Pathway forthe Synthesis Triacylglycerols … · Effect of ozone fumigation on 14C distribution in acyl moieties of neutral lipid (A), galactolipid (B), and phospholipid (C) classes

Plant Physiol. (1990) 94, 773-7800032-0889/90/94/0773/08/$01 .00/0

Received for publication October 25, 1989Accepted June 5, 1990

Pathway for the Synthesis of Triacylglycerols fromMonogalactosyldiacylglycerols in Ozone-Fumigated

Spinach Leaves

Takeshi Sakaki*, Noriaki Kondo, and Mitsuhiro YamadaDivision of Environmental Biology, The National Institute for Environmental Studies, Onogawa,

Tsukuba, Ibaraki 305, Japan (T.S., N.K.); and Department of Biology,University of Tokyo, Komaba, Meguro-ku, Tokyo 153, Japan (M.Y.)

ABSTRACT

When the upper leaf surface of spinach (Spinacia oleracea L.)plants was treated with [1-14C]acetate and grown for 2 days, 14Cwas effectively incorporated into acyl moieties of leaf lipids inratios approximately their composition by mass. Fumigation ofthe plants with ozone (0.5 microliter per liter) caused a redistri-bution of 14C among lipid classes, i.e. a marked increase of 14Ccontent in triacylglycerol (TG) and 1,2-diacylglycerol (1,2-DG) anda decrease of label in monogalactosyldiacylglycerol (MGDG) with-out affecting 14C distribution in leaf fatty acids. Label in both TGand 1,2-DG was found predominantly in their polyene molecularspecies. Since MGDG consists of similar polyene molecular spe-cies, the results indicate the synthesis of TG from MGDG via 1,2-DG. Label was also accumulated in tri- and tetragalactosyldiacyl-glycerol, products of galactolipid:galactolipid galactosyltransfer-ase (GGGT). Moreover, there was a close relation between in-creases in the amounts of TG and the oligogalactolipids in ozone-treated leaves. These results indicate that MGDG was convertedto 1,2-DG by GGGT and then to TG. In intact chloroplasts isolatedfrom ozone-treated leaves, there was an enhanced production offree fatty acid (FFA), which was diminished by the addition ofcoenzyme A (CoA) and ATP, indicating that ozone stimulated thehydrolysis of MGDG to liberate FFA, which was in turn convertedto acyl-CoA. The final step of TG synthesis, acylation of 1,2-DGwith acyl-CoA, was confirmed by feeding with [1-14C]linolenicacid in leaf discs excised from ozone-fumigated leaves; 14C waseffectively incorporated into TG but not into 1,2-DG. These resultsdemonstrate the synthesis of TG from 1,2-DG and FFA whichwere liberated from MGDG in ozone-fumigated spinach leaves.

In previous papers (27, 28), we have examined the effect ofozone fumigation on the lipid composition of spinach leavesand demonstrated a decrease in MGDG,' DGDG, and PC,

'Abbreviations: MGDG, DGDG, TGDG, and TTGDG, mono-,di-, tri-, and tetragalactosyldiacylglycerol, respectively; PC, phospha-tidylcholine; TG, triacylglycerol; 1,2-DG and 1,3-DG, 1,2- and 1,3-diacylglycerol, respectively; GGGT, galactolipid:galactolipid galacto-syltransferase; FFA, free fatty acid; 18:3, a-linolenic acid; 18: 1, oleicacid; 16:0, palmitic acid; 16:1, 3-trans-hexadecenoic acid; 16:3, hex-adecatrienoic acid; 18:0, stearic acid; 18:2, linoleic acid; S, M, D, andT, saturated, monoenoic, dienoic, and trienoic fatty acid residuesesterified to glycerol, respectively; PG, phosphatidylglycerol; PA,phosphatidic acid; PE, phosphatidylethanolamine.

and a concomitant increase in TG. The results suggested themetabolic conversion of MGDG and PC to TG in ozone-fumigated leaves (27). In addition, a comparison of the mo-lecular species between MGDG and the increased TG (28)indicated the direct conversion of 1,2-DG backbone ofMGDG to TG.

This paper presents labeling studies demonstrating the syn-thesis ofTG from MGDG in ozone-fumigated spinach leaves.The results confirm the operation of the pathway fromMGDG to TG via 1,2-DG and also demonstrate that MGDGis converted to 1,2-DG by GGGT. We also report that ozonestimulates the hydrolysis of MGDG to release FFA, mainlya-linolenic acid (18:3), and that the 18:3 is converted to itsacyl-CoA form and then acylated to 1,2-DG to yield TG.

MATERIALS AND METHODS

Plant Materials and Ozone Fumigation

Spinach (Spinacia oleracea L., cv New Asia) plants werecultivated from seeds in a glasshouse as described previously(25). In [1-'4C]acetate-labeling experiments, expanding sec-ond leaves of rapidly growing spinach plants (4 weeks old)were used for the better incorporation of acetate into endog-enous fatty acids. In the remaining experiments, youngestmature leaves of spinach plants (6-8 weeks old) were used(25, 27, 28). Ozone-induced lipid changes in the leaves atthese different stages ofdevelopment were essentially the sameas those described in the previous reports (27, 28). Thefumigation of the plants with ozone (0.5 gL/L) was carriedout in an environment-controlled growth cabinet as describedpreviously (25).

