INCOKBOKATION OF C14 OR Ba INTO THE BHOSPIIAkTI%)ES OF RIJNNER BEAN LEAVES'

Abstract 'The phosphatide content of primary leaves of runner bean increased li~learly

with time, between the 8th ant1 20th day of devcloprnent, a t a rate proportional to the growth of the leaves.

Pletached leaves incorporated C1<rlto the total lipids (pigments, 11011- phosphatidcs, and phosphntit-les) when supplied with C'140? in the light for 1 minute followed by a 30-minute period in the light or dark in tracer-free air and when supplied with pyruvate-2-C14 or acetate-l-C-.I4 in the light. WLXS

incorporatert into the phosphatides after orthophosphate-P" was supp1;ed both in the light and dark. \Vith each precursor, radioisotope was incorporated into the four phosphatide fractions obtairietl hy chro~natography of the total lipids: inositol-carbohydrate phosphatides (Fraction I ) , phosphatidyl ethanolami~re - phosphatidyl seri~le (Fraction 111, an unkraourn phosphztide (Fraction 111), and lecithin (Fr;iction IV).

Distribution of radioactivity among these phosphatide fractions arid among the phosphatide nloicties varied greatly with the precursor supplied. Thus, with C:lIO.I as prec~lrsor~ Fractio~i I and I11 hati the highest specific activities, nrld C'+elitered both the water-solttble xrloieties arld the f , ~ t t y acids in a11 of the phosphatitfe f~;ictions. \;Vith pyruvate-C1"or acetate-CM, lecithin was rmost highly labellect, and C14 was found almost exc111sivt:ly in the fatty acids of the 1,hqsphatidc.s. iVith orthophosphate-P3", Fraction I11 had the highest specific ;ictivitv. The bearing of these filldings on the nletabolism of phosphatides in leaves is discnssed.

Introduction

Iia the precediiag paper (7) , methocls were describetl for the extractioil of leaf lipids ailcl their separation by chrom:ttography into a non-phosphatide fraction (i~lcluding pigments) , a11tf four phssphatide fractions. A1 though complete resol~ation of the phos1)hatitIcs into individu;il coralponents was not achieved, it was considered worth while employing this fraction:~tion pro- cedure i11 conjui~ction with the isotope tracer technique to stutly phospllatitfe metabolis~n in 1e:tves.

Relatively few esperimeimts with 1:tbelliilg agents tlesig~led to elucidate phosphatitle met:it1olism in plants have bee11 carried out. Arnoff ~f al. (2) observed a small irlcorporation of ("'5irlt o the lipid fraction of t~nrIey seedlings after photosynthesis for 1 hour in the presence of C:I4O2. Kapid i11cor1>0r;~tio11 sf ('I4 illto the fatty acid moieties ol the lipids of Scenedesnzus obl iyuz~s after 40-seco~ld exposures to C ' 1 4 0 2 w;ts reported by Clendennirig (4). Milhnud, Renson, rtild Calvi11 (1 3) observed a considerable incorporation of CI4 illto the lipids (irmcludi~lg phosphatides) of Scenedesmu.~ after metabolism o f labelled pyruvate or hydroxypyruvate in the light. Incorporation of P" from ortho-

lManuscript receiveti July 15, 1957. Contribimtion frorxl the Divisio~l of Applied Biology, National l<eseardl I,aboratories,

Ottawa. Canada. I'rescnted in part a t thc. nleetirig of the American Society of Plant I'hysiologists, Storrs, Connecticut, August 26-30, 1056; see Plant Physiol. 31, xxx~rii (1956).

Issued as N.K.C. No. 4404. Waticsnal Research Cc~uncil of Canada Postdoctorate Fellow, 1055-56. Present address:

Botanisches Irlstitut, ?'iit)i~igen, Germariy.

Can. J. Botany, 35 (1957)

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908 CANAIIItPN JOURNAL OF BOTANY. VOE. 35, 1957

phosphate-PS2 into the phosphatides of peanut ~nitocho~idria (12), and of kidney bean chloroplasts (14) has recently been reported.

The present paper deals with a study of the incorporation of radioisotopes into the phospllatides of primary leaves of runner hean (Phaseolus multi$orus), usiiig C1402, pyr~vate-2-C:'~, acetate-l-C'4, and orthophosphate-P3? as prccur- sors. Runner bean was selected as plant material because its growth was fairly rapid, and the two primary leaves, being physiologicallv similar, were s~aitable for use in coiltrolled experiments. Furthermore, the extel~sive irives- tigations of Jordan and Chibriall (6) indicated thai there was 311 accumulation of phosphatides in runner hean leaves during the periocl of rapit1 growth. In the present work, attenipts were clirectec-l towi~rd determining the effect of light or1 the i~~corporatiori of radioisotope into the phosphatides, and also the distribution of radioactivity ~tn-nong the phosphatide fractions arrd among tlae various phosphdtide moieties. I t was hopeti, thereby, to 011t:iin an over-all pictul-e on which further siudies of the mechanisrri of phosphaticle synthesis in plants could be based.

Materials and Methods Blnnt Material

'The results reported were obtained with primarv leaves of scarlet runner bean (Pl7aseolus ~nult<Rorus), grown under greenho~ase conclitions, i11 soil containing- verrnic~alite. After 14 days o f gro~vth, the tops of the runner bean plants were removed to prevent formation of pinnate leaves.

Ay~alyt ical AJethod.s Phosphorus was determined by King's procedure (9). Chlorophylls a

ant1 b were separatecl chromatogr;lphi(:ally on a coluinil of sucrose (4), and determined spectrophc~tometrically in acetone solution, using fi/li~cki~lliey's coilstants (1 1).

