comparative studies enzymes related serine metabolism ... · reagen ts and substrates. crystalline...

Plant Physiol. ('1968) 13, 1813- 120

Comparative Studies of Enzymes Related to Serine Metabolismin Higher Plants'

Geoffrey P. Cheung2, I. Y. Rosenblum3, and H. J. SallachDepartment of Physiological Chemistry, UJniversity of Wisconsin Medical School,

Madison, Wisconsin 53706

Received July 1, 1968.

Abstract. The following enzymes related to serine metabolism in higher plants have beeninvestigated: 1) D-3.phosphoglycerate dehydrogenase, 2) phosphohydroxypyruvate :L-glutamatetransaminase, 3) D-glyeerate dehydrogenase, and 4) hydroxypyruvate :i.-alanine transaminase.Comparative studies on the distribution of the 2 dehydrogena,ses in seeds and leaves fromvarious plants revealed that D-3-phosphoglyccrate dehydrogenase is widely distributed in seedsin contrast to D-glycerate dehydrogenase, whioh is either absent or present at low levels, andthat the reverse pattern is observed isn green leaves.

The levels of activity of the 4 enzymes listed above were followed in different tissues ofthe developing pea (Pisum sativum, var. Alaska). In the leaf, from the tenth to seventeenthday of germination, the specific activity of D-glycerate dehydrogenase increased markedly tandwas much higher than D.3-phosphoglycerate dehydrogenase which remained relatively constantduring this time period. Etiolation resulted in a decrease in D-glycerate dehydrogenase andan increase in D-3.phosphoglycer-ate dehydrogenase activities. In apical meristem, on the otherhand, the level of D-3-plhosphoglycerate dehydrogenase exceeded that of D-glycerate dehydro-genase at all time periods studied. Low and decreasing levels of both dehydrogenases werefound in epicotyl and cotyledon. The specific activities of the 2 transaminases remainedrelatively constant during development in both leaf and apical meristem. In general, however,the levels of phosphohydroxypyruvate :m.-glutamate transaminase were comparable to thoseof D-3-phosphoglycerate dehydrogen-ase in a given tissue as were those for hydroxypyruvate:.-alanine transaminase and i)-glycerate dehydrogenase.

Two separate pathways for serine biosynthesisin animal systems have been established (9,18, 29,31). The individual reactions in the pathway uti-lizing phosphorylated in,termediates (phosphorylatedpathway) are given by reactions I to 3 and thoseinvolving nonphosphorylated compounds (nonphos-phorylated pathway) are shown in reactions 4 and 5:1) D-3-P-glycerate + DPN+ < > P-hydroxypyru-vate + DPNH d- H+, 2) P-hydroxypyruvate -1i-glutamate < > L-P-serine -T- !a-ketoglutarate,3) L-P-serine -± H.,O - L-serine + Pi, 4) D-glyc-erate + DPN+ < > hydroxypyruvate + DPNH± H+, 5) hydroxypyruvate + L-alanine <---* Lserine+ pyruvate. Evidence for the occurrence of bothl

1 This study was supported in part by Grant AM-00922 from the National Institute of Arthritis and Meta-bolic Diseases, National Institutes of Health, UnitedStates Public Health Service and by Research ContractNo. AT(11-1)-1631 from the United States AtomicEnergy Commission.

2 Present Address: Department of Pediatrics, Uni-versity of Colorado Medical Center, Denver, Colorado80220.

3 Predoctoral Fellow of the United States PublicHealth Service.

4Abbreviations used are: PGDH = D)-3-phosphogly-,cerate dehydrogenase; GDH = D-glycerate dehydro-genase.

of the above palthw%rays for serine nmetabolism inplant systems has been reporte(l and is discussedbelow. Relatively few of these studies, however.have been carried out on the individual enzyi-Maticreactions in either of the 2 rouites.

The phosphorylated pathway, described above inreactions 1 to 3, was fir-st demonistrated in highelplants by Hanfor(d and Davies (5) who showxed theover-all conversion of n-3-P-glycerate to i.-P-serineand L-serine in 1homogenates of pea epicotyls. Inatddition, the results of i11 ivo() '4CO2 labeling experi -ments during short term photosynthesis are consis-tent with a functional phosphorvlate(d pathway forserine formation in many plant preparatiolls incluld-ing spinach chloroplasts (3), tobacco leaves (6), andalgae (7). Furthermore, the occurrence in wheatgerm of all of the enzymes of the phosphorvlatedpathway, i.e. PGDH', P-hydroxypyruvate :L-gluta-mate transaminase, and phosphoserine phosphatase,has been demonstrated in this laboratory (unpub-lished observations).

