Carnitine is associated with fatty acid metabolism in plants

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Plant Physiology and Biochemistry 45 (2007) 926e931 www.elsevier.com/locate/plaphy

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Carnitine is associated with fatty acid metabolism in plants Benoıˆte Bourdin, Herve´ Adenier, Yolande Perrin* UMR-CNRS 6022 Ge´nie Enzymatique et Cellulaire, Centre de Recherche Royallieu, Universite´ de Technologie de Compie`gne, BP 20529, 60205 Compie`gne Cedex, France Received 23 March 2007 Available online 29 September 2007

Abstract The finding of acylcarnitines alongside free carnitine in Arabidopsis thaliana and other plant species, using tandem mass spectrometry coupled to liquid chromatography shows a link between carnitine and plant fatty acid metabolism. Moreover the occurrence of both mediumand long-chain acylcarnitines suggests that carnitine is connected to diverse fatty acid metabolic pathways in plant tissues. The carnitine and acylcarnitine contents in plant tissues are respectively a hundred and a thousand times lower than in animal tissues, and acylcarnitines represent less than 2% of the total carnitine pool whereas this percentage reaches 30% in animal tissues. These results suggest that carnitine plays a lesser role in lipid metabolism in plants than it does in animals. Ó 2007 Elsevier Masson SAS. All rights reserved. Keywords: Carnitine; Lipid metabolism; Plant development; Arabidopsis thaliana; Mass spectrometry

1. Introduction Carnitine, a quaternary ammonium amino acid, occurs in the microbial, plant and animal kingdoms, and is involved in energy metabolism, hormonal action and adaptation to stress. Although its presence in plant tissue has been reported five decades ago [1,2] only one review was published since then on its possible metabolic significance [3]. Carnitine is present in the tissues and organs of several species including cereals and legumes, in dry and germinating seeds, and in leaves (for review see Ref. [3]). Enzymatic activities related to carnitine transfer on acyl moieties have also been measured in plant tissues and localised in mitochondria [4,5] and plastids [6,7]. The recent identification and characterisation of an Arabidopsis thaliana knockout mutant for a putative carnitine transmembrane carrier (BOU) suggest that carnitine may have a much more critical role than expected Abbreviations: CAT, carnitine acyltransferase; CACT, carnitine acylcarnitine translocase; DW, dry weight; LACS, long-chain acyl-CoA synthase; LCFA, long-chain fatty-acid; MRM, multiple reaction monitoring; HPLC, high performance liquid chromatography; RT, retention time. * Corresponding author. Tel.: þ33 344 234 416; fax: þ33 344 203 910. E-mail address: [email protected] (Y. Perrin). 0981-9428/$ - see front matter Ó 2007 Elsevier Masson SAS. All rights reserved. doi:10.1016/j.plaphy.2007.09.009

[8]. BOU is similar to the mitochondrial carnitine/acylcarnitine translocases from animal and yeast. The Arabidopsis bou knockout mutant shows impaired post-germinative growth linked to a deficiency in the use of fatty acid degradation products. In mammals, carnitine is known for its involvement in fatty acid metabolism, for the peroxisomal to mitochondrial trafficking of activated fatty acids destined to b-oxidation [9]. The carnitine shuttle system involves carnitine acyltransferases (CAT), which allows the reversible exchange of coenzyme A and carnitine onto fatty acids, and carnitine/acylcarnitine translocases (CACT), in charge of the transport of carnitine esters across intracellular membranes. Carnitine has been ascribed additional roles in animals such as in coenzyme-A homeostasis, energy storage in the form of acetylcarnitine, secretion of poorly metabolised acyl residues and regulation of hormonal action [10]. In yeast, a carnitine shuttle system takes part in an alternative pathway of fatty acid catabolism that bypasses the glyoxylate cycle, allowing the peroxisomal to mitochondrial trafficking of activated acetate to feed the Krebs cycle [11]. Hypothesising a comparable metabolic role in plants as in mammals and yeast, we have initiated studies on the possible

