thebiosynthesis of a-aminolevulinic higher plantsradiochromatogram scanner, model 7201, and the...

Plant Physiol. (1974) 53, 297-303

The Biosynthesis of a-Aminolevulinic Acid in Higher Plants

II. FORMATION OF 14C-6-AMINOLEVULINIC ACID FROM LABELED PRECURSORS IN GREENINGPLANT TISSUES'

Received for publication August 2, 1973 and in revised form October 5, 1973

SAMUEL I. BEALE' AND PAUL A. CASTELFRANCODepartment of Botany, University of California, Davis, California 95616

ABSTRACT

5-Aminolevulinic acid was accumulated by greening cucum-ber (Cucumis sativus L. var. Alpha green) cotyledons, barley(Hordeum sativum var. Numar) leaves, and bean (Phaseolusvulgaris L. var. Red Kidney) leaves in the presence of various"C-labeled precursors and levulinic acid, a competitive inhibitorof 5-aminolevulinic acid dehydrase. The radioactivity in the ac-cumulated 8-aminolevulinic acid was measured.The most effective labeled precursors were the 5 carbon

dicarboxylic compounds glutamate, glutamine, and a-keto-glutarate. "C-Labeled glycine and succinate were relativelypoor. The carboxyl and the methylene carbons of glycine wereincorporated into 5-aminolevulinic acid to about equal extent.The carboxyl carbon of glutamate was incorporated almost aswell as the internal carbons of the same compound. These re-sults are inconsistent with the succinyl CoA-glycine succinyltransferase (5-aminolevulinic acid synthetase) mode of 5-amino-levulinic acid production.'When the same experiments were performed on turkey

blood (which, as avian blood in general, possesses 5-amino-levulinic acid synthetase), 5-aminolevulinic acid was labeledmost effectively from glycine-2-"C, moderately well from gly-cine-1-VC and glutamate-3,4-"C and not at all from glutamate-1-14C.

It appears probable that greening higher plant tissues possessan alternate route to 5-aminolevulinic acid in which the carbonskeleton of glutamate (and a-ketoglutarate) is incorporatedintact into the first committed metabolite of the chlorophyllpathway.

It is generally accepted that the biosynthesis of Chl followsa pathway identical to that of heme from ALA,' the first com-pound unique to the heme biosynthetic pathway through pro-toporphyrin IX, the immediate precursor of heme. In photo-synthetic bacteria, heme and bacteriochlorophyll arise from acommon pool of intermediates and share a common sequenceof enzymes. Lascelles (9) has discussed a feedback regulationscheme whereby the common part of the pathway is regulated

IThis work was supported by National Science FoundationGrant GB 31261.

2 Present address: Department of Plant Pathology, University ofCalifornia, Davis, Calif. 95616.

'Abbreviations: ALA: 8-Aminolevulinic acid; ALA pyrrole: 2-methyl-3-carbethoxy-4-(3-proprionic acid)pyrrole.

at the step of formation of ALA from glycine and succinylCoA catalyzed by the enzyme succinyl CoA-glycine succinyltransferase (ALA synthetase).

Considerably less is known about heme and Chl biosynthesisin plants. First, there is no information concerning the degreeto which the two pathways share common pools of interme-diates and enzymes. Second, the source of ALA in plants ispresently unknown. No unequivocal evidence exists for thepresence of ALA synthetase in plants (including algae), and thesuggestive evidence which does exist does not indicate a rolefor this enzyme in Chl biosynthesis, since the enzymic activityis not correlated with changing rates of Chl synthesis (17, 23).

Because we were unsuccessful in demonstrating ALA syn-thetase activity in extracts of greening plant tissues, we turnedto an in vivo method of measuring the accumulation of ALAwhen the enzyme ALA dehydrase was inhibited by levulinicacid. We showed that the ALA which accumulated in the pres-ence of levulinic acid was the ALA destined for Chl synthesis(2). We now report that the incorporation of label into thislevulinic acid-induced ALA from a number of 14C precursorshas been accomplished, and that the pattern of incorporationindicates an alternate route of ALA formation.A preliminary account of portions of this work has been

published (1).

MATERIALS AND METHODS

Plant Materials. Cucumber, barley and bean seedlings weregrown as described elsewhere (2).Avian blood. Blood was withdrawn from the subclavian

vein of a 14-month-old broad-breasted white hen turkey into asterile syringe containing 1 ml of sodium heparin (1000 U.S.P.units/ml) per 50 ml of blood to be withdrawn. The blood wasimmediately cooled in an ice bath, and experimental incubationwas begun no more than 30 min thereafter.

