Planta
Planta 147, 384-388 (1980)
0 by Springer-Verlag 1980
Fatty-Acid Composition and Biosynthesis in Cell Suspension Cultures of Glycine max (L.) Merr., Catharanthus roseus G. Don and Nicotiana tabacum L. J.J. MacCarthy and P.K. Stumpf Department of Biochemistry and Biophysics, University of California, Davis, CA 95616, USA
Abstract. The fatty-acid composition of C. r o s e u s and N . t a b a c u m cell suspension cultures was unaffected by subculture on W o o d and Braun, Murashige and Skoog, or G a m b o r g B5C media. However, placing the cultures - which were normally grown at 2 5 ~ - at 15~ reduced growth but resulted in enhanced formation of oleic and linolenic acids in C . r o s e u s cultures and increased levels of linoleic and linolenic acids in cultures of G. m a x and N . t a b a c u m , respectively. The incorporation of p*C]acetate into [14C]linoteic acid was more rapid in N . t a b a c u m cells than in G. m a x cells, but was very poor in C. r o s e u s where the [~C] label was distributed mainly between palmitic and oleic acids. Key words: Acetate incorporation - C a t h a r a n t h u s Cell suspension cultures - Fatty acids - G l y c i n e N i c o t i a n a - Temperature and fatty-acid synthesis.
Don ( V i n c a
r o s e a L.) from the Apocynaceae, and
t a b a c u m (L.), cv. Wisconsin 38, from the Solanaceae. G. m a x cell cultures had been reported
Nicotiana
to have relatively high levels of linolenic acid (Stearns and Morton, 1975; Tattrie and Veliky, 1973) and Solanaceae species in cell culture had relatively high levels of linoleic acid (Siebertz et al., 1978; Tattrie and Veliky, 1973). The fatty-acid composition of C. r o s e u s was unknown. In this communication, fatty-acid compositions of several cell suspension cultures in response to medium formulation and temperature are reported. Comparisons of the synthesis of [14C]fatty acids from [14C]ace_ tate by each culture are also given.
Materials and Methods Celt Suspension Cultures
Introduction Cell suspension cultures provide useful and interesting biochemical systems for studies in plant cell metabolism (Street, 1975). Such cultures exhibit a characteristic fatty-acid composition which may resemble that of some organ of the original plant, though not necessarily the organ from which the culture was derived (Tattrie and Veliky, 1973). Cell suspension cultures usually exhibit a rapid rate of growth, with production of new cells during an exponential phase that endures for several days. The phase of rapid and increasing cell production must be accompanied by fatty-acid synthesis correlated with membrane development. With this in mind, cell suspension cultures were employed which represented widely different plant groups. These were cultures of G l y c i n e m a x (L.) Merr., cv. Mandarin, from the Leguminosae (Gamborg and Wetter, 1975), C a t h a r a n t h u s r o s e u s G .
The Glycine max cell suspension culture was very kindly provided by K.G. Lark, University of Utah, Salt Lake City, Utah, USA; the Nieotiana tabacum callus culture was provided by T. Murashige, University of California, Riverside, California, USA ; and the Catharanthus roseus cell suspension culture was initiated in this laboratory. Cell suspension cultures of N. tabacum and C. roseus were initiated from callus cultures and maintained on Murashige and Skoog medium (MS; Murashige and Skoog, 1975) and on a modified Wood and Braun medium (WB; Wood and Braun, 1961), respectively (Table 6). G. max cell suspension cultures were maintained on Gamborg's casein-supplemented B5C medium (Table 6). All three cultures were grown in 125-ml Erlenmeyer flasks on a G-10 model gyrotory shaker (New Brunswick Scientific Co., New Brunswick, N.J., USA) rotating at 131 rpm, under continuous illumination (General Electric fluorescent cool-white lamps; illuminance 455 lumen m 2) and at a temperature of 25_+0.5~C. The cultures of N. tabacum and G. m a x were subcultured every 7days and the C. roseus culture every 14days. Each celI suspension culture was grown and subsequently incubated on the medium on which it had been initiated so as to eliminate selection of ceil types or genotypes by transferring actively growing suspension culture onto medium of different composition. Since
0032-0935/80/0147/0384/$01.00
J.J. MacCarthy and P.K. Stumpf: Fatty-Acids in Cell Suspension Cultures fatty-acid synthesis may be affected by medium composition, N. tabacum and C. roseus were grown on WB and B5C, and MS and B5C, respectively, over two subcultures. The G. max culture could not be grown on either WB or MS medium. For studying the effects of culture at 15~ C, cells grown at 25~ were inoculated into fresh media and grown at 15~ in the light for 10 days, at 131 rpm. Dry weight, cell number and packed cell volume were routinely determined.
385
was determined in 200 gl of incubation medium (dissolved in 10 ml of Phase Combining System (PCS) scintillation fluid (Amersham Corp., Arlington Heights, Ill., USA), and using a Beckman LS-230 liquid scintillation counter (Beckman Instruments, Fullerton, Cal., USA). The sedimented cells were resuspended and washed 2 times in deionized water. The final pellet was extracted and analyzed for fatty acids as described above. All data presented in this paper have been duplicated.
Results and Discussion Determinations of Dry Weight, Cell Number, and Packed Cell Volume
For determining dry weight, the suspension culture (1.0 ml) was filtered with suction through a 2.4-cm GFIC glass microfibre filter paper (Whatman, Maidst0ne , Kent, U.K.). The filtered cells were washed 3 times in deionised water, and dried at 80 ~ C to constant weight. For cell counts, the suspension culture (1.0 ml) was macerated with 1.0 ml of 15% chromium trioxide. N. tabacum and C. roseus cells were macerated at 70 ~ C for 20 min, G. max cells at 70 ~ C for 45 rain. The macerate was cooled, diluted, and counted. For determinations of packed cell volumes, the suspension culture (15 ml) was centrifuged at 1,000 • g for 2 rain. An International Clinical bench-top centrifuge equipped with a No. 809 fixed angle head (International Equipment Corp., Needham Heights, Mass., USA) was used. The volume of sedimented cells was given as percentage of the total culture volume.
