224
Biochimica et Bdophysica Acta, 398 (1975) 224-230 @ Elsevier Scientific Publishing ~~rnpan3~, Amsterdam
BBA
Printed
in The ~~therIands
56646
SYNTHESIS OF SULPHOQUINOVOSYL SIGHER PLANTS
JOHN
-
DIACYLGLYCEROL
BY
L. HARWOOD
Department (Received
of Biochemistry, February
University
College, P.O. Box 78, Cardiff CFl
1X1, (U.K.1
7th, 1975)
Summary The biosynthesis of sulphoquinovosyl diacylglycerol in germinating alfalfa seeds has been examined. Some incorporation of [ ’ ‘S] sulphate into the lipid occurs before chlorophyll production and this is unaffected by chloramphenicol. Cysteic acid, molybdate, sulphite and sulpholactic acid all reduce incorporation of 1 .i ‘S] sulphate into sulphoquinovosyl diacylglycerol. Some comparisons are made with other seed types. The results indicate that sulphoquinovosyl diacylglycerol synthesis in alfalfa probably proceeds by a pathway similar to that in Er@etla.
Introduction The plant sulpholipid, more correctly called sulphoquinovosyl diacylglycer01, was discovered by Benson and co-workers [ I] when ’ ‘SO:- was rapidly incorporated into it. Daniel et al. [2] showed that the structure was that of 6-deoxy-6-sulphoa-D -glucopyranosyl diacylglycerol. Since that date, sulphoquinovosyl diacylglycerol has been found in all green plants examined, and it contains high amounts 6 palmitic, anoleic and linolenic acids [3,4f. Sulphoqu~novosyf diacylglycerol is primarily Iocalised in chloroplasts [ 5,6] and correlations have been found between chlorophyll and sulpholipid content in higher plants [7,8]. The phytoflagellate, Ochromonas danica, contains sulphoquinovosyl diacylglycerol when photosynthesising but not when it is grown in a sucrose medium (91. These results have been interpreted [6] to imply a direct role for sulphoqu~ovosyl diacylglycerol in photosyntllesis and Weier and Benson [IO] have suggested that the plant sulpholipid may help to orientate chlorophyll within the chloroplast membranes. Despite its widespread occurrence, little is known of the biosynthesis of sulphoquinovosyl diacylglycerol. Benson and Shibuya [ll] obtained evidence for the occurrence of small amounts of sulpholactaldehyde, sulpholactate and
225
sulphopropanediol in Chlorella and Davies et al. [12] showed that molybdate and cysteic acid reduced the incorporation of ’ ‘SO, ‘- into sulphoquinovosyl diacylglycerol in Euglena. These results led to proposal of a pathway for 6sulphoquinovose synthesis [ 121. Other alternatives for the origin of 6-sulphoquinovose have been put forward [6,13--151 but no more direct evidence has been obtained. Results are now reported on the synthesis of sulphoquinovosyl diacylglycerol in higher plants which confirm and extend the data obtained by Davies et al. 1121 using Euglena. Materials
and Methods
Chemicals. [ ” S] Sulphate (carrier-free) was purchased from t,he Radiochemical Centre, Amersham, U.K. Chloramphenicol was from Sigma and Lcysteic acid from B.D.H. Chemicals Ltd, Poole, U.K. All other reagents used were of analar grade. Sulphoquinovosyl diacylglycerol was prepared from alfalfa var. Caliverde by the method of O’Brien and Benson [ 161. Sulpholactic acid was prepared from cysteic acid by HNOl treatment. Anions were exchanged for sulphate using Amberlite IRA-400 (B.D.H. Chemicals Ltd) and sulphate removed with Ba(OH), . The purity of the compound and the absence of cysteic acid was checked by thin-layer chromatography (see below), infrared spectroscopy using a Perkin-Elmer 257 spectrophotometer and mass spectroscopy using a Varian CH5D mass spectrometer on line with a Varian 620i computer. [ ’ ‘S] Cysteic acid was prepared from ’ ‘S-labeled protein (alfalfa) by performic acid oxidation [ 171. Following protein hydrolysis with HCl, [ ’ 'S] cysteic acid was purified by chromatography on Dowex l-X8, 400 mesh (Sigma). Seeds. Alfalfa (Medicago satiua var. Caliverde and Sabilt) seeds were a generous gift from Dr W. Ellis Davies, Welsh Plant Breeding Station, Plas Gogerddan, Aberystwyth, U.K. Brussels sprout seeds (Brassica oleracea var. gemmifera (cv Exhibition)) were purchased from Samuel Dobie and Sons Ltd, Llangollen, U.K. and perennial rye grass seeds (Lolium perenne) from Noah Rees and Griffins, Working Street, Cardiff, U.K. Germination. Germination was carried out at room temperature with surface-sterilised seeds using autoclaved distilled water in sterile test tubes. .’ ‘SO, *- was added at appropriate times. Normally 15 alfalfa var. Sabilt or brussels sprout seeds and 10 rye grass seeds were used per 0.5 ml of water. Extraction and identificatiorz of sulpholipid. To terminate germination, seeds were rinsed in several changes of distilled water and then rapidly ground in 1 ml of distilled water using a pestle and mortar at 1°C. 3.75 ml of chloroform/methanol (1 : 2, v/v) were then added and the mixture left at room temperature for at least 1 h. 1.25 ml of chloroform and 1.25 ml of 2 M KC1 in 0.5 M phosphate buffer, pEI 7.4, were then added, the extracts shaken and allowed to separate, This is essentially the method of Garbus et al. 1181 which was found to give a quantitative extraction of sulphoquinovosyl diacylglycerol into the lower phase. The resultant lower phase was washed with fresh upper phase and samples taken for counting and chromatography. Tests showed that
226
98 i 1% of radioactive sulphoquinovosyl diacylglycerol were recovered with this procedure with no contamination by other ’ “S-labeled compounds. The .’ ’ S-labeled lipid co-chromato~aplled with authentic sulphoquinovosyl diacylglycerol in a number of solvent systems. No [’ ‘S] sulphate was liberated on acid hydrolysis indicating the absence of that group in the lipid. After mild alkaline hydrolysis, the ’ ‘S-labeled component co-chromatographed with 6sulphoquinovosyl glycerol. The labelled lipid was, therefore, assumed to be sulphoquinovosyl diacylglycerol. ~~l~~~~~og~~~~~~. Sulphoquinovosy~ diacylglyeerol (RF 0.28) was separated from other lipids by chromatography on silica gel G (E. Merck, Darmstadt, Germany) plates using chloroform/methanol/glacial acetic acid/water (170 : 30 : 20 : 7, by vol.). For purity checks solvent systems containing chloroform/methanol/water (65 : 25 : 4, by vol.) and chloroform/methanol~a~etone/water (65 : 35 : 4 : 4, by vol.) were also used. Lipids were visualised using iodine vapour or 0.001% aqueous Rhodamine 6G spray. Plates containing radioactive lipid were also scanned using a Packard model 7201 Radiochromatogram scanner and examined with a Radiochromatogram Spark Chamber (Birchover Instruments Ltd, Hitchin, Herts, U.K.). When necessary, sulphoquinovosyl diacylglycerol was eluted quantitatively from the plates using ~hloroform/methanol/acetic acid (200 : 100 : 1, by vol.) followed by chloroform/methanol in the proportions (2 : 1, v/v) and then (1 : 1, v/v). [ 3 ’ S ] Cysteic acid, [ ’ ‘S] sulpholactic acid and other aqueous-soluble J ’ S-labelled compounds were separated on Cellulose CC41 powder (Whatman) thin-layer chromatography plates using methanol or diethyl ether/formic acid/water (7 : 2 : 1, by vol.) as solvent. R, values for cysteic and sulpholactic acids in the first solvent were 0.48 and 0.63 and in the second 0.12 and 0.45, respectively . Scintillation counting. Samples were routinely counted in a scintillant consisting of PCS (Amersham-Searle)/water (6 : 4, v/v). This scintillant was >92% efficient with both aqueous and lipid samples. Little quenching was noted during direct counting of silica gel provided that the amount of gel was not more than 2.5 m&ml of scintillant. A Beckman LS-100 counter was used. Quench corrections were made by the channels ratio method. Results and Discussion Time course of l~beliing. The time courses of labelling for two plants, rye grass (1;. perenne) and alfalfa (I?!. sat&a var. Sabilt) are shown in Fig. 1. In both cases, there appear to be two increases in the rate of sulphoquinovosyl diacylglycerol synthesis. The first results in a small amount of synthesis corresponding to root and shoot emergence but before leaf greening takes place. The second is synchronous with chlorophyll synthesis. In each case the increase in sulphoquinovosy1 diacylglycerot formation is preceded by enhanced uptake of water (and [ ’ iiS] sulphate). The effect of chloramphenicol, which effectively prevents chloroplast protein synthesis [ 191 is shown in Table 1. It has little effect on the early synthesis of sulphoquinovosyl diacylglycerol but begins to cause more inhibition at later stages of germination.