Incubation Conditions. Plant tissues were incubated as de-scribed previously (2). Whole heparinized blood was incubatedat 37 C, in shallow layers with moderate shaking to insuregood aeration.Measurement of Respired '4CO2 Plant tissue was incubated

in 250-ml Erlenmeyer flasks fitted with rubber stoppers fromwhich were suspended 2.1-cm glass fiber filter discs wetted with0.1 ml of 1 N NaOH. The filter discs were replaced every 30min, and the trapped "4CO, on the discs was assayed by liquidscintillation. Radioactivity was measured with a Tricarb scin-tillation spectrometer Model 3310 using 5 ml of scintillationcocktail (0.3 g of POPOP, 15 g of PPO, 150 g of naphthalene,720 ml of absolute ethanol, 1140 ml of toluene, 1140 ml ofp-dioxane) per vial. The filter discs were allowed to remain inthe cocktail for at least 1 hr before counting.

Purification of 34C-ALA. At the end of the incubations, plant297

www.plantphysiol.orgon March 15, 2020 - Published by Downloaded from Copyright © 1974 American Society of Plant Biologists. All rights reserved.

http://www.plantphysiol.org

BEALE AND CASTELFRANCO

tissues were homogenized in 3 ml of 5% HClO4 per g of freshtissue with a Polytron tissue homogenizer. Turkey blood in-cubations were terminated by addition of an equal volume of10% HC104. After centrifugation to remove the precipitatedprotein and cell debris, the supernatant solutions were neu-

tralized to pH 4.5 with KOH, allowed to stand overnight at 4C, then centrifuged to remove the precipitated KClI4. Thesupernatant solutions were than applied to columns of Dowex-50W-X8, 100 to 200 mesh, which had been previously washedwith 1 N HCl, and H20. Two ml of resin bed volume were

allowed for every 15 ml of supernatant solution to be added.The columns were then washed with water, and cationic ma-

terials (including ALA) were eluted with 5 ml of 1 M am-

monium acetate (pH 7.0) per ml of resin bed volume. Thecationic fraction was adjusted to pH 6.8 with Na2PO,, thenheated for 10 min in a boiling water bath in the presence of1% ethylacetoacetate, to convert the ALA to 2-methyl-3-car-bethoxy-4-(3-propionic acid) pyrrole (ALA-pyrrole) (11). Aftercooling, the pH was adjusted to 7.2 with NaOH, and thesolution was extracted with three 1-volume portions ofchloroform, which removed the excess ethylacetoacetate. Thenthe pH was lowered to 3.0 with HCl, and the solution was ex-

tracted with three one volume portions of diethyl ether. Thecombined ether extracts which contained the ALA-pyrrolewere washed with three 1-volume portions of deionized waterto remove excess acetic acid.

Radiopurity of 14C-ALA. The ether extract was storedovernight in the freezer; the ether was decanted before the icehad a chance to melt and concentrated under N2. Chromatog-raphy of ALA-pyrrole was performed on Whatman No. 1paper in a solvent of 1-butanol-l-propanol-5 % aqueous am-

monia (2: 1: 1). Radioactive spots were located with a Packardradiochromatogram scanner, Model 7201, and the ALA-pyrrole was visualized by spraying the paper with the follow-ing mixture: 200 mg of p-dimethylaminobenzaldehyde dis-solved in 8 ml of ethanol and 2 ml of 12 N HCl.

Gas-liquid chromatography of ALA-pyrrole was performedafter methylation with diazomethane in ether. Samples were

injected onto a 2-foot column of 1.5% OV-210 on 100 to 120mesh Gas-Chrom Q (Applied Science Laboratories, Inc., StateCollege, Pa.), operated at 180 C on an Aerograph Model A-90-P gas chromatograph (Wilkins Instrument and Research),equipped with a Nuclear Chicago Model 4998 gas flowradiation counter.

Reagents. Sodium heparin (1000 U.S.P. units per ml) was ob-tained from Invenex Pharmaceuticals, San Francisco, Calif.

Radiochemicals were purchased from the following sources:sodium formate-'4C, glycine-1-14C, glycine-2-14C, L-proline-U-4C and ALA-4-'4C from Schwarz Bioresearch; succinic acid-2,3-`4C, glyoxylic acid-1-'4C, glyoxylic acid-2-"C, L-glutamine-U-"C, and DL-glutamic acid-l-"4C from Amersham/Searle; a-

ketoglutaric acid-U-"C, and DL-glutamic acid-3, 4-14C fromICN; acetic acid-i-"4C, acetic acid-2-"C, ALA-4-'4C, DL-glu-tamic acid-1-2C and DL-glutamic acid-3,4-'4C from New Eng-land Nuclear.

RESULTS

Incorporation of '4C-Labeled Compounds into ALA byCucumber Cotyledons. Six-day-old etiolated cucumber cot-yledons were illuminated at 28 C in 240 ft-c of cool-whitefluorescent light for 4 hr. Samples of 100 cotyledon pairs (100cotyledon pairs consistently weighed between 5.75 and 5.9 g)were placed in 9.0-cm dishes and bathed with 5 ml of testsolution composed of 100 mm levulinic acid, 10% dimethyl-sulfoxide and approximately 10 /1.c of one of the '4C-labeled

compounds listed in Table I. After 4 more hr of incubationin the light at 28 C, the samples were ground in 5% HCIO, andALA was purified as described under "Materials and Methods."Of the compounds tested, those which labeled ALA most

effectively were glutamate, ae-ketoglutarate, and glutamine.Of particular interest was the incorporation of label intoALA from C1-labeled glutamate. Glycine, whether labeled in

C1 or in C2, was particularly poor as a labeled precursor ofALA, and succinate was also relatively poor.

