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Acyl lipids in photosynthetically active tissue cultures of tobacco
Plant Science Letters, 12 (1978) 119--126
119
© Elsevier/North-Holland Scientific Publishers Ltd.
ACYL LIPIDS IN PHOTOSYNTHETICALLY ACTIVE TISSUE CULTURES OF TOBACCO
H.P. SIEBERTZ, E. HEINZ and L. BERGMANN Botanisches Institut der Universiti~t zu K~ln, Gyrhofstrasse 15,D-5000 Koln 41 (G.F.R.)
(Received October 27th, 1977) (Accepted February 14th, 1978)
SUMMARY
Lipid mixtures extracted from green leaves, green tissue cultures, etiolated cotyledons and heterotrophic cultures were subjected to quantitative deterruination o f the major phospho- and glycolipids. Fatty acid analyses including positional distribution at the glycerol moiety were carried out for individual lipid components. The results demonstrate tnat green cultures occupy a position between green leaves and heterotrophic cultures. The major membrane lipids known from photoautotrophic tissues are present in green cultures. Components concentrated in chloroplasts such as monogalactosyl-(MGD) and sulphoquinovosyl diacylglycerol (SQD) and the fatty acids C,6 • 3 and 3-transC16 : 1 occur at markedly reduced levels. A general increase in the proportion of Cls .. 2 at the expense o f C18 : 3 is observed in tissue cultures. The positional distribution of significant fatty acids is identical in the lipids from green cultures and leaves.
INTRODUCTION
Tissue cultures are established tools for research in various fields o f plant physiology. An increasing n u m b e r of investigations is directed towards the elucidation o f composition and metabolism o f the various lipid classes present in these cultures (summarized in ref. 1). With one exception [2], all these experiments have been carried out with non-green cultures, the lipids o f which differ in several aspects from those o f photosynthetic tissues of the m o t h e r plant. Therefore it seemed worthwhile to analyze the lipids from a photosynAbbreviations: APE, acylated phosphatidyl ethanolamine; ASG, acylated steryl glycoside;
BPA, bis-phosphatidic acid; DGD, digalactosyl diacylglycerol; GLC, gas-liquid chromatography; MGD, monogalactosyl diacylglycerol; PA, phosphatidic acid; PC, phosphatidyl choline; PE, phosphatidyl ethanolamine; PG, phosphatidyl glycerol; PI, phosphatidyl inositol; SQD, sulfoquinovosyl diacylglycerol; TLC, thin layer chromatography.
120
thetically active culture. Furthermore the comparison between a green culture and a photosynthetic tissue o f the parent plant should explore a possible use of green cultures as model systems for experimentation in the field of lipid metabolism. For this purpose we used a well established green culture from t o b a c c o [ 3 ] , which as m e m b e r o f the Solanaceae should be an interesting plant for studies in fatty acid biosynthesis. Leaves of plants from Solanaceae and several other families [4] are characterized b y the occurrence of hexadecatrienoic acid C16 : 3, which is an interesting fatty acid from several points of view. It is usually localized rather exclusively in the sn-2-position of MGD and represents a marker for a chloroplast-concentrated lipid. Previous experiments with Anthriscus leaves (belonging to the Apiaceae) were interpreted as showing that C~6 : 3 was derived from C~6 : 0 b y sequential desaturation [5]. On the other hand C~6 : 3 has also been proposed [6] to be derived from C~4 • 3 and to be the immediate precursor of Cla : 3. Although C~8 .. 3 is the most abundant polyenoic acid in leaf lipids, its exact biosynthesis has n o t yet been clarified. For further studies in this direction an easily manipulatable system such as a tissue culture would be of great advantage. From this point of view we were specifically interested in the occurrence of C16 • 3 in the lipids from green cultures of tobacco. EXPERIMENTAL
Growth o f tissue cultures The SCI line of t o b a c c o cells, derived from stem pith of Nicotiana tabacum var. Samsun [3] was grown at 25°C in a modified liquid Murashige-Skoogmedium as described in detail before [7]. After 10--12 days o f culture cells were harvested at the end of the logarithmic growth phase, washed twice with fresh sterile medium and lyophilized. Lipid extraction and analysis Lipids from lyophilized cultures were extracted with h o t chloroform/methanol/isopropanol 20/10/5. Lipid extracts were purified b y Solvent partition and analyzed after thin layer chromatography {TLC) separation o f individual c o m p o n e n t s b y previously described methods [ 5] including gas-liquid chromatography (GLC) of fatty acid methyl esters, quantitation b y inclusion of C17 : 0 as internal standard and positional analysis b y lipase or phospholipase A2 hydrolysis. GLC peak assignments were confirmed b y rechromatography after hydrogenation. F a t t y acids in Tables I and III are characterized b y n u m b e r of carbon atoms and double bonds. Percentages below 0.1% are denoted as tr. For chromatographic comparison reference c o m p o u n d s were prepared for phosphatidic acid (PA) {hydrolysis o f phosphatidyl choline (PC) b y phospholipase D), acylated phosphatidyl ethanolamine (APE) (acylation of phosphatidyl ethanolamine (PE) with oleoyl chloride according to ref. 8) and bis-phosphatidic acid (BPA) (by acylation o f phosphatidyl glycerol (PG) with oleoyl chloride). Cardiolipin was a gift from Prof. Dr. W. Fischer, University of Erlangen.