227
2
3
4
5
6 7 Germination
0 (days)
9
10
Fig. 1. Time copse of [ 35Slsulphate uptake and sulphoquinovosyl diacylglycerol labelling. Germination, extraction and separation were carried out as described under Materials and Methods. -, sulphoquinovosyl d:scylglycerol labelling and .- - - - - -1:. sulphate uptake from alfalfa seeds. O---O, sulphoquinovosyl diacylglycerol labelling and n - - - - - -a. sulphate uptake from rye grass seeds.
Cysteic acid as a precursor. [ ’ ‘S] Cysteic acid was incorporated into sulphoquinovosyl diacylglycerol by germinating alfalfa seeds with a higher efficiency than [ 3 ‘S] sulphate. In addition, unlabelled cysteic acid, when added to the germination medium, decreased incorporation of [ 3 ‘S] sulphate into sulphoquinovosyl diacylglycerol of alfalfa and brussels sprout seeds. The results were very similar to data obtained with Euglena [12] and show that cysteic acid can also serve as a precursor of the sulpholipid in higher plants. Effect of molybdate on incorporation of [” ’ S] sulphate into lipid. Inclusion of molybdate in the germination medium dramatically lowered incorporation of [ 3 ‘S] sulphate into alfalfa lipid. It also resulted in an inhibition of sulphate uptake. However, the percentage of incorporated 3 ‘S in sulphoquinovosyl diacylglycerol was lowered some 4096, a similar result to that obtained with Euglena [ 121 . Since molybdate inhibits ATP sulphurylase in a TABLE
I
SYNTHESIS PHENICOL Figures
OF SULPHOQUINOVOSYL
are means 2 S.D. (three
Seed
Chloramphenicol
Alfalfa
DIACYLGLYCEROL
IN THE
35S-labelled
0 50 50 40 50
*
100 115 ? 16 109 ? 21 58+ 7 74* 12
0 40
10 10
* Controls
(mg/ml)
4 5 8 8
were carried
OF CHLORAM-
determinations). Germ. time (days) .--
Brussels sprout
PRESENCE
out at each germination
100 66-+ time.
6
lipid (dpm)
(percent
of control)
.,\I 1:,1 fa
I-, ‘,C) _ti,
was in lipid with 0.1 mM sulphite plus 0.1 mM su!phate than with 1 mhl sulphate (Table II). These results, therefore, lend some support for the pathna!, suggested by Haines [6] though demonstration of the enzyme involved must tit’ made before sulphite can be included as a definite participant. Effect of sulpholactic acid. Sulpholactic acid is a postulated intermediat.e in the conversion of sulphopyruvate to sulphoquinovose. It has been found in small amounts in Chlorella [ll] . [’ “S] Sulpholactic acid prepared I’rc~m [ A‘S] cysteic acid was taken up by germinating alfalfa and brussels sprout SCY~C!S and t,he effect of addition of unlabelled sulpholactic acid was therefore testracl. The results are shown in Table III. In both seed types inhibition of sulphoq~i;novosyl diacylglycerol labelling from [ .’ 5S] sulphate was found. In brus~t~l~ sprout, sulphate uptake was similarly reduced. These results would agree \tlitIi ;I proposal for sulpholactic acid as an intermediate in sulphoquinovosgl di;icylglycerol synthesis in alfalfa. Conclusions
The results described above have confirmed that higher plants show similar properties to Euglena for the biosynthesis of sulphoquinovosyl diacylglyccar01 from sulphate. These indicate the involvement of 3’-phosphoadenosinr 5 ‘phosphosulphate and cysteic acid and, in addition, sulphite and sulpholactic acid. Direct demonstration of individual enzyme steps will now have to btb made in order to determine the exact contribution of each substance and suc,h experiments are now in progress. Acknowledgements
The author would like to thank Drs J. Kay and A. Olavrsen of this department for help with isolation of [“S] cysteic acid and with infrared spectroscopy, respectively. Dr N. Evans of the Chemistry Department kindly carried out the mass spectrometry. Mrs M.E. Fletcher provided skilled technic4 assistance. References 1
Benson.
