Phosphatidylcholine Synthesis - Plant Physiology

Plant Physiol. (1988) 88, 854-8610032-0889/88/88/0854/08/$01.00/0

Phosphatidylcholine SynthesisDIFFERING PATTERNS IN SOYBEAN AND CARROT

Received for publication March 14, 1988 and in revised form June 20, 1988

ANNE H. DATKO AND S. HARVEY MUDD*'Laboratory ofGeneral and Comparative Biochemistry, National Institute ofMental Health,Bethesda, Maryland 20892

ABSTRACr

The methylation steps in the biosynthesis of phosphatidylcholine bytissue culture preparations of carrot (Daucus carota L.) and soybean(Glycine max), and by soybean leaf discs, have been studied. Preparationswere incubated with tracer concentrations of L-[H3Cqmethionine and thekinetics of appearance of radioactivity in phosphomethylethanolamine,phosphodimethylethanolamine, phosphocholine, phosphatidylmethyl-ethanolamine, phosphatidyldimethylethanolamine, phosphatidylcholine,methylethanolamine, dimethylethanolamine, and choline followed atshort incubation times. With soybean (tissue culture or leaves), an initialmethylation utilizes phosphoethanolamine as substrate, forming phos-phomethylethanolamine. The latter is converted to phosphatidylmeth-ylethanolamine, which is successively methylated to phosphatidyldi-methyethanolamine and to phosphatidylcholine. With carrot, again, aninitial methylation is of phosphoethanolamine. Subsequent methylationsoccur at both the phospho-base and phosphatidyl-base levels. Both ofthese patterns differ qualitatively from that previously demonstrated inLemna (SH Mudd, AH Datko 1986 Plant Physiol 82: 126-135) in whichall three methylations occur at the phospho-base level. For soybean andcarrot, some added contribution from initial methylation of phosphati-dylethanolamine has not been excluded. These results, together withthose from similar experiments carried out with water-stressed barleyleaves (WD Hitz, D Rhodes, AD Hanson 1981 Plant Physiol 68: 814-822) and salinized sugarbeet leaves (AD Hanson, D Rhodes 1983 PlantPhysiol 71: 692-700) suggest that in higher plants some, perhaps all,phosphatidylcholine synthesis occurs via a common committing step(conversion of phosphoethanolamine to phosphomethylethanolamine)followed by a methylation pattern which differs from plant to plant.

In higher plants the quantitatively dominant utilization ofmethionine methyl groups is for the synthesis of PtdCho.2 Forexample, we have shown that, in Lemna paucicostata, the meth-yls used for this purpose exceed by more than two-fold the totalmethyls in protein methionine, and are more than the sum ofthe methyls in all other methylated end products combined (16).Recent studies in this laboratory produced evidence that, inLemna, the substrates for the methylations involved in PtdCho

' Reprint requests should be addressed to the authors at Building 36,Room 3D06, National Institute of Mental Health, Bethesda, MD 20892.

2 Abbreviations: phosphate esters are designated by the prefix, P- (e.g.P-EA or P-Cho); the corresponding phosphatidyl derivatives, by theprefix, Ptd (e.g. PtdEA or PtdCho); and the corresponding glycerylphos-pho-bases by the prefix, GP- (e.g. GP-EA or GP-Cho). AdoMet, S-adenosyl-L-methionine; EA, ethanolamine; MEA, methylethanolamine;DMEA, dimethylethanolamine; Cho, choline.

biosynthesis are almost exclusively water-soluble derivatives ofP-EA. Intact plants were incubated under normal growth con-ditions with methionine labeled with radioactivity in the methylgroup. After relatively short incubations (e.g. 1 min) at least 99%of the total radioactivity in all methylated EA derivatives wasfound in P-MEA, P-DMEA, and P-Cho. After longer incuba-tions, increasing proportions of such radioactivity was found inPtdCho until, finally, 89% was in the latter compound withvirtually all the remainder in soluble Cho (7%) and P-Cho (4%)(17). Similar experiments have now been extended to suspensioncultures of both soybean and carrot, and to leaves of soybean.The labeling patterns observed were qualitatively different foreach of these plants, and different also from the pattern previ-ously observed with Lemna (17). We are led to suggest that thepathway for PtdCho biosynthesis in each of the three plantsinvestigated is significantly different. These results, and ourinterpretations, are presented here. Some of these experimentshave been reported in preliminary form (18).

MATERIALS AND METHODS

Plant Tissue Culture. Soybean (Glycine max cv Peking, ob-tained from Dr. Lowell Owens, USDA/ARS, Beltsville, MD20705) cell suspension cultures were maintained at about 27C,with shaking, in Gamborg's B5 medium containing 1 mg/L of2,4-D. Stock cultures were transferred weekly. Cell density forinitiation of experimental cultures was estimated as wet weight(by filtration on Miracloth, vacuum applied for 1 min), or as cellvolume (hematocrit tube, gentle centrifugation at 45 on Inter-national centrifuge, 5 min). Separate experiments showed that0.1 mL packed cells was equivalent to 5.6 mg wet weight.Cultures to be used for labeling with methionine were initiatedat about 13 mg wet weight/mL of growth medium, and grownfor 4 days, at which time the wet weight had increased to about30 to 35 mg/mL.