121 I n s o m e cases w h e n fresh c u l t u r e s w e r e d i r e c t l y p l a c e d i n t o h o t e x t r a c t i o n m e d i u m large q u a n t i t i e s o f a n u n u s u a l p h o s p h o l i p i d w e r e o b s e r v e d in t h e extracts. This substance c o c h r o m a t o g r a p h e d with phosphatidyl m e t h a n o l (prep a r e d as r e f e r e n c e c o m p o u n d a c c o r d i n g t o ref. 9, R f - v a l u e o f 0.4 in c h l o r o f o r m / m e t h a n o l / N H 4 O H c o n c . 7 0 / 2 0 / 1 . 5 o n p r e c o a t e d silica gel plates} a n d p r o b a b l y originated from phospholipase D-catalyzed transphosphatidylation reactions between phospholipids and methanol [ 10,11]. RESULTS AND DISCUSSION T h e t o t a l lipids f r o m leaves, g r e e n a n d h e t e r o t r o p h i c c u l t u r e s a m o u n t e d t o 9 . 7 , 6.3 a n d 1.7%, r e s p e c t i v e l y , o f t h e d r y w e i g h t ( T a b l e IIa). C o r r e s p o n d i n g values f r o m a v a r i e t y o f h e t e r o t r o p h i c c u l t u r e s c o v e r e d a r a n g e o f 2 . 6 - . 8 . 1 % (1). In T a b l e I t h e f a t t y acid a n a l y s e s f r o m t o t a l lipid e x t r a c t s are s u m m a r i z e d . W h e n g o i n g f r o m leaves via g r e e n t o n o n - g r e e n c u l t u r e s a general increase in t h e p r o p o r t i o n o f C 1 6 : 0, Cls : 0, C,s : 2 a n d a d e c r e a s e in C18 : 3 c a n b e observed. M o s t significant are t h e r e d u c e d levels o f C,6 : 3 a n d 3-trans-C16 : 1, b o t h o f w h i c h are c o n n e c t e d in p h o t o s y n t h e t i c tissues w i t h t h e c h l o r o p l a s t lipids M G D a n d P G , r e s p e c t i v e l y . F o r c o m p a r i s o n t h e f a t t y acids f r o m e t i o l a t e d c o t y l e d o n s are also i n c l u d e d ( T a b l e I) s h o w i n g similarities w i t h g r e e n cultures. TABLE I FA'I'rY ACID MIXTURES IN MOL % OBTAINED BY TRANSESTERIFICATION OF TOTAL LIPID EXTRACTS FROM FOUR DIFFERENT TISSUES
Leaves Green culture Heterotr. culture Etiol. cotyledons
16:0
3-tr 16:1 16:1
16:2
16:3
18:0
18:1 18:2
18:3
20:0
16.2 20.3 26.1 17.9
1.2 0.4 0.3 2.0
0.8 0.5 n.a. n.a.
7.0 0.4 tr 0.8
1.8 4.3 4.6 7.7
1.7 5.9 3.5 8.2
55.1 32.9 11.3 27.3
tr 0.9 1.2 0.7
ASG a a
ba
ca
0.9 3.7 1.8 1.7
1.24 1.62 1.89 1.73
13 2.4 2.7 --
2.6 0.3 tr tr
14.1 34.6 53.1 35.4
TABLE II ACYL LIPIDS IN MOL% FROM FOUR DIFFERENT TISSUES
Leaves Green culture Green culture
Heterotr. culture
MGD
DGD
SQD PG
PE
PI
47.1 22.2 25.2 10.3
25.4 17.0 23.4 27.1
5.7 2.5 2.8 1.6
3.9 19.3 12.8 13.4
1.1 8.7 4.2 25.9 5.9 25.0 7.6 36.2
7.1 5.2 4.8 2.5
PC
9.7 6.3 6.1 1.7
a a, rag lipid/100 rag dry weight; b, ~mol ester/rag lipid; c, mg chlorophyll/100 rag lipid.
122 T A B L E III F A T T Y A C I D C O M P O S I T I O N IN MOL% O F I N D I V I D U A L LIPIDS F R O M F O U R DIFFERENT TISSUES a = leaves, b = green c u l t u r e , c = h e t e r o t r o p h i c c u l t u r e , d = e t i o l a t e d c o t y l e d o n s , n in t h e last c o l u m n r e p r e s e n t s n u m b e r o f i n d e p e n d e n t p r e p a r a t i o n s a n d analyses used t o calculate m e a n values, w h i c h are given in t h e table. T h e s e p a r a t e d e t e r m i n a t i o n s s h o w e d o n l y m i n o r d e v i a t i o n f r o m means. 16:0
16:1
MGD
a b c d
3.5 3.6 11.9 6.6
DGD
a b c d
3-tr16:1
16:3
18:0
18:1
18:2
18:3
1.0 0.2 0.3 1.7
16.0 2.5 tr 0.7
0.8 0.9 1.4 3.4
1.3 1.4 0.4 2.5
5.4 8.5 27.8 8.1
71.6 82.8 58.5 77.0
15.8 16.9 33.0 17.4
0.4 tr 0.4 2.9
2.3 tr
1.7 4.3 6.8 6.4
1.7 2.7 2.3 5.5
5.4 10.3 39.7 8.9
71.3 65.5 17.1 58.9
48.7 40.4 56.0 35.8
1.4 0.7 0.8 7.2
0.4
SQD
a b c d
2.9 4.7 3.4 12.3
3.1 4.4 1.8 10.5
8.1 16.7 17.3 9.7
35.5 32.9 19.7 24.5
PG
a b c d
24.6 46.7 64.6 35.3
tr tr 0.4 4.2
2.2 5.2 2.8 8.9
5.6 4.9 0.9 5.2
11.4 19.9 22.0 31.3
22.4 16.1 4.0 13.'7
PE
a b c
31.4 21.7 24.6
tr 0.2 0.2
4.6 4.0 2.5
2.1 3.9 0.4
41.9 58.2 66.1
PC
a b c d
26.6 19.6 23.3 29.3
0.9 0.2 tr 1.6
4.7 4.6 3.8 11.2
3.4 8.9 1.6 5.1
PI
a b c d
38.8 42.2 45.6 23.7
1.6 tr 0.2 2.5
5.1 2.5 3.1 11.9
ASG
a b b b c c c c
47.2 26.3 40.8 39.9 43.4 40.9 41.2 40.5
tr 0.7 0.6 1.2 2.1 tr 5.3 4.7
12.2 52.8 11.5 12.4 10.5 23.4 10.1 21.4
tr
34.0 7.3 4.0 0.8
20:0
n
4 5 4 1
tr 1.0 tr
3 4 3 1 4 4 2 1
0.7
2 3 3 1
20.1 11.6 5.8
tr 1.4 0.5
2 3 4
39.1 50.4 62.6 30.5
25.0 16.0 8.5 22.3
tr 1.5 tr tr
3 4 4 1
4.6 3.4 0.6 4.5
24.3 35.0 42.7 36.1
25.7 17.0 6.8 21.4
tr tr tr
2 2 4 1
8.6 1.8 8.9 10.2 7.9 tr 10.8 17.2
19.5 13.0 28.8 25.4 27.0 32.4 33.8 8.1
10.7 3.2 6.4 8.0 6.1 3.3 3.3 8.1
1.8 2.1 2.9 3.0 2.9 3.0 1.5 tr
1 1 1 1 1 1 1 1
tr
123
Our values for the heterotrophic culture differ from previously published data from t o b a c c o cultures [12] b y a low level of C18 : i and an increased proportion o f C18 : 2. But as shown for other cultures fatty acid and lipid proportions m a y vary with differences in conditions and age of cultures [ 1, 13--16]. Therefore we carried o u t an analysis o f the total fatty acids from lipids of green tissues cultured for 4, 10, 13 and 16 days. The last three cultures had fatty acid patterns n o t differing from those in Table I. Also the values for pmole ester/ mg lipid (1.75--1.62) were similar to the figure reported in Table I. In contrast the tissue cultured for four days had less pmole ester/mg lipid (1.42) and also showed a slightly changed pattern of fatty acids, since the proportion of Cls: 2 was higher (46%) and that of C~s :3 lower (21%) than the corresponding values from Table I. The usual acyl lipid constituents o f cellular membranes found in leaves are also present in green and non-green cultures (Table II) as was expected from previous