The second route for serine formation in plantsis via the glycolate pathway, also known as theglyoxylate-serine pathway (16, 28). This pathwayby which glycolate is converted to suigars in photo-synthesizing tissues, has been prolose(l oni the basisof in vivo sttudies with 1 C-labeled sutbstrates and

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181PLANT PHYSIOLOGY

involves the following sequence of reaction*>: gly-colate = gl oxylate *-> glycine < serine <->i)-glycerate D-3-P-glycerate i-hexoses.

Much of thle literatture onl the metabolism of inter-mediates of this pathway in plants has been reviewedin recent papers from the laboratories of Cossins(2()) and Tolbert (23).

Early studies on glycolate and glyoxvlate metabo-lismii in leaf tissues were reported in 1962 (16, 28).Since that time, numerous detailed studies tutilizingdifferent 1 4C-labeled intermediates of the lyvcolatepathway have been carried out with a wide -varietyof plant tissues including wheat leaves (25, 26, 27).pea leaves (15), tobacco leaves (6) pea roots. peacotvledons, corn roots, corn coleoptiles. and sunI-flower cotvledons (20). In all of these investiga-tions serine has been implicated as a key intermediatein the over-all path of carbon from glycolate tocarbohydrate. Available evidence from the aboveiln vivo studies suggests that the pathway fromserine to sucrose is vzia hvbdroxvpvruvate. n- glcverateand D-3-P-glycerate.

Although the noniphosphorvlated pathway (de-scribed above in reactions 4 and 5) has not beenimplicated as a functional route for serine formationfrom carbohydrate in plants, both enzymes involved,i.e. GDH (21) and hydroxypyruvate :i-alaninetransaminase (30) have been demonstrated in avariety of plant tissues. Both enzymes show essen-

tiallv the same distribution of activity in that thev.are widely distributed in green leaves with little or11o activity in seeds or tubers. Developmentalsttudies carried out with peas and wheat have coni-firmed the fact that GDH is primarily a leaf enzymeand that the main inerease in enzymatic activityduring growth is associated with maximal leaf de-velopment (22).

In view of the alternate routes for serine metabo-lism in plants, a comparative studv of the enzymes

of the phosphorylated and nonphosphorylated path-ways was carried out to investigate the relativesignificance of the 2 routes. The distribution ofPCGDH in seeds and leaves from various plantsources has been compared to that of GDH. Tnaidditioni, the relative levels of activity of the dehy-(Irogenases and transaminases in different parts ofthe developing pea plant have been determined andalre reported.

Materials and Methods

Reagen ts and Substrates. Crystalline lactate de-hydrogenase, glutamate dehydrogenase. pvruvatekinase, DPN, DPNH, ATP, D-3-P-glycerate, pyru-vate, pyridoxal phosphate, tris-HCl, and ignited sea

sand were obtainled from Sigma Chemical Company.Crystalline phosphoglycerate mutase and enolasewere purchased from C. F. Boehringer and Soehne.GimbH, Mannheim. GDH was purified from spinachleaves to step 4 as outlined elsewhere (19); aliquotsof the ammonium sulfate residuies which were stored

ait -200, were (lissolve(l in 0..1 M1t)hosphate buffer.pH 7.4. as needed and freed of ammonium ioIns bydialvsis against the same Ibuffet before use. D-Glyc-erate and hvdiroxypyruvate were plrel)ared andassayed as described previously (29). The cyclo-hexvlaminnionitumi salt of the dimethyl ketal of P-by-droxypyruvate (Cal,biochem.) was converted to thefree acidI tusitig the method of Ballou (1). Otherreagents were commercial preparations of the highestpurity available. Stock solutions of all rieagenltswere prepared at neutrality as their potassiumni saltsunless otherwise indicated.