Adnzinisfration of 1,nbclled P~cc?~rsors* C?Q2 was admi~~istered to iildividual leaves in a lucite chamber, described

by Towers and nilortimer (15). ISach leaf was preillunliilated for 5 minutes a t 24" and ai ;L light intensity of 2000 ft.-candles a t tlw leaf surface, whereupon C1Q2 (generated by addition of 70%) perchloric acid to 50-100 pc. of sodium carbonate-C14) was i11t roducecl ill to the chamber. After photosy~i thesis for 1 minute, the leaf was either immediately extracted (initial co~rtrol), imme- diately placetl in the dark in 11-acer-free air, or kept in the light (2000 ft-c.) in tracer-free air for various periods of tinae. In the latter two cases, ihe petioles were immersed in tap water.

Sodium acetate-1-C1", sodium pyruvate-2-('I4, or orthophosphate-I'3z were supplied to the leaf in diffuse light (30 ft-c.) a t 24-25', by immersing the petiole in 0.05 IW solutiorl of the labelled compound (acljustetl to pH 5-6). \;hen the solution had been completely talteii up, the leaf was either imrnetli- ately extractetl (initial control), placed in the dark (~e t io le immersed in

"All labelled comporinds were obtained from Atomic Energy of Canada, Ltd.

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EREKPIAKDT AXD KATES: P~IOSPWATIDES IN LEAVES 909

waterj, or kept in the light (2000 ft-c., petiole i~n~llersed in water) for various periotls of time. I,arge differences in the rate of uptake of the precursor by leaves of the same plant were sometimes observed.

Extraction sf Tstul Lipids

"Unt:tgged" leaves were extracted in l>ullc as described in another paper

(7). 'I'he P content of the total lipit1 extract (washed chloroform extract) was taken ,is n measure of the phosphatide content, since all of the P was found to be associated with the phosphatide fractions obtained after chronlntography on silicic rtcid - ('elite (7).

'"Tagged" leaves were extracted as follows. Each lamina (2.5-3.0 g. fresh weight) was ground to a powdcr ennder liquid nitrogen in a mortar, and the powder wrts imrnetlidtely added to 12 ml. of boiling i-propanol in a 100 mal. bealter. 'I'he mixture was tr:tnsfcrrcd to a 15 rnl. conical centrifuge tube, stirred for ;L few rrli~iutes a t 80°, and ccntrifugetl. The inso1uble residue was extr:tcted successively in i he centrilugc tuhc with two more 12-ml. portions of boiling i-propanol, followed by three 12-m1. portions of hot i-propanol- chloroform (1 :I). The centrifuged extracts were coinbined, concentrated i n a n c ~ ~ o (Islath teillperature, 40'; nitrogel1 stream) to about 5 ml., asnd diluted with 25 ml. of chloroform. 'The chloroforn~ extract frorn leaves suppliecl with CI4-labelled precursors was wc~shetl five times with 15-1111. portions of water, as dcscribcd previouslj~ (7) ; the extract from P:Q-fed lcaves was washed three times with 15 ml. portions of 0.5 A'lil tlisodium phosphate in 2 3f potassium chloride (12) , ant1 twice with water. 'The final water-wash in all cases contained less than 171; of the radioactivity irk the washed extract. 'Phe chloroforrll extract (coniainirig the total lipids and pigments) was then made to a ~ I ~ o \ ' c ' T ~ volume, ant1 alicluots were taken for determination of total P and for cotmnting. The rensaiilclcr of the solution was brought to dryness in a stream of nitrogen a t 30°, ancl the resiclue was dissolvetl in 0.5 -1 ml. of chloroform and sul-ijected to chromatography.

Fractionation of Lipids on Stlicic Acid - Celite Columns Chromatography of the lipids of "tagged" leaves was carried out as

described previously for the "'untagged" 1c:tves (7), using a columr~ of silicic acid - Celite mixture (411 ; 1.25 g./lOO -150 pg. P) in a 10-12 mm. diameter tube. Elution with 10 column volumes eac-h of chloroform, chloroforrn- methanol (5 : I ) , and chloroform-methanol (I :1) yielcled, as described previoixsl 57 (73, a non-phosphatide fraction (cont airling glycerides, pigments, and unsaponifia1)lcs) , an i~rositol-carbohydrate phosphatide fraction (Frac- tion I ) , a phosphatitlyl ethanolamine - phosphatidyl serirle frac-tion (Fraction 11), an ixnknown phosphatide (Fraction I I I ) , and lecithin (Fraction IV). Similar results were obtained, in some early experiments, using the solvent sequence: chloroforn~, ch1orolorn1-rnethanol (4:1), and methrt~lol, but the separation of Fraction I11 from Fraction 1V was not complete (cf. Figs. 2 and 3).

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910 CANADIAN JOURNAL OF BOTANY. VOL. 35. 1957

Clhrornatography of the lipids on silicic-:icici-impregnt~ted paper accordirlg to the method of Lea, Rhodes, :tnd Stoll (10) was also attempted (using 20(% methanol in chloroform), but the separation of the phosphatide fractions was not as sharp as on silicic acid - Celite columns.

1Fydrolysi.s l'hosphatide fractioils were hydrolyzed with acid or alkali, and the

hydrolyzates were separated into ether-soluble and water-soluble products, as described previously (7). The ether extracts were washed with water and concentrated to dryness in a streann of ilitrogen (hxth temperature, <40°); the fatty acid residue was weighed (and in some cases titrated with alkali), rrlade to ;L knotvn concentration in chloroform, ailti an ;aliquot was taken for countiiig. 'The water-soluble part of the hydrolyzate was freed from hydrochloric acid as described previously (7), dissolve(l in water to a known volunlc, it11 t~liquot was taken for counting, and the remainder was subjected to chrol1aatogr;~phic analysis.