investigations [1,2,13,17]. We could n o t detect BPA, which had been observed in heterotrophic cultures [ 1 8 ] . PA and cardiolipin as well as another phospholipid (referred to as u n k n o w n in ref. 17) were present only in trace amounts. Differences b e t w e e n the three tissues are found in relative proportions, although t h e y are n o t as drastic as in the t w o unsaturated C~6 fatty acids mentioned above. The decrease in chloroplast lipids (especially MGD and SQD) and the increase in acylated steryl glycoside (ASG) and phospholipids, in agreem e n t with previou§ results [1,2,13,17], should be pointed out. T h e y parallel the reduction in chlorophyll c o n t e n t (Table IIc) and signify the predominance of extraplastidic m e m b r a n e systems in cultured tissues. The fatty acids in individual lipid c o m p o n e n t s from the different tissues are presented in Table III. Most trends observed in fatty acids from total extracts (Table I), i.e. increase in Cls : o, Cls : 2 (Fig. l e ) and reduction in C~s : 3 (Fig. l c ) , can mainly be attributed to changes in level and fatty acid composition of chloroplast lipids MGD, DGD, SQD and PG. Similar generalisations cannot be made for the proportion o f C~6 : 0 in glyco- and phospholipids, since its level shows great variation. MGD is exceptional, since even in non-green cultures C~s : 3 is the predominant f a t t y acid (Table III). Also in the phospholipids PC, PE and PI, which are localized mainly in extraplastidic membrane systems, the reduction in the proportion o f Cls : 3 (Fig. l d ) and the increase in Cls : 2 (Fig. l f ) are observed, whereas ClS : 0 was present in similar proportions in these lipids from the different tissues. The f a t t y acid composition o f individual lipids from etiolated cotyledons resembles the green culture with respect to proportions o f C , 6 : 0, C~s : 2 and C~s : 3, whereas in contrast only trace amounts o f C ~ : 3 and 3-trans-C,6 :, are present. The fatty acids in ASG varied in different experiments, b u t the high proportions of C16:0, C~s :0 and C:0:0 should be mentioned (Table III). The most important changes are f o u n d again in the levels of C16 : 3 (Fig. la) and 3-trans-C16 :, (Fig. l b ) . As expected t o b a c c o is a typical 16:3-plant with nearly all C16 : 3 in MGD (Table III). This concentration in MGD is even more p r o n o u n c e d in green cultures, b u t here the proportion has drastically decreased
124
O/o
C 16:3
~- trans- C 164
(3
b
C18:3
C18:3
C18:2
C
d
e
C18:2
100-
! In
50iii
H~ 12 12
I 1234
1234
55
5
123/-
1 2
6
Fig. 1. C o m p a r i s o n o f f a t t y acid p r o p o r t i o n s in individual lipids f r o m leaves, g r e e n . a n d h e t e r o t r o p h i c cultures. In (a) t h e p r o p o r t i o n s o f C~6 : 3 in MGD = 1 a n d DGD = 2, in (b) t h o s e o f 3-trans-C,6 : ~ in PG = 4 are c o m p a r e d ; (c) a n d (e) s u m m a r i z e t h e results f o r Cls : 3 and C~s : 2, r e s p e c t i v e l y , in t h e p r e d o m i n a n t c h l o r o p l a s t lipids MGD = 1, DGD = 2, SQD = 3 and PG = 4; (d) a n d (f) s h o w t h e t r e n d s f o r t h e s a m e f a t t y acids in t h e m a i n l y e x t r a p l a s t i d i c p h o s p h o l i p i d s PE = 5 a n d PC = 6. Black bars in a - d r e p r e s e n t t h e f a t t y acid p r o p o r t i o n in individual lipids f r o m leaves set as 100%. In (e) a n d (f) t h e p r o p o r t i o n o f C~8 : 2 in lipids f r o m h e t e r o t r o p h i c c u l t u r e s is set as 100%. F o r each f a t t y acid t w o sets o f bars w i t h t h e s a m e n u m b e r s are s h o w n , t h e left o n e f o r green and t h e right o n e for h e t e r o t r o p h i c cultures in (a)--(d). In (e) and (f) t h e left set r e p r e s e n t s leaves, t h e right o n e g r e e n cultures.;
from 16 to 2.5%. The MGD from heterotrophic cultures contains only traces of C16 : 3. This is in agreement with differences in fatty acid composition between leaves and non-green cultures from other 16:3-plants, which also do not produce C16 : 3 in non-green tissue cultures [1] or etiolated organs of the parent plants. Similar differences are seen in the proportion o f 3-trans-C16 : 1 from PG, which is another fatty acid typical for chloroplasts. In chloroplasts from C4-plants this fatty acid has been suggested to be related t o grana formation [ 1 9 ] , which may also be of relevance in the present context. Both C~6 : 3 and 3-trans-C~6 : ~ have been proposed to be derived from C,6 : 0 b y desaturations specifically connected to chloroplasts [ 5,20]. The results from Table III indicate the reduced capacity in fatty acid desaturation of chloroplasts from green cultures placing them as expected from chlorophyll levels between green leaves and heterotrophic cultures. In contrast to desaturating systems the enzymes responsible for the assembly o f typical plastid lipids and for directing available fatty acids into the right positions (see below) operate at not t o o different rates also in green cultures. A glycerolipid m a y furthermore be characterized b y the positional distribution o f fatty acids b e t w e e n C-1 and C-2 of the glycerol moiety, for which in higher plants the following generalisations may be made [21] : in mainly extraplastidic phospholipids (PC, PE, PI) C,6 : 0 is localized at C-l; 3-trans-C,6 : in PG and C~6 .. 3 in MGD are found at C-2; C,6 : 0 in DGD and SQD predomin-
125 so
~
I
5 o
.GD I
C-1
I i
I i
C-1
o
r
C-2
,: i
~
I - L ] ~ l
I
I
I
1
;
~
u
i
i
,
Ii
50. . . . . ID,~DI
C-2
i
i
i"
I
C-1
C-1 I
b
I
I
'
I I
r p
I
1 '
C-2 50-
i
i
t I
J
i
i
50........ PC'
C-1
'
h
,
,
!