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input of carnitine in the metabolism of fatty acids as metabolic energy source or building blocks of membrane lipids. Studying carnitine-dependent metabolism on the model plant Arabidopsis thaliana could answer remaining questions in the area of fatty acid metabolism, such as the way they circulate between the different cellular compartments involved in their biosynthesis, modification and utilization. Analytical methods used so far to assess carnitine in plants, such as Tenebrio bioassay [1] DTNB spectrometric assay [12] and radioactive assay [13] have enabled the detection and quantification of free carnitine. Knowing the state of the carnitine pool in plants is of key importance to define its role in plant physiology and in this regard the detection of acylcarnitines is of prime interest. In order to precisely identify and quantify carnitine and acylcarnitines in plants we have adapted a method used for carnitine quantification on human fluids and tissues. This method is based on high performance liquid chromatography coupled to electrospray tandem mass spectrometry (LC-MS/MS) which is today considered to be the most sensible and accurate way of quantifying carnitine and acylcarnitines [14e16]. This article describes primary evidence of the presence of acylcarnitines alongside free carnitine in Arabidopsis and other species opening a new field of research regarding plant lipid metabolism. 2. Materials and methods 2.1. Plant material and cultivation The study included Arabidopsis thaliana (ecotype Columbia), Nicotiana tobaccum (tobacco, cultivar SR1), Linum usitatissinum (flax, cultivar Barbara) and Brassica napus (rapeseed, cultivar Westar RV31). Seeds were surface sterilised before in vitro cultivation that was performed on solid Murashige and Skoog medium [17] without sucrose (MS medium). Low nitrogen MS medium was made according to [18]. Mature seeds were invariably imbibed and stratified on MS medium at 4  C for 4 days. Culture conditions were of 22  C temperature, 50% relative humidity, 150 mmole/m2 per s light intensity and 16 h photoperiod. 2.2. Analysis of carnitine and acylcarnitines by tandem mass spectrometry Carnitine quantifications were performed according to Refs. [15,16] with some modifications. The equipment consists in a Famos autosampler and an Ultimate 3000 LC Packing capillary HPLC (Dionex) combined to a Quattro Micro triple quadruple mass spectrometer (Waters, France). In vitro seedlings were ground under liquid nitrogen. One mL of acetonitrile/water (8/2, v/v) was added to the tissue powder obtained from 50 to 100 mg seedlings. The suspension was sonicated for 30 s before being centrifuged at 16,000  g for 10 min at 4  C. The supernatant was evaporated under nitrogen at 42  C. The dry residue was stored at 20  C and taken up in 50e100 ml of methanol/water (8/2, v/v) on the

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day of analysis. The analysis was carried out on the aqueous methanol supernatant after centrifugation at 16,000  g for 10 min at 4  C. The HPLC separation was performed on a 150  0.5 mm ˚ pore size) Biobasic-4 C4 capillary (5 mm particules, 300 A column (ThermoElectron Corporation, France) maintained at 35  C. The elution gradient was performed according to Ref. [14] 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 10 min and lowered to 10% from 10 to 25 min. The flow was set at 8 mL/min and the injected sample volume was 1 mL. Mass spectrometry analysis was performed in the positive MRM mode. Collision energies, of 20 eV for carnitine, acetyl- and medium chain acylcarnitines and of 27 eV for long chain acylcarnitines, were set using standards. Standards were L-carnitine, O-acetyl-L-carnitine and racemic DL form for octanoylcarntine, decanoylcarnitine, lauroylcarnitine, myristoylcarnitine, palmitoylcarnitine, stearoylcarnitine and arachidoylcarnitine, all purchased from Sigma. L-linoleoylcarnitine was from Larodan AB (Sweden). Standards were used to assess transitions: 162 > 85 and 162 > 103 for carnitine, 204 > 85 and 204 > 145 for acetylcarnitine, 288 > 85 for octanoylcarnitine, 316 > 85 for decanoylcarnitine, 344 > 85 for lauroylcarnitine, 372 > 85 for myristoylcarnitine, 400 > 85 for palmitoylcarnitine, 424 > 85 for linoleylcarnitine, 428 > 85 for stearoylcarnitine and 456 > 85 for arachidoylcarnitine. Quantification of carnitine and acylcarnitines was done by measuring the area under each specific peak using MassLynx 4.0 (Waters, France). Samples were run without internal standards (peak area A), then with a mix of standards at known concentrations (peak area As, concentration Cs). The equation ‘‘C ¼ A  Cs/(As  A)’’ was used to calculate the concentration of carnitine and acylcarnitines in the sample solutions. Each data on carnitine and acylcarnitine contents is expressed as a mean and standard error calculated from at least 3 experiments performed independently. 2.3. Fatty acid analysis by gas chromatography Total lipids were extracted from 500 mg of 6-day-old seedlings using Folch method [19]. Following trans-methylation for 1 h at 70  C with a mixture containing methanol/ sulphuric acid (100/2.5, v/v) fatty acid methyl esters were extracted by heptane. Their quantification was performed on a Hewlett Packard 5890 capillary gas chromatograph with flame ionization detector, equipped with a capillary column CpWax 52 B (VARIAN) (30 m  0.32 mm, 0.50 mm ID dimensions). Hydrogen was used as carrier gas (pressure in the cavity was 0.7 bar). Following injection, the oven temperature was raised from 120  C to 240  C at 10  C/min and held at this temperature for 10 min. Quantitative determinations of fatty acid methyl esters were based on the response of external standard solution (Fatty Acid Methyl Ester Mix, FAME, Supelco).

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B. Bourdin et al. / Plant Physiology and Biochemistry 45 (2007) 926e931

3. Results The extraction method was efficient in recovering simultaneously highly polar carnitine and less polar acylcarnitines from plant materials. Three independent experiments led to over 96% recovery when standards, ranging from carnitine to arachidoylcarnitine, were added to plant tissue samples at the beginning of the extraction procedure. Previous assays were carried out using separate extraction methods for polar carnitine and short-chain acylcarnitines (based on amino acid extraction procedure) and for less polar medium- and long-chain acylcarnitines (based on Folch lipid extraction method). We chose the single extract method not only because it is less time-consuming and requires less plant tissue, but also because of a slightly better efficiency in the recovery of medium-chain acylcarnitines. HPLC conditions give a good partition except for carnitine and acetylcarnitine that show very close retention times on the capillary column (Fig. 1). The selected MRM transitions allow excellent detection on standards and plant samples. A nonspecific signal is seen on the channel corresponding to palmitoylcarnitine but at a retention time (RT) very distinct from the standard. The same phenomenon occurs on the channel of octanoylcarnitine, decanoylcarnitine and myristoylcarnitine (not shown). Because these unknown compounds show very distinct RT from the carnitine esters their presence does not lower the quality of the analyses. Detection sensitivity is lower for carnitine and acetylcarnitine than for medium-chain and long-chain acylcarnitines. Transition 162 > 103 gives a slightly better signal than transition 162 > 85 for carnitine. Carnitine and acetylcarnitine in plant extracts (endogenous compounds and added standards) present a shorter RT than the corresponding standards in methanol/water (8/2, v/v) indicating that plant extracts interfere with the retention of carnitine and acetylcarnitine on the capillary column. Analyses carried out on Arabidopsis seedlings show that free carnitine is present in nanogram amounts per mg dry weight (Fig. 2A). Our results are in accordance with the published results about the quantification of free carnitine in plant tissues, using the DTNB spectrometric assay and showing levels ranging from 1 ng/mg DW (peanut seed) to 48 ng/mg DW (avocado mesocarp) [12]. Carnitine content increases during the first stage of seedling development. It tends to be unevenly distributed in six-day-old seedlings, with a slightly higher content in roots than in rosettes. The global content of quantifiable acylcarnitines averages 20e30 pg per mg dry weight (Fig. 2B). No significant change of this content is observed during seedling growth. As for carnitine, the roots contain slightly more acylcarnitines than the rosettes in six-day-old seedlings. An analysis performed on two-week-old greenhouse seedlings gave results similar to in vitro seedlings (6 ng/mg DW for carnitine and 38 pg/mg DW for the quantifiable acylcarnitines). The pool of carnitine esters is mainly made of long-chain acylcarnitines, predominantly palmitoylcarnitine, stearoylcarnitine and linoleylcarnitine (Fig. 2C) which reflects the predominance of long chain fatty acids (LCFA) in the corresponding plant material (Fig. 2D). Acetylcarnitine is not