Chromatographic Idenf<ficntion of Water-soluble IIydrolysis Yroducts Seprtration of hydrolysis products by two-directional chromatography was

carried out 011 Whatman No. 1 paper (acid-w;~shed, 12 X 16 in.) using n-11utanol - acetic acid - water (5:3:1) in the first direction (16 hours desccirdiilg), and n-butanol - 9596 ethanol -- water (210:128:62) in the secoild direction (1 6 hours desceiiding). Separation of the following compouilds expected in the hydrolyzntes was achieved: glycerophosph:ete, i~lositol phos- phate, hexoses, pentoses, glycerol, iriositol, choline, ethanolalnint., aird serine. The separated spots were visualized by autoradiogrrtphy. For further identification, the spots corresponding to glycerophosphate, choline, c~nd hexoses were eluted with water and rechromritographed on Whatman No. 1 paper, in one direction, together with appropriate staiitiards (solvent for phosljhate esters and cholirie: n-t~utanol - acetic acid - water, 5 :3 : I ; for sugars: pyridine - ethyl acetate -- water, 4:10:10). Visualization of the spots was achieved by sg~rayiilg with the specific sprays described previously (7 ) , and by autoradiography.

Separation of the hydrolysis products was in some cases carried out by nlearis of paper electrophoresis, followed by papcr chromatography of the anionic, neutral, and cationic substances in orie directiori (7).

Plating and Counting rill radioa(:tivt material was plated on alurnilrium plailchets over ;t circular

area of 4 cm.VLip ids were plateci a t infiriitc thinness fro111 chlorofornn solu- tion, allowing the solvent to evaporate a t rooin temperature. Aqueous solutiorls were plated on alkali-etched aluminum pl;unchets, and were dried down under an infrared larnp. Radioactivity iincorporated illto the i-prop;t-lnol-insoluble portion of leaves was determined by combustion to carl~on dioxide according to Raker et al. (3) and counting as barium carbon:~te. Samples were usually courated in triplicate with ail end-window GM-tube, and the results in that case are given as averages with mean deviations.

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EBEKIIAKDT AND KATES: PkIOSPHATIDES I N LEAVES 91 1

Results

F~rln~t ioTL of Ptzosphatides Dz~ring ~ e ~ ~ l ~ p m e n t and Growth of Leaves Before proceeding with studies or1 the incorporation of radioisotopes into

the phosphatides, infor~mr-ltion concernirlg the rate of accumu1:ttion of phosphatitfcs by leaves was required. 'The changes observed ill the phos- phrttitle I' content and in the length of primary leaves of ru~-iiler bean during tlevelopnlent fro111 the embryo stage arc illustrated i11 Fig. 1. During the first 4 (lays of growth, very little change of phosphatide P content occurred.

AGE, BAYS

Frc. 1. Forrliation of phosphatides during development arid growth of primary leaves of runner bean (Plzaseolus mulCz:fiorus). Phosphatide P content determined on the total lipids of 200 enibryonic leaves (dissected frsni 100 seeds), 40 leaves of 4-day-old plants, or 20 leaves of 8- to 20-day-old plants. Data expressed as P content per lead, or average length of lanaina (aloi~g rnediari vein).

Between the fourth and eighth dav both the phosphatide T' contc~l t and the leaf lcirgt h inc.re;escci rapidly; between the 8th arid 20th day thc iricreases were 111~~xi1nal and linear with time, and paralleled each other. During this latter period there was a linear relationship between the phosphatide content antl the c:orrespo1ltIi11g leaf length, sriggestirig that phosphatide accumulation is direct1 y concerilctl ill the growth of the leal.

For the tracer studies, leaves from 2- to 3-week-old plants were uscd, since they had a sufficierntly high corntent of phosphatides, and the rate of x<:cumula- tion of phosphatides was maximal.

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912 CANADIAN JOURNAL OF BOTANY. VOL. 35, 1957

hcorpora.tiola o j CL4 or P32 into Phssphatides ,4fter Feeding T'arious Precursors d~f lue~zcl : o j Light and Dark Very little radioactivity was iricorporated into the totaI lipids (+pigments)

of detaclied runner bear1 leaves after photosynthesis for 1 minute in the presence of (:lQ2, but increasing amourlts of activity appeared in the total lipids after subserluent photosynthesis in tracer-free air for 30 minutes or longer ('Table I). Activity was also incorporated into the lipids refter a 30 minute clarlc period followi~lg 1 rninute photosynthesis in CIQOa. Because of the lack of a proper control, however, i t was not c~scertained whether the activity incorporateci in the dark was greater or less than that incorporated in ail equivalent light period.

INCORPORATION OF C14 IKlYl T H E TOTAL LIPIDS O F RUNNER REAN LEAV~.:S AFTER T J I ~ T A ~ ~ E OF c140s

TIae leaves from two 22-clay-old rurilaer bean plants were i~idividr~ally fed C1Q2 (gclieratetl frorn 100 PC. of NasC140a) in the light (2000 ft_c.. j. for 1 minute, then kcpt in &racer-free air in the light (2000 ft-c.j for the ~ndicatetl periods of time. '6hc results obtained with leaves from the sarne g~la~l t are to be compn red.