I
I
I
' C-2
, ~
1
i
I
n , 1
I , i
I
' a
'
i
I
I , i
r
i I
~P - ,,
C-2
so
16:016:116:318:018:118:218:3
50 16016118018 1182183
Fig. 2. Positional distribution of fatty acids between C-1 and C-2 at the glycerol moiety of individual lipids from leaves (bar at the left side for each fatty acid), green (bar in the middle) and heterotrophic cultures (right bar). PG and SQD from heterotrophic cultures were not analyzed.
ates either at C-1 or is found in considerable proportions at C-2 as in 16:3plants. The positional analyses from the green tissues (fig. 2) are in general agreement with these predictions, although the proportion o f C~6 : 0 at C-2 in D G D and SQD from both green tissues is lower than expected. But it is evident that green leaves and green cultures basically do not differ in the positional distribution o f fatty acids in phospho- and glycolipids. This indicates that the specificities o f enzymatic systems introducing asymmetric distributions are not altered in green cultures. In non-green cultures the uniformity in fatty acid composition o f individual lipids (Table III) is reflected by a uniformity in positional distribution. This distribution is characterized by a general preference o f C16 : 0 for C-1 in all lipids as usually only found in extraplastidic phospholipids. Especially DGD resembles closely phospholipids in its diglyceride portion. In su mm ar y our investigation in the lipid field shows that green cultures contain the characteristiclipids of photosynthetic tissues from the parent tobacco plant regarding individual lipid components as well as occurrence and positional distribution of fatty acids. O n the other hand, some chloroplast components such as MGD, SQD, C 1 6 : 3 and 3-trans-C,e :, occur at largely reduced levels.
126
The surprisingly low level of C~6 : 3 in green cultures and its high proportion in leaves may provide a clue for investigations on the metabolic position o f this particular fatty acid. ACKNOWLEDGEMENTS
The authors gratefully acknowledge the support by the Deutsche Forschungsgemeinschaft. REFERENCES 1 S.S. Radwan and H.K. Mangold, Adv. Lipid Res., 14 (1976) 171. 2 W. Hiisemann, S.S. Radwan, H.K. Mangold and W. Barz, in preparation as reported by H.K. Mangold, in W. Barz, E. Reinhard and M.H. Zenk (Eds.), Plant Tissue Culture and its Bio-technological Application, Springer Verlag, Berlin-Heidelberg-New York, 1977, p. 55. 3 L. Bergmann, J. Gen. Physiol., 43 (1960) 841. 4 G.R. Jamieson and R.H. Reid, Phytochemistry, 10 (1971) 1837. 5 H.P. Siebertz and E. Heinz, Z. Naturforsch., 32c (1977) 193. 6 P.K. Stumpf, in T. Galliard and E.I. Mercer (Eds.), Recent Advances in the Chemistry and Biochemistry o f Plant Lipids, Academic Press, London-New York - San Francisco, 1975, p. 95. 7 L. Bergmann, W. Grosse and P. Koth, Z. Pflanzenphysiol., 80 (1976) 60. 8 R.M.C. Dawson, N. Clarke and R.H. Quarles, Biochem. J., 114 (1969) 265. 9 P. Comfurius and R.F.A. Zwaal, Biochim. Biophys. Acta, 488 (1977) 36. 10 R. Douce, M. Faure and J. Mar~chal, Compt. Rend., 262 (1966) 1549. 11 S.F. Yang, S. Freer and A.A. Benson, J. Biol. Chem., 242 (1967) 477. 12 J.D. Weete, Lipids, 6 (1971) 684. 13 M. Song and N. Tattrie, Can. J. Bot., 51 (1973) 1893. 14 S.S. Radwan and H.K. Mangold, Chem. Phys. Lipids, 14 (1975) 87. 15 E.M. Stearns and W.T. Morton, Phytochemistry, 14 (1975) 619. 16 R. Bligny, Plant Physiol., 59 (1977) 502. 17 S.S. Radwan, F. Spener, H.K. Mangold and E.J. Staba, Chem. Phys. Lipids, 14 (1975) 72. 18 E.M. Stearns and W.T. Morton, Lipids, 12 (1977) 451. 19 C. Tuquet, T. Guillot-Salomon, M.de Lubac and M. Signol, Plant Sci. Lett., 8 (1977) 59. 20 C.T. Bartels, A.T. James and B.W. Nichols, Eur. J. Biochem., 3 (1967) 7. 21 E. Heinz, in M. Tevini and H.K. Lichtenthaler (Eds.), Lipids and Lipid Polymers in Higher Plants, Springer-Verlag, Berlin-Heidelberg, 1977, p. 102.
