The incorporation of [1-14C]acetate into lipids during embryogenesis in oil palm tissue cultures

Phytochemwy,

Pnntadm Great

Vol 23, No 1, pp 35-39, 1984

0031~9422/84s3oo+ooa 0

Bntam

1984 Pergamon Press Ltd

THE INCORPORATION OF [1-‘4C]ACETATE INTO LIPIDS DURING EMBRYOGENESIS IN OIL PALM TISSUE CULTURES ELAINE TURNHAM* and D H NORTHCOTE Department of Bmchemstry, Umverslty of Cambndge, Cambridge CB2 lQW, U K (Recewed 25 May 1983) Key Word Index-Elaezs

guzneensts, Palmae, 011 palm, trtacylglycerol synthesis, phosphohpld synthesis, cell

dlfferentlatlon

Abstract-A tissue culture line of 011palm produced embryoids and during the embryogenesis large quantities of hpld were stored m the cells The synthesis of the lipid was monitored by measuring mcorporatlon of [1-14C]acetate, under optimum conditions, mto the total hpld and separation by TLC mto neutral and polar hpld Both synthesis of trlacylglycerol and polar hpld increased during embryoid formation A rapid increase m the formation of polar hpld occurred m the penodjust before the embryoids became vtslble and this probably corresponded with the increased rate of cell dlvislon that occurred at that tune

phohplds and tnacylglycerols of embryold cells During a time course of embryogenesis the rate of incorporation of acetate mto both total lipids and tnacylglycerols increased Just before the appearance of embryoids m the cultures

INTRODUCTlON Undifferentiated callus cultures of oil palm dlfferenuate to form hard globular embryoids [l] These embryoids resemble embryos developed w UIUOm their ability to deposit triacylglycerols m oleosomes [Z] The zn ~ztro embryoids may develop root and shoot pnmordia and m some cases plantlet regeneration from somatic embryoids occurs [3] The mcorporatlon of radloactlve precursors into fatty acids zn uzuohas been widely employed m studres of lipid synthesis m both developing 011seeds and m plant tissue cultures Gurr et al [4] showed that, m Crambe abyssznzca seeds, radloactlve acetate was incorporated preferentially mto phosphohplds, but the proportion of radioactivity incorporated mto tnacylglycerol increased as the seed developed Almost all the acetate was incorporated mto the fatty acyl moieties of the lipids, whereas [U14C]glucose was Incorporated mto the glycerol moiety In developing sunflower seeds acetate incorporation mto fatty acids increased rapidly from ten days after flowering durmg the time at which tnacylglycerol accumulation began [5,6] Slmllar results have been obtamed with developmg seeds of soybean [7,8] and Nasturtium [9] Radloactlve acetate 1salso readily incorporated into lipids of plant tissue cultures [10-l 51 Dunng embryogenesls m tissue cultures of 01 palm, an Increase m hpld content of the cells was observed [ 161 In culture lines m which embryogenesis did not occur there was no nse m total lipid Electron microscopic exammatton showed that stored hpld was a spectic product of differentiated embryold cells A rise m the activity of acetyl CoA carboxylase zn uztro was also correlated with the increase m lipid In this work the mcorporatlon of [l14C]acetate mto lipids ln uzuo during a time course of embryogenesls was studied Acetate was incorporated mto phosphohplds of oil palm callus and into phos-

RESULTSAND

DISCUSSION

Optzmzsatzon of zncubatzon condztzonsfor zncorporatzon of [1-14C]acetate znto 011palm lzpzds The optimum pH of the mcubatlon buffer was determined for maximum mcorporatlon of [ 1-14C]acetate A mixture of 011palm callus and chopped embryold tissue was incubated with 10 nmol [1-14C]acetate (21 7 kBq) m buffers at pH between 5 0 and 8 5 for 4 hr at 25” No nonradioactive acetate was included m the mcubatlon mixture The amount of acetate taken up by the cells was determined by sampling the mcubatlon medium before and after mcubatlon (Fig la) The amount of radloactlvlty incorporated mto total hpld (chloroform-methanol soluble material, Fig lb) and into trlacylglycerol separated by TLC (Fig lc) was also measured In these optlmlsatlon experiments duplicate samples were assayed and the results did not deviate from the mean value by more than 5 % The amount of acetate taken up by the tissue decreased with increasing pH Incorporation of radloactlvity mto total hpld increased up to pH 6 5 m MES buffer, but was highest at pH 6 0 m phosphate buffer Acetate mcorporatlon into trlacylglycerols was highest at pH 7 3 m phosphate buffer, although only slight differences m mcorporatlon mto total hpld or trlacylglycerols were observed between pH 6 0 and 7 3 m phosphate buffer Acetate incorporation mto lipids was low at pH 8 &8 5 At pH 6 5 ca 25 % of the radioactivity taken up by the cells was incorporated into total lipid and of this, 44% was present m trlacylglycerols For further experiments 0 7 M phosphate buffer pH 6 5 was used Increasing the concentration of acetate in the external medium resulted m an increase m acetate incorporation mto total hpld up to a