Activity in rZctivit~7 in Activity ill Post- z-propanol total lipids, water-washi~~gs,

illulrnirnatican extract, counts % of i-propanol % of i-propariol Plant Leaf time. miri. X 10-3!rnin. activity rtctivity

-- - - A- - -- -- -. -

I 1 0 44 .7 -t 3 .0 0 . 5 + 0 4 104 + 3 . 5 2 30 54.0 + 3.5 14.7 k 0.4 87.5 1 .3

T l ~ c effect of light and dark oil the ii-mcorporatiori of CP4 intc) the lipids was better ascertained when pyruqate-2-(?"was used as precursor (Table TI). After supplg-iilg the precursor for 23 minutes in diffuse light (controls), about 27-30% of the activity taken up by the leaf was incorporated into the 2-propanol-soluble material, about one-fifth to one-sixth of which was associated with the total lipids (phosphaticles and pigrnents plus 11011- phosphatides), and the re~~iaiilder with the uiater-so1ut)le part of the extract. After a subsequent period of 60 minutes in the light, the activity of the i-propanol extract was uncharmged, but the activity of the total lipids (both the phosphatides aild tlze pigments plus non-phosphatides) was increased threefold, a t the expense of the activity in the water-soluble fraction. When the leaf was kept in the dark for 60 ~llinutes inste;td of ill the light, the activity of the i-propano1 extract tlecreased greatly, presumably because

'1 1011. much of the r~ctivity ill the water-soluble fraction was lost by respir- t ' However, 110 significant change in the activity of either the phosphatides or the pigments plus rlon-phosphatides was ol~serverl with respect to the control. I t is also interesting that both the light and the dark treatment led to an increase in the specific activity of the i-propailol-insoluble residue, the

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TABLE I1

INCORPORATION OH: CIQNTO FRACTIONS OF RCNNER BRAN I-EAVES AFTER UPTAKE OF P P R U V X ' ~ E - ~ - C ~ ~

l[-let,~checl prirnary leaves frorn two 16-day-old plants were each fecl 24 pc. of socIiurn p~7r1rvate-2-C""(0.15 1n1. of 0.05 hf solution, p I I 5.0) during 23 ~ n i n ~ ~ t e s untlcr 30 ftlc.-; one leaf from tach plant w,is irnrnediately extracted (controls); o f the reinainlng Icnves one was kept in the !ight (2000 ft-c-.) for 60 minutes, the othcr \\-as kept ill the tlarlc for 60 rninutes. Leaves were fractiorlatetl as described in the text. Total activity taken up was 930 X 1 O3 counts/mirl./leaf; phosphntide P contents: Plant 1, co~itrol leaf, 124 pg., Iight leaf, 139 pg. ; Plant 11. cc~ntrol, 1,10 pg.; dark, 124 pg.

Activity in fraction, Ojo of total activity taken up

I'lant I Plant I I

F r a c t i o ~ ~ Cor~trol leaf Light Ieaf Control leaf Dark leaf

i-F'ropanol extract 28.6 t 2 .2 27.2 _+ 1 . 3 26.9 f 0 .2 12.1 + 0 . 3

Water-sol ilble 25.1 2 0 . 8 14.6 k 0 . 1 20.1 k 0 . 9 8.2 k 0 . 4 Total lipids 5 . 1 + 0.2 14.5 i- 0 . 2 5 .4 _+ 0.2 4.9 2 0.1

FPhosphatidcs 3 .4 9 .9 3 .9 3.8 Pigments f

noii-phosphnticies 0 . 8 2 . 3 0 .7 0 .7

Specific activity, counts/rniil./prg. C --

Residue ilisolul~le after i-propa~iol extractioil 1 1 1 1 . 1 17.0r t0 .2 11.81k0.1 113.6k0.1

TABLE I I I

INCOKPORA'~ION OF C ~ ~ N T O LIPlD FRACTIONS OF KUXNER BRAN LEAVES AFTER UPTAKE OF .~CETA'TI~~-~-C'~

Leaves from a 16-day-old plant werc each fed 167 prc. of sodiulml acetate-1-Cil (0.64 1111. o f 0.05 M solution, pH 5 ) during 94 minutes a t 24Oa1ld in diffuse light (30 ft-c.); oilc leaf was then kept in the light (2000 it-c.) for 91 minutes, and the other kept i11 the dark for 92 nlinutes. Phosphatide P content: light, 200 pg.; dark, 209 pg.

Activity in fraction, C O U K I ~ S x 10-3/min.

Fraction Light leaf Dark leaf

i-Propanol extract 1,080 k 8 540 I_+ 30 Total lipids 667 + 6 330 rt_ 5 Phosphatides 2 88 109 I'igments + non-phosphatides 160 93

light treatment being: more effective in this respect. These results show that light is required for the incorporation of CI1'& into the pkosphatides and other lipids whcn pyruvate-2-C1"is administered.

The effect of Bight oln the iilcc~rporation of CM from acetate-1-C1"into the lipid fraction was not ascertaixled with certainty, again because of the Back of n suitcthle initial control. Ilowever, the results given in Table 111 suggest that light does stimulate the ii~corporatiorl of CI4 into the phosphatide and the pigrncrlt plus non-phosphatide fractions.

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CANADIAN JOURNAL OF BOTANY. VOL. 35, 1959

TABLE IV

Leaves frona 17-day-old plarats were each fed orthophosphate-1':'3 during 30 nai~l~ltes in the light (2000 ft-c.); 'control' leaf was inamediately extracted; 'light' leaf kept in light (2000 ft-c.) for 60 minutes; "lark' leaf kept in dark for 60 minutes.

Co~ntrol leaf Light leaf Dark leaf

Activity in phosphatides, counts X 10-3/min. 11 .7+0 .6 2 3 . 8 5 1 . 2 3 3 . 8 3 ~ 0 . 7

Total P in phosphatides, pg. 97.2 91.4 120 Specific activity of phosphatides,

cou~ats/rnin./pg. P 120 262 282

The effect of light and (lark on the illcorporatiorl of P32 from orthophosphate- P35irlto the phosphnticte fraction is shown by the data in 'Table IV. Both the light arid the dark treat~neilt resulted in a large increase in the activity of the phosphatides over that of the (:olitrol. 011 the basis of the specific :activities, light and tlark treat nlerlts would appear to be equally effective for the incorporation of P35rito the phosphatides.

