Plant & CellPhysiol. 21 (8): 1595-1606 (1980)
Glycerolipid synthesis in Avena leaves during greening of etiolated seedlings I. Lipid changes in leaves Jun-ichi Ohnishi and Mitsuhiro Yamada
(Received September 24, 1980)
Etiolated seedlings of Avena sativa L. were illuminated under fluorescent light of 1,500 lux at 25°C and the lipid changes in their first leaves and isolated plastids were followed during 24 hr of greening. Lipid changes were divided into a degradation phase [0 hr of illumination (hr-L) to 3 hr-L] and a synthetic phase (3 hr-L to 24 hr-L). In the degradation phase, which paralleled prolamellar body transformation in the plastids, both plastidic lipids, monogalactosyldiglyceride (MGDG), digalactosyldiglyceride (DGDG), sulfoquinovosyldiglyceride (SQDG) and phosphatidylglycerol (PG), and extraplastidic lipids, phosphatidylcholine (PC), phosphatidylethanolamine (PE) and phosphatidylinositol (PI), were partially degraded. Plastidic lipids (except DGDG) began to increase at 1.5 hr-L without the de novo synthesis offatty acids, at the same time that there was a temporary accumulation of diglyceride, whose fatty acids were similar to the fatty acids of phospholipids such as PC, PE and PI, but different from the fatty acids of DGDG. This suggests that there is conversion of phospholipids to plastidic lipids during the later degradation phase. During the later degradation phase and the early synthetic phase (until 6 hr-L), plastid division occurred, resulting in a 30% increase in the plastid number per cell. Plastidic lipids were synthesized rapidly during the synthetic phase, in accordance with the beginning of light-enhanced fatty acid synthesis and thylakoid proliferation. In addition, the fatty acid composition of the plastidic lipids changed markedly throughout the synthetic phase: in MGDG, DGDG and SQDG, a-linolenate increased as linoleate decreased at both the C-l and C-2 positions of snglycerol; in PG, a-linolenate increased in compensation for the decrease in linoleate at C-l and hexadecenoate (3t) appeared and increased in compensation for the decrease in palmitate at C-2. This is evidence of the parallel desaturation of linoleate to linolenate at both C-l and C-2 of MGDG, DGDG and SQDG, and also of the position-specific desaturation of linoleate to linolenate at C-I of PG and of palmitate to hexadecenoate at C-2 of PG. The formation of hexadecenoate was entirely light-dependent. Extraplastidic lipids increased slightly in the early synthetic phase (3 hr-L to 6 hr-L), but declined later. Key words: Avena sativa - Chloroplast development - Fatty acid composition Greening leaves - a-Linolenic acid - Lipid changes. Abbreviations: BSA, bovine serum albumin; DG, diglyceride; DGDG, digalactosyldiglyceride; ER, endoplasmic reticulum; GLC, gas-liquid chromatography; Hepes, N-2-hydroxyethylpiperazine-N'-ethanesulfonic acid; MGDG, monogalactosyldiglyceride; PC, phosphatidylcholine; PE, phosphatidylethanolamine; PG, phosphatidylglycerol; PI, phosphatidylinositol; PLB, prolamellar body; SQDG, sulfoquinovosyldiglyceride; TLC, thin-layer chromatography; x hr-L, x hr of illumination; 16:0, plamitic acid; 16:1, 3-trans-hexadecenoic acid; 18:0, stearic acid; 18:1, oleic acid; 18:2, linoleic acid; 18:3, a-linolenic acid. 1595
Downloaded from http://pcp.oxfordjournals.org/ at University of Sussex on July 27, 2015
Department of Biology, College of General Education, The University of Tokyo, Komaba, Meguro-ku, Tokyo 153,japan
1596
J. Ohnishi and
M. Yamada
Materials and rnethods
Seeds of oat, Avenasativa L. var. Victory I (The General Swedish Seed Company, Limited) were germinated in the dark at 25°C on wet vermiculite. Whole seedlings continued to grow for ten days, although the first leaves which had emerged from the coleoptiles stopped growing after reaching a length of 8 em on the 7th day. The 8-day-old seedlings were illuminated with 1,500 lux of fluorescent light (5 W/m 2) for 24 hr, during which the first leaves were collected at intervals. No further increase in the length and fresh weight of the first leaves was observed during 24 hr of greening. Therefore, the changes in lipids determined on the basis of fresh weight of the leaves reflect the lipid changes per leaf cell in greening leaves. Monocotyledonous leaves show an age sequence for cells from the base to the tip, owing to the activity of their basal meristems. Because the amounts of lipids in different-aged cells varied (14), two-centimeter sections, 4 to 6 em from the base of the leaf, were excised with a razor blade and used in the lipid analysis. These sections greened most rapidly on illumination. Plastids were isolated according to Nakamura and Yamada (16) with a slight modification. About 20 g of the leaf sections were cut into 5 mm pieces with a razor blade then homogenized in a Waring blendor three times for 3 sec with a semi-frozen slurry of 300 ml of the grinding medium, consisting of 10 m.M sodium pyrophosphate/ HCI, pH 7.0, 0.4 Msorbitol, 5 mM MgCI 2, 1 mM EDTA, 2 mM sodium ascorbate and 0.1 % (w/v) BSA. The resulting slurry was squeezed through two layers of Miracloth (Chicopee Mills, Inc.). The filtrate was centrifuged at 2,000 xg for 4 min to precipitate crude plastids. The crude plastids were suspended in a small volume of 50 mM Hepes/NaOH, pH 8.0, 0.4 M sorbitol, 5 mMMgCI 2 , 1 mu MnCI 2 ,
Downloaded from http://pcp.oxfordjournals.org/ at University of Sussex on July 27, 2015
When etiolated leaves develop into green leaves under illumination, etioplasts in the leaves differentiate into chloroplasts, accompanied by thylakoid biogenesis within the plastids (5). Lipid changes in greening leaves have been reported in several plants such as barley (25, 29), maize (13, 14), wheat (3), pea (23, 31) and bean (23). The generalization made from these observations is that galactolipids in etiolated leaves partially decrease during the initial stages of greening, but markedly increase during later stages, parallel to the rapid synthesis of chlorophyll. The increase in galactolipids was characterized by an increase in a-linolenate, reflecting the synthesis of a-linolenoyl-galactolipids in greening leaves (14, 29). However, little has been reported on phospholipid changes in greening leaves. Tevini (29) examined lipid changes in barley leaves during retarded greening and reported the conversion of galactolipids to phospholipids during the initial stages of greening. In relation to the lipid changes in greening leaves, tracer experiments have indicated that [14CJacetate is incorporated mainly into sterols and unsaponifiable lipids by detached leaves during early stages (2), but during later stages acetate incorporation is channelled to fatty acids and acyl lipids (2, 11, 19), in close connection with the appearance of photosynthetic activity (19). The present report deals with changes in glycerolipids in greening Avena leaves, in connection with the development of etioplasts to chloroplasts. Attention is focussed on changes in the constituent unsaturated fatty acids of the plastidic lipids.