Plant Materials. In the distribution studies.plants were either grown in the laboratory or ob-tained from commercial sources. All seeds werepurchased from local dealers. For the developmentalstudies, pea seeds, Pisitrn sativiinm (var., Ala-ka).were soaked overnight in tap water and tlhen planltedabout 3 cm deep in vermiculite for growth. Tem-perature was kept constant at 300 and a daily photo-period- of 12 hr was maintained except for dark-,grown plants. Water was supplied daily to allplants. \t the indicated times after plantinZ, thepeas were harvested and their mature leaves, apicalneristems, cotyTledons, and epicotyls (the latter beingdefined as that area of the shoot extending from itsattachment to the cotyledons to the level directlybelow the first lateral shoot) were dissected out andimm-ediately frozen ill a container immersed in anaceetone-dry ice hath.

Enz-vine Preparationts. All operations were car-ried out at 4' unless otherwise stated.

In the distributioni studies, plant material; wereprepared as described by Staifford et al., (21) exceptthat the extractions were made vith 0.1 M phosiphatebuffer, pH 7.4, containing 0.00)3 M E:DTA and0.0001 M 2-mercaptoethanol.

The following conditions were used in the devel-opmenital studies. Frozen tisstues were ground withsea sand in a mortar and pestle with 1:3 (w/v) ratioof 0 01 M tris-HCI buffer, pH 7.4, containing 0.01 MrEDTA and 0.0001 M 2-mereaptoethanol. The slurrywas filtered through cheese cloth prior to centrifuga-tionl at 35,O0Og for 30 min. The resultant super-niatant soluition ras centrifuged at 105,000g for 30min and the final supernatant brought to 80 %saturation by the slow addition, with stirrinig, of thecalculated amount of solid ammonium sulfate. Theprecipitate was collected by cenltri fugation and re-dissolved in 0.01 M phosphate buffer, pH 7.4, con-taining 0.001 M EDTA and 0.0001 M 2-mercapto-ethanol, in a 1 :1 (v/w) ratio of the original tissueweight. This preparation was used for the assaysof the dehydrogenases. Before use in the assaysfor the transaminases and for D-glycerate kinase, theenzyme preparation was dialyzed against the samephosphate buffer with regular changes until thedialysate gave a negative reaction to Nessler'sreagent.

In control experiments, the supernatant solutionsfrom the 80 % ammonium sulfate fraction were

CHEI'NG, ET Al,. SERTNE METABOTISM IN HIGHER PLANTS

assayed for dehydrogenase and transalm inase activi-ties. None were detected. All enzymle assays wereperformned on the same dlay that the plants werehalrvested. -Protein conicelntrations were (letermllin(d1w the method of Lowry et ail., ( 13) uising l)bovineser-umiii all)umin as the standard.

Standard Assays for GDH aiid PGDH. Thestandard assay conditions (lescribed previously wereuised for GDH (29) and PGDTI (31). Ilitialvelocities were measured uisinig a Gilford 2000 re-cording spectrophotometer. Assavs were carried outin triplicate on eaclh saample and the average valtuereported.

Standard Assay for- P-Hydroxvpv, it-natc :i.-Gluta -nate Transamiiinase. Detailed conditions for thedeterminiationi of this transaminase have been pub-lished previously (24). The present stuidies werecar-ried out essentially in the same manner exceptthat the com)plete reaction mixture contained: so-dium bor,ate butffer, pH 8.2, 100 pumoles; pyridoxalphosiphate. 2.5 X 10-2 ',moles; EDTA, 50 ,imoles:L-glutamate, 10 tmoles: 2-mercaptoethanol. 0.o0,umoles; and P-hydroxypyruvate. 5 tmoles. in a totalvolume of 1.4 ml. The same controls as used pre-viouslv, i.c. zero time control, internal a-ketoglu-tarate standard, and complete reaction mixtulres inwhich either P-hydroxypvruivate or L-glutamate wasomitted (cf. 24). were carried out under the abovecondition,s with water added where substrates wer-eomitted. The reaction was stopped by tlle additionof 0.1 ml of 4.5 N perchloric acid and precipitatedprotein was remove(l bv centrifugation. .Aliquots ofthe deproteinized solutionis (1.0 ml) were dilutedwitlh 0.25 ml of water before the addition, witlhvigorous stirring, of 0.25 ml of 1 N KOH. Thesoltitionis were chilled and( insoluble potassiuim per-chlorate wvas removed by centrifugation. Aliquotsof the resulting supernatant solutions were assavedfor a-ketoglutarate. The determinationi of thisa-keto acid and all other coneditions wer-e identicalto those ouitlined earlier (24).

