Molecular species composition of plant cardiolipin determined by liquid chromatography-mass

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1 Molecular species composition of plant cardiolipin determined by liquid chromatography-mass spectrometry Yonghong Zhou, Helga Peisker and Peter Dörmann Institute of Molecular Physiology and Biotechnology of Plants (IMBIO), University of Bonn, Karlrobert-Kreiten-Straße 13, Bonn, Germany Yonghong Zhou, Helga Peisker, Peter Dörmann, Corresponding author: Peter Dörmann, phone , fax , Running title: Plant Cardiolipin Abbreviations: DGDG, digalactosyldiacylglycerol; DW, dry weight; FAME, fatty acid methyl ester; MGDG, monogalactosyldiacylglycerol; MS, mass spectrometry; PC, phosphatidylcholine; PE, phosphatidylethanolamine; PG, phosphatidylglycerol; PI, phosphatidylinositol; Q-TOF, quadrupole time-of flight; SQDG, sulfoquinovosyldiacylglycerol; WT, wild type; Fatty acids are abbreviated as X:Y with X, number of carbon atoms, and Y, number of double bonds in the acyl chain. 1

2 ABSTRACT Cardiolipin (CL), an anionic phospholipid of the inner mitochondrial membrane, provides essential functions for stabilizing respiratory complexes and it is involved in mitochondrial morphogenesis and programmed cell death in animals. The role of CL and its metabolism in plants are less well understood. The measurement of CL in plants, including its molecular species composition, is hampered by the fact that CL is of extremely low abundance, and that plants contain large amounts of interfering compounds including galactolipids, neutral lipids and pigments. We used solid phase extraction (SPE) by anion exchange chromatography to purify CL from crude plant lipid extracts. Liquid chromatography-mass spectrometry was employed to determine the content and molecular species composition of CL. Thus up to 23 different molecular species of CL were detected in different plant species, including Arabidopsis, mung bean, spinach, barley and tobacco. Similar to animals, plant CL is dominated by highly unsaturated species, mostly containing linoleic and linolenic acid. During phosphate deprivation or exposure to an extended dark period, the amount of CL decreased in Arabidopsis, accompanied with an increased degree in unsaturation. The mechanism of CL remodeling during stress, and the function of highly unsaturated CL molecular species, remains to be defined. Free-form Keywords: Arabidopsis, anion exchange chromatography, solid phase extraction Supplementary Keywords: cardiolipin, lipidomics, mass spectrometry, mitochondria, phospholipids 2

3 INTRODUCTION Cardiolipin (CL) is an anionic membrane phospholipid with bipartite structure containing four acyl groups bound to two glycerol moieties which are linked via two phosphate groups to a third glycerol. CL is distributed in the cytoplasmic membrane in prokaryotes and almost exclusively located in the inner mitochondrial membrane of eukaryotes. A growing body of evidence indicates that CL is required for different mitochondrial functions. CL is involved in maintaining the mitochondrial membrane potential which is important for protein import (1, 2). CL is also required for the activity and structural integrity of complexes of the electron transport chain, including NADH ubiquinone reductase (complex I), ubiquinone-cytochrome c reductase (complex III) and cytochrome c oxidase (complex IV) and the mitochondrial F 1 F -ATP synthase (complex V) (3 5). CL binds to the ADP/ATP carrier protein influencing its catalytic and structural properties (6, 7). Moreover, CL plays an essential role in the regulation of apoptotic cell death (8 12). CL serves as an anchor for cytochrome c on the outer leaflet of the inner mitochondrial membrane. Stress signals induce the peroxidation of CL by cytochrome c, and peroxidized CL releases cytochrome c from inner membrane. CL also interacts with B-cell/lymphoma 2 family proteins thereby increasing the permeability of the outer mitochondrial membrane. The increased permeability facilitates cytochrome c release into the cytosol and stimulates the activation of caspase (8 12). Although not all of the regulatory proteins are conserved in animals and plants, a number of molecular characteristics including the caspase-like protease activity, Bcl-2-like family members and mitochondrial proteins are highly related (13). In plants, CL is crucial for maintaining mitochondrial ultrastructure and for stabilizing the respiratory complex I/complex III supercomplexes, as well as the major mitochondrial fission factor DRP3 complex (14, 15). CL depletion in the Arabidopsis cls mutant leads to defects in mitochondrial morphogenesis and stress responses, resulting in enhanced sensitivity to programmed cell death effectors including UV light, heat shock and darkness (14 16). The de novo biosynthesis pathway of CL in plants is similar to other eukaryotes, but is different from most prokaryotes. In E. coli, CL synthase transfers a phosphatidyl group from phosphatidylglycerol (PG) or phosphatiylethanolamine (PE) to another PG to form CL (17, 18). In Actinobacteria (19), yeast 3

4 (2), mammals (21) and plants (16), CL is synthesized from cytidine diphosphate diacylglycerol (CDP- DAG) and PG, using CDP-DAG as a phosphatidyl donor. Microscopic analysis of Arabidopsis leaf cells stained with the CL-specific fluorescent dye 1-N-nonyl-acridine orange showed that CL is enriched in the so-called CL-enriched membrane domains. Arabidopsis CL synthase is targeted to the inner mitochondrial membrane with its C-terminus facing the intermembrane space (14). In vitro assays showed that plant CL synthases prefer both CDP-DAG and PG esterified to oleic acid (18:1) over CDP-DAG and PG esterified to palmitic acid (16:) (22, 23). It is possible that the enzymatic specificity of plant CL synthase leads to the enrichment of specific CL species which could play an essential role in mitochondria. However, this scenario remains elusive because the molecular species composition of CL in plants is unknown. Previously, CL from plants was determined by chromatographic methods, including thin layer chromatography (TLC), gas chromatography (GC) or high performance liquid chromatography (HPLC). TLC separation, in some cases combined with radiochemical labeling, leads to determination of the relative abundance of CL in comparison with other lipids (15, 22). Combination of TLC and GC or GC- MS provides the means for quantification of individual fatty acids in CL (24). HPLC was employed to separate and quantify major lipid classes in plants, and trace amount of CL can be detected in some plant tissues (25). Tandem mass spectrometry (MS/MS) with liquid chromatography separation or direct infusion was developed for profiling plant membrane lipids, providing detailed information on most lipids except CL (26, 27). LC-MS was used to analyze CL in Arabidopsis, but only the relative abundance for a single selected molecular species was obtained (14). In contrast to plants, mass spectrometry has successfully been used to determine the CL molecular species in bacteria and animals. Direct infusion mass spectrometry ( shotgun lipidomics ) resulted in the quantification of CL molecular species from mouse (myocardium, liver, skeletal muscle) (28). Multidimensional MS-based shotgun lipidomics was also employed to analyze membrane lipids including CL in purified mitochondria (29). Normal-phase LC-ESI-MS led to the quantification of CL molecular species in lipid extracts of E. coli (3). More LC-MS methods have been developed for CL analysis from animal or human samples (31 35). 4