2
Daniel,
A..4., H..
Am. Chem. 3
Tutloch.
4
Kates,
Daniel.
Mivno, Sot.
A.P..
5
Davies,
W.H.,
Hainrs.
T.H.
Acadcmlr Rosrnlwr~.
8
Thomas.
1j.R.
9
Miyachi.
S.,
Weivr,
11
Benson.
A.A.
Davies,
W.H.,
13
Lvhmann,
14
Schiff,
K.1.
(1 973)
Llpids
Res.
and
Prcker,
and
Yari,
W. 8,
Proc. I’..
Natl.
Acad.
Lrpagr,
Sci.
M..
t1.S.
Shlbuva.
45.
1582
I. and
(1973)
Hoppe-Srylrr’s
T.W.
(lHG5)
Blomembrancs
M. (1 9G4) A.K.
S. and
Benson.
and
1587
Bt~nu~n.
A..\.
(I :Jlil ) .1.
%. Phvsiol.
Chc.m.
J’hytochen~istrr
IIf
Eukarvotic
Shibuya.
Mrrcer,
E.J.
Bcwwn, Hodson,
Biochemistrv
(1970)
J.
3.
Exp.
Bat.
4,
741
274~285
A.A.
(1 OGG) .I. Prc~to/ool.
(1967)
Am.
.J. lb>t.
I. (19Gl)
Iced.
and A.A. R.C.
Cioodwin. (1964) (1973)
879
XX!1
~749
Mlrruorranianrs
254---25&l G7,
Brnson,
A.A.
-354.
225-265
Croodwin. and
Stobart.
Miyachi,
and
(1 959)
London
.J. and J.A.
R. K.O.,
Fischer,
Lipid
Mercrr,
and
12
E. and
Adv.
A. and
T.E.
Wrsw.
Mumma.
1765~~17tiG
Hein/.
Press,
7
IO
R3.
M. (1970)
(i
H. and M..
J.
Pror.
54. 20.
3X9
(1 !lGG)
Am.
Chrm.
Rrv.
7(ip78
79
‘J’.W. Ann.
13,
402
Plant
Biorhcm. Sot.
83.
Phvsiol.
.J. 98.
3li!J
I57 24.
381~413
373
(I’:r\\ln.
.t..\..
Biochimica et Bdophysica Acta, 398 (1975) 224-230 @ Elsevier Scientific Publishing ~~rnpan3~, Amsterdam
BBA
Printed
in The ~~therIands
56646
SYNTHESIS OF SULPHOQUINOVOSYL SIGHER PLANTS
JOHN
-
DIACYLGLYCEROL
BY
L. HARWOOD
Department (Received
of Biochemistry, February
University
College, P.O. Box 78, Cardiff CFl
1X1, (U.K.1
7th, 1975)
Summary The biosynthesis of sulphoquinovosyl diacylglycerol in germinating alfalfa seeds has been examined. Some incorporation of [ ’ ‘S] sulphate into the lipid occurs before chlorophyll production and this is unaffected by chloramphenicol. Cysteic acid, molybdate, sulphite and sulpholactic acid all reduce incorporation of 1 .i ‘S] sulphate into sulphoquinovosyl diacylglycerol. Some comparisons are made with other seed types. The results indicate that sulphoquinovosyl diacylglycerol synthesis in alfalfa probably proceeds by a pathway similar to that in Er@etla.