Stanidard Assay for- HvidroxAvpvrz'lt atc : -.-4lan inceTransaminiase. The method usede for the determina-tioni of this transaminiase is de-cribed in detail belowv.In gener,al, the procedure involves the measurementof the p1-rodutct of the reactioni, lpyruvate. in thepresence of tinreacted hydroxypyruvate. Both ofthese a-keto acids are substrates for lactate (lel-drogenase (14), but only the latter compountd is a-substrate fo- slpinach GDH (8). In the assay pro--cedure, uii reacted hydroxypvruvate is quantitatively-reduced in the presence of excess DPNI-I and spinachG;DH prior to the spectrophotonietric determinlationof pyruivate withl lactate dehvdrogenase.

In the standard assay for the transaminase, var\r-ing levels of enzyme were preincubated for 10 minat 37° with sodiunm borate buffer, pH 8.2., 100 umoles;pyridoxal phosphate, 2 X 10-2 ,nuoles; and L-alanine.10 Mkmoles. The reaction was started bv the additionof 6 ,nioles of hydroxypyruivate to give a finalvoltumie of 1.4 ml. The reaction wias terminated

after a 60 mmit incutbation at 37' by the addlition of0.1 ml of 4.5 N perchloric acid.

The enzyimatic activities of all tissuies werer> ron-tinel\ (leterminie(l at 4 levels of enzymie and(1 thelinear relationship betwveen enzylmie concentration an(lpyruvate produce(d wvas used in the calculation ofspecific activity. A complete reactioni niiixtur-e tlhitwvas stopled(at zero tinie w\as used to miiollitor for-the presence of pvruvate in the enzyme solutioni.Additional controls includ(le( comiiplete reaction inix-tures except for the followving: A) enzyme, to de-terumiine possible nonenzymatic transamination a.l wellas nonenzvmiatic loss of hydroxypvruvate: B) ala-nine, to nieasuire enzvnatic loss of hvdroxvpvrnvate:and C) hydroxyp) ruvate, to (leternmine productionof pyruvate from alanine by other possible reactions.Finally, a complete reaction mixture, in \hich bothsubstrates wAere omitted and 1.0 pumole of P!yruvat-wvas added, was used as an internald standard tomoniitor for pyruvate recovery.

Following deproteinization, 0.25 nml of 1 N KOHIwas added, with vigorous stirring, to 1.0( ml aliquotsof the supernatant solutions. After chilling, insoluble potassium perchlorate was renmoved bv centrifiu-gation. Aliquots of the resulting supernatant solu-tioIns were diluted 1 :1 (v/v) withl 1.0( M phosphatebuffer, pH 7.4, prior to the spectropllotometric de-termination of unr-eacted hvdroxypyruvate and of thlepyruvate produced bhy transamination.

Aliquots (0.5 ml) of the above diluited soluitioliswere transferred to cuvettes wlhiclh contaille(d 10Mmoles of phosphate buffer, pH 7.4, and 1.22 tmolesof DPNH in a filial volumile of 28 niil. The ab-sorbancy at 340 rmpt was measured wvith a Gilford2000 slpectrophotometer usinlg a water blank beforeaand after the addition of 0.1 ml of spinach GDH(40 X l103 units). Due to the favorable eqiuilibriumiiiof this reaction, Keq -- 0.33 X 1012 (8 ), any bly-droxypvruvate present is reduced immediately with aconcomitant decrease in DPNH concentration. Acontrol assay mixture containing only buffer, wvater,DPNH, and sipinachl GD()H \was used to monitor forany iionspecific oxidation of DPNH. After correc-tion.s for volume chaniges and any changes in al-sorb)aicy of contr-ol assay mlixtulres, un reacted h\ -droxypyruvate concentrationi is calculated froml theobserved chanige in absorbancy at 340 mni using themolar extinctionl coefficienit of DPNH.

Following the above step, 0.1 ml of a soluttioni ofcrystalliine lactate dehydrogenase (0.25 nig) wasadded to the contents of the control and experimentalcuvettes. Since excess DPNH is still present, alnvpyruvate in the reaction mixtures is reduced to lac-tate with the simultaneous oxidation of DPNHThe changes in absorbancv at 340 m.u with controland experimental reaction mixtures are used for thecalculation of pyruvate concentration as oultlinedabove for hydroxypyruvate.