Endogenous Lipids of Cell Cultures
As indicated in Table 1, the fatty-acid compositions of N. tabacum and C. roseus cell cultures grown on B5C, WB and MS media for 7 days and taken over two subcultures were essentially constant. With the N. tabacum cells, the major unsaturated fatty acid was linoleic acid which constituted about 60% of the total fatty acids of these ceils. Oleic acid made up only about 2 3% of the total fatty acids. In contrast, with C. roseus cells, oleic and linoleic acids comprised ca. 30 and 40%, respectively, of the total fatty acids of these cells regardless of medium formulation and subculturing. The fatty-acid composition of these two cultures could therefore be attributed
Fatty Acid Extraction and Analysis
Lyophilized cells (100 mg) were saponified with 20% K O H in methanol (w/v) for 30 rain at 85 ~ C. The lipid extract was cooled, acidified with 6N HCt, and extracted 3 times with petroleum ether (b.p. 35-60 ~ C). The combined petroleum-ether phases were evaporated to dryness at room temperature under a stream of N2. The fatty-acid extract was redissolved in ca. 70% diazomethane in diethyl ether freshly prepared (de Boer and Backer, 1963) from N-methyl-N-nitroso-p-tolulenesulfonamide (Diazald, Aldrich Chemical Co., Milwaukee, Wis., USA). Following methylation at 0 ~ for 30 rain, excess diazomethane in diethyl ether was blown off with a stream o f N2- The methylated derivatives were redissolved in 100 pl of toluene for gas-liquid chromatography (GLC). A Model 920 GLC (Varian Aerograph, Walnut Creek, Cal., USA) equipped with a stainless-steel column (152 cm long, inner diameter 0.64cm) and packed with 10% diethylene glycol succinate: (Supelco Inc., Bellefonte, Pa., USA) was used. Radioactivity in the sample was detected with a Biospan Model 4998 detection'gystem (Nuclear-Chicago Corp., Des Plaines, Ill., USA). Individuafff'atty-acids were identified by comparison with standards of known fatty acid composition and by argentation thin-layer chromatography followed by ozonolysis of the fatty acids (Stein and Nicolaides, 1962). The fatty-aldehyde products of ozonolysis were identified using GLC.
Incubation with
[14C] Substrates
Freshly harvested cells (6 days old, 0.5 g fresh weight) were added to 1.5 ml sterile medium (pH 7.3), containing 1.0 pCi sodium [14C]acetate (New England Nuclear, Boston, Mass., USA; 54 mCi/ mmol) and gently agitated until mixed. The 25-ml scintillation vials were capped and incubated on a gyrotory shaker (131 rpm) at 25 ~ C in the light. Following incubation, cells were separated from medium by centrifugation. Residual [x4C]acetate
Table 1. The fatty-acid composition of N. tabacum and C. roseus cell cultures grown on B5C, WB and MS media for 7 days and taken over 2 subcultures Medium a Subculture
Dry wt. (g/liter)
Fatty acid b 16:0
18:0
18:1
18:2
18:3
N. tabacum ~
B5C
1st 2nd
7.6 t0.2
19.1 20.7
2.5 1.6
3.3 2.4
61.7 61.0
13.5 14.4
WB
1st 2nd
5.6 6.0
23.4 19.1
2.0 2.4
2.0 2.6
61.2 63.2
11.4 12.8
MS
1st 2nd
4.7 5.3
23.2 21.2
1.4 2.1
1.9 4.2
57.8 57.2
15.8 15.4
B5C
1st 2nd
9.2 10.2
20.0 18.2
1.8 1.4
26.8 30.8
40.6 41.3
11.0 8.5
WB
1st 2nd
8.5 8.2
18.7 18.2
1.5 1.3
27.3 28.7
43.0 43.1
9.6 8.7
MS
1st 2nd
13.2 13.4
18.3 18.8
0.91 1.8
32.7 29.3
39.4 42.9
8.8 7.6
C. roseus d
a B 5 C = G a m b o r g ' s B5C medium; W B = W o o d and Braun medium; M S = M u r a s h i g e and Skoog medium b 16:0, palmitic acid; 18:0, stearic acid; 18:1, oleic acid; 18:2, linoleic acid; 18 : 3, linolenic acid Inoculum was 1.7 g/liter and 2.4 g/liter dry weight at the 1st and 2rid transfers, respectively a Inoculum was 1.6 g/liter and 1.7 g/liter dry weight at the 1st and 2nd transfers, respectively
386
J.J. MacCarthy and P.K. Stumpf: Fatty-Acids in Cell Suspension Cultures
16-
12-
N.tabacum ~
:
5
-4-6"8 "40"60"80
5
Table 2. The effect of increasing temperature on endogenous fattyacid levels of suspension-cultured N. tabacum cells. See Materials and Methods for extraction and analysis of fatty acids Day
~ i
}15
olI
C.roseus
-6
25
815
.
"=
5 -- -]s 25
~
--
O- ~ -
--,,~-
G.max 8-
~
/
--15
---
4
8
10
18:1
18:2
18:3
4.6 2.9 4.1
52.3 49.8 50.8
15.5 16.8 14.2
11.3 13.3 21.0
2.8 2.8 1.3
50.4 55.4 58.8
9.6 7.9 9.7
23.2 22.7 14.9
Cells grown at 15 ~ C
4 7 10
5 8 12
2 _s =
0
0
18:0
Total fatty acids (mg/g dry wt)
24.4 27.5 28.4
3.3 3.1 2.7
Cells grown at 25 ~ C
-8 -20
6
-='
r-4 '
j
~
~t 2
-30
-12 -30
O0
--
/ } 2 5 '
16:0
-2
15
O.
Fatty acids as % of total
29.8 32.2 28.5
1.9 1.7 2.3
Table 3. The effect of increasing temperature on endogenous fattyacid levels of suspension-cultured cells of C. roseus. See Materials and Methods for extraction and analysis of fatty acids
.0
12
Day
Fatty acids as % of total 18:1
18:2
18:3
Total fatty acids (mg/g dry wt.)