5 In the present work, we present a new strategy for the quantification of a full set of molecular CL species from plants. This method is based on the enrichment of acidic glycerolipids including CL via anion exchange chromatography, and its quantification by LC-MS. Employing this method, it was for the first time possible to identify and quantify ~ 23 molecular species of CL in different plant tissues, and to observe changes in absolute amounts and composition of CL during different environmental stress conditions. MATERIALS AND METHODS Lipid standards and gas chromatography Tetramyristoyl cardiolipin (14:) 4 CL and tetraoleoyl cardiolipin (18:1) 4 CL, were obtained from Avanti Polar Lipids. (14:) 4 CL was used as internal standard for the quantification of CL. Bovine heart CL, mostly containing (18:2) 4 CL, was from Sigma. (18:) 4 CL was generated by hydrogenating (18:1) 4 -CL with hydrogen gas/platinum(iv)oxide as described (36). CL isolated from plants and standards ((14:) 4 CL, (18:) 4 CL, (18:1) 4 -CL, bovine heart CL) were quantified after conversion into fatty acid methyl esters by gas chromatography using pentadecanoic acid as internal standard (15:, Sigma) (37). The amount of 15: was accurately determined by weight. The response factors for the detection of methyl esters with different chain lengths or number of double bonds were determined experimentally using a rape seed fatty acid methyl ester standard (Supelco). GC measurements were done at least in triplicate and mean and SD calculated. The SD was always below 1 %. Plants and growth conditions Arabidopsis thaliana (ecotype Columbia-) was grown under short day conditions with 8 hours light, 23 C/16 hours dark, 2 C, on pots containing soil and vermiculite (2:1, v/v), or on agar-solidified Murashige and Skoog (MS) medium containing 1 % sucrose, or in hydroponic medium (38). Phosphate deprivation experiments were done by replacing 2.5 mm KH 2 PO 4 with 1.25 mm K 2 SO 4 in the hydroponic 5

6 medium (38). The pho1-2 mutant (39) was obtained from the Nottingham Arabidopsis Stock Center (stock N857, Nottingham, UK). Barley (Hordeum vulgare L., cv. Barke) and tobacco (Nicotiana benthamiana) were grown in pots containing soil and vermiculite (2:1, v/v) under long day condition (16 hours light 23 C/8 hours dark 2 C) or in the dark (etiolated). Spinach leaves and mung bean sprouts were purchased from a local supermarket. Extraction of total lipids and purification of cardiolipin by anion exchange chromatography Plant material (~ 5 mg fresh weight) was placed in boiling water for 15 min to inactivate lipases. After removing the water and addition of 5. nmol (14:) 4 CL per sample as internal standard, total lipids were successively extracted with 6 ml of chloroform/methanol (1:2, v/v) and 6 ml of chloroform/methanol (2:1, v/v). The remaining plant material was dried at 12 C for 1 hours used for dry weight (DW) determination. The combined lipid extracts were mixed with 6 ml chloroform and 4.5 ml.3 M ammonium acetate (final ratios: chloroform/methanol/.3 M ammonium acetate, 2:1:.75, v/v/v), and centrifuged at 1 x g for 5 min to obtain phase separation. The lower phase containing lipids was collected and evaporated under an air stream. Diethylaminoethyl (DEAE) cellulose (Whatman) was activated by washing with 1 M ammonium acetate until no chloride was detected, and stored in methanol (1:1, v/v) at 4 C before usage. Total lipids were dissolved in chloroform/methanol/water (3:7:1, v/v/v) and loaded on the DEAE column (1 ml bed volume). Neutral and basic lipids, including MGDG, DGDG, PE and PC, were eluted with chloroform/methanol/water (3:7:1, v/v/v). Acidic lipids including CL, PG, PI and SQDG were eluted with chloroform/methanol/.8 M ammonium acetate (3:7:1, v/v/v). Chloroform and.3 M ammonium acetate were added to a final ratio of chloroform/methanol/ammonium acetate (2:1:.75, v/v/v), and the lipids harvested in the chloroform phase to remove excessive salt, then dried again under a stream of air, and dissolved in 2 µl tetrahydrofuran/methanol (1:1, v/v) for LC-MS analysis. Quantification of cardiolipin by LC-MS The HPLC system was composed of an Agilent 12 Series quaternary pump with an autosampler and a degasser (12 Series, Agilent Technologies). Solvents for LC-MS analysis, including chloroform and 6

7 methanol, were HPLC grade. Tetrahydrofuran (LiChrosolv, Merck Millipore) for LC-MS was gradient grade. The lipid extract purified by DEAE chromatography (3 µl) was injected onto a Nucleodur C18 Gravity analytical column (5 mm x 4.6 mm, 1.8 µm particle size, Macherey-Nagel). The binary gradient consisted of solvent A (tetrahydrofuran/methanol/5 mm ammonium acetate, 3:2:5, v/v/v) and solvent B (tetrahydrofuran/methanol/5 mm ammonium acetate, 7:2:1, v/v/v) (4). The solvent gradient was formulated as follows: 4 % solvent B for.5 min, 4 % to 75 % in 9.5 min, hold at 75 % for 5 min, 75 % to 1 % in 15 min, hold at 1 % for 5 min, 1 % to 4 % in 2 min, hold at 4 % for 1 min. The chromatography was performed at room temperature and at a flow rate of 2 µl/min. A 653 Accurate-Mass Quadrupole Time-of-Flight (Q-TOF) LC-MS instrument (Agilent) was used in the negative electrospray ionization mode. The instrument parameters were set as described (41). The energy for collision induced dissociation was 6 V. Mass spectra were recorded at a rate of 5 spectra sec -1. The extracted ion chromatograms ( MS only mode of unfragmented ions) were used for quantification after integration using the Agilent MassHunter Qualitative Analysis software. The data were further processed in Microsoft Office Excel 213. Values are given in nmol mg -1 DW. Because the CL molecular species were separated by chromatography, a correction for isotopic overlap was not required. Validation studies To study within-day precision, lipids were extracted from spinach leaves with three different fresh weights of (25, 5, 75 mg; 3-5 replicas each) and CL molecular species were quantified by LC-MS. To determine between-day precision, the lipid extraction and quantification was repeated in the same way 7 days later. An add-back study was performed by adding different amounts of (18:1) 4 CL (1.1 to 5.5 nmol) to 5 mg of spinach leaf tissue at the beginning of the lipid extraction. The amounts of (18:1) 4 CL were determined by LC-MS and compared with the amounts of (18:1) 4 CL quantified by GC. The recovery was determined by adding 5. nmol of (14:) 4 CL to 5 mg of spinach leaf tissue. After lipid extraction and DEAE column chromatography, the peak area of (14:) 4 CL was determined by LC-MS. The peak area of (14:) 4 CL was also directly measured by LC-MS without lipid 7

8 extraction and DEAE column chromatography. Because a large loss of (14:) 4 CL in the strongly diluted solution was observed in the latter experiment, (14:) 4 CL was mixed with spinach leaf lipids (as a "carrier") immediately before LC-MS measurement. The recovery was determined as the ratio of the peak areas of (14:) 4 CL extracted with spinach leaf lipids versus the peak area of (14:) 4 CL directly measured by LC-MS. RESULTS Cardiolipin from plants can be purified by anion exchange chromatography Many different glycerolipids can be analyzed by mass spectrometry of total lipids extracted from plants with the exception of CL (42). Attempts to analyze CL from total leaf lipid extracts by LC-MS or LC- MS/MS using a variety of solvents and C18 or a C8 reverse phase columns were not successful. The signal intensity for CL was too low, presumably due to the low abundance and interferences with other lipids, and therefore this method was not suitable for quantitative analysis of CL. Solid phase extraction (SPE) was employed to purify CL prior to LC-MS analysis. SPE with anion exchange columns was previously used to separate neutral and basic phospholipids (e.g. PE, PC) from anionic lipids including CL from rats (43). After testing several SPE columns and eluents, including normal and reverse phases and anion exchange columns, SPE on a DEAE cellulose column was selected because this method resulted in a satisfactory separation with the enrichment of CL in the acidic lipid fraction, accompanied with the removal of neutral and basic lipids (Fig. 1). Fig. 2A shows the separation of total lipids from Arabidopsis callus and leaves by DEAE column chromatography. The neutral (MGDG and DGDG) and basic lipids (PE and PC) as well as some unidentified lipids were removed in the neutral/basic fraction F1. CL together with PI, PG, SQDG and other unidentified anionic lipids were eluted in acidic fraction F2. Different amounts of CL standards, i.e. (14:) 4 -CL, (18:) 4 -CL and (18:1) 4 -CL, were mixed and chromatographed on the DEAE cellulose column as described above. Then the relative amounts of CL standards were measured by quantification of the acyl groups by GC. The presence of different acyl chain 8

9 lengths and degrees of unsaturation in CL did not affect the isolation via the DEAE column (Fig. 2B). In a second experiment, CL was purified from total spinach leaf lipids by DEAE chromatography. After additional purification by TLC, a single band for CL was obtained and the acyl composition was quantified by GC (Fig. 2C). About 9 % of the acyl groups in spinach CL have a chain length of 18:X. The most abundant fatty acids are α-linolenic (18:3) and linoleic acid (18:2), amounting to 4 % and 37 %, respectively. About 1 % of the fatty acids are 16:X, mostly palmitic acid (16:). The acyl chain composition of CL in spinach was similar to that of purified spinach mitochondria (24). Based on the acyl chain composition, we predicted that 72:X, i.e. (18:X) 4 CL, represents the most abundant CL molecular species in spinach with a high degree of unsaturation. Identification of cardiolipin molecular species by liquid chromatography-mass spectrometry Previous studies indicated that due to the presence of two phosphate groups, CL molecular species can be observed in the negative mode by mass spectrometry as CL anions carrying one or two negative charges (molecular ions [M-H] - or [M-2H] 2- ). Indeed, MS direct infusion experiments with CL standards confirmed that CL signals are higher in the negative mode as compared to the positive mode. Because CL contents in plant tissues are considered to be extremely low, we selected LC-MS in the negative mode as a means to quantify the different CL molecular species. The LC-MS chromatogram of (18:1) 4 CL shows a peak at min for the extracted ion of m/z (Fig. 3A) corresponding to the singly negatively charged molecular ion [M-H] -. The MS spectrum at min reveals the presence of a second ion at m/z which corresponds to the double negative ion [M-2H] 2- (Fig. 3B). The ratio of the [M-H] - and [M-2H] 2- signals was similar for the different standards (14:) 4 CL, (18:) 4 CL and (18:1) 4 CL, and the singly negative signal was always higher. The MS/MS spectrum of the [M-H] - anion of (18:1) 4 CL is depicted in Fig. 3C. Fragmentation of (18:1) 4 CL results in a number of anions, including m/z for glycerolphosphate, m/z for the 18:1 free fatty acid anion, m/z for monoacylglycerolphosphate, and m/z for diacylglycerolphosphate. The fragmentation pattern of the parental ion of (18:1) 4 CL can be explained by dissociation of the ester bond of the fatty acids (1), or of the phosphate ester linkages to glycerol (2, 3, 4) (Fig. 3D). Based on the presence of the ions for free fatty acid anions and of mono- and 9

10 diacylgylcerolphosphates, the acyl composition of CL molecular species can be exactly determined via MS/MS experiments (Table 1, Table 2). Separation and quantification of CL molecular species Fig. 4A shows the extracted ion chromatograms for the [M-H] - ions of a mixture of the three CL standards containing 15 pmol each of (14:) 4 CL, (18:) 4 CL and (18:1) 4 CL. All three peaks were eluted from the reverse phase column in the range from 75 % to 1 % solvent B (between 14 and 26 min), in the order of (14:) 4 CL, (18:1) 4 CL and (18:) 4 CL. The exact amounts of the three CL standards in the mixture were determined by GC of fatty acid methyl esters. The ratios of the LC-MS [M-H] - peak areas to the amounts of CL quantified by GC were calculated (Fig. 4A). These ratios were very similar for (14:) 4 CL, (18:) 4 CL and (18:1) 4 CL, indicating that the chain length and degree of unsaturation had no large impact on the amounts of CL detected as [M-H] -. Therefore, no correction factor for the different CL molecular species was required, and (14:) 4 CL was selected as an internal standard because its retention time was expected not to overlap with authentic plant CL molecular species. Next, CL from bovine heart was analyzed by LC-MS to address the questions whether isotopologic and isomeric forms of CL can be separated. The presence of two 13 C carbons in a CL molecule results in the increase in the m/z of the [M-H] - ion by 2, corresponding to the mass difference originating from one double bond (2H). Therefore, it is important to consider the existence of different isotopologs of CL derived from the presence of one or more 13 C isotopes in the molecule, and to correct for possible overlap with molecular species differing by the presence of one double bond in one of the acyl chains. Fig. 4B shows the extracted ion chromatograms of bovine heart CL for m/z (black), (light grey), (dark grey). The different peaks eluting at , , and min were further analyzed by MS/MS experiments to determine their acyl composition. The most abundant molecular species of bovine heart CL is 72:8 with four 18:2 fatty acids, followed by 72:7 and 72:6 molecular species. MS/MS analysis of the peak eluting at min revealed that it contains 72:8CL with four 18:2 groups, i.e. (18:2) 4 CL. In addition to the m/z of corresponding to [M-H] - containing 12 C atoms ( 12 C-72:8CL), two isotopologs eluting at the same time with two ( ) or four 13 C carbons ( ) can be identified. The peak areas of 1

11 the different isotopologs of 72:8CL eluting at min correspond to the calculated isotope distribution of (18:2) 4 CL. The isobaric peaks of m/z at min (containing 13 C 2-72:8CL) and at min ( 12 C-72:7CL) are clearly separated. Similarly, the peaks of m/z at min ( 13 C 4-72:8CL), at min ( 13 C 2-72:7CL) and at min ( 12 C-72:6CL) are separated by LC-MS. Therefore, isotopologs with two 13 C carbon atoms are separated from CL molecular species differing by one double bond, and thus a correction for isotopic overlap was not required. Different isomeric molecular species of CL can have the same m/z, for example 72:6CL with the two isomers (18:2) 2 (18:1) 2CL and (18:2) 3 (18:)CL. Indeed, two isomers of 72:6CL were identified by LC-MS of bovine heart CL, (18:2) 2 (18:1) 2 CL eluting at min, and (18:2) 3 (18:)CL at min. Therefore, isomeric CL molecular species carrying acyl groups with different degrees of unsaturation can be separated by LC-MS, but oftentimes elute at very similar retention times. To determine the limits of detection for CL, different amounts of (18:1) 4 CL were quantified employing (14:) 4 CL as internal standard. Fig. 4C shows the double logarithmic plot of the amount of (18:1) 4 CL measured by LC-MS versus the amount of (18:1) 4 CL quantified by GC of acyl groups after methylation. The quantification of (18:1) 4 CL by LC-MS was linear in the range from ~5 to ~1 pmol. Taken together, the method of choice for the determination of CL from plants encompasses the purification of CL from crude lipid extracts by DEAE column chromatography and subsequent quantification by LC-MS of the extracted ion chromatogram for [M-H] - using (14:) 4 CL as internal standard. For exact identification, the acyl compositions of all molecular CL species detected by LC-MS need to be verified by MS/MS spectra (Table 2). Validation of CL quantification from spinach leaves Anionic lipids including CL were isolated from spinach leaves using DEAE column chromatography and separated by LC-MS analysis. As the DEAE column fraction F2 contains PG, PI and SQDG, in addition to CL (Fig. 2A), it was of interest to compare the retention times of the different lipids on the LC-MS column. Fig. 5A shows that the main molecular species of the lipids (34:4 PG, 34:3 PI, 34:3 SQDG) are eluted at 6 to 8 min, well before 72:12 CL (14 min). The other molecular species of PG, PI 11

