Plant & CellPhysiol. 21 (8): 1607-1618 (1980)
Glycerolipid synthesis in Avena leaves during greening of etiolated seedlings II. a-Linolenic acid synthesis Jun-ichi Ohnishi and Mitsuhiro Yamada
(Received September 24, 1980)
To determine the synthesis of a-linolenic acid esterified to galactolipids, etiolated leaves from Avenasativa L. were fed with [1-14C]acetate for the first 3 hr of greening, and the redistribution of 14C incorporated into the fatty acid moieties of lipid classes was examined during a 21-hr chase. Phosphatidylcholine (PC) was most heavily labeled, but lost a large portion of its 14C during the chase. Galactolipids, such as monogalactosyldiglyceride (MGDG) and digalactosyldiglyceride (DGDG), were only slightly labeled at the start, but gradually gained 14C during the chase. When greening Avena leaves were pulse-labeled with [2- 3H]glycerol and chased in the same manner, a similar incorporation and redistribution of 3H were observed in the glycerol moieties of lipid classes. A [1-14C]0Ieic acid-feeding experiment also showed the same redistribution of 14C from PC to galactolipids and little change of the 14C incorporated into phosphatidylglycerol and phosphatidylethanolamine. These results are evidence that galactolipids were synthesized from PC in greening Avena leaves. Time courses for the 14C in the fatty acid moieties of lipid classes in both [1-14C]acetate- and [1-14C]0Ieic acid-feeding experiments showed dominant labeling of oleoylPC during the early hours and the subsequent transfer of the label from oleoyl-PC to linoleoyl-PC, linoleoyl-MGDG and finally to a-linolenoyl-MGDG. From these results, the major pathway of a-linolenic acid synthesis in greening Avena leaves is proposed: oleate synthesized de novo is first acylated to PC then desaturated to linoleoyl-PC, which is in turn converted to MGDG and desaturated to a-linolenoyl-MGDG. Because of the slow incorporation of 14C into a-linolenoyl-DGDG, in contrast to the rapid incorporation of 14C into a-linolenoyl-MGDG, in the [1-14C]acetate- and [l-14C]linoleic acidfeeding experiments, the former lipid class seems to be formed by the galactosylation of the latter rather than by the desaturation of linoleoyl-DGDG. The involvement of PC-exchange protein in the transfer of linoleoyl-PC from ER to the plastid is discussed. Key words: Avenasativa- Fatty acid desaturation - Greening leaves - a-Linolenic acid synthesis - Monogalactosyldiglyceride - Phosphatidylcholine.
Abbreviations: DG, diglyceride; DGDG, digalactosyldiglyceride; ER, endoplasmic reticulum; GLC, gas-liquid chromatography; MGDG, monogalactosyldiglyceride; PC, phosphatidylcholine; PE, phosphatidylethanolamine; PG, phosphatidylglycerol; PI, phosphatidylinositol; SQDG, sulfoquinovosyldiglyceride; TG, triglyceride; TLC, thin-layer chromatography; x hr-L, x hr of illumination; 16:0, palmitic acid; 16:3, hexadec-trienoic acid; 18:1, oleic acid; 18:2, linoleic acid; 18:3, a-linolenic acid; 18:1f16:0-MGDG, 1-0Ieoyl-2-palmitoyl-MGDG. 1607
Downloaded from http://pcp.oxfordjournals.org/ at Deakin University Library on March 15, 2015
Department of Biology, College of General Education, The University of Tokyo, Komaba, Meguro-ku, Tokyo 153,]apan
1608
]. Ohnishi and M. Yamada
plast with ER for the synthesis of 18:3.
