oni - Plant Physiology · digests in which glyoxylate and cofactors were supplied to those containing acetate-1-C14. NoATPor CoAwas included in those digests in which acetyl-CoA was

PLANT PHYSIOLOGY

10. RAPPAPORT, L. Factors affecting flowering in let-tuce. Doctoral Thesis. Michigan State Univer-sity, East Lansing 1956.

11. RAPPAPORT, L. and BONNER, J. Quantitative plantresponses to gibberellin and some climatic factors.Plant Physiol. 33 suppl.: 43. 1958.

12. RAPPAPORT, L. and WITTWER, S. H. Stimulation offlowering by vernalization of endive seedlings-Apreliminary report. Proc. Amer. Soc. Hort. Sci.67: 438-439. 1956.

13. RAPPAPORT, L. and WITTWER, S. H. Flowering inhead lettuce as influenced by seed vernalization,temperature, and photoperiod. Proc. Amer. Soc.Hort. Sci. 67: 429-437. 1956.

14. RAPPAPORT, L., WITTWER, S. H. and TUKEY, H. B.Seed vernalization and flowering in lettuce. (Lac-tutca sativa). Nature 178: 51. 1956.

15. SARKAR, S. Versuche zur Physiologie der Vernalisa-tion. Biol. Zentr. 77: 1-49. 1958.

16. VAN DEN MUIJZENBERG, E. W. B. The influenceof the air conditioned glasshouse on the growth ofplants. 13th Int. Hort. Cong. Rept. Pp. 895-903.Roy. Hort. Soc., London. 1952.

17. WENT, F. NV. The experimental control of plantgrowth. Chronica Botanica Co., Waltham, MIass.1957.

18. NVITTWER, S. H. and BUKOVAC, M. J. Gibberellineffects on temperature and photoperiodic require-ments for flowering of some plants. Science 126:30-31. 1957.

19. \WITTWER, S. H. anid BUIKOVAC, M. J. The effectsof gibberellin oni economic crops. Econ. Botany12: 213-255. 1958.

20. WVITTWER, S. H., BUKOVAC, A. J., SELL., H. AI. andA?ELLER, L. E. Some effects of gibberelliin onlflowering and fruit settinig. Plant Physiol. 32:39-41. 1957.

MALATE SYNTHETASE IN HIGHER PLANTS.'YUKIO YAMAMOTO 2 AND HARRY BEEVERS

DEPARTMENT OF BIOLOGICAL SCIENCES, PURDUE UNIVERSITY, LAFAYETTE, INDIANA

Malate synthetase brings about the condensation ofglyoxylate and acetyl CoA to form malate, accordingto reaction 1.CO-SCoA 4 COOH

CH, Mialate synthetase 3 CH,+

CHO H.,O 2 CHOH+CoA-SH

COOH 1 COOH

The enzyme was first discovered in microorgan-isms by Wong and Ajl (14) and is one of the key en-

zymes of the glyoxylate cycle described by Kornbergand Krebs (6). In earlier work we have shown thatthe enzymes of the glyoxylate cycle are present in theendosperm of the germinating castor bean (Kornbergand Beevers 8) and one of these, isocitritase, has beenexamined in some detail (Carpenter and Beevers 5).In this report a comparable investigation has beencarried out on malate synthetase. Its distribution ina variety of plant tissues has been determined andsome of the properties of the castor bean enzyme havebeen investigated. The results reinforce the earliersuggestions that malate synthetase and isocitritase, as

components of the glyoxylate cycle are an essentialpart of the machinery by which fats are converted tocarbohydrate.

1 Received May 11, 1959.2 Recipient of Fulbright Travel Grant. Permanent

Address: Biological Institute, Faculty of Science, NagoyaUniversity.

-MATERIALS AND MIETHODSPLANT MIATERIALS Germination of the castor

beans and the preparation of crude extracts (castorbean preparation) were carried out by methods de-scribed previously (8). Other seeds were germinatedin a similar fashion on vermiculite and grown in thedark at 300 C for the stated periods. Mature partswere cut from plants growing in the greenhouse.

Lactobacilluts arabin osuis, adaptedl to malate, wasgrown by the method of Nossal (9) as mo(lifie(d byStiller (11). LYophilized cells were storedl at-15o C.

Speciall chemiiicals Sodium glyoxylate was ob-taimed from M\Tann Biochemicals. Acetate-l-C'4 andacetic-i-C'4 anhvdride at a rated specific activity of1 millicurie per millimole were supplied by Nuclear ofChicago.

Acetyl-1-CI4-CoA was prepared from the anhy-dride by the method of Simon and Shemin (10). Allmeasurements of radioactivity were made on BaCO,in a windoowless gas flow counter andl the results arecorrected for background and self absorption. Com-bustions to determine the C'4 content of substrateswere carried out according to Stutz and Burris (13).

Aceto-CoA-kinase was prepared from yeast usingBerg's (3) method to the stage of the first ammoni-um sulfate precipitation. It was stored in the frozencondition and maintained its activity over severalmonths. The kinase was shown to have a low butdetectable malate synthetase activity. In the resuiltsof those experiments in wlhichi it was used tllese blankvalues (150-250 cpm) have been subtracted.

All enzvme incubations were carried otut at 300 C.

102

Copyright (c) 2020 American Society of Plant Biologists. All rights reserved.

YAMAMOTO AND BEEVERS-MALATE SYNTHETASE

RESULTS

ASSAY OF MALATE SYNTHETASE: In earlier workmalate synthetase activity was detected by allowingthe reaction to proceed in the presence of acetate-2-C14, separating the products on a two-dimensionalchromatogram and counting the C14 in malate. Iso-citritase was present in the extracts, and isocitrateusually was used as the source of glyoxvlate for themalate synthetase reaction (8).

This method suffers as an assay system for malatesynthetase itself because, in addition to its slowness,the synthesis of malate depends on the ability of theextracts to convert acetate into acetyl-CoA. and on

the activity of isocitritase. In the earlier work (8)it was shown that isocitritase activity was higher thanthat of the acetate activating system and of the malatesynthetase in the crude extracts. In addition, itseemed that glyoxylate in substrate amounts was in-ferior to isocitritase + isocitrate as a source of gly-oxylate.

In the present work, with different reagents, itwas shown that glyoxylate was just as effective as

isocitrate + isocitritase. The progress of acetate-1-C14 incorporation into malate is shown in figure 1.In all subsequent work, therefore, glyoxylate was

added as the acceptor for acetyl CoA, and the synthesisof malate was thus independent of isocitritase activity.

In an attempt to make the assay independent ofthe acetate activating activity of the extracts, acetyl-

303

MIN S GLYOXYLATE

z

40

FIG. 1. Progress of acetate incorporation into malate.

Standard assay conditions (table II) were used, except

that an equimolar amount of D-isOcitrate was substituted

for glyoxylate in one of the digests.

TABLE IDEGRADATION OF MALATE SAMPLES PRODUCED FROM

AcErYL-1-C14 CoA AND ACETATE-1-C'4BY CASTOR BEAN PREPARATIONS

C14 (CPM/MG BACOq) ININDIVIDUAL CARBONS

COOH-CHOH-CH2-COH goO oSUBSTRATE O-H -C2-COOH % F o1 2 AND 3 4 TAL INC-4

Acetyl-CoA 1) 2.1 1.0 38.1 93 %O2) 1.3 0 12.0 90 %

Acetate 1) 0.9 1.5 35.8 94 %O2) 5.6 0.4 151.0 96%

The malate was isolated chromatographically fromdigests in which glyoxylate and cofactors were suppliedto those containing acetate-1-C14. No ATP or CoA wasincluded in those digests in which acetyl-CoA was thesubstrate. One and two are separate experiments.

1-C14CoA was employed. As shown in table I, mal-ate synthesis was achieved with this substrate, in theabsence of ATP and CoA. However, the instabilityof the acetyl-CoA and the necessity for chromato-graphic separation immediately before use make it anundesirable substrate for assay of malate synthetase.The use of aceto-CoA-kinase from yeast to augmentthe native acetate activating enzyme is described ina later section.

An important fact which was established by theacetyl-CoA experiment and the parallel experimentswith acetate-1-C'4 (table I) is that the C14 is almostexclusively confined to the C-4 COOH group of mal-ate. This establishes that the condensation occurs asindicated in reaction 1 and further, that. once formed,the malate is stable; there is a negligibly small amountof randomization between the COOH groups of malateas might be expected if fumarase were active in theextracts. In addition, of course, the maintenance ofthis inequality of labelling in the COOH groups ofmalate precludes any possibility of its having arisensecondarily from succinate in a hypothetical directback to back condensation of acetate.

