I958

Studies in Carotenogenesis25. THE INCORPORATION OF 14CO2, [2-14C]ACETATE AND [2-14C]MEVALONATE

INTO fl-CAROTENE BY ILLUMINATED ETIOLATED MAIZE SEEDLINGS*

By T. W. GOODWINDepartment of Biochemistry, The University, Liverpool 3

(Received 28 May 1958)

It has been known for some time that etiolatedseedlings of many plants (e.g. maize, wheat, barleyand sunflower) synthesize carotenoids when germi-nated under the most stringent conditions of light-exclusion (Beck, 1937a; Strain, 1938). Xantho-phylls generally predominate and the individualpigments are usually the same as those xantho-phylls found in green leaves (Beck, 1937b; Strain,1938); fl-carotene is normally present only intraces (Blaauw-Jansen, Komen & Thomas, 1950;Kay & Phinney, 1956).

According to Kay & Phinney (1956) illuminationof etiolated maize seedlings results within the first24-48 hr. in a rapid synthesis of fl-carotene, withno commensurate increase in the xanthophyllfraction. Because of this observation it was con-sidered that illuminated etiolated maize seedlingsshould provide good experimental material forexamining the biosynthesis of ,8-carotene in photo-synthetic tissues of higher plants. The presentpaper is a report of an investigation of the in-corporation of labelled materials known to becarotenoid precursors in other organisms (Braith-waite & Goodwin, 1957), into fl-carotene synthe-sized by illuminated etiolated maize seedlings.

Part of this work has already been briefly re-ported (Goodwin, 1958b).

EXPERIMENTAL

Culturing of seedling8. Maize (South African HorseTooth) seeds were soaked overnight in water, then plantedin trays containing a 3-5 cm. layer of damp, acid-washedsilver sand and kept in the dark in a warm room at 280.The seedlings were watered each morning, during whichtime (1-2 min.) they were exposed to a weak red light,just sufficiently intense to see to carry out the watering.When required the seedlings were removed from the sand,washed with water, placed in distilledwater in small beakersand exposed in the warm room to the light from a 100wtungsten lamp at a distance of 50 cm. Excised seedlingswere prepared simply by cutting off the roots at the nodeconnecting root and stem. The excised stems were thenplaced in water and illuminated in the same way as wholeseedlings.

* Part 24: Goodwin (1958a).

Uptake of carbon dioxide by seedlings. The seedlings,either intact or excised, were placed in water in smallbeakers which were then placed in a 3 1. glass vessel(vacuum desiccator). The requisite amount of Bal4CO3 wasweighed into a small ignition tube and then sufficient un-labelled BaCO3 added to give an atmosphere containing0-3% (v/v) of CO2. The tube was then lowered by a piece ofthread through the neck of the vessel into a beaker con-taining lactic acid, which slowly liberated the C02. Thebung of the vessel was replaced immediately after tippingthe BaCO3 into the acid, and the vessel was illuminated for24 hr. under the conditions described above.

Extraction of pigment8. About 6-10 seedlings (afterremoval of their roots) or excised stems were cut into smallpieces which were immediately dropped into a mortar con-taining acid-washed silver sand and acetone (about 20 ml.).The tissues were thoroughly ground up and the mixturewas transferred to a G4 sintered-glass filter and filtered.The vacuum was removed, further acetone (10-15 ml.)added, the mixture extracted in 8itu by stirring and grind-ing with a glass rod with a flattened end and then filtered.This process was repeated (usually 3-4 times) until thefiltrate was colourless. An equal volume of ether wasadded to the combined acetone extracts in a separatingfunnel, followed by water dropwise until two layers formed.The aqueous layer was discarded and the ether layer twicewashed with small volumes ofwater to free it from acetone.The ethereal solution was dried by standing for 0.5 hr. overanhydrous Na2SO4, filtered through a G4 sinter, and eitherevaporated to dryness at 30° in a stream of N2 or, if thetotal chlorophyll content was required, first made up to asuitable volume for spectrophotometric examination beforeevaporation to dryness.The dry residue was then saponified, the unsaponifiable