Based on a dual labeling experiment supporting the transfer of both 14C-acyl and 3H-glycerol moieties from 18:2/18:2-PC to 18:3/18:3-MGDG in developing maize leaves (32), Roughan et al. (27) proposed a pathway in which 18 :2-PC desaturated from 18:1-PC in ER is degraded to 18:2-DG, which in turn is transferred to the chloroplast and undergoes galactosylation and desaturation to 18 :3MGDG (eukaryotic pathway). Recently, Roughan et al. (28) demonstrated that chloroplasts isolated from young spinach leaves possess the ability to successively desaturate 18:1jI6:0-MGDG to 18:3jI6:3-MGDG. This leads to an alternative pathway for 18:3 synthesis in the chloroplast alone (prokaryotic pathway) (27), which is consistent with the view of Siebertz and Heinz (30). Under these circumstances, the synthesis of 18:3 in leaves is confusing with respect to both the path and its subcellular localization. In a previous paper (23), we showed that MGDG which mostly contains 18:3 increased two-fold in greening Avena leaves during 24 hr. Thus, these leaves provide excellent materials for the investigation of the path and the site of 18:3 synthesis. Our present study on the labeling of greening Avena leaves was undertaken to determine the path of 18:3 synthesis in vivo.
Downloaded from http://pcp.oxfordjournals.org/ at Deakin University Library on March 15, 2015
Intensive studies from Stumpf's laboratory have determined the path of 18: 1CoA synthesis from acetyl-CoA in the chloroplast (15, 16, 22, 29, 37). However, details of the polyunsaturation of 18: 1 are obscure, although the sequential desaturation of 18: 1 to 18:3 is proved (5, 11). Two types of desaturation reactions have been proposed: desaturations in the form of acyl-CoAs and acyl-glycerides. Vijay and Stumpf (43) reported that 18: l-CoA was desaturated directly to 18 :2-CoA by microsomes from developing safflower seeds. The same conclusion was made by Mazliak's group for microsomes of potato (1) and pea leaves (8, 41). In contrast, early experiments with Chlorella indicated the rapid incorporation and turnover of fatty acids in PC and MGDG, evidence of the involvement of these lipids in polyunsaturation (2, 10, 21). In higher plants, Roughan first suggested that PC is involved in the desaturation of both 18:1 and 18:2 in pumpkin leaves (25, 26). Results of pulse-chase experiments with leaves of maize (31,32) and broad bean (47) support the desaturation of 18:1 in PC. Moreover, desaturation of 18:1-PC to 18:2-PC has been demonstrated in the microsomes of various microorganisms (3, 18, 24, 39, 48), developing safflower seeds (33, 35) and pea leaves (34). Further desaturation of 18:2-PC to 18:3-PC was suggested from the results of labeling experiments with alfalfa leaves (45) and the homogenate of developing soybean cotyledons (36), but labeling experiments with the leaves of spinach (30), broad bean (13) and some Gramineae plants (4,44) were not favorable for the synthesis of 18 :3 in PC. On the other hand, Siebertz and Heinz (30) and Heinz and Harwood (13) proposed the desaturation of 18:1 to 18:3 in MGDG, based on the time course of tracer incorporation into spinach and broad bean leaves. I t is puzzling that isolated chloroplasts are not capable of synthesizing 18:2 and 18:3 (37), in spite of the predominance of 18:3 in the fatty acids of chloroplasts. Tremolieres and Mazliak (41) showed the localization of 18: 1 desaturation in ER and 18:2 desaturation in the chloroplast, indicative of the cooperation of the chloro-
a-Linolenic acid synthesis in greening Avena leaves
1609
Materials and :methods
Results
Pulse-chase experiments with [1-14C]acetate and [2-3H ]glycerol When etiolated Avena leaves were pulse-fed with [1-14C]acetate for 3 hr in the light then chased with unlabeled acetate for the next 21 hr in the light, 14C was incorporated into the fatty acid moieties of polar lipids and pigments during the 3 hr of labeling and the first 3 hr of the chase. The bulk of the label in the polar lipids (78°h» was retained in greening leaves during the later part of the chase. The distribution of radioactivity in the lipid classes during the chase is shown in Fig. IA. At the start of the chase (3 hr-L), the label in PC was the largest among the polar lipids, reaching a maximum (about 40°h> of the total polar lipids) at 6 hr-L. This was followed by a marked decrease until 24 hr-L, in which 40% of the maximum label was lost. The label in PG and PE, respectively, contained about half the label in PC at 3 and 6 hr-L, and reached a maximum at 9 hr-L. Thereafter, PG reduced about 40% of the label until 24 hr-L, although the label in PE slightly decreased during the chase. In contrast, MGDG and DGDG contained little label at 3 hr-L, but gradually gained label throughout the chase. Other lipids such as SQDG and PI showed little label.