Phospho- and glycolipid changes in greening Avena leaves
1597
Downloaded from http://pcp.oxfordjournals.org/ at University of Sussex on July 27, 2015
and 1 mn EDTA and layered on a discontinuous gradient consisting of 1.5 and 1.0 M sucrose in the suspending buffer without sorbitol. After centrifugation at 800 X g for 20 min, intact plastids (Class I) which banded at the interface were collected in a Hitachi density gradient fractionator DGF-U. The purified plastids were more than 90% intact when viewed by phase-contrast and electron microscopy, and they were free from the mitochondrial enzyme, succinate dehydrogenase (data not shown). Fig. 3a shows the typical profile of an isolated etioplast containing PLB. The plastid number was counted with a Thoma haemocytometer. Prior to lipid extraction, about 1 g of the leaf sections was steamed for 10 min to minimize lipid degradation by phospholipase D during extraction (24). Lipids were extracted from the leaf sections or from isolated plastids, according to Bligh and Dyer (4). Lipid classes were separated by silica gel TLC. Neutral lipids were developed with petroleum etherjchlorofromjacetone (4 : I : 1, vjv) (12) and polar lipids were developed with acetonejbenzenejwater (91 : 30 : 8, vjv) (20) or with chloroformjmethanoljwater (65 : 25 : 4, vjv) in the first direction and with chloroformjmethanoljammoniajisopropylamine (60 : 30 : 4 : 0.5, vjv) in the second direction (12). Lipid spots were located under UV light after spraying the plate with 0.01 % (wjv) primuline in 800/0 (vjv) acetone (33). Each lipid was identified by cochromatography with known standards and by spraying the plate with reagents specific for the lipid classes (26, 34). To quantify each lipid, the spot separated on a plate was scraped off and heated at 80°C for 2 hr in a screw-capped tube containing 2 ml of3% (wjw) RCI in methanol and a given amount of pentadecanoic acid as an internal standard. The fatty acid methyl esters were extracted with petroleum ether and analyzed by GLC in a Shimadzu GC-6AMF with glass columns (3 mm X 2 m) of 100~ (wjw) diethylene glycol succinate on Celite 545. The content of each lipid was determined from the quantities of its constituent fatty acids (1). The separated lipid sample was treated with the lipase from Rhizopus delemar (Seikagaku Kogyo Co., Ltd.) to yield 2-acyllysolipid and fatty acid composition at the C-2 position of sn-glycercl was determined (8). The composition at the C-l position was calculated from the total and C-2 compositions. The activity of fatty acid synthesis in the crude plastids was measured by the incorporation of [1-14C]acetate into the fatty acid moieties according to Nakamura and Yamada (16). Pigments in the leaf sections were extracted with acetone in a mortar and pestle and those in isolated plastids with 80% (vjv) acetone.· Chlorophylls and carotenoidswere determined photometrically in 800/0 (vjv) acetone (15, 28). Succinate dehydrogenase (EC 1.33.99.1) activity was determined according to Riatt (10). The plastid number per cell was determined in the leaf sections which had been fixed in 5% (wjv) glutaraldehyde, according to Possingham and Smith (21). At least 20 mesophyll cells were counted and averaged. Electron microscopy of the isolated plastids and the leaves was performed as reported previously (18). Tri-, di- and mono-olein, PG, PI, pentadecanoic acid and BSA, Fraction V, were purchased from Sigma. Sodium [1-14C]acetate (60.2 mCijmmole) was obtained from The Radiochemical Center.
]. Ohnishi and M. Yamada
1598
Results
Lipid composition of greening leaves andfatty acidsynthesis by isolated plastids
o
2
MGDG
Fig. 1. Polarlipid content of etiolated and greening Avena leaves. Values are the means of duplicate samples, provided that the deviations are less than 5% of the means.
3 6 12 24 Illumination (hr)
Downloaded from http://pcp.oxfordjournals.org/ at University of Sussex on July 27, 2015
Changes in the polar lipid content of Avena leaves during 24 hr of greening is shown in Fig. 1. Major lipids were glycolipids, such as MGDG, DGDG and SQDG, as well as phospholipids such as PG, PC, PE and PI. There was an initial decrease in all lipids, indicative of a partial breakdown of all the lipid classes during the early stages of greening. The three glycolipids and PG, of plastid origin (see Table 2), showed two types of diphasic changes: MGDG, SQDG and PG decreased for the first 1.5 hr and thereafter increased, whereas DGDG decreased for the first 3 hr then increased. During the 24 hr, MGDG almost doubled and DGDG, SQDG and PG increased by about 20, 40 and 20%, respectively. In contrast, extraplastidic lipids such as PC, PE and PI showed triphasic changes; after decreasing for the first 3 hr, they increased up to 6 hr-L and then decreased until 24 hr-L. Fig. 2 shows the incorporation of [1-14C]acetate into fatty acid moieties by the plastids isolated from greening Avena leaves. The activity of fatty acid synthesis in the dark was very low throughout greening. Light-enhanced fatty acid synthesis began at 3 hr-L and increased continuously during greening. The activity in 12 hr-L etiochloroplasts was comparable to that in chloroplasts isolated from lightgrown leaves. Since the site offatty acid synthesis in leaves seems to be limited to plastids (22), the simultaneous increase in plastidic lipids (Fig. 1) together with the induction of
Phospho- and glycolipid changes in greening Avena leaves
~3
1599
o
o
~
s:
........... (J)
~
o
/0
2
E
c
/0
u
§ u
I
,0
a-e· e -e -
e-
e_
e
« 00 3 6
12
24
c
Illumination ( hr ) Fig. 2. Incorporation of [1-14C]acetate into fatty acid moieties by the crude plastids isolatedfrom etiolated and greening Avena leaves. Isolated plastids were incubated with [1-14C]acetate in the light (0) or in the dark (e). Chloroplasts (C) were isolated from 8-day-old, light-grown leaves.