The enzyme assay system which was developedmakes use of this localization of C14 in the malateand the specific decarboxylation at C-4 which isbrotught about by malate adapted Lactobacillus arabi-niosuts. Acetate-i-C'4 was used as substrate and atthe end of the incubation the malate production wasestimated by first stopping the reaction by boiling andthen adding the incubation mixture (or an appropriatealiquot) with 50 micromoles of unlabeled malate(pH 5.7) to a Warburg vessel. A suspension of Lac-tobacillus cells was added after filling with N. TheCO, from C-4 was collected in KOH and the C'4content of the carbonate was determined. The totalradioactivity in the carbonate was a measure of malateproduction from acetate; since the specific activitv ofthe acetate-i-C'4 was known, malate formation inmicromoles can also be calculated. It was establishedthat residual acetate was not decarboxylated by theLactobacillus. Malate synthetase activity of Lacto-

103


PLANT PHYSIOLOGY

TABLE IICo-FACTOR REQUIREMENTS FOR MALATE SYNTHESIS

ACETATE-1-C'4 INCORPORATED INTOMALATE (CPM)

FRESH AFTER 16 HRDIALYSIS AT 30 C.PREPARATION A (STILL) B (STIRRED)

Completesystem' 31,700 8,750 6,140- CoA 122 115 85- ATP 371 127-Mg++ 10,121-Glyoxylate 10,452 1,740 379-GSH 23,172

1 The complete system (standard assay conditionis)contained the following constituents in the stated amountsin micromoles. Potassium phosphate, pH 7.6, 60;glutathione, (GSH) 3.3; MgCl,, 10; CoA, 1.3;CH3C140ONa 2.5 (1.7 X 10; cpm), ATP, 2.7; glyoxyl-ate, 3; castor bean preparation 0.25 mnl in a final volumeof 1.5 ml. Incubation time 30 min.

bacillus preparations was shown to be virtually zero.The sensitivity of the method (lepenids on the initialspecific activity of the acetate. In most of our ex-periments 2.5 FM acetate containiing about 100,000cpm C14 were adde(l. Since malate decarboxylationis quantitative, the formation of more than 0.001 AMof malate can be readily detected.

MALATE SYNTHETASE FROM CASTOR BEANS: a)Co-factors for intcorporation, of acetate into mitalate:Previously (7, 8) the incubations were carried out inthe presence of CoA, ATP, Mg++ and glutathione(GSH). The essentiality of these components isestablished by the results in table II. The effects ofomitting CoA or ATP, components of the activatingsystem, are particularly striking; in the absence ofeither, the incorporation was reduced to less than 1 %of the control rate. The omission of glyoxylate fromthe digests did not completely abolish nmalate synthesisby the fresh preparation, Nwhich presumably containedsome endogenous source of glyoxylate (7). The es-

sentiality of the glyoxylate was clearly established bythe results with the (lialyze(l enzyme. in which malateformation was eventually reduce(d to about 6 % of thecontrol rate. The decline in the activity of malatesynthetase during dialysis was not appreciahly greaterthan that which occurred when the enzyme was storedat the same temperature for a similar period.

The influence of CoA and ATP is shown in figure2. The curves relating malate synthesis to co-factorconcentration show a linear phase at concentrationslower than 1 mg/ml; at levels greater than this, sat-uration of the enzyme is approached. In the subse-quent experiments 1.5 mig. ATP (2.7 micromoles)and 1 mg CoA (1.3 micromoles) were added to eachreaction mixture.

b) Effect of enzyme concentration: The lowercurve in figure 3 shows that malate synthesis is pro-portional to the amount of enzyme added over a wide

range of concentrations. The amount of enzymeusually added in the experiments was 0.25 ml (about2.5 mig protein).