*Present address MRC Bone Research Untt, Nuffield Orthopaedtc Centre, Headtngton, Oxford OX3 7LD, UK 35

36

E TURNHAMand D H NORTHCOTE

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Ag 1 Optmwatlon of mcubatlon condltlons for mcorporatlon of [l-“CJacctate mto llplds m 011 palm cultures Effect of mcubatlon buffer pH on [1-“C]acetate uptake by cells (a), and mcorporatlon mto total bplds (b) and trwylglycerols (c) (O+, 007 M MES buffer, O--O, 007 M sodium phosphate buffer, A-A, 0 07 M Tns-HCI buffer) Effect of acetate concentration in mcubation nurture on rate of incorporation mto total hplds (d) and time course of mcorporatlon (e)

oL5

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of 5 mM acetate (Fig Id) Above substrate conccntratlon this concentration the incorporation began to decline slowly A substrate concentration of 5 mM was therefore used m subsequent expenments A time course of acetate mcorporatlon into total hpld IS shown m Fig le An mcubatlon time of 2 hr was chosen for time course experiments

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Callus or embryold tissues were mcubated separately m 0 07 M phosphate buffer, pH 6 5, containing 10 nmol [l14C1acetate for 4 hr at 25” No non-radloactlve acetate was included m the mcubatlon buffer The extracted hplds were analysed by TLC and the amount of radloactlvlty mcorporated mto the different hpld classes determmed Radloactlve profiles of the chromatograms of callus and embryold lipids run m heptane+ilethyl ether-acetlc acid and chloroform-methanol-water are shown m Figs 2 a-d In callus tissue 82% of the radloactlvlty mcorporated mto hpld remained at the origin when heptaneether-acetic acid was used, this probably represented mcorporatlon mto polar hplds Only 8% of applied radloactlvlty cochromatographed with tnacylglycerols Recovery of radloactlvlty from the TLC plate was When applied 787; of the total radloactlvlty chloroform-methanol-water was used 76% of the apphed radloactlvlty cochromatographed with phosphatidy1 choline and other polar hplds Chromatography, using heptane-ether-acetic acid, of radloactlvely labelled hpld from embryold tissue showed that 40 y0 of the radloactlvlty remained at the ongm and a further 40% cochromatographed with trlacylglycerols When analysed with chloroform-methanol-water, 58 % of the total radloactlvlty cochromatographed wrth neutral hplds Samples of hpld from callus and embryolds were saponified and rechromatographed with heptane-ether-

Llpld synthesis dunng embryogenesls m 011palm cultures

37

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Fig 2 Rad:oactlvlty profiles of thm layer chromatograms of od palm callus and embryold hpld samples (a) Callus, heptane-dlethyl ether-acetlc acid (15 10 l), (b) EmbryoId, TLC solvent as (a), (c) Callus, chloroformmethanol-water (65 25 4), (d) EmbryoId, TLC solvent as (c) The posmons ofmarker hpld samples are mdxated on (b) and (d)

acetic acid (Fig 3a, b) The maJonty of the radloactlvlty ran at the posltlon of free fatty acids The ratlo of radloactlvlty present m the chloroform extract compared to the aqueous phase after sapomficatlon was 11 1 for callus and 135 1 for embryold tissue This indicated that the radloactlvlty from [l- 14C]acetate was incorporated mostly into the fatty acyl chains of the hpld and not into glycerol moletles The recovery of radloactlvlty after sapomficatlon was 94 % (callus) and 116 “/ (embryolds) lncorporatton of [1-14C]acetate into lzplds durmg a tune course of embryogenesls of or1palm tissue culture he JH Callus of hne JH when grown on medium contammg 2 5 mg 3-napthyl acetic acid/l spontaneously dlfferentlated to give rise to embryoids During a time course of growth of 63 days, samples of tissue (0 5 g) were mcubated m 10 ml 0 07 M phosphate buffer pH 6 5 containing 30 pm01 potassmm hydrogen carbonate, 5 pmol sodium acetate and 10 nmol [l- 14C]acetate (21 7 kBq) for 2 hr at

