Plant Cell Reports (1983) 2:7-10

Plant Cell Reports © Springer-Verlag 1983

Evidence for an Alternative Route from Sterol to Sapogenin in Suspension Cultures from Trigonellafoenumgraecum K. R. Brain and M. H. Williams Welsh School of Pharmacy, UWIST, CardiffCF1 3NU, UK Received September 14, 1982/November 15, 1982

Abstract Incorporation of [4-14C]cholesterol, [26-14C]cholesterol, [4-14C]sitosterol and [22,23-3H]sitosterol into sapogenin in suspension cultures from Trigonella foenumgraecum differed when substrates were added at subculture and 10 d after subculture, suggesting alternative biosynthetic routes were in operation.

Abbreviation 2,4-D

(2,4-dichlorophenoxyacetic

acid)

(1973), Khanna et al. (1975a,b) and Brain and Lockwood (1976). The level of sapogenin varied widely and was clearly influenced by hormonal factors (Brain and Lockwood 1976). Observations on the ratio of diosgenin (25-R) to yamogenin (25-L) in stored seeds of T__~.foenumgraecum (Hardman and Brain 1972) showed that this varied quite widely with time, and during callus induction there were marked variations in levels of free and bound sterols and sapogenin with time and hormonal supplementation (Lockwood and Brain 1976), presumably due to turnover of the sterol and sapogenin pools. It was therefore of interest to determine whether the route of sapogenin synthesis in cultures from this species was consistent with that reported for D_~. deltoidea.

Introduction Experimental The biosynthesis of diosgenin from sterol in plant tissue culture has been shown to follow the same pathway as in intact plants. Stohs et al. (1969) claimed equal incorporation of label into dios$~nin glycosides from [4-''C]cholesterol and [26-'~C]cholesterol in 10 d old undifferentiated suspension cultures from Dioscorea deltoidea, and concluded that the cholesterol molecule was incorporated intact. Tomita and Uomori (1971, 1974) obtained incorporation of a number of sterols and hydroxylated derivatives into sapogenins in cultures from Dioscorea tokoro and demonstrated that hydroxylation at positions 16, 22 and 26 must occur before cyclization.

Suspension cultures were initiated, maintained and extracted for free and bound sterol and sapogenin as reported previously (Brain and Lockwood 1976). Cultures were grown on Murashige and Skoog's medium (Flow Labs, Irvine, UK) supplemented with 0.25 mg -I 2,4-D or with 0.Smg -I 2,4-D and 0.5 mg -I kinetin. All radiochemicals were from Amersham International, and 14.8 kBq added in diethyl ether solution in each case (Brain and James 1982).

Results and Discussion Stohs et ai,~1974) further examined incorporation of [4-14C-22,23-JH]sitosterol into diosgenin glycosides by cultures from Dioscorea deltoidea when the substrates were added 10 d after subculture and cultures harvested after 30 d. They found incorporation of label from . [22,23-~H]sitosterol (0.41%) was half that from [4-14C~itosterol (0.92%) (com~red with about 7% from [4-'~C]cholesterol and [26-" C]cholesterol under the same conditions). This was consistent with oxygenation at C26 or direct hydroxylation at C22, rather than oxygenation at C22 via a ~-22,23 intermediate. The lower xncor from • p oratxon " [4- 14 C]sitosterol as compared to [4-14C]cholesterol was attributed to dilution of the substrate by the larger pool of endogenous sitosterol, and the need to remove the C24 ethyl group before cyclisation. ~roduction of diosgenin by cultures oenumgraecum has Seen reported by

of Trigonella Brain et al.

In a. preliminary exp@ciment "'[4-14CJcholesterQ1, [26-14C]cholesterol, [4-]4C]sitosterol and [22,23-JH] sitosterol were added at subculture to suspension cultures of Trigonella foenumgraecum on medium containing kinetin and these harvested and extracted after 10 d (Table I). In both cases most of the activity was found in the cell fraction. With cholesterol addition most of this was in bound form, and both [4-14C]cholesterol and [26-14C]cholesterol behaved similarly. In each case about 8% of the activity added was recovered in the diosgenin/yamogenin fraction. Where sitosterol was added, even more of the activity was taken up by the cells, but less of this was in bound form. 1.3% of the activity from [4-14C]sitosterol, ~d 0.6% of the activity from [22,23~C]sitosterol was incorporated into the sapogenin

%

%

2,4- D + kinetin

2,4-D alone 80 ¸

80

~ 0 ..i.u

p

mediumtotal

60"

60"

',4--

E CHOLESTEROL to_ >, •*" 40.

cell bound

0

b 0

~

20

20

~o

0

~

b

2o

3'o

days after addition O

%

~..~

\\~'0.....~.~.~ \

80

%

2A-D alone [22,23-3H_~],..I ~

Evidence for an alternative route from sterol to sapogenin in suspension cultures from Trigonella foenumgraecum.

Incorporation of [4-(14)C]cholesterol, [26-(14)C]-cholesterol, [4-(14)C]sitosterol and [22,23-(3)H]-sitosterol into sapogenin in suspension cultures f...
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