Plant Physiol. (1980) 65, 171-1750032-0889/80/65/0171/05/$00.50/0

Intracellular Localization of Two Enzymes Involved in CoumarinBiosynthesis in Melilotus alba1

Received for publication May 7, 1979 and in revised form September 4, 1979

JONATHAN E. POULTON2, DUNCAN E. MCREE, AND ERIC E. CONNDepartment of Biochemistry and Biophysics, University of California, Davis, California 95616

ABSTRACT

The localization of phenylalanine ammonia-lyase [EC 43.1.51 withinsweet clover (Melilotus alba) leaves was investigated. Apical buds andaxillary leaves contained 15 to 30 times more enzyme activity than didmature leaves. Mesophyil protoplasts were prepared by digesting youngleaves with Celiulysin and Macerase and were gently ruptured yieldingintact chloroplasts. These chloroplast preparations exhibited neither phen-ylalanine ammonia-lyase nor o-coumaric acid O-glucosyltransferase activ-ities. The general enzymic properties of sweet clover leaf phenylalanineammonia-lyase were similar to those described for this enzyme isolatedfrom other plant species. The conversion of L-phenylalanine to trans-cinnamic acid, which occurred at an optimum pH of about 8.7, was stronglyinhibited by the metabolites trans-cinnamic and o-coumaric acids. In con-trast, o-coumaric acid glucoside, coumarin, p-coumaric acid, and meliloticacid had no significant effect on the reaction rate.

During the last 2 decades, the biosynthesis ofcoumarin in sweetclover leaves (Melilotus alba) has been studied extensively usingin vivo tracer techniques and cell-free enzyme preparations (14,19). This compound exists in this tissue as the /8-glucoside of 2-hydroxy-cis-cinnamic acid (coumarinic acid) and probably ap-pears in its free form only when the cells are crushed and theglucoside undergoes hydrolysis. The biosynthetic pathway leadingto coumarin has been recently reviewed (4) and is summarized inFigure 1. Increasing attention is now being paid in the field ofphenylpropanoid and flavonoid metabolism toward the control ofthese pathways and toward the localization of the enzymes con-cerned within the cell. Evidence has accumulated showing thatthese enzymes may be associated with distinct organelles (1). Thelocalization of the enzymes of the coumarin biosynthetic pathwayitself, however, has been less well studied. The unique enzyme ofthe pathway, cinnamic acid o-hydroxylase, was demonstrated byGestetner and Conn (6) to be located within the chloroplast. Wewere therefore interested to determine whether the other enzymesof this pathway, namely PAL3 and o-coumaric acid O-glucosyl-transferase, are also associated with this organelle. Although PALwas originally regarded as being a soluble enzyme (5), it hasrecently been demonstrated in microsomal, peroxisomal, glyoxy-somal, and chloroplast fractions from higher plants (1). Thedistribution of PAL in various parts of the sweet clover plant andits relationship to the o-hydroxycinnamic acid content have been

1 This work was supported by United States Public Health ServiceGrant GM 05301.

2Present address: Department of Botany, University of Iowa, IowaCity, Iowa.

3Abbreviations: PAL: phenylalanine ammonia-lyase; UDP: uridinediphosphate.

171

reported by Kleinhofs et al. (11). We report here an extension ofthese studies, indicating some of the more important properties ofthe sweet clover PAL and also information pertaining to itssubcellular localization. In addition, the possibility of a chloro-plastic location for the o-coumaric acid glucosyltransferase hasbeen investigated.

MATERIALS AND METHODS

Chemicals. L-[U-"C]Phenylalanine (460 mCi/mmol) and L-phenyl[l-'4CJalanine (carboxyl (59 mCi/mmol)) were obtainedfrom Schwarz/Mann and Amersham/Searle, respectively, andwere diluted with unlabeled L-phenylalanine Aldrich ChemicalCompany. UDP-D-[U-14C]glucose (292 mCi/mmol) was pur-chased from ICN Pharmaceuticals Inc., and diluted with unla-beled UDP-glucose from Sigma Chemicals. The following com-pounds were recrystallized from water before use: ortho, para-,and meta-coumaric acids, coumarin and DL-m-tyrosine. Cellulysinand Macerase were purchased from Calbiochem.