Introduction The plant sulpholipid, more correctly called sulphoquinovosyl diacylglycer01, was discovered by Benson and co-workers [ I] when ’ ‘SO:- was rapidly incorporated into it. Daniel et al. [2] showed that the structure was that of 6-deoxy-6-sulphoa-D -glucopyranosyl diacylglycerol. Since that date, sulphoquinovosyl diacylglycerol has been found in all green plants examined, and it contains high amounts 6 palmitic, anoleic and linolenic acids [3,4f. Sulphoqu~novosyf diacylglycerol is primarily Iocalised in chloroplasts [ 5,6] and correlations have been found between chlorophyll and sulpholipid content in higher plants [7,8]. The phytoflagellate, Ochromonas danica, contains sulphoquinovosyl diacylglycerol when photosynthesising but not when it is grown in a sucrose medium (91. These results have been interpreted [6] to imply a direct role for sulphoqu~ovosyl diacylglycerol in photosyntllesis and Weier and Benson [IO] have suggested that the plant sulpholipid may help to orientate chlorophyll within the chloroplast membranes. Despite its widespread occurrence, little is known of the biosynthesis of sulphoquinovosyl diacylglycerol. Benson and Shibuya [ll] obtained evidence for the occurrence of small amounts of sulpholactaldehyde, sulpholactate and
225
sulphopropanediol in Chlorella and Davies et al. [12] showed that molybdate and cysteic acid reduced the incorporation of ’ ‘SO, ‘- into sulphoquinovosyl diacylglycerol in Euglena. These results led to proposal of a pathway for 6sulphoquinovose synthesis [ 121. Other alternatives for the origin of 6-sulphoquinovose have been put forward [6,13--151 but no more direct evidence has been obtained. Results are now reported on the synthesis of sulphoquinovosyl diacylglycerol in higher plants which confirm and extend the data obtained by Davies et al. 1121 using Euglena. Materials
and Methods
Chemicals. [ ” S] Sulphate (carrier-free) was purchased from t,he Radiochemical Centre, Amersham, U.K. Chloramphenicol was from Sigma and Lcysteic acid from B.D.H. Chemicals Ltd, Poole, U.K. All other reagents used were of analar grade. Sulphoquinovosyl diacylglycerol was prepared from alfalfa var. Caliverde by the method of O’Brien and Benson [ 161. Sulpholactic acid was prepared from cysteic acid by HNOl treatment. Anions were exchanged for sulphate using Amberlite IRA-400 (B.D.H. Chemicals Ltd) and sulphate removed with Ba(OH), . The purity of the compound and the absence of cysteic acid was checked by thin-layer chromatography (see below), infrared spectroscopy using a Perkin-Elmer 257 spectrophotometer and mass spectroscopy using a Varian CH5D mass spectrometer on line with a Varian 620i computer. [ ’ ‘S] Cysteic acid was prepared from ’ ‘S-labeled protein (alfalfa) by performic acid oxidation [ 171. Following protein hydrolysis with HCl, [ ’ 'S] cysteic acid was purified by chromatography on Dowex l-X8, 400 mesh (Sigma). Seeds. Alfalfa (Medicago satiua var. Caliverde and Sabilt) seeds were a generous gift from Dr W. Ellis Davies, Welsh Plant Breeding Station, Plas Gogerddan, Aberystwyth, U.K. Brussels sprout seeds (Brassica oleracea var. gemmifera (cv Exhibition)) were purchased from Samuel Dobie and Sons Ltd, Llangollen, U.K. and perennial rye grass seeds (Lolium perenne) from Noah Rees and Griffins, Working Street, Cardiff, U.K. Germination. Germination was carried out at room temperature with surface-sterilised seeds using autoclaved distilled water in sterile test tubes. .’ ‘SO, *- was added at appropriate times. Normally 15 alfalfa var. Sabilt or brussels sprout seeds and 10 rye grass seeds were used per 0.5 ml of water. Extraction and identificatiorz of sulpholipid. To terminate germination, seeds were rinsed in several changes of distilled water and then rapidly ground in 1 ml of distilled water using a pestle and mortar at 1°C. 3.75 ml of chloroform/methanol (1 : 2, v/v) were then added and the mixture left at room temperature for at least 1 h. 1.25 ml of chloroform and 1.25 ml of 2 M KC1 in 0.5 M phosphate buffer, pEI 7.4, were then added, the extracts shaken and allowed to separate, This is essentially the method of Garbus et al. 1181 which was found to give a quantitative extraction of sulphoquinovosyl diacylglycerol into the lower phase. The resultant lower phase was washed with fresh upper phase and samples taken for counting and chromatography. Tests showed that
226