[1-14C]Acetate Labeling

Spinach plants, 4 weeks old, were transferred from a glass-house to a growth cabinet maintained at a temperature of20°C, a relative humidity of 75%, a light intensity of 430 to580 juE/m2's PAR at leaf level and a photoperiod of 10 h,and grown for 2 d in the cabinet. Then a 10 1AL aliquot of 1.7mm sodium [1-'4C]acetate (100 ,Ci/mL) in 75% ethanol wasapplied with a microsyringe to the upper surface ofthe secondleaf of each treated plant. Treated leaves were approximately5 to 7 cm2 in area. Two hours later, the surface was wipedwith moistened filter paper to remove unabsorbed acetate

773

Dow

nloaded from https://academ

ic.oup.com/plphys/article/94/2/773/6086143 by guest on 07 August 2021

Plant Physiol. Vol. 94, 1990

60

20 - 41

to COClio0 - c.>

0 L- 2201

0

-2 -2m

Ozone

-° O__ °_=ij->

v ,J6:0Q

o 30-103 ft5

-A0- 116:0 ~ 1:1

18:2 0

18:10An in,A A_ -V- - v

,v -v-v°'\-v-Yv-wVY v Y01 2 4 6 "7.50 2 4 6 It7.5

m

Time, h

60 containing lipid spots were scraped from the plate, methano-lyzed, and quantified by GLC (27).

40 - 20

u EX l10cQ

20 - 0

0

Figure 1. Effect of ozone fumigation on 14C distribution in fatty acidsisolated from spinach leaves prelabeled with [1-14C]acetate. Ozone(0.5 ML/L) treatment was started 2 h after the onset of photoperiod.Open symbols, untreated; closed symbols, ozone-treated; -2 m and7.5 m, distribution of the amounts of endogenous fatty acids (mol%)in leaves at -2 and 7.5 h, respectively.

(31). Two days after the labeling, the plants were fumigatedwith ozone in the same cabinet. Ozone treatment was started2 h after the onset of photoperiod. At the times indicated,seven labeled leaves were removed for each measurement.Under these conditions, 25 to 35% of '4C applied to the leaveswas recovered in fatty acids of total lipid extract.

[1-14C]18:3 Labeling

Spinach plants, 6 to 8 weeks old, were fumigated withozone for 2 h. Then leaf discs (15 mm in diameter) were

excised from the interveinal areas of the treated leaves, and a

5 ,uL aliquot of [ I- 4C] 18:3 in ethylene glycol monoethyl ether(50 pCi/mL) was spread on the upper surface of each disc(21). The discs were floated on distilled water in Petri dishesand incubated at 20°C in the dark. At the times indicated,eight labeled discs were removed for each measurement.

Determination of Oligogalactolipid Content

After spinach plants, 6 to 8 weeks old, were fumigated withozone for various periods of time (0-8 h), leaf lipids were

extracted as described previously (27). The lipids were firstfractionated on a silica gel column (100-200 mesh; BW-80,Fuji Davison Chemicals, 1 cm x 4 cm), successively, with 10mL ofchloroform, 50 mL ofacetone, and 30 mL ofmethanol.TGDG and TTGDG, completely recovered in the methanolfraction, were concentrated and further separated by silica gelTLC (0.25-mm thick, Merck) developed with chloroform/methanol/acetic acid/water (170:30:20:7, v/v) as described byWebster and Chang (34). TGDG and TTGDG were locatedwith primuline (27) and identified by cochromatography withthe labeled TGDG and TTGDG (26). Regions of silica gel

Analysis of Lipids Isolated from Labeled Leaves andLeaf Discs

Lipids in the labeled leaves and leaf discs were isolated afterboiling in isopropanol for 5 min. TGDG and TTGDG were

separated as described above, and other polar lipids were

separated by two-dimensional TLC (27). Neutral lipids were

separated by one-dimensional TLC (27) with carriers of tri-olein, trilinolenin, 18:1, 1 ,2-DG, and 1 ,3-DG (10 ,g each).

In ['4C]acetate-labeled samples, the lipids separated by TLCwere supplied with 18:1 (100 ,ug) as carriers and methano-lyzed. Aliquots of the total lipids were also methanolyzedwithout carriers. The fatty acid methyl esters formed were

extracted three times with petroleum ether. When the esterswere contaminated with pigments, they were purified by TLC(28). Radioactivity in the esters was determined by liquidscintillation counting (3255, Packard) with a scintillator ofAquasol-2. Part of the methyl esters from the total lipids wasalso subjected to radio-GLC to determine '4C distribution inthe fatty acid species.

In ['4C]18:3-labeled samples, silica gel zones containinglipid spots separated on the TLC plate were scraped off intoAquasol-2/water (9:1, v/v) for scintillation counting.