C) Effect of exogenlouts aceto-CoA-kinase: Thecapacity of the systeml to produce malate from acetatecan be strikingly increased by a(ldition of the acetateactivating enzyme froml y3east (figure 3 ). In thepresence of a fixed amount (0.2 ml) of aceto-CoA-kinase (determined to be aIn a(lequate amount in pre-liminary experimiients) hiiglher rates of malate forma-tion were obtained; the rate of the r-eaction wvas againproportional to the amiiouniit of castor bean preparationwrhich was adde(l. These resuilts confirm-n that thenative acetate activating systemii is less vigorous thanthe malate sv-nthetase; the nmaximum ability to pro-duce malate was lnot imieasure(d in the previouis assaysVstem. as suspectedl earlier (8).

d) Effect of pH: When the malate synthesisreaction was carrie(l out at different pH levels thedata in table III were obtained. A broad optimumin the region of pH 6.8 to 8.0 is apparent. Effectson the acetate activating enzyme anid the mlalate syn-thetase are compounded in this curve for the over allreaction. The stan(lardl enzyme assay w,as carriedout at pH 7.6.

e) Effect of age of seedlings: Ratpid fat break-down begins only after 3 to 4 (lays of germination;several of the enzymes of the endospernm (1) as wellas isocitritase (5) show very striking ilncreases inactivity at this time. A sinilar, thouglh smlaller riseis exvidelt in the malate synthetase activity, measuredin the absence of exogenous aceto-CoA-kinase (lowercurve of figure 4). In the presence of the acetateactivating enzyme a miiore striking rise is evident be-tween days 1 and 3; the activity per seedling remainsat this high level, in contrast to the system in which

x Co A ATP

050 100-X

040 1 0 0

Z

aw30 ~~~~~~~60-

0.00 20- 40-Z

10- 204

4

0 05 10 15 20 0 0.5 10 15 20

MG. Co A MG. ATP

FIG. 2. CoA and ATP requirements for the produc-tion of malate from acetate and glyoxylate. In the ex-periment in which CoA concentration was varied, 1.5 mgATP was added to all digests. In the (separate) ATPexperiment, 1 mg CoA was added to all digests. Otherconditions as in table II. Acetate: Sp.Act.: 5 X 104cpm/,u'M.

104



h'U

0

x

0

4

4

z

0

I-

W)

>.o15C

-J

xE

z

(L0

< oo

cn a

z

LJ q

0

105

1 2 3 4 5 6 7

ENZYME CONCENTRATION (MG. PROTEIN ADDED) AGE OF SEEDLING IN DAYS

FIG. 3 (left). Effect of enzyme concentration on malate production. The lower curve was obtained underthe conditions shown in table II, except that the amount of enzyme was varied as indicated. The upper curve wasobtained in a parallel series of digests to which 0.2 ml aceto-CoA-kinase were also added. Acetate: Sp.Act.:2.7 X 104 cpm/,M.

FIG. 4 (right). Variation in activity of malate synthetase during germination of castor beans. Extracts weremade at the times indicated; activity was measured under the standard assay conditions (lower curve). The uppercurve was obtained when 0.2 ml aceto-CoA-kinase were also present. Acetate: Sp.Act.: 5 X 104 cpm/4M.

only native aceto- CoA-kinase is present. This latterfall can thus be ascribed to a progressive failure inthe native acetate activating system, rather than toa decline in malate synthetase. It is important tonote that malate synthetase can be extracted from un-germinated seeds, and that this can be done in theearly stages of growth. Isocitritase was not detect-able at these times (5).

f) Intracellular localization of malate synthetase.Enzyme extracts were usually prepared in dilute phos-phate and centrifuged at 10,000 X G for 15 minutes.However, if the blending was carried out in the medi-um employed earlier for preparing mitochondria andmicrosomes (1) the final supernatant had very feebleactivity. (table IV). On the other hand, the par-ticulate fractions were highly active in malate syn-thesis when aceto-CoA-kinase was provided. Thestriking effect of the exogenous acetate activating en-zyme indicates that there had been a separation ofendogenous activating enzyme and malate synthetaseduring the centrifuging (table IV). Although themicrosomes have a high specific activity of malate

synthetase, the total amount of protein in these frac-tions is very small, (1) and a much higher percentage(about 60 %) of the total activity in the originalhomogenate was recovered with the mitochondrialfraction (which bad been washed by resuspension insucrose phosphate). The enzyme so recovered canbe readily displaced or eluted from the particles bysimply transferring them to 0.01 M potassium phos-phate, pH 7.6.