98 i 1% of radioactive sulphoquinovosyl diacylglycerol were recovered with this procedure with no contamination by other ’ “S-labeled compounds. The .’ ’ S-labeled lipid co-chromato~aplled with authentic sulphoquinovosyl diacylglycerol in a number of solvent systems. No [’ ‘S] sulphate was liberated on acid hydrolysis indicating the absence of that group in the lipid. After mild alkaline hydrolysis, the ’ ‘S-labeled component co-chromatographed with 6sulphoquinovosyl glycerol. The labelled lipid was, therefore, assumed to be sulphoquinovosyl diacylglycerol. ~~l~~~~~og~~~~~~. Sulphoquinovosy~ diacylglyeerol (RF 0.28) was separated from other lipids by chromatography on silica gel G (E. Merck, Darmstadt, Germany) plates using chloroform/methanol/glacial acetic acid/water (170 : 30 : 20 : 7, by vol.). For purity checks solvent systems containing chloroform/methanol/water (65 : 25 : 4, by vol.) and chloroform/methanol~a~etone/water (65 : 35 : 4 : 4, by vol.) were also used. Lipids were visualised using iodine vapour or 0.001% aqueous Rhodamine 6G spray. Plates containing radioactive lipid were also scanned using a Packard model 7201 Radiochromatogram scanner and examined with a Radiochromatogram Spark Chamber (Birchover Instruments Ltd, Hitchin, Herts, U.K.). When necessary, sulphoquinovosyl diacylglycerol was eluted quantitatively from the plates using ~hloroform/methanol/acetic acid (200 : 100 : 1, by vol.) followed by chloroform/methanol in the proportions (2 : 1, v/v) and then (1 : 1, v/v). [ 3 ’ S ] Cysteic acid, [ ’ ‘S] sulpholactic acid and other aqueous-soluble J ’ S-labelled compounds were separated on Cellulose CC41 powder (Whatman) thin-layer chromatography plates using methanol or diethyl ether/formic acid/water (7 : 2 : 1, by vol.) as solvent. R, values for cysteic and sulpholactic acids in the first solvent were 0.48 and 0.63 and in the second 0.12 and 0.45, respectively . Scintillation counting. Samples were routinely counted in a scintillant consisting of PCS (Amersham-Searle)/water (6 : 4, v/v). This scintillant was >92% efficient with both aqueous and lipid samples. Little quenching was noted during direct counting of silica gel provided that the amount of gel was not more than 2.5 m&ml of scintillant. A Beckman LS-100 counter was used. Quench corrections were made by the channels ratio method. Results and Discussion Time course of l~beliing. The time courses of labelling for two plants, rye grass (1;. perenne) and alfalfa (I?!. sat&a var. Sabilt) are shown in Fig. 1. In both cases, there appear to be two increases in the rate of sulphoquinovosyl diacylglycerol synthesis. The first results in a small amount of synthesis corresponding to root and shoot emergence but before leaf greening takes place. The second is synchronous with chlorophyll synthesis. In each case the increase in sulphoquinovosy1 diacylglycerot formation is preceded by enhanced uptake of water (and [ ’ iiS] sulphate). The effect of chloramphenicol, which effectively prevents chloroplast protein synthesis [ 191 is shown in Table 1. It has little effect on the early synthesis of sulphoquinovosyl diacylglycerol but begins to cause more inhibition at later stages of germination.
227
2
3
4
5
6 7 Germination
0 (days)
9
10
Fig. 1. Time copse of [ 35Slsulphate uptake and sulphoquinovosyl diacylglycerol labelling. Germination, extraction and separation were carried out as described under Materials and Methods. -, sulphoquinovosyl d:scylglycerol labelling and .- - - - - -1:. sulphate uptake from alfalfa seeds. O---O, sulphoquinovosyl diacylglycerol labelling and n - - - - - -a. sulphate uptake from rye grass seeds.
Cysteic acid as a precursor. [ ’ ‘S] Cysteic acid was incorporated into sulphoquinovosyl diacylglycerol by germinating alfalfa seeds with a higher efficiency than [ 3 ‘S] sulphate. In addition, unlabelled cysteic acid, when added to the germination medium, decreased incorporation of [ 3 ‘S] sulphate into sulphoquinovosyl diacylglycerol of alfalfa and brussels sprout seeds. The results were very similar to data obtained with Euglena [12] and show that cysteic acid can also serve as a precursor of the sulpholipid in higher plants. Effect of molybdate on incorporation of [” ’ S] sulphate into lipid. Inclusion of molybdate in the germination medium dramatically lowered incorporation of [ 3 ‘S] sulphate into alfalfa lipid. It also resulted in an inhibition of sulphate uptake. However, the percentage of incorporated 3 ‘S in sulphoquinovosyl diacylglycerol was lowered some 4096, a similar result to that obtained with Euglena [ 121 . Since molybdate inhibits ATP sulphurylase in a TABLE
I
SYNTHESIS PHENICOL Figures
OF SULPHOQUINOVOSYL
are means 2 S.D. (three
Seed
Chloramphenicol
Alfalfa
DIACYLGLYCEROL
IN THE
35S-labelled
0 50 50 40 50
*
100 115 ? 16 109 ? 21 58+ 7 74* 12
0 40
10 10
* Controls
(mg/ml)
4 5 8 8
were carried
OF CHLORAM-
determinations). Germ. time (days) .--
Brussels sprout
PRESENCE
out at each germination
100 66-+ time.