matter extracted by our standard procedures (Goodwin,1955) and dissolved in light petroleum (the fraction withb.p. 40-60' was used throughout this investigation). Thepigments of the unsaponifiable extract were first separatedby chromatography on icing sugar (Goodwin, 1958a);fl-carotene and most of the sterols are not held by thisadsorbent and run straight into the filtrate. The fl-carotenewas then further purified by chromatography on a columnof alumina weakened with methanol (Goodwin, 1955). Thefl-carotene zone was eluted with light petroleum containing1% (v/v) of ethyl ether and diluted with light petroleum toa volume appropriate for spectrophotometric assay. Afterassay the solution was concentrated at 30° under N2 to3-5 ml. and quantitatively transferred to a 15 ml. taperedcentrifuge tube ready for crystallization.

Crystallization of the fl-carotene. As only about 100 g. of,-carotene was usually isolated from the seedlings, it was

612

STUDIES IN CAROTENOGENESIScrystallized after addition of unlabelled carrier material.Crystalline ,-carotene (synthetic: Hoffman-La Roche andCo., Basel, Switzerland) (3-5 mg.) dissolved in ethyl ether(5-10 ml.) was added to the labelled ,B-carotene extract inthe centrifuge tube. After thorough mixing of the contents,the centrifuge tube was placed in a warm-water bath (30°)and gently rotated as a stream of N2 was directed into thetube. When the volume of solvent had been reduced byapproximately half, the tube was tightly bunged and placedin a cold room ( - 25') overnight. The following day theresulting crystals were centrifuged down (low-speedM.S.E. Minor centrifuge; 5 min.) at 0° and the supernatantwas decanted. The crystals were then washed twice withcold (0°) methanol (5 ml.), being again centrifuged aftereach washing. The crystals were finally dissolved in ethylether or CHC13 (5-25 ml. according to activity) for 14C-assay, and a small volume (0-1-0.2 ml.) was diluted appro-priately for spectrophotometric assay. After the samplehad been counted, the solvent was removed at 300 underN2 from the residual solution and the ,B-carotene recrystal-lized in the manner just described. This process wasrepeated until a constant specific activity was attained.

Preparation and extraction of chloroplasts. Chloroplastswere prepared as described previously (Goodwin, 1958a).The chloroplast suspension was centrifuged down, thesupernatant removed, and the chloroplast pellet extractedaccording to the procedure just described for whole tissues.The residual pigments, if any, present in the supernatantwere extracted by adding an equal volume of ethanol,heating on a boiling-water bath for 2 min., cooling andshaking twice with light petroleum, each time with avolume equal to that of the original supernatant. Thecombined extracts were then reduced at 300 under N2 to avolume suitable for spectrophotometric assay. The residueremaining after extracting the chloroplasts were extractedin the same way as whole tissues.

Spectrophotometric assay. The standard methods forchlorophylls (Smith & Benitez, 1955) and carotenoids(Goodwin, 1955) were used throughout, with a UnicamSP. 600 photo-electric spectrophotometer.

Labelled materials. [2-14C]Acetate and BaL4CO3 werepurchased from A.E.R.E., Harwell, Bucks, and DL-[2-14C]-mevalonate was very kindly synthesized by Dr R. Y. Corn-forth.

14C Assays. The materials were plated on planchets byusing small disks of lens paper (Glover, 1956) and countedby means of a proportional counter (Nuclear MeasurementsCorp., Indianapolis, U.S.A.) (50% efficiency). The counts

were continued to give an accuracy of ± 5%. The amountsof material on the planchets were so small that errors dueto self-absorption were insignificant (less than 10%) andcorrections were never made.