Carrot (Daucus carota L. cv Danvers, obtained from Dr.Benjamin F. Matthews, USDA/ARS, Beltsville, MD 20705) cellsuspension cultures were maintained at about 27°C, with shaking,in a defined liquid medium (12). Stock cultures were transferredweekly. Cell density for initiation of experimental cultures wasdetermined by weight as described for soybean, or by measure-ment of cell volume (centrifugation at a setting of 30, Interna-tional centrifuge, 5 min). Separate experiments showed that 0.1mL packed cells was equivalent to about 30 mg wet weight.Cultures which were to be used for labeling with methioninewere initiated with 6 to 7 mg wet weight/mL medium, andallowed to grow for 3 d, at which time the weight had increasedto about 25 mg/mL.Lemna Growth in the Dark. Dark-grown plants of Lemna

paucicostata Hegelm. 6746 were produced by growth under ourusual standard conditions (2) except that the medium contained1 gM cytokinin, the growth chamber was darkened, and the

854

Dow

nloaded from https://academ

ic.oup.com/plphys/article/88/3/854/6083332 by guest on 28 N

ovember 2021

PHOSPHATIDYLCHOLINE SYNTHESIS

plants received 15 to 20 min red light each 4 h. Plants weresubcultured at d 6, taking care to avoid use of colonies stillcontaining original green fronds. At d 11, 150 fronds were usedfor labeling with [3H3C]methionine. Growth rate in the dark wasminimally slower (doubling time perhaps 40 h) than in the light(doubling time 34-36 h).

Labeling with L-I3H3ClMethionine. Standard Procedures.Lemna plants growing under steady state conditions were labeledcontinuously with L-[3H3C]methionine and processed to yield awashed methanol-chloroform-insoluble pellet and componentssoluble in either methanol-water or chloroform-methanol, all aspreviously described (16, 17).

Suspension cultures of soybean or carrot were labeled contin-uously with L-[3H3C]methionine while maintained in freshbatches of their respective growth media. At the end of eachincubation, cells were harvested by rapid filtration under verymild suction and quickly washed several times on the filter padwith ice-cold growth medium. The cake of washed cells wastransferred to a wide-mouthed glass homogenizing tube andhomogenized in methanol:chloroform:2 M formic acid (12:5:3),initially cooled almost to dry-ice temperature (1). The resultinghomogenate was centrifuged to yield a pellet and a supernatantfraction which was transferred to a 12 mL conical centrifugetube and evaporated to dryness at room temperature. The pelletwas extracted with 3.9 mL methanol:chloroform (2: 1), while thesmall residue resulting from evaporation of the original super-natant fluid was extracted with a further 0.9 mL of metha-nol:chloroform (2: 1). After centrifugation, the two supernatantswere combined and separated into methanol-water soluble andchloroform-methanol-soluble fractions (3). The two pellets werecombined by transfer of the smaller one with methanol washes.The combined pellet was washed, and the washes combined withthe methanol-water-soluble fraction, obtained above, for analy-sis, all as previously described (16, 17).Soybean (Glycine max cv Peking) seedlings were maintained

in a greenhouse, in summer, with natural light. Young, expand-ing leaves were detached from the plant and cut into 3x3 cmsquares. These were placed on filter paper thoroughly moistenedwith a medium containing sorbitol, 0.3 M; HEPES buffer, pH6.5, 50 mM; polyvinylpyrrolidone, 0.5%; BSA, 1%; DTT, 2 mM(22); and L-[3H3C]methionine, 350 nM; and sliced through thelower cuticle (attempting to leave the upper cuticle largely intact)with a sharp razor at intervals to "dice" the leaf into approxi-mately 1 mm squares. The diced leaf tissue was placed, lowerside down, in a vacuum desiccator between two layers of filterpaper moistened with the same medium. After several shortperiods (approximately 30 s each) ofevacuation and readmissionof air, the infiltrated tissue was incubated under a light until atotal of 10 min had elapsed from the beginning of infiltration.After rapid transfer to a mortar, the tissue was washed threetimes with cold, nonradioactive medium which was removedeach time by aspiration. A small amount of liquid nitrogen wasadded to freeze the tissue, then allowed to evaporate. The tissuewas ground with methanol:chloroform:2 M formic acid (12:5:3),and the homogenate worked up as described above for thehomogenates of cultured cells.

Chromatography. Except as otherwise noted, paper chroma-tography was carried out on Whatman No. 1 paper using thedescending method. Solvents were: solvent A, phenol, 125 g: 1-butanol, 125 mL:88% HCOOH, 6.8 ml:H20, 25.7 mL (chro-matography paper predipped in 1 N KCI and dried); solvent B,2-propanol:88% HCOOH:H20 (7:1:2) (v/v); solvent C, 2-pro-panol:29% NH40H:H20 (7:1:2) (v/v); solvent D, methanol:88%formic acid:H20 (80:14.8:5.2) (v/v) (ascending development forapproximately 6 h); solvent E, chloroform:methanol:aceticacid:H20 (65:35:8:4) (v/v) (ascending development, overnight,on Whatman SG 81 silica gel chromatography paper); solvent F,

chloroform:methanol:28% NH40H (65:35:5) (v/v) (ascendingthin layer chromatography on plates of silica gel H for 90 min)(19). (The above solvents have been named to conform to thedesignations used previously [ 17]).

Other Methods. Methods have been described for paper elec-trophoresis, phosphatase treatment, mild alkaline deacylation,acid hydrolysis of phosphatidyl derivatives or glycerylphospho-bases to the free bases, methylation with CH3I, location ofradioactive compounds on chromatograms and electrophoreto-grams, and elution of such materials (16, 17). For location andquantitative elution of radioactive materials from thin layerplates of Silica Gel H, the gel from 0.5 cm sections of thedeveloped chromatograms were scraped into individual centri-fuge tubes and eluted successively with chloroform:methanol(2:1), twice with chloroform:methanol:H20 (200:97:3), and oncewith methanol (21). Radioactivity was determined in aliquots ofthe combined eluates to locate and quantitate peaks of radioac-tivity. Authentic internal standards of radioactive markers (3H-labeled when the unknowns were ''C-labeled;'4C-labeled whenthe unknowns were 3H-labeled) were used to locate unknownpeaks of interest and to monitor recoveries. In general these wereexcellent (75-95%), with most losses occurring during elutions.To prevent such losses from affecting the calculated amount ofradioactivity in a compound which had been subjected to mul-tiple sequential chromatographic purification steps, the fractionof each compound in the starting material for any given step wasexpressed as a fraction of the total radioactivity on the chroma-togram for that step. The portion of the total radioactivity in theinitial extract which was present as the compound of interestcould then be calculated by multiplication of these fractions forthe entire purification sequence.