40.4 41.4 39.8
27.5 30.3 29.7
11.4 8.7 10,2
16.1 19,7 19.7
28.9 33.5 31.8 29.9
35.8 31.6 31.5 27.7
8.0 7.2 7.3 7.8
8.1 9.8 19.3 18.2
time I d l Fig. 1. The effect of temperature on dry-weight yields, cell number, and packed cell volume of N. tabacum, C. roseus and G. m a x cell suspension cultures
to the different genotypes rather than to different medium formulations, and this conclusion is supported by the results of Tattrie and Veliky (1973) who have demonstrated, for 8 cell lines cultured on specific media, that fatty-acid composition is related to the species. The G. max culture was maintained only on B5C medium since it did not grow on WB or MS media. In contrast to the other two cultures on B5C medium, linolenic acid comprised up to 40-50% of the fatty acids (Table 4).
Growth and Fatty-Acid Composition at Different Temperatures
Changes in dry weight, cell number and packed cell volume of the suspension cells of the three different species as a function of time and temperature are summarized in Fig. 1. There were no marked differences in these parameters with C. roseus cells grown at 25 ~ and 15 ~ C. In contrast, decided increases in packed cell volume and dry weight were noted with the N. tabacum, and in all three parameters with the G. max cells at 25 ~ as compared to 15 ~ C. These results indicate that these parameters should be monitored in order to define these tissue culture systems
16:0
18:0
Cells grown at 15 ~ C
4 7 10
20.7 19.5 20.4
T T T
Cells grown at 25 ~ C
5 11 15 20
21.5 22.3 22.1 29.9
3.7 1.3 T T
more precisely since the response of the three cultures as a function of time and temperature differed considerably, Tables 2, 3 and 4 summarize the shifts in fatty-acid composition when N. tabacum, C. roseus, and G. max were grown at 15~ and 25 ~ C for varying periods of time. Although the results were not analyzed statistically, certain trends could be readily detected. In terms of percent composition, the level of palmitic acid in the three cultures remained essentially constant; in all cultures, stearic acid appeared as a trace fatty acid. However, marked differences were observed in the levels of C18 unsaturated fatty acids. With G. max (Table 4) and N. tabacum (Table 2), the levels of endogenous oleic acid were quite low; when G. max was grown at 15 ~ C for a 10-day period,
J.J. MacCarthy and P.K. Stumpf: Fatty-Acids in Cell Suspension Cultures
387
Table 4. The effect of increasing temperature on endogenous fattyacid levels of suspension-cultured cells of G. max. See Materials and Methods for extraction and analysis of fatty acids
Table 6. Composition of nutrient media for cell suspension cultures (rag/liter)
Day
NH4NO3
Fatty acids as % of total 16:0
18:0
18:l
18:2
18:3
Total fatty acids (mg/g dry wt.)
2.2 2.3 3.2
16.8 7.8 5.5
16.0 23.0 23.2
43.8 47.2 48.6
20.8 27.3 28.2
9.1 7.8 4.3
20.3 14.6 15.0
49.6 53.4 52.6
48.4 27.0 20.9
~grownatl5~ 4 7 10
21.4 19.8 19.7
~
grownat25~
4 7 10
18.2 20.5 25.2
2.7 3.7 3.5
Table 5. Incorporation of [14C]acetate into fatty acids of suspension culture cells. See Materials and Methods for incubation conditions. Initial concentration of [14C]acetate was 18.5 nmol Time (h)
[~4C]acetate incorporated
Incorporated into fatty acids (as %)
Total (nmol)
%
16:0
18:0
18:1
18:2
18:3
12.1 14.6 17.2 23.4 15.l
33.3 27.1 29.6 30.7 30.4
6.l 8.6 4.9 6.9 7.6
22.7 17.1 18.5 14.9 7.6
36,4 44.3 44.4 46.5 50.6
1.5 2.9 2.5 1.0 3.8
1.8 3.4 2.7 3.6 3.0
9.5 18.2 14.7 19.2 16.3
39.2 35.3 45.3 36.8 34.4
3.9 9.8 4.0 5.3 1.6
54.9 51.0 44.0 48.7 56.3
1.9 3.9 5.3 6.6 6.3
0 0 1.3 2.6 1.6
0.9 1.0 1.4 1.2 1.8
5.0 5.2 7.6 6.3 9.5
47.1 46.4 35.0 35.1 28.3
8.8 10.7 12.5 10.8 6.7
41.2 39.3 45.0 37.8 35.0
2.9 3.6 7.5 13.5 23.3
talcum 1 2 3 4 5.5
2.2 2.7 3.4 4.3 2.8
C. FOSeMS
1 2 3 4 5.5 G. m a x
1
2 3 4 5.5
0
0 0 2.7 6.7
t h e o l e i c - a c i d level d r o p p e d m a r k e d l y ( T a b l e 4). I n C. roseus ( T a b l e 3), t h e levels w e r e m u c h h i g h e r initially. W i t h r e g a r d t o l i n o l e i c a c i d , t h e G. m a x c u l t u r e s s h o w e d i n c r e a s e d a c c u m u l a t i o n a t 15 ~ C. I n G. m a x a n d C. roseus, l i n o l e i c a c i d d e c l i n e d w i t h t i m e o f cult u r e a t 25 ~ C. T h e l i n o l e i c l e v e l s i n N . tabacum w e r e
Nutrient
(NH4)2SO
MS
WB
B5C
16,500
4
KNO3 KCI KH2PO 4 NaNO3 NaH2PO4. H20 Na2SO 4. 10H20 CaClz. 2H20 Ca(NO3)2.4H20 MgSO4.7H20 MnSO4.HEO ZnSO4.7H20 H3BO3 KI Na2MoO4 92H20 H2MoO4-H20 CuSO4.5HaO COC12.6H20 Na2EDTA FeSO4.7H20 Sucrose Glycine Nicotinic acid Aneurine- HC1 2,4-D Kinetin Inositol Casein hydrolysate L-asparagine hydrate L-glutamine pH
790 134 1,900 80 2,500 910 170 1,800 364 150 454 440 150 288 370 738 250 15 5 10 8.6 2.7 2.0 6.2 1.5 3.0 0.83 0.75 0.75 0.25 0.002 0.25 0.0016 0.025 0.0195 0.025 0.025 0.025 37.3 37.3 37.3 27.9 27.9 27.9 30,000 20,000 20,000 2.0 3,0 3.0 0.5 0.50 0.50 0.10 0.10 0.10 1.0 1.0 1.0 0.12 100 100 100 2,000 200 -200 5.7 5.5 5.5 -
somewhat higher than those observed in the other t w o c u l t u r e s y s t e m s a n d w h e n t h e s e cells w e r e g r o w n a t 25 ~ C, t h e r e w a s a f u r t h e r i n c r e a s e i n t h e l i n o l e i c percent levels as a function of days of growth. Finally, t h e e n d o g e n o u s l i n o l e n i c a c i d l e v e l s o f t h e t h r e e cult u r e s y s t e m s v a r i e d w i d e l y . W i t h G. m a x , a l m o s t half of the total fatty acids was linolenic acid whereas w i t h C. roseus t h i s a c i d a c c o u n t e d f o r o n l y 7 - 1 1 % a n d w i t h N. tabacum, f o r 9 - 1 6 % o f t h e t o t a l f a t t y - a c i d content. T h u s , i n cell c u l t u r e s o f t h r e e d i f f e r e n t s p e c i e s widely divergent fatty-acid compositions were observed; these were perturbed only to a limited extent when the cultures were grown at different temperatures.