Plant Materials. Healthy leaves were harvested immediatelybefore use from sweet clover plants (M. alba var. White Blossom),which were grown in greenhouses. These plants exhibited ,B-glucosidase activity and contained high levels of the glucosides ofo-coumaric acid. Generally, all enzyme preparations and chloro-plast fractions were derived from the apical buds and the axillaryleaves from the first two to three macroscopic nodes, where highestPAL activities were observed. In localization studies with theglucosyltransferase, however, the youngest fully expanded leaves,from which the lower epidermis could be peeled off, were used.Enzyme Assays. PAL activity was usually assayed by measuring

the rate of formation of ['4CJcinnamate from L-[U-'4C]phenylala-nine, which were separated by paper chromato raphy. The reac-tion mixture consisted of 0.32 tmol of L-[U- 4C]phenylalanine(containing 360,000 dpm) and 14.7 tsmol sodium borate (pH 8.8),which was incubated with up to 100 ,ul of enzyme preparation ina total volume of 150,l. Incubation was carried out at 37 C fortime periods up to 30 min. Control tubes, in which boiled enzymepreparation or homogenization buffer alone replaced the activeextract, were included. The reaction was terminated by the suc-cessive additions of 20 yt 50 mm NaOH containing 20 Ag trans-cinnamic acid and 20,l 40o (w/v) trichloracetic acid. The result-ant mixture was cooled to 4 C, centrifuged in a Beckman microfugeB centrifuge, and the supernatant liquid was applied to the originof a 3.8-cm-wide strip ofWhatman 3MM chromatography paper.Trans-cinnamic acid (1.25 ymol, dissolved in methanol) was ap-plied to the origin as carrier and the chromatogram was developedusing benzene-acetic acid-H20 (2:2:1, v/v). After thoroughlydrying the chromatogram, the zone containing cinnamic acid,which was detectable under UV light (254 nm) as a darklyabsorbing zone, was cut out and counted by scintillation spectrom-etry. The scintillation fluid contained 0.5% (w/v) PPO and 0.02%(w/v) POPOP in a mixture (2:1, v/v) of toluene and Triton X-100.

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POULTON, McREE, AND CONN

,CH2CHNH2COOH ,H COOH H ,COOH

HHA OH

PHENYLALANINE TRANS-CINNAMIC o-COUMARIC ACIDACID // c

H\ ,H H ,COOHc=c~ ~~'--C=C'COOH 'H

K~O-GWUCOSE D -GLUCOSEP-GLUCOSIDE OF P-GLUCOSIDE OFCOUMARINIC ACID o-COUMARIC ACID,E

H

C= U F

OHCOUMARINIC ACID

H

COUMARIN

FIG. 1. Biosynthesis of coumarin from L-phenylalanine. Enzymes in-

volved in this pathway are: A: PAL; B: cinnamic acid o-hydroxylase; C:

UDPG: o-coumaric acidO-glucosyltransferase; E: fl-glucosidase. The cis:

trans isomerization of o-coumaric acid glucoside (D) is mediated by UV

light. Coumarin is formed by the spontaneous lactonization of coumarinic

acid (F).

The above assay was modified for determining the PAL activ-

ity of fractions after sucrose density centrifugation. The sensitivity

of the assay was increased by reducing the assay phenylalanine

concentration from 2.13 to 0.6 mm. Additionally, L-phenyl[1-"4CJalanine replaced L-[U-'4C]phenylalanine, while borate was

replaced by 14.7,mol Tris-HCl (pH 8.8). The reaction was

terminated as described above. Thereafter, [14C]cinnamate was

separated from residual L-[14CJphenylalanine by extraction into1

ml toluene (15). Following centrifugation, an aliquot (0.5 ml) of

the toluene phase was added to 10 ml scintillation fluid and the

radioactivity estimated by scintillation spectrometry.