12 and SQDG elute around 5 to 1 min (not shown). Therefore, LC-MS chromatography resulted in the separation of CL from other anionic lipids, thereby contributing to the enhanced sensitivity of CL quantification. Similar to the separation of CL molecular species from bovine heart (Fig. 4B), the molecular species from spinach leaves were separated by LC-MS (Fig. 5B; Table 1). In addition, the molecular species from spinach were also separated from isotopologs (see Fig. 4B). For most molecular species, only one acyl composition could be attributed (depicted by black traces/ms only in Fig. 5B). For some molecular species, different acyl compositions were detected. Therefore, the MS/MS traces for three different fragments (acyl anions of 18:1, 18:2 or 18:3) are shown for 72:9 CL, 72:8 CL, 72:7 CL, 72:6 CL and 7:9 CL. For clarity, in the following these molecular species were quantified based on their MS only chromatograms, disregarding the presence of molecules with different acyl compositions. To test the within-day and between-day precision, the main molecular species of CL from spinach leaf were quantified on day 1, and again 7 days later, by LC-MS. Three different amounts of fresh weight (25, 5 or 75 mg) were employed for CL extraction. Extraction from all three different amounts of fresh weight gave similar CL contents, but 5 mg fresh weight provided the most reproducible results (Fig. 6A). The within-day precision can be deduced from the relative SD (ratio of SD to the mean; n=5; in %) of the CL contents measured on day 1 (Fig. 6A). The relative SDs (for 5 mg fresh weight, day 1) for the two most abundant molecular species were 14.3 % for 72:11 CL and 13.8 % for 72:1 CL. For the between day precision, we calculated the ratio of the difference between the means measured at day 1 and day 8, to the mean of day 1. These values were 8.7 % for 72:11 CL and 9.1 % for 72:1 CL. Therefore, measurements of CL molecular species via LC-MS are characterized both by high within-day and between-day precision. Next, an add-back study was performed by adding different amounts of (18:1) 4 CL to 5 mg of spinach leaves. The amounts of (18:1) 4 CL were quantified by LC-MS and plotted versus the amounts determined by GC of fatty acid methyl esters. Fig. 6B shows that the (18:1) 4 CL amounts measured by LC-MS in a spinach extract are very similar to those determined by GC. Linear regression indicates that the values are highly correlated (R 2 =.967) and that the regression line is close to the diagonal. 12

13 The recovery was determined as the ratio of the peak area of (14:) 4 CL added to 5 mg of spinach leaf tissue prior to lipid extraction, versus the peak area of (14:) 4 CL directly measured by LC- MS. The recovery for (14:) 4 CL was 83.7 ± 5.4 % (n=3). Characterization of CL molecular species from plant tissues Quantification of the most abundant molecular species of CL from spinach leaf is depicted in Fig. 7A. Spinach CL is dominated by 72:X molecular species with four 18:X acyl chains, amounting to 96 mol % of total CL. A minor amount of 7:X with three 18:X and one 16: was also present, as well as 68:X molecular species with two 18:X and two 16: acyl groups. Among the 72:X, species, 72:1 and 72:9 are the most abundant molecules, followed by other highly unsaturated molecular species. Calculation of the acyl composition of spinach CL based on its molecular species composition (Fig. 7A) revealed that it contains about 44 % 18:3, 42 % 18:2, 9 % 18:1 and 4 % 16:. This is in agreement with the acyl composition of spinach CL as determined by GC (Fig. 2C). Lipids were extracted from different plants including tobacco leaves, mung bean hypocotyls and barley leaves, in addition to spinach leaves (Fig. 7A-D). The total CL contents differ between the different plants. The molecular species distribution is similar, indicating that the CL pattern is conserved. The average numbers of double bonds per CL molecule are ~ 1 in the different plants. The major molecular species are 72:X CL, followed by 7:X CL and 68:X CL. The 18:X acyl groups are usually highly desaturated (18:3, 18:2), while the 16:X acyl groups are almost exclusively saturated (16:). To determine whether the acyl pattern of CL differs between green and non-green tissues, CL was isolated from green and etiolated leaves of barley (Fig. 7D). The total amounts of CL from green and etiolated barley leaves are similar (.193 ±.5 and.2 ±.31 nmol mg -1 DW, respectively; mean ± SD, n=3). Etiolated barley leaves contain slightly more highly unsaturated (72:12 CL) molecular species than green barley leaves. In agreement with this finding, the average number of double bonds per CL molecule was slightly increased in etiolated leaves. Changes in cardiolipin composition during phosphate deprivation 13

14 Under phosphate deprivation, the amounts of the non-phosphorous galactolipid digalactosyldiacylglycerol (DGDG) and of the sulfolipid sulfoquinovosyldiacylglycerol (SQDG) increase, while the contents of phospholipids decrease, to save phosphate for other essential cellular processes including DNA and RNA synthesis (44). The extra pool of DGDG that increases during phosphate deprivation was located to the chloroplast, plasma membrane, tonoplast, and it was even detected in the mitochondrion (44 46). The contents of phosphatidylcholine (PC) and phosphatidylglycerol (PG) strongly decrease under phosphate deprivation, but it was unclear whether the CL content or composition are also changed. We tested the impact of phosphate deprivation on CL in leaves or roots by growing Arabidopsis plants in hydroponic culture in the presence or absence of phosphate (Fig. 8A, B). The total CL content in leaves decreases by 17 %, from.28 ±.32 to.174 ±.14 nmol mg -1 DW after phosphate deprivation, while in roots, it decreases by 67 %, from.399 ±.5 to.132 ±.6 nmol mg -1 DW (mean ± SD, n=3). The degree of unsaturation of CL in leaves is higher than in roots, regardless of phosphate availability, because CL in leaves (+P) contains an average of 9.91 ±.2 double bonds per CL molecule, while CL in roots (+P) it contains 9.46 ±.6 (mean ± SD, n=3). The average numbers of double bonds per CL molecule are increased in both leaves and roots during growth on low phosphate medium. 72:12 CL shows the strongest change in leaves where it increases 2 fold during phosphate deprivation. Apart from 72:12 and 72:11, other 72:X molecules are decreased in leaves (-P). The increase in the degree of unsaturation of CL in roots is reflected by the finding that highly unsaturated molecular species 72:12 and 72:11 show a moderate decrease, while the more saturated molecules 72:1 and 72:9 are more strongly reduced. The phosphate-deficient Arabidopsis pho1-2 mutant represents an alternative, genetic model to study phosphate limitation, because the leaves of pho1-2 are permanently phosphate-deprived due to the block in the root phosphate transporter PHO1 (47). As a consequence, the contents of phospholipids are decreased in pho1-2 leaves while the amounts of DGDG and SQDG are increased (44). CL was measured by LC-MS in leaves of Arabidopsis WT and pho1-2 (Fig. 8C). The total content of CL in pho1-2 ( nmol mg -1 DW) was slightly reduced as compared to WT ( nmol mg -1 DW; mean ± SD, n=3). Differences in the degree of CL desaturation were detected, because CL in pho1-2 is more highly unsaturated, as the contents of 72:12 and 72:11 are increased, while the amounts 14

