Lipid molecular species composition of thylakoid membranes

12

Biochimica et Biophysics @ Elsevier/North-Holland

Acta, 617 (1980) Biomedical Press

12-19

BBA 57492

LIPID MOLECULAR MEMBRANES

MASATERU Research

NISHIHARA,

Institute

(Received

SPECIES

KAZUSHIGE

for Food Science,

June 18th,

COMPOSITION

OF THYLAKOID

YOKOTA

Kyoto

and MAKOTO

University,

Uji, Kyoto

KIT0 611 (Japan)

1978)

Key words: Lipid composition; (Chloroplast membrane)

Molecular

species; Galactosyl

acylglycerol;

Phospholipid;

Summary Lipid molecular species compositions of chloroplast thylakoid membranes of mesophyll cells from Spinacia oleracea, Glycine max, Oryza sativa and Zea mays and of bundle sheath cells from Zea mays have been quantitatively determined. No significant difference in the lipid molecular species composition was found among the five membrane sources. The predominant molecular species of monogalactosyldiacylglycerol was the l-linolenoyl1.2linolenoyl species. The l-linolenoyl12-linolenoyl and l-palmitoylQ2-linolenoyl species were the major molecular species of digalactosyldiacylglycerol. 6-Sulfoquinovosyldiacylglycerol was mainly composed of the palmitoyl//linolenoyl and palmitoyl// linoleoyl species. Almost all of the C-2 position of phosphatidylglycerol were esterified with the palmitoyl or A3-trans-hexadecenoyl residue. The molecular species compositions of phosphatidylcholine and phosphatidylinositol were basically similar to those of membranes in non-photosynthetic tissues.

Introduction Lipid composition of chloroplasts is extremely different from those of other intracellular organelles. Glycolipid is the major lipid class, and phospholipid is a minor component. Chloroplasts are composed of thylakoid and envelope membranes. Although monogalactosyldiacylglycerol and digalactosyldiacylglycerol

.4bbreviations: 16

:

3,

a-linolenic

14

: 0.

myristic

7,10,13-hexadecatrienoic acid.

acid; acid;

16 18

: 0, : 0.

palmitic stearic

acid; acid;

A3-trans-16 18

: 1, oleic

:

1,

acid;

A3-trans-hexadecenoic 18

: 2.

linoleic

acid;