Downloaded from http://pcp.oxfordjournals.org/ at Deakin University Library on March 15, 2015
Etiolated leaves were excised from oat (Avena sativa L. var. Victory I) seedlings grown in the dark at 25°C for 8 days as previously reported (23). The upper halves (4 em long) of the leaves were removed with a razor blade, and their bases were dipped in sodium [1-14C]acetate solution (200/lCi/ml water). These leaf sections were incubated for 3 hr at 25°C under 1,500 lux fluorescent light. After a brief washing with water, the sections were transferred to 5 mM unlabeled acetate solution and chased for another 21 hr in the light. At intervals during the chase, leaf sections were withdrawn and heated in boiling isopropanol for 5 min. In a similar experiment with [2- 3H]glycerol, an 80 /lCi/ml isotope solution was used for the 3-hr pulse labeling and 10 rna unlabeled glycerol solution for the 21-hr chase. In other experiments leaf sections were labeled with [1_14C]0Ieic acid or [1-14C]linoleic acid. A 5 /ll drop of isotope solution (30/lCi/ml diethylene glycol monoethyl ether) was absorbed from the surface of each section. The bases of the leaf sections were then dipped -in water after which the sections were kept in the light at 25°C for 24 hr. The extraction of lipids from the leaf sections and the separation of lipid classes by TLC were performed as reported previously (23). For the analysis of both the polar and neutral lipids on the same plate, the lipid sample on the plate was first developed half-way with acetone/benzene/water (91 : 30 : 8, v/v) , immediately transferred to petroleum ether/chloroform/acetone (4 : I : I, v/v) and developed to the top. Separated lipids were counted with a Beckman LS 9000 scintillation system. Fatty acid methyl esters were prepared from each lipid class (23) and analyzed by radio-GLC (50). Sodium [1-14C]acetate (60.2 mCi/mmole), [1_14C]0Ieic acid (58 mCi/mmole) and [1-14C]linoleic acid (52 mCi/mmole) were purchased from The Radiochemical Center. [2- 3H] Glycerol (200 mCijmmole) was obtained from New England Nuclear.
1610
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Glycerolipid synthesis in Avena leaves during greening of etiolated seedlings II. a-Linolenic acid synthesis Jun-ichi Ohnishi and Mitsuhiro Yamada
(Received September 24, 1980)
To determine the synthesis of a-linolenic acid esterified to galactolipids, etiolated leaves from Avenasativa L. were fed with [1-14C]acetate for the first 3 hr of greening, and the redistribution of 14C incorporated into the fatty acid moieties of lipid classes was examined during a 21-hr chase. Phosphatidylcholine (PC) was most heavily labeled, but lost a large portion of its 14C during the chase. Galactolipids, such as monogalactosyldiglyceride (MGDG) and digalactosyldiglyceride (DGDG), were only slightly labeled at the start, but gradually gained 14C during the chase. When greening Avena leaves were pulse-labeled with [2- 3H]glycerol and chased in the same manner, a similar incorporation and redistribution of 3H were observed in the glycerol moieties of lipid classes. A [1-14C]0Ieic acid-feeding experiment also showed the same redistribution of 14C from PC to galactolipids and little change of the 14C incorporated into phosphatidylglycerol and phosphatidylethanolamine. These results are evidence that galactolipids were synthesized from PC in greening Avena leaves. Time courses for the 14C in the fatty acid moieties of lipid classes in both [1-14C]acetate- and [1-14C]0Ieic acid-feeding experiments showed dominant labeling of oleoylPC during the early hours and the subsequent transfer of the label from oleoyl-PC to linoleoyl-PC, linoleoyl-MGDG and finally to a-linolenoyl-MGDG. From these results, the major pathway of a-linolenic acid synthesis in greening Avena leaves is proposed: oleate synthesized de novo is first acylated to PC then desaturated to linoleoyl-PC, which is in turn converted to MGDG and desaturated to a-linolenoyl-MGDG. Because of the slow incorporation of 14C into a-linolenoyl-DGDG, in contrast to the rapid incorporation of 14C into a-linolenoyl-MGDG, in the [1-14C]acetate- and [l-14C]linoleic acidfeeding experiments, the former lipid class seems to be formed by the galactosylation of the latter rather than by the desaturation of linoleoyl-DGDG. The involvement of PC-exchange protein in the transfer of linoleoyl-PC from ER to the plastid is discussed. Key words: Avenasativa- Fatty acid desaturation - Greening leaves - a-Linolenic acid synthesis - Monogalactosyldiglyceride - Phosphatidylcholine.