Incorporation of "C Compounds into ALA by GreeningBean Leaves. Ten-day-old etiolated kidney bean seedlings were

illuminated for 5 hr at 30 C, then the primary leaves were

excised, separated, and placed in a single layer in Petri dishes,1.55 g of tissue per dish. Five ml of 25 mm levulinic acid con-

taining 10 ,c of "C-labeled substrate were added to eachPetri dish. After 3 more hr of illumination, the tissue was

homogenized and analyzed for total ALA content and radio-activity of the ALA. As with cucumber cotyledons, glutamatelabeled the ALA much more effectively than glycine (TableII).

Incorporation of "C Compounds into ALA by GreeningBarley Leaves. Seven-day-old etiolated barley leaves were

illuminated for 4 hr at 30 C, then the leaves were cut into 10-mm segments, and 1-g portions of tissue were placed in 50-mlbeakers containing 5 ml of 20 mm levulinic acid and 25 ,ucof "C compound. After 4 more hr of illumination, the tissuewas homogenized. and the radioactivity in the purified ALA-pyrrole was determined. As with cucumber cotyledons andbean leaves, glutamate was much more effective than glycinein labeling ALA (Table III).

Table 1. Iincorporationi of"Cfrom Exogenious Compounilds inlto ALAby Greenintg Cucuimber Cotyledons

These are the pooled results from five separate experiments inwhich 6-day old etiolated cucumber cotyledons were preillumi-nated for 4 hr, then incubation was continued for 4 more hr in thepresence of 100 mm levulinic acid, lO%10lc of the indicated 'IC compound.

dimethylsulfoxide, and

Labeled Precursor ALA

Name Specific Total Radioactivityradioactivity

~sc'Mmole nmoles/g Cpm/1lO6 Cpml,Ac,,'1A?;oIe .fresh wt supplied*g

Acetate-U-1AC 47.5 302 166Formate-'4C 52.5 320 35DL-Glutamate-1-14C 25 229 232DL-Glutamate-1-14C 274 436DL-Glutamate-1-14C 5.3 234 231DL-Glutamate-3,4-14C 55.5 251 262DL-Glutamate-3,4-14C 277 308DL-Glutamate-3,4-14C 245 491DL-Glutamate-3,4-14C 14.2 253 392L-Glutamine-U-_4C 45 258 405Glyoxylate-1-14C 7.4 219 49Glyoxylate-2-14C 7.4 232 140Glycine-1-'4C 46.5 289 12Glycine-2-14C 36.7 188 13Glycine-2-'4C 277 16a-Ketoglutarate-U- 4C 200 198 229a-Ketoglutarate-U-14C 239 277Succinate-1,4-14C 20.4 253 48L-Proline-U-14C 200 272 65

298 Plant Physiol. Vol. 53, 1974



PVC-ALA FROM LABELED PRECURSORS

Table II. Inicorporationi of 'IC ilito ALA by Greeniing Beanz LeavesTen-day-old etiolated bean seedlings were illuminated for 5 hr,

then excised primary leaves were illuminated for 3 more hr in thepresence of 25 mm levulinic acid and 10 Ac of the indicated 'ICcompound.


Name Specific Total Radioactivityradioactivity

tsyr/iole nntoles/g cs/106cppi ;freshwi fresh wt

Glycine-1-"4C 46.5 314 117Glycine-2-14C 36.7 271 264DL-Glutamate-1-"4C 25 242 742DL-Glutamate-3,4-14C 55.5 262 2640

Table III. Incorporationi of 1"C i/ito ALA by GreeniilngBarley Leaves

Seven-day-old etiolated barley was illuminated for 4 hr, then10-mm leaf segments were illuminated for 4 more hr in the presenceof 20 mm levulinic acid and 25 ,uc of the indicated 14C compound.


Name Specific Total Radioactivity

tir/osmole nmoles/g rpm/JO' rpm,Ac/;"mole fresh wt supplieds gfresh wi

Glycine-1-"4C 46.5 153 75Glycine-2-"4C 36.7 163 66DL-Glutamate-1-"4C 25 167 739DL-Glutamate-3, 4-14C 55.5 148 900

Respiration of "C-Labeled Compounds by Greening Cucum-ber Cotyledons. Six-day-old etiolated cucumber cotyledonswere illuminated for 4 hr; then they were placed in Erlenmeyerflasks, 50 cotyledon pairs per flask, and bathed with 2 ml ofsolution containing 100 mm levulinic acid, 10% dimethyl-sulfoxide, and 5 /ic of labeled compound. The flasks werestoppered and placed in the light at 28 C; the respired CO.was measured as described under "Materials and Methods."All of the labeled compounds were respired at comparablerates (Fig. IA) based on expectation from position of label,indicating that they all entered the cells.