fatty acid synthesis suggests that the plastidic lipids were supplied with fatty acids which were light-dependently synthesized in the plastid per se. Moreover, plastidic lipids such as MGDG, SQDG and PG seem to be formed from other lipids, such as DGDG and the other phospholipids, because of the decrease in the latter lipids in contrast to the increase in the former between 1.5 hr-L and 3 hr-L. Correspondingly, at 1.5 hr-L, there was a temporary accumulation of DG (0.1 pmolefg fresh weight), the fatty acid composition of which is similar to the compositions of PC, PE and PI, but not to the composition of DGDG (Table 1). This is evidence that plastidic lipids such as MGDG, SQDG and PG were produced from such phospholipids as PC, PE and PI through DG, before the induction of light-dependent fatty acid synthesis. Lipid content of isolated plastids and plastid division
The electron microscopic examination of Avena etiochloroplasts often showed profiles of plastid division at 3 hr-L and 6 hr-L. Fig. 3b shows a typical division of Table 1 Fatry acid compositions of various lipids at 1.5 hr-L Fatty acid
% Composition
DG
PC
PE
PI
DGDG
16:0
34
27
22
48
14
18:0
3
Glycerolipid synthesis in Avena leaves during greening of etiolated seedlings I. Lipid changes in leaves Jun-ichi Ohnishi and Mitsuhiro Yamada
(Received September 24, 1980)
Etiolated seedlings of Avena sativa L. were illuminated under fluorescent light of 1,500 lux at 25°C and the lipid changes in their first leaves and isolated plastids were followed during 24 hr of greening. Lipid changes were divided into a degradation phase [0 hr of illumination (hr-L) to 3 hr-L] and a synthetic phase (3 hr-L to 24 hr-L). In the degradation phase, which paralleled prolamellar body transformation in the plastids, both plastidic lipids, monogalactosyldiglyceride (MGDG), digalactosyldiglyceride (DGDG), sulfoquinovosyldiglyceride (SQDG) and phosphatidylglycerol (PG), and extraplastidic lipids, phosphatidylcholine (PC), phosphatidylethanolamine (PE) and phosphatidylinositol (PI), were partially degraded. Plastidic lipids (except DGDG) began to increase at 1.5 hr-L without the de novo synthesis offatty acids, at the same time that there was a temporary accumulation of diglyceride, whose fatty acids were similar to the fatty acids of phospholipids such as PC, PE and PI, but different from the fatty acids of DGDG. This suggests that there is conversion of phospholipids to plastidic lipids during the later degradation phase. During the later degradation phase and the early synthetic phase (until 6 hr-L), plastid division occurred, resulting in a 30% increase in the plastid number per cell. Plastidic lipids were synthesized rapidly during the synthetic phase, in accordance with the beginning of light-enhanced fatty acid synthesis and thylakoid proliferation. In addition, the fatty acid composition of the plastidic lipids changed markedly throughout the synthetic phase: in MGDG, DGDG and SQDG, a-linolenate increased as linoleate decreased at both the C-l and C-2 positions of snglycerol; in PG, a-linolenate increased in compensation for the decrease in linoleate at C-l and hexadecenoate (3t) appeared and increased in compensation for the decrease in palmitate at C-2. This is evidence of the parallel desaturation of linoleate to linolenate at both C-l and C-2 of MGDG, DGDG and SQDG, and also of the position-specific desaturation of linoleate to linolenate at C-I of PG and of palmitate to hexadecenoate at C-2 of PG. The formation of hexadecenoate was entirely light-dependent. Extraplastidic lipids increased slightly in the early synthetic phase (3 hr-L to 6 hr-L), but declined later. Key words: Avena sativa - Chloroplast development - Fatty acid composition Greening leaves - a-Linolenic acid - Lipid changes. Abbreviations: BSA, bovine serum albumin; DG, diglyceride; DGDG, digalactosyldiglyceride; ER, endoplasmic reticulum; GLC, gas-liquid chromatography; Hepes, N-2-hydroxyethylpiperazine-N'-ethanesulfonic acid; MGDG, monogalactosyldiglyceride; PC, phosphatidylcholine; PE, phosphatidylethanolamine; PG, phosphatidylglycerol; PI, phosphatidylinositol; PLB, prolamellar body; SQDG, sulfoquinovosyldiglyceride; TLC, thin-layer chromatography; x hr-L, x hr of illumination; 16:0, plamitic acid; 16:1, 3-trans-hexadecenoic acid; 18:0, stearic acid; 18:1, oleic acid; 18:2, linoleic acid; 18:3, a-linolenic acid. 1595