Radioactive Compounds. Sources or methods for preparationof most radioactive compounds have been specified (17). L-[3H3C]Methionine, 184 to 188 x 106 dpm/nmol, was purchasedfrom Amersham. Before use it was purified by dissolving in 0.3mL potassium phosphate buffer, pH 6.9, 0.01 M, and passagethrough a column of Dowex 50-NH4', 0.8 x 1.5 cm. The flow-through was collected at 0°C in a tube to which had been added0.3 mL containing 0.1 mM 2-mercaptoethanol and 0.1 mMformic acid. The column was washed with cold water until thetotal volume in the collecting tube was 3.0 ml. Small aliquots ofthis purified preparation were stored frozen and thawed individ-ually immediately prior to use in a labeling experiment.A mixture of [3H3C]PtdMEA/PtdDMEA was used in a recov-

ery experiment. The mixture was prepared by incubation ofsoybean cells for 3 min with L-[3H3C]methionine. The resultingchloroform-methanol-soluble fraction was chromatographedpreparatively with solvent E, and the combined peak of labeledPtdMEA/PtdDMEA eluted and used.

Purification of Compounds Derived from Labeling Experi-ments. Individual methylated EA derivatives derived from plantsor tissues labeled with L-[3H3C]methionine were purified, andthe amount of radioactivity in each determined, as follows.Methylated derivatives ofP-EA were purified from the methanol-water-soluble fraction by sequential electrophoresis at pH 7 andchromatography with solvent B. The individual methylated com-pounds did not resolve during these steps, but did so duringsubsequent chromatography with solvent C. The resulting indi-vidual phospho-bases were hydrolyzed to free bases by treatmentwith phosphatase and their purity and identity confirmed bychromatography with solvent A, a procedure which separates theindividual EA derivatives.

Methylated derivatives of PtdEA were purified from the chlo-roform-methanol-soluble fraction by chromatography with sol-vent E, which separated PtdCho from an area to which PtdMEAand PtdDMEA travelled together. These two areas were elutedseparately and subjected to acid hydrolysis to form the free bases.

855

Dow



ovember 2021

Plant Physiol. Vol. 88, 1988

The latter were electrophoresed at pH 7.0 (in one early experi-ment chromatography with solvent B was substituted for thisstep), then chromatographed with solvent A to separate theindividual free bases.

Free bases were purified from the methanol-water-soluble frac-tion by electrophoresis at pH 7.0, chromatography with solventB (the latter step was omitted in some experiments), and finallyseparated from one another by chromatography with solvent A.

Determination of the Amount of Each PtdEA Derivative. Todetermine the amount of each PtdEA derivative in cultured cells,suspension cultures of carrot or soybean were grown virtually toisotopic equilibrium (4.4 doublings for carrot and at least 2.3doublings for soybean) in media containing 32P-inorganic phos-phate of known specific activity. To provide convenient internal3H markers of the methylated PtdEA derivatives, each prepara-

tion was incubated with L-[3H3C]methionine, 6 to 7 nm, for 15min immediately prior to harvest. The chloroform-methanol-soluble fractions from each were chromatographed with solventE. Three areas of the resulting chromatograms were elutedseparately:

PtdCho. Material in the area containing the peak of PtdChowas eluted and subjected to mild alkaline deacylation to yieldthe water-soluble GP-Cho derivative. The latter was chromato-graphed with solvent D, giving a single 32P,3H-containing peak,the 32p content of which permitted calculation of the PtdChocontent of the original preparation of suspension culture.PtdDMEA. The more slowly moving portion of the combined

[3H3C]PtdMEA/PtdDMEA peak contained relatively little 32p.After elution and preparative chromatography with solvent F,this material was found to contain virtually all the [3H3C]Ptd-DMEA as well as a minor amount of [3H3C]PtdMEA. Mildalkaline deacylation of the PtdDMEA derivative, followed bychromatography of the resulting GP-DMEA with solvent Cyielded a single 32P,3H-containing peak from which the tissuecontent of PtdDMEA was calculated.PtdMEA and PtdEA. The more rapidly moving portion of the

combined [3H3C]PtdMEA/PtdDMEA peak was eluted togetherwith a major peak of 32p which overlapped and ran as far forwardas the beginning of the peak of 3H-labeled neutral lipid. Themajor 32P-labeled peak was known to contain PtdEA. Chroma-tography with solvent F did not resolve PtdMEA from PtdEA.Accordingly, after addition back of the minor amount ofPtdMEA from the chromatography with solvent F of the Ptd-DMEA fraction (see above), the mixture of PtdMEA and PtdEAwas subjected to mild alkaline deacylation. To the water-solubleproducts was added a marker of authentic ['4C]GP-EA, and thewhole chromatographed with solvent C. The resulting peaks ofpartially, but incompletely, resolved [3H,32P]GP-MEA andI14C,32PJGP-EA were each eluted separately and chromato-graphed with solvent D. The resulting peaks of these two com-pounds were sufficiently resolved that an unambiguous calcula-tion could be made of the original contents of both PtdMEA andPtdEA.

RESULTS

Preliminary Experiments. As a first step in developing methodsand conditions suitable for experiments with tissue culturedmaterial, a pilot experiment was performed with a soybeansuspension culture incubated for 10 min in the presence of 2.5nM L-[3H3C]methionine. In an effort to minimize the action of a

solvent-activated phospholipase D which has been reported inplant tisues or cells incubated with aqueous methanol (9, 20),the cells were extracted with isopropanol heated almost to boiling(8). The resulting homogenate was worked up essentially asdescribed for the standard procedure in "Materials and Meth-ods." The results obtained differed in several ways from thoseexpected on the basis of similar experiments with Lemna (17).