Respiration of "C Compounds by Greening Bean Leaves.Ten-day-old etiolated kidney bean seedlings were illuminatedfor 5 hr, then the primary leaves were excised, separated,placed in Erlenmeyer flasks, 1 g of tissue per flask, and bathedwith 2.5 ml of 25 mm levulinic acid containing 5 /uc of the "C-labeled compound. During the next 3 hr of illumination, CO2samples were collected as described under "Materials andMethods." As with cucumber cotyledons, glycine and gluta-mate are both respired, but the C1 of glycine or glutamate arerespired to a much greater extent than are the C. of glycine orthe C, and C, of glutamate (Fig. 1B). Also the rate of "CO2formation was in general much greater in bean leaves than incucumber cotyledons.

In Table IV, the relative abilities of glycine-1-"C, glycine-2-4C, DL-glutamate-1-"'C, and DL-glutamate-3 ,_14C to labelALA and CO, are compared for both cucumber cotyledonsand bean leaves. Note that DL-glutamate 3,44-"C was the leasteffective precursor of 14CO2 but the most effective precursorof 14C-ALA in both tissues.

RADIOPURITY OF C"-ALA FRACTIONS

Paper Chromatography. Portions of four samples fromcucumber cotyledon preparations, containing purified "C-

ASUCCINATE 1,.4- 1c

o GLYCINE I1c1 GLYCINE- 2 I C

OL GLUTAMATE 1 14c

0 ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~14KEoOEGtUTAEATE U c

o GLYOXYLATE C

*GLYOXYLATE 2

i

x

FIG. 1. Formation of "CO2 from labeled precursors by green-ing cucumber cotyledons (A) and kidney bean leaves (B). Six-day-old etiolated cucumber cotyledons were illuminated for 4 hr, thenbathed in a solution of 100 mm levulinic acid, 10% dimethylsulf-oxide, and 5 ,uc of the indicated "C compounds. Ten-day-oldetiolated bean seedlings were illuminated for 5 hr, the primaryleaves were excised and bathed in a solution of 25 mm levulinicacid and 5 ,Ac of the indicated "C compound. Evolution of "CO2during continued illumination was measured as described in thetext. Cumulative respired "CO2 is plotted against hours of expo-sure to the test solutions.

Plant Physiol. Vol. 54, 1973 299



300 BEALE AND C

ALA-pyrrole labeled from L-proline-U-`C, DL-glutamate-3,4-4C, L-glutamine-U-`C, or acetate-U-14C, were chromato-graphed on paper as described under "Materials and Methods."After chromatography, the paper was cut into vertical stripsand passed through the radiochromatogram scanner. Eachsample yielded a single major radioactive component, whichcoincided with the only Ehrlich-positive spot. The radio-activities of the peaks were proportional to the total radioac-tivities in the samples (Table V). If the ether extracts werenot carefully washed with H20 and the dissolved H20 re-moved by freezing out, both radioactivity and Ehrlich-positivematerials streaked during chromatography.Gas Chromatography. Portions of the above four samples

were methylated and injected into the gas chromatograph asdescribed under "Materials and Methods." All samples had asingle mass peak corresponding to authentic methylated ALA-pyrrole and a single radioactivity peak corresponding to theALA-pyrrole mass peak. The relative radioactivities of thepeaks were proportional to the total radioactivities of thesamples (Table VI).

Table IV. Relative Label Incorporationi inlto ALA anid Respired C02by Greeninig Cucumber Cotyledonis anid Beani Leaves

In each column the data are normalized with respect to gly-cine-1 -14C.

Cucumber Cotyledons Bean LeavesLabeled Precursor

C02 ALA C02 ALA

Glycine-1-'4C 1.0 1.0 1.0 1.0Glycine-2-ttC 0.9 1.1 0.23 I 2.4DL-Glutamate-1-l4C O.9 27 1.25 6.5DL-Glutamate-3,4-54C 0.13 27 0.20 23

AS- TELFRANCO Plant Physiol. Vol. 53, 1974

Table VII. Effect of Antaerobiosis oni ALA Accumulationi anidLabelinig of ALA by Glutamic Acid 3,4-14C in Greeninlg

Cucumber CotyledonsSix-day-old etiolated cucumber cotyledons were illuminated for

4 hr in air, then illumination was continued for 3 more hr in thepresence of 100 mm levulinic acid, 10%c dimethylsulfoxide, 9 ,tc ofglutamate-3,4-14C, and under the indicated atmosphere.

Accumulated ALA

Atmosphere Ratio of B/AA. Total B. Radioactivity

accumulated

n,noles/g fresh uwt cpn/ g fresh wiAir 312 5440 17.4O2 450 8485 19.1N2 67 1151 17.1

Table VIII. Incorporation of 'IC in2to ALA by Whole HeparinizedTuirkey Blood

Freshly drawn blood was incubated for 60 min in the presenceof 100 mm levulinic acid (neutralized to pH 7.5 with NaOH) and5,uc of the indicated 14C compound.


