appl. Environ. Microbiol. 69 (2003) 7492e7498. [6] M. Canovas, V. Bernal, A. Sevilla, J.L. Iborra, Salt stress effects on the central and carnitine metabolisms of Escherichia coli, Biotechnol. Bioeng. 96 (2007) 722e737. [7] E. Arrigoni-Martelli, V. Caso, Carnitine protects mitochondria and removes toxic acyls from xenobiotics, Drugs Exp. Clin. Res. 27 (2001) 27e49. [8] J. Franken, S. Kroppenstedt, J.H. Swiegers, F.F. Bauer, Carnitine and carnitine acetyltransferases in the yeast Saccharomyces cerevisiae, a role for carnitine in stress protection, Curr. Genet. 53 (2008) 347e360. lu, H. Satiroglu, I. Alican, A. Kaçmaz, A. Sakarcan, L[9] G. Sener, K. Paskalog carnitine ameliorates oxidative damage due to chronic renal failure in rats, J. Cardiovasc. Pharmacol. 43 (2004) 698e705. [10] D. Silva-Adaya, V. Pérez-De La Cruz, M.N. Herrera-Mundo, K. Mendoza-Macedo, J. Villeda-Hernández, Z. Binienda, S.F. Ali, A. Santamaría, Excitotoxic damage, disrupted energy metabolism, and oxidative stress in the rat brain: antioxidant and neuroprotective effects of L-carnitine, J. Neurochem. 105 (2008) 677e689. [11] C. Wood, M. Noh Hj Jalil, A. Ariffin, B.C.S. Yong, D.R. Thomas, Carnitine shortchain acyltransferase in pea mitochondria, Planta 158 (1983) 175e178. [12] C. Wood, M. Noh Hj Jalil, I. McLaren, B.C.S. Yong, A. Ariffin, P.H. McNeil, N. Burgess, D.R. Thomas, Carnitine long-chain acyltransferase and oxidation of palmitate, palmitoyl coenzyme A and palmitoylcarnitine by pea mitochondria preparations, Planta 161 (1984) 255e260. [13] H. Gerbling, B. Gerhardt, Carnitine-acyltransferase activity of mitochondria from mung-bean hypocotyls, Planta 174 (1988) 90e93. [14] H. Schwabedissen-Gerbling, B. Gerhardt, Purification and characterization of carnitine acyltransferase from higher plant mitochondria, Phytochemistry 39 (1995) 39e44. [15] C. Masterson, C. Wood, Pea chloroplast carnitine acetyltransferase, Proc. Biol. Sci. 267 (2000) 1e6. [16] C. Masterson, C. Wood, Carnitine palmitoyltransferases in pea leaf chloroplasts: partial purification, location, and properties, Can. J. Bot. 78 (2000) 328e335. [17] B. Bourdin, H. Adenier, Y. Perrin, Carnitine is associated with fatty acid metabolism in plants, Plant Physiol. Biochem. 45 (2007) 926e931.
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