MALATE SYNITHETASE IN OTHER PLANT TISSUES:A variety of plant materials, listed in table V was

tested for malate synthetase activity in the standardassay system (table II). The materials are listed indecreasing order of effectiveness in inducing malatesynthesis in the presence of exogenous aceto-CoA-kinase. The ability to produce malate without benefitof this addition is shown in the first column of figures.Clearly, the enzyme is widespread, but there are verylarge differences in the malate synthetase activity inthe tissues examined. It is noteworthy that thosewhich gave extracts with high specific activity are

TABLE IIIEFFECTr OF PH ON MALATE SYNTHESIS

PH 5.6 6.0 6.4 6.8 7.2 7.6 8.0Malate formation(cpm acetate-C14 111 964 2,330 3,740 2,990 3,290 3,280incorporated)

Standard assay conditions (table II) were used, except that the potassium phosphate- component was adjustedto the pH levels indicated. Acetate: Sp.Act.: 5X104 cpm/micromole.

40

.. ° o

1 o

)l


PLANT PHYSIOLOGY

TABLE I\

MALATE SYNTHETASE ACTIVITY OF CELL FRACTIONS OFCASTOR BEAN ENDOSPERMI

FRACTION

\Vashedmitochondria

NIicrosomesSupernatant

MN ALATE SY NTTHFTASE ACTIVITY(CPMI ACETATE-C0 4 INCORPORATED

/MG PROTEIN)- A( 1ETO-COA- +ACETO-COA-

KINASE KINASF (0.2 AII

393169172

3,4507,175290

Standard assay conditions were u-sedI (table II).Mitochondria were obtained by centrifuginig for 30

min. at 10,000 X G after an initial 10 mnii. spin at800 x G. The nmicrosomal fraction was that sediiment-ing in 60 min. at 54,000 X G after removal of the mito-chondria. Acetate: Sp. Act.: 2.7 X 104 cpvn/micromole.

largely ones which have a higlh fat colnLent a(n 1 inwhich, furthermore, conversion to carbohydrate oc-curs. In some of the closely neighboring tissues. suchas the castor bean embryo, malate svnthetase activityis very low, and it is uniformly low Inimature leaves.

HoNxxever, several other non-fatty tissues showclearly detectable activity, e.g., corn embryo and thepea cotyledon. It is of interest too that activity wasstill quite strong in the watermelon cotyledon evenafter eight (lays of growth in the light, but after alonger period it had virtually (lisappeared from thesunflower cotyledlon. The enzyme is present in thecastor bean endosperm wlhen it is mlaturing on theparent plant.

It can be concludedl from the (lifferent degrees ofstimulation induced bv the exogenous aceto-CoA-kinase that the relative amounts of en(logenious activa-ting enzyme andl nialate synthetase w-ere not the sanmein each tissue. The imiost strikiiig stimulation (70-fold) was that in sunflower seed w-hiclh ha(l just begunto germinate, but sizeable increases w\ere observedin most of the other tissues.

It should he emilphasized that tissuies other tb-rn thecastor bean endospermi were not invxestigated intein-sively to find the age for best vieWls of malate svntlhe-tase. It is quite possible theni that at solmie stage inits development the sunflowver or even the peanutmight be a superior source.

DiscussioxNTIn the present work, by the use of acetyl-1-C' '-CoA

it has been establishedl that the miialate synthetase re-action occurs as written in eqluatioln 1. and that theC14 in the malate whicl is l)ro(luced remlailns almlostexclusively in C-4. Parallel resuilts of a similar kindhave recently been describe(d by Bradbeer andl Stunmpf(4) for peanut and stinfloxNver preparations. G61-oxylate an(l (isocitritase + isocitrate) are nowt shownto be equally effective as acetyl acceptors in the malatesynthetase reaction. Absolute requirements for CoAandl ATP were establislhed wvhen acetate, rather thanacetyl-CoA was inclu(ledl.

It was shown that the endogenous acetate activat-ing system in the castor bean extracts was incapableof prodlucing acetyl-CoA at a sufficiently rapid rate tosaturate the malate synthetase reaction; the ad(litionlof yeast aceto-CoA-kinase greatly improve(d the ratesof malate synthesis andl allowed a more realistic pic-ture of the capacity an(d distribution of miialate sy-n-thetase itself.