20

ii

E '

~07C

21

25

20

1cIe 70

?5

0o

0o

a0

ao

a0

0

0 10 20 30

DISTANCE FROM ORIGIN (cm)

E

CL

40

FIG. 1. Representative chromatograms of phospho-bases. In eachcase, the material chromatographed contained the combined phospho-base fraction prepurified by electrophoresis at pH 7.0, followed bychromatography with solvent B. Chromatography with solvent C isillustrated. In this figure, as in all others in this paper, radioactivity dueto authentic internal standards (added as appropriate before each step) isindicated by dashed lines (and referred to the right-hand ordinate). Eachsuch peak is named. Radioactivity due to the material under study isindicated by solid lines (and referred to the left-hand ordinate). Toppanel, 1 min incubation with Lemna (see Ref. 17); middle panel, 3 minincubation with soybean suspension culture (see Table I); bottom panel,10 min incubation with carrot suspension culture (see Table II). Theslight displacement between the '4C and the3H peaks in the P-DMEAarea (bottom panel) is due to isotope effects.

First, rather than all three of the P-EA derivatives, P-MEA, P-DMEA, and P-Cho, becoming labeled with radioactive methylgroups, with soybean suspension culture only P-MEA was solabeled, containing 0.7% of total tissue radioactivity (with P-DMEA and P-Cho each containing less than 0.01%). Second,radioactivity entered the PtdEA derivatives relatively rapidly,and substantial amounts were present not only in PtdCho (ashad been the case with Lemna), but also in both PtdMEA andPtdDMEA. The results with soybean were similar to thosewith Lemna in that the free EA bases contained relatively littleradioactivity.To further confirm these unexpected results, the identities of

the crucial compounds from soybean were confirmed:

0-a

Carrot Cells

aC P-MEaC-P-DMEA

a L~~~~~~~~~~~~~~~~~~-4

1C~~~~~~~~~~~~~~~~~~~~~~~~~~~~~1ori - ~~~~~~~~~~~~~~~~~ 0U~~~~~~~~~~~~~~~~~~~ l0

856 DATKO AND MUDD

0

0

0

50

Dow



ovember 2021


P-MEA. A portion of the free base obtained by the action ofphosphatase on this material was treated with CH3I. The newlyformed methylated products moved with the expected com-

pounds, DMEA and Cho, during chromatography withsolvent A.MethylatedPtdEA Derivatives. The PtdCho and the combined

PtdMEA/PtdDMEA peaks resulting from chromatography withsolvent E were each chromatographed preparatively with solventF. The latter solvent resolves the three PtdEA derivatives inquestion. After elution of the separated peaks, and acid hydrol-ysis, the radioactive free bases obtained moved with the expectedauthentic compounds during chromatography with solvent A.During the course ofthese, and other, preliminary experiments

we found that the use of hot isopropanol to extract the tissueswas causing some breakdown of phosphatidyl bases to the cor-

responding free bases, accompanied by the appearance of artifac-tual radioactive material which travelled close to the combinedpeak of PtdMEA/PtdDMEA during chromatography with sol-vent E. These difficulties were avoided in subsequent experi-ments by use of methanol:chloroform:2 M formic acid, cooled todry-ice temperature as extractant, a procedure developed tominimize phosphatase activities in plant tissues (1).

Labeling Experiments with Soybean Suspension Cultures.After standardization of methodology, as described above, a

series of experiments were performed in which soybean suspen-sion cultures were incubated for various periods with L-[3H3C]methionine, and the amount of radioactivity in each EA deriv-ative determined. Representative illustrations of the results ob-

tained, and the purifications achieved, for the most importantcompounds are shown in Figures 1 to 4. In these figures, typicalresults obtained previously with Lemna (17) are shown also toillustrate the qualitative differences between the two plants.Figure 1 illustrates that during brief incubations, whereas withLemna 3H entered P-MEA, P-DMEA, and P-Cho in substantialamounts, with soybean only P-MEA became labeled. Figure 2shows that, after phosphatase treatment of the putative [3H3C]P-MEA from soybean, a single radioactive product was obtained,moving, as expected, with MEA in a solvent which resolves EAand its three methylated derivatives. In Figure 3 it is seen that,whereas with Lemna very little radioactivity entered any phos-phatidyl derivative at early incubation time, with soybean 3Hlabeled not only PtdCho, but also a faster moving peak with RFof 1.4 to 1.5 relative to PtdCho (and running just behind thepeak of neutral lipid). Figure 4 shows that after elution of the

1250

1000

750E

I-

2'500

250

600

400

200

0

1800

1500

1200

E 900

-a

600

300

400

300

200

100

0

100

50

E

-0

50(3j

25

0

50

25

10 20 30

DISTANCE FROM ORIGIN (cm)0

FIG. 3. Representative chromatograms of chloroform-methanol-sol-uble fractions. Aliquots of these fractions from the same experimentsillustrated in Figure I were chromatographed with solvent E.

E

I.

20,00

15,0C

10,0(

50C

E

ci


FIG. 2. Chromatography of phosphatase-treated P-MEA from soy-

bean. The material eluted from the area containing the P-MEA peak (cm20-25) shown in the middle panel of Figure 1 was treated with phospha-tase and chromatographed with solvent A. The very small peaks movingin front of each major peak (e.g. "C at cm 6-8, in front of ['4C]EA atcm 3-5) are due to minor "twinning" of each free base during chroma-tography in this system.