Incorporation o f [ 1 4 C ] A c e t a t e into Lipids Table 5 shows the incorporation of [t4C]acetate by c u l t u r e s o f G. m a x , N. tabacum, a n d C. roseus o v e r a 5.5-h period.
388
ca
J.J. MacCarthy and P.K. Stumpf: Fatty-Acids in Cell Suspension Cultures
2,0 j
1.6,,r
1.2-
0'8-
G. max~
=_ 0"4,,r
0
I
I
I
I
I
1
2
3
4
5
time
[hi
Fig. 2. The ratio of unsaturated to saturated fatty acids of N. tabacum, C. roseus and G. m a x as a function of time. Culture temperature = 25 ~ C
Several significant observations derive from a comparison of the data in Table 4 and Table 5. Firstly, the G. m a x cells incorporated [14C]acetate initially into [14C]oleate which converted slowly to [t4C]linoleate only. [t4C]Acetate incorporation into linolenate over the 5.5-h incubation period was negligible. In sharp contrast, about 50% of the endogenous fatty acids in this cell culture was linolenate, about 15 20% linoleate, and about 10% oleate. Evidently, the incorporation data bear no relationship to endogenous fatty-acid levels in this cell line. Secondly, the C. roseus cell cultures exhibited a markedly different biosynthetic pattern. The major [14C]fatty acid synthesized was [t~C]oleate, with only traces of [14C]linoleate being formed; the [14C]palmitate level did not vary appreciably. And thirdly, the N. tabacum cell cultures incorporated [t4C]acetate principally into [14C]linoleate; with the exception of a lack of linolenate formation, the [14C]fatty-acid labelling pattern was quite similar to the endogenous fatty-acid composition of these cells. All three cell lines showed lack of [t4C]linolenate synthesis from [t~C]acetate. Figure 2 presents in summary form the trend of [14C]labe!ling of the total unsaturated and saturated fatty acids by these three different cell cultures as a function of time. It is quite clear that in general the C. roseus and the G. m a x cells were fairly similar in the [arC] pattern after 3 h of incubation, but the N. tabacum cells were markedly different (Table 5). In conclusion, these results clearly indicate that each of the three cell cultures studied had its lipid biosynthesis little affected by variations of the growth media, as measured in terms of the fatty acids found as components of their total lipid. Each cell culture accumulated fatty acids in a consistent, characteristic
pattern, and these patterns were quite different when compared with each other. Thus, in terms of fattyacid composition, these cell cultures do not revert to a generalized pattern that would primarily reflect/ be determined by acyl requirements of the membrane system of growing or stationary cells; their fatty-acidcomposition patterns are rather determined by the specific genotype of the specific cell. Finally, the [14C]acetate-incorporation studies show that while the labelling of palmitic acid was about the same in all species, marked differences occurred in the labellling patterns of oleic, linoleic and linolenic acids. The explanation for these differences must await the elucidation of the pathways for the biosynthesis of these unsaturated fatty acids, and of the interaction of the various cellular compartments wherein these activities are presumably localized. We would like to thank Dr. S. Yang, Mann Laboratory, University of California, Davis, for the use of a low temperature facility, Dr. M. Pollard for help with ozonolysis, and Billie Gabriel for help in preparing the manuscript. This investigation has been supported in part by grant PCM7601495 from the National Science Foundation.
References de Boer, Th. J., Backer, H.J.: Diazomethane. In: Organic Synthesis, Coll. Vol. 4, pp. 250-253, Rabjohn, N., ed. London: John Wiley and Sons, Inc. 1963 Gamborg, O.L. : Callus and cell culture. In: Plant Tissue Culture Methods, pp. 1-10, Gamborg, O.L., Wetter, L.R., eds. Saskatoon, Canada: National Research Council 1975 Murashige0 T., Skoog, F.: A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol. Plant. 15, 473497 (1962) Stein, R.A., Nicolaides, N.: Structure determination of methyl esters of unsaturated fatty acids by gas-liquid chromatography of the aldehydes formed by triphenyl phosphine reduction of the ozonides. J. Lipid Res. 3, 476~478 (1962) Siebertz, H.P., Heinz, E., Bergmann, L.: Acyl lipids in photosynthetically active tissue cultures of tobacco. Plant Sci. Lett. 12, 119-126 (1978) Stearns, E.M., Morton, W.T.: Biosynthesis of fatty acids from acetate in soybean suspension cultures. Lipids 10, 597 601 (1975) Street, H.E. : Plant cell cultures: Present and projected applications for studies in cell metabolism. In: Genetic Manipulations with Plant Materials, pp. 211-229, Ledoux, L., ed. New York: Plenum Press 1975 Stumpf, P.K., Weber, N. : Uptake and metabolism of fatty acids by soybean suspension cells. Lipids 12, 120-124 (1977) Tattrie, N.H., Veliky, I.A.: Fatty acid composition of lipids in various plant cell cultures. Can. J. Bot. 51, 513-516 (1973) Wood, H.N., Brown, A.C.: Studies on the regulation of certain essential biosynthetic systems in normal and crown gall tumor cells. Proc. Nat. Acad. Sci. USA 47, 1907 1913 (1961)
Received 31 January; accepted 27 August 1979
Planta 147, 384-388 (1980)
0 by Springer-Verlag 1980
Fatty-Acid Composition and Biosynthesis in Cell Suspension Cultures of Glycine max (L.) Merr., Catharanthus roseus G. Don and Nicotiana tabacum L. J.J. MacCarthy and P.K. Stumpf Department of Biochemistry and Biophysics, University of California, Davis, CA 95616, USA
Abstract. The fatty-acid composition of C. r o s e u s and N . t a b a c u m cell suspension cultures was unaffected by subculture on W o o d and Braun, Murashige and Skoog, or G a m b o r g B5C media. However, placing the cultures - which were normally grown at 2 5 ~ - at 15~ reduced growth but resulted in enhanced formation of oleic and linolenic acids in C . r o s e u s cultures and increased levels of linoleic and linolenic acids in cultures of G. m a x and N . t a b a c u m , respectively. The incorporation of p*C]acetate into [14C]linoteic acid was more rapid in N . t a b a c u m cells than in G. m a x cells, but was very poor in C. r o s e u s where the [~C] label was distributed mainly between palmitic and oleic acids. Key words: Acetate incorporation - C a t h a r a n t h u s Cell suspension cultures - Fatty acids - G l y c i n e N i c o t i a n a - Temperature and fatty-acid synthesis.