acid; 18

: 3,

13

are the major components of these membranes [ 1,2], 6-sulfoquinovosyldiacylglycerol and phosphatidylglycerol are localized in the thylakoid membranes [l-3]. It is interesting to compare the lipid molecular species composition of the thylakoid membranes from several plant species for an understanding of the function of the membranes in photosynthesis. In this paper, we describe the lipid molecular species compositions of the thylakoid membranes prepared from Spinacia oleracea, Glycine max, Oryza sativa and Zea mays. Materials and Methods Chemicals. Phospholipase C (Bacillus cereus) was purchased from Calbiothem. Phospholipase AZ (Crotalus admanteus) was from Boehringer Mannheim. Heptadecanoic acid was the product of Applied Science. Thin-layer plates (Art. 5721, 5724 and 11845) were purchased from Merck. Plant material. Spinach (S. oleracea L. var. Viroflay, 42 days old), soybean (G. max L. merr. var. Tsurunoko, 42 days old), rice plant (0. sativa L. var. Nipponbare, 40 days old) and maize (2. mays L. var. Honey Buntum, 45 days old) were used for experiments. These plants were grown in the experimental farm of the Research Institute for Food Science, Kyoto University. Preparation of thylakoid membranes and lipid extraction. The leaves of S. oleracea, G. max and 0. satiua and 2. mays leaves were homogenized at 3°C according to Jensen and Bassham [ 41 and Woo et al. [ 51, respectively, just after the leaves were picked off the plants. Intact chloroplasts were prepared from mesophyll cells [4] and bundle sheath cells [ 51. The envelope membranes were removed by osmotic shock [6] and the thylakoid membranes were purified by differential centrifugation [2]. Lipids were extracted by the method of Bligh and Dyer [ 71. Lipid extract was washed twice with 2 M KCl. Separation and purification of lipid classes. Lipid classes were purified by using DEAE-cellulose column chromatography [ 81 followed by thin-layer chromatography. The solvent systems used for the purification of each lipid by thin-layer chromatography were as follows: monogalactosyldiacylglycerol by CHC1&H30H/CH3COCH3/H,0/CH$OOH (200 : 50 : 60 : 5 : 2, v/v), digalactosyldiacylglycerol, phosphatidylcholine, phosphatidylinositol and phosphatidic acid by CHC1&H30H/CH3COOH (65 : 25 : 8, v/v), 6-sulfoquinovosyldiacylglycerol and phosphatidylglycerol by CHC13/CH30H/28% NH,OH (200 : 120 : 15, v/v). Phospholipase reaction. Phospholipids were hydrolyzed by phospholipase C [ 91 or phospholipase AZ [lo]. Analysis of molecular species. Analysis of phospholipid molecular species composition was carried out as previously [9,11]. Further resolution of the phosphatidylglycerol molecular species composition was performed by using phospholipase AZ [lo]. The molecular species of monogalactosyldiacylglycerol and digalactosyldiacylglycerol were separated on AgNO,-plates [9] by using CHC1&H30H/Hz0 (120 : 47 : 8, v / v ) as a developing solvent. Separation of the 6sulfoquinovosyldiacylglycerol molecular species on AgN03-plate was performed by using a solvent system CHC1&H30H (250 : 100, v/v) after the lipid had been acetylated by acetic anhydride and pyridine [9]. The visualiza-

14

tion of the subfractions on the plates was performed as previously [9]. Lipid was extracted from each subfraction by using CHC13/CH30H (2 : 1, v/v) after the subfraction had been scraped off from the plate. Fatty acid methyl esters were prepared from the lipid extract and analyzed by gas-liquid chromatography [ 111. For the quantitative analysis, a known amount of heptadecanoic acid had been added to each subfraction before it was scraped off from the plate. Positional distribution of fatty acids in the molecular species was determined by gas-liquid chromatography/mass spectrometry analysis of monoacetyldiacylglycerol [ 91. The monoacetyldiacylglycerol was prepared from 1,2diacylglycerol [9] which was the product of 2% H,S04-hydrolysis of galactolipids [ 121. For the determination of the 6-sulfoquinovosyldiacylglycerol molecular species, fatty acid composition of each subfraction was analyzed. Determination of lipid composition. An appropriate amount of lipid extract was applied on a thin-layer plate and separated by two-dimensional chromatography [ 111. Sugar [ 131, phosphorus [ 141 and sulfur [15] were determined in separated subfractions. Results Lipid composition

Thylakoid membranes in mesophyll cells of S. oleracea, G. max, 0. sativa and 2. mays are composed of stroma and grana lamellae, but the membranes of 2. mays bundle-sheath cells lack grana lamellae. These thylakoid membranes had similar lipid compositions except for those from the bundle-sheath cells (Table I). Major lipids were monogalactosyldiacylglycerol (38-44%), digalactosyldiacylglycerol (24-30%), 6-sulfoquinovosyldiacylglycerol (14-18s) and phospholipids (15-19s). Phosphatidylglycerol was the major class of phospholipids. The lipid composition of bundle-sheath cells is different from those of mesophyll cells. The most remarkable difference is the high content of phosphatidic acid. It is unlikely that phosphatidic acid was produced by hydrolysis of phospholipids by phospholipase D in leaves during preparation of lipids as we will describe afterwards. Although unidentified glycolipid subfractions were TABLE LIPID ND,

I COMPOSITION

not

detected;

OF T. trace.