Certain points about the assay proce(lllre shouldbe noted. The suIml of the pyruvate pro(luced aindIthe unreacted hydroxvpvruvate recovered shotuld be

1(81 5i

1 Pl,A91T i4'41SIOLOGY

equal to the 6 ,umoles of hydroxypyruvate added atthe start of the reaction. In most experiments, thisrecovery was 90 %. The controls carried out in thetransaminase assay that permitted a measure ofhydroxypyruvate recovery, i.e. nonenzyme blank, zerotime control, and complete reaction mixture minusalanine, demonstrated that under the conditions ofthe assay there was routinelv a small nonenzvmaticloss of hydroxypyruvate. In the control in whichsubstrates were omitted and pyruvate added in thetransaminase assay, recoveries of pyruvate by thespectrophotometric assay were greater than 90 %.

Assay for D-Glycerate Kinase. The spectropho-tometric procedure described bv Lamprecht et al.,(11) was used for the assav of this enzyme.

Specific Activities of Enzyme. One unit ofenzyme activitv for both dehydrogenases and trans-aminases is defined as that a-mount of enzyme thatcatalyzes the formation of 1 mpumole of product permin under the standard conditions of assav. Spe-cific activitv is units per mg of protein.

Results

Distribution Studies. The levels of activity ofPGTDH and GDH found in a variety of seeds andleaves are summarized in table I. PGDH is widelydistributed in seeds in contrast to GDH which iseither absent or present at very low levels. In thoseseeds in which both activities were detected, thelevel of PGDH greatly exceeds that of GDH. Incontrast to seeds, mature leaves show the oppositedistribution of the 2 dehydrogenases. Since PGDHis primarily associated with seeds and GDH withleaves, the activities of the 2 dehydrogenases indifferent tissues were compared in gerninating peas.

Developmental Studies on GDH and PGDH.The levels of activity of both dehydrogenases inleaves during development of peas are given in tableTT. When plants are grown in the light, PGDH

Table II. Specific Activities of 3-PhosphoglyceratC andD-Glycerate Dehydroqtenases in Pea Leaves

During DevelopmentExperimental condition.s as described in table I.

Age and growth PGDH:conditions GDH PGDH GDH

nifAsmoles per 1m1g protcinl ratio

ber inin

10 days, light11 " "12 " "14 ' "15)17 "12 days, dark16 " "

10.23.33.31.36.38.7'7.

.4 4- 1.6' 3.5 + 0.90 + 1.8 3.3 + 0.3.7 ± 3.2 3.1 ± 0.5.4 - 1.8 2.2 + 0.5. -+- 1.1 2.2 + 0.4.9 ± 1.6 2.1 + 0.3.7 + 0 6.9 + 0.4 + 0.3 5.8 + 0

0.340.140.090.070.060.050.900.78

Mean deviation of results from at least 3 separategrowth experiments.

activity remains relatively constant within the timeperiod studied. On the other hand, the specificactivity of GDH shows a marked increase between10 and 12 days, after which time there is a gradualincrease to a relatively constant level. These re-

sults confirm observations made by Stafford andMagaldi (22) who reported similar changes in GDHactivity at this stage of development. The over-alltrend of increasing activities of GDH, as comparedto PGDH, during leaf development is seen more

clearly when the ratios of the respective activitiesare used as the criterion of change. In contrast to

the results obtained in the light, a reciprocal trendis observed in etiolated pea leaves as reflected by an

increase in the PGDH:GDH ratios.The levels of activity of the 2 dehydrogenases in

apical meristem during development (table III)differ significantly from those in leaf. PGDH ac-

tivity is several fold higher and GDH is significantlylower in apical meristem than in leaf. However, as

in leaf, GDH shows an increase between 10 and 12

Table 1. Distribution of 3-Phosphoglycerate and D-Glycerate Dehydrogenases in Plant Seeds and Leaves

'rie procedures used for the enzyme preparation, standard conditions for assay and other experimental details-re described under Materials and Methods.

Source

Pea (Pisum sativum)Soybean (Glycine mnax)Snap Bean (Phaseolus vulgarus)Mung Bean (Phaseolus aureus)Oat (Avena sativa)Wheat (Triticutn aestivum)Wheat GermBeet (Beta vulgaris)Parsley (Petroselinum crispum)Spinach (Spinct' ia ol. racca)Radish (Raphanus sativas)Endive (Cichoriun en(liva)

SeedSpecific activities off:

PIGDH GDH PGDH GDH

m,rntoles per mtq protein per min10.4 <0.3 2.1 40.03.4 0.4 5.8 24.2

17.0 1.2 2.6 9.44.5 0 <0.3 8.92.5 0 <0.3 18.1

<0.3 <0.37.6 <0.3

<0.31.5

<0.3O.. .i...< .3... ~ <0.31

. . . . .