Don ( V i n c a
r o s e a L.) from the Apocynaceae, and
t a b a c u m (L.), cv. Wisconsin 38, from the Solanaceae. G. m a x cell cultures had been reported
Nicotiana
to have relatively high levels of linolenic acid (Stearns and Morton, 1975; Tattrie and Veliky, 1973) and Solanaceae species in cell culture had relatively high levels of linoleic acid (Siebertz et al., 1978; Tattrie and Veliky, 1973). The fatty-acid composition of C. r o s e u s was unknown. In this communication, fatty-acid compositions of several cell suspension cultures in response to medium formulation and temperature are reported. Comparisons of the synthesis of [14C]fatty acids from [14C]ace_ tate by each culture are also given.
Materials and Methods Celt Suspension Cultures
Introduction Cell suspension cultures provide useful and interesting biochemical systems for studies in plant cell metabolism (Street, 1975). Such cultures exhibit a characteristic fatty-acid composition which may resemble that of some organ of the original plant, though not necessarily the organ from which the culture was derived (Tattrie and Veliky, 1973). Cell suspension cultures usually exhibit a rapid rate of growth, with production of new cells during an exponential phase that endures for several days. The phase of rapid and increasing cell production must be accompanied by fatty-acid synthesis correlated with membrane development. With this in mind, cell suspension cultures were employed which represented widely different plant groups. These were cultures of G l y c i n e m a x (L.) Merr., cv. Mandarin, from the Leguminosae (Gamborg and Wetter, 1975), C a t h a r a n t h u s r o s e u s G .
The Glycine max cell suspension culture was very kindly provided by K.G. Lark, University of Utah, Salt Lake City, Utah, USA; the Nieotiana tabacum callus culture was provided by T. Murashige, University of California, Riverside, California, USA ; and the Catharanthus roseus cell suspension culture was initiated in this laboratory. Cell suspension cultures of N. tabacum and C. roseus were initiated from callus cultures and maintained on Murashige and Skoog medium (MS; Murashige and Skoog, 1975) and on a modified Wood and Braun medium (WB; Wood and Braun, 1961), respectively (Table 6). G. max cell suspension cultures were maintained on Gamborg's casein-supplemented B5C medium (Table 6). All three cultures were grown in 125-ml Erlenmeyer flasks on a G-10 model gyrotory shaker (New Brunswick Scientific Co., New Brunswick, N.J., USA) rotating at 131 rpm, under continuous illumination (General Electric fluorescent cool-white lamps; illuminance 455 lumen m 2) and at a temperature of 25_+0.5~C. The cultures of N. tabacum and G. m a x were subcultured every 7days and the C. roseus culture every 14days. Each celI suspension culture was grown and subsequently incubated on the medium on which it had been initiated so as to eliminate selection of ceil types or genotypes by transferring actively growing suspension culture onto medium of different composition. Since
0032-0935/80/0147/0384/$01.00
J.J. MacCarthy and P.K. Stumpf: Fatty-Acids in Cell Suspension Cultures fatty-acid synthesis may be affected by medium composition, N. tabacum and C. roseus were grown on WB and B5C, and MS and B5C, respectively, over two subcultures. The G. max culture could not be grown on either WB or MS medium. For studying the effects of culture at 15~ C, cells grown at 25~ were inoculated into fresh media and grown at 15~ in the light for 10 days, at 131 rpm. Dry weight, cell number and packed cell volume were routinely determined.
385
was determined in 200 gl of incubation medium (dissolved in 10 ml of Phase Combining System (PCS) scintillation fluid (Amersham Corp., Arlington Heights, Ill., USA), and using a Beckman LS-230 liquid scintillation counter (Beckman Instruments, Fullerton, Cal., USA). The sedimented cells were resuspended and washed 2 times in deionized water. The final pellet was extracted and analyzed for fatty acids as described above. All data presented in this paper have been duplicated.
Results and Discussion Determinations of Dry Weight, Cell Number, and Packed Cell Volume
For determining dry weight, the suspension culture (1.0 ml) was filtered with suction through a 2.4-cm GFIC glass microfibre filter paper (Whatman, Maidst0ne , Kent, U.K.). The filtered cells were washed 3 times in deionised water, and dried at 80 ~ C to constant weight. For cell counts, the suspension culture (1.0 ml) was macerated with 1.0 ml of 15% chromium trioxide. N. tabacum and C. roseus cells were macerated at 70 ~ C for 20 min, G. max cells at 70 ~ C for 45 rain. The macerate was cooled, diluted, and counted. For determinations of packed cell volumes, the suspension culture (15 ml) was centrifuged at 1,000 • g for 2 rain. An International Clinical bench-top centrifuge equipped with a No. 809 fixed angle head (International Equipment Corp., Needham Heights, Mass., USA) was used. The volume of sedimented cells was given as percentage of the total culture volume.