:\ameSpecific .X. Total B- RadioRatio of B/A-Name radio- A. Total B.cRadio-

activity activity

Mc/,C/mole ntnoles sz/ppliedGlycine-1_i4C 46.5 61.8 135 2Glycine-2-14C 36.7 44.5 1092 25DL-Glutamate-1-'4C 25 67.8 0 0DL-Glutamate-3,4-'4C 55.5 58.4 248 4

Table V. Paper Chromatography of ALA-pyrroleRadiopurity of '4C-ALA-pyrrole was determined as described

in the text.

C. RelativeA. RF of B. RF Of Peak Height D. CPM RaLabeled Precursor Ehrsich-e Majioriv Of Major in Total D/CSpostiv Radioantiv Radioactive Sample

SpotCompnentComponent

L-Proline-U-'4C 0.43 0.43 4 1311 330DL-Glutamate- 0.48 0.48 90 28200 310

3,4-14CL-Glutamine- 0.41 0.42 98 32500 340U 14C

Acetate-U-'4C 0.40 0.42 83 20700 250

Table VI. Gas Chromatography of Methylated ALA-pyrroleRadiopurity of the 14C-ALA-pyrrole was determined as de-

scribed in the text.

Labeled Precursor

L-Proline-U-1 4C

DL-Glutamate-3,4i14CL-GI utamine-U-i 4C

Acetate-U-iiCAuthentic ALA-pyrrole

A.RetentionTime

ntin

9.259.299.029.259.02

B. Radio- C. Radio-

activity in activity inWekAhole

Sample

cpm

100 13112100 282002200 325001500 20700

Ratio ofC/B

13.113.414.813.8

02 Requirement for '4C-ALA Formation. Six-day-old etio-lated cucumber cotyledons were illuminated for 4 hr, thenplaced, 100 cotyledon pairs per sample, in 250-ml Erlenmeyerflasks and bathed with 4.5 ml of solution (100 mm levulinicacid, 10% dimethylsulfoxide) containing 9 ,uc of glutamate-3, 4-'4C. One flask was flushed continuously with N2, anotherwith 02, and a third was left in contact with air. After 3 morehr of illumination, the samples were ground and analyzed fortotal ALA and radioactivity in the ALA fractions. Table VIIshows that anaerobiosis inhibited total ALA accumulationwhile the specific radioactivity in the ALA fraction remainedthe same.ALA Accumulation in Turkey Blood Treated with Levulinic

Acid. Four and one-half ml portions of freshly drawn turkeyblood were placed in 25-ml Erlenmeyer flasks containing 1.5ml of various concentrations of levulinic acid neutralized to pH7.5 with NaOH. The flasks were shaken at 37 C, and sampleswere taken periodically from ALA determination. One hundredmM levulinic acid was the most effective concentration underthese conditions, and most of the ALA was accumulatedwithin the first 30 min.

Larger samples of fresh blood (47.5 ml) were incubatedunder identical conditions in 125-ml Erlenmeyer flasks con-taining 2.5 ml of neutralized levulinic acid (final concentra-tion 100 mM) and 5 [cc of "C compound. After 60 min of in-cubation, equal volumes of 10% HCIO4 were added to theflasks to stop the incubations. The ALA-pyrrole fractionswere purified, and their radioactivities determined. Unlikethe results with plant tissues, glycine-2-'4C was a much more

I



14C-ALA FROM LABE

Table IX. Comparisoni of Relative Label Ilicorporationi iiito ALAIn each column the data are normalized with respect to glycine-

1-14C.

Labeled Precursor Cucumber Barley TurkeyCotyledons Leaves Leaves Blood

Glycine-1-14C 1.0 1.0 1.0 1.0Glycine-2-14C 1.1 2.4 2.4 8.1DL-Glutamate-1-14C 27 6.5 10 0DL-Glutamate-3,4-14C 27 23 12 1 .8

effective precursor of '4C-ALA than the other compoundstested (Table VIII).

Table IX is a summary of the relative abilities of the fourspecifically labeled amino acids (glycine-l-"4C, glycine-2-'4C,glutamate-l-"lC, and glutamate-3, 4-'4C) to label ALA incucumber cotyledons, barley and bean leaves, and turkey blood.It shows clearly the marked differences in the abilities of thefour specifically labeled compounds to label ALA in planttissues and in blood.

DISCUSSION

,LED PRECURSORS 301

since these tissue cultures were incapable of producing Chl, therelevance of this enzyme to Chl biosynthesis remains unknown.

In a recent report by Wellburn and Wellburn (22), glycine-2-"C was found to be incorporated into Chl by chloroplastpreparations which were also able to incorporate ALA intoChl. The authors concluded that ALA synthetase was pres-ent in their chloroplast preparations, although the exclusionof the C1 of glycine from the product was not reported, and noevidence was given that the label was incorporated into thetetrapyrrole nucleus, in contrast to the phytol and the methylester groups of Chl. This claim to the detection of ALA syn-thetase was therefore premature.The most recent report of ALA synthetase from a plant

source describes the enzyme activity found in the green skinsof potatoes stored at low temperature and dim light (17).Again, the preferential incorporation of the C2 over the C1 ofglycine was not shown. Although the enzyme was purifiedfrom the original homogenate in such a way as to remove smallmolecules, some ALA was synthesized from glycine andsuccinate even without the addition of pyridoxal phosphate,CoA, ATP, Mg2+, or succinyl-CoA synthetase. There was nocorrelation between the observed enzyme activity and the rateof Chl synthesis in the tissue. Evaluation of the significanceof this ALA-forming system must await further research.