CHLOROFORM----*cGHLOROFORM - METHANOL 64 I ) +4------METHANOL --------

ELUATE VOLUME, ml.

FIG. 2. Chromatographic separatioii of 1;lhclled lipids obtained from runner bean leaves after fcetling C1'Oz. 'Three 16-day-old leaves were adrni~~istcred CL4(ls (generated from 54 pc. Na2C1403) for 1 n~inute in the liqht as tiescribed in the test; leaves were extracted aiter a sut~sequent period of 30 ini~iutes i l l the light. Cornhined total lipitis were chro- matographeti o l r a cc,lurnn of 2.5 g. of m i ~ e d at1sorb;lnt; columil volunle, 4.2 ~ n l . Total acti~rit y and appliecl to column are given in 'l'able V.

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EBERHAKDT AND KATES: PEIOSPIIATIDES IN LEAVES

Distribution of Radioactivity Among the Various Lipid Fractions The tfistributiorl of radioactivity among the lipid fractions after atlmin-

istration of various precursors in the light is illustrated in Figs. 2 and 3, and summarized in Tnblc V.

With C140r as precursor, more than three-quarters of the activity lixrd by the total lipids was found in the pigment plus non-phosphatide fraction;

ELUATE VOLUME. ml.

FIG. 3. Chromatographic sep:iratioa~ of labellecl lipicls oLtailled from rurlmer bear, leaves after uptake of pyrtivate-2-C'I, acetate-1-C'" or orthophosphate-1'32 in the light. 'Tiigging conditio:~s for 1>yruvat.e-2-C" and acctate-l-(:lk:~rt. gi\-en in 'I'ables I 1 and 111 (light-treated leaves), respectively; for phospbr-ite-Pu tagging, two 15-day-old 1e;~ves were adrninisterecl orthophos~)l~ntl-:-I'= (ahoul 15 P C . ) for 4 h o ~ ~ r s ill diffuse light (30 ft.- ca~ldles), ant1 the lipicls were iln~netiiatel): estracteti. \ITeight of luixecl :itlsorbant (g.), and colrlnln voliltlze (rill.), respectively: pyr~~vate-2-C:'" 1.25, 1.3; acetate-l-C:l< 1.25, 8 . 7 ; I'32, 2.5, 3.6. Total radioactivity and P applied to colums are give11 in 'I'l~hIe V.

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CANADIAN JOTJRNAL O F BOTANY. VOL. 35. 1957

TABLE V DISTRIBUTION OF KADIOACTIITYTY i1MON(i TAPID FRACTIONS AFTER UPTAKE OF

VARIOIiS I,ABELI,ED PRECUIISOIIS r . Iota1 lipids, obtained from leaves after uptake of lal~elled precursors in the

light, were fractionated chrornatogra~~hicall~ (see Figs. 2 alrcl 3). TotaI ratIio- activity (cour~ts/min.) and total I' ( p g . ) appIied to colunlns: CIQOR, 96.4 X lo3, 344; pyrusrat-e-2-eli, 117 X 10" 112; acetate-1-CI1, 574 X 103, 180; ortho- phosphate-Pa, 42.2 X lo3, 251

_I--------- _---.-P--.--~ __l_l_.-l___ -------- ._ _.lll__ll__P-

Fraction

Pigrrrents f non-

-- phosphatidrs I I I 111 IV Kccoverics - -----

C'402 % of total counts/min. 76.7 10.8 2 - 9 0 . 8 9.9 101.1 '% of total I-' 0 33.3 17.9 2 .2 34.8 88.2 Specific activity.

courits/rnin. /patom P - 2,#20 1,400 3,2003 2,450 .-

Ppravate-2-("'4 O/o of total counts/min. 15.8 2.5.5 7.3 1 . 5 34.0 84.lt (z, of total P 0 3 8 . 0 20.1 5 . 9 36.6 100.6 Specific activity,

corrnts/n~iri./patorrr P - 20,100 10.800 7.400* 27,900 -

Acetate-l-CI4 % of total counts/init~. 24 .0 9 .6 6 .6 2 . 4 24.5 67 .6 t % of totai l'l 0 38 20 h 37 201 Specific. activity,

counts/rnin./patom P - 25,000 33,000 39, OOO* ri6.000 --

Orthophos~llate-I'32 ?(-, of total cou!~ts/rnin. 4 . 4 13.2 18.6 31.2 32.4 99.8 %; of total !' 2 . 4 17.2 18.7 1 7 . 5 34.5 YO. J Specific. activity,

counts/min./paton~ P (9,300) 4.010 5,170 9,300 4,900 -

*Sgecific activity- only approximate, because of thc low activity in this fraction. ?Low recoverii-s of radioactivity may be diie to loss of labt4led r~olatilit n!nterial during concentratioi~ of

clllorsforln so l~~ t i on of thp lipids priijr to chroniatography. $L)istrib~~tion of P was uot determined, b u t assiin~ed identical with valuvs for the pyruvatc-fed Icaves.

the remainder distributed among the four phosphatide fractions, chiefly in I:rac:tions I ancf IV. On the hasis of their specific activities (c.p.rn./,u atom P), the degree of incorporation of ('14 into the phosphatitle fractiolls was in the order, Fraction 1 and Fraction I11 > Fraction IV > Fraction TI. i'ltternpts were also ~nade to determine which of the pigment plus non-phos- phaticle constittients were radio,lctive. Both chlorop11~-11 n and b were found r;dioactivc (1050 an(] 680 c.p.rn./micrornole, respectively), and together with pheophytills accountetl for about 10% of the activity in the non-phosphatide frac-tion; fatty acids (obtained after saponification) containecl ahout ")'j (of the activity (specific activity, 800 c.~).m./microe(~uiv:~1ent j ; anti u~~saponifiable material (c-hiefly car-otenoids) acconnnted for about 75y0 of the activity.