Do

00 E

I-

Q

0 10 20 30 40


FIG. 4. Chromatography of acid-hydrolyzed PtdMEA/PtdDMEAfrom soybean. The material eluted from the combined PtdMEA/Ptd-DMEA peak (cm 20-26) shown in the middle panel of Figure 3 was

subjected to acid hydrolysis, and electrophoresed at pH 7.0. More than96% ofthe 3H radioactivity migrated to the area to which the methylatedEA free bases move as a group. Material in the latter area was eluted andchromatographed with solvent A, producing the results illustrated here.

peak in question and acid hydrolysis the major portion of theradioactivity now moved with DMEA, with, however, a readilydetected smaller peak moving with MEA. It was concluded thatthe peak on the original chromatogram (Fig. 3) had been com-posed of a mixture of PtdMEA and PtdDMEA.

857

Soybean Cells

14C-DMEA

114C-MEA~ I 2(DO 14~~~~~IC-Choline

| 4C-EA

r --o 0

Dow



ovember 2021

Plant Physiol. Vol. 88, 1988

The results obtained in all such labeling experiments carriedout with soybean suspension cultures are summarized in TableI. At all times up to 15 min, methyl groups originating inmethionine labeled P-MEA only among the phospho-bases, butall three of the methylated PtdEA derivatives. Radioactivity inany methylated EA free base was minimal at such times. In thistable, radioactivity in any given compound has been expressedrelative to that in PtdCho. This is convenient because Choderivatives, especially PtdCho, are the end products into whichalmost all of the radioactive methyls entering this group ofcompounds finally flow. This is shown by the results in the lastcolumn ofTable I. After an experiment in which a 3 min labelingperiod had been followed by a 26 h period ofgrowth in standard,nonradioactive medium, virtually all detected radioactivity wasin Cho derivatives, with PtdCho containing some 83% of thetotal, and far lesser amounts in free Cho and P-Cho.3

Labeling Experiment with Soybean Leaf. To examine whethersuch results were characteristic of a more differentiated soybeantissue, an experiment was performed with soybean leaf discsinfiltrated with [3H3C]methionine and incubated for 10 min. Theresults were as follows (expressed as radioactivity in the specifiedcompound relative to that in [3H]PtdCho which, itself, contained0.54% of total tissue radioactivity): P-MEA, 39%; P-DMEA, notdetected (<0.5%); P-Cho, not detected (<0.5%); PtdMEA, 19%;

Table I. Labeling ofEA Derivatives in Soybean Suspension Cultureswith L-PH3C]Methionine

Suspension culture material was incubated in growth medium for thetimes indicated in the presence of the following concentrations of L-[3H3C]methionine, 186 x 106 dpm/nmol: 0.63 min, 8.3 nM; 1.1 min, 8.3nM; 3 min, 2.2 nM; 15 min, 7.0 nM; 3 min (with 26 h chase innonradioactive medium), 3.1 nM. In the chase experiment, after thelabeling period the cells were washed free of radioactive medium priorto beginning the chase incubation. At the ends of the incubations, tissueswere processed as described under "standard procedures" in "Materialsand Methods," except that the material from the chase experiment, only,was extracted with hot isopropanol. Total dpm in the washed tissueswere 711, 1230, 2920, 6830, and 654 (each x 103) in the order listed.Radioactivity in each EA derivative was determined as described in"Materials and Methods."

Time of Labeling (min)Metabolite 1.1 3.0 15 3 (with 26

h chase)

radioactivity, % ofthat in PtdChoP-MEA 74 70 76 23 1P-DMEA 0 0 0 0 0P-Cho 0 0 0 0 4PtdMEA <1 3 8 16 0PtdDMEA 134 82 81 79 0PtdChoa 100 100 100 100 100MEA 0 0 <1 <1 0DMEA 0 1 <1 < 1 0Cho 0 <1 2 <1 16

a The radioactivity in PtdCho as a percent of the total tissue radioac-tivity in each experiment was the following: 0.63 min, 0.14; 1.1 min,0.25; 3 min, 3.1; 15 min, 3.5; 3 min (with 26 h chase), 12.2.

3In distinction to all other experiments reported in Table I, hotisopropanol was used as extractant in the chase experiment (which wascarried out early in the course of these studies). The results obtained arethought to be so unequivocal in demonstrating that choline derivativesare the end product of the methyl flow in question that it was notnecessary to repeat this experiment using the standard extractant of coldmethanol:chloroform:2 M formic acid.

PtdDMEA, 84%; PtdCho, 100%; combined methylated EA freebases, <3%.

Labeling Experiment with Bleached Lemna. To further ex-amine whether the results with soybean suspension cultures wereaffected by the fact that such cultures are nonphotosynthetic, anexperiment was carried out with Lemna plants bleached byhaving been grown in the dark. Following a 3 min incubationwith L-[3H3C]methionine, the results were very similar to thosepreviously reported for unbleached Lemna (17). Each methylatedP-EA derivative was strongly labeled, with P-MEA, P-DMEA,and P-Cho containing, respectively, 1.1, 1.3, and 1.2% of totaltissue radioactivity. Radioactivity in phosphatidyl derivatives wasminimal (<0.07% of total tissue radioactivity in any such com-pound). Radioactivity in methylated EA free bases was alsominimal (0.01, 0.09, and 0.13% of total tissue radioactivity inMEA, DMEA, and Cho, respectively).

Labeling Experiments with Carrot Suspension Cultures. Inview of the striking differences in the labeling patterns observedbetween Lemna and soybean, it was decided to extend thesestudies to a third plant tissue. The results of a series of experi-ments with carrot suspension cultures, similar in design to thoseperformed with the soybean suspension cultures, are illustratedin Figures 1 and 3, and fully summarized in Table II. Surpris-ingly, the pattern with carrot was different from that with eitherLemna or soybean: Methyl groups from methionine not onlyrapidly labeled all three methylated P-EA derivatives, as hadbeen observed with Lemna, but not with soybean, but alsorapidly labeled all three methylated PtdEA derivatives, as hadbeen observed with soybean, but not with Lemna. Again, labelingof any EA-containing free base was minimal. As with soybean,a chase experiment (Table II, last column) demonstrated thatCho derivatives, especially PtdCho, are the end-products intowhich all these methyl groups eventually flow.