119
© Elsevier/North-Holland Scientific Publishers Ltd.
ACYL LIPIDS IN PHOTOSYNTHETICALLY ACTIVE TISSUE CULTURES OF TOBACCO
H.P. SIEBERTZ, E. HEINZ and L. BERGMANN Botanisches Institut der Universiti~t zu K~ln, Gyrhofstrasse 15,D-5000 Koln 41 (G.F.R.)
(Received October 27th, 1977) (Accepted February 14th, 1978)
SUMMARY
Lipid mixtures extracted from green leaves, green tissue cultures, etiolated cotyledons and heterotrophic cultures were subjected to quantitative deterruination o f the major phospho- and glycolipids. Fatty acid analyses including positional distribution at the glycerol moiety were carried out for individual lipid components. The results demonstrate tnat green cultures occupy a position between green leaves and heterotrophic cultures. The major membrane lipids known from photoautotrophic tissues are present in green cultures. Components concentrated in chloroplasts such as monogalactosyl-(MGD) and sulphoquinovosyl diacylglycerol (SQD) and the fatty acids C,6 • 3 and 3-transC16 : 1 occur at markedly reduced levels. A general increase in the proportion of Cls .. 2 at the expense o f C18 : 3 is observed in tissue cultures. The positional distribution of significant fatty acids is identical in the lipids from green cultures and leaves.
INTRODUCTION
Tissue cultures are established tools for research in various fields o f plant physiology. An increasing n u m b e r of investigations is directed towards the elucidation o f composition and metabolism o f the various lipid classes present in these cultures (summarized in ref. 1). With one exception [2], all these experiments have been carried out with non-green cultures, the lipids o f which differ in several aspects from those o f photosynthetic tissues of the m o t h e r plant. Therefore it seemed worthwhile to analyze the lipids from a photosynAbbreviations: APE, acylated phosphatidyl ethanolamine; ASG, acylated steryl glycoside;
BPA, bis-phosphatidic acid; DGD, digalactosyl diacylglycerol; GLC, gas-liquid chromatography; MGD, monogalactosyl diacylglycerol; PA, phosphatidic acid; PC, phosphatidyl choline; PE, phosphatidyl ethanolamine; PG, phosphatidyl glycerol; PI, phosphatidyl inositol; SQD, sulfoquinovosyl diacylglycerol; TLC, thin layer chromatography.
120
thetically active culture. Furthermore the comparison between a green culture and a photosynthetic tissue o f the parent plant should explore a possible use of green cultures as model systems for experimentation in the field of lipid metabolism. For this purpose we used a well established green culture from t o b a c c o [ 3 ] , which as m e m b e r o f the Solanaceae should be an interesting plant for studies in fatty acid biosynthesis. Leaves of plants from Solanaceae and several other families [4] are characterized b y the occurrence of hexadecatrienoic acid C16 : 3, which is an interesting fatty acid from several points of view. It is usually localized rather exclusively in the sn-2-position of MGD and represents a marker for a chloroplast-concentrated lipid. Previous experiments with Anthriscus leaves (belonging to the Apiaceae) were interpreted as showing that C~6 : 3 was derived from C~6 : 0 b y sequential desaturation [5]. On the other hand C~6 : 3 has also been proposed [6] to be derived from C~4 • 3 and to be the immediate precursor of Cla : 3. Although C~8 .. 3 is the most abundant polyenoic acid in leaf lipids, its exact biosynthesis has n o t yet been clarified. For further studies in this direction an easily manipulatable system such as a tissue culture would be of great advantage. From this point of view we were specifically interested in the occurrence of C16 • 3 in the lipids from green cultures of tobacco. EXPERIMENTAL
Growth o f tissue cultures The SCI line of t o b a c c o cells, derived from stem pith of Nicotiana tabacum var. Samsun [3] was grown at 25°C in a modified liquid Murashige-Skoogmedium as described in detail before [7]. After 10--12 days o f culture cells were harvested at the end of the logarithmic growth phase, washed twice with fresh sterile medium and lyophilized. Lipid extraction and analysis Lipids from lyophilized cultures were extracted with h o t chloroform/methanol/isopropanol 20/10/5. Lipid extracts were purified b y Solvent partition and analyzed after thin layer chromatography {TLC) separation o f individual c o m p o n e n t s b y previously described methods [ 5] including gas-liquid chromatography (GLC) of fatty acid methyl esters, quantitation b y inclusion of C17 : 0 as internal standard and positional analysis b y lipase or phospholipase A2 hydrolysis. GLC peak assignments were confirmed b y rechromatography after hydrogenation. F a t t y acids in Tables I and III are characterized b y n u m b e r of carbon atoms and double bonds. Percentages below 0.1% are denoted as tr. For chromatographic comparison reference c o m p o u n d s were prepared for phosphatidic acid (PA) {hydrolysis o f phosphatidyl choline (PC) b y phospholipase D), acylated phosphatidyl ethanolamine (APE) (acylation of phosphatidyl ethanolamine (PE) with oleoyl chloride according to ref. 8) and bis-phosphatidic acid (BPA) (by acylation o f phosphatidyl glycerol (PG) with oleoyl chloride). Cardiolipin was a gift from Prof. Dr. W. Fischer, University of Erlangen.