15 of 72:1, 72:9 and 72:8 are decreased (Fig. 8C). This increase in highly unsaturated molecular CL species in pho1-2 is also reflected by the higher average number of double bonds per CL molecule and is consistent with the results obtained with phosphate-deprived WT leaves (Fig. 8A). Cardiolipin response to heat shock and darkness In animals, CL is known to play an important role during programmed cell death (11), but its function in plant cell death is less well understood. Cell death in plants can be induced by applying a short heat stress, or by growing the plants in complete darkness (14). Exposure to a short heat shock, as well as the growth in darkness resulted in senescence and cell death in the CL-deficient cls mutant of Arabidopsis, while the WT was not affected (14). The responses of CL content and composition to heat shock and growth under dark conditions were studied to unravel the effect of programmed cell death on CL. First, Arabidopsis plants were exposed to 65 C for 1 min and subsequently grown at room temperature under short day conditions for 1, 2 or 3 days. The CL content and pattern was compared to control plants not exposed to heat stress. No significant changes in CL content or composition were observed in the different plants (Fig. 9A). The number of double bonds per CL molecule was 1.12 ±.1 in control plants and did not change after the heat shock (mean ± SD, n=3). Therefore, the heat shock of 1 min did not affect CL content or composition. Next, CL was measured in Arabidopsis plants exposed to darkness for 2, 4 or 6 days, to observe the changes in CL during dark-induced cell death. The plants were grown on soil under short day condition. The total CL content decreased from.252 ±.11 at days to.2 ±.8 at 4 days and.177 ±.1 nmol mg -1 DW (mean ± SD, n=3) at 6 days of growth under dark conditions. The degree of unsaturation of CL increased continuously, from 9.74 ±.3 double bonds per molecule at day to 1.18 ±.3 nmol mg -1 DW (mean ± SD, n=3) after 6 days. This result is in line with the finding that the highly unsaturated molecular species 72:12 and 72:11 increased, while other more saturated molecular species decreased (72:1, 72:9, 72:8) (Fig. 9B). 15

16 DISCUSSION Cardiolipin, a crucial phospholipid in bacterial cells and in mitochondria of eukaryotes, has previously been analyzed in plants by chromatographic methods, including TLC, GC and HPLC (15, 22, 24, 25). These techniques provided data on the content of plant CL, but failed to give the information of molecular species composition. In contrast to plants, the total amount and molecular species composition of CL from animal tissues has previously been studied by mass spectrometry in great detail. In most studies, crude lipid extracts were obtained from tissue samples, cell cultures or isolated mitochondria employing methanol/chloroform/aqueous buffer system (48, 49). Subsequently, CL was measured by separation of lipid extracts via LC-MS using different tandem mass spectrometers (3, 31, 34, 35, 5) (Table 3). The determination of CL molecular species from animal cells is straight forward, as the relative proportion of mitochondrial membranes in animal cells is considerably higher than in plant cells which contain the chloroplast as an additional organelle. Plastids in plant cells harbor large amounts of membrane lipids and pigments which interfere with LC-MS quantification of minor lipid classes like CL. Therefore, detailed information on the molecular species composition of plant CL was lacking. Here, we report an LC-MS approach for the determination of CL from plants. Anion exchange chromatography on a DEAE column was essential for measuring CL by LC-MS. After removing the major basic and neutral lipids (MGDG, DGDG, PE, PC) separation and identification of a large number of molecular CL species, including minor molecular species, became feasible (Fig. 1). Only acidic lipids remained present in the purified CL fraction, and they were separated by the subsequent LC-MS step (Fig. 5A). DEAE column separation did not lead to the differential enrichment of CL molecular species carrying acyl groups with different chain lengths or degrees of unsaturation (Fig. 2B, C). Previously, CL was enriched from animal lipids before quantification by mass spectrometry using silica/normal phase chromatography (32, 33, 51) (Table 3). CL molecular species from animal tissues can be measured using a MALDI ion source, or by direct infusion into the mass spectrometer with an electrospray ion source (ESI) (28, 32). An increase in sensitivity can be obtained after separation of CL molecular species by LC, e.g. using a normal phase column (3, 34, 35). We used reversed phase LC for separation as this method is highly robust and 16

17 reproducible (Fig. 4, 5). The elution of CL molecular species from the reverse phase column depended on the acyl chain length and degree of unsaturation as previously described (31, 33, 5, 51). CL molecular species with shorter acyl chains or with a higher degree of unsaturation were eluted earlier (Fig. 4, 5). CL can lose one or two protons, producing singly and double negatively charged ions during ionization in the negative mode. During LC-MS analysis, singly charged parental ions [M-H] - were the main products, and were therefore selected for MS/MS fragmentation and quantification (Fig. 3). The higher abundance of [M-H] - versus [M-2H] 2- ions might depend on the solvent system and the characteristics of the ionization source, and is in agreement with other studies (33, 34, 5, 51) (Table 3). On the other hand, double negative anions of CL, [M-2H] 2-, were employed for quantification in alternative approaches (28, 3, 31, 35). CL can also be recorded in the positive mode ([M+Na] + or [M+NH 4 ] + ). CL molecules show a wide range of isotope distribution with isotopologs containing only 12 C atoms, or one, two, three or more 13 C atoms. All isotopologs of a given molecular species of CL elute at the same retention time, and the peak containing only 12 C carbons is the most abundant one. No isotopic correction was required, because the 12 C peak with the m/z of [M-H] - could clearly be distinguished from ions containing one or more 13 C atoms (Fig. 4, 5). Because the peak areas during LC-MS of equimolar mixtures of (14:) 4 CL, (18:) 4 CL and (18:1) 4 CL are similar, no correction factor was employed for the different molecular species of CL, and (14:) 4 CL was selected as internal standard with single-point calibration, similar to previous studies on quantification of animal CL (28, 31, 34, 35, 5). On the other hand mass dependent correction factors were used to quantify CL from E. coli (3) (Table 3). The total amount of CL determined in different plant species and organs varied between.64 and.32 nmol mg -1 DW in tobacco and spinach leaves, respectively (Fig. 7). Arabidopsis leaves and roots contain around.2 and.399 nmol mg -1 DW of total CL (Fig. 8). Previously, CL was quantified in Arabidopsis leaves by TLC combined with densitometry. The total amount of CL was found to be around 34 ± 12 ng mg -1 fresh weight (15). This amounts to about.24 nmol mg -1 DW (molecular weight of CL ca. 14 ng nmol -1 ; 1 mg fresh weight.1 mg DW), in agreement with data presented here. The total content of glycerolipids in Arabidopsis leaves and roots was previously determined to be around 17

18 165 and 64 nmol mg -1 DW, respectively (52). Therefore, CL amounts to only.12 % and.62 % of total lipids in leaves and roots, respectively. The molecular species composition of CL in plants is conserved, as 72:X CL species with four 18:X fatty acids are predominant in the different plants, including spinach, tobacco, mung bean, barley and Arabidopsis leaves and roots (Fig. 7, 8, 9). The most abundant molecular species 72:12, 72:11, 72:1, 72:9, 72:8 contain mostly 18:2 or 18:3 fatty acids, and the average number of double bonds per CL molecule is in the range of 9 to 1 (Fig. 7). The molecular species composition of CL from green tissues (leaves of spinach, tobacco, Arabidopsis) or nongreen tissues (mung bean hypocotyls, Arabidopsis roots) are very similar. Furthermore, monocots (barley leaf) have a similar set of highly unsaturated CL molecules as dicots, and the molecular species composition is also similar in green and etiolated barley leaves. Therefore, plant CL is highly unsaturated. These results are consistent with in vitro CL synthase assays with mung bean mitochondrial inner membranes, or with a protein extract containing CL synthase purified from Arabidopsis. The two CL synthase preparations displayed a preference for both CDP-DAG and PG esterified with unsaturated 18:X acyl groups (22, 23). The high degree of unsaturation renders plant CL distinct from bacterial and yeast CL, but similar to animal CL. The most abundant CL species in E. coli were 64:2, 66:2, and 68:2, and no CL molecular species containing more than 4 total double bonds was detected (3). Similarly, CL from yeast harbored 4 or less double bonds, and the major species yeast were 66:4, 68:4, 7:4 and 72:4 (29). Mammalian CL, e.g. from bovine heart, is rich in 72:8 with four 18:2 acyl groups (33) (Fig. 4B). Under phosphate deprivation, a certain proportion of phospholipids is replaced with DGDG in the chloroplast and in extraplastidial membranes (44). Mitochondrial lipids also participate in the phosphate response, because the amounts of PC and PE decrease, while the DGDG content increases in mitochondria during phosphate starvation (45). Previous measurements in Arabidopsis cell cultures and in isolated mitochondria by TLC in combination with fatty acid quantification by GC suggested that CL slightly increases after phosphate deprivation (45). We measured CL in extracts from whole Arabidopsis leaves and roots. Arabidopsis leaves showed a slight reduction of CL after phosphate deprivation (WT 18