Glucosyltransferase activity was assayed by measuring the rate

of o-coumaric acid glucoside formation from o-coumaric acid and

UDP-['4C]glucose. The assay mixture contained 150 nmol o-cou-

maric acid, 75 nmol UDP-D-[U-'4C]glucose (containing 180,000

dpm), 10,umol K-phosphate buffer (pH 8.0), 5,tmol f-mercapto-

ethanol, 0.3 mg BSA, and 0.1 ml enzyme preparation in a total

volume of 0.15 ml. Incubation was carried out at 35 C for h and

the reaction was terminated by the addition of 10,ul glacial acetic

acid. The resultant mixture was cooled to 4 C, centrifuged in a

Beckman microfuge B centrifuge, and the supernatant liquid was

applied to Whatman 3MM chromatography paper. Ortho-cou-

maric acid glucoside (0.2 ttmol, dissolved in 50%o ethanol) was

applied to the origin as carrier, and the chromatogram was devel-

oped using 1-butanol-acetic acid-H20 (4:1:5, v/v). The glucoside

zone, which was detected under UVlight (254 nm), was cut out

and counted by scintillation spectrometry. Control tubes, in which

either enzyme or o-coumaric acid were absent, were included with

all incubations.NADPH-dependent triose-P dehydrogenase activity was meas-

ured by the method of Heber etal. (8).

Chromatograpbk Identification of Reaction Products. Cin-

namic acid was identified as the product of the reaction by co-

chromatography of an ether extract of a terminated reaction

mixture with an authentic sample on Whatman 3MM chromatog-

raphy paper using the following solvent systems: (I) benzene-

acetic acid-H20 (2:2:1, v/v); (II)I -propanol-concentrated NH40H(7:3, v/v); (III) 1-butanol-acetic acid-H20 (4:1:1.8, v/v); and (IV)

2% acetic acid. Furthermore, since solvent system IV allows the

efficient separation of cis- and trans-cinnamic acids, it was shown

that only the trans-isomer was produced by the enzyme under the

described reaction conditions.The presence in crude extracts of enzymes which catalyze the

formation of phenylacetate from L-phenylalanine has been re-cently reported to interfere with certain PAL estimations (20).Using a modification of the "double-decker two-dimensional"technique of Stafford (20), it was demonstrated here that Melilotusextracts catalyzed the formation of phenylacetate at less than 4%of the rate of trans-cinnamic acid synthesis.

Preparation of Crude Homogenate for PAL Kinetic Studies.Leaves were collected and weighed (3.5 g), washed twice with colddeionized H20, and blotted dry. These were homogenized with alittle quartz sand in a mortar at 4 C with 10 ml 50 mm sodiumborate (pH 8.8). The homogenate was centrifuged at 30,900g for20 min. Dowex I-X2 (0.3 g/ml, equilibrated with this buffer) wasadded to the resultant supernatant liquid and the mixture wasstirred slowly at 4 C for 10 min. The ion exchanger was removedby filtration through glass-wool and, when necessary, by furthercentrifugation in a clinical centrifuge. The supernatant liquid wasused immediately for kinetic studies.