RESULTS

Effect of illuminating etioklted seedlingsExperiments were usually carried out on etiolatedseedlings between 6 and 9 days old; preliminaryinvestigations with seedlings of various ages showedthat such seedlings had very similar xanthophylllevels and that the pattern of fl-carotene synthesison illumination was also similar. Table 1 recordssome typical figures for the carotenoid content of7-day etiolated seedlings before and after illumina-tion for 24 hr. The pigments from the etiolatedplants are almost entirely xanthophylls and theirconcentration is much less than that in normalgreen leaves [total carotenoids about 30 mg./100 g. fresh wt. (Goodwin, 1952)]. Illumination ofthe seedlings results in the synthesis of both ,B-carotene and xanthophylls, and the ratio of light-synthesized #-carotene to light-synthesized xan-thophylls is typical of normal green leaves. Evenafter 24 hr. illumination the total concentration ofcarotenoids is much less than that in matureleaves. The observations of Kay & Phinney (1956)that, during the first 24 hr. of illuminating maizeseedlings, only #-carotene is synthesized was notconfirmed. This may be due to varietal differences;they used a single cross-strain (L 289 x L 205).

Excised etiolated seedlings also synthesize caro-tenoids on illumination and the results obtained(Table 1) were very similar to those found withwhole seedlings.

Incorporation of [2-14C]acetate andDL-[2-_4C]mevalonate into etiolated seedlings

The labelled acetate or mevalonate was addedwithout dilution with inert material to the waterin which the seedlings were placed for exposureto light. Preliminary experiments showed that

Table 1. Carotenoid synthesis by illuminated etiolated m-aize seedling

7-day seedlings were illuminated for 24 hr. under a 100w tungsten lamp at 50 cm. distance. Temp. 280.

Intact seedlingsBefore illuminationAfter illuminationIncrease in concn. after illumination

Excised seedlingsBefore illuminationAfter illuminationIncrease in concn. after illumination

Conen. of pigment(mg./100 g. fresh wt.)

,8-Carotene Xanthophylls0-070-940-87

Trace0-740-74

1-193-131-94

1-192-561-37

fl-Carotene as% of totalpigments

5.923-131-0

022-435-1

Vol. 70 613

T. W. GOODWIN

Table 2. Incorporation of [2-14C]acetate and DL-[2-14C]mevalonate into the unsaponiftable matter ofilluminated, excised etiolated maize seedlings

6-day excised seedlings were illuminated for 24 hr. in thepresence of labelled substrate under conditions given inTable 1. Counts added: [2-1"C]acetate, 2 x 106/min.; DL-[2-14C]mevalonate, 1-8 x 106/min.

Total activity(counts/min.)

FractionTotal unsaponifiable matter,8-Carotene

1st cryst.2nd cryst.3rd cryst.4th cryst.

Counts expected assuming,B-carotene is 0-5% of totalunsaponifiable matter

[2-14C].Acetate405 000

DL-[2-14C]_Mevalonate506 000

500 9200 350

- 320410

2 000 2 500

no significant uptake of either compound wasobserved on illuminating intact etiolated seedlings,i.e. they were unable to penetrate the intact rootsystem. Marked uptake was, however, obtainedwith excised seedlings and these were used in allfurther experiments. Table 2 gives the results oftypical experiments with both substrates. Al-though in both cases there is considerable in-corporation into the unsaponiflable material of theseedlings, the incorporation into fl-carotene is notdetectable with the labelled acetate and, although,definite, only very slight with mevalonate. Theincorporation from mevalonate is much less thanwould be expected even on the basis of the per-centage of the total unsaponifiable matter repre-sented by fl-carotene, even though on illuminationfl-carotene is being rapidly synthesized, whereassynthesis of the remaining unsaponifiable material,mainly steroids, is negligible. Many experimentsshow that in 24 hr. illuminated seedlings f,-carotene represents between 0-3 and 0-5 % ofthetotal unsaponiflable matter. The same results areobtained if excised seedlings are placed in contactwith labelled acetate or mevalonate for 24 hr. inthe dark, before being brought into the light. It

was considered that if there were a pool of 'dark'precursors of ,B-carotene present, the acetate ormevalonate would equilibrate with them during the24 hr. contact in the dark, but again there was nosignificant incorporation into the p-carotene.The distribution of activity in the unsaponifiable

matter is different with the two labelled substrates.Table 3 shows that much more activity (about 30%of the total) appears in the weakly adsorbed 'pre-,B-carotene fraction' when labelled mevalonate isused than when labelled acetate is used (about5 %). This type of distribution was confirmed inother experiments and also on paper chromato-graphy with the solvent system of Loeffler (1955).It was also demonstrated that none of this labellingwas due to trace contamination with mevalonateitself. This aspect of the problem was not pursuedfurther but an infrared spectrum of the crude 'pre-fl-carotene fraction' indicated that it was anoxygen-free, long-chain, paraffin-like compound.

