Chapter 17
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Effect of Tridemorph on Sterol Synthesis in Algae Glenn W. Patterson Department of Botany, University of Maryland, College Park, MD 20742
Tridemorph was added to the culture medium of Chlorella sorokiniana at concentrations of 1 ppm and 2.5 ppm (3.3 and 8.3 x 10 Μ), reducing the growth rate by approximately 50% and 75%, respectively. Tridemorph at a concentration of 1 ppm completely inhibited the production of ergosterol, the normal end product of sterol synthesis in this organism. 24-Methylpollinastanol, 24-methylenepollinastanol, and ergost-8(14)-enol accumulated in a 2:3:1 ratio, respectively. At a tridemorph concentration of 2.5 ppm desmethyl sterols were not detected, but 24-methylpollinastanol, 24-methylenepollinastanol, and 24-methylenecycloartanol accumulated in a ratio of 5:4:3, respectively. The results demonstrate that the lack of C-24 ethyl sterols in Chlorella is not due to its inability to produce the 24-methylene precursor. -6
T r i d e m o r p h (4-tridecyl-2,6-dimethylmorpholine) is a n ergosterol biosynthesis inhibitor i n many fungi (1). It has been shown to inhibit the Δ to Δ isomerase reaction i n Botrytis cinerea (2), and several Oomycetes (3), but it apparently inhibits primarily t h e A * reductase i n U s t i l a g o mavdis (4). I n higher plants w h i c h use the cycloartenol pathway i n contrast to the lanosterol pathway o f fungi, tridemorph inhibits the opening of the 9,19-cyclopropane ring characteristic o f this pathway (5,6,7). A study of the tridemorph inhibition o f cultures of C h l o r e l l a sorokiniana was attractive for several reasons. C . sorokiniana is a m e m b e r o f G r o u p I I I A o f C h l o r e l l a based o n sterol composition (8). T h i s group is distinguished by p r o d u c t i o n of ergosterol as the principal sterol (like most fungi) but u n l i k e fungi, this group synthesizes ergosterol through the cycloartenol pathway o f higher lants. C . sorokiniana is also unusual i n that it synthesizes sterols w i t h a methyl ut not a n ethyl at C-24. It is not k n o w n whether the lack o f a second alkylation reaction is due to lack of the enzyme for the second alkylation or whether the product o f the first alkylation could not serve as a precursor for the second alkylation. T r i p a r a n o l and A Y - 9 9 4 4 both produced a n accumulation of 24methylene compounds i n other species of C h l o r e l l a (9,10), but neither inhibitor 1
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resulted i n the accumulation of 24-methylene compounds i n C h l o r e l l a sorokiniana (11,12), giving some reason to believe that C . sorokiniana could not produce the 24-methylene group. T r i d e m o r p h treatment o f C h l o r e l l a w o u l d be expected to inhibit formation of the same end product (ergosterol) as i n fungi. H o w e v e r , the cycloartenol pathway used by C h l o r e l l a w i l l present a potential site of inhibition not present i n fungi - the opening o f the 9,19-cyclopropane ring. T h i s biosynthetic step usually occurs soon after the first alkylation reaction, meaning that inhibition o f the opening o f the cyclopropane ring should produce 24-methylene sterols i f they are synthesized by C h l o r e l l a .
Methods A l g a l cultures were grown o n a standard glucose m e d i u m (8) i n 1 p p m and 2.5 p p m o f tridemorph (3.3 a n d 8.3 χ 10"" M ) with rates of growth inhibition at 50% and 75%, respectively. L i p i d was extracted from lyophilized cultures by extraction with c h l o r o f o r m / m e t h a n o l (2:1) followed by saponification o f the l i p i d and isolation o f the sterol fraction from a l u m i n a c o l u m n chromatography (8). T h e sterol fraction was analyzed by capillary gas chromatography o n a 30 m c o l u m n o f SPB-1 (15) and by capillary gas chromatography-mass spectroscopy o n a F i n n i g a n - M A T m o d e l 4512 instrument as previously described (15).