Concentrations of PtdEA and Its Methylated Derivatives. Bylabeling suspension cultures of soybean or carrot virtually toisotopic equilibrium with [32P]inorganic phosphate of knownspecific activity, and purifying PtdEA and each of its methylatedderivatives, first in the phosphatidyl form, then as the corre-sponding glycerylphospho-base, the tissue contents of each of thephosphatidyl compounds was determined. The results are sum-

Table II. Labeling ofEA Derivatives in Carrot Suspension Cultureswith L-[3H3C]Methionine

Experimental conditions and procedures were similar to those de-scribed in the legend to Table I. The concentrations of L-[3H3C]methio-nine were as follows: 3 min, 5.5 nM; 10 min, 5.5 nM; 15 min, 6.2 nM; 10min (with 26.6 h chase in nonradioactive medium), 5.6 nM. All sampleswere extracted in the standard way. Total dpm in the washed tissueswere 996, 841, 2010, and 2420 (each x 103) in the order listed.

Time of Labeling (min)Metabolite 10 (with 26.6

3.0 10 15 h chase)radioactivity, % ofthat in PtdCho

P-MEA 103 62 46 <0.1P-DMEA 47 34 22 <0.1P-Cho 22 14 11 0.5PtdMEA 7 10 19 0.9PtdDMEA 74 70 49 1.2PtdChoa 100 100 100 100MEA <1 <1 2 <0.1DMEA <1 <1 <1 <0.1Cho <1 <1 1 7.8

a The radioactivity in PtdCho as a percent of the total tissue radioac-tivity in each experiment was the following: 3.0 min, 1.3; 10 min, 2.5;15 mim, 5.7; 10 mi (with 26.6 h chase), 23.6.

858 DATKO AND MUDD

Dow



ovember 2021


marized in Table III, and compared there to values reportedpreviously for Lemna (16).

DISCUSSION

During the course of extending studies of the kinetics oftransfer of the methyl groups originating in methionine into thenetwork of methylated derivatives of EA, we have observedstriking qualitative differences for the patterns between Lemna(17), soybean, and carrot. Although in each plant PtdCho isquantitatively the overwhelmingly dominant end product intowhich methyls flow once they have entered this network, theintermediates in the process appear to vary. Various possiblecauses were considered as explanations of these differences: (a)Evidence against the possibility that the difference was due tosuspension cultures being non-photosynthetic, whereas Lemnaplants were photosynthetic, was provided by the demonstrationsthat infiltrated leaf discs of soybean gave patterns very similar tosoybean suspension cultures, and that bleached Lemna gavepatterns very similar to green Lemna. (b) Evidence against thepossibility that the pattern initially observed with soybean sus-pension cultures was a property of undifferentiated tissue,whereas that seen with Lemna was a property of differentiatedtissue, was provided, again, by the demonstration that the patternfor soybean suspension culture was the same as that for soybeanleaf, and further, by the subsequent finding that the two undif-ferentiated suspension cultures of soybean and carrot differedstrikingly from one another. We conclude, therefore, that eachof the three plants in question has a characteristic, and different,biosynthetic route for PtdCho formation.What conclusions do the present experiments permit as to the

details of these biosynthetic routes? The pattern for Lemna, inwhich at early times virtually all newly introduced methyl groupsare found in P-MEA, P-DMEA, and P-Cho seems quite une-quivocally to suggest that methylations occur almost exclusivelywith the phospho-bases as substrates, ending with P-Cho. Thelatter is then presumably converted successively to CDP-choline(by the action of CTP:P-Cho cytidylyltransferase), and finally toPtdCho (by the action of CDP-Cho: 1,2-diacylglycerol P-Cho-transferase). This interpretation has been put forth previouslyand discussed (17), and is diagrammed in the top panel of Figure5. In experiments with cell-free extracts of Lemna, we haverecently demonstrated the phospho-base N-methyltransferase ac-tivities called for by this scheme (18).The results with soybean seem also to be amenable to a

straightforward interpretation. We suggest that in this tissue, asin Lemna, the initial methylation is of P-EA, forming P-MEA.However, P-MEA, rather than being further methylated, insteadis transferred (via the CDP-MEA derivative and the actions of

Table III. Concentrations ofPtdEA and Methylated PtdEA DerivativesDeterminations were made of phosphatidyl derivatives after labeling

virtually to isotopic equilibrium with 32P-inorganic phosphate, as de-scribed in "Materials and Methods". Values for Lemna have beenpreviously published (16), and are shown here for comparison. Valuesare shown relative to PtdCho. The approximate absolute concentrationsof PtdCho were as follows: Lemna, 0.8 nmol/frond, or 1.6 nmol/mg wetweight (16); soybean, 7.0 nmol/mg wet weight, or 720 nmol/,umol P inthe washed pellet; carrot, 5.0 nmol/mg wet weight, or 340 nmol/jimol Pin the washed pellet.