detected in the majority of plant extracts. However its detection in some rare cases, as shown in Fig. 1B, suggests that acetylcarnitine occurs in Arabidopsis seedlings but at levels close to the detection threshold. Transitions corresponding to oleoylcarnitine (426 > 85), linolenylcarnitine (422 > 85) and eicosenoylcarnitine (454 > 85) have been included in our analyses despite the unavailability of standards. In most plant extracts, a compound was detected that can relate to eicosenoylcarnitine. It gives a clear signal at the transition 454 > 85 with a RT of 14.97 min, very close to arachidoylcarnitine RT. Quantification of this compound based on the arachidoylcarnitine standard gives levels similar to myristoylcarnitine. Surprisingly transitions that corresponded to oleoylcarnitine and linolenylcarnitine did not show any clear signals at retention times close to stearoylcarnitine and linoleylcarnitine. This is unexpected for linolenylcarnitine since linolenic acid is the most abundant LCFA quantified in our plant material (Fig. 2D). Three-day-old Arabidopsis seedlings grown on a modified low nitrogen MS medium that totally abolishes storage lipid utilisation [18] show a moderate increase of free carnitine content as compared to seedlings grown on normal MS medium whereas the global acylcarnitine content is not affected by such a defect in storage lipid catabolism (Table 1). Analyses on 6-day-old in vitro seedlings of rapeseed, flax and tobacco at the stage of emergence of the first pair of leaves, corresponding to 5-day-old Arabidopsis seedlings, show that free carnitine (Fig. 3 insert) and acylcarnitine contents (Fig. 3) do not significantly differ between the four species. 4. Discussion LC-MS/MS analysis has made possible for the first time, the detection and quantification of acylcarnitines alongside free carnitine in Arabidopsis and other plant species. This result is significant as it confirms the link between carnitine and fatty acid (FA) metabolism in plants that was previously suggested by acylcarnitine transferase activities measured in isolated mitochondria and plastids [3]. The increase in carnitine level measured in Arabidopsis seedlings that are impaired in their storage lipid utilisation is possibly a further indication of the connection between carnitine and lipid metabolism. As with mammals the carnitine pool in plants is mostly made of free carnitine but carnitine is also found esterified to FA of various chain lengths with predominance to LCFA. The unavailability of acylcarnitine standards hinders with an exhaustive analysis but the detection of C16:0 palmitoylcarnitine, C18:0 stearoylcarnitine, C18:2 linolenylcarnitine and C20:0 arachidoylcarnitine indicates that the acylcarnitine pool involves most if not all LCFAs present in plant cells. However, it has to be noted that, as shown for stearoylcarnitine, the relative abundance of a specific acylcarnitine does not necessarily correlates to the relative abundance of its fatty acid counterpart. The levels of free carnitine and acylcarnitines are respectively a hundred and a thousand times lower in plants than

B. Bourdin et al. / Plant Physiology and Biochemistry 45 (2007) 926e931 15.76 10188

A 100 %

Arachidoylcarnitine

0 15.54 4378

100 %

Stearoylcarnitine

0 15.26 5993

100 %

Palmitoylcarnitine

0 6.38 9593

100 %

Acetylcarnitine

0 6.26 6777

100 %

Carnitine

0

929 2: MRM of12 Channels ES+ 456.42 > 85.02 3.66e4 Area 2: MRM of12 Channels ES+ 428.4 > 85.02 1.57e4 Area

2: MRM of 12 Channels ES+ 400.3 > 85.02 2.25e4 Area

1: MRM of 4 Channels ES+ 204.36 > 85 4.49e4 Area 1: MRM of 4 Channels ES+ 162.2 > 103 3.70e4 Area