Separation of Molecular Species in TG and 1,2-DG

Labeled 1,2-DG was acetylated in acetic anhydride/pyri-dine (5:1, v/v) overnight at 30°C in the dark (4) and purifiedby silica gel TLC developed with hexane/diethyl ether/aceticacid (70:30:1, v/v). To separate the molecular species, labeledTG and acetyl-DG (4,000 dpm each) were supplied withspinach TG and acetyl-DG (0.1 smol each) as carriers andsubjected to argentation TLC with chloroform/methanol(96:4, v/v) and chloroform/methanol (97:3, v/v) as the sol-vents, respectively (28). For autoradiography, the plate was

exposed to x-ray film for 30 d. Silica gel regions containingeach TG molecular species were scraped off into Bray's scin-tillator/water (9:1, v/v) (3) for scintillation counting.

Preparation of Intact Chloroplasts

Spinach plants, 6 to 8 weeks old, were fumigated withozone for 2 and 4.5 h, and the leaves were excised and chilledon ice for 10 to 20 min. The leaves were deveined, cut intosmall pieces and homogenized in an ice-cold grinding mediumcontaining 0.33 M sorbitol, 50 mM Hepes (pH 7.6 with Tris),2 mm EDTA, and 1 mM MgCl2 (9). The homogenate was

filtered through four layers of gauze and the filtrate was

centrifuged at 2,000g for 90 s. The pellet was suspended ingrinding medium and centrifuged again. The resulting pelletwas then layered on top of discontinuous Percoll gradients ingrinding medium consisting, from the bottom, of 90% (v/v),4 mL and 40%, 10 mL. After centrifugation at 7,000g for 15min (RPS 27-2(3) rotor, Hitachi), intact chloroplasts re-

covered at the 40/90% interface were collected and washedtwice with the grinding medium. The chloroplasts obtainedfrom both untreated and ozone-treated leaves were more than

Cu0

CI

C-,

774 SAKAKI ET AL.

10

Dow



PATHWAY FOR TRIACYLGLYCEROL SYNTHESIS FROM MONOGALACTOLIPIDS

AS

TG

Ozone

o /I

1,2-OG

FFA S

o00-° -o_O_O.- --

-2 0 2 4 6 8Time, h

B0.8

TGDG- v

14 - Ozone / 7-* 4 /---TTG06

0 0-0-o4-9=:Lo0-!

0--O 8A=A=A=As°I-

X 40

; 30

20*0 20

*' 1

MGOGI--

I DGDGI_o-AS&A

-2 0 2 4 6 8

Z 15

X, _ 10

._,'a-

Time, h

C

0-0\10 PC

Ozone P

E I-13 0 ,! -PE

I-<=S--v,V.-20 2 -4 v

-2 0 2 4 6 8

Figure 2. Effect of ozone fumigation on 14Cdistribution in acyl moieties of neutral lipid (A),galactolipid (B), and phospholipid (C) classesisolated from spinach leaves prelabeled with [1-14C]acetate. The conditions for ozone treatmentwere the same as those in Figure 1. Open sym-bols, untreated; closed symbols, ozone-treated.

Time. h

90% intact as estimated by the method of Lilley et al. (14).Chl content was determined by the method of Arnon (2).

Measurement of FFA Production in Intact Chloroplasts

The intact chloroplasts isolated from untreated and ozone-

treated leaves were suspended in a medium (0.4 mL) of 0.33M sorbitol and 25 mM Hepes (pH 7.2 with KOH) with andwithout 0.5 mm CoA and 1.0 mM ATP in a final concentrationof 1.0 mg Chl/mL. The suspension was incubated with shak-ing at 25°C in darkness. ATP of 1.0 mm was additionallysupplied at every 1 h. FFA in the chloroplast lipid extract wasseparated by silica gel TLC developed with hexane/diethylether/acetic acid (70:30:1, v/v), methanolyzed and quantifiedby GLC (27).

Chemicals

Sodium [1-"'C]acetate (58 mCi/mmol), [1-"'C]18:3 (56.2mCi/mmol) and x-ray film (Hyperfilm-Beta Max) were pur-chased from Amersham, Aquasol-2 from New England Nu-clear and Percoll from Pharmacia Fine Chemicals. Trioleinand trilinolenin were obtained from Nakarai Chemicals, 1,2-DG and 1,3-DG (pig liver) from Serdary Research Laborato-ries and CoA (sodium salt) from Sigma.

RESULTS

Effect of Ozone on Leaf Lipids Prelabeled with [1-14C]Acetate

The previous paper (28) suggested that TG was mainlyderived from polyunsaturated molecular species of MGDGin ozone-treated leaves. To confirm this metabolism, expand-ing spinach leaves were fed with ['4C]acetate and grown for 2d in a growth cabinet until ['4C]acetate was thoroughly incor-porated into polyunsaturated fatty acids in leaf lipids. Figure1 shows that the distribution of '4C in fatty acids reached thelevel approximately proportional to the composition of theendogenous fatty acids. Treatment of the leaves with ozone

did not affect the 14C distribution for at least 7 h (Fig. 1),consistent with the quantitative conservation of endogenousfatty acids in ozone-treated leaves (27).