The possible linmitation of the endogenous activat-ing system was recognize(d in an earlier report (8)in which the rates of mnalate synthesis wvere consi(ler-ably loxN-er than those which would be recluire(l invivo if the malate syvthetase an(d glyoxvlate cyclewere major steps in the conversion of fat to carbo-lhydrate. The presient results show that this is in factso, an(l whell this limitatioln was remove(l, iniitial rates

TrAB1LE V

AIALATE SYNTHETASE AC rIVITY' OF PLANT JISSi ES

ACETATE-C'4 INCORPORATEDINTO MALATE (CPMI/MG

PROTEINT)

PLANT MATERIAL WrITHOUTEXOGENOUSACETo-CoA-KINASE

Castor bean endosperm, 5-day-oldseedling 2,500

Soybean cotyledoni, 5-day-oldseedling 598

Sunflower seed, 24 hr germination 33Watermelon cotyledon, 5-day-old

seedling 173Pea cotyledon, 5-day-old

seedling 497Peanut cotyledon, 5-day-old

seedling 128Watermelon cotyledoni, 8 days

in light 213Vignia sesquiipedalis cotyledon,2-day-old seedling 445

Corn embryo, 5-day-old seedling 82\Watermelon hypocotyl, 5-day-old

seedling 16Castor Bean, maturing seed 84Vignta sesquiipedalis, hypocotyl

5-day-old seedling 193Wheat coleoptile,

5-dav-old seedling 56Castor bean embryo, 5-day-old

seeclling 50Sunflower cotyledon,

3 weeks in light 96Castor bean, mature leaf 2Tomato, mature leaf 60Tobacco, mature leaf 38Barley, mature leaf 18Soybean, mature leaf 7Sunflower, mature leaf 1

ADDITIONALINCORPORATION'ON ADDING

YEAST ACETO-COA-KI NASE

2,700

4,0722,2117

1,917

1,383

1,694

1,149

840488

325148

0

62

0

02900000

1 The various plant extracts were prepared by grind--ing a measured weight with an appropriate volume of-0.05 M potassium phosphate pH 7.6.

0.25 nml. of the supernatant solution obtained aftercentrifuging at 10,000 x G for 15 nmin was used in thestandard assay conditions (table II). 0.2 ml of aceto-CoA-kinase was added as required.

Acetate: Sp.Act.: 2.7 X 104 cpmn/M.

106



of malate synthesis more than ten times those reportedearlier, i.e., (about 10 micromoles/hr individual bean)were achieved. While this rate is still not in excessof that required to be completely convincing, it is suf-ficiently high to warrant the view that it is of impor-tance and we regard it as subject to improvement bydifferent extraction procedures. Using a differentprocedure, high rates of malate synthesis have beenachieved in extracts of peanut cotyledons, (Marcus,personal communication). The limitation imposed bythe endogenous activating system when the malatesynthetase reaction is measured in crude extractswould not be a consideration in vivo, when acetateunits, already in the activated (CoA) form, are pre-sumably provided during p-oxidation of the long chainfatty acids (12).

Malate synthetase is present at the earliest stagesof germination but increases strikingly during days1 to 3. This increase is similar to that observed inmitochondrial enzymes (1) and in isocitritase (5).In contrast to isocitritase, however, the level of mal-ate synthetase per seedling does not decline in thelater stages of germination, and furthermore, malatesynthetase is present in the ripening and ungerminatedseed.

A substantial portion (60 %) of the enzyme pres-ent in a sucrose phosphate homogenate can be re-covered with the separated mitochondria. Althoughthis association with the particles persists throughwashing in sucrose phosphate and sedimenting. it isreadily broken on transfer to dilute phosphate(0.01 M) and the enzyme is found in the clear super-nate. When homogenates are prepared in dilutephosphate. the enzyme is recovered in the solublephase. At the present time, the association of the en-zyme with the mitochondria is regarded merely as afortunate circumstance in purification attempts(Yamamoto and Beevers, unpublished) ; other criteriafor regarding the enzyme as a genuine mitochondrialcomponent have not yet been met.

From the experiments on distribution of the en-zyme, it is clear that germinating fatty seeds are thebest sources of the enzyme (table V). However,other tissues such as pea cotyledons and corn seedlingshave definite enzyme activity, and in general the dis-tinction between fatty and non-fatty tissues, thoughquite marked, is not as complete as it was for iso-citritase (5). The enzyme appears to persist longerin germinating seeds of watermelon than does iso-citritase, and it is present in the ripening and ungermi-nated castor bean. Nevertheless, it is clear that bothisocitritase and malate synthetase are present togetherin the most active conditions in all of those tissueswhich are converting fat into carbohydrate and thusthe present results are in accord with the proposedimportance of the glyoxylate cycle in this conversion.In those non-fatty tissues in which it occurs, malatesynthetase may play a role by replenishing C-4 di-carboxylic acids at the expense of glyoxylate gener-ated in some reaction other than that catalyzed by iso-citritase.