Abbreviations: DG, diglyceride; DGDG, digalactosyldiglyceride; ER, endoplasmic reticulum; GLC, gas-liquid chromatography; MGDG, monogalactosyldiglyceride; PC, phosphatidylcholine; PE, phosphatidylethanolamine; PG, phosphatidylglycerol; PI, phosphatidylinositol; SQDG, sulfoquinovosyldiglyceride; TG, triglyceride; TLC, thin-layer chromatography; x hr-L, x hr of illumination; 16:0, palmitic acid; 16:3, hexadec-trienoic acid; 18:1, oleic acid; 18:2, linoleic acid; 18:3, a-linolenic acid; 18:1f16:0-MGDG, 1-0Ieoyl-2-palmitoyl-MGDG. 1607
Downloaded from http://pcp.oxfordjournals.org/ at Deakin University Library on March 15, 2015
Department of Biology, College of General Education, The University of Tokyo, Komaba, Meguro-ku, Tokyo 153,]apan
1608
]. Ohnishi and M. Yamada
plast with ER for the synthesis of 18:3.
Based on a dual labeling experiment supporting the transfer of both 14C-acyl and 3H-glycerol moieties from 18:2/18:2-PC to 18:3/18:3-MGDG in developing maize leaves (32), Roughan et al. (27) proposed a pathway in which 18 :2-PC desaturated from 18:1-PC in ER is degraded to 18:2-DG, which in turn is transferred to the chloroplast and undergoes galactosylation and desaturation to 18 :3MGDG (eukaryotic pathway). Recently, Roughan et al. (28) demonstrated that chloroplasts isolated from young spinach leaves possess the ability to successively desaturate 18:1jI6:0-MGDG to 18:3jI6:3-MGDG. This leads to an alternative pathway for 18:3 synthesis in the chloroplast alone (prokaryotic pathway) (27), which is consistent with the view of Siebertz and Heinz (30). Under these circumstances, the synthesis of 18:3 in leaves is confusing with respect to both the path and its subcellular localization. In a previous paper (23), we showed that MGDG which mostly contains 18:3 increased two-fold in greening Avena leaves during 24 hr. Thus, these leaves provide excellent materials for the investigation of the path and the site of 18:3 synthesis. Our present study on the labeling of greening Avena leaves was undertaken to determine the path of 18:3 synthesis in vivo.
Downloaded from http://pcp.oxfordjournals.org/ at Deakin University Library on March 15, 2015
Intensive studies from Stumpf's laboratory have determined the path of 18: 1CoA synthesis from acetyl-CoA in the chloroplast (15, 16, 22, 29, 37). However, details of the polyunsaturation of 18: 1 are obscure, although the sequential desaturation of 18: 1 to 18:3 is proved (5, 11). Two types of desaturation reactions have been proposed: desaturations in the form of acyl-CoAs and acyl-glycerides. Vijay and Stumpf (43) reported that 18: l-CoA was desaturated directly to 18 :2-CoA by microsomes from developing safflower seeds. The same conclusion was made by Mazliak's group for microsomes of potato (1) and pea leaves (8, 41). In contrast, early experiments with Chlorella indicated the rapid incorporation and turnover of fatty acids in PC and MGDG, evidence of the involvement of these lipids in polyunsaturation (2, 10, 21). In higher plants, Roughan first suggested that PC is involved in the desaturation of both 18:1 and 18:2 in pumpkin leaves (25, 26). Results of pulse-chase experiments with leaves of maize (31,32) and broad bean (47) support the desaturation of 18:1 in PC. Moreover, desaturation of 18:1-PC to 18:2-PC has been demonstrated in the microsomes of various microorganisms (3, 18, 24, 39, 48), developing safflower seeds (33, 35) and pea leaves (34). Further desaturation of 18:2-PC to 18:3-PC was suggested from the results of labeling experiments with alfalfa leaves (45) and the homogenate of developing soybean cotyledons (36), but labeling experiments with the leaves of spinach (30), broad bean (13) and some Gramineae plants (4,44) were not favorable for the synthesis of 18 :3 in PC. On the other hand, Siebertz and Heinz (30) and Heinz and Harwood (13) proposed the desaturation of 18:1 to 18:3 in MGDG, based on the time course of tracer incorporation into spinach and broad bean leaves. I t is puzzling that isolated chloroplasts are not capable of synthesizing 18:2 and 18:3 (37), in spite of the predominance of 18:3 in the fatty acids of chloroplasts. Tremolieres and Mazliak (41) showed the localization of 18: 1 desaturation in ER and 18:2 desaturation in the chloroplast, indicative of the cooperation of the chloro-