Z0.314.427.812.75.4

CHE,N(G TLE-SERIN MIETABOLISM IN- HIGHER PLANTS11'Fable 111. Spccific Activities of 3-Plhosphoglyccrate (tia

)-Glveerate Delhxdrogenascs in7 Apical MeristeJiiDuring Pea Dezelopment

Experimental conditions as described in table 1.

\ge and growthconditions

10 days, light11 " "1314

16 "17 II

12 days. dark14 "16 " "

G(DH P(DH

in,pamoics Per iug proteinlper litinl

2.8 ± 0.21 12.9 ± 08.1 ± 1.7 14.4 ± 1.68.3 ± 1.1 1.5.4 + 1.36.4 + 1.0 16.0 ± 0.9

.; ± 0.9 1.5.4 + 0.64.5 -f- 0.5 14.5 -4- 0.34.3 ± 0 16.0 ± 0.12.7 ± 0.1 14.6 ± 0.42.4 ± 0.3 10.4 ± 0.43.2 - 0.1 11.4 ± 0.5

PGDH:GDH

4.61.81.92.;2.83.23.75.44.33.6

Mean deviation of results from at least 3 separategrowth experimetnts.

Table IV. Spccifit Activities of 3-Phosphoglycerate (oldD-Glycerate Dchydrogenatises in Pea Fpicotls

Durinig DevelopenttExperimental conditions as described in table 1.

Age and growthconditions GDH

l'GDH:PGDH GDH

days. Etiolation appears to have little or no effecton PGI)H whereas GDH activity decreases to valueslower thlla those correspoIldinig to the samile tilimep)eriod wVhen pl)laIts are grown in the lighit.

In epicotyls durin,g dlevelopment (table I-V).(GDH remains constant throughout the timie )erio(lstudied. HoNwever, PGDH shows a ldecrease illactivity after the eleventh day, problal)ly reflectingthe cessatioin of growth in this region of the plantas development proceeds. Tlle last tisstue studiedwith respect to the activities of the 2 (elev(ll-ogeialsesduring growth was the cotyledon (table V). Thelevels of GDH activity remlain essentialily constanituntil the seventeenth day of growth after which tinieactivity of this enzyme could not be detected. Thelevel of PGDHI activity deecreased withi timle untiltulndetectable after 15 days of growth.

The levels of both (lehydrogenases observed illel)icotyl and cotyledn(lo udrinig development reflectthe physiological role of these tisstues. The epicotylis not a 1)art of tlle l)lant's storage systemii and wouldnot be exp)ected to show a rapid turnover- of starch.fIn accordance, both enzyimies were low in this tissuetlhroughout the timiie period investigated. The coty-ledons, which supply reserve food to the growingplant, age anId die after an iniitial period of metabolicactivity. Accordingly, the enizyme levels remainedrelatively coinstant or decreased, and after the seven-teenith dav neither activitv could be detecte(l.

days, ligh.1I. .

hl!AIIl(eS per lllfJ prtetinper Jllinj

0.7 + 0.11 1.3 + 0.51.0 + 0.1 3.3 ± 0.60.8 ± 0.2 2.8 + 0.40.8 + 0.1 1.9 ± 0.20.7 + 0.1 1.2 ± 0.3

1.93.33.52.41.7

MMean deviation of results from at least 3 separategrowth experiments.

Table V. Specific Activities of 3-Phlosphoglvccrate (anidD-Glvcerate Dehydrogeniases in Pea Cotyledons

During DezvelopmiewnExperimental conditions as described in table I.

Age and growtthconditions

10 days, light 1.11 " "112 1.:

13 " 1.,1415 "~ " 1.

16 " " 1

17 L" 1

Mean deviation of rgrowth experiments.

GDH PGDH

1m,umoles per mytg protein.pernlin

A4 ± 0.31.6 ±+ 0.2

7 0.33 0.35 0.3

-+- 0.3.5 + 0.3.5 0.3

4.72.91.81.20.5

results from at I

0.7+ 0.5-+- 0.44- 0.4-+: 0.1

PGDH:GDH

r-atio

3.41.81.10.90.3

Table Vt. Specific Activities of Hydroxypyruzvate:L-Vmlaninc an1d Phosphzohvdro.rvpvrniva(te:L-Gllltahllatc

Ti7lrmsawitiases I)nring Pea DcvelopnienetExperimental cotnditions as described in table I.