SUMMARYThe malate produced from acetate-i-C'4 (or

acetyl-1-C'4 CoA) and glyoxylate by malate synthe-tase from castor bean endosperm contains C'4 almostexclusively in C-4. An assay system for malate syn-thetase was developed which makes use of this factand of the ability of Lactobacillus cells to release C-4of the malate so produced as C1402. Absolute re-quirements for CoA and ATP were demonstrated formalate synthesis when acetate was used as substrate.

The capacity of crude extracts of castor bean endo-sperm and other plant tissues to produce malate fromacetate and glyoxylate is limited by their ability toconvert acetate to acetyl-CoA. When aceto-CoA-kinase prepared from yeast was added, the rates ofmalate synthesis were considerably enhanced. Mal-ate svnthetase was shown to be present in widely dif-ferent amounts in a variety of plant materials. Al-though the enzyme is not confined to those tissuesconverting fats in sugars, the best sources are fattyseeds in which this change is occurring, and whichare known to contain isocitritase.

During the second and third days of germinationof the castor bean, the malate synthetase, which ispresent in the ripened seed, shows a striking increasein activity; it is maintained at this level for severaldays. The bulk of the malate synthetase activity ofcastor bean homogenates can be recovered with themitochondria.

The results reinforce earlier suggestions about theimportance of malate synthetase and the glyoxvlatecycle in the conversion of fats to carbohydrates.

ACKNOWLEDGENIENTSWe gratefully acknowledge the support of a grant

from the National Science Foundation (G-5072) insupport of this work. We are indlebted to Dr. M.Zenk for help and advice in preparing CoA deriva-tives, and to Dr. MIary Stiller for the Lactobacillusculture.

LITERATURE CITED1. AKAZAWA, T. and BEEVERS, H. Mitoclhondria in the

endosperm of the germinating castor bean: a de-velopmental study. Biochem. Jour. 67: 115-118,1957.

2. BEEVERS, H. and WALKER, D. A. The oxidative ac-tivity of particulate fractions from germinatingcastor beans. Biochem. Jour. 62: 114-120. 1956.

3. BERG, P. Acvl adenylates: an enzymatic mechanismof acetate activation. Jour. Biol. Chem. 222:991-1013. 1956.

4. BRADBEER, C. and STUMPF, P. K. Fat metabolismin higher plants. XI. The conversion of fat intocarbohydrate in peanut and sunflower seedlings.Jour. Biol. Chem. 234: 498-501. 1959.

5. CARPENTER, W. D. and BEEVERS, H. The distribu-tioln and properties of isocitritase in plants. PlantPhysiol. 34: 403-409. 1959.

107


PLANT PHYSIOLOGY

6. KORJNBER(., H. L. al1d KREBS, H. A. Synthesis ofcell constituents from C-2 uniits by a modified tri-carboxylic acid cycle. Nature 179: 988-991.1957.

7. KORNBERG, H. L. and BEEVERS, H. A Mechanisnmof conversion of fat to carbohydrate in castor beans.Nature 180: 35-36. 1957.

8. KOwNBERG, H. L. anld BEEVERS, H. The glyoxylatecycle as a stage in the conversion of fat to carbo-hydrate in castor beanis. Biochem. Biophys. Acta26: 531-537. 1957.

9. NOSSAL, P. MI. Estimationi of i-malate and fuma-rate by malic decarboxylase of Lactobacillus arabi-nosius. Bioclhem. Jour. 50: 349-355. 1952.

10. STi\roN\, E. J. anid SHFI\IiN. D. Tlle preparation of

S-succinyl coenzyme A. Jour. Amer. Chem. Soc.15: 2520. 1953.

11. STILLER, M. L. The mechanism of malate synthesisin crassulaceani leaves. Thesis, Purdue Uniiver-sity. 1959.

12. STUCMPF, P. K. an1d BARBER, G. A. Fat imietabolismin higher plants. VII :-oxidation of fatty acidsby peanut miiitochonldria. Plant Physiol. 31: 304-308. 1956.

13. STUTZ, R. L. and BURRIS, R. H. Photosyn1thesis andmetabolism of orgainic acids in higher planits.Plant Phvsiol. 26: 226-243. 1951.

14. WONG, D. T. 0. and Aji,, S. J. Conversion of ace-tate and glyoxylate to mlalate. Jour. Amer. Chem.Soc. 78: 3230-3231. 1956.