Incorporation of 14C02 into etiolated seeingsWhen 14CO2 is used as a substrate with either

intact or excised seedlings, there is a marked

Table 4. Incorporation of 14C02 into the unsaponi-fiable matter of illuminated, intact and excizedmaize seedlings

6-day seedlings were illuminated for 24 hr. under theconditions given in Table 1, and exposed in glass vessels toan atmosphere containing 0-3% (v/v) of 14CO,. Countsadded, 6 x 106/min. -.

Total unsaponifiable matterfl-Carotene

1st cryst.2nd cryst.3rd cryst.4th cryst.

Counts expected assumingp-carotene is 0.5% of totalunsaponifiable matter

-xozai &cTiviTy(counts/min.)

Intact Excisedseedlings seedlings125 000 142 000

4 500*8 2007 2008 500650

7 0006 0004 8004 600700

* Unexpectedly low.

Table 3. Distribution of labelling in the unsaponifiablermatter of excised etiolated maize seedlingsilluminated in the presence of [2-14C]acetate and DL-[2-14C]mevalonate

6-day excised seedlings were illuminated as in Table 1. Unsaponifiable matter was chromatographed on weakenedalumina; the developer was light petroleum (b.p. 40-60°) containing varying amounts of ethyl ether.

Fractions in order ofincreasing adsorptive

affinity1. Unknown2. Unknown3. ,8-Carotene (crude)4. Sterols

Total activity (counts/min.)

Solvent required for elutionLight petroleumLight petroleum + 1-2% (v/v) of etherLight petroleum + 2% (v/v) of etherLight petroleum + 20% (v/v) of ether

[2-14C]Acetate

4300700

4 00071 400

DL-[2-l4C]Mevalonate59 00010 0001 800

144 000

614

STUDIES IN CAROTENOGENESIS

Table 5. Intra- and extra-plaetidic di8tribution of carotenoid8 in illuminatedexci8ed etiolated maize seedling8

6-day seedlings were illuminated for 24 hr. under the conditions described in Table 1; the chloroplast was prepared asdescribed under Experimental. Concn. of carotenoid

(mg./100 g. fresh wt.)

Before illuminationTotal carotenoids

After illuminationTotal carotenoids,B-Carotene,B-Carotene as percentage oftotal carotenoids

Chloroplasts Residue

1-19

1-610-61

38-2

0-85Trace

preferential incorporation of label into ,B-carotenecompared with that appearing in the other un-saponifiable constituents; up to ten times thatexpected from simple quantitative analysis of theunsaponifiable matter is observed (Table 4).

Distribution of f-carotene in chloroplastsand whole seedlings

The results just described strongly suggestedthat ,B-carotene was being synthesized from photo-synthetically-fixed C02. If this were so synthesiswould most likely occur into the chloroplasts, thedevelopment of which are stimulated by light, andin which photosynthesis takes place. Further,the carotenoids of normal green leaves are entirelyconcentrated in the chloroplasts (Goodwin, 1952,1958a).Table 5 gives the results of a typical carotenoid

analysis of the chloroplasts and extra-plastidicresidue of illuminated etiolated maize seedlings.They show that (i) the extra-plastidic carotenoidsrepresent quantitatively and qualitatively thepigments present in unilluminated etiolated seed-lings (compare Table 1); (ii) all the fl-caroteneformed on illumination is in the chloroplasts; and(iii) all the xanthophylls formed on illumination are

similarly situated.It is never possible to remove all the chloroplasts

from the residual extra-plastidic material, and theresults given in Table 5 were obtained by using a

calculation based on the fact that the chlorophyllsexist only in the chloroplasts. First, the concentra-tion of chlorophylls and carotenoids are determinedin the chloroplast fraction and in the residue;secondly, the 'chloroplast carotenoids' present inthe residue are calculated from the concentrationof chlorophylls in the residue and the chlorophyll/carotenoid ratio for the pure chloroplasts fraction;finally, this calculated value is subtracted fromthe observed total carotenoid concentration andadded to the chloroplast-carotenoid figure to give thegross amount of carotenoids in the chloroplasts.