Lipids

THYLAKOID AU values

MEMBRANES are mol%.

s. oleracea

G. max

0.

satiua

z. mays Mesophyll

Bundle

Monogalactosyldiacylglycerol

38

39

44

40

30

Digalactosyldiacylglycerol

29

28

24

30

23

14

17

14

10

11

11

8

10

10

Phosphatidylcholine

3

3

3

1

6

Phosphatidylinositol

1

2

1

1

T

Phosphatidylethanolamine

ND

ND

Phosphatidic

T

6-Sulfosuinovosyldiacylglycerol lB Phosphatidylglycerol

acid

3

T

ND 3

4

2 19

sheath

15

TABLE

II

MOLECULAR

SPECIES

Molecular 2-18 ND,

species

: 1,

: O//2-18

1-16

not

detected;

Molecular

species

C-l 16 18 18

less

COMPOSITION than

5%

: 2,

1-18

T. trace.

All

s.

OF

were

: O//2-18 values

MONOGALACTOSYLDIACYLGLYCEROL

omitted

from

: 2.

the

Table.

: O//2-18

1-18

: 3,

These 1-18

molecular

: l//2-18

and

oleracea

G. nwx

1-16

: O//

:

2//2-18

: 3.

z. moys

sotiua

0.

MesophyU

:3 :3 :3

were

1-18

are mol%.

c-2

: O//18 : 3//18 : 3//16

species

:3

Bundle

2

6

55

82

89

92

83

36

ND

ND

ND

ND

1

4

sheath

I

detected on a thin-layer plate, the sum of sugar content of the subfractions was less than 5% of that of the known glycolipids. Phosphorus could not be detected in any unidentified subfractions. Sulfur was only detected in the subfraction of 6-sulfoquinovosyldiacylglycerol. Galactolipid molecular species composition The galactolipid molecular species compositions are shown in Tables II and III. The predominant molecular species of monogalactosyldiglyceride of the five thylakoid membranes was the 1-18 : 312-18 : 3 species. The lipid from 5’. olerucea contained the molecular species of which C-2 position was esterified with 16 : 3, a lower homologue of 18 : 3 [16]. Thus, the molecular species of monogalactosyldiacylglycerol was basically composed of the 1-18 : 312-18 : 3 species except that of S. oleracea. The 1-18 : 312-18 : 3 and 1-16 : 012-18 : 3 species were the most abundant species in digalactosyldiacylglycerol. However, the amount of the 1-18 : 31216 : 3 species in digalactosyldiglyceride of S. olerucea was much smaller than that in monogalactosyldiglyceride. 6-Sulfoquinovosyldiacylglycerol molecular species composition It was impossible to convert 6-sulfoquinovosyldiacylglycerol to monoacetyldiacylglycerol, since diacylglycerol was not formed from this lipid by 2% TABLE

III

MOLECULAR

SPECIES

Molecular 18

: 1.

species l-18

less

: O//2-18

COMPOSITION than : 2,

5%

1-18

were

:

l//2-18

OF

DIGALACTOSYLDIACYLGLYCEROL

omitted

from

:3

and

1-18

the

Table.

:

2112-18

These

: 3.

molecular ND.

not

species

detected;

were

: O//2-

1-16

T. trace.

All

values

are mol%. Molecular C-l 16 16 18 18 18

species

s.

oleraceo

G. max

satiua

0.

c-2

: O//18 : O//18 : O//18 : 31118 : 3//16

z. mays Mesophyl

:2 :3 :3 :3 :3

6 13 1 64 6

T 24 7

11 13 4

1 24 2

Bundle 8 19 T

64

66

69

64

ND

ND

ND

ND

sheath

16

TABLE

IV

MOLECULAR Molecular 18

SPECIES species

less

:3

18

: l//18

Molecular

and

COMPOSITION than

:

2//18

5%

:

were

OF

3. T, trace.

from

All

the

values

Table,

These

molecular

species

were

0.

sativa

16 16 16 18

: O//16 : O//l8 : O//18 : O//18 : O//18

: 0

T

T

:1 :2 :3 :3

7

3

15

10

75

71

T

: O//18

:

2,

%. mays Mesophyll

16

18

are mol%.

