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J. Biol. Chem., Vol. 281, Issue 32, 22684-22694, August 11, 2006
Separation and Identification of Major Plant Sphingolipid Classes from Leaves* 1![]() ![]()
From the
Received for publication, April 27, 2006 , and in revised form, June 9, 2006.
Sphingolipids are major components of the plasma membrane, tonoplast, and other endomembranes of plant cells. Previous compositional analyses have focused only on individual sphingolipid classes because of the widely differing polarities of plant sphingolipids. Consequently, the total content of sphingolipid classes in plants has yet to be quantified. In addition, the major polar sphingolipid class in the model plant Arabidopsis thaliana has not been previously determined. In this report, we describe the separation and quantification of sphingolipid classes from A. thaliana leaves using hydrolysis of sphingolipids and high performance liquid chromatography (HPLC) analysis of o-phthaldialdehyde derivatives of the released long-chain bases to monitor the separation steps. An extraction solvent that contained substantial proportions of water was used to solubilized >95% of the sphingolipids from leaves. Neutral and charged sphingolipids were then partitioned by anion exchange solid phase extraction. HPLC analysis of the charged lipid fraction from A. thaliana revealed only one major anionic sphingolipid class, which was identified by mass spectrometry as hexose-hexuronic-inositolphosphoceramide. The neutral sphingolipids were predominantly composed of monohexosylceramide with lesser amounts of ceramides. Extraction and separation of sphingolipids from soybean and tomato showed that, like A. thaliana, the neutral sphingolipids consisted of ceramide and monohexosylceramides; however, the major polar sphingolipid was found to be N-acetyl-hexosamine-hexuronic-inositolphosphoceramide. In extracts from A. thaliana leaves, hexosehexuronic-inositolphosphoceramides, monohexosylceramides, and ceramides accounted for 64, 34, and 2% of the total sphingolipids, respectively, suggesting an important role for the anionic sphingolipids in plant membranes.
Sphingolipids are recognized as universal components of eukaryotic membranes with a diverse array of functions (13). Recent interest in sphingolipids from plants has been stimulated by the realization that they may form a significant proportion of the plasma membrane (4), potentially as lipid rafts (5, 6), are involved in signaling a plant's response to drought (7, 8), and regulate the ultimate fate of plant cells through programmed cell death (9, 10). In order to understand the role of plant sphingolipids in this diverse array of already discovered roles and to determine what other biological functions sphingolipids may have in plants, it is necessary to be able to measure the sphingolipid content in a qualitative and quantitative way (11). Sphingolipid signaling is thought to be a complex multifactorial signal derived from the interaction of several different sphingolipids (12), making the examination of all sphingolipids a critical factor in the dissection of sphingolipid function. Thus, the emerging field of sphingolipidomics has received much attention in animal biology but remains neglected in plants (13). Previous studies on plant sphingolipids have exclusively concentrated on single sphingolipid classes (1417). Neutral sphingolipids, such as ceramide and monohexosylceramide, are easily purified from plants. Since they are soluble in chloroform and resistant to mild-base hydrolysis, they can be purified to near homogeneity with relative ease (18). This has made them a prime target for study by a variety of methods from a wide array of species (18, 19). Glycosylinositolphosphoceramides (GIPCs)2 are not so amenable to purification, however, and little research has been carried out on their prevalence or occurrence in plants since their initial characterization some 3040 years ago (20, 21). GIPCs (also referred to as phytoglycolipid) isolated from corn, soybean, and tobacco were found to have the general structure (N-acetyl)glucosamine-glucuronic-inositolphosphoceramide with the addition of additional hexoses and pentoses at either the inositol or glucosamine residues (22, 23).
The proportion of total sphingolipids that the GIPCs represent is currently not known. Release of long-chain bases (LCBs) from sphingolipids by hydrolysis of an entire tissue sample is substantially different from that released by hydrolysis of monohexosylceramides purified from the same source (24). The implication is that the nonmonohexosylceramide component of plant sphingolipids is a substantial portion of the total sphingolipid pool. The problems addressed in this study are as follows. 1) What is the total sphingolipid content of Arabidopsis thaliana; 2) what is the relative contribution of individual sphingolipid species to the total sphingolipid content; and 3) what is the nature of the different sphingolipid species? Answering these questions required the development of protocols for the complete extraction of all sphingolipids from plants and their separation by chromatography. To demonstrate general applicability of the protocols used to answer these problems and to serve as a comparison, a similar analysis of the sphingolipid content of tomato and soybean was also performed. In all cases, the GIPCs were shown to represent a substantial proportion of the total sphingolipid.
Materials Except where noted, all chemicals were of high performance liquid chromatography (HPLC) grade or the highest grade available from Sigma. Methanol and tetrahydrofuran were Omnisolv grade, obtained from EMD Biosciences (San Diego, CA). Propan-2-ol was HPLC grade, and hexanes were optima grade, both obtained from Fisher.
Hydrolysis of Sphingolipids and Identification of Long-chain Bases
Solubilization of Sphingolipids Method I (after Bligh and Dyer (27))To the frozen tissue, 2 ml of methanol, 1 ml of chloroform, and 0.35 ml of water were added. The sample was vortexed for 1 min before centrifuging at 500 x g for 10 min. The supernatant was removed to a second tube, and the pellet was extracted again with 1 ml of chloroform. After centrifuging as before, the supernatant was removed and combined with the first. The insoluble material was retained for analysis. To the combined supernatants, 1 ml of 0.88% KCl in water was added. After vortexing for 1 min, the sample was spun as before to affect phase separation. The two phases were removed to fresh tubes and dried under nitrogen for analysis. Insoluble material present at the interphase was added to the pellet remaining after extraction and dried under nitrogen.
Method II (after Nichols (28) and Christie (29))The frozen tissue was extracted with 50 ml of propan-2-ol by macerating for 1 min at 3000 rpm with an ULTRATURRAX T-25 fitted with a S25N-18G dispersing element. After centrifugation for 10 min at 500 x g, the supernatant was removed. The pellet was further extracted by shaking overnight with 50 ml of chloroform-propan-2-ol (1:1, v/v). After centrifugation as before, the supernatant was removed, combined with the previous extract, and dried under nitrogen for analysis. Method III (after Hanson and Lester (30))To the frozen tissue, 5 ml of solvent E (ethanol/water/diethylether/pyridine/ammonia (15:15:5:1:0.018, v/v/v/v/v)) was added. The tissue was transferred to a DUALL glass homogenizer and homogenized until fully disrupted. The sample was transferred back to a Pyrex tube, capped, and incubated at 60 °C for 15 min with occasional shaking. The extract was spun at 500 x g while still warm, and the supernatant was transferred to a fresh tube. The pellet was extracted twice more, each time with 5 ml of solvent E, and the supernatants were combined and dried under nitrogen for analysis.
Method IV (after Toledo et al. (31))The sample was processed exactly as for Method III, except 5 ml of solvent H was used (lower phase of propan-2-ol/hexane/water, 55:20:25 (v/v/v)) in place of solvent E.
Extraction of Sphingolipids
Solid Phase Extraction of Sphingolipid Extract The C18 eluate was applied to 2 ml of AG4X-X4 acetate resin (Bio-Rad) supported in a 6-ml glass syringe with upper and lower Teflon frit and allowed to flow by gravity. The column was washed with chloroform/methanol/water (16:16:5, v/v/v) until the eluate ran clear. The column flow-through (neutral lipids) was dried under nitrogen and redissolved in 2.8 ml of chloroform/acetic acid (99:1), and 100 µl was removed for analysis. The anionic charged lipids were eluted from the column with 6 ml of chloroform/methanol/water/ammonia (16:16:5:1) containing 0.1% triethylamine. The eluate (anionic lipids) was dried under nitrogen and redissolved in 280 µl of propan-2-ol/hexane/water (3:1:1, v/v/v), and 10 µl was removed for analysis.
The neutral fraction was applied to a SepPak Silica cartridge equilibrated with chloroform/acetic acid (99:1, v/v) and allowed to drain by gravity flow. The cartridge was washed with 15 ml of chloroform/acetic acid (99:1, v/v), which was discarded. Sphingolipids were sequentially eluted with 4 ml of acetone and 4 ml of methanol, dried under nitrogen, and redissolved in 300 µl of chloroform and stored at 30 °C for HPLC analysis.
Preparative HPLC of Anionic Sphingolipids
Preparative HPLC of Neutral Sphingolipids
Electrospray Ionization and Mass Spectrometry of Sphingolipids
Assay for SphingolipidsA prerequisite for purification of any compound is an ability to measure it both qualitatively and quantitatively. Sphingolipids are unique compared with other lipids, since each molecule contains a 2-aminooctadecane backbone or LCB. Hence, hydrolysis and measurement of the liberated LCBs is a quantitative measure of the sphingolipid content. LCBs were liberated by hydrolyzing the sphingolipids under conditions that minimized the formation of artifacts (18, 25), permitted good separation and quantification of the products (Fig. 1), and could be performed with minimal sample handling and cleanup. The identity of each peak was assigned by the following: (a) comparison with known standards (t18:0, d18: 1(4E), and d18:0); (b) mass spectrometry (t18:1, t18:0, d18:2, d18:1-Glc, d18:1, 1,4-anhydro-t18:1, and d18:0); (c) inference from elution time (t18:1-Glc, d18:2-Glc, 1,4-anhydro-t18:0, and E/Z isomers); and (d) comparison with previously published data (4, 19, 32). The hydrolysis reaction was found to be essentially complete by 8 h, although overnight hydrolysis was usually more convenient, but became increasingly nonlinear with respect to the amount of starting material at values greater than 10 mg dry weight (data not shown). Consequently, the equivalent of 10 mg dry weight ( 100 mg fresh weight) was used for the majority of analyses. Solubilization of SphingolipidsPrevious studies have shown that standard lipid extraction techniques are poor at solubilizing plant sphingolipids (4, 24). Hydrolysis of total tissue samples from Arabidopsis indicated a sphingolipid content of 192 ± 8.5 nmol g fw1 (Fig. 2 and Table 1). Measurement of sphingolipids in Method I (see "Experimental Procedures") showed that 43% of the total sphingolipids remained insoluble, with the soluble fraction divided between 48% in the lower chloroform phase and 9% in the upper aqueous phase. The total amount of sphingolipids recovered was 173 ± 12 nmol g fw1. Method II, based around extraction of lipids into large volumes of propan-2-ol to inhibit lipases, solubilized 40% of the total sphingolipids. The total amount of sphingolipids measured in this instance was 281 ± 9 nmol g fw1. Method III used a basic, hydrophilic solvent containing substantial amounts of water that was developed for the extraction of inositolphosphoceramides from yeast (30). A similar solvent mix has also been used to extract phosphoinositol-containing sphingolipids from plants (21). Using this solvent, 96% of the 281 ± 6 n mol g fw1 of sphingolipid measured could be solubilized, indicating that a relatively small proportion of sphingolipids are unextractable. Method IV, based upon a mix of propan-2-ol, water, and hexane, was equally efficient, extracting 98% of the 282 ± 6 nmol g fw1 of sphingolipid and was the preferred method for extracting sphingolipids for purification due to the presence of lipase inhibiting propan-2-ol and the neutral pH of the solvent. The efficacy and general applicability of the extraction method was tested by extracting sphingolipids from leaf tissue of Arabidopsis, tomato, and soybean (Fig. 3), where the total amount of sphingolipid solubilized was 98, 96, and 87% of the total in each species, respectively. These data indicate that propan-2-ol/hexane/water mixes may be useful and efficient in extracting sphingolipids from a broad range of samples.
Separation of Neutral and Charged SphingolipidsA clear functional division between the classes of plant sphingolipids is the presence or absence of a charged head group. Ion exchange chromatography has been used to purify phosphoinositol-containing sphingolipids from yeast (33), and this was adapted to the plant extracts. In all three species examined, an enrichment of different LCBs was found in each of the lipid classes. In each species, the anionic sphingolipids consisted of >95% trihydroxy LCBs, with the majority of that as t18:1(8E) (Fig. 4 and Table 2). The neutral fractions contained more variation between species with regard to LCB content. The neutral sphingolipids from Arabidopsis contained mostly t18:1 LCBs (65%) with a greater proportion of t18:1(8Z) than t18:1(8E). The neutral sphingolipids from tomato contained almost entirely d18:2 (>80%), with almost 4 times as much d18:2(4E/8E) as d18:2(4E/8Z). In contrast, soybean neutral sphingolipids were divided between t18:1 (39%) and d18:2 (48%) with approximately equal ratios of 8 E and Z.
HPLC Separation of SphingolipidsIn order to discern how many different species of sphingolipid were present in each class of neutral and anionic lipids, the lipids were separated by normal phase HPLC, from which fractions were collected and analyzed for sphingolipid content (Fig. 5). Both the anionic and neutral sphingolipid classes contained one major sphingolipid. Neutral sphingolipids were identified as ceramide, 2-hydroxy-ceramide, and monohexosylceramide by comparison with purified standards. Mass spectra of the purified neutral sphingolipids matched the existing mass spectra for these compounds and that of the standards. No standards exist for GIPCs, however, and the nature of these compounds was investigated by mass spectrometry. Electrospray Ionization/Mass Spectrometry of Charged Plant SphingolipidsSphingolipids are a diverse class of compounds containing multiple LCBs and fatty acids with varying degrees of saturation and hydroxylation. Fatty acids usually differ by 2 carbon units with an m/z of 28; hence, sphingolipids were identified in each sample as a group of compounds that differed by an m/z of 28. In each case, the fractions containing the majority of the charged sphingolipids (fractions 16 and 17) were infused into the mass spectrometer and a profile typical for sphingolipids identified (Figs. 6A and 7A). The fragmentation scheme for GIPCs is shown in Fig. 8. From Arabidopsis, the major parent ion detected was m/z 1284.8 [M + Na]+ (Fig. 6A). Product ion scans of this ion created a major product ion of m/z 621.1 and m/z 459.1 with lesser fragments of m/z 361.1 and m/z 283.0 (Fig. 6B). Precursor ion scans for m/z 621.1 revealed six sphingolipid species with varying acyl-chain length and degrees of desaturation, indicating that the m/z 621.1 ion represents a common head group fragment (Fig. 6C). The fragmentation of m/z 1284.8 to m/z 621.1 represents a neutral loss of 663.7 atomic mass units (Z0 fragment) and this can be seen as a minor [Z0 + H]+ species at m/z 664.7 in the product ion scan (Fig. 6B). This neutral loss corresponds to the molecular weight of t18:1h24:0 ceramide (N-2-hydroxylignoceroyl-8-phytosphingenine) and is the major ceramide backbone in the charged sphingolipids. Neutral loss scans for loss of 664 found one major species at m/z 1284.9 [M + Na]+ and additional adduct species at m/z 1300.9 [M + K]+ and m/z 1306.9 [M + Na2]+ (Fig. 6D). Using the information provided from these scans, the molecular structure of the major charged sphingolipid from Arabidopsis was proposed to be hexose-hexuronic-inositolphosphoceramide (Fig. 8). The exact identity of the hexose and hexuronic acid could not be assigned based on the mass spectrum. The atomic masses corresponding to particular fragments are tabulated in Table 3.
Mass spectrometry of the equivalent fraction from tomato revealed that the major charged sphingolipid in tomato is not the same as in Arabidopsis. The major peak was at m/z 1320.9 with an associated pattern of peaks at 28 atomic mass units difference (Fig. 7A). The product ion spectrum of m/z 1321 showed that the m/z 1304 ion is rapidly generated from the m/z 1320.9 ion, corresponding to the loss of an ammonium adduct (m/z 17). Two other fragments at m/z 664.7 and m/z 640 are potentially the ceramide and head group, respectively (Fig. 7B). Precursor ion scans of the m/z 640 fragment reveal a cluster of ions separated by 28 atomic mass units, indicating that this is the head group fragment (Fig. 7C). MS3 of the m/z 640 ion produced a spectrum consistent with a parent ion structure N-acetylhexosamine-hexuronic acid-inositolphosphoceramide (Fig. 7D). MS3 of the m/z 664.7 fragment established this as the t18:1-h24:0 ceramide backbone (Figs. 7E and 9). Precursor ion scans with the m/z 664.7 fragment revealed the major peaks at m/z 1321.0 as expected and additional peaks at m/z 1304.0 and 1262.1 (Fig. 7F). This is consistent with the m/z 1321.0 peak being an ammonium adduct [M + NH4]+, the m/z 1304 peak being the protonated ion [M + H]+ and the m/z 1262.1 peak the nonacetylated hexosamine-hexuronic-inositolphosphoceramide. Fraction 16 from soybean produced identical results to tomato (data not shown). The soybean HPLC profile also showed a significant amount of a second charged sphingolipid peak in fraction 22. Mass spectrometry identified numerous compounds in this fraction, but none were pure enough for conclusive identification. In light of this, reexamination of fraction 22 from the HPLC of Arabidopsis anionic sphingolipids revealed a species with an m/z of 1447 that lost m/z 664, consistent with a dihexosyl-hexuronic-IPC species, but the signal was too weak to obtain conclusive spectra (data not shown). Quantification of Sphingolipid ClassesIn theory, it should be possible to separate the sphingolipid classes and quantify the differing amounts in each class, thereby determining the relative proportions in the original tissue. In practice, however, losses occur during the separation process, which, without the addition of internal standards for each compound at the start of the separation, makes absolute quantification impossible. The relative proportion of each class may be calculated based on the quantification of each sphingolipid peak, however (Table 3), and for Arabidopsis this was calculated to be ceramide 1.7%, monohexosylceramide 33.9%, and GIPC 64.4%, making GIPCs the predominant sphingolipid. This percentage is only valid assuming equal losses of each class of compound during the procedure, which does not appear to be the case. Due to the unique distribution of LCBs between the classes, selective losses from one class relative to another cause a change in the molar ratio of the LCB content. This can be seen most dramatically in the tomato and soybean sphingolipid samples (compare totals in Figs. 3 and 4), where the steps involved had been optimized only for Arabidopsis, leading to greater losses from the tomato and soybean samples. Calculation of the proportion of monohexosylceramide to GIPC using the different ratios of t18:1(8Z) and t18:1(8E) in these separated sphingolipids compared with the ratio in total tissue yields a sphingolipid composition of monohexosylceramide 37% and GIPCs 63%. Calculation based on the proportions of t18:1 E to Z in the crude neutral and anionic fractions gives a similar composition of monohexosylceramides 31% and GIPCs 69%. Allowing for the experimental errors involved, these data indicate that, in Arabidopsis, GIPCs are mole for mole approximately twice as abundant as monohexosylceramides.
Plant sphingolipids are receiving increased attention due to the recognition of their roles in a number of fundamental plant processes. Given the large amount of data on sphingolipids in animals and yeast, it is only reasonable to make comparisons between these two systems and plants to look for similarities. Caution needs to be exercised when making such comparisons if the exact sphingolipid content of the organism of interest is unknown, since organisms may vary substantially in their sphingolipid classes and LCB profile.
In this respect, plants are uniquely different from animals and yeast in that the major LCBs t18:1(8E/Z) and d18:2(4E/(E/Z)) are not found in these organisms. The proportion of d18:2 found in a particular species appears to segregate along taxonomic lines, with the Solanacae (tomato and tobacco) having large proportions of d18:2, the Fabacae (pea and soybean) an intermediate amount of d18:2, and the Brassicacae very low to nonexistent levels of d18:2 (Arabidopsis and other brassicas). This may be a reflection of a difference in 4 desaturase activity between the different taxonomic groups. Indeed, all d18:1 in tomato contained the 4 desaturation in contrast to soybean and A. thaliana, where little or no d18:1 4 was detected respectively. In all cases examined, the d18:2 appears largely confined to the glucosylceramide fraction. The functional significance of this division remains to be assessed. A more striking division of the LCBs and one that appears to be conserved between species is the high level of trihydroxy-LCBs in the GIPCs. The uniformity of this feature in the face of diversity in other sphingolipid classes suggests a conserved functional role for the 4-hydroxy of the LCB in GIPCs. Interestingly, the ceramide fraction of neutral lipids is also highly enriched in trihydroxy-LCBs with a t18:1(8E) to 18:1(8Z) ratio similar to GIPCs. This is consistent with free ceramide originating from GIPC hydrolysis, suggesting that GIPCs are turned over much faster than glucosylceramides. Consistent with this, labeling of tomato sphingolipids by feeding labeled serine to leaf discs almost exclusively labels GIPCs and not monohexosylceramides (34). The significance of the increased turnover of GIPCs is not known but may be a result of their recycling between the Golgi apparatus and the plasma membrane, where they are apparently enriched (4). Solubilization of sphingolipids has been a major obstacle to their study. Most lipid extraction techniques utilize extraction into chloroform/methanol mixes and phase partition into chloroform to remove nonlipid contaminants. This does not work well for sphingolipids with large amounts of GIPCs unextracted from the insoluble tissue. Extraction into chloroform-methanol-water mixes is possible if the water content is high. A ratio of 16:16:5 (v/v/v) proves an excellent solvent for GIPCs; however, upon phase partition, the GIPCs become distributed between the chloroform and aqueous phases and the interphase precipitate. A solvent mixture based on propan-2-ol, water, and hexane was finally chosen, because the primary alcohol is known to inhibit lipases and the mixture is relatively innocuous, making handling easier. Sphingolipids are thought to have a higher phase transition temperature than other lipids and are traditionally extracted at higher temperatures; 60 °C used in this study did not affect the lipids themselves and gave adequate solubilization. The vital components of the sphingolipid extraction procedure are effective cell disruption through the use of a close fitting glass homogenizer, the use of solvents containing a significant proportion of water, and incubation at high temperature. The GIPC class of sphingolipids from Arabidopsis consists of just one major species, hexose-hexuronic-inositolphosphoceramide. Under the conditions used here, it naturally formed a sodium adduct in electrospray ionization-mass spectrometry, although no sodium was added to the sample and ammonium acetate was present in the solvent. Previous characterization of GIPCs from fungi has shown that increased sensitivity can be achieved by forming lithium adducts (35). Attempts to displace the sodium by adding lithium iodide or lithium acetate failed to produce significant amounts of lithium adduct up to 5 mM final concentration, and higher concentrations caused ion suppression, indicating a high affinity of the Arabidopsis GIPC for sodium. Despite this, fragmentation and identification of the sodium adduct fragments was possible, although the presence of additional potassium and disodium adducts may represent a complicating factor for future quantification of these lipids by electrospray ionization-tandem mass spectrometry. The major GIPCs from soybean and tomato behaved quite differently from the Arabidopsis GIPC naturally forming ammonium or hydrogen adducts, presumably due to the difference in structures of the GIPCs between these species. In all cases, the plant GIPCs appear to most readily fragment at the Z0 position, which requires MS3 to extract further information about the ceramide backbone. This can easily be achieved by MS3 in the Q-TRAP 4000 or by setting the declustering potential high enough to cause in-source fragmentation of the parent ion followed by conventional tandem mass spectrometry (35). In summary, procedures necessary to determine the sphingolipid content of a target, model plant species, in this case A. thaliana, have been established, and it has been shown that they are generally applicable to other species. This will provide the opportunity to establish an Arabidopsis sphingolipidomics program for separating and identifying all of the major sphingolipid species. Future high-throughput screening procedures should enable detailed characterization of sphingolipid and sphingolipid signaling mutants and increase our understanding of the role of sphingolipids in Arabidopsis.
* This work was supported by National Science Foundation 2010 Grant 0312559. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1 To whom correspondence should be addressed: Donald Danforth Plant Science Center, 975 N. Warson Rd., Saint Louis, MO 63132. Tel.: 314-587-1644; Fax: 314-587-1744; E-mail: jmarkham{at}danforthcenter.org.
2 The abbreviations used are: GIPC, glycosylinositolphosphoceramide; HPLC, high performance liquid chromatography; fw, fresh weight; LCB, long-chain base; IPC, inositolphosphoceramide; MS3, hybrid tandem mass spectrometry with linear ion trap fragmentation.
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