Downloaded from http://pcp.oxfordjournals.org/ at University of Sussex on July 27, 2015
Department of Biology, College of General Education, The University of Tokyo, Komaba, Meguro-ku, Tokyo 153,japan
1596
J. Ohnishi and
M. Yamada
Materials and rnethods
Seeds of oat, Avenasativa L. var. Victory I (The General Swedish Seed Company, Limited) were germinated in the dark at 25°C on wet vermiculite. Whole seedlings continued to grow for ten days, although the first leaves which had emerged from the coleoptiles stopped growing after reaching a length of 8 em on the 7th day. The 8-day-old seedlings were illuminated with 1,500 lux of fluorescent light (5 W/m 2) for 24 hr, during which the first leaves were collected at intervals. No further increase in the length and fresh weight of the first leaves was observed during 24 hr of greening. Therefore, the changes in lipids determined on the basis of fresh weight of the leaves reflect the lipid changes per leaf cell in greening leaves. Monocotyledonous leaves show an age sequence for cells from the base to the tip, owing to the activity of their basal meristems. Because the amounts of lipids in different-aged cells varied (14), two-centimeter sections, 4 to 6 em from the base of the leaf, were excised with a razor blade and used in the lipid analysis. These sections greened most rapidly on illumination. Plastids were isolated according to Nakamura and Yamada (16) with a slight modification. About 20 g of the leaf sections were cut into 5 mm pieces with a razor blade then homogenized in a Waring blendor three times for 3 sec with a semi-frozen slurry of 300 ml of the grinding medium, consisting of 10 m.M sodium pyrophosphate/ HCI, pH 7.0, 0.4 Msorbitol, 5 mM MgCI 2, 1 mM EDTA, 2 mM sodium ascorbate and 0.1 % (w/v) BSA. The resulting slurry was squeezed through two layers of Miracloth (Chicopee Mills, Inc.). The filtrate was centrifuged at 2,000 xg for 4 min to precipitate crude plastids. The crude plastids were suspended in a small volume of 50 mM Hepes/NaOH, pH 8.0, 0.4 M sorbitol, 5 mMMgCI 2 , 1 mu MnCI 2 ,
Downloaded from http://pcp.oxfordjournals.org/ at University of Sussex on July 27, 2015
When etiolated leaves develop into green leaves under illumination, etioplasts in the leaves differentiate into chloroplasts, accompanied by thylakoid biogenesis within the plastids (5). Lipid changes in greening leaves have been reported in several plants such as barley (25, 29), maize (13, 14), wheat (3), pea (23, 31) and bean (23). The generalization made from these observations is that galactolipids in etiolated leaves partially decrease during the initial stages of greening, but markedly increase during later stages, parallel to the rapid synthesis of chlorophyll. The increase in galactolipids was characterized by an increase in a-linolenate, reflecting the synthesis of a-linolenoyl-galactolipids in greening leaves (14, 29). However, little has been reported on phospholipid changes in greening leaves. Tevini (29) examined lipid changes in barley leaves during retarded greening and reported the conversion of galactolipids to phospholipids during the initial stages of greening. In relation to the lipid changes in greening leaves, tracer experiments have indicated that [14CJacetate is incorporated mainly into sterols and unsaponifiable lipids by detached leaves during early stages (2), but during later stages acetate incorporation is channelled to fatty acids and acyl lipids (2, 11, 19), in close connection with the appearance of photosynthetic activity (19). The present report deals with changes in glycerolipids in greening Avena leaves, in connection with the development of etioplasts to chloroplasts. Attention is focussed on changes in the constituent unsaturated fatty acids of the plastidic lipids.