Sterols of Chlorella sorokiniana cultured in presence of 1 ppm tridemorph. C o n t r o l cultures o f C . sorokiniana contained ergosterol as the p r i n c i p a l sterol as previously reported (8) with smaller amounts o f ergost-7-enol a n d ergosta-5,7dienol (Table I). A tridemorph concentration o f 1 p p m completely inhibited synthesis o f ergosterol with a n accumulation o f 24-methylpollinastanol and 24methylenepolhnastanol (Table I). T h e mass spectrum o f 24-methylpollinastanol is presented i n T a b l e II and is identical to that previously reported (11). T h e mass spectrum o f 24-methylenepollinastanol is also presented o n T a b l e II and is identical to that o f the authentic compound (9). T h e data show that at 1 p p m of tridemorph, sterol biosynthesis is inhibited primarily at the opening o f the cyclopropane ring since over 80% o f the sterols from such cultures contain 9,19cyclopropane rings. These data are i n accord with w o r k o n carrot, tobacco, soybean (7) and bramble cell cultures (5) and with corn seedlings (6), a l l o f w h i c h have sterol synthesis inhibited at the opening o f the 9,19-cyclopropane ring. A third sterol detected i n cultures inhibited by 1 p p m tridemorph was 5a-ergost-8(14)-enol, a sterol not previously reported from tridemorph-inhibited tissues. T h e capillary gc relative retention times o f this sterol were similar to those o f campesterol, 5a-ergost-8(9)-enol, 5a-ergost-8(14)-enol, 5a-ergosta8(9),14-dienol, and 5a-ergosta-8(9), 24(28)-dienol. H o w e v e r , the mass spectrum clearly showed the presence o f a monounsaturated C?Q c o m p o u n d with few other distinguishing features, which is characteristic ο ϊ » ( 9 ) or 8(14) monoenes (13). A n examination o f the mass spectra (data not shown) o f authentic 5aergost-8(14)-enol, 5a-ergost-8(9)-enol, 5a-stigmast-8(9)-enol, a n d 5a-stigmast8(14)-enol showed that the spectra o f the two C 2 g compounds and the two C^g compounds were practically identical except that the 8(9)-compounds h a d a Base peak o f m / z 43 while the 8(14)-compounds had a base peak o f m / z 400 (or m / z 414). T h e compound from C h l o r e l l a had a base peak o f m / z 400 suggesting its identity w i t h 5a-ergost-8(14)-enol. B o t h packed c o l u m n and capillary gas chromatography can distinguish between the two sterols, with the 8(14) c o m p o u n d having the shorter retention time o n both polar and non-polar
17. PATTERSON
Effect of Tridemorph on Sterol Synthesis in Algae
233
Table I. Gas Chromatographic Relative Retention Times and Quantitative Distribution of Sterols ofC. sorokiniana Cultured with and without Tridemorph
Sterol D i s t r i b u t i o n
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SPB-1
RRT
b
Tridemorph
0
lppm
2.5 p p m
Ergosterol
1.21
85
Ergosta-5,7-dienol
1.36
6
Ergost-7-enol
1.38
9
Ergost-8(14)-enol
1.27
18
24-Methylpollinastanol
1.41
32
41
24-Methylenepollinastanol
1.38
50
35
24-Methylenecycloartanol
1.87
L
24
as % of total sterol
^compared to cholesterol at 1.00
columns (14,15). T h e C h l o r e l l a compound had a R R T o f 1.27 o n SPB-1, identical with the R R T of 5a-ergost-8(14)-enol. T h e corresponding R R T of 5aergost-8(9)-enol was 1.33. T h e quantity of sample available d i d not permit N M R analysis. T h e data available indicate that one site o f i n h i b i t i o n is at the opening of the 9,19-cyclopropane ring i n agreement w i t h previous w o r k w i t h plants using the cycloartenol pathway (5,6,7). T h i s b l o c k is not complete at a n inhibitor concentration o f 1 p p m since some sterol is metabolized past the 9,19ring opening step only to accumulate as 5a-ergost-8(14)-enol. It appears that little or no sterol is metabolized past this point with culture concentrations of tridemorph at 1 p p m .
Sterols of Chlorella sorokiniana cultured in the presence of 2.5 ppm tridemorph. W h e n cultures are grown i n the presence of 2.5 p m of tridemorph, n o desmethyl sterols were detected. T h e major sterols accumulating were 24methylpollinastanol and 24-methylenepollinastanol w h i c h were accompanied by 24-methylenecycloartenol rather than 5a-ergost-8(14)-enol (Table I). 24Methylenecycloartanol was identified by its characteristic mass spectrum and R R T (Table II). A t 2.5 p p m of tridemorph, the opening of the cyclopropane ring appears to be completely b l o c k e d so that 5a-ergost-8(14)-enol cannot accumulate as it d i d at 1 p p m .