Intracellular Localization of PAL. Young leaves (0.6 g) werecut into approximately 0.5-mm slices using a sharp razor blade.The slices were placed in a covered 9-cm Petri dish with 20 mlmedium A, containing 3% (w/v) Cellulysin, 0.75% (w/v) Macer-ase, and 0.05% (w/v) BSA. The tissue was quickly evacuated twice(40 mm Hg) with a vacuum pump. Incubation was then under-taken at 34 C in a shaking water bath at 45 oscillations/min. After2 h, the contents of the Petri dish were fltered through a44-,umnylon net, and the released protoplasts were harvested by centri-fuging the filtrate at lOOg for 3 min in a bench-top swingingbucket centrifuge. The protoplast pellet was washed twice usingmedium B and finally resuspended in 1.1 ml medium B. Theprotoplasts were gently ruptured by drawing them up and down10 times through a 25-gauge needle using a l-ml syringe. Analiquot (0.85 ml) of the resultant chloroplast suspension was thenloaded onto the top of alinear 30-60o (w/w) sucrose gradient (40ml). The gradient was centrifuged for 1 h at 25,000 rpm in aBeckman SW 27 rotor using a Sorvall OTD-50 ultracentrifuge at4 C. Fractions (1.35 ml) were collected using an ISCO gradientfractionator and subjected to rapid freeze-thaw treatment byimmersion in liquid N2 to rupture the chloroplast membraneseffectively.

Intracellular Localization ofO-Glucosyltransferase. The isola-tion of protoplasts and chloroplasts described above was modifiedslightly in view of the known tissue localization and f8-mercapto-ethanol requirement of the glucosyltransferase (12). The lowerepidermis was carefully removed from the youngest fully ex-panded leaves by peeling, before the tissue was placed upon thesurface of the digestion medium. Tissue infiltration by evacuationwas not necessary. Digestion of the leaves and subsequent isolationof chloroplasts and protoplasts were undertaken as before, exceptthat media D and E replaced media B and C, respectively.

Media. The following media were used: (A): 25 mm Mes wasmixed with 25 mm Tris to reach pH 5.5. To this solution was thenadded 0.7M mannitol, 0.05M sodium ascorbate, and 0.05mmMgCl2; (B): 20 mm Tricine-NaOH (pH 7.5) containing 0.4Mmannitol and1 mg/ml BSA; (C): 20 mm Tricine-NaOH (pH 7.5)containing1 mg/ml BSA; (D); medium B plus 50 mM,f-mercap-toethanol; (E): medium C plus 50 mm fl-mercaptoethanol.

Protein and Chl Estimations. The protein content of fractionscontaining negligible Chl was determined by the sulfosalicylicacid method (16). Total Chl was determined by the method ofArnon (2).

RESULTS

DETERMINATION OF THE GENERAL KINETIC PROPERTIESOF M. alba PAL

Preparation of Enzyme Extract. A crude preparation possessingPAL activity could be obtained by maceration of sweet clover leaftissue with borate buffer and quartz sandin a mortar, followed by

172 Plant Physiol. Vol. 65, 1980

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COUMARIN BIOSYNTHESIS IN MELILOTUS ALBA

centrifugation. Removal of phenolic inhibitors was achieved bytreatment with Dowex I-X2 ion exchanger, leading to a 40-50%oincrease in the PAL specific activity. This method was usedroutinely in preference to treatment with polyvinylpolypyrroli-done, since the latter did not remove inhibitors as efficiently(about 20% increase in PAL specific activity observed) at this pH.No reaction could be observed when boiled enzyme extract re-placed the active preparation in the assay mixture.

Stability. Storage of the Dowex-treated extract for 24 h eitherat 4 C or deep-frozen at -20 C resulted in the loss of 61 and 63%,respectively, of the initial PAL activity. Due to this instability, theenzyme extract was prepared freshly for each experiment.pH Optima. The pH optimum for the deamination of L-phen-

ylalanine, determined in several buffer systems, was found to bein the range of 8.5-8.8. Over 50%o of the maximum rate wasrealized between pH 6.8 and 10.3. Sodium borate (at pH 8.8) waschosen as standard buffer for these studies in preference to K-phosphate, since it exhibits a greater buffering capacity within theoptimum pH range. A pH optimum ofabout 8.5 was also observedin glycine-NaOH buffer, but the rate of reaction was here about30%o lower than that in borate buffer.