19 leaves -P versus +P; pho1-2 versus WT) (Fig. 8A, C). The retention of CL in Arabidopsis leaf mitochondria might be due to the fact that CL is essential for mitochondrial functions and therefore cannot be removed. On the other hand, the CL content was strongly decreased in roots after phosphate deprivation (Fig. 8B), in accordance with the decrease in other phospholipids (PC, PE) in roots (52). These changes might be caused by the strong increase in root biomass caused by root proliferation during phosphate deprivation. The degree of desaturation of CL under phosphate deprivation was increased (Fig. 8). In leaves, the total amounts of 72:12 and 72:11 were increased, while 72:1, 72:9 and 72:8 were decreased. In roots, the amounts of 72:12 and 72:11 were only slightly increased, while 72:1 and 72:9 were strongly decreased during phosphate deprivation. These results point towards the remodeling of CL under phosphate starvation. CL plays a critical role in programmed cell death in animal cells (8 12). Previous results indicated that CL also plays a role in programmed cell death in Arabidopsis, because CL deficient cls mutant plants displayed a higher sensitivity to cell death inducing stress factors including heat shock and exposure to prolonged darkness (14). We employed the same two stress conditions to record the impact of cell death on the CL content or composition. A short heat shock of 1 min at 65 C had no effect on the total CL amount or degree of unsaturation in Arabidopsis plants (Fig. 9A), possibly because this stress was rather mild. However, growth under darkness resulted in the decrease in CL content and a shift towards higher unsaturated molecular species in Arabidopsis leaves. Therefore, the latter results suggest that changes in the content and composition of the molecular species of CL might be relevant for cell death. Several enzymes have been proposed to be involved in remodeling of CL in yeast and animals, including tafazzin (21, 53), acyl-coa:lysocardiolipin-acyltransferase (54), calcium-independent phospholipase A2 (55), and the trifunctional enzyme (56). The high abundance of highly unsaturated molecular species of CL, and their increase during phosphate deficiency and growth in darkness, point towards a critical role of these unsaturated CL forms in the inner mitochondrial membranes. It is possible that highly unsaturated CL molecules are required to maintain the activity of the respiratory chain complexes, and this might become even more relevant during phosphate deficiency stress or growth under darkness. The establishment of the LC-MS method for CL measurements from plants 19

20 along with the results on the shift in CL molecular species composition under phosphate deprivation and growth under darkness provide the means to investigate CL remodeling mechanisms in plants in more detail. ACKNOWLEDGMENTS The authors thank Vera Wewer and Katharina vom Dorp (University of Bonn) for help with mass spectrometry analysis and for critically reading the manuscript. Seeds of barley (Hordeum vulgare L., cv. Barke) were obtained from Prof. Jens Léon (University of Bonn). This work was supported by a grant from University of Bonn (to P.D.). 2

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24 different solvents: Verification of the concentration of chlorophyll standards by atomic absorption spectroscopy. Biochim. Biophys. Acta 975: Folch J., M. Lees, and G. H. Sloane Stanley A simple method for the isolation and purification of total lipides from animal tissues. J. Biol. Chem. 226: Bligh E. G., and W. J. Dyer A rapid method of total lipid extraction and purification. Can. J. Biochem. Physiol. 37: Bird S. S., V. R. Marur, and M. J. Sniatynski, and H. K. Greenberg, and B. S. Kristal Lipidomics profiling by high resolution LC MS and high energy collisional dissociation fragmentation: focus on characterization of mitochondrial cardiolipins and monolysocardiolipins. Anal. Chem. 83: Samhan Arias A. K., J. Ji, and O. M. Demidova, and L. J. Sparvero, and W. Feng, and V. Tyurin, and Y. Y. Tyurina, and M. W. Epperly, and A. A. Shvedova, and J. S. Greenberger, and H. Bayır, and V. E. Kagan, and A. A. Amoscato Oxidized phospholipids as biomarkers of tissue and cell damage with a focus on cardiolipin. Biochim. Biophys. Acta 1818: Li M., R. Welti, and X. Wang. 26. Quantitative profiling of Arabidopsis polar glycerolipids in response to phosphorus starvation. Roles of phospholipases Dζ1 and Dζ2 in phosphatidylcholine hydrolysis and digalactosyldiacylglycerol accumulation in phosphorus starved plants. Plant Physiol. 142: Vaz F. M., R. H. Houtkooper, and F. Valianpour, and P. G. Barth, and R. J. A. Wanders. 23. Only one splice variant of the human TAZ gene encodes a functional protein with a role in cardiolipin metabolism. J. Biol. Chem. 278: Cao J., Y. Liu, and J. Lockwood, and P. Burn, and Y. Shi. 24. A novel cardiolipin remodeling pathway revealed by a gene encoding an endoplasmic reticulum associated acyl CoA:lysocardiolipin acyltransferase (ALCAT1) in mouse. J. Biol. Chem. 279: Mancuso D. J., H. F. Sims, and X. Han, and C. M. Jenkins, and S. P. Guan, and K. Yang, and S. H. Moon, and T. Pietka, and N. A. Abumrad, and P. H. Schlesinger, and R. W. Gross. 27. Genetic ablation of calcium independent phospholipase A2gamma leads to alterations in mitochondrial lipid metabolism and function resulting in a deficient mitochondrial bioenergetic phenotype. J. Biol. Chem. 282: Taylor W. A., and G. M. Hatch. 29. Identification of the human mitochondrial linoleoyl coenzyme A monolysocardiolipin acyltransferase (MLCL AT 1). J. Biol. Chem. 284: Kim J., and C. L. Hoppel Comprehensive approach to the quantitative analysis of mitochondrial phospholipids by HPLC MS. J. Chromat. B 912:

25 FIGURE LEGENDS Fig. 1. Flow chart for cardiolipin analysis from plants. Total lipids are extracted from ~ 5 mg plant material with chloroform/methanol. Lipids are fractionated by anion exchange chromatography on a DEAE column. Neutral and basic lipids are eluted with chloroform/methanol/water (3:7:1, v/v/v), and acidic lipids are eluted with chloroform/methanol/.8 M ammonium acetate (3:7:1, v/v/v). CL, cardiolipin; DGDG, digalactosyldiacylglycerol; LC-MS, liquid chromatography-mass spectrometry; MGDG, monogalactosyldiacylglycerol; PC, phosphatidylcholine; PE, phosphatidylethanolamine; PG, phosphatidylglycerol; PI, phosphatidylinositol; SQDG, sulfoquinovosyldiacylglycerol. TLC, thin-layer chromatography. Fig. 2. Purification of cardiolipin by DEAE column chromatography A: Total lipids from Arabidopsis callus and leaves were separated by DEAE column chromatography and fractions F1 (chloroform/methanol/water, 3:7:1, v/v/v) and F2 (chloroform/methanol/.8 M ammonium acetate, 3:7:1, v/v/v) chromatographed on a TLC plate. Lipids were stained with iodine vapor. CL is enriched in the anionic lipid fraction F2. B: Isolation of different molecular species of CL by DEAE column chromatography. A mixture of the CL standards (14:) 4 CL, (18:) 4 CL and (18:1) 4 CL was chromatographed on a DEAE column, and fraction F2 harvested. Fatty acid methyl esters were prepared from the CL lipids before (-) and after (+) chromatography, and quantitated by gas chromatography. The relative abundance of CL molecular species in not affected by DEAE column chromatography. C: Gas chromatography analysis of fatty acids derived from spinach leaf CL. CL was purified by DEAE column chromatography and TLC from spinach leaves. The CL band was harvested from the TLC plate, fatty acids converted into methyl esters, and quantified by GC. Data represent mean and standard deviation of at least 3 measurements. Fig. 3. Separation and characterization of (18:1) 4 CL by LC-MS/MS 25

26 (18:1) 4 CL was separated on a reverse phase column and detected by LC-MS and LC-MS/MS experiments on a Q-TOF mass spectrometer in the negative mode. A: Extracted ion chromatogram for (18:1) 4 CL ([M-H] -, m/z ). B: MS spectrum of (18:1) 4 CL recorded at min in A. The singly negatively charged ion ([M-H] -, m/z ) is higher than the double negatively charged ion ([M-2H] 2-, m/z ). C: MS/MS spectrum of (18:1) 4 CL (m/z ) , [PO 3 ] - ; , [GroP] - ; , [18:1] - ; , [18:1GroP] - ; , [di18:1grop] - ; , [di18:1gropgrop] - ; , [monolysocl] -, , [M-H] -, molecular ion of (18:1) 4 CL. D: Structure of (18:1) 4 CL with fragmentation sites. Fragmentation of the oleoyl ester bond (1) or at the phosphate ester bonds (2, 3, 4) results in the generation of the fragments as depicted in C. Fig. 4. Quantification of CL molecular species with internal standards A: An equimolar mixture of the standards (14:) 4 CL, (18:1) 4 CL and (18:) 4 CL was separated by LC- MS. The extracted ion chromatograms of the [M-H] - anions were integrated. The ratios of the peak areas to the amounts (quantified by GC of methyl esters) is comparable for the three CL molecular species. B: LC-MS separation of bovine heart CL which is rich in 72:8 CL. The different molecular species are separated. Black, m/z ; light grey, m/z ; dark grey, m/z C: Linear range for the quantification of (18:1) 4 CL. The double logarithmic plot shows different amounts of (18:1) 4 CL quantified by LC-MS using (14:) 4 CL as internal standard versus the amounts of (18:1) 4 CL as determined by GC of methyl esters. Quantification of (18:1) 4 CL by LC-MS is linear in the range of 5 to 1 pmol. Data points represent mean and standard deviation of at least 3 measurements (n=3). Fig. 5. Separation of anionic spinach leaf membrane lipids on reverse phase LC-MS Anionic lipids were isolated by DEAE column chromatography and separated by LC-MS. A: Separation of 72:12 CL (m/z ), 34:4 PG (m/z ), 34:3 PI (m/z ) and 34:3 SQDG (m/z ) from spinach leaf. The anionic lipids PG, PI and SQDG are eluted between 5 and 8 min, while CL is eluted starting around 14 min. 26

27 B: Separation of different CL molecular species from spinach leaves. The panels show the chromatograms of [M-H] - for the different molecular species. The arrows point to the respective molecular species, with isotopolog peaks of another molecular species carrying two 13 C and harboring one additional double bond eluting ca. 1 min earlier. For the molecular species that consist of molecules with different fatty acid combinations, the MS/MS trace data for the different acyl anions are shown (18:1, red; 18:2, green; 18:3, yellow). Fig. 6. Validation for the quantification of CL molecular species A: Lipids were isolated from spinach leaves by DEAE chromatography and quantified by LC-MS. The bar diagram shows the molecular species of 72:12 CL, 72:11 CL, 72:1 CL, 72:9 CL, 72:8 CL and 72:7 CL as quantified on day 1 (left three bars) and day 8 (right three bars). On each day, lipids were isolated from three different fresh weights of spinach leaves (25, 5 and 75 mg tissue; from left to right). The bars show mean and SD of at least 3 measurements. B: Add-back study for (18:1) 4 CL. The amount of (18:1) 4 CL added to 5 mg spinach leaf tissue before lipid extraction and purification was compared to the amount of that measured by GC. Data points show mean and SD of 5 measurements. The diagonal indicating perfect matches is shown as dashed line. Fig. 7. The cardiolipin composition in green and non-green plant tissues CL composition in spinach leaves (A), tobacco leaves (B), mung bean hypocotyls (C) and green and etiolated barley leaves (D). CL in total lipid extract was isolated by DEAE column chromatography and quantified by LC-MS using (14:) 4 CL as internal standard. The bars on the right indicate the total amounts of CL. Numbers depict the average numbers of double bonds calculated per CL molecule. Data represent mean and standard deviation of at least 3 measurements (n=3). Fig. 8. Effect of phosphate deprivation on CL content and composition A: CL composition from Arabidopsis leaves. 27

28 B: CL composition from Arabidopsis roots (B) of plants grown in hydroponic culture with or without phosphate. C: CL composition from Arabidopsis WT and the pho1-2 mutant plants grown on soil. The bars on the right indicate the total amounts of CL. Numbers depict the average numbers of double bonds calculated per CL molecule. The data show mean and SD of at least 3 measurements (n=3-5). Values significantly different to the +P or WT control are indicated by asterisks (Student's t-test, p <.5). Fig. 9. Change of CL composition in response to heat shock and darkness A: CL composition in Arabidopsis leaves after heat-shock. Plants grown on MS medium were exposed to a heat shock of 65 C for 1 min and then at room temperature for the time as indicated. C, Control, plants without heat shock. B: CL composition in Arabidopsis leaves of plants grown in darkness. Plants were grown on soil in darkness for the times as indicated. The bars on the right indicate the total amounts of CL. Numbers depict the average numbers of double bonds calculated per CL molecule. The data show mean and SD of at least 3 measurements (n=3). Values significantly different to the d control are indicated by asterisks (Student's t-test, p <.5). 28

