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

Comparative Studies of Enzymes Related to Serine Metabolism in Higher Plants'

Geoffrey P. Cheung2, I. Y. Rosenblum3, and H. J. Sallach Department 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 been investigated: 1) D-3.phosphoglycerate dehydrogenase, 2) phosphohydroxypyruvate :L-glutamate transaminase, 3) D-glyeerate dehydrogenase, and 4) hydroxypyruvate :i.-alanine transaminase. Comparative studies on the distribution of the 2 dehydrogena,ses in seeds and leaves from various plants revealed that D-3-phosphoglyccrate dehydrogenase is widely distributed in seeds in contrast to D-glycerate dehydrogenase, whioh is either absent or present at low levels, and that the reverse pattern is observed isn green leaves. The levels of activity of the 4 enzymes listed above were followed in different tissues of the developing pea (Pisum sativum, var. Alaska). In the leaf, from the tenth to seventeenth day of germination, the specific activity of D-glycerate dehydrogenase increased markedly tand was much higher than D.3-phosphoglycerate dehydrogenase which remained relatively constant during this time period. Etiolation resulted in a decrease in D-glycerate dehydrogenase and an increase in D-3.phosphoglycer-ate dehydrogenase activities. In apical meristem, on the other hand, 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 were found in epicotyl and cotyledon. The specific activities of the 2 transaminases remained relatively constant during development in both leaf and apical meristem. In general, however, the levels of phosphohydroxypyruvate :m.-glutamate transaminase were comparable to those of 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 biosynthesis of the above palthw%rays for serine nmetabolism in in animal systems have been established (9,18, 29, plant systems has been reporte(l and is discussed 31). The individual reactions in the pathway uti- below. Relatively few of these studies, however. lizing phosphorylated in,termediates (phosphorylated have been carried out on the individual enzyi-Matic pathway) are given by reactions I to 3 and those reactions in either of the 2 rouites. involving nonphosphorylated compounds (nonphos- The phosphorylated pathway, described above in phorylated pathway) are shown in reactions 4 and 5: reactions 1 to 3, was fir-st demonistrated in highel 1) D-3-P-glycerate + DPN+ < > P-hydroxypyru- plants by Hanfor(d and Davies (5) who showxed the vate + DPNH d- H+, 2) P-hydroxypyruvate -1 over-all conversion of n-3-P-glycerate to i.-P-serine i-glutamate < > L-P-serine -T- !a-ketoglutarate, and L-serine in 1homogenates of pea epicotyls. In 3) L-P-serine -± H.,O - L-serine + Pi, 4) D-glyc- atddition, the results of i11 ivo() '4CO2 labeling experi- erate + DPN+ < > hydroxypyruvate + DPNH ments during short term photosynthesis are consis- ± H+, 5) hydroxypyruvate + L-alanine <---* Lserine tent with a functional phosphorvlate(d pathway for + pyruvate. Evidence for the occurrence of bothl serine formation in many plant preparatiolls incluld- ing spinach chloroplasts (3), tobacco leaves (6), and algae (7). Furthermore, the occurrence in wheat 1 This study was supported in part by Grant AM- germ of all of the enzymes of the phosphorvlated 00922 from the National Institute of Arthritis and Meta- pathway, i.e. PGDH', P-hydroxypyruvate :L-gluta- bolic Diseases, National Institutes of Health, United States Public Health Service and by Research Contract mate transaminase, and phosphoserine phosphatase, No. AT(11-1)-1631 from the United States Atomic has been demonstrated in this laboratory (unpub- Energy Commission. lished observations). 2 Present Address: Department of Pediatrics, Uni- versity of Colorado Medical Center, Denver, Colorado The second route for serine formation in plants 80220. is via the glycolate pathway, also known as the 3 Predoctoral Fellow of the United States Public glyoxylate-serine pathway (16, 28). This pathway Health Service. by which glycolate is converted to suigars in photo- 4Abbreviations used are: PGDH = D)-3-phosphogly- ,cerate dehydrogenase; GDH = D-glycerate dehydro- synthesizing tissues, has been prolose(l oni the basis genase. of in vivo sttudies with 1 C-labeled sutbstrates and 1813 1814 181PLANT PHYSIOLOGY