Tissue PtdEA PtdMEA PtdDMEA PtdCho% (relative to PtdCho)

Lemna plants 38 NDa ND 100Soybean culture 80 11 0.6 100Carrot culture 46 8 1 100

the appropriate cytidylyltransferase and P-MEAtransferase) toform PtdMEA. The latter is successively methylated to Ptd-DMEA and PtdCho (Fig. 5, middle panel). This working modelaccounts for the appearance of labeled methyls in P-MEA onlyamong the phospho-bases, and for the rapid appearance of labelin PtdMEA and PtdDMEA, as well as in PtdCho. Note that thepool size of PtdMEA, and especially that of PtdDMEA, is smallrelative to that of PtdCho (Table III), so that the labeled methylsmight make their way to PtdCho relatively quickly. In its simplestform, this model predicts that PtdMEA should be relativelyhighly labeled at early times compared to PtdDMEA andPtdCho, and become less so as incubation times progress. Be-cause this prediction is not borne out by the experimental results(Table I), we suggest the pool of PtdMEA may not be uniform,but rather is composed of at least a rapidly turning-over portionthrough which methyls pass rapidly to PtdDMEA, and a moreslowly turning-over portion which progressively accumulatesradioactivity during the first minutes of incubation.An alternative route for formation of PtdMEA, compatible

with the in vivo labeling data, would involve the N-methylationof PtdEA. This is the dominant pathway in animals and micro-organisms (4, 6). In experiments with cell-free extracts of soy-bean, we have demonstrated the presence of an AdoMet:P-EAN-methyltransferase activity, but failed to detect either P-MEAor P-DMEA N-methyltransferases. Further, we found bothAdoMet:PtdMEA and PtdDMEA N-methyltransferases, but didnot detect a PtdEA N-methyltransferase (18). These findings areconsistent with the sole route to Ptd-MEA being through P-MEA, as shown in Figure 5, but certainly do not rule out someparticipation of an as-yet-to-be demonstrated PtdEA N-methyl-transferase. It is noted that such a pathway would encounter thesame difficulty and necessitate the same assumption as to thenonhomogeneity of PtdMEA as was made above.The results with carrot are most equivocal in terms of the

methylation pathway(s) utilized. As a tentative working model,we propose the scheme shown in Figure 5, bottom panel. Again,the initial methylation is placed at P-EA. The resulting P-MEAis partially converted to PtdMEA (the dominant pathway insoybean), partially methylated to P-DMEA (the dominant path-way in Lemna). The P-DMEA also is partitioned between con-version to PtdDMEA and methylation to P-Cho. The P-Cho isconverted to PtdCho. Of course each partially methylated phos-phatidyl derivative is subjected to further methylation, so thatfinally almost all is used to form PtdCho. It is difficult to specifythe relative importance of the three cytidylyltransferase/methyl-ated P-EA-basetransferase sequences postulated in this model.For the moment, in Figure 5 each has been tentatively assignedroughly equal proportions of the total flux.

Studies with cell-free carrot extracts have demonstrated eachof the N-methyltransferase activities called for by the schemeillustrated in Figure 5, and, again, we have been unable to detectan AdoMet:PtdEA N-methyltransferase activity (18). Neverthe-less, the possible contribution of some methylation of PtdEA toPtdMEA can not be regarded as conclusively ruled out. Withrespect to this possibility, it is of interest that Marshall and Kates(10, 11) were able to obtain cell-free preparations from spinachleaves catalyzing the AdoMet-dependent methylation of Ptd-MEA to PtdDMEA, and of PtdDMEA to PtdCho, whereas theycould not demonstrate methylation of PtdEA in this system.Moore (13) found that incorporation of the methyl group ofAdoMet into lipid-soluble material by extracts of castor beanendosperm was markedly stimulated by PtdMEA or PtdDMEA,but much less so (probably 'not significant') by PtdEA. Identifi-cation of the products of the methylation reaction revealedPtdDMEA and PtdCho, but no PtdMEA.A great deal of experimental attention has been devoted to

studies of the biosynthesis of PtdCho in other plants (reviewed,aND, not determined.

859

Dow



ovember 2021

DATKO AND MUDD Plant Physiol. Vol. 88, 1988

- P-Choline

CDP-Choline

PtdCho

SOYBEAN CDP-EA

PtdEA

P-EA P-MEA

+ $~~~~CDP-MEA

PtdMEA

P-DMEA

CDP-DMEA

PtdDMEA

P-EA P-MEA -*- P-DMEA

FIG. 5. Tentative working models ofthe pathways for entry of methyl groups

P-Choline into the network of methylated deriva-tives ofEA leading to PtdCho. Models forLemna, soybean, and carrot are shown.

CDP-Choline Heavy arrows indicate major fluxes; solid-line arrows, significant fluxes; and dashed

PtdCho arrows, minor or trace fluxes. In soybeanand carrot, some participation ofN-meth-ylation ofPtdEA to PtdMEA has not beenconclusively ruled out.

P-Choline

CARROT CDP-EA

+PtdEA

CDP-MEA CDP-DMEA

PtdMEA PtdDMEA

CDP-Choline

.. PtdCho

for example, in Refs. 14 and 15). However, to our knowledgeonly two previous series of experiments have followed the kinet-ics of incorporation of radioactivity originating in methioninemethyl (or, in the instances to be discussed below, that offormate,a precursor of this methyl group) by intact plant tissues into bothphospho-base and phosphatidyl methylated derivatives of EA.Such an experimental design permits analysis in much the sameway as the studies reported here: (a) Hitz, Rhodes, and Hanson(7) administered pulses of ['4C]formate to attached water-stressedbarley leaves. At 15 min, P-MEA and P-Cho were at least asheavily labeled as was PtdCho. PtdMEA was detectably labeled.Free methylated EA bases acquired very little label. Radioactivitychased into PtdCho. Analysis of these results, as well as a varietyof others, by computer modeling led to the conclusion that themethylation steps in the biosynthesis of PtdCho "take place atthe phosphoryl base (i.e. phospho-base) level, and at the phos-pholipid (i.e. phosphatidyl) level." A firm conclusion as to therelative importance of these pathways was felt not to be justified(7). We suggest that, as far as can be judged, the situation inthese barley leaves may be not unlike that in carrot. (b) Hansonand Rhodes (5) infiltrated salinized sugarbeet leaf discs with [14C]formate. At 2 to 5 min P-MEA, P-DMEA, and P-Cho "weremajor labeled products," but "the corresponding free bases andphosphatidyl derivatives were not appreciably labeled." Again,label chased into PtdCho. These results, and others, were inter-preted as indicating the operation of a methylation pathway atthe level of phospho-bases (5). A quantitative limit upon anycontribution of a pathway involving methylation at the phospha-tidyl level was not set, but the authors did indicate that theyjudged such a pathway not to be 'significant' in the sugarbeetleaf system. In our opinion, as far as can be judged, the situationin these sugarbeet leaves closely resembles that subsequentlyfound with intact Lemna (17).