a-Linolenic acid synthesis in greening Avena leaves
1609
Materials and :methods
Results
Pulse-chase experiments with [1-14C]acetate and [2-3H ]glycerol When etiolated Avena leaves were pulse-fed with [1-14C]acetate for 3 hr in the light then chased with unlabeled acetate for the next 21 hr in the light, 14C was incorporated into the fatty acid moieties of polar lipids and pigments during the 3 hr of labeling and the first 3 hr of the chase. The bulk of the label in the polar lipids (78°h» was retained in greening leaves during the later part of the chase. The distribution of radioactivity in the lipid classes during the chase is shown in Fig. IA. At the start of the chase (3 hr-L), the label in PC was the largest among the polar lipids, reaching a maximum (about 40°h> of the total polar lipids) at 6 hr-L. This was followed by a marked decrease until 24 hr-L, in which 40% of the maximum label was lost. The label in PG and PE, respectively, contained about half the label in PC at 3 and 6 hr-L, and reached a maximum at 9 hr-L. Thereafter, PG reduced about 40% of the label until 24 hr-L, although the label in PE slightly decreased during the chase. In contrast, MGDG and DGDG contained little label at 3 hr-L, but gradually gained label throughout the chase. Other lipids such as SQDG and PI showed little label.
Downloaded from http://pcp.oxfordjournals.org/ at Deakin University Library on March 15, 2015
Etiolated leaves were excised from oat (Avena sativa L. var. Victory I) seedlings grown in the dark at 25°C for 8 days as previously reported (23). The upper halves (4 em long) of the leaves were removed with a razor blade, and their bases were dipped in sodium [1-14C]acetate solution (200/lCi/ml water). These leaf sections were incubated for 3 hr at 25°C under 1,500 lux fluorescent light. After a brief washing with water, the sections were transferred to 5 mM unlabeled acetate solution and chased for another 21 hr in the light. At intervals during the chase, leaf sections were withdrawn and heated in boiling isopropanol for 5 min. In a similar experiment with [2- 3H]glycerol, an 80 /lCi/ml isotope solution was used for the 3-hr pulse labeling and 10 rna unlabeled glycerol solution for the 21-hr chase. In other experiments leaf sections were labeled with [1_14C]0Ieic acid or [1-14C]linoleic acid. A 5 /ll drop of isotope solution (30/lCi/ml diethylene glycol monoethyl ether) was absorbed from the surface of each section. The bases of the leaf sections were then dipped -in water after which the sections were kept in the light at 25°C for 24 hr. The extraction of lipids from the leaf sections and the separation of lipid classes by TLC were performed as reported previously (23). For the analysis of both the polar and neutral lipids on the same plate, the lipid sample on the plate was first developed half-way with acetone/benzene/water (91 : 30 : 8, v/v) , immediately transferred to petroleum ether/chloroform/acetone (4 : I : I, v/v) and developed to the top. Separated lipids were counted with a Beckman LS 9000 scintillation system. Fatty acid methyl esters were prepared from each lipid class (23) and analyzed by radio-GLC (50). Sodium [1-14C]acetate (60.2 mCi/mmole), [1_14C]0Ieic acid (58 mCi/mmole) and [1-14C]linoleic acid (52 mCi/mmole) were purchased from The Radiochemical Center. [2- 3H] Glycerol (200 mCijmmole) was obtained from New England Nuclear.
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