In the classic studies of Shemin and co-workers (16) on thebiosynthesis of heme, use was made of specifically labeledprecursors. It was shown that all the nitrogen atoms of hemewere derived from the amino nitrogen of glycine, while thecarbon atoms of heme were derived from the methylene car-bons of glycine and the four carbons of succinate. The car-

boxyl carbon of glycine was completely excluded. A condensa-tion of glycine and succinate to form CO2 and ALA wasproposed and later an enzyme, succinyl CoA-glycine succinyltransferase (ALA synthetase) was detected in avian erythro-cytes (6), mammalian tissues (3, 10), photosynthetic bacteria(8), and more recently, in yeast (14, 15) and nonphotosyntheticbacteria (21).

Figure 2A shows how in the ALA synthetase reaction glycineand succinyl CoA condense to form ALA, with loss of the C1of glycine as CO2. Note also how succinyl CoA can be formedfrom a-ketoglutarate or glutamate with loss of the C1 of thesecompounds as CO2.

Early feeding studies with labeled Chl precursors to green-ing higher plant (18, 19) and algal (4) tissues failed to show thepredicted labeling pattern: (a) glycine was no more effectivethan acetate as a labeled Chl precursor; (b) the carboxyl carbonof glycine was incorporated into Chl to an appreciable extent;(c) a high percentage of the 14C label derived from either gly-cine-1l-wC or 2-14C turned out to be in the methyl ester group,which is added to the tetrapyrrole moiety at a relatively latestep in the Chl biosynthetic pathway, and is in no case derivedfrom ALA (4).

There are several reports of the presence of ALA syn-thetase in plant tissue extracts. The earliest of these (12) is an

abstract which was not followed by a publication sufficientlydetailed to allow evaluation of the claim. Another group re-ported the detection of ALA synthetase in a crude homogenatefrom nongreening soybean callus cultures (23). No data weregiven on the specific requirements for glycine, succinyl CoA,and pyridoxal phosphate. The preferential incorporation ofthe C2 over the C1 of glycine was not demonstrated. The ob-served ALA synthetase activities were not correlated with invivo changes in porphyrin production. No attempt was made toexclude the possibility that the enzyme activity might be dueto microbial contamination of the tissue cultures. Finally,

a- keto-gi utarate

COOH

CH2

CH2

C=o**COOH

ami noacid

trar

CoASH -

Mg++ a-ketoglutarateLiPS2 dehydrogenase

**CO2 k--

COOH

succinyl- CHH2CoA CH2

CO-SCc

L-)COC

coo

CHrc-keto- CH

CH.glutarate 2

C=C

coC

reduction

COI

y,o-dioxo- CH,valerate CH.

CH(

keto COOHacid

CH2

PALP CH2nsaminase 2

CH-NHO**COOH

glutamate

A

C(

ALA synthetasePALP .>

)A C

CH NH *Co2 C*COOH

glyci ne

H

transaminase2 PALP

)H amino ketoacid acid

B

amino ketoDH acid acid

2 V PALP2 transaminase

:OOH

'H2H2

=o

:H2-NH2

ALA

COOH

CH2

CH2 glutamate

CH-NH2

COOH

possibleI cyclic

i ntermedi ate

'ICOOH

CH2

CHi2C-=o

CH2-NH2

ALA

FIG. 2. A: ALA formation from succinyl CoA and glycinecatalyzed by ALA synthetase, and succinyl CoA formation fromglutamate or a-ketoglutarate, illustrating the loss of C1 of glycine,a-ketoglutarate, and glutamate. B: Hypothetical formation of ALAfrom glutamate or a-ketoglutarate, either via -y,S-dioxovalerate or

another, unknown route, where the C1 of glutamate of a-ketoglu-tarate is incorporated into ALA. LipS2: lipoic acid; PALP: pyri-doxyl phosphate; TPP: thiamin pyrophosphate.