DIFFERENCES BETWEEN LIGNIN-LIKE POLYMIERS FORMIED BYPEROXIDATION OF EUGENOL AND FERULIC ACID

IN LEAF SECTIONS OF PHLEUM'HELEN A. STAFFORD2

BIOLOGY DEPARTMENT, REED COLLEGE, PORTILAND, OREGON

Coniferyl alcohol, eugenol, aiid ferulic aci(l can beconverted to lignin-like polymers in the presence ofH20. via a peroxidase-catalyzed oxidation. Thisreaction can be brought about by tissue sections orcell-free extracts of wood-y and herbaceous plants(6, 10, 14). Furthermore, Neish an(d his coworkers(5) have shown that ferulic acid-_3C"4 is an efficientprecursor of the guaiacyl imioiety of lignin of wheat,presumably by conversion to coniferyl alcohol.

Since the position of eugenol in the scheme oflignin biosynthesis hias been (luestioned by Higuchi(10), the ligniin-like polymers l)ro(ducedl 1b peroxida-tion in the presence of eugenol and(l ferulic aci(1 halvebeen restudied by means of differelnt ancalytical tech-niques. The quantitative mletlhodl for lignin deter-minations used in the previous studlies (10, 14) isbased on the weight of the residue insoluble in 72 %H,S04, a technique originally (levised for woody tis-sues. In order to obtain more reliable dlata foryoung. herbaceous tissues, (leterminations used in thepresent investigatioln are base(d on extraction of thelignin in 2 % NaOH (4). followed by an estimationof the free phenolic groups (7), and determinationof the ultraviolet difference spectra of the lignin ex-tracts (1, 2. 8). Timothy grass, Phleiur pratense,was chosen as the test material because it is easy togrow and to study anatomically and biochemically,and mature shoots have been reporte(d to contain asmuch as 30 % of their drv weiglht as lignin (13).Results were obtained which show that there are bothqualitative alnd quantitative (lifferences in productsfrom eugenol and ferulic acid.

Received for publication May 22, 1959.2 Guggenheim Fellowr at Harvard University, 1958

to 1959.

A \TERIALS AND MIETHODS

Tissue sanmples were dried first in an oven at 70° Cand then in a vacuum desiccator over CaCl2 andH.SO4. Dry weight values were either (letermined(Iirectlv or were calculated froml comlparable samples.Lignin was (letermined as follows: after mloisteningwith (listilled water, the samples, containing 30 to 40nmg (dry weiglht, were ground in a miiortar with etlheruntil all the chlorophyll was removed, and(1 then wverethoroughly extractedl Nwith (listilled w\ater. The resi-(dIe was extractedl for about 16 hours in 2 to 3 m1l of0.5 N NaOH at about 700 C. Tlis technique is basedon the method of Bondi and AMeyer (4), who considerthe extraction to be quantitative for y,oung annuials.The supernatant and washes of the centrifuged resi-(lue were combined, neutralized to about pH 8.5 to 9,an(l were analvzed within 3 hours for their phenolicaui(I ultraviolet absorbing contents. Some of thesecomponents were unstable after about 24 hours untderthese alkaline conditions. Re-extraction of the resi-(lue yiel(led no significant amount of ultraviolet ab-sorbing coImpounds. The resi(lue w\\as dried, weighedand teste(d for materials reacting withl phloroglucinolandI with Cl,-Na.,SO, (9, 10. 14).

Ultraviolet absorption spectra were determined onaliquots. one diluted with 0.05 N NaOH anld the otherwith 0.05 Al phosphate buffer at pH 7, the differencespectrunm being obtained by- subtractionl (1. 2). Op-tical (lensity readings were nmade in a Beckman spec-trophotonmeter at intervals of 5 to 10 nmi from 230 to450 mA.

Phenol analyses w-ere made by a modification ofthe miiethod of Gierer for native lignin preparations(7). Suitable aliquots (containing 1 to 3 ug phenol)of the extract in 0.55 ml of distilled water were addedto 0.4 ml of 0.5 MA tris (hydroxymethvl)anminomethaniebuffer at pH 9.0 and(I 0.05 ml of a freshly pr-epared

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oni - Plant Physiology · digests in which glyoxylate and cofactors were supplied to those containing acetate-1-C14. NoATPor CoAwas included in those digests in which acetyl-CoA was

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