1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 12.00 13.00 14.00 15.00 16.00 17.00 18.00 19.00 15.76 631

B 100 %

Arachidoylcarnitine

Time

2: MRM of12 Channels ES+ 456.42 > 85.02 2.39e3 Area

0 15.54 446

100 %

Stearoylcarnitine

0 100 %

15.26 551

Palmitoylcarnitine

0 100

5.76 144

Acetylcarnitine

%

2: MRM of12 Channels ES+ 428.4 > 85.02 1.55e3 Area

2: MRM of 12 Channels ES+ 400.3 > 85.02 8.79e3 Area

1: MRM of 4 Channels ES+ 204.36 > 85 718 Area

0 100

5.70 1580

%

Carnitine

1: MRM of 4 Channels ES+ 162.2 > 103 7.46e3 Area

0 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 12.00 13.00 14.00 15.00 16.00 17.00 18.00 19.00

Time

Fig. 1. Example of chromatograms obtained with a mix of standards (A); and with an extract from Arabidopsis seedlings at 3 day post-imbibition (B). The mix of standard contained 20 ng/mL of each acylcarnitine and 200 ng/mL of carnitine and acetylcarnitine. MRM transitions of carnitine (162 > 103), acetylcarnitine (204 > 85), palmitoylcarnitine (400 > 85), stearoylcarnitine (428 > 85) and arachidoylcarnitine (456 > 85) are shown. Retention times and pic area values are given for every specific signal (italics).

in skeletal muscles, liver, heart and kidneys of mice [16] whereas Arabidopsis seedlings and rodent skeletal muscles present similar levels of long-chain acyl-CoA [20,21]. Moreover whereas the percentage of acylated carnitine reaches around 30% in rodent skeletal muscle, liver and plasma, our results show that in plant tissues the pool of acylcarnitines represents only 1e2% of the total carnitine content [22]. Our results indicate that plants do not require large pools of acylcarnitines for metabolic function. In mammalian cells, long-chain acylcarnitines are not only transportable form of fatty acids but, also represent a considerable stock of readily available activated acyl groups that cells use for membrane repair when lacking the energy to activate fatty acids [9]. It is possible that due to reliance on photosynthetic energy and on activated FAs from de novo synthesis plants do not need to implement such an emergency supply of activated FAs.

LCFA b-oxidation is a major energy source for the heart and skeletal muscles of mammals, a process in which carnitine plays a significant role by allowing the import of activated LCFAs within mitochondria [23]. In plants FA b-oxidation can also supply energy especially during the early germination process of oilseed species when seedlings are not yet photoautotrophous. Acylcarnitines are present in tobacco, a nonoilseed species, and in the aerial and root systems of fully photoautotrophous seedlings from all tested species. This clearly signifies that carnitine is not solely linked to LCFA b-oxidation of germinating oilseeds. Moreover the abolition of storage FA utilisation in Arabidopsis seedlings grown on low nitrogen MS medium does not result in the modification of their acylcarnitine contents. FA b-oxidation happens in non-fatty plant tissues possibly for the removal of toxic short-chain FAs and the recycling of acyl-CoA molecules

B. Bourdin et al. / Plant Physiology and Biochemistry 45 (2007) 926e931

930

B

A 14

40 35

pg/mg DW

10 8 6 4

20 15 5 0

rosettes

0-3 days

24:0

22:1

18:1

18:0

16:1

2 1

0

18:2

16:0

3

14:0

20:0

14:0

2

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8:0

10:0

6

4

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8

18:2

18:0

16:0

10

µg/mg DW

5

12

4

6

rosettes

20:1

root system rosette

14

roots

22:0

D

16

4-6 days

18:3

roots

4-6 days

0-3 days

20:0

0

ric aci d rist ic a c id Pa lmi tic Pa aci lmi d tole ic a cid Ste aric a Ole cid ic a cid Lin ole ic a cid Lin ole nic aci Ara d chi doi ca Eic cid ose noi ca Be hen cid ic a cid Eru cic Lig aci noc d eric aci d

ine

e

rnit

itin Ara

chi

doy

lca

arn

Lin ole

arn ylc aro Ste

lca toy lmi Pa

ylc

itin

e

ine rnit

niti

ne

ine

car

rnit

oyl rist My

lca roy Lau

car oyl can

De

tan

oyl

car

niti

niti

ne

ne

0

Lau

pg/mg DW

25

10

2

Oc

30

My

ng/mg DW

12

C

45

Fig. 2. Analyses of carnitine, acylcarnitines and fatty acids on in vitro Arabidopsis seedlings. Carnitine (A); and global acylcarnitine (B) contents in seedlings aged 0e3 day and 4e6 day post-imbibition, and in roots and rosettes harvested on 6-day-old plantlets. (C) Content of all detectable carnitine esters in roots and rosettes of 6-day-old plantlets. (D) Fatty acid composition of 6-day-old plantlets. Each value is given as mean  standard error of results obtained from three independent analyses.

from membrane lipid turnover [24]. Carnitine could therefore be related to FA b-oxidation of mature tissues which would explain the presence of medium-chain acylcarnitines alongside long-chain ones. The prevalence of long-chain acylcarnitines could as well indicate the involvement of carnitine in de novo lipid synthesis. LCFA b-oxidation in animal cells occurs in mitochondria whereas peroxisomes are dedicated to partial degradation of very long chain FAs [9]. Both organelles rely on the carnitine system for FA import, implementing transferases for the reversible transfer of activated acyl groups from membrane impermeable CoA to mobile carnitine, and translocases that act as carriers [9]. The precise mechanisms by which FAs transit

Table 1 Variation in the contents of free carnitine and overall quantifiable acylcarnitines between 3-day-old seedlings grown on normal MS medium and modified low nitrogen MS medium

Carnitine content (ng/mg DW) Global acylcarnitine content (ng/mg DW)

MS medium

Modified MS medium

1.1  0.2

1.7  0.3

0.021  0.003

0.017  0.002

Each value is given as mean  standard error of results obtained from three independent analyses.

in plant cells between their sites of synthesis, modification or degradation have not been fully elucidated. In plants, longchain acyl-CoAs are assumed to be imported into peroxisomes for b-oxidation by an ATP binding cassette transporter (PXA1) as in yeast cells. Still the presence of long-chain acyl-CoA synthase (LACS) activities within the peroxisomal matrix in Arabidopsis and in yeast cells raises the question of an alternative transport pathway parallel to PXA1 [20]. The details of fatty acid exportation from plastids following de novo synthesis are not well understood but, the contribution of LACS activities present on the outer envelope suggests the reactivation to acyl-CoA after passage of free FAs through the inner membrane by an unknown carrier. It is worth mentioning that with animals, LCFA import into mitochondria is thought to implicate both the activation of acyl groups by LACS in the outer membrane and the carnitine shuttle system in the inner membrane [23]. The presence of free carnitine and acylcarnitine esters raises the question of their metabolic significance in plant cells. Today no data allows us to associate with certainty the carnitine pool to FA intracellular trafficking but considering the existence of a carnitine shuttle system in the animal and yeast kingdoms, it is tempting to propose that a carnitine shuttle system, associated or not to LACS activities, could be one of the missing links in the understanding of intracellular fatty acid trafficking in plant cells.

B. Bourdin et al. / Plant Physiology and Biochemistry 45 (2007) 926e931

931

25 Carnitine content (ng/mg DW): 3.2 ± 2.1 in rapeseed 5.1 ± 3.1 in flax 2.7 ± 1.7 in tobacco

pg/mg DW

20

rapeseed flax tobacco

15

10

5

arn itin e Ara ch ido ylc

arn itin e Lin ole ylc

arn itin e Ste aro ylc

arn itin e Pa lm ito ylc

arn itin e ris toy lc My

arn itin e La uro ylc

arn itin e De ca an oy lc

Oc

tan oy lc

arn itin e

0

Fig. 3. Amounts of free carnitine (figure insert) and quantifiable acylcarnitines in rapeseed, flax and tobacco 6-day-old in vitro seedlings.

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