Plant Physiol. Vol. 54, 1973



302BEALE AND CASTELFRANCO

In the experiments reported here, "C has been incorporatedinto the ALA which accumulates in greening tissues in re-sponse to levulinic acid treatment. In all three plant tissuestested (cucumber cotyledons, barley and bean leaves), the mosteffective "C precursor was glutamate, while glycine was avery poor "C donor. Of particular significance were the in-corporations of the C, of glutamate to a comparable extentas theC3 andC4, and the C, of glycine to about the same ex-tent as the C2. Clearly, this pattern is incompatible with thesuccinyl transferase mode of ALA formation (Fig. 2A).Our experiments with turkey blood were done in order to

test our methods on a type of system which is known to formALA via the succinyl transferase route. We have demonstratedthat, like plants, whole turkey blood can accumulate ALA inthe presence of levulinic acid, but that, unlike plants, the labelis most easily accumulated from the methylene carbon ofglycine, and to a much lesser degree from the carboxyl carbonof glycine (Table VIII). The incorporation of label fromglutamate-3, 4-"C probably occurs via transamination to a-ketoglutarate, entry into the tricarboxylic acid cycle, and con-version to succinyl CoA. The inability of glutamate-l-"C tolabel ALA is in agreement with this scheme (Fig. 2A), for C,is lost during the conversion of a-ketoglutarate to succinylCoA. We conclude that our methods give the expected labelingpattern in an avian blood system of the type known to formALA via ALA synthetase (16).

In an attempt to test the possibility that the ALA-labelingresults obtained with plant tissues might be caused by thedifferential entry of the "C compounds into the cells, therespiration of a number of test compounds was studied in bothcucumber cotyledons and bean leaves (Fig. 1, Table IV). Notonly were all the test compounds respired, but the one whichwas respired the least, glutamate-3, 4-"C, was the most effectivedonor of "C to ALA. This finding suggests that permeabilitybarriers are not responsible for the differences in the labelingabilities of the test compounds, and that the labeling patternis due to a mode of ALA formation in which glutamate is amore immediate precursor of ALA than glycine. Furthermore,the respiration patterns indicate that the label appearing inALA is not due to refixation of respired "CO, from the testcompounds; for in that case, those compounds which form themost "CO2 would also be expected to form the most "C-ALA.

There are interesting differences among the various planttissues studied. While cucumber cotyledons incorporate gluta-mate-l-"C into ALA to the same extent as glutamate-3,4-14C,bean leaves incorporate glutamate-3,4-"C more readily thanglutamate-1-1C (Table IV). This difference could be the re-sult of a higher tricarboxylic acid cycle activity in the beantissue (as indicated by the faster respiration rates in Fig. 1)combined with a rapid equilibration between the glutamateand the a-ketoglutarate pools. In the tricarboxylic acid cycle,the radioactivity of a-ketoglutarate-l-"C is lost during the firstturn, while the radioactivity of a-ketoglutarate-3,4-"C is re-distributed within the molecule during the first two cyclesand decays hyperbolically thereafter.

Alternative routes of ALA biosynthesis have been suggestedby various authors when ALA synthetase could not be de-tected in plant tissue extracts (5, 20). These alternatives all in-volve the transamination of y, 8-dioxovaleric acid (a-keto-glutaraldehyde), a reaction which can be carried out by en-

zymes present in Rhodopseudomonas spheroides (13) andChlorella (5). Although some authors have discounted the im-portance of this route because the transaminase levels do notfollow changes in Chl biosynthetic rate (5), it is important toremember that the transamination would not be the first stepunique to this pathway, and regulation might be effected at an

earlier step (Fig. 2B). Although the biosynthesis of -y, 8-dioxo-valeric acid has not been demonstrated, its formation by re-duction of a-ketoglutarate could occur via steps analogous toknown enzymatic reactions (20). ALA synthesis utilizing theintact carbon skeleton of a-ketoglutarate via -y, -dioxovalericacid is compatible with our "C-labeling results (Fig. 2B).

Another hypothetical route of ALA formation which iscompatible with our results is the cyclization of glutamatethrough the 5-semialdehyde to A'-pyrroline-5-carboxylic acid,followed by the addition of an oxygen atom at C2 and thecleavage at the original C-N bond. An enzyme has been iso-lated from Clostridium which is capable of reductively cleavingthe appropriate C-N bond Of D-proline to yield 8-aminova-leric acid (7).

The presence of a pathway of ALA formation in plants thatis different from ALA synthetase may be significant both fromevolutionary and cell-physiological viewpoints, because it couldprovide a basis for explaining the differential regulation ofheme and Chl biosynthesis within the various subcellularorganelles of plant cells.

Acknoulledginentsq-We are indebted to P.AI . Rich for her skillful technicalassistance. to P. K. Stumpf and E. E. Conn of the Department of Biochemistryand Biophysics for the use of their radiochromatogram, scanning and gas chro-matography equiipment, and to F. X. Ogasawara and L. Fuqua of the Departmentof Avian Sciencese, for the turkey blood.

LITERATURE CITED

1. BEALE. S.I. A-ND P. A. CASTELFRA'NCO. 1973.14C incorpoiation from exogenouscompoundis into 6-aininole,ulinicacili by greeningcuctumber cotyledons.Biochemii. Biophys. Res. Commun. 52: 143-149. (corrected version publishedin vol. 53).

2. BEALE,. S. I. AND P. A. CASTELFRANCO. 1974. Thebiosvntlhesis of 6-aminolevu-linic acid in higlier plants. I. Accuinuklati, n ofiB-aninole\ ulinic acid in green-ingplanttissues. Plant Physiol. 53: 291-296.