6
lipid (dpm)
(percent
of control)
.,\I 1:,1 fa
I-, ‘,C) _ti,
was in lipid with 0.1 mM sulphite plus 0.1 mM su!phate than with 1 mhl sulphate (Table II). These results, therefore, lend some support for the pathna!, suggested by Haines [6] though demonstration of the enzyme involved must tit’ made before sulphite can be included as a definite participant. Effect of sulpholactic acid. Sulpholactic acid is a postulated intermediat.e in the conversion of sulphopyruvate to sulphoquinovose. It has been found in small amounts in Chlorella [ll] . [’ “S] Sulpholactic acid prepared I’rc~m [ A‘S] cysteic acid was taken up by germinating alfalfa and brussels sprout SCY~C!S and t,he effect of addition of unlabelled sulpholactic acid was therefore testracl. The results are shown in Table III. In both seed types inhibition of sulphoq~i;novosyl diacylglycerol labelling from [ .’ 5S] sulphate was found. In brus~t~l~ sprout, sulphate uptake was similarly reduced. These results would agree \tlitIi ;I proposal for sulpholactic acid as an intermediate in sulphoquinovosgl di;icylglycerol synthesis in alfalfa. Conclusions
The results described above have confirmed that higher plants show similar properties to Euglena for the biosynthesis of sulphoquinovosyl diacylglyccar01 from sulphate. These indicate the involvement of 3’-phosphoadenosinr 5 ‘phosphosulphate and cysteic acid and, in addition, sulphite and sulpholactic acid. Direct demonstration of individual enzyme steps will now have to btb made in order to determine the exact contribution of each substance and suc,h experiments are now in progress. Acknowledgements
The author would like to thank Drs J. Kay and A. Olavrsen of this department for help with isolation of [“S] cysteic acid and with infrared spectroscopy, respectively. Dr N. Evans of the Chemistry Department kindly carried out the mass spectrometry. Mrs M.E. Fletcher provided skilled technic4 assistance. References 1
Benson.
2
Daniel,
A..4., H..
Am. Chem. 3
Tutloch.
4
Kates,
Daniel.
Mivno, Sot.
A.P..
5
Davies,
W.H.,
Hainrs.
T.H.
Acadcmlr Rosrnlwr~.
8
Thomas.
1j.R.
9
Miyachi.
S.,
Weivr,
11
Benson.
A.A.
Davies,
W.H.,
13
Lvhmann,
14
Schiff,
K.1.
(1 973)
Llpids
Res.
and
Prcker,
and
Yari,
W. 8,
Proc. I’..
Natl.
Acad.
Lrpagr,
Sci.
M..
t1.S.
Shlbuva.
45.
1582
I. and
(1973)
Hoppe-Srylrr’s
T.W.
(lHG5)
Blomembrancs
M. (1 9G4) A.K.
S. and
Benson.
and
1587
Bt~nu~n.
A..\.
(I :Jlil ) .1.
%. Phvsiol.
Chc.m.
J’hytochen~istrr
IIf
Eukarvotic
Shibuya.
Mrrcer,
E.J.
Bcwwn, Hodson,
Biochemistrv
(1970)
J.
3.
Exp.
Bat.
4,
741
274~285
A.A.
(1 OGG) .I. Prc~to/ool.
(1967)
Am.
.J. lb>t.
I. (19Gl)
Iced.
and A.A. R.C.
Cioodwin. (1964) (1973)
879
XX!1
~749
Mlrruorranianrs
254---25&l G7,
Brnson,
A.A.
-354.
225-265
Croodwin. and
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Miyachi,
and
(1 959)
London
.J. and J.A.
R. K.O.,
Fischer,
Lipid
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and
12
E. and
Adv.
A. and
T.E.
Wrsw.
Mumma.
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Press,
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M. (1970)
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J.
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