Linearity with Protein and with Time L. The rate of cinnamicacid production at pH 8.8, catalyzed by 0.33 mg of protein, waslinear for about 30 min. The extent of this reaction after 30-minincubation at this pH was proportional to the protein amountadded up to at least 0.33 mg.

Michaelis Constant for L-Phenylalanine. The results of meas-urements of initial velocity as a function of L-phenylalanineconcentration showed a pronounced downward deviation close tothe 1/v axis, when plotted according to the Lineweaver-Burkmethod (Fig. 2). Two Km values could be determined from thesedata: Km, 154 ,pM and KmL, 78 ,UM (23).

Inhibition by Metabolites. The inhibition of PAL activity byvarious metabolites of the coumarin pathway and their analogswas studied (Table I). Among those investigated, only trans-cin-namic acid and o-coumaric acid exhibited significant inhibitory

0.20

0b. 0.15

Ex

J-c 0.10x 00

E>

__ 0.05

5

L- Phenylolonine concentration (mm-')0 p- I p-0 20 40 60 80

L-PHENYLALANINE CONCENTRATION(mMvU1)FIG. 2. Lineweaver-Burk plot for the deamination of L-phenylalanine

by sweet clover PAL. Inset describes the variation in initial reactionvelocity (V) within the concentration range 0.1-3 mM.

Table I. Inhibition ofPAL Activity by MetabolitesThe activity ofthe crude PAL preparation was measured at two different

L-phenylalanine concentrations. The effects observed by the inclusion ofcertain metabolites at 0.5 mm concentration are shown below.

Inhibition Observed

L-Phenyl- L-Phenyl-Addition 0.5 mm alanine alanine

Concen- Concen-tration 2 tration 0.2mM mM

Trans-cinnamic acid 24 63Dihydrocinnamic acid 4 15Coumarin 12 9o-Coumaric acid glucoside 12 10Melilotic acid 14 21DL-m-Tyrosine 17 22m-Coumaric acid 25 27p-Coumaric acid 15 15o-Coumaric acid 26 55L-Tyrosine 10 9

z 100

280

so

t 60

< 40--J0120z

0"e0

l00o IB

80

60

40

20-

0

000-

_\-

I I I1 I I I I I500 1000 1500 2000 500 1000 1500 2000

INHIBITOR CONCENTRATION (iLM)FIG. 3. Inhibition of PAL activity by cinnamic and o-coumaric acids.

The enzyme preparation (0.3 mg protein) was incubated for 15 min with0.3 mM L-[U-'4C]phenylalanine either alone or in the presence of variousconcentrations of either cinnamic acid (A) or o-coumaric acid (B). Thedata have been expressed as the percentage of the control activity remain-ing versus the inhibitor concentration.

properties. Such inhibition could be markedly reduced by a 10-fold increase in phenylalanine concentration. The potency of theinhibition was further tested by maintaining the L-phenylalanineconcentration constant (300 ,UM) and varying the inhibitor concen-tration in the range 0-2 mm (Fig. 3). The rate of cinnamic acidproduction could be halved by the addition of either 330 ,UMcinnamic acid or 380,UM o-coumaric acid.

TISSUE AND CELLULAR LOCALIZATION OF PAL

Tissue Localization. The activity of PAL was determined inextracts from three parts of the sweet-clover plant: (a) apical budsand axillary leaves from the first macroscopic node; (b) axillaryleaves from the second to fourth macroscopic nodes; and (c) fullymature leaves. Table II indicates that whether the data are ex-pressed in terms of the fresh weight of tissue or with respect toprotein levels, the fully mature leaves had only about 4% of theactivity of the very young tissue. The older axillary leaves (b)showed an intermediate activity.

Effect of 24-h Dark Period on PAL Activity. The above planttissues were also assayed for PAL activity from comparable plantsthat had been kept at the same temperature but in the dark for 24h. Table II shows that the PAL activity in all fractions was lessthan 20%o of the control plants which had been kept in the light.

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POULTON, McREF, AND CONN

Intracellular Localization of PAL. Mesophyll protoplasts wereprepared in high yield from active tissue and ruptured gently toobtain chloroplast preparations which were 50 to 85% intact asjudged by Chl and triose-P dehydrogenase distribution studies.Figure 4 shows a typical fractionation profile, illustrating theseparation of intact chloroplasts from broken chloroplasts bysucrose density centrifugation. The undamaged chloroplasts, rep-resented by the coinciding peaks of triose-P dehydrogenase activ-ity and Chl at p = 1.22, did not exhibit PAL activity. Indeed, 100%oof the PAL activity applied to the gradient was recovered in thesoluble fraction at the top of the gradient. The failure to detectPAL activity in the intact chloroplasts was not due to high assaysucrose concentrations tr to freeze-thaw treatment of the organ-elles. Control experim its shoWed that 1.06 M sucrose (32%, w/w)reduced the PAL activity of a crude PAL preparation by only20%o. Furthermore, the PAL activity of such a preparation was notaltered by freeze-thaw treatment in the presence of 48% (w/w)sucrose. We concluded that PAL is not associated to any signifi-cant degree with the chloroplasts of M. alba var. White Blossom.Identical results were also obtained with chloroplasts from M.alba var. Spanish.

Intracellular Localization of O-Glucosyltransferase. In theseexperiments, chloroplasts were prepared from protoplasts and

Table II. Influence ofIllumination on Activity ofPAL in Various LeafFractionsfrom M. alba

Control Leaves' Leaves Kept 24 hTissuea PAL Activity Darkness PAL

Activitynmol cin- nmol cin- nmol cin- nmol cin-namic namic namic namic

acid/h g acid/he acid/h.g acid/h.fresh mgpro- fresh mgpro-weight tein weight tein

Apical buds and first axillaryleaf 790 67.7 117 11.1

Axillary leaves from secondto fourth nodes 412 43.6 55.5 5.8

Fully mature leaves 28 3.2 NSb NS

a See text for explanation.b Not significant.

0 5 10 15 20 25TOP Gradient Volume (ml I

-60 -

3:

50 Oe

40 QU)

30

FIG. 4. Distributions of PAL and NADPH-dependent triose-P dehy-drogenase activities following sucrose density gradient centrifugation, asdescribed in the text. Undamaged chloroplasts, represented by coincidingpeaks of triose-P dehydrogenase activity ( *), and Chl (O-0),did not exhibit PAL activity (A- - -A). Sucrose concentration:(Z )-

separated by sucrose density centrifugation in the presence of /8-mercaptoethanol. Neither the intact nor the broken chloroplastpreparations possessed glucosyltransferase activity. In contrast, allof the glucosyltransferase activity applied to the gradient wasregularly recovered in the soluble fraction at the top of thegradient. Control experiments showed that 1.06 M sucrose (32%,w/w) reduced the glucosyltransferase activity of a crude prepara-tion by only 7%. Moreover, freeze-thaw treatment of this prepa-ration in the presence of 48% (w/w) sucrose led to only an 18%decrease in its glucosyltransferase activity. Thus, the failure todetect glucosyltransferase activity within the gradient fractionswas not due to high assay sucrose concentrations or to their priorfreeze-thaw treatment. Ortho-coumaric acid glucosyltransferaseactivity is therefore not associated with chloroplasts of M. albavar. White Blossom; identical data were obtained with chloroplastsfrom M. alba var. Spanish.