25” Incorporation of radloactlvlty mto total hpld (chloroform-methanol soluble material) and tnacylglycerols was measured (Fig 4) At each time pomt two tissue samples were assayed separately The results did not deviate from the mean value by more than 10% Embryoids were vlslble m the cultures from 35 days Over the time course of 63 days changes m the rate of incorporation of [1-‘4C]acetate into total lipids and triacylglycerols were observed After an initial drop m the rate of acetate mcorporatlon mto both trlacylglycerols and total hpld, there was a rapid increase from day 21 to day 35 There was 3 Cfold increase m mcorporatlon mto total hpld over this penod and the mcorporatlon mto trmcylglycerols increased 5-fold, the mcorporatlon then dechned The peak of mcorporatlon mto tnacylglycerol or total lrpld comclded with the time at which embryoids became vlslble m the cultures Although mcreases m acetate mcorporatlon mto both total hpld and trlacylglycerol occurred, the mcorporatlon mto total hpld always remamed much higher than m-

E TIJRNHAMand D H NORTHC~TE 15

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Fig 3 RadIoactIvIty profiles of thm layer chromatograms of 011palm callus and embryold hpld samples after sapomficatlon (a) Callus, heptanedlethyl etheracetIc acid (15 10 l), (b) Embryoid, TLC solvent as (a) The posmon of a marker fatty acid sample IS Indicated on (b)

tures are sterols, steryl esters and steryl glycosldes [17] Phosphohplds are synthesized at a high rate during periods of rapid cell division [18] Trlacylglycerols are usually present at a level of 5 ‘A or less [ 19,203 During in uztro embryogenesls of 011palm cultures, storage hpld 1s deposited m the embryold cells [2] This deposition of trlacylglycerol was preceded by an increase m the activity of a&y1 CoA carboxylase m the tissue cultures [ 163 The results presented here demonstrate that the incorporation of labelled acetate mto lipids zn uwo increased concomitant with embryold appearance However, the Increase m hpld synthesis was not due entirely to increases m trlacylglycerol synthesis A rapid increase m mcorporatlon of radioactive acetate mto other hpld classes, especially polar hplds, occurred Just before embryoids became visible Tumham and Northcote [ 161 showed that I I an increase m the rate of cell dlvlslon and pnmary wall 0 60 30 40 10 20 50 70 deposltlon occurred, together with the appearance of Time after subculture (days1 embryoids m the tissue cultures and the me in acetate mcorporatlon mto polar lipids shown here may reflect Fig 4 Changes in the rate of mcorporation of [l-14C]acetate increases m cell membrane phosphohpld synthesis during mto total hplds (-0) and trxqlglycerols (O--0) durmg this phase of rapid cell dlvlslon As the embryoids a time course of embryogenesls of 011 palm tissue cultures developed m the tissue cultures and increased m size from EmbryoIds became vlslble m the cultures on day 35 (arrowed) 35 days onwards, hpld synthesis declined but the proportion of radloactlve acetate incorporated mto triacylglycerols compared to other hpld classes increased, probably corporation into triacylglycerol Incorporation of acetate reflecting the contmumg deposition of storage tmcylglyInto other hpld classes e g polar hplds, also occurred m 011 cerol m the embryold cells palm tissue cultures and the mcorporatlon into these lipids increased during the time course However, the proportion of radloactlvlty from [1-14C]acetate mcorEXPERIMENTAL porated into tnacylglycerol compared to other hpld All chemicals used were of A R quahty wherever possible classes varied during the tune course, from 7 y0at day 21 to 11% at day 35, with a further increase to 22 % at day 49, Murashlge and Skoog medmm was obtamed from Flow and 17 y0 at day 63 This reflects an increasing synthesis of Laboratorres, Irvme, Ayrshlre, Scotland Agar Noble and casammo acids were from Difco Laboratones, Detroit, MI, tIlacylglycero1 compared to other hplds as embryogenesls USA [l-%JAcetx acid, sodmm salt (sp radmactlvlty occurred 2 17 GBq/mmol) was purchased from Amersham Internatlonal, The promment hplds of most heterotrophlc cell culI