Figure 2 shows the changes in the distribution of "1C in acylmoieties of neutral lipid, galactolipid and phospholipidclasses. In agreement with the quantitative changes in endog-enous lipids (27, 28), the radioactivity in MGDG decreased,whereas the radioactivity in TG and 1,2-DG was increased byozone treatment. Figure 3 shows that "1C distribution in TGand 1,2-DG from ozone-fumigated leaves shifted to the highlyunsaturated molecular species: T3 species in TG and T2 speciesin 1,2-DG, consistent with the results of unlabeled TG (28).Since MGDG is composed almost entirely of T2 molecularspecies (6), these results support the conversion ofMGDG to1,2-DG, and then to TG in ozone-fumigated leaves.As shown in Figure 2C, the level of "1C in PC was gradually

reduced in non-fumigated leaves, probably due to the conver-

sion ofPC to galactolipids (21, 23). Ozone treatment inhibitedthe reduction of 14C in PC, indicative of the suppression ofPC metabolism. The level of "1C in PG was slightly reduced4 h after the fumigation, whereas "1C in PA increased soon

after the ozone fumigation (27). The label in PE was un-

changed throughout the period of ozone fumigation.

TGDG and TTGDG in Ozone-Treated Leaves

Figure 2B shows a marked increase of 14C in TGDG andTTGDG, products ofGGGT. Since GGGT produces 1,2-DGfrom MGDG in addition to these oligogalactolipids (5, 33,35), it is probable that the conversion of MGDG to 1,2-DGis catalyzed by this enzyme. To investigate further the involve-ment ofGGGT in the TG synthesis from MGDG, content ofTG was compared with that of TGDG and TTGDG inspinach leaves treated with ozone (Fig. 4). In this experiment,the amount of TG derived from MGDG was estimated on

the basis of 16:3 content in TG, because its 16:3 was solelyderived from MGDG (27, 28). The increases in both TGDGand TTGDG were closely correlated with the increase of 16:3in TG (Fig. 4), confirming that MGDG was converted to 1,2-

4

2

2

0

0.6

0.3

0

775

8

6tom

a

C,-a

lim

z.s

Q2

PA

a

I1

Dow




i. fi ,,-i

MA

vi

Figure 3. Effect of ozone fumigation on 14Cdistribution in the molecular species of TG and1,2-DG isolated from spinach leaves prelabeledwith [1-14C]acetate. Spinach plants were fumi-gated with ozone (0.5 ,uL/L) for 5 h. The molec-ular species of TG and acetylated 1,2-DG wereseparated by argentation chromatography andtheir radioactivities were shown by autoradiog-raphy. S, M, D, and T, saturated, monoenoic,dienoic, and trienoic fatty acid residues esterifiedto glycerol, respectively.

". i i

rP(:

DG by GGGT reaction and then to TG during ozone fumi-gation.

Increase in FFA in Intact Chloroplasts Isolated fromOzone-Treated Leaves

Figure 2A also shows that 14C in FFA increased soon afterthe start ofozone treatment. When intact chloroplasts isolatedfrom ozone-fumigated spinach leaves were incubated in thedark, the production ofFFA in the chloroplasts was markedlystimulated (Fig. 5), although the level ofthe stimulation variedwith the chloroplast preparations. The species of FFA accu-mulated were mainly 18:3 and 16:3 (Table I), indicating thatozone enhanced the hydrolysis of MGDG to FFA, probablyby the stimulation of the galactolipid-hydrolyzing enzyme(galactolipase) activity (11, 36). On the other hand, littleaccumulation of endogenous FFA in leaves during the fumi-gation with ozone (27) suggests that FFA liberated in thechloroplasts by ozone is actively metabolized in leaf cells.As shown in Figure 6, FFA production was effectively

diminished by the addition of CoA and ATP to the chloro-plasts isolated from both non- and ozone-treated leaves. Thisis due to the synthesis of acyl-CoA from FFA by acyl-CoAsynthetase in the envelope membranes (13). Fatty acids denovo synthesized from ['4C]acetate by the isolated chloroplastsare also converted to acyl-CoA (23). The fatty acid speciesreduced by the addition of CoA and ATP were mainly 18:3(Table II), indicating the synthesis of 18:3-CoA by the chlo-roplasts.

Metabolism of [1-14C]18:3 in Ozone-Treated Leaf Discs

Based on the structural analysis ofTG, we have shown thatthe fatty acid species esterified to 1,2-DG to form TG ismainly 18:3 in ozone-treated leaves (28). Thus, it is conceiv-able that 18:3 released from complex lipids in the chloroplastsin response to ozone (Fig. 5; Table I) is the fatty acid acylatedto 1,2-DG via 18:3-CoA (Fig. 6; Table II). To clarify this

4

Ca

-cc=

cu

m

C=

Ca

C.)