Table 6. Uptake of 14CO2 into unsaponifiable matterby iuminated etioloted maize 8eedltings in thepresence and absence of hydroylakmine

7-day seedlings were illuminated under the conditionsgiven in Table 1; chloroplasts were prepared as describedunder Experimental; hydroxylamine concn., 0-001 m.

ChloroplastsResidue

Total

Activity(counts/min./g. wet wt.)

Without Withhydroxylamine hydroxylamine

740 120140 150880 270

Action of hydroxylamine

According to Bandurski (1949), 0-001 m-hydroxyl-amine inhibits photosynthesis without inhibitingrespiration. It has been found that etiolatedmaize seedlings, placed in O-OOl M-hydroxylamineand then illuminated, fail to produce any increasein their carotenoid levels and also fail to producechlorophylls. In other words, development of thephotosynthetic apparatus is inhibited. In an

experiment in which uptake of "4CO2 by illumi-nated etiolated seedlings was measured in thepresence and absence ofhydroxylamine, the uptakewas 3-4 times as great in the absence of the in-hibitor, but there was a significant uptake in thepresence of the inhibitor (Table 6). As this occurs

in the absence of photosynthesis, these resultsindicate that a carboxylation reaction can proceedwhich is not dependent on photosynthesis.

D'istribution of total unsaponifiable matterin chloroplasts and whole seedlings

Two determinations of the distribution of un-

saponifiable matter (mainly sterols) between thechloroplosts and extra-plastidic material of maizeseedlings illuminated for 24 hr. indicated that 89

Total

1-19

2-560-61

23-8

Vol. 70 615

616 T. W. GOODWIN I958(86-92) % is present in the chloroplasts; thisagrees with the distribution of 14CO2 recorded inTable 6.

DISCUSSION

The present investigation demonstrates that theillumination of etiolated seedlings results in thedevelopment of chloroplasts in which chlorophylland carotenoids are synthesized; the ratio ,B-carotene/xanthophylls in these chloroplasts isthesame as that observed in mature leaves. As thedark carotenoids are entirely xanthophyllic innature, illumination appears at first sight to bringabout a specific synthesis of ,B-carotene. However,when the xanthophylls synthesized in the dark areallowed for, there is no such preferential synthesisof fl-carotene; the fl-carotene and xanthophylls arebeing synthesized in the chloroplast in about thesame ratio as in normal leaves (Goodwin, 1952).These observations differ from those of Kay &Phinney (1956), who found that on illuminatingetiolated seedlings of their strain of maize onlyf-carotene was synthesized over the first 24 hr.The reason for this difference may be due to thedifferent strains used. The marked incorporation of14CO2 into the fl-carotene produced on illuminationindicates that the pigment is produced afresh in thenewly developed chloroplast by the photosyn-thetically fixed C02. This conclusion is confirmedby the action of 0 001M-hydroxylamine, whichinhibits both photosynthesis and formation of ,B-carotene. There is no indication whatever from thepresent experiments that illumination triggers offthe conversion of dark-formed precursors into f,-carotene. If this were so, a significant incorporationof 14CO2 into the molecule would not be expected,and certainly there would not be an enrichment ofthe isotope in the pigment compared with theremainder of the unsaponifiable material. Such alight-triggering action in carotenogenesis does,however, occur in Neuro&pora (Zalokar, 1955).The failure of labelled acetate and mevalonate