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Lipid synthesis durmg embryogenesu m 011palm cultures Amersham, Bucks, U K Growth of od palm nssue cultures Tissue cultures of 011palm (Elaezs 9umeensts Jacq ) line JH were obtamed from Umlever Research, Shambrook, Bedford, U K The cultures were mamtamed on half-strength Murashlge and Skoog medium [21] with the addmon of 1 g casammoaclds/l,25%sucroseand25mg3napthylacetlc acid/l The medium was sobdmed by the addmon of 1% Agar Noble The cultures were mamtamed at 25” and dlummated at a light mtensny of 100 lux Hrlth an 18 hr photopermd They were routinely subcultured every 8-10 weeks All mampulatlons were carned out on a sterile lammar an-flow bench Media and mstruments were stenhzed by autoclavmg at 120” and 103 kPa for 20nun Under these culture condmons, cultures of 011 palm lme JH ddferentlated spontaneously to produce small hard globular embryolds Incubation oftwsue cultures wtth [ 1-‘%Z]ocetate Samples of 011 palm cultures were incubated with [I-‘*C]acetate m VIUO, followed by extraction and analysu of lipids The optnnum condmons for the mcorporatlon of acetate mto lipids were determined The standard mcubatlon mature contamed 007 mm01 NaPl buffer, pH 6 5, 30~01 KHCO,, 5 pm01 NaOAc and 10 nmol [l-‘*C]acetate (217 kBq) m a total vol of 10 ml Tissue (co 500 mg) was added and the samples incubated at 25” for up to 6 hr At the end of the mcubatlon the tissue was removed and the mcubatlon medium sampled to determme the amount of radioactivity remauung The tissue was washed wnh buffer and hplds extracted L~pld extractIon and analysts At the end of the mcubatlon, the tusue was homogenized m propan-2-01 at 80” (20 ml/g tissue) to mhlbn hpase activity After filtration the residue was extracted overnight at 4” with CHCl,-MeOH (3 2) (20 ml/g tusue) and then refiltered The propanol extract was rotary evaporated to dryness, taken up m CHCl,-MeOH (3 2) and recombined with the first CHCl,-MeOH (3 2) extract The extracted lipids were punfied by washing by the method of Folch et al [22], rotary evaporated to dryness and taken up m a small vol of CHCI, Samples of the extracted hplds were saponified The CHCI, solvent was removed by evaporation and the hplds redusolved m 10 % KOH m 90 % EtOH followed by mcubauon at 75”for 2 hr The samples were acldnied to pH 10 usmg HCI and free fatty acids extracted mto CHCl, The CHCI, extract was washed with an equal vol of 1 y0 HOAc plus 1 y0 malomc acid Samples of sapom6ed or unsapom&d bpids were analysed by TLC on s&a gel plates m heptane-Et+HOAc (15 10 1) or in CHCl,-MeOH-Hz0 (65 25 4) Both solvent systems contamed 0 005 % butylated hydroxytoluene to prevent lipid oxutanon Lipids were vrsuahxed by exposure of the an-dned plates to I2

39

vapour Llpld markers were run m parallel with the samples The TLC plates were cut mto 1 cm stnps and radmactlvlty determined m the strops using a Searle Mark III bqmd scmtdlatlon counter, after unmerslon of the stnps m scmtdlatlon fluid (15 I toluene, 0 75 I Tnton X-100,6 g 2,5_dlphenyloxaxole) Acknowledgements-We gratefully acknowledge the SERC and Undever for CASE award and also Dr L Jones, Undever, for supplymg the tissue culture

REFERENCES 1 Jones, L H (1974) 011 Palm News 17, 1 2 Jones, L H (1974) m Industrtal Aspects of Bmchemzstry (Spencer, B, ed) pp 813-833 Proceedmgs of FEBS Specutl Meeting, Dublin, 1973, North Holland, Amsterdam 3 Corley, R H V, Barrett, J N and Jones, L H (1978) 011 Palm News 22,2 4 GUT, M I, Blades, J , Appleby, R S , Smith, C G , Robmson, M P and Nichols, B W (1974) Eur J Bwchem 43,281 5 McMahon, V and Stumpf, P K (1966) Plant Phys~ol 11,

148 6 Ichdtara, K and Noda, M (1980) PhytochemIstry 19,49 7 Rmne, R W and Canvm, D T (1971) Plant Cell Physrol 12, 387 8 Porra, R J and Stumpf, P K (1976) Arch BIochem B~ophys 176, 53 9 Pollard, M R and Stumpf, P K (1980) Plant Phys~ol 66, 649

10 Fletcher, J S and Beevers, H (1970) Plant Phys~ol 45,765 11 Steams, E M Jr and Morton, W T (1975) LIpIds 10, 597 12 Stumpf, P K and We&, N (1977) LIP& 12, 120 13 Wdson, A C and Kates, M (1978) Lzpufs 13, 504 14 McCarthy, J J and Stumpf, P K (1980) Planta 147, 384 15 Tsar, C H and Kmsella, J E (1982) LIP& 17,367 16 Turnham, E and Northcote, D H (1982) Btochem J 208, 323 17 Radwan, S S (1975) Fette, Selfen, Anstnchm 77, 181 18 Song, M and Tattne, T H (1973) Can J Botany 51,1893 19 Radwan, S S , Spener, F, Mangold, H K and Staba, E J (1975) Chem Phys LIP& 14,72 20 Shin, B S, Mangold, H K and Staba, E J (1971) m L.es Cultures de TISSUSde Plantes Colloque No 193, Pans,

CNRS 21 Murashlge, T and Skoog, F (1962) Phystol Plant 15,473 22 Folch, J, Lees, M and Sloane-Stanley, S H (1957) J BIO~ Chem 226,497