-C

E

4-

co

Cu-Cu-

C

2B

C

72=

3 6 9 1:

Content of 16:3 in TG

(nmol /pmol leaf fatty acid)Figure 4. Relationship between oligogalactolipid content and 16:3content in TG in spinach leaves fumigated with ozone (0.5 AL/L) forvarious periods of time (0-8 h). Values are shown in terms of nmol/Amol leaf fatty acid, because the content of total leaf fatty acidsremained unchanged during the fumigation with ozone (27, 28).Circles and triangles represent different experimental preparations.

776 SAKAKI ET AL.

dim"_iai.

0..

.A*e.-

:j.?,.54i.."I D

ownloaded from

https://academic.oup.com

/plphys/article/94/2/773/6086143 by guest on 07 August 2021


_0.2-E©

E

0

U-

o~ ~~~n

0.1

0 2.5 5

Time, h

Figure 5. Time course of increase of FFA in intact chloroplastsisolated from spinach leaves fumigated with ozone (0.5 jLIL) for 0(0), 2 (0), and 4.5 (0) h.

point, ["4C] 18:3 was exogenously applied to leaf discs excisedfrom ozone-fumigated spinach leaves and its metabolism was

examined. As shown in Figure 7, 14C in FFA sharply decreasedwithin 8 h, accompanied by a concomitant increase of labelin glycerolipids, including TG, both in control and ozone-treated leaf discs. In control discs, "4C accumulated in TGwith the maximum at 4 h and gradually decreased thereafter(Fig. 7A). The temporary accumulation of exogenously ap-plied FFA in TG has been also observed in maize (8) andspinach (24) leaves. In ozone-treated discs, on the other hand,the label in TG continuously increased for 8 h; thereafter, thelevel was maintained for an additional 12 h (Fig. 7A). At 20h of incubation, the label in TG in ozone-treated discs was

almost four times that in the control. These results suggestthat ozone stimulated the incorporation of [14C] 18:3 into TG.Both T3-TG and other TG were labeled to a greater extent inozone-treated discs than in the control (Fig. 8).

Figures 7B and 7C show the labeling of polar lipids. PC wasthe most heavily labeled polar lipid in both control and ozone-

treated discs. In control discs, the label in PC sharply increasedand reached the maximum at 8 h, whereas in ozone-treateddiscs the label in PC similarly increased up to 2 h but thereafterdeclined. The difference of label in PC between control andozone-treated discs after 2 h could be accounted for by thedifference in TG. However, the fact that 14C in 1,2-DG wasmaintained at a low level with no difference between controland ozone-treated leaf discs indicates that PC would not beinvolved in TG synthesis via 1,2-DG. In this respect, we

propose that PC is synthesized by the acylation with tempo-rarily increased FFA and subsequently supplies acyl-CoA forthe TG synthesis by the acyl-exchange between sn-2 positionof PC and acyl-CoA (32).

In MGDG, the label gradually increased up to 20 h incontrol and ozone-treated discs (Fig. 7B). However, lack of'4C accumulation in either 1,2-DG or TGDG suggests that['4C] 18:3-MGDG would not supply 1,2-DG for the TG syn-thesis. This means that ['4C] 18:3 is incorporated into TG atthe acylation step of 1,2-DG derived from unlabeled MGDG.The level of '4C in PA was constant (less than 1%) throughoutthe experimental period in the discs irrespective of ozonetreatment (Fig. 7C), excluding the contribution of ['4C]PA tothe TG synthesis. The label also accumulated in PE and PGwith little difference between control and ozone-treated discs(Fig. 7C), suggesting that these lipids would not be involvedin the TG synthesis caused by ozone.

DISCUSSION

Based on the previous (27, 28) and the present results, wepropose the pathway for the lipid changes in ozone-treatedspinach leaves:

1. MGDG -- 1,2-DG, galactolipid:galactolipid galactosyl-transferase

2. MGDG -- FFA, galactolipase

3. FFA -- acyl-CoA, acyl-CoA synthetase

4. 1,2-DG + acyl-CoA -- TG, diacylglycerol acyltransferase.

Our previous paper (28) indicated that the 1,2-DG moietiesof MGDG were converted to TG in ozone-fumigated leaveson the basis of the close similarity in the structure ofMGDGand TG. The present tracer experiments directly demon-strated the operation of this pathway in ozone-fumigatedspinach leaves (Figs. 2 and 3). In addition, the involvement

Table I. FFA Species in the Intact Chloroplasts Isolated from Ozone-Fumigated Spinach LeavesThe isolated intact chloroplasts were suspended in a concentration of 1.0 mg Chl/mL and incubated

at 250C in darkness.