29 TABLE 1. Cardiolipin molecular species CL Acyl composition Molecular Species Formula (M) [M-H] - (calculated mass) Retention time (min) a Occurrence 1 14: 14: 14: 14: 56: C 65 H 126 O 17 P St 2 18:3 18:3 18:3 18:3 72:12 C 81 H 134 O 17 P AC, AL, AR, BL, MH, SL, TL 3 18:3 18:3 18:3 18:2 72:11 C 81 H 136 O 17 P AC, AL, AR, BL, MH, SL, TL 4 18:3 18:3 18:2 18:2 72:1 C 81 H 138 O 17 P AC, AL, AR, BL, MH, SL, TL 5 18:3 18:2 18:2 18:2 72:9 C 81 H 14 O 17 P AC, AL, AR, BL MH, SL, TL 6 18:3 18:3 18:2 18: AC, AL, AR, MH, SL, TL 7 18:3 18:2 18:2 18:1 72:8 C 81 H 142 O 17 P AC, AL, AR, BL, MH, SL, TL 8 18:2 18:2 18:2 18: BH, SL 9 18:3 18:2 18:1 18:1 72:7 C 81 H 144 O 17 P AC, AL, AR, BL, MH, SL, TL 1 18:2 18:2 18:2 18: BH 11 18:3 18:1 18:1 18:1 72:6 C 81 H 146 O 17 P AC, AR, BL, MH, SL, TL 12 18:2 18:2 18:1 18: BH 13 18:2 18:2 18:2 18: BH 14 18:1 18:1 18:1 18:1 72:4 C 81 H 15 O 17 P St 15 18: 18: 18: 18: 72: C 81 H 158 O 17 P St 16 18:3 18:3 18:3 16:1 7:1 C 79 H 134 O 17 P AC, AL, AR, BL, SL, TL 17 18:3 18:3 18:2 16:1 7:9 C 79 H 136 O 17 P AC, AL, AR, BL, MH, SL, TL 18 18:3 18:2 18:2 16:1 7:8 C 79 H 138 O 17 P AR, MH 19 18:3 18:3 18:2 16: AC, AL, AR, BL, SL, TL 2 18:3 18:2 18:2 16: 7:7 C 79 H 14 O 17 P AC, AL, AR, BL, MH, SL, TL 21 18:3 18:3 18:1 16: AC, AL, AR, BL 22 18:2 18:2 18:2 16: 7:6 C 79 H 142 O 17 P AC, AL, AR, BL, MH, SL, TL 23 18:3 18:1 18:1 16: 7:5 C 79 H 144 O 17 P AC, AR, MH 24 18:2 18:2 18:1 16: AC, AR, SL, TL 25 18:2 18:1 18:1 16: 7:4 C 79 H 146 O 17 P AC, AR, MH 26 18:3 18:3 16: 16: 68:6 C 77 H 138 O 17 P AC, AR, BL, MH, SL, TL 27 18:3 18:2 16: 16: 68:5 C 77 H 14 O 17 P AC, AR, BL, MH, SL, TL 28 18:3 18:1 16: 16: 68:4 C 77 H 142 O 17 P AC, AR, MH, SL, TL 29 18:3 16: 16: 16: 66:3 C 75 H 14 O 17 P AC, AR 3 18:2 16: 16: 16: 66:2 C 75 H 142 O 17 P AC, AR All CL molecular species were characterized by LC-MS/MS analysis. Occurrence of the CL molecular species: AC, Arabidopsis callus; AL, Arabidopsis leaf; AR, Arabidopsis root; BH, bovine heart (commercial CL); BL, barley leaf; MH, mung bean hypocotyl; SL, spinach leaf, TL, tobacco leaf; St, standards (14:) 4CL, (18:1) 4 CL, (18:) 4CL (hydrogenation of (18:1) 4 CL).. 29

30 TABLE 2. Product ion peaks after fragmentation of singly negatively charged molecular ions of CL Abbreviation Formula of fragment Calculated mass (u) Abbreviation Formula of fragment Calculated mass (u) Phosphate (-H 2 O) Diacylglycerol phosphate PO di14:grop C 31 H 6 O 8 P Glycerolphosphate (-H 2 O) di16:grop C 35 H 68 O 8 P GroP C 3 H 6 O 5 P : 16:1GroP C 35 H 66 O 8 P Fatty acid anion di16:1grop C 35 H 64 O 8 P : C 14 H 27 O : 18:GroP C 37 H 72 O 8 P : C 16 H 31 O : 18:1GroP C 37 H 7 O 8 P :1 C 16 H 29 O : 18:2GroP C 37 H 68 O 8 P : C 18 H 35 O : 18:3GroP C 37 H 66 O 8 P :1 C 18 H 33 O :1 18:1GroP C 37 H 68 O 8 P :2 C 18 H 31 O :1 18:2GroP C 37 H 66 O 8 P :3 C 18 H 29 O :1 18:3GroP C 37 H 64 O 8 P Monoacylglycerol phosphate (-H 2 O) di18:grop C 39 H 76 O 8 P :GroP C 17 H 32 O 6 P : 18:1GroP C 39 H 74 O 8 P :GroP C 19 H 36 O 6 P : 18:2GroP C 39 H 72 O 8 P :1GroP C 19 H 34 O 6 P : 18:3GroP C 39 H 7 O 8 P :GroP C 21 H 4 O 6 P di18:1grop C 39 H 72 O 8 P :1GroP C 21 H 38 O 6 P :1 18:2GroP C 39 H 7 O 8 P :2GroP C 21 H 36 O 6 P :1 18:3GroP C 39 H 68 O 8 P :3GroP C 21 H 34 O 6 P di18:2grop C 39 H 68 O 8 P :2 18:3GroP C 39 H 66 O 8 P di18:3grop C 39 H 64 O 8 P

31 TABLE 3: Mass spectrometry-based methods for cardiolipin measurements Samples Analyzed Lipid Extraction MS Human platelets Folch None NP HPLC- QQQ Rat heart tissue, mitochondria Bovine heart inner mitochondria complexes Mouse heart, liver, skeletal muscle Bligh & Dyer Chloroform/ Methanol Bligh & Dyer Mouse Chloroform/ macrophage Methanol RAW tumor cells Rat liver,mouse Folch heart, dog heart mitochondria Rat liver mitochondria E. coli K-12 strain W311 Human blood lymphocytes, rat brain, lung, small intestine Rat and mouse heart mitochondria Arabidopsis leaf, root, mung bean, spinach, barley, tobacco Bligh & Dyer, using C 2 H 2 Cl 2 Bligh & Dyer Folch Chloroform/ Methanol Chloroform/ Methanol None Silica/NP HPLC NP HPLC- QQQ MALDI- TOF MS Ions Measured Quantification Purification Reference [M-2H] 2- Single-point calibration from IS a (35) [M-H] - Single-point calibration from IS (34) [M+Na] + None reported (32) None ESI QQQ [M-2H] 2- Single-point calibration from IS (28) None Silica/ NP HPLC None None NP HPLC NP RP HPLC- [M-2H] 2- QTOF MS, MS/MS RP HPLC- Ion trap MS, MS 2, MS 3 RP HPLC- Orbitrap MS NP HPLC- QTOF MS [M-H] - [M+NH 4 ] + [M-H ]- Single-point calibration with mass dependent correction factors Relative quantification of molecular species (31) (33) Single-point calibration from IS (5) [M 2H] 2- Single-point calibration with mass dependent correction factors (3) RP HPLC- [M-H] - None reported (51) QTOF MS, MS/MS NP HPLC- MS Anion RP HPLC Exchange QTOF MS [M-H] - [M-H] - Multiple-point standard curves with IS a Single Point Calibration from IS a (57) This manuscript a Including within-day and day-to-day validation. ESI, electrospray ionization; IS, internal standard; MALDI, matrix assisted laser desorption ionization; MS, mass spectrometry; NP, normal phase; QQQ, triple quadrupole; QTOF, quadrupole time-of-flight; RP, reverse phase 31

32 Plant Material Lipid Extraction (chloroform/methanol) Anion Exchange Chromatography (DEAE column) Neutral/Basic Lipids (PC, PE, MGDG, DGDG) Acidic Lipids (PA, PI, PG, SQDG, CL) TLC LC-MS (CL) Figure 1 Zhou et al. (216)

33 A MGDG CL PE PG PC DGDG SQDG PI Origin Acyl Composition (mol %) Acyl Composition (mol %) B (DEAE) C F1 F2 F1 F2 Bovine Callus Leaves Heart CL (14:) 4 CL (18:) 4 CL (18:1) 4 CL : 18: 18:1 Spinach CL 16: 16:1 18: 18:1 18:2 18:3 Fatty Acid Figure 2 Zhou et al. (216)