Tissue

SeedLeaf

Apicalineristem

Hydroxy-p)yruvate:L-alanine

Age transaminase

10111315;16

11131516

deviationexperiments.

P-Hydroxy-pyruvate:i.-glutamatetran saminase

inpAn10oles per m1i.l pr-oteinl pernin<0.1 1.8 01

8.2 -+ 0.5 4.6 0.18.5 + 1.6 4.7 + 0.48.8 1.6 4.2 + 0.2

13.1 0.2 4.5 + 0.611.1 + 0.7 4.0 0.6

9.4 + 0.69.5 1.18.6 + 1.18.5 + 0.68.3 + 0.9

of results from 2

8.7 + 0.711.1 0.410.7 + 0.412.4 + 0.811.7 0.3

separate growtli

Developmental Studies oni Transamninases. The*.. levels of activity of hydroxypyruvate :L-alanine and

*P-hydroxvpyruvate :L-glutamate transaminases foundduring development of the pea plant are given in

least 3 separate table VI. Because of the amounts of enzyme prep-aration required for the transaminase assavs. only

1011131516

1818PLAN'T PHYSIOLOGY

Table VII. . Issaj for D-Glyceratc Kiiiase inPea Leaves

Pea leaves from plants grown in the field w-eretreated and assayed as described under 'Materials andMlethods.

Comnponients ofassay system

CompleteCompleteMinus D-glycerateMinus ATPMinus enzyme

Phosphoglycerateformation

in,uiolses per nig protein per nin15.015.50

seed, leaf and a-pical meristemn were investigated.II contrast to the dehydrogenases, the activities ofboth transaminases remained relatively constant inboth leaf and apical meristem over the time periodstudied. However, the levels of activity of a giventransaminase are generally comparable to those ofthe dehydrogenase of the corresponding pathwav inthe tissues investigated. In seeds, for example. hy-droxypyruvate :L-alanine transaminase is barely de-tectable, as is GDH, whereas the 2 enzymes of thephosphorylatecd pathway are readily demonstrable.I-n leaf, the activity of hydroxypyruvate :L-alaninetransaminase is higher than that of P-hydroxvpyru-vate :L-glutamate transaminase in agreement withthe relative levels of activity of the 2 dehydrogenasesin this tissue. Although there is no decrease in thelevel of activity of hydroxypyruvate :L-alanine trans-aminase in apical meristem as compared to leaf (aswas observed with GDH), the activity of P-hydroxy-pyruvate :L-glutanmate transaminase is higher in thistissue than it is in leaf which is consistent with thechanges observed for PGDH in these 2 tissues.

In view of the relationship of D-glycerate togl-colytic intermediates and of the high levels ofactivity of the enzymes of the nonphosphorvlatedpathwway in leaves, studies wvere carried out to de-termine if glvcerate kinase is present in pea leaves.The results (table VII) clearly establish the presenceof this enzvme in this tissue.

Discussion

'lihe miiaini plupose of this sttidv was to gain in-.>i;g,ht into the relativ e sigilificainice of the phos-hlorylated anid nonphosphorylated lathways for

serinie mnetabolism during development. Since germi-nia,ting see(l consists of an organized collection ofmiany tissues which differ widely in ftunction and

fate. it was necessary to investigate the individual

parts of the developing plant if meanlingful resultswere to be obtained. On the basis of the relativelevels of activity of dte individual enzymes, thepresent studies indicate that the phosphorylated path-way is of major importance for serine metabolism intissues associated with rapid cell proliferation, e.g.seed and apical meristem, and that the nonphos-

plhohylated pathway plays a predominant role ing-reen leaves. The functional role of both pathwaysmust be closely associated with carbohydrate metabo-lism in view of the intermediates involved.