DISCUSSION

The conversion of L-phenylalanine to cinnamic acid by PAL,the first enzyme of the coumarin pathway, was demonstrated incrude extracts from sweet clover (11). We have here confirmedthat the highest PAL activities in M. alba are exhibited by fractionsfrom the actively growing tissue of the plant. It is precisely herethat the level of o-coumaric acid O-glucosyltransferase (10) andthe over-all rate of production of bound coumarin (13) are at theirmaxima. It might be proposed that the enzymic properties ex-hibited by one particular PAL may be in part determined by thenature of the biosyntheses (e.g. lignin, coumarin, flavonoid) beingundertaken by that specific tissue. Investigation of M. alba PALin crude extracts and also with a more purified preparation has,however, indicated that it possessed properties very similar toother PALs from tissues engaged in lignin or flavonoid biosyn-thesis (7, 23). PAL, as the first enzyme of phenylpropanoidmetabolism, often shows end product inhibition by cinnamic acids(5) and by flavonoids (3). The effect of certain intermediates incoumarin biosynthesis on the in vitro activity of M. alba PAL wastested. Whereas coumarin and o-coumaric acid glucoside did notsignificantly affect the reaction rate, the enzyme was potentlyinhibited by relatively low concentrations of the metabolites cin-namic acid and o-coumaric acid (cf. 17). If PAL activity weresubject to similar regulatory constraints in vivo, optimal reactionrates would be promoted only if the levels of these metabolites inthe vicinity of the enzyme were kept as low as possible. This couldbe achieved by the following mechanisms: (a) when PAL wouldcatalyze the rate-limiting step in the coumarin biosynthetic path-way; or (b) when cinnamic and o-coumaric acids would be quicklyremoved in some way from the vicinity of the enzyme. Thisprocess might be aided if PAL and the cinnamic acid o-hydrox-ylase were in different intracellular compartments.The second proposal was tested by investigating the intracellular

localization of the coumarin biosynthetic enzymes. In view of thechloroplastic location of cinnamic acid o-hydroxylase (6, 9), chlo-roplasts were isolated from M. alba leaves by gently rupturingmesophyll protoplasts. These chloroplast preparations were up to80%o intact as judged by Chl and marker enzyme studies, butshowed neither PAL nor o-coumaric acid glucosyltransferase ac-tivities. The absence of PAL in sweet clover chloroplasts is insharp contrast with data obtained for the monocotyledonousplants Hordeum vulgare (18) and Avena sativa (22), where the highchloroplastic PAL activities have been correlated with the exist-ence of large concentrations of C-glycosylflavones in the organelle(22). Further experimentation should determine whether chloro-plastic PAL activity is a typical feature of monocotyledonous butnot of dicotyledonous plants.That the M. alba chloroplasts possess little if any PAL activity

was confirmed by the isolation of chloroplasts by standard tech-niques involving gentle homogenization of leaf tissue (data notshown). These chloroplast preparations, which were 60 to 70%

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COUMARIN BIOSYNTHESIS IN MELILOTUS ALBA

intact as judged by phase contrast microscopy, exhibited only veryweak PAL activities, ranging from zero to a maximum of 17 nmolcinnamic acid/h mg Chl. These activities were not always repro-ducible and were extremely small when compared with the rate ofo-hydroxylation of cinnamic acid catalyzed by sweet clover chlo-roplast preparations (1.48-1.67 umol/h.mg Chl) (6); they mayhave been due to contamination of the chloroplast fraction. More-over, when these PAL activities were related to the total PALactivity present in the tissue, it was conlcuded that a maximum of1 to 2% resides within the chloroplast.Since the M. alba PAL appears not to be located within the

chloroplasts, one is faced with the interesting question as to howeasily cinnamic acid may pass through the chloroplast membranesto reach the chloroplastic o-hydroxylase. Similar problems mayapply to the o-coumaric acid formed within the chloroplast, whichmust then traverse the same permeability barrier to reach theextrachloroplastic glucosyltransferase. Although the transport ofprimary metabolites into and out of this organelle, which ofteninvolves certain translocators, has been investigated (21), thepassage of cinnamic acids and flavonoids across these membranesis poorly understood. The permeability of the M. alba chloroplastmembranes toward cinnamic and o-coumaric acids is under in-vestigation in this laboratory.

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