121 I n s o m e cases w h e n fresh c u l t u r e s w e r e d i r e c t l y p l a c e d i n t o h o t e x t r a c t i o n m e d i u m large q u a n t i t i e s o f a n u n u s u a l p h o s p h o l i p i d w e r e o b s e r v e d in t h e extracts. This substance c o c h r o m a t o g r a p h e d with phosphatidyl m e t h a n o l (prep a r e d as r e f e r e n c e c o m p o u n d a c c o r d i n g t o ref. 9, R f - v a l u e o f 0.4 in c h l o r o f o r m / m e t h a n o l / N H 4 O H c o n c . 7 0 / 2 0 / 1 . 5 o n p r e c o a t e d silica gel plates} a n d p r o b a b l y originated from phospholipase D-catalyzed transphosphatidylation reactions between phospholipids and methanol [ 10,11]. RESULTS AND DISCUSSION T h e t o t a l lipids f r o m leaves, g r e e n a n d h e t e r o t r o p h i c c u l t u r e s a m o u n t e d t o 9 . 7 , 6.3 a n d 1.7%, r e s p e c t i v e l y , o f t h e d r y w e i g h t ( T a b l e IIa). C o r r e s p o n d i n g values f r o m a v a r i e t y o f h e t e r o t r o p h i c c u l t u r e s c o v e r e d a r a n g e o f 2 . 6 - . 8 . 1 % (1). In T a b l e I t h e f a t t y acid a n a l y s e s f r o m t o t a l lipid e x t r a c t s are s u m m a r i z e d . W h e n g o i n g f r o m leaves via g r e e n t o n o n - g r e e n c u l t u r e s a general increase in t h e p r o p o r t i o n o f C 1 6 : 0, Cls : 0, C,s : 2 a n d a d e c r e a s e in C18 : 3 c a n b e observed. M o s t significant are t h e r e d u c e d levels o f C,6 : 3 a n d 3-trans-C16 : 1, b o t h o f w h i c h are c o n n e c t e d in p h o t o s y n t h e t i c tissues w i t h t h e c h l o r o p l a s t lipids M G D a n d P G , r e s p e c t i v e l y . F o r c o m p a r i s o n t h e f a t t y acids f r o m e t i o l a t e d c o t y l e d o n s are also i n c l u d e d ( T a b l e I) s h o w i n g similarities w i t h g r e e n cultures. TABLE I FA'I'rY ACID MIXTURES IN MOL % OBTAINED BY TRANSESTERIFICATION OF TOTAL LIPID EXTRACTS FROM FOUR DIFFERENT TISSUES
Leaves Green culture Heterotr. culture Etiol. cotyledons
16:0
3-tr 16:1 16:1
16:2
16:3
18:0
18:1 18:2
18:3
20:0
16.2 20.3 26.1 17.9
1.2 0.4 0.3 2.0
0.8 0.5 n.a. n.a.
7.0 0.4 tr 0.8
1.8 4.3 4.6 7.7
1.7 5.9 3.5 8.2
55.1 32.9 11.3 27.3
tr 0.9 1.2 0.7
ASG a a
ba
ca
0.9 3.7 1.8 1.7
1.24 1.62 1.89 1.73
13 2.4 2.7 --
2.6 0.3 tr tr
14.1 34.6 53.1 35.4
TABLE II ACYL LIPIDS IN MOL% FROM FOUR DIFFERENT TISSUES
Leaves Green culture Green culture
Heterotr. culture
MGD
DGD
SQD PG
PE
PI
47.1 22.2 25.2 10.3
25.4 17.0 23.4 27.1
5.7 2.5 2.8 1.6
3.9 19.3 12.8 13.4
1.1 8.7 4.2 25.9 5.9 25.0 7.6 36.2
7.1 5.2 4.8 2.5
PC
9.7 6.3 6.1 1.7
a a, rag lipid/100 rag dry weight; b, ~mol ester/rag lipid; c, mg chlorophyll/100 rag lipid.
122 T A B L E III F A T T Y A C I D C O M P O S I T I O N IN MOL% O F I N D I V I D U A L LIPIDS F R O M F O U R DIFFERENT TISSUES a = leaves, b = green c u l t u r e , c = h e t e r o t r o p h i c c u l t u r e , d = e t i o l a t e d c o t y l e d o n s , n in t h e last c o l u m n r e p r e s e n t s n u m b e r o f i n d e p e n d e n t p r e p a r a t i o n s a n d analyses used t o calculate m e a n values, w h i c h are given in t h e table. T h e s e p a r a t e d e t e r m i n a t i o n s s h o w e d o n l y m i n o r d e v i a t i o n f r o m means. 16:0
16:1
MGD
a b c d
3.5 3.6 11.9 6.6
DGD
a b c d
3-tr16:1
16:3
18:0
18:1
18:2
18:3
1.0 0.2 0.3 1.7
16.0 2.5 tr 0.7
0.8 0.9 1.4 3.4
1.3 1.4 0.4 2.5
5.4 8.5 27.8 8.1
71.6 82.8 58.5 77.0
15.8 16.9 33.0 17.4
0.4 tr 0.4 2.9
2.3 tr
1.7 4.3 6.8 6.4
1.7 2.7 2.3 5.5
5.4 10.3 39.7 8.9
71.3 65.5 17.1 58.9
48.7 40.4 56.0 35.8
1.4 0.7 0.8 7.2
0.4
SQD
a b c d
2.9 4.7 3.4 12.3
3.1 4.4 1.8 10.5
8.1 16.7 17.3 9.7
35.5 32.9 19.7 24.5
PG
a b c d
24.6 46.7 64.6 35.3
tr tr 0.4 4.2
2.2 5.2 2.8 8.9
5.6 4.9 0.9 5.2
11.4 19.9 22.0 31.3
22.4 16.1 4.0 13.'7
PE
a b c
31.4 21.7 24.6
tr 0.2 0.2
4.6 4.0 2.5
2.1 3.9 0.4
41.9 58.2 66.1
PC
a b c d
26.6 19.6 23.3 29.3
0.9 0.2 tr 1.6
4.7 4.6 3.8 11.2
3.4 8.9 1.6 5.1
PI
a b c d
38.8 42.2 45.6 23.7
1.6 tr 0.2 2.5
5.1 2.5 3.1 11.9
ASG
a b b b c c c c
47.2 26.3 40.8 39.9 43.4 40.9 41.2 40.5
tr 0.7 0.6 1.2 2.1 tr 5.3 4.7
12.2 52.8 11.5 12.4 10.5 23.4 10.1 21.4
tr
34.0 7.3 4.0 0.8
20:0
n
4 5 4 1
tr 1.0 tr
3 4 3 1 4 4 2 1
0.7
2 3 3 1
20.1 11.6 5.8
tr 1.4 0.5
2 3 4
39.1 50.4 62.6 30.5
25.0 16.0 8.5 22.3
tr 1.5 tr tr
3 4 4 1
4.6 3.4 0.6 4.5
24.3 35.0 42.7 36.1
25.7 17.0 6.8 21.4
tr tr tr
2 2 4 1
8.6 1.8 8.9 10.2 7.9 tr 10.8 17.2
19.5 13.0 28.8 25.4 27.0 32.4 33.8 8.1
10.7 3.2 6.4 8.0 6.1 3.3 3.3 8.1
1.8 2.1 2.9 3.0 2.9 3.0 1.5 tr
1 1 1 1 1 1 1 1
tr
123
Our values for the heterotrophic culture differ from previously published data from t o b a c c o cultures [12] b y a low level of C18 : i and an increased proportion o f C18 : 2. But as shown for other cultures fatty acid and lipid proportions m a y vary with differences in conditions and age of cultures [ 1, 13--16]. Therefore we carried o u t an analysis o f the total fatty acids from lipids of green tissues cultured for 4, 10, 13 and 16 days. The last three cultures had fatty acid patterns n o t differing from those in Table I. Also the values for pmole ester/ mg lipid (1.75--1.62) were similar to the figure reported in Table I. In contrast the tissue cultured for four days had less pmole ester/mg lipid (1.42) and also showed a slightly changed pattern of fatty acids, since the proportion of Cls: 2 was higher (46%) and that of C~s :3 lower (21%) than the corresponding values from Table I. The usual acyl lipid constituents o f cellular membranes found in leaves are also present in green and non-green cultures (Table II) as was expected from previous