Incubation Content of FFASample Tm

SampleTime 16:0 16:1 16:3 18:0 18:1 18:2 18:3

h nmol/mg ChiNone 0 17.4 0 3.0 4.4 4.1 0 5.3

2 24.1 0 13.4 6.5 5.2 1.7 40.6Difference 6.7 0 10.4 2.1 1.1 1.7 35.3

Ozone (2 h) 0 17.7 0 3.9 4.4 4.5 1.8 4.12 29.9 0 30.6 4.7 6.6 2.1 82.3

Difference 12.2 0 26.7 0.3 2.1 0.3 78.2

777

Dow




0.2 - * _03,2h

C.) 0s g-- 32

cm

E

wO /F_--0~~ None

= r

-

0 13

Time, h

Figure 6. Time course of production of FFA by intact chloroplastsisolated from non- and ozone (03)-fumigated spinach leaves in thepresence (broken line) and absence (solid line) of CoA and ATP.

of GGGT in TG synthesis, i.e. the production of 1,2-DGfrom MGDG, was demonstrated by the marked accumulationof label in TGDG and TTGDG in ozone-fumigated leaves(Fig. 2B). When the oligogalactolipid content was comparedwith 16:3 content in TG (Fig. 4), 0.36 nmol TGDG and 0.21nmol TTGDG were formed for 1.0 nmol 16:3 in TG, respec-

tively. Assuming that each molecule ofTGDG and TTGDGis synthesized from 3 and 4 molecules of MGDG with theproduction of 2 and 3 molecules of 1,2-DG, respectively (35),then 1.35 nmol 1,2-DG per 1.0 nmol 16:3 in TG are synthe-sized for the production of both oligogalactolipids (2 x 0.36nmol + 3 x 0.21 nmol). Since the molecular species of TGincreased by ozone was predominantly T3-TG consisting ofapproximately an equal molar ratio of sn- 1,3-18:3-2-16:3 andsn-1,2,3-18:3 (28), 1.0 nmol 16:3 in TG corresponds to about2 nmol TG. Therefore, about 70% ofTG synthesized can beexplained by the reaction producing TGDG and TTGDGfrom MGDG, provided that all of 1,2-DG derived from

MGDG is metabolized to T3-TG. Furthermore, there is an

additional production of 1,2-DG which corresponds toDGDG synthesis by GGGT. On the basis of the above cal-culations, we concluded that 1,2-DG moieties of TG were

formed from MGDG by GGGT reaction during ozone fu-migation.GGGT was first reported by Van Besouw and Wintermans

(33) to be the enzyme responsible for DGDG synthesis.However, the synthesis of unnatural oligogalactolipids suchas TGDG and TTGDG by this enzyme casts some doubt on

the involvement of the enzyme in DGDG synthesis in vivo(12). The present results established another role of GGGT,that is, the supply of 1,2-DG backbone for the synthesis ofTG. In support, GGGT is located in the chloroplast envelopes(5), where the diacylglycerol acyltransferase responsible forthe final step ofTG synthesis is also associated in spinach leafcells (15).We have also found that the fatty acid species acylated to

1,2-DG was predominantly 18:3 (28). Although 18:3 is a

major fatty acid constituent in leaf lipids, the path for thesynthesis of 18:3-CoA as the precursor of TG in ozone-

fumigated leaves remained uncertain (28). In the presentwork, we showed that the production of FFA, mainly 18:3,by the hydrolysis ofMGDG was stimulated in the chloroplastsisolated from ozone-treated leaves within 2 h, and that the18:3 was effectively converted to acyl-CoA (Figs. 5 and 6;Tables I and II). Furthermore, when ['4C] 18:3 was exoge-

nously applied to the ozone-treated leaf discs, the label was

rapidly metabolized to TG by the acylation to 1,2-DG (Fig.7). All these findings confirmed the view that FFA liberatedin the chloroplasts was converted to acyl-CoA and then in-corporated into TG.We have also shown that ['4C] 18:3 was actively incorpo-

rated into TG in control leaf discs as well as in ozone-treateddiscs (Figs. 7A and 8). An exceptionally large amount ofincorporation from exogenously applied FFA into TG hasbeen already observed in intact leaves and cotyledons (8, 19,21, 24). Therefore, leaf cells can actively scavenge temporarilyaccumulated FFA by their metabolic conversion to TG. Be-cause FFA act as potent inhibitors of the Hill reaction, un-

couplers of photophosphorylation (17, 30) and mediators of

Table II. Effect of Exogenously Applied CoA and ATP on the Production of FFA by Intact ChloroplastsIsolated from Ozone-Fumigated Spinach Leaves

The intact chloroplasts were suspended in a concentration of 1.0 mg ChI/mL and incubated at 250Cin darkness for 4 h.

Content of FFASample CoA + ATP"

16:0 16:1 16:3 18:0 18:1 18:2 18:3

nmol/mg Chl

None - 15.7 0 26.6 1.9 3.2 3.1 80.8+ 9.4 0 23.9 1.8 2.4 1.7 45.0

Difference 6.3 0 2.7 0.1 0.8 1.4 35.8

Ozone (2 h) - 24.2 0 45.7 4.5 6.1 3.1 117.7+ 16.0 0 33.8 4.3 5.6 1.2 52.7

Difference 8.2 0 11.9 0.2 0.5 1.9 65.0a CoA (0.5 mM) and ATP (1.0 mM) was initially supplied, and 1.0 mM of ATP was additionally supplied

at every 1 h.