Phospho- and glycolipid changes in greening Avena leaves
1597
Downloaded from http://pcp.oxfordjournals.org/ at University of Sussex on July 27, 2015
and 1 mn EDTA and layered on a discontinuous gradient consisting of 1.5 and 1.0 M sucrose in the suspending buffer without sorbitol. After centrifugation at 800 X g for 20 min, intact plastids (Class I) which banded at the interface were collected in a Hitachi density gradient fractionator DGF-U. The purified plastids were more than 90% intact when viewed by phase-contrast and electron microscopy, and they were free from the mitochondrial enzyme, succinate dehydrogenase (data not shown). Fig. 3a shows the typical profile of an isolated etioplast containing PLB. The plastid number was counted with a Thoma haemocytometer. Prior to lipid extraction, about 1 g of the leaf sections was steamed for 10 min to minimize lipid degradation by phospholipase D during extraction (24). Lipids were extracted from the leaf sections or from isolated plastids, according to Bligh and Dyer (4). Lipid classes were separated by silica gel TLC. Neutral lipids were developed with petroleum etherjchlorofromjacetone (4 : I : 1, vjv) (12) and polar lipids were developed with acetonejbenzenejwater (91 : 30 : 8, vjv) (20) or with chloroformjmethanoljwater (65 : 25 : 4, vjv) in the first direction and with chloroformjmethanoljammoniajisopropylamine (60 : 30 : 4 : 0.5, vjv) in the second direction (12). Lipid spots were located under UV light after spraying the plate with 0.01 % (wjv) primuline in 800/0 (vjv) acetone (33). Each lipid was identified by cochromatography with known standards and by spraying the plate with reagents specific for the lipid classes (26, 34). To quantify each lipid, the spot separated on a plate was scraped off and heated at 80°C for 2 hr in a screw-capped tube containing 2 ml of3% (wjw) RCI in methanol and a given amount of pentadecanoic acid as an internal standard. The fatty acid methyl esters were extracted with petroleum ether and analyzed by GLC in a Shimadzu GC-6AMF with glass columns (3 mm X 2 m) of 100~ (wjw) diethylene glycol succinate on Celite 545. The content of each lipid was determined from the quantities of its constituent fatty acids (1). The separated lipid sample was treated with the lipase from Rhizopus delemar (Seikagaku Kogyo Co., Ltd.) to yield 2-acyllysolipid and fatty acid composition at the C-2 position of sn-glycercl was determined (8). The composition at the C-l position was calculated from the total and C-2 compositions. The activity of fatty acid synthesis in the crude plastids was measured by the incorporation of [1-14C]acetate into the fatty acid moieties according to Nakamura and Yamada (16). Pigments in the leaf sections were extracted with acetone in a mortar and pestle and those in isolated plastids with 80% (vjv) acetone.· Chlorophylls and carotenoidswere determined photometrically in 800/0 (vjv) acetone (15, 28). Succinate dehydrogenase (EC 1.33.99.1) activity was determined according to Riatt (10). The plastid number per cell was determined in the leaf sections which had been fixed in 5% (wjv) glutaraldehyde, according to Possingham and Smith (21). At least 20 mesophyll cells were counted and averaged. Electron microscopy of the isolated plastids and the leaves was performed as reported previously (18). Tri-, di- and mono-olein, PG, PI, pentadecanoic acid and BSA, Fraction V, were purchased from Sigma. Sodium [1-14C]acetate (60.2 mCijmmole) was obtained from The Radiochemical Center.