G. max

s. oleracea

species

6-SULFOQUINOVOSYLDIACYLGLYCEROL

omitted

29 T 8 63

14

T

Bundle

T

T

T

T

33

33

62

67

T

sheath

T

H,S04-hydrolysis. Therefore, we could not determine the positional distribution of fatty acids in the molecular species subfractions obtained by thin-layer chromatography (Table IV). No similarity was detected between the molecular species compositions of 6-sulfoquinovosyldiacylglycerol and galactolipids. The 18 : 3118 : 3 species was not detected. Major molecular species were the 16 : 0118 : 3 and 16 : 0118 : 2. The 16 : 0116 : 0 species was one of the major molecular species of 6*ulfoquinovosyldiacylglycerol from 0. sutiua. Phosphatidylglycerol molecular species composition Table V shows the molecular species composition of phosphatidylglycerol. The molecular species composition was different from those of other phospholipids in the thylakoid membranes. The species of which C-2 position was esterified with 16 : 0 and A3-trans-16 : 1 were the major molecular species. Generally, saturated and unsaturated fatty acids are distributed in phospholipid molecules in such a way that a saturated fatty acid is esterified to C-l position and unsaturated fatty acid to C-2 position [9,11,17-191. However, TABLE

V

MOLECULAR

SPECIES

Molecular 16 1-18

: 0.

species 1-18

: l//2-18

Molecular

16

: O//16 : 0 : O/lA3-trans-16 : O//A3-trans-16 : l//16 : 0 : l//A3-trans-16 : 21116 : 0 : 2//63-trans-16 : 31116 : 0 : 3/lA3-trans-16 : 21118 : 3

18 18 18 18 18 18 18

5%

s.

species c-2

18

COMPOSITION than

were

OF

oleracea

PHOSPHATIDYLGLYCEROL

omitted

: O//2-18 : 1, 1-18 : O//2-18 : 3, 1-18 : 2112-18 : 2 and

C-l

16

less

from

:

2,

1-18

the

Table.

These

: l//2-18 : 1, : 3112-18 : 3 T, trace. l-18

G. l?mY

0. satiua

molecular

l-18 All

z.

values

4

: :

1 1

:1

10

T 43

18 19

T

15

T

3

3

5

1

28

5

T 39 13 7 14

: 3,

Bundle 34 22 1 T 1

1

17

5

1

5

6

2

8

3

2

T

2

4

10 T

4

1

T

3

10 T

l-18 : O//2: l//2-18 : 2,

are mol%.

5

16

were l-18

mays

Mesophyll

:1 :1

species

: O//2-18

4 21

sheath

17 TABLE

VI

MOLECULAR Molecular 18

: 1,

:

1-18

Molecular C-l

SPECIES COMPOSITION

species

less than

l//2-18

species

:

l,l-18

s.

OF PHOSPHATIDYLCHOLINE

5% were omitted

:

l//2-18

:2

from

and 1-18

G. max

oleracea

the Table.

: l//2-18

These molecular

: 3.

0. satiua

z.

C-2

16 : O//18 16 : O//18 18 : O//l8 16 : O//18 18 : O//18 18 : 21118 18 : 21118 18 : 3//18

:1 :2 :2 :3 :3 :2 :3 :3

10 18 2 21 3 1 13 8

4 22 18 20 4 13 9 3

species

were 1-18

: O//2-

T, trace. All values are molW.