investigations [1,2,13,17]. We could n o t detect BPA, which had been observed in heterotrophic cultures [ 1 8 ] . PA and cardiolipin as well as another phospholipid (referred to as u n k n o w n in ref. 17) were present only in trace amounts. Differences b e t w e e n the three tissues are found in relative proportions, although t h e y are n o t as drastic as in the t w o unsaturated C~6 fatty acids mentioned above. The decrease in chloroplast lipids (especially MGD and SQD) and the increase in acylated steryl glycoside (ASG) and phospholipids, in agreem e n t with previou§ results [1,2,13,17], should be pointed out. T h e y parallel the reduction in chlorophyll c o n t e n t (Table IIc) and signify the predominance of extraplastidic m e m b r a n e systems in cultured tissues. The fatty acids in individual lipid c o m p o n e n t s from the different tissues are presented in Table III. Most trends observed in fatty acids from total extracts (Table I), i.e. increase in Cls : o, Cls : 2 (Fig. l e ) and reduction in C~s : 3 (Fig. l c ) , can mainly be attributed to changes in level and fatty acid composition of chloroplast lipids MGD, DGD, SQD and PG. Similar generalisations cannot be made for the proportion o f C~6 : 0 in glyco- and phospholipids, since its level shows great variation. MGD is exceptional, since even in non-green cultures C~s : 3 is the predominant f a t t y acid (Table III). Also in the phospholipids PC, PE and PI, which are localized mainly in extraplastidic membrane systems, the reduction in the proportion o f Cls : 3 (Fig. l d ) and the increase in Cls : 2 (Fig. l f ) are observed, whereas ClS : 0 was present in similar proportions in these lipids from the different tissues. The f a t t y acid composition o f individual lipids from etiolated cotyledons resembles the green culture with respect to proportions o f C , 6 : 0, C~s : 2 and C~s : 3, whereas in contrast only trace amounts o f C ~ : 3 and 3-trans-C,6 :, are present. The fatty acids in ASG varied in different experiments, b u t the high proportions of C16:0, C~s :0 and C:0:0 should be mentioned (Table III). The most important changes are f o u n d again in the levels of C16 : 3 (Fig. la) and 3-trans-C16 :, (Fig. l b ) . As expected t o b a c c o is a typical 16:3-plant with nearly all C16 : 3 in MGD (Table III). This concentration in MGD is even more p r o n o u n c e d in green cultures, b u t here the proportion has drastically decreased
124
O/o
C 16:3
~- trans- C 164
(3
b
C18:3
C18:3
C18:2
C
d
e
C18:2
100-
! In
50iii
H~ 12 12
I 1234
1234
55
5
123/-
1 2
6
Fig. 1. C o m p a r i s o n o f f a t t y acid p r o p o r t i o n s in individual lipids f r o m leaves, g r e e n . a n d h e t e r o t r o p h i c cultures. In (a) t h e p r o p o r t i o n s o f C~6 : 3 in MGD = 1 a n d DGD = 2, in (b) t h o s e o f 3-trans-C,6 : ~ in PG = 4 are c o m p a r e d ; (c) a n d (e) s u m m a r i z e t h e results f o r Cls : 3 and C~s : 2, r e s p e c t i v e l y , in t h e p r e d o m i n a n t c h l o r o p l a s t lipids MGD = 1, DGD = 2, SQD = 3 and PG = 4; (d) a n d (f) s h o w t h e t r e n d s f o r t h e s a m e f a t t y acids in t h e m a i n l y e x t r a p l a s t i d i c p h o s p h o l i p i d s PE = 5 a n d PC = 6. Black bars in a - d r e p r e s e n t t h e f a t t y acid p r o p o r t i o n in individual lipids f r o m leaves set as 100%. In (e) a n d (f) t h e p r o p o r t i o n o f C~8 : 2 in lipids f r o m h e t e r o t r o p h i c c u l t u r e s is set as 100%. F o r each f a t t y acid t w o sets o f bars w i t h t h e s a m e n u m b e r s are s h o w n , t h e left o n e f o r green and t h e right o n e for h e t e r o t r o p h i c cultures in (a)--(d). In (e) and (f) t h e left set r e p r e s e n t s leaves, t h e right o n e g r e e n cultures.;
from 16 to 2.5%. The MGD from heterotrophic cultures contains only traces of C16 : 3. This is in agreement with differences in fatty acid composition between leaves and non-green cultures from other 16:3-plants, which also do not produce C16 : 3 in non-green tissue cultures [1] or etiolated organs of the parent plants. Similar differences are seen in the proportion o f 3-trans-C16 : 1 from PG, which is another fatty acid typical for chloroplasts. In chloroplasts from C4-plants this fatty acid has been suggested to be related t o grana formation [ 1 9 ] , which may also be of relevance in the present context. Both C~6 : 3 and 3-trans-C~6 : ~ have been proposed to be derived from C,6 : 0 b y desaturations specifically connected to chloroplasts [ 5,20]. The results from Table III indicate the reduced capacity in fatty acid desaturation of chloroplasts from green cultures placing them as expected from chlorophyll levels between green leaves and heterotrophic cultures. In contrast to desaturating systems the enzymes responsible for the assembly o f typical plastid lipids and for directing available fatty acids into the right positions (see below) operate at not t o o different rates also in green cultures. A glycerolipid m a y furthermore be characterized b y the positional distribution o f fatty acids b e t w e e n C-1 and C-2 of the glycerol moiety, for which in higher plants the following generalisations may be made [21] : in mainly extraplastidic phospholipids (PC, PE, PI) C,6 : 0 is localized at C-l; 3-trans-C,6 : in PG and C~6 .. 3 in MGD are found at C-2; C,6 : 0 in DGD and SQD predomin-
125 so
~
I
5 o
.GD I
C-1
I i
I i
C-1
o
r
C-2
,: i
~
I - L ] ~ l
I
I
I
1
;
~
u
i
i
,
Ii
50. . . . . ID,~DI
C-2
i
i
i"
I
C-1
C-1 I
b
I
I
'
I I
r p
I
1 '
C-2 50-
i
i
t I
J
i
i
50........ PC'
C-1
'
h
,
,
!