3. BOTTOMILEY. S. S. AND G. A. S.MITHEE. 1968. Characterization and measurementofA-aminoaevulnate synthetase in bone marrow cell mitochiondria. Bio-

csinm. Biophys. Acta 159: 27-37.4. DELLA ROSA, R. J., K. I. ALT.MAN, AND K. SALONION. 1953. Thebiosynthesis

of chlorophyll as studied witli labeled glycine and acetic acid. J. Biol. Chem.202: 771-779.

5. GASSMANS ,'A., J. PLUSCEC, AND L. BOGORAD. 1968.b-Aminolevulinic acid trans-

aminase in Chlorella vulgaris. Plant Phvsiol. 43:1411-1414.6. GIBSON-, K. D., W. D. LAVER, AND A. NEUBERGER. 1958. Initial stages in the

biosynthesis of porphyrins. 2. The formation of6-amiiinolaevulic acid fronmglycineancl succinyl-coenzyme A by particles from chicken erythrocytes.Biochens. J. 61: 8-6 29.

7. HODGI.-S, D. S. AND R. H. ABELES. 1969. Studies of the mechanism of action

of D-proline reductase: the presence on covalently bound pyruvate and itsrole in the catalytic process. Arch. Biochem. Biophys. 130: 274-285.

8. KIKUeCHI, G., A. KUMAR. P. TALMAGE, AND D. SHEMIN. 1958. The enzmaticsynthlesis of 3-aminolexvulinic acid. J. Biol. Chem. 233:1214-1219.

9. LASCELLES, J. 1968. Regulation of heme and chlorophyll synthesis. In: Good-win, T. W., ed. Porphyrins and Related Compounds. Biochem. Soc. Symp.-No. 28. Academic Press, New York. pp. 49-59.

10. MARVER, H. S., A. COLLI-Ns, D. P. TsCHrUDY AND 'M. RECHCIGL, JR. 1966.

6-Aminole-ulinic acid svnthetase. II. Induction in rat liver. J. Biol. Chem.241: 4323-4329.

11. MAUZERELL, D. AN-D S. GRANICIK. 1956. The occurrence and accumulation of3-aminolevulinic acid and porphobilinogen in urine. J. Biol. Chem. 219: 435-446.

12. 'MILLER, J.5X. AND D. TENG. 1967. The purification and kinetics of amino-levulinic acid synthetase from higher plants. 7tlh International Cong. Bio-chem. Tokyo. p. 1059.

13. NEUBERGER, A. AND J. A. TURNER. 1963. -j.8-Dioxovalerate aminotransferaseactivity in Rhodopseudomonas spheroides. Biochim. Biophys. Acta 67:342-345.

14. PORRA, R. J., R. BARNES, AND 0. T. G. JONES. 1972. The level and sub-cellulardistribution of 8-aminolaevulinate synthase activity in semi-anaerobic andaerobic yeast. Hoppe-Seyler's Z. Physiol. Chem. 353: 1365-1368.

15. PORRA, R. J., E. A. IRVING, AND A. M. TEN-NICK. 1972. The detection of3-aminolaevulinic acid synthetase in anaerobically grown Torulopsis utilis.Arch. Biochem. Biophys. 149: 563-565.

16. RADIN, N. S., D. RITTENBERG, AND D. SHEMIN. 1950. The role of glycine inthe biosynthesis of heme. J. Biol. Chem. 184: 745-753.

302 Plant Physiol. Vol. 53, 1974



14C-ALA FROM LABELED PRECURSORS

17. RAMASWAMY, N. K. AND P. M. NAIR. 1973. &-Aminolevulinic acid synthetasefrom cold-stored potatoes. Biochim. Biophys. Acta 293: 269-277.

18. ROBERTS, D. W. A. AND H. J. PERKNs. 1962. Chlorophyll biosynthesis andturnover in wheat leaves. Biochim. Biophys. Acta 58: 499-506.

19. ROBERTS, D. W. A. AND H. J. PERINS. 1966. The incorporation of the twocarbons of acetate and glycine into the phorbide and phytol moieties ofchlorophylls a and b. Biochim. Biophys. Acta 127: 42-46.

20. TAIT, G. H. 1968. General aspects of haem synthesis. In: Goodwin, T. W.,

303

ed. Porphyrins and Related Compounds. Biochem. Soc. Symp. No. 28.Academic Press, New York. pp. 19-34.

21. TAIT, G. H. 1972. 5-Aminolaevulinate synthetase of Micrococcus denitrificans.Biochem. J. 128: 32p.

22. WELLBURN, F. A. M. AND A. R. WELLBURN. 1971. Chlorophyll synthesis byisolated etioplasts. Biochem. Biophys. Res. Commun. 45: 747-750.

23. WIDER DE XIFRA, E. A., A. M. DEL C. BATTLE, AND H. A. TIGIER. 1971.6-Aminolaevulinate synthetase in extracts of cultured soybean cells. Bio-chim. Biophys. Acta 235: 511-517.

Plant Physiol. Vol. 54, 1973



thebiosynthesis of a-aminolevulinic higher plantsradiochromatogram scanner, model 7201, and the...

Documents