to become incorporated into fl-carotene in theexcised illuminated etiolated seedlings is almostcertainly due to their failure to reach the site ofcarotenoid biosynthesis in the chloroplast ratherthan to the existence of an entirely novel pathwayof biosynthesis. At least this is the simplifyingassumption which must be made at the moment,and it is supported by the fact that both acetateand mevalonate are incorporated into the sterolsof the seedlings and it is known that the basicpathways of sterol and carotenoid synthesis inother organisms are similar. The present observa-tion that most of the sterols (80-90%) of theilluminated seedlings are in the chloroplasts isconsistent with the views of Chibnall (1939), whofound a high lipid content in the chloroplast com-

pared with that of the cytoplasm, and Menke &Jacob (1942), who found that 20% of the chloro-plast lipids were sterols. These chloroplast sterolsbecome highly active in the presence of labelledacetate and mevalonate, and the question ariseswhy these and not the carotenoids are labelled.This cannot be answered at the moment but twopossibilities exist: (a) acetate and mevalonatecannot enter the chloroplast as such, but in thecytoplasm are rapidly converted into trace amountsof labelled sterols, which are then transferred tothe chloroplasts; (b) the acetate and mevalonatecan penetrate the chloroplast but are very rapidlytaken up by the sterol-synthesizing system beforethey can reach the carotene-synthesizing site.

SUMMARY

1. The carotenoids of etiolated maize seedlingsare almost entirely xanthophylls, only traces of,-carotene being present.

2. Intact or excised etiolated seedlings synthe-size ,B-carotene and xanthophylls when illuminated.All the light-synthesized carotenoids are present inthe chloroplasts which also develop on illunmination.

3. [2-14C]Acetate and [2-14C]mevalonate cannotpenetrate the root system of intact maize seedlings,and are thus not incorporated into the unsaponi-flable matter. They are rapidly incorporated intothe unsaponiflable matter of excised seedlingskept either in the dark or illuminated. There is,however, only an insignificant incorporation into,B-carotene in illuminated excised seedlings.

4. 14C02 is rapidly incorporated into the un-saponifiable matter of illuminated intact andexcised seedlings, and there is a very marked andpreferential incorporation into ,B-carotene.

5. Hydroxylamine (0 001M) inhibits the syn-thesis of ,B-carotene, chlorophyll and, presumably,chloroplast development in illuminated excisedseedlings.

6. It is concluded that the synthesis of ,B-carotene in illuminated seedlings occurs in thechloroplasts and utilizes photosynthetically-fixedC02; this is an integral part of the process ofturning an etiolated plant into a photosyntheticplant.Thanks are due to Hoffmann-La Roche (Basel, Switzer-

land) for generous gifts of synthetic ,B-carotene, to Dr R. Y.Cornforth for the synthesis of DL-[2-14C]mevalonate and toMiss B. M. Eales and Miss B. Begnett for skilled technicalassistance.

REFERENCES

Bandurski, R. S. (1949). Bot. Gaz. ill, 95.Beck, W. A. (1937a). Protoplasma, 28, 273.Beck, W. A. (1937 b). Plant Phy8iol. 12, 885.Blaauw-Jansen, G., Komen, J. G. & Thomas, J. B. (1950).

Biochim. biophy8. Acta, 5, 179.

Vol. 70 STUDIES IN CAROTENOGENESIS 617Braithwaite, G. D. & Goodwin, T. W. (1957). Biochem. J.

66, 31PChibnall, A. C. (1939). Protein Metabolism in Plants, p. 153.New Haven: Yale University Press.

Glover, J. (1956). In Modern Methods of Plant Analysis,vol. 1, p. 325. Ed. by Paech, K. & Tracey, M. V.Heidelberg: Julius Springer.

Goodwin, T. W. (1952). The Comparative Biochemistry ofthe Carotenoids, p. 292. London: Chapman and Hall.

Goodwin, T. W. (1955). In Modern Methods of PlantAnalysis, vol. 2, p. 272. Ed. by Paech, K. & Tracey,M. V. Heidelberg: Julius Springer.