I
I
I
' C-2
, ~
1
i
I
n , 1
I , i
I
' a
'
i
I
I , i
r
i I
~P - ,,
C-2
so
16:016:116:318:018:118:218:3
50 16016118018 1182183
Fig. 2. Positional distribution of fatty acids between C-1 and C-2 at the glycerol moiety of individual lipids from leaves (bar at the left side for each fatty acid), green (bar in the middle) and heterotrophic cultures (right bar). PG and SQD from heterotrophic cultures were not analyzed.
ates either at C-1 or is found in considerable proportions at C-2 as in 16:3plants. The positional analyses from the green tissues (fig. 2) are in general agreement with these predictions, although the proportion o f C~6 : 0 at C-2 in D G D and SQD from both green tissues is lower than expected. But it is evident that green leaves and green cultures basically do not differ in the positional distribution o f fatty acids in phospho- and glycolipids. This indicates that the specificities o f enzymatic systems introducing asymmetric distributions are not altered in green cultures. In non-green cultures the uniformity in fatty acid composition o f individual lipids (Table III) is reflected by a uniformity in positional distribution. This distribution is characterized by a general preference o f C16 : 0 for C-1 in all lipids as usually only found in extraplastidic phospholipids. Especially DGD resembles closely phospholipids in its diglyceride portion. In su mm ar y our investigation in the lipid field shows that green cultures contain the characteristiclipids of photosynthetic tissues from the parent tobacco plant regarding individual lipid components as well as occurrence and positional distribution of fatty acids. O n the other hand, some chloroplast components such as MGD, SQD, C 1 6 : 3 and 3-trans-C,e :, occur at largely reduced levels.
126
The surprisingly low level of C~6 : 3 in green cultures and its high proportion in leaves may provide a clue for investigations on the metabolic position o f this particular fatty acid. ACKNOWLEDGEMENTS
The authors gratefully acknowledge the support by the Deutsche Forschungsgemeinschaft. REFERENCES 1 S.S. Radwan and H.K. Mangold, Adv. Lipid Res., 14 (1976) 171. 2 W. Hiisemann, S.S. Radwan, H.K. Mangold and W. Barz, in preparation as reported by H.K. Mangold, in W. Barz, E. Reinhard and M.H. Zenk (Eds.), Plant Tissue Culture and its Bio-technological Application, Springer Verlag, Berlin-Heidelberg-New York, 1977, p. 55. 3 L. Bergmann, J. Gen. Physiol., 43 (1960) 841. 4 G.R. Jamieson and R.H. Reid, Phytochemistry, 10 (1971) 1837. 5 H.P. Siebertz and E. Heinz, Z. Naturforsch., 32c (1977) 193. 6 P.K. Stumpf, in T. Galliard and E.I. Mercer (Eds.), Recent Advances in the Chemistry and Biochemistry o f Plant Lipids, Academic Press, London-New York - San Francisco, 1975, p. 95. 7 L. Bergmann, W. Grosse and P. Koth, Z. Pflanzenphysiol., 80 (1976) 60. 8 R.M.C. Dawson, N. Clarke and R.H. Quarles, Biochem. J., 114 (1969) 265. 9 P. Comfurius and R.F.A. Zwaal, Biochim. Biophys. Acta, 488 (1977) 36. 10 R. Douce, M. Faure and J. Mar~chal, Compt. Rend., 262 (1966) 1549. 11 S.F. Yang, S. Freer and A.A. Benson, J. Biol. Chem., 242 (1967) 477. 12 J.D. Weete, Lipids, 6 (1971) 684. 13 M. Song and N. Tattrie, Can. J. Bot., 51 (1973) 1893. 14 S.S. Radwan and H.K. Mangold, Chem. Phys. Lipids, 14 (1975) 87. 15 E.M. Stearns and W.T. Morton, Phytochemistry, 14 (1975) 619. 16 R. Bligny, Plant Physiol., 59 (1977) 502. 17 S.S. Radwan, F. Spener, H.K. Mangold and E.J. Staba, Chem. Phys. Lipids, 14 (1975) 72. 18 E.M. Stearns and W.T. Morton, Lipids, 12 (1977) 451. 19 C. Tuquet, T. Guillot-Salomon, M.de Lubac and M. Signol, Plant Sci. Lett., 8 (1977) 59. 20 C.T. Bartels, A.T. James and B.W. Nichols, Eur. J. Biochem., 3 (1967) 7. 21 E. Heinz, in M. Tevini and H.K. Lichtenthaler (Eds.), Lipids and Lipid Polymers in Higher Plants, Springer-Verlag, Berlin-Heidelberg, 1977, p. 102.