With pyruvate-2-C1I4 as precursor, rriost of the activity (70'3;) fixed by the total lipids of the light-treated leaf wits associ;tted with the phosphatides, of which Fraction IV (lecithin) had the highest proportioil, followed by I;r,ic-tions I , 11, and 111, in that order. The specific activities were atso in the same order. 'I'he control and dark-treated leaves (see Table I I) had the same distributioal of activity among the lipid fractiotls as the light-treated leaf.

With acetate- 3 -C1"s precursor, again most of the activity in the total lipicls of the light-treated leaf was associated with the phosphatides, arnong

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F:HF:KIIAKDT A N D KATES: PEIOSPIIATIDES IN LEAVES 917

which the activity was distributecl in about the same nlaniier as with pyruvate-2-C14, except that the lecithin fraction had an eve11 higher pro~)ortian of the activity. The specific activities (calculated on the basis of the same distributiorl of P as with pyruvate) showed that the lecithin frnction was about twice as active as any of the other fractions. The dark-treated leaf (see 'I'able 111) had a distribution of activity among the lipid fractions simi1:ir to that of the light-treated leaf.

i ihei l 01-thop11osph:ite-lm \vris supplied for 4 hours in diffuse light, the radio- activity was found to be distributed almost entirely (except for a few per cent in the pigment pl~as non-yhosphatide fr:tction) amolig the four phosphatide fractions (Table V). Surprisingly, Fraction 111, which previously was fo1111d to contain only a minor proportion of C1" (from cru-bon-labelled precursors) ant1 of the total phosphorus, now contained about one-third of the 1'" "1

the total lipids and about 18y0 of the total p, and had about twice the specific activity of arny of the other phosphatide fractions. Furthermore, the pigment plus 11011-phosphatidc fraction cont;~ined n sn~a l l but significant amount of radioactivity allcl P, which would corrcspoild to a phosphntide with specific activity comparable to that of Fraction 111. 'I'liese cliffcreilces are difficult to explain, but perhaps are a rcflcction of the abnormally high concerltratioil of orthophosphate available to the leaf under the conclitions of this experiment.

Distribution qf IZl~dionct ivity A m o n g JIydrolysis Products of tJzc Y h o s p l ~ a t ide Fractions

After acid hydrolysis of the phosphaticle fractioils obtained from leaves tagged with C1402, pyruvate-2-C13! or acetate-1-C1", the radioactivity was fount1 distributed between the water-soluble and ether-soluble constituents as given in Tablc VI.

With ('1402 as precursor, the activity of Fractioil I was distribute(1 almost eclually between et hcr- and water-soluble constitaients, while tha t of E'ract ions I1 anti IV was :issociatetl more with the ether-soluble constituents, although a (:onsicicrable proportion (about 207i,) was foulld in the water-solubles. LtJith pyruvate-2-C1" as precursor, hotvever, oinly a few per cent of the activity of each fraction appeared in the water-soluble constituents, while more than 9091, of the activity appeared ill the ether-soluble yro(Iucts. With acetate-1- (:I4 ;is precursor, the activity- of each fraction was associated almost elltirely with the ether-soluble products, the ainount in the water-soluble pro(1ucts being less than 1 C;ro.

Radioactive Water-.solublr. f3yd~oLysis Products The water-soluble products of hydrolysis of each of the phosphatide fractio~ls

(except Fraction 111, the radioactivity of which was too low) obtltined from leaves supplie(1 with (31">2 in the light (see Fig. 2) were subjected to two-directiorlal chromatog-raphy, and autoradiogr:rms of the resulting chromatograms were made. The radioactivity in the products of acid hydrolysis of Fraction I was associated mostly with hexoses (identified

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9 18 CANADIAN JOURNAP, OF BOTANY. VOI,. 35 10.57

TABLE VI

~)ISTRIBUTIC)N OF 1ZADIOAC'~IVITY I N HYDROLYSIS PRODUCTS OF PHOSPHATIDE FRACTIONS

Phosphatide fractions were hydrolyzed with 3 N hydrochloric acid, and the products were separated illto ether-soluble and water-soluble constituents, as described in the text

--

;\ctivity in yo of activity in fractivi~ fractiori -- -- -

lt'hosphatide hydrolyzed, Water- Ether- Precursor fraction counts/m~n. soluble soluble

Acetate- l -C14 I 2 7 , 2 0 0 I I 19,300 I I I 5,230 I V 62,500

*I~lsufficient rriaterial for hydrolysis.

chronlstographically as glucose and galactose) ; spots corresponding to inositol, inositol phosphate, pentose, and glycerol also were present oti the autoratliogrsm, but that for glycerophosphate was absent. In contrast, allnost all the radioactivity in the products of alkaline hydrolysis of Fraction I was associated with a slow-movirlg spot corresponding to glycosidic material, but 110 spots corresponding to free hexoses or pentoses were evident; the autoradiogram also showed spots coi-rcspondi~lg to glycerophosphate, to glycerol, a i d to two unidentified substances (cf. the hydrolysis protiucts of unlabelled Fraction I given in the previous paper (7)).

'I'he acid hytlrolyzate of Fraction I1 coiltairled radioactive glycerophospl~~~te, glycerol, and eth:inolamirle, but also considerable amourlts of radioactive hexoses, probably arising from the inositol-carbohydrate phosphatides still present (7). l 'he acid hydrolyzate of Fraction 1V contained radioactive glycerophosl)h:itc, glycerol, and choline, but also contained some radioactive hexoses.

I t is important to note tha t none of the above radior-~ctive products can possibly be water-soluble cont;lminants associated with the phosphatide fr:~ctions, since they were completely abse~l t in autoradiograms of similar cht-om:itograms obtained with the u~zhydro!yzecl ~)hosphatide fractions.

'The water-soluble products of the aciti hydrolysis of the phosphatitle fractions obtained from leaves supplied with pyruvate-2-C1' (see Fig. 3) were seyaratecl by electrophoresis followed by paper chromatography. Radioactive glycerophosphate was found in the hydrolyzate o f each of the four phosphatide fractions, and radioactive sugars were fourid in that of

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EBERHARDT AND KATES: PI-IOSPPPATIDES IN LEAVES 969

Fraction I ; no radioactive bases were found in any of the fractions. The low radioactivity in the hydrolyzates prevented further work on the identification of the radioactive hydrolysis products.

The water-soluble hyrlrolysis products of the phosphatides fl-or11 leaves supplied with acetate-1-(:I4 were not investigated because of the very low radioactivity present.

Discussion

Jordan ant1 Chihnall (6) observed a large increase in the total phosphatides of primary leaves of runner beall between the 8th and 30th days after germination. ?'he present I-esults (Fig. 1) are in accord with this, and show further that the increase in phosphatide content is linear with time and parallels the growth of the leaves. Jord:~n am1 Chibniell also fourid (6) that the accumu1;ttion of phosphatides in the priinary leaves was very much slower than the disappearance of phosphatidcs from the cotyledolis; they colicludcd that the seed phosphatides functioned as reserve food. The possibility that the leaf phosphatides arise by tral~slocation of seed phosphatides woul(1 thus appear remote. I t is much more likely that the accumulation of phosphatides in growing leaves is due to sylithesis in situ, since C14 is i~ncorporated rela- tively rapidly into the phosphatides of detached leaves durilig photosynthesis in the presence of C1'*02 (Table I and Fig. 2). However, leaves rnay utilize molecular fragments derived from seed phosphatides or other seed conlporients to synthesize phosphaticles, since they illcor~)orate CIt or PZ2 into the phos- phatides when supplied with labelled pyruvate, acetate, or orthophosphate (Tables 11, 111, and IV).

'The eflect of light and dark on the incorporati011 of radioisotope into the phosphatides of leaves varies with the precursor used. Duri~lg photosynthesis for one ~ninute in C1402, 110 radioactivity appears in the lipids, but subsecrhnerlt photosynthesis in tracer-free air for 30 mi~lutes or loliger leads to i~lcorpor;ition of <'I4 into the lipicls ('Table I). IIowever, (:I4 is also irlcorporatecI into the lipicls after ec~uivalent periods in the dark. I t would appear the11 that phosphatides are syilthesized in the leaf both during photosynthesis and during dark nzetabolisrn, but lurther work will be necessary to establish this with certainty.

By contrast, light is required for the incorporation of (:I4 into the phosy~hatidcs when pyruvate-2-C1"s supplied, whereas darkness cornpletely blocks the incorporation ('l'ablc TI). I t is interesting that Milhaud, Benson, and (13) observed a much greater i~lcorporatior~ of C1" illto the lipids of Scenedesmus during light metabolism of pyruvate-2-CB4 than during dark metabolism of this precursor. When acetate-1-C1" is supplied to leaves, much more radioactivity appears in the phosphatides during a light period than during an equivalent tiark period ('Table IIT). Further work is necessary, however, to establish whether light is essential for the fixation of C14 from acetate- 1 -CB4 illto the phosphatides.

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920 CANADIAN JOURNAL OF BOTANY. VOL. 35, 1957

The light rcc~uirement with pyruvate may be defined more precisely, inas- much as almost all tlle C'14 incorporated into the phosphatides from labelled pyruvate (or acetate) in the light is associated with the fatty acid rnoieties (Table VI). Pyruvate is most likely converted 10 fatty acid by the sequence of reactions : pyruvate -- acctyl-Coenzyme A fatty acid-Coenzyme A. The first reaction would occur both in the light a n d the dark, but the second step would require :t. high level of reducing power which could orlly be supplied in the light-. Thus, as in other photosynthetic processes, the light requirement for the co~lversiorl of pyralvate to fatty acid would be ecluivalent to a recluirement of rcduci~lg power.

Incorporation of P32 from orthophosphate-I'N into the phosphaticles proceeds equally well in tlle light ant1 in the dark (Table IV). The aderlosirle triphos- phate required for the i~~corpor:ation of P3"il?to phosphatides (8, 12, 14) is thus supplietl equally well i r~ the light by photosynthetic p%mospllorylation (I), and in the dark by oxidative phosphorylation (5). I t is interesting that in leaves supplied with orthophosphate-P" Fraction I11 has about twice the specific activity of I he other phospl~atide fractions, and its absolute amount is increased several-fold over that found in leaves under ilorrrlal condiiions (scc 'Fable HI in Ref. 7). This fraction (which appears to cont:tin a proteolipid-type of phosphatide (7)) may perhaps be the precursor of some of the other phosphatides present in leaves, but further study is necessary to deternsine whether this hypothesis is correct. Mazelis and Stumpf (12) fourlcl that the phosphatide which became labelled ~vi th P" in peanus mitochondri:al systems was neither lecithin, nor cephalin, nor phosphatidic acid; its very low R f vr-tlue in ethanol-water suggests thltt i t might be similar to Fractiorn TII, which has a low solubility in ethanol.