]. Ohnishi and M. Yamada
1598
Results
Lipid composition of greening leaves andfatty acidsynthesis by isolated plastids
o
2
MGDG
Fig. 1. Polarlipid content of etiolated and greening Avena leaves. Values are the means of duplicate samples, provided that the deviations are less than 5% of the means.
3 6 12 24 Illumination (hr)
Downloaded from http://pcp.oxfordjournals.org/ at University of Sussex on July 27, 2015
Changes in the polar lipid content of Avena leaves during 24 hr of greening is shown in Fig. 1. Major lipids were glycolipids, such as MGDG, DGDG and SQDG, as well as phospholipids such as PG, PC, PE and PI. There was an initial decrease in all lipids, indicative of a partial breakdown of all the lipid classes during the early stages of greening. The three glycolipids and PG, of plastid origin (see Table 2), showed two types of diphasic changes: MGDG, SQDG and PG decreased for the first 1.5 hr and thereafter increased, whereas DGDG decreased for the first 3 hr then increased. During the 24 hr, MGDG almost doubled and DGDG, SQDG and PG increased by about 20, 40 and 20%, respectively. In contrast, extraplastidic lipids such as PC, PE and PI showed triphasic changes; after decreasing for the first 3 hr, they increased up to 6 hr-L and then decreased until 24 hr-L. Fig. 2 shows the incorporation of [1-14C]acetate into fatty acid moieties by the plastids isolated from greening Avena leaves. The activity of fatty acid synthesis in the dark was very low throughout greening. Light-enhanced fatty acid synthesis began at 3 hr-L and increased continuously during greening. The activity in 12 hr-L etiochloroplasts was comparable to that in chloroplasts isolated from lightgrown leaves. Since the site offatty acid synthesis in leaves seems to be limited to plastids (22), the simultaneous increase in plastidic lipids (Fig. 1) together with the induction of
Phospho- and glycolipid changes in greening Avena leaves
~3
1599
o
o
~
s:
........... (J)
~
o
/0
2
E
c
/0
u
§ u
I
,0
a-e· e -e -
e-
e_
e
« 00 3 6
12
24
c
Illumination ( hr ) Fig. 2. Incorporation of [1-14C]acetate into fatty acid moieties by the crude plastids isolatedfrom etiolated and greening Avena leaves. Isolated plastids were incubated with [1-14C]acetate in the light (0) or in the dark (e). Chloroplasts (C) were isolated from 8-day-old, light-grown leaves.
fatty acid synthesis suggests that the plastidic lipids were supplied with fatty acids which were light-dependently synthesized in the plastid per se. Moreover, plastidic lipids such as MGDG, SQDG and PG seem to be formed from other lipids, such as DGDG and the other phospholipids, because of the decrease in the latter lipids in contrast to the increase in the former between 1.5 hr-L and 3 hr-L. Correspondingly, at 1.5 hr-L, there was a temporary accumulation of DG (0.1 pmolefg fresh weight), the fatty acid composition of which is similar to the compositions of PC, PE and PI, but not to the composition of DGDG (Table 1). This is evidence that plastidic lipids such as MGDG, SQDG and PG were produced from such phospholipids as PC, PE and PI through DG, before the induction of light-dependent fatty acid synthesis. Lipid content of isolated plastids and plastid division
The electron microscopic examination of Avena etiochloroplasts often showed profiles of plastid division at 3 hr-L and 6 hr-L. Fig. 3b shows a typical division of Table 1 Fatry acid compositions of various lipids at 1.5 hr-L Fatty acid
% Composition
DG
PC
PE
PI
DGDG
16:0
34
27
22
48
14
18:0
3