778 SAKAKI ET AL.

Dow




A

5 10 15Time, h Time, h

C2I ipg-ii PA -

I

- 40

-a

30

cS 20

I - -

z \ PC

101- PE

I- PG= --L

0 5 10 15 20Time, h

thylakoid disintegration (22, 30), the pathway from FFA toTG may play a role in diminishing these toxic functionsinduced by FFA.

Recently, Nouchi and Toyama (20) have reported an in-crease in the content of several phospholipid classes in ozone-fumigated morning glory leaves. This might be partly due tothe net synthesis of these phospholipids from FFA liberatedby ozone fumigation. As shown in Figure 7, exogenouslyapplied [14C] 18:3 was incorporated not only into TG but alsointo some phospholipid classes. It is likely that the synthesisof the phospholipids may also play a scavenging role of FFAin leaf cells.

Galactolipid-hydrolyzing enzyme (galactolipase) has beenstudied with various plant species (7), including spinach (10,29). The enzyme is associated, at least partly, with chloroplasts(1, 1 1) and is generally assumed to be involved in the lipidcatabolism (7). Although the mode of stimulative action of

0 I

Control Ozone-treatedI ,T~~otalITG

0 4 81 62 0 4 8 1216_0

\~"o -/ TI-TG'To----------

0 4 8 12 16 20 0 4 8 12 16 20

60

40 "

I-20 c

1-I-1

0

Time, h

Figure 8. Time course of [1-14C]18:3 incorporation into the molecularspecies of TG in leaf discs excised from ozone-fumigated spinachplants. Duration of ozone (0.5 IAL/L) fumigation was 2 h. The molecularspecies were separated by argentation chromatography as describedin "Materials and Methods."

Figure 7. Time course of [1-'4C]18:3 incorpo-ration into neutral lipid (A), galactolipid (B), andphospholipid (C) classes in leaf discs excisedfrom ozone-fumigated spinach plants. Durationof ozone (0.5 yL/L) fumigation was 2 h. Opensymbols, untreated; closed symbols, ozone-treated.

ozone remains to be determined, the enzyme activity is re-ported to be stimulated by various conditions, such as achange in pH (10, 17), addition of salts (36) and detergents(1, 16) to the suspending medium, and aging (10, 11, 17). Inaddition, a cystine residue is important for the activity ofgalactolipase purified from rice bran (16). In this respect,ozone has been shown to oxidize cysteine to form cystine(18).

LITERATURE CITED

1. Anderson MM, McCarty RE, Zimmer EA (1974) The role ofgalactolipids in spinach chloroplast lamellar membranes. I.Partial purification of a bean leaf galactolipid lipase and itsaction on subchloroplast particles. Plant Physiol 53: 699-704

2. Arnon DI ( 1949) Copper enzymes in isolated chloroplasts. Poly-phenoloxidase in Beta vulgaris. Plant Physiol 24: 1-15

3. Bray GA (1960) A simple efficient liquid scintillator for countingaqueous solutions in a liquid scintillation counter. AnalBiochem 1: 279-285

4. Christie WW (1982) Lipid analysis, Ed 2. Pergamon Press,Oxford

5. Dorne AJ, Block MA, Joyard J, Douce R (1982) The galactolipid:galactolipid galactosyltransferase is located on the outer surfaceof the outer membrane of the chloroplast envelope. FEBS Lett145: 30-34

6. Douce R, Joyard J (1980) Plant galactolipids. In PK Stumpf, ed,The Biochemistry of Plants, Vol 4. Academic Press, New York,pp 321-362

7. Galliard T (1980) Degradation of acyl lipids: hydrolytic andoxidative enzymes. In PK Stumpf, ed, The Biochemistry ofPlants, Vol 4. Academic Press, New York, pp 85-116

8. Hawke JC, Stumpf PK (1980) The incorporation of oleic andlinoleic acids and their desaturation products into the glycer-olipids of maize leaves. Arch Biochem Biophys 203: 296-306

9. Heemskerk JW, Bogemann G, Wintermans JFGM (1983) Turn-over of galactolipids incorporated into chloroplast envelopes.An assay for galactolipid:galactolipid galactosyltransferase.Biochim Biophys Acta 754: 181-189

10. Helmsing PJ (1967) Hydrolysis of galactolipids by enzymes inspinach leaves. Biochim Biophys Acta 144: 470-472

11. Hirayama 0, Matsui T (1976) Effects of lipolytic enzymes onthe photochemical activities of spinach chloroplasts. BiochimBiophys Acta 423: 540-547

1-

a

4D

'a0

cJ

Cs

31

'a 21

-a

-,

779

Dow




12. Joyard J, Douce R (1987) Galactolipid synthesis. In PK Stumpf,ed, The Biochemistry of Plants, Vol 9. Academic Press, Or-lando, FL, pp 215-274

13. Joyard J, Stumpf PK (1981) Synthesis of long-chain acyl-CoAin chloroplast envelope membranes. Plant Physiol 67: 250-256