In the strictest sense it may be that the results with barley andsugarbeet are applicable only to stressed tissues stimulated toform betaine. However, when these results are taken togetherwith those for Lemna (17), and those detailed here for soybeanand carrot, it seems far more likely that higher plants possess acommon committing step in PtdCho synthesis at the methylationof P-EA to P-MEA. This reaction, which accounts for some,

perhaps all, of the initial methylation in this pathway, is accom-panied by an unusual diversity in the subsequent methylationreactions leading to more highly methylated derivatives. In thisconnection, we note that the systems we have studied wereselected chiefly because they offered favorable experimental ma-terials, not with any foreknowledge that there might be differ-ences in the pathways under study. It might be of interest toextend studies ofthe sort reported here to a variety of additionalhigher plants, chosen in hopes ofrevealing a phylogenetic patternin the pathways utilized. Soybean suspension cultures and soy-bean leaves gave virtually identical patterns, suggesting thatfuture studies may be carried out with either of these types ofsystems, chosen for experimental convenience, and that theresults with either will be equally valid in describing the meth-ylation pattern characteristic of the plant under investigation.

LITERATURE CITED

1. BIELESKI RL, RE YOUNG 1963 Extraction and separation of phosphate estersfrom plant tissues. Anal Biochem 6: 54-68

2. DATKO AH, SH MUDD, J GIOVANELLI 1980 Lemna paucicostata Hegelm.6746: development of standardized growth conditions suitable for biochem-ical experimentation. Plant Physiol 65: 906-912

3. GIOVANELLI J, SH MUDD, AH DATKO 1985 Quantitative analysis of pathwaysof methionine metabolism and their regulation in Lemna. Plant Physiol 78:555-560

4. HANSON AD, WD HITz 1982 Metabolic responses of mesophytes to plantwater deficits. Annu Rev Plant Physiol 33: 163-203

5. HANSON AD, D RHODES 1983 14C Tracer evidence for synthesis ofcholine andbetaine via phosphoryl base intermediates in salinized sugarbeet leaves. PlantPhysiol 71: 692-700

6. HARWOOD JL, NJ RUSSELL 1984 Lipids in Plants and Microbes. George Allen& Unwin, New York, p 99

7. HITz WD, D RHODES, AD HANSON 1981 Radiotracer evidence implicatingphosphoryl and phosphatidyl bases as intermediates in betaine synthesis bywater-stressed barley leaves. Plant Physiol 68: 814-822

8. KATES M 1972 Techniques of Lipidology. In TS Work, E Work, eds, Labora-tory Techniques in Biochemistry and Molecular Biology, Vol 3. AmericanElsevier Publishing Co, New York, p 348

9. KLEINIG H, C Kopp 1978 Lipids, lipid turnover, and phospholipase D in plantsuspension culture cells (Daucus carota). Planta 139: 61-65

10. MARSHALL MO, M KATES 1973 Biosynthesis of phosphatidyl ethanolamineand phosphatidyl choline in spinach leaves. FEBS Lett 31: 199-202

11. MARSHALL MO, M KATES 1974 Biosynthesis of nitrogenous phospholipids inspinach leaves. Can J Biochem 52: 469-482

12. MATTHEWS BF, JM WIDHOLM 1978 Regulation of lysine and threonine syn-

860

LEMNA

P-EA P-MEA N P-DMEA

CDP-EA

PtdEA

CDP-MEA

iPtdMEA

CDP-DMEA

iPtdDMEA

Dow



ovember 2021

PHOSPHATIDYLCHOLINE SYNTHESIS 861

thesis in carrot cell suspension cultures and whole roots. Planta 141: 315- 18. MUDD SH, AH DATKO 1987 Patterns of methylation in phosphatidylcholine321 synthesis (abstract 681). Plant Physiol 83: S-1 13

13. MOORE JR TS 1976 Phosphatidylcholine synthesis in castor bean endosperm. 19. RENKONEN 0, A LUUKKONEN 1976 Thin-layer chromatography of phospho-Plant Physiol 57: 383-386 lipids and glycolipids. In GV Marinetti, ed, Lipid Chromatographic Analysis,

14. MOORE JR TS 1982 Phospholipid biosynthesis. Annu Rev Plant Physiol 33: Ed 2, Vol 1, Dekker, Inc, New York, pp 1-58235-259 20. ROUGHAN PG, CR SLACK, R HOLLAND 1978 Generation of phospholipid

MB 1980 Phospholipid biosynthesis. In PK Stumpf, ed, The Biochem- artefacts during extraction of developing soybean seeds with methanolic15. MUDD sovets Lipid13:pohl497-503snPKSumf e,Te lchmistry of Plants, Vol 9. Academic Press, New York, pp 249-282 21. RYtrER DJ, WE CORNAZER 1970 Biosynthesis of liver microsomal phospha-

16. MUDD SH, AH DATKO 1986 Methionine methyl group metabolism in Lemna. tidylcholines during the development of choline deficiency. Proc Soc ExpPlant Physiol 81: 103-114 Biol Med 134: 630633

17. MUDD SH, AH DATKO 1986 Phosphoethanolamine bases as intermediates in 22. SERVAITES JC, WL OGREN 1977 Rapid isolation of mesophyll-cells from leavesphosphatidylcholine synthesis by Lemna. Plant Physiol 82: 126-135 of soybean for photosynthetic studies. Plant Physiol 59: 587-590

Dow



ovember 2021

Documents

Phosphatidylcholine Synthesis - Plant Physiology