Endogenous Lipids of Cell Cultures
As indicated in Table 1, the fatty-acid compositions of N. tabacum and C. roseus cell cultures grown on B5C, WB and MS media for 7 days and taken over two subcultures were essentially constant. With the N. tabacum cells, the major unsaturated fatty acid was linoleic acid which constituted about 60% of the total fatty acids of these ceils. Oleic acid made up only about 2 3% of the total fatty acids. In contrast, with C. roseus cells, oleic and linoleic acids comprised ca. 30 and 40%, respectively, of the total fatty acids of these cells regardless of medium formulation and subculturing. The fatty-acid composition of these two cultures could therefore be attributed
Fatty Acid Extraction and Analysis
Lyophilized cells (100 mg) were saponified with 20% K O H in methanol (w/v) for 30 rain at 85 ~ C. The lipid extract was cooled, acidified with 6N HCt, and extracted 3 times with petroleum ether (b.p. 35-60 ~ C). The combined petroleum-ether phases were evaporated to dryness at room temperature under a stream of N2. The fatty-acid extract was redissolved in ca. 70% diazomethane in diethyl ether freshly prepared (de Boer and Backer, 1963) from N-methyl-N-nitroso-p-tolulenesulfonamide (Diazald, Aldrich Chemical Co., Milwaukee, Wis., USA). Following methylation at 0 ~ for 30 rain, excess diazomethane in diethyl ether was blown off with a stream o f N2- The methylated derivatives were redissolved in 100 pl of toluene for gas-liquid chromatography (GLC). A Model 920 GLC (Varian Aerograph, Walnut Creek, Cal., USA) equipped with a stainless-steel column (152 cm long, inner diameter 0.64cm) and packed with 10% diethylene glycol succinate: (Supelco Inc., Bellefonte, Pa., USA) was used. Radioactivity in the sample was detected with a Biospan Model 4998 detection'gystem (Nuclear-Chicago Corp., Des Plaines, Ill., USA). Individuafff'atty-acids were identified by comparison with standards of known fatty acid composition and by argentation thin-layer chromatography followed by ozonolysis of the fatty acids (Stein and Nicolaides, 1962). The fatty-aldehyde products of ozonolysis were identified using GLC.
Incubation with
[14C] Substrates
Freshly harvested cells (6 days old, 0.5 g fresh weight) were added to 1.5 ml sterile medium (pH 7.3), containing 1.0 pCi sodium [14C]acetate (New England Nuclear, Boston, Mass., USA; 54 mCi/ mmol) and gently agitated until mixed. The 25-ml scintillation vials were capped and incubated on a gyrotory shaker (131 rpm) at 25 ~ C in the light. Following incubation, cells were separated from medium by centrifugation. Residual [x4C]acetate
Table 1. The fatty-acid composition of N. tabacum and C. roseus cell cultures grown on B5C, WB and MS media for 7 days and taken over 2 subcultures Medium a Subculture
Dry wt. (g/liter)
Fatty acid b 16:0
18:0
18:1
18:2
18:3
N. tabacum ~
B5C
1st 2nd
7.6 t0.2
19.1 20.7
2.5 1.6
3.3 2.4
61.7 61.0
13.5 14.4
WB
1st 2nd
5.6 6.0
23.4 19.1
2.0 2.4
2.0 2.6
61.2 63.2
11.4 12.8
MS
1st 2nd
4.7 5.3
23.2 21.2
1.4 2.1
1.9 4.2
57.8 57.2
15.8 15.4
B5C
1st 2nd
9.2 10.2
20.0 18.2
1.8 1.4
26.8 30.8
40.6 41.3
11.0 8.5
WB
1st 2nd
8.5 8.2
18.7 18.2
1.5 1.3
27.3 28.7
43.0 43.1
9.6 8.7
MS
1st 2nd
13.2 13.4
18.3 18.8
0.91 1.8
32.7 29.3
39.4 42.9
8.8 7.6
C. roseus d
a B 5 C = G a m b o r g ' s B5C medium; W B = W o o d and Braun medium; M S = M u r a s h i g e and Skoog medium b 16:0, palmitic acid; 18:0, stearic acid; 18:1, oleic acid; 18:2, linoleic acid; 18 : 3, linolenic acid Inoculum was 1.7 g/liter and 2.4 g/liter dry weight at the 1st and 2rid transfers, respectively a Inoculum was 1.6 g/liter and 1.7 g/liter dry weight at the 1st and 2nd transfers, respectively
386
J.J. MacCarthy and P.K. Stumpf: Fatty-Acids in Cell Suspension Cultures
16-
12-
N.tabacum ~
:
5
-4-6"8 "40"60"80
5
Table 2. The effect of increasing temperature on endogenous fattyacid levels of suspension-cultured N. tabacum cells. See Materials and Methods for extraction and analysis of fatty acids Day
~ i
}15
olI
C.roseus
-6
25
815
.
"=
5 -- -]s 25
~
--
O- ~ -
--,,~-
G.max 8-
~
/
--15
---
4
8
10
18:1
18:2
18:3
4.6 2.9 4.1
52.3 49.8 50.8
15.5 16.8 14.2
11.3 13.3 21.0
2.8 2.8 1.3
50.4 55.4 58.8
9.6 7.9 9.7
23.2 22.7 14.9
Cells grown at 15 ~ C
4 7 10
5 8 12
2 _s =
0
0
18:0
Total fatty acids (mg/g dry wt)
24.4 27.5 28.4
3.3 3.1 2.7
Cells grown at 25 ~ C
-8 -20
6
-='
r-4 '
j
~
~t 2
-30
-12 -30
O0
--
/ } 2 5 '
16:0
-2
15
O.
Fatty acids as % of total
29.8 32.2 28.5
1.9 1.7 2.3
Table 3. The effect of increasing temperature on endogenous fattyacid levels of suspension-cultured cells of C. roseus. See Materials and Methods for extraction and analysis of fatty acids
.0
12
Day
Fatty acids as % of total 18:1
18:2
18:3
Total fatty acids (mg/g dry wt.)