REGULATION OF ISOPENTENOID METABOLISM
234 3
Table II. Mass Spectral Data of some Sterols from Tridemorph Inhibited Chlorella sorokiniana Sterol
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Fragmentation
[M]
1
+
[M-CH ]
+
3
[M-H 0]
+
2
[M-33]
+
[M-side c h a i n ]
+
[ M - r i n g A + part o f Β ]
+
[M-side c h a i n + w a t e r ]
+
base peak
c
2
b
3
412 (14)
4
440 (18)
400 (100)
414 (25)
385 ( 28)
399 (65)
397 (20)
425 (26)
382 ( 5)
396 (34)
394 (21)
422 (33)
367 ( 5)
381 (44)
379 (21)
407 (42)
273 ( 19)
287 (32)
287 (12)
315 ( 6)
302 (18)
300 (11)
300 (28)
269 (31)
269 (15)
297 (11)
— 255 ( 9)
400
43
55
55
R e p o r t e d as m / z (relative intensity) D
l 2 3 4
c
d u e to cleavage of 9,19-cyclopropane ring and ring Β
= ergost-8(14)-enol = 24-methylpollinastanol = 24-methylenepollinastanol = 24-methylenecycloartanol
T r i d e m o r p h inhibits the opening of the 9,19-cyclopropane ring C h l o r e l l a sorokiniana as it has been reported to do i n other plants synthesizing sterols by the cycloartenol pathway (5,6,7). It apparently inhibits the metabolism o f 5aergost-8(14)-enol at a concentration o f 1 p p m , an effect not previously reported. A l t h o u g h 5a-ergost-8(14)-enol can be easily distinguished from campesterol o n mass spectroscopy, the two can be easily confused i n gas chromatography. T h e frequent occurrence of campesterol i n higher plants may have obscured the presence of ergost-8(14)-enol.
Significance of research results. T h i s w o r k provides some additional insight into sterol synthesis o f C h l o r e l l a sorokiniana. an organism differing from most plants and most other C h l o r e l l a species i n that it apparently is unable to synthesize the C-24 ethyl group. T h i s could be due to the lack of the enzyme necessary for the second alkylation reaction or it could be due to the lack of the necessary 24-methylene precursor for the second alkylation reaction. Previous w o r k with other inhibitors w i t h
17.
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Effect of Tridemorph on Sterol Synthesis in Algae
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C h l o r e l l a sorokiniana have produced n o accumulation o f 24-methylene precursors as they have i n other species o f C h l o r e l l a leading to the speculation that C h l o r e l l a s o r o k i n i a n a cannot synthesize the 24-methylene group. T h i s w o r k shows clearly that w h e n inhibited with tridemorph. C h l o r e l l a accumulates 24methylene compounds at both inhibitor concentrations tested. T h e current w o r k suggests that the enzyme for the second alkylation is either missing or is ineffective i n C h l o r e l l a sorokiniana.
Literature Cited 1. Mercer, E.I. In: Sterol Biosynthesis Inhibitors, Berg, D, Plempel, M, Eds; Ellis Horwood, Ltd., Chichester, England. 1988. pp. 120-150. 2. Kato, T. Shoami, M. and Kawase, Y. J. Pest. Sci. 1980. 5, 69-70. 3. Berg, L.R., Patterson, G.W., and Lusby, W.R. Lipids 1983. 18, 448-452. 4. Kerkenaar, Α., Uchiyama, M., and Versluis, G.G. Pestic. Biochem. Physiol. 1981. 16, 97-104. 5. Schmitt, P., Benveniste, P., and Leroux, P. Phytochemistry 20, 2153-2159. 6. Bladocha, M. and Benveniste, P. Plant Physiol. 1983. 71, 756-762. 7. Hosokawa, G., Patterson, G.W., and Lusby, W.R. Lipids 1984. 19, 449-456. 8. Holden, M.J. and Patterson, G.W. Lipids 1982. 17, 215-219. 9. Doyle, P.J., Patterson, G.W., Dutky, S.R., and Thompson, M.J. Phytochemistry 1972. 11, 1951-1960. 10. Dickson, L.G. and Patterson, G.W. Lipids 1973. 8, 443-445. 11. Chan, J.T., Patterson, G.W., Dutky, S.R., and Cohen, C.F. Plant Physiol. 1974. 53, 244-249. 12. Chiu, P.L., Patterson, G.W. and Dutky, S.R. Phytochemistry 1976. 15, 1907-1910. 13. Galli, G. and Maroni, S. Steroids 1967. 10, 189-192. 14. Patterson, G.W. Anal. Chem. 1971. 43, 1165-1170. 15. Itoh, T., Tani, H., Fukushima, K., Tamura, T., and Matsumoto, T. J. of Chromatog. 1982. 234, 65-76. RECEIVED March 20, 1992