Goodwin, T. W. (1958a). Biochem. J. 68, 503.Goodwin, T. W. (1958b). Biochem. J. 68, 26P.Kay, R. E. & Phinney, B. (1956). Plant Physiol. 31,

226.Loeffler, J. E. (1955). Yearb. Carneg. ln8tn, 54, 159.Menke, W. & Jacob, E. (1942). Hoppe-Seyl. Z. 272,

227.Smith, J. H. C. & Benitez, A. (1955). In Modern Methods of

Plant Analy8is, vol. 4, p. 142. Ed. by Paech, K. &Tracey, M. V. Heidelberg: Julius Springer.

Strain, H. H. (1938). Publ. Carneg. Instn, no. 490.Zalokar, M. (1955). Arch. Biochem. Biophya. 56, 318.

Canavanine and Related Compounds in Leguminosae

BY E. A. BELLDepartment of Biochemi8try, King'8 College, London, W.C. 2

(Received 14 May 1958)

Canavanine (o-amino-8-guanidinoxybutyric acid)occurs in the free state in the seeds of the jackbean, (anavalia en8iformi8, and in the seeds of C.tineata and C. obtusifoltia (Kitagawa & Tomiyama,1929; Kitagawa, 1937; Damodaran & Narayanan,1939). Fearon (1946) showed that compounds con-taining the guanidoxy grouping,

-0NHI*C(:NH)*NH2,react with trisodium pentacyanoarmnonioferrate inaqueous solution at pH 7, to give colours whichrange from orange-red with N-methoxyguanidineto magenta with canavanine itself. With thisreagent a representative selection of plants weretested for the presence of canavanine, and positiveresults were obtained with species of Medicago,Ornithopu8 and Colutea. Canavanine was isolatedfrom C. arborescen8 (Fearon & Bell, 1955).The inhibition of the pentacyanoammonioferrate

colour reaction by such compounds as ascorbic acidand creatinine (Fearon & Bell, 1955) makes itsdirect application to biological tissues or extractsof limited value. This difficulty has been overcomeby using paper chromatography and paper iono-phoresis to separate pentacyanoammonioferrate-reacting compounds from others which inhibit ormask the colour reaction.The occurrence of canavanine in concentrations

of 3-5% of dry weight in the seeds of Canavaliaen8iformi8 and Colutea arborescen8 suggests itsfunction as a nitrogen-storage product and hencethe existence of an enzyme system associated withits utilization. Damodaran & Narayanan (1940)reported the existence in the seeds of Canavatiaensiformis of an enzyme which brought about thehydrolysis of canavanine to canalin and urea. Theyalso showed that this enzyme was identical with

jack-bean arginase, though the optimum pH ofhydrolysis was 9-4 for arginine and 7-5 for cana-vanine. It was suggested that canavanine mightbe the natural substrate of this arginase. In viewof these facts, and of the occurrence of canavaninein plants other than Canavalia, it seemed likelythat other known sources of arginase in the Legu-minosae, such as vetch (Vicia 8ativa) and redclover (Trifolium praten8e), might also prove to besources of canavanine. This is now shown to be so.It is also shown that canavanine occurs in theseeds of many leguminous plants; quantitativedeterminations have been made on those seedswhich give the strongest colour reaction withtrisodium pentacyanoammonioferrate, and theamino acid has been isolated from two of them. Thepresence in leguminous seeds of compounds otherthan canavanine which also react with the penta-cyanoammonioferrate at pH 7 has been demon-strated.

EXPERIMENTALPentacyanoammonioferrate reagent (PCAF). A 1% (w/v)

solution of PCAF (Fearon, 1946) in Cu-free distilled waterwas used.

Pho8phate buffer (pH 7). This was prepared by mixingequal volumes of 0 066M-NaH2PO4 and 0 066M-Na2HPO4.

Determination of m-amino nitrogen. This was determinedas a-amino acid carboxyl by the method of Van Slyke,MacFadyen & Hamilton (1941).

Specificity of the PCAF colourreaction for canavanine

Fearon (1946) found that, of the substituted guanidineswhich he tested, only the guanidoxy compounds reactedwith PCAF to give orange and red colours in the rangepH 5-7-5, and of these only canavanine gave a vividmagenta.