The distributiorl of (.'I4 among the lipid fractions and among the phosphatide moieties varies greatly with the labelled precursor supplied (Tables V and VI). Under the relatively ~lormal corltiitiolls of photosynthesis irl the presence of Ci402, a much higher proportion of CI4 is incorporated into the pignlents plus rion-phosphatides than into the phosphaticles, whereas the proportions are reversed when acetate-('I4 or pyi-uvate-CI4 is supplied. Furthermore, with C1402 both the water-soluble :tild ether-soluble phosphatide constituents become labelled, wllereas with acetate or pyruvate the fatty acid moieties appear tcs be lak~ellecl allnost exclusively. TFhus, with the latter precursors, the differences i11 the specific activities of the phosphatide fractions indicate that the fatty acid moieties in each of the fractions are labelled to different extents. From the data in Table V, the f ~ t t y acids of Fraction IV (lecithin) would appear to be about twice as radioactive as those from I'raction I1 (phosphatid~-l ethanolamine-serine). Kennedy (8) has shoWra that lecitlli~l and phosphdtitlyl ethanolanline are synthesized in cell-free enzyme systems by esterificatioi~ uf a 11-a,/?-diglyceride with phosphorylcholirle ailtl phospl~oryl- eth:~r~olarniile, respectively. The above observation is difficult to reconcile with this mecha~lism unless one assumes that the diglycericfes which form lecithin are no1 in equilibrium with those that form phosphatidyl ethanolamine.

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EBEKIPLZRDT ANL) KATES: PIIOSFIIATIDES IN LEAVES 92 1

All of the water-soluble moieties of the phosphatide fractions (see (7)) become labelled during photosyi~thesis in the presence of C:1402, the glucosc and galactose (in that order) of Fraction I being most highly labelled. I,acking specific activity data , however, it is not possible to deierrnirie product-precursor relationships for the water-soluble constituents. Further studies along these lilies are conte~~~pl;ttecl.

Acknowledgment

The authors are iildebted to Dr. L). C. Laortimer and Dr. C:. D. Nelson for helpful suggcstio~ls corlcerilirlg the tracer experiments. The exc-ellelit technical :tssistarac.e of bliss Fay Inglis, h4r. I-larold Hre~lcler, and NIrs. Alma Harvey is gratefully acknowledged.

Ref erences 1. ARKON, 1). I., \ % r ~ ~ ~ ~ , ~ ~ , F. R., ;111d ALLEN. M. R . I'hotosynthesis by isolated

chloroplasts. 11. Photosynthetic phosphorylation, the conversion oh light into phosphdte honti energy. J. Anl. Chenl. Soc. 76, 6324-6.32")1954).

2. IZRONOFF, S., BENSON, il., ~ I A S S I D , 1%'. Z., and CALVIN, M. Ilistribution oh Cli in photosyi~thesizing hLarley seetllings. Sciellce, 105, 654-665 (1947).

3. BAKER, X., FEIN~EKG, H.. anti I ~ I L L , R. Analytical procedures using a co~nbined combustion-diffusion vessel. Simple wet cornl)ustion method suitable for routine cnrhon-14 arinlyses. Anal. Chcm. 26, 1504 -1506 (1953).

4. CI,ESI)ENXING, K. A. Distrit)utioil of tracer carbon among thc lipids of the alga Scenedesmz~s during brief photosp~lthetic exposures. Arch. Bioche~n. 27, 75-88 (1950).

5. GCKJT~MRN, M., BRAIII~EY, TI. F., anti CAI'VIN, NI. I'hosphorus aild photosynthesis. I. I>iffcrences in the light and dark incorporation of ratfiophosplaorus. J. Am. Chem. FOC. 75, 1962-1967 (1953).

5. T O R U A N , R. C:. and C ~ r x n ~ ~ r , r , . A. C. Observations on the fat illetnbolisin of leaves. 11. Fats and phosphatidcs of the rrlllller bean (Pht~seolus ntu2fiJEorus). Ann. Dotany, 47, 163-186 (1933).

7. KATES, R1. and EREKHAKIIT, F. M. Isoldtion and fractionlati011 of leaf phosphatides. Can. J. Botany, 35, 895-90.5 (1957).

8. KEN;VE,DT;, E. 1'. Biological syilthesis of phospholipids. Can. J. Diochem. Physiol. 34, 334-347 (1956).

9. KING, El. J. ?'lie colori~netric determination of phosphorus. Niochem. J. 26, 292-297 (1032).

80. LEA, C. Il., Iir~onr.:~, T). N., ancl STOI-L, It. D. On [he chromatographic separation of gl~cerophospholipids. Eiochem. J. 60, 353 -363 (195.5).

3 1. MACI~INNEY, (;. Criteria for purity oh chlorophyll preparations. J. Biol. Chern. 132, 91-109 (8940).

32. M,tz~,r.~s, G. and STUMPP, F'. K. Fat metabolism in higher plants. 1.1. Incorporation of P" into peanut rnitochondrial phospholipids. Plant F'llysiol. 30, 237 -243 ( 1 955).

13. R'IILHAUT), G., HENSON, A. A., and CALVIN, M. Rletabolisrn of pyruvic acid-2-C14 and hvdroxypyruvic acid-2-C1$ in algae. J. Diol. Chem. 218, 599-606 (1956).

14. SISAKYAN, 3 . M. and SMIRNOV, B. P. Patlns of synthesis of phospholipides i~n chloropldsts in slitro. Lloklady Akad. Nauli S.S.S.R. 107, 449-451 (19.561.

1.5. TOWERS. G. 11. N. and ILIORTIMER, D. C. The role of keto ;~cicls in photo~~~nt lnet ic carbon dioxicle assirnil,+tion. Can. J . Hiochem. Physiol. 34, 511-519 (1956).

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