6 26 11 17 10 10 5 5

mays

Mesophyl

Bundle sheath

6 31 2 26 5 8 6 4

6 39 1 23 6 7 10 3

phosphatidylglycerol was composed for a considerable amount of the molecular species esterified with 16 : 0 at C-2 position and an unsaturated fatty acid at C-l position. Molecular species compositions of phosphatidylcholine and phosphatidylinositol The molecular species compositions of phosphatidylcholine and phosphatidylinositol (Tables VI and VII) were similar to those from non-photosynthetic tissues [11,17,18]. Fatty acid composition of phosphatidic acid No remarkable differences were found in the fatty acid composition of phosphatidic acid (Table VIII). It is unlikely that phosphatidic acid was formed by hydrolysis of phospholipids by phospholipase D in leaves, since A3-trans-16 : 1 was not detected.

TABLE

VII

MOLECULAR

SPECIES

COMPOSITION

OF PHOSPHATIDYLINOSITOL

Molecular species less than 5% were omitted from the Table. These molecular 18 : 1. l-18 : l//2-18 :l, 1-18 : l//2-18 : 2, 1-18 : l//2-18 : 3, 1-18 : 2//2-18

species were and 1-18

:2

:

1-18 : O//22//2-18 : 3.

T, trace. All values are mol%. Molecular C-l 16 16 18 16 18 18

species

s. oleracea

G. max

0. sativa

c-2

: O//18 : O//l8 : O//l8 : O//18 : O//l8 : 3//18

:1 :2 :2 :3 :3 :3

* Phosphatidylinositol

13 40 2 32 T 8 content

T 35 10 42 13 T of the lipids

T 10 1 58 10 20 from

bundle-sheath

z. mays Mesophyll

Bundle

4 40 9 35 7 T

-

cells was trace as described

sheath *

in Table

I.

18 TABLE

VIII

FATTY

ACID

ND,

detected;

not

Fatty

COMPOSITION T, trace.

OF All

s. oleracea

acid

PHOSPHATIDIC

values

ACID

are %.

*

G. max

0.

sativa

z.

may.5 Bundle

Mesophyll

14

:0

16

: 0

A3-tram-16

I

:I

-

18: 0 18 : 1 18:

2

18

:3 :3

16

* Phosphatidic

21

37

34

31

ND

ND

ND

ND

9

6

6

2

4

7

7

32

acid

content

of lipids

from

T

T

T

33

2

29

38

27

17

24

27

ND

ND

ND

ND

S. oleracea

was trace

as described

sheath

in Table

I.

Discussion The positional specificity of 16 : 0 in galactolipids of spinach was determined to be the C-2 position by using pancreatic [20] and Rhizopus [21] lipase, but specific positional distribution of this fatty acid at C-l position was also described by using pancreatic lipase [22]. We determined the galactolipid molecular species by gas-liquid chromatography/mass spectrometry analysis as described in Methods, and 16 : 0 was found to be in C-l position. The discrepancy may be due to the differences between analytical methods. The molecular species composition of phosphatidylglycerol was unique. Haverkate and van Deenen [lo] described the molecular species composition of phosphatidylglycerol extracted from the leaves of S. olerucea. In their experiments, A3-truns-16 : 1 was located at C-2 position and the 1-18 : 312-A3truns-16 : 1 species was the most abundant one. Our results are consistent with theirs. The other major molecular species in their results were the 1-18 : 31216 : 0 and 1-16 : 002-18 : 2 species. In our experiments, the molecular species containing unsaturated fatty acids and 16 : 0 were only 1-unsaturated12-16 : 0 species. Probably, the 1-16 : O//2-unsaturated fatty acyl species which they detected might be obtained from phosphatidylglycerol of the other intracellular organelles in the leaf cells. It has been assumed that A3-truns-16 : 1 is produced from the esterified 16 : 0 of phosphatidylglycerol [23]. The species containing 16 : 0 in Table V may be precursors of the species containing A3tram-16 : 1. Therefore, we may conclude that almost all of the C-2 positions of the molecular species of phosphatidylglycerol are esterified with A3-truns16 : 1 and its precursors. Probably, phosphatidylglycerol esterified with A3truns-16 : 1 at C-2 position may be required for thylakoid membrane function. Acknowledgement This work was supported by a grant for Scientific Research from the Ministry of Education of Japan.

19

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