14. Lilley RM, Fitzgerald MP, Rienits KG, Walker DA (1975)Criteria of intactness and the photosynthetic activity ofspinachchloroplast preparations. New Phytol 75: 1-10

15. Martin BA, Wilson RF (1984) Subcellular localization of triac-ylglycerol synthesis in spinach leaves. Lipids 19: 117-121

16. Matsuda H, Hirayama 0 (1979) Enzymatic properties of agalactolipase from rice bran. Agric Biol Chem 43: 697-703

17. McCarty RE, Jagendorf AT (1965) Chloroplast damage due toenzymatic hydrolysis of endogenous lipids. Plant Physiol 40:725-735

18. Mudd JB, Leavitt R, Ongun A, McManus TT (1969) Reactionof ozone with amino acids and proteins. Atmos Environ 3:669-682

19. Murphy DJ, Stumpf PK (1980) In vivo pathway of oleate andlinoleate desaturation in developing cotyledons of Cucumissativits L. seedlings. Plant Physiol 66: 666-671

20. Nouchi I, Toyama S (1988) Effects of ozone and peroxyacetylnitrate on polar lipids and fatty acids in leaves of morningglory and kidney bean. Plant Physiol 87: 638-646

21. Ohnishi J, Yamada M (1980) Glycerolipid synthesis in Avenaleaves during greening of etiolated seedlings. II. a-Linolenicacid synthesis. Plant Cell Physiol 21: 1607-1618

22. Okamoto T, Katoh S, Murakami S (1977) Effects of linolenicacid on spinach chloroplast structure. Plant Cell Physiol 18:551-560

23. Roughan PG, Holland R, Slack CR (1980) The role of chloro-plasts and microsomal fractions in polar-lipid synthesis from[1-'4C]acetate by cell-free preparations from spinach (Spinaciaoleracea) leaves. Biochem J 188: 17-24

24. Roughan PG, Thompson GA Jr, Cho SH (1987) Metabolism ofexogenous long-chain fatty acids by spinach leaves. ArchBiochem Biophys 259: 481-496

25. Sakaki T, Kondo N, Sugahara K (1983) Breakdown of photosyn-

thetic pigments and lipids in spinach leaves with ozone fumi-gation: role of active oxygens. Physiol Plant 59: 28-34

26. Sakaki T, Kondo N, Yamada M (1990) Free fatty acids regulatetwo galactosyltransferases in chloroplast envelope membranesisolated from spinach leaves. Plant Physiol 94: 781-787

27. Sakaki T, Ohnishi J, Kondo N, Yamada M (1985) Polar andneutral lipid changes in spinach leaves with ozone fumigation:triacylglycerol synthesis from polar lipids. Plant Cell Physiol26: 253-262

28. Sakaki T, Saito K, Kawaguchi A, Kondo N, Yamada M (1990)Conversion of monogalactosyldiacylglycerols to triacylglyc-erols in ozone-fumigated spinach leaves. Plant Physiol 94: 766-772

29. Sastry PS, Kates M (1964) Hydrolysis of monogalactosyl anddigalactosyl diglycerides by specific enzymes in runner-beanleaves. Biochemistry 3: 1280-1287

30. Siegenthaler PA (1973) Change in pH dependence and sequentialinhibition of photosynthetic activity in chloroplasts by unsat-urated fatty acids. Biochim Biophys Acta 305: 153-162

31. Slack CR, Roughan PG (1975) The kinetics of incorporation invivo of ['4C]acetate and ['4C]carbon dioxide into the fatty acidsof glycerolipids in developing leaves. Biochem J 152: 217-228

32. Stymne S, Stobart AK (1987) Triacylglycerol biosynthesis. In PKStumpf, ed, The Biochemistry of Plants, Vol 9. AcademicPress, Orlando, FL, pp 175-214

33. Van Besouw A, Wintermans JFGM (1978) Galactolipid forma-tion in chloroplast envelopes. I. Evidence for two mechanismsin galactosylation. Biochim Biophys Acta 529: 44-53

34. Webster DE, Chang SB (1969) Polygalactolipids in spinachchloroplasts. Plant Physiol 44: 1523-1527

35. Wintermans JFGM, Heemskerk JWM (1987) On the synthesisof digalactosyldiacylglycerol in chloroplasts, and its relation tomonogalactolipid synthesis. In PK Stumpf, JB Mudd, WDNes, eds, The Metabolism, Structure, and Function of PlantLipids. Plenum Press, New York, pp 293-300

36. Wintermans JFGM, Helmsing PJ, Polman BJJ, Van GisbergenJ, Collard J (1969) Galactolipid transformations and photo-chemical activities of spinach chloroplasts. Biochim BiophysActa 189: 95-105

780 SAKAKI ET AL.

Dow



Documents

Pathway forthe Synthesis Triacylglycerols … · Effect of ozone fumigation on 14C distribution in acyl moieties of neutral lipid (A), galactolipid (B), and phospholipid (C) classes