In general, the routes for carbohydrate utilizationior formation in plants vary not only with the stageof development but also with different types of plants.Since the pea is a starch-storing seed, upon germi-nation there is a rapid turnover of this material asevidenced by respiratory quotients of approximatelyone. Although starch is the major reserve in theendosperm, fat and protein also play an importantrole in seed development. As germination proceeds.the plant begins to replenish its sugars concomitantwith the onset of photosynthesis. In green tissues.hexose phosphates are the net prodticts of carbonlaIssimilation by the photosynthetic carbon cycle.Another route for sugar formationl involves theglyoxylate cycle in which a net conversion of carbonfrom fatty acids to carbohydrate is effected. ThisPathway is known to be of importance in fat-storingseeds during germination whenl massive breakdownof stored fat and its conversion to carbohvdrateoccurs (2). The key enzymes of this pathway, i.e.isocitritase and malate svnthetase, are knowin to beabsent from seed until germination when de novosynthesis occurs (12, 17). A third route by whichl)Iants can form precursors of hexose is the glycolate,or glyoxvlate-serine, pathway. -GGlycerate has beeni(leimonstrated as an interme(liate in the pathway fromilglvcine or serine to sucrose in pea leaves (15).

Trhe einzymes of the noniplhosphorvlated plathway,GTDH and hydroxypyrtluvate :1.-alaninie transaiminase.mllust function in the glycolate pathway and thus pro-vide the enzymatic link between serine and D-glVc-errate. The role of these 2 enzymes in this gluco-neogenic route is further sulpported by the presentdemonstration of a D-glycerate kinase in pea leaves.This activity wvas shown to require D-glvcerate andto be dependent upon ATP. The presence of thiskinase completes the circuit -of enzymies necessaryfor the conversion of serine to triose-P precursorsof sugars. That these enzymes are inivolved in agluconeogenic role in leaf is further indlicated by theresults of the light vs. dark growth experimiients.\When peas -were grown in the dark, leaves werefoulnd to cotntain reduiced levels of GDH over those)resent in light-grow6,n plants; a sinmilar resuilt wasobtaiined with apical meristem. These observationscorrelate well with the findings of Miflin et atl. ( 15)who showed that in pea leaves the conversion of,erine to sucrose is light dependent.

The present studies do not rule ouit the ilossi-b)ilitv that in certain tissues, e.g. leaf, apical meristem.the nonphosphorylated pathway mnay also serve as aroute for serine biosynthesis from carbohvdrate in-termediates. In fact, the labeling patterns in D-glyc-erate, formed in tobacco leaves during 14CO0 photo-synthesis, suggest that this compound can be formedint vivo directly from intermediates in the reductivepentose phosphate cycle as well as via the glycolate

CHF,UNG, ET AL.-SERINE METABOLISM IN IIIGHER PLANTS1pathway (6). By analogy to animal s)ystems, D-glyc-erate cotuld arise either from D-2-P-glycerate bv theactioIl of a specific phosphatase (4) or more in-directly from fructose by the following conversions:fructose fructose-4-P -4 D-glyceraldehyde > D-glycerate (10). In plant tissues, sucrose wouldprovide a reads' soturce of fructose bv the action ofinvertase (ssucrase).

The fact that ungerminiated seeds contaiin signifi-cant levels of PGDH anld P-hydroxypyruvate:i.-glu-tamate transaminase, while those of GDH and hy-droxvpyruvate :iL-alanine transaminase are eitherabsent or very low, is indicative that the phosphoryl-aited pathvay is titilized for serine formation in thistissule. In addition, the results suggest that in thosetissues associated with rapid cell proliferation, suchas apical imieristem, the same pathway is of greaterimportance. Since D-3-P-glycerate is one of theearly products of CO., fixation in the photosyntheticcarbon cycle. the first substrate for the phosphoryl-aited pathway is readily available in tissues carryingotut photosvntlhesis. Although the levels of activityof GDH anid hydroxypyruvate :i-alanine transami-niase exceed those of PGDH and P'-hydroxypyruvate:L.-glutamnate transaminase in leaf, the latter enlzymesalre readily (lemonstrable in this tissue. These re-sulIts are consistent \\-ith those from in vivo 14CO.,studies whiclh incdicate that both cytoplasmic (glylco-late pathway) and chloroplastic (phosphorvlatedl)patway) routes funct.ion for ser-inie biosynthesis inlplant leavres (cf. 6).

Acknowledgments

The authors are indebted to Professor F. K. Skoog,lepartmnent of Botany, for the generous use of the green-house and its facilities durilng the course of these studies.The technical assistanice of Miss Julie Appert is grate-ft1ilx acknowledged.

Literature Cited

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PLA.\NT Il1 \ SiOI.O&;\

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39-54.

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