40.4 41.4 39.8
27.5 30.3 29.7
11.4 8.7 10,2
16.1 19,7 19.7
28.9 33.5 31.8 29.9
35.8 31.6 31.5 27.7
8.0 7.2 7.3 7.8
8.1 9.8 19.3 18.2
time I d l Fig. 1. The effect of temperature on dry-weight yields, cell number, and packed cell volume of N. tabacum, C. roseus and G. m a x cell suspension cultures
to the different genotypes rather than to different medium formulations, and this conclusion is supported by the results of Tattrie and Veliky (1973) who have demonstrated, for 8 cell lines cultured on specific media, that fatty-acid composition is related to the species. The G. max culture was maintained only on B5C medium since it did not grow on WB or MS media. In contrast to the other two cultures on B5C medium, linolenic acid comprised up to 40-50% of the fatty acids (Table 4).
Growth and Fatty-Acid Composition at Different Temperatures
Changes in dry weight, cell number and packed cell volume of the suspension cells of the three different species as a function of time and temperature are summarized in Fig. 1. There were no marked differences in these parameters with C. roseus cells grown at 25 ~ and 15 ~ C. In contrast, decided increases in packed cell volume and dry weight were noted with the N. tabacum, and in all three parameters with the G. max cells at 25 ~ as compared to 15 ~ C. These results indicate that these parameters should be monitored in order to define these tissue culture systems
16:0
18:0
Cells grown at 15 ~ C
4 7 10
20.7 19.5 20.4
T T T
Cells grown at 25 ~ C
5 11 15 20
21.5 22.3 22.1 29.9
3.7 1.3 T T
more precisely since the response of the three cultures as a function of time and temperature differed considerably, Tables 2, 3 and 4 summarize the shifts in fatty-acid composition when N. tabacum, C. roseus, and G. max were grown at 15~ and 25 ~ C for varying periods of time. Although the results were not analyzed statistically, certain trends could be readily detected. In terms of percent composition, the level of palmitic acid in the three cultures remained essentially constant; in all cultures, stearic acid appeared as a trace fatty acid. However, marked differences were observed in the levels of C18 unsaturated fatty acids. With G. max (Table 4) and N. tabacum (Table 2), the levels of endogenous oleic acid were quite low; when G. max was grown at 15 ~ C for a 10-day period,
J.J. MacCarthy and P.K. Stumpf: Fatty-Acids in Cell Suspension Cultures
387
Table 4. The effect of increasing temperature on endogenous fattyacid levels of suspension-cultured cells of G. max. See Materials and Methods for extraction and analysis of fatty acids
Table 6. Composition of nutrient media for cell suspension cultures (rag/liter)
Day
NH4NO3
Fatty acids as % of total 16:0
18:0
18:l
18:2
18:3
Total fatty acids (mg/g dry wt.)
2.2 2.3 3.2
16.8 7.8 5.5
16.0 23.0 23.2
43.8 47.2 48.6
20.8 27.3 28.2
9.1 7.8 4.3
20.3 14.6 15.0
49.6 53.4 52.6
48.4 27.0 20.9
~grownatl5~ 4 7 10
21.4 19.8 19.7
~
grownat25~
4 7 10
18.2 20.5 25.2
2.7 3.7 3.5
Table 5. Incorporation of [14C]acetate into fatty acids of suspension culture cells. See Materials and Methods for incubation conditions. Initial concentration of [14C]acetate was 18.5 nmol Time (h)
[~4C]acetate incorporated
Incorporated into fatty acids (as %)
Total (nmol)
%
16:0
18:0
18:1
18:2
18:3
12.1 14.6 17.2 23.4 15.l
33.3 27.1 29.6 30.7 30.4
6.l 8.6 4.9 6.9 7.6
22.7 17.1 18.5 14.9 7.6
36,4 44.3 44.4 46.5 50.6
1.5 2.9 2.5 1.0 3.8
1.8 3.4 2.7 3.6 3.0
9.5 18.2 14.7 19.2 16.3
39.2 35.3 45.3 36.8 34.4
3.9 9.8 4.0 5.3 1.6
54.9 51.0 44.0 48.7 56.3
1.9 3.9 5.3 6.6 6.3
0 0 1.3 2.6 1.6
0.9 1.0 1.4 1.2 1.8
5.0 5.2 7.6 6.3 9.5
47.1 46.4 35.0 35.1 28.3
8.8 10.7 12.5 10.8 6.7
41.2 39.3 45.0 37.8 35.0
2.9 3.6 7.5 13.5 23.3
talcum 1 2 3 4 5.5
2.2 2.7 3.4 4.3 2.8
C. FOSeMS
1 2 3 4 5.5 G. m a x
1
2 3 4 5.5
0
0 0 2.7 6.7
t h e o l e i c - a c i d level d r o p p e d m a r k e d l y ( T a b l e 4). I n C. roseus ( T a b l e 3), t h e levels w e r e m u c h h i g h e r initially. W i t h r e g a r d t o l i n o l e i c a c i d , t h e G. m a x c u l t u r e s s h o w e d i n c r e a s e d a c c u m u l a t i o n a t 15 ~ C. I n G. m a x a n d C. roseus, l i n o l e i c a c i d d e c l i n e d w i t h t i m e o f cult u r e a t 25 ~ C. T h e l i n o l e i c l e v e l s i n N . tabacum w e r e
Nutrient
(NH4)2SO
MS
WB
B5C
16,500
4
KNO3 KCI KH2PO 4 NaNO3 NaH2PO4. H20 Na2SO 4. 10H20 CaClz. 2H20 Ca(NO3)2.4H20 MgSO4.7H20 MnSO4.HEO ZnSO4.7H20 H3BO3 KI Na2MoO4 92H20 H2MoO4-H20 CuSO4.5HaO COC12.6H20 Na2EDTA FeSO4.7H20 Sucrose Glycine Nicotinic acid Aneurine- HC1 2,4-D Kinetin Inositol Casein hydrolysate L-asparagine hydrate L-glutamine pH
790 134 1,900 80 2,500 910 170 1,800 364 150 454 440 150 288 370 738 250 15 5 10 8.6 2.7 2.0 6.2 1.5 3.0 0.83 0.75 0.75 0.25 0.002 0.25 0.0016 0.025 0.0195 0.025 0.025 0.025 37.3 37.3 37.3 27.9 27.9 27.9 30,000 20,000 20,000 2.0 3,0 3.0 0.5 0.50 0.50 0.10 0.10 0.10 1.0 1.0 1.0 0.12 100 100 100 2,000 200 -200 5.7 5.5 5.5 -
somewhat higher than those observed in the other t w o c u l t u r e s y s t e m s a n d w h e n t h e s e cells w e r e g r o w n a t 25 ~ C, t h e r e w a s a f u r t h e r i n c r e a s e i n t h e l i n o l e i c percent levels as a function of days of growth. Finally, t h e e n d o g e n o u s l i n o l e n i c a c i d l e v e l s o f t h e t h r e e cult u r e s y s t e m s v a r i e d w i d e l y . W i t h G. m a x , a l m o s t half of the total fatty acids was linolenic acid whereas w i t h C. roseus t h i s a c i d a c c o u n t e d f o r o n l y 7 - 1 1 % a n d w i t h N. tabacum, f o r 9 - 1 6 % o f t h e t o t a l f a t t y - a c i d content. T h u s , i n cell c u l t u r e s o f t h r e e d i f f e r e n t s p e c i e s widely divergent fatty-acid compositions were observed; these were perturbed only to a limited extent when the cultures were grown at different temperatures.
Incorporation o f [ 1 4 C ] A c e t a t e into Lipids Table 5 shows the incorporation of [t4C]acetate by c u l t u r e s o f G. m a x , N. tabacum, a n d C. roseus o v e r a 5.5-h period.
388
ca
J.J. MacCarthy and P.K. Stumpf: Fatty-Acids in Cell Suspension Cultures
2,0 j
1.6,,r
1.2-
0'8-
G. max~
=_ 0"4,,r
0
I
I
I
I
I
1
2
3
4
5
time
[hi
Fig. 2. The ratio of unsaturated to saturated fatty acids of N. tabacum, C. roseus and G. m a x as a function of time. Culture temperature = 25 ~ C
Several significant observations derive from a comparison of the data in Table 4 and Table 5. Firstly, the G. m a x cells incorporated [14C]acetate initially into [14C]oleate which converted slowly to [t4C]linoleate only. [t4C]Acetate incorporation into linolenate over the 5.5-h incubation period was negligible. In sharp contrast, about 50% of the endogenous fatty acids in this cell culture was linolenate, about 15 20% linoleate, and about 10% oleate. Evidently, the incorporation data bear no relationship to endogenous fatty-acid levels in this cell line. Secondly, the C. roseus cell cultures exhibited a markedly different biosynthetic pattern. The major [14C]fatty acid synthesized was [t~C]oleate, with only traces of [14C]linoleate being formed; the [14C]palmitate level did not vary appreciably. And thirdly, the N. tabacum cell cultures incorporated [t4C]acetate principally into [14C]linoleate; with the exception of a lack of linolenate formation, the [14C]fatty-acid labelling pattern was quite similar to the endogenous fatty-acid composition of these cells. All three cell lines showed lack of [t4C]linolenate synthesis from [t~C]acetate. Figure 2 presents in summary form the trend of [14C]labe!ling of the total unsaturated and saturated fatty acids by these three different cell cultures as a function of time. It is quite clear that in general the C. roseus and the G. m a x cells were fairly similar in the [arC] pattern after 3 h of incubation, but the N. tabacum cells were markedly different (Table 5). In conclusion, these results clearly indicate that each of the three cell cultures studied had its lipid biosynthesis little affected by variations of the growth media, as measured in terms of the fatty acids found as components of their total lipid. Each cell culture accumulated fatty acids in a consistent, characteristic
pattern, and these patterns were quite different when compared with each other. Thus, in terms of fattyacid composition, these cell cultures do not revert to a generalized pattern that would primarily reflect/ be determined by acyl requirements of the membrane system of growing or stationary cells; their fatty-acidcomposition patterns are rather determined by the specific genotype of the specific cell. Finally, the [14C]acetate-incorporation studies show that while the labelling of palmitic acid was about the same in all species, marked differences occurred in the labellling patterns of oleic, linoleic and linolenic acids. The explanation for these differences must await the elucidation of the pathways for the biosynthesis of these unsaturated fatty acids, and of the interaction of the various cellular compartments wherein these activities are presumably localized. We would like to thank Dr. S. Yang, Mann Laboratory, University of California, Davis, for the use of a low temperature facility, Dr. M. Pollard for help with ozonolysis, and Billie Gabriel for help in preparing the manuscript. This investigation has been supported in part by grant PCM7601495 from the National Science Foundation.
References de Boer, Th. J., Backer, H.J.: Diazomethane. In: Organic Synthesis, Coll. Vol. 4, pp. 250-253, Rabjohn, N., ed. London: John Wiley and Sons, Inc. 1963 Gamborg, O.L. : Callus and cell culture. In: Plant Tissue Culture Methods, pp. 1-10, Gamborg, O.L., Wetter, L.R., eds. Saskatoon, Canada: National Research Council 1975 Murashige0 T., Skoog, F.: A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol. Plant. 15, 473497 (1962) Stein, R.A., Nicolaides, N.: Structure determination of methyl esters of unsaturated fatty acids by gas-liquid chromatography of the aldehydes formed by triphenyl phosphine reduction of the ozonides. J. Lipid Res. 3, 476~478 (1962) Siebertz, H.P., Heinz, E., Bergmann, L.: Acyl lipids in photosynthetically active tissue cultures of tobacco. Plant Sci. Lett. 12, 119-126 (1978) Stearns, E.M., Morton, W.T.: Biosynthesis of fatty acids from acetate in soybean suspension cultures. Lipids 10, 597 601 (1975) Street, H.E. : Plant cell cultures: Present and projected applications for studies in cell metabolism. In: Genetic Manipulations with Plant Materials, pp. 211-229, Ledoux, L., ed. New York: Plenum Press 1975 Stumpf, P.K., Weber, N. : Uptake and metabolism of fatty acids by soybean suspension cells. Lipids 12, 120-124 (1977) Tattrie, N.H., Veliky, I.A.: Fatty acid composition of lipids in various plant cell cultures. Can. J. Bot. 51, 513-516 (1973) Wood, H.N., Brown, A.C.: Studies on the regulation of certain essential biosynthetic systems in normal and crown gall tumor cells. Proc. Nat. Acad. Sci. USA 47, 1907 1913 (1961)
Received 31 January; accepted 27 August 1979