involves the following sequence of reaction*>: gly- ait -200, were (lissolve(l in 0..1 M1t)hosphate buffer. colate = gl oxylate *-> glycine < serine <-> pH 7.4. as needed and freed of ammonium ioIns by i)-glycerate D-3-P-glycerate -hexoses.i dialvsis against the same Ibuffet before use. D-Glyc- Much of thle literatture onl the metabolism of inter- erate and hvdiroxypyruvate were plrel)ared and mediates of this pathway in plants has been reviewed assayed as described previously (29). The cyclo- in recent papers from the laboratories of Cossins hexvlaminnionitumi salt of the dimethyl ketal of P-by- (2()) and Tolbert (23). droxypyruvate (Cal,biochem.) was converted to the Early studies on glycolate and glyoxvlate metabo- free acidI tusitig the method of Ballou (1). Other lismii in leaf tissues were reported in 1962 (16, 28). reagents were commercial preparations of the highest Since that time, numerous detailed studies tutilizing purity available. Stock solutions of all rieagenlts different 1 4C-labeled intermediates of the lyvcolate were prepared at neutrality as their potassiumni salts pathway have been carried out with a wide -variety unless otherwise indicated. of plant tissues including wheat leaves (25, 26, 27). Plant Materials. In the distribution studies. pea leaves (15), tobacco leaves (6) pea roots. pea plants were either grown in the laboratory or ob- cotvledons, corn roots, corn coleoptiles. and sunI- tained from commercial sources. All seeds were flower cotvledons (20). In all of these investiga- purchased from local dealers. For the developmental tions serine has been implicated as a key intermediate studies, pea seeds, Pisitrn sativiinm (var., Ala-ka). in the over-all path of carbon from glycolate to were soaked overnight in tap water and tlhen planlted carbohydrate. Available evidence from the above about 3 cm deep in vermiculite for growth. Tem- iln vivo studies suggests that the pathway from perature was kept constant at 300 and a daily photo- serine to sucrose is vzia hvbdroxvpvruvate. n- glcverate period- of 12 hr was maintained except for dark- and D-3-P-glycerate. ,grown plants. Water was supplied daily to all Although the noniphosphorvlated pathway (de- plants. \t the indicated times after plantinZ, the scribed above in reactions 4 and 5) has not been peas were harvested and their mature leaves, apical implicated as a functional route for serine formation neristems, cotyTledons, and epicotyls (the latter being from carbohydrate in plants, both enzymes involved, defined as that area of the shoot extending from its i.e. GDH (21) and hydroxypyruvate :i-alanine attachment to the cotyledons to the level directly transaminase (30) have been demonstrated in a below the first lateral shoot) were dissected out and variety of plant tissues. Both enzymes show essen- imm-ediately frozen ill a container immersed in an tiallv the same distribution of activity in that thev aceetone-dry ice hath. .are widely distributed in green leaves with little or Enz-vine Preparationts. All operations were car- 11o activity in seeds or tubers. Developmental ried out at 4' unless otherwise stated. sttudies carried out with peas and wheat have coni- In the distributioni studies, plant material; were firmed the fact that GDH is primarily a leaf enzyme prepared as described by Staifford et al., (21) except and that the main inerease in enzymatic activity that the extractions were made vith 0.1 M phosiphate during growth is associated with maximal leaf de- buffer, pH 7.4, containing 0.00)3 M E:DTA and velopment (22). 0.0001 M 2-mercaptoethanol. In view of the alternate routes for serine metabo- The following conditions were used in the devel- lism in plants, a comparative studv of the enzymes opmenital studies. Frozen tisstues were ground with of the phosphorylated and nonphosphorylated path- sea sand in a mortar and pestle with 1:3 (w/v) ratio ways was carried out to investigate the relative of 0 01 M tris-HCI buffer, pH 7.4, containing 0.01 Mr significance of the 2 routes. The distribution of EDTA and 0.0001 M 2-mereaptoethanol. The slurry PCGDH in seeds and leaves from various plant was filtered through cheese cloth prior to centrifuga- sources has been compared to that of GDH. Tn tionl at 35,O0Og for 30 min. The resultant super- aidditioni, the relative levels of activity of the dehy- niatant soluition ras centrifuged at 105,000g for 30 (Irogenases and transaminases in different parts of min and the final supernatant brought to 80 % the developing pea plant have been determined and saturation by the slow addition, with stirrinig, of the alre reported. calculated amount of solid ammonium sulfate. The precipitate was collected by cenltri fugation and re- Materials and Methods dissolved in 0.01 M phosphate buffer, pH 7.4, con- taining 0.001 M EDTA and 0.0001 M 2-mercapto- Reagen ts and Substrates. Crystalline lactate de- ethanol, in a 1 :1 (v/w) ratio of the original tissue hydrogenase, glutamate dehydrogenase. pvruvate weight. This preparation was used for the assays kinase, DPN, DPNH, ATP, D-3-P-glycerate, pyru- of the dehydrogenases. Before use in the assays vate, pyridoxal phosphate, tris-HCl, and ignited sea for the transaminases and for D-glycerate kinase, the sand were obtainled from Sigma Chemical Company. enzyme preparation was dialyzed against the same Crystalline phosphoglycerate mutase and enolase phosphate buffer with regular changes until the were purchased from C. F. Boehringer and Soehne. dialysate gave a negative reaction to Nessler's GimbH, Mannheim. GDH was purified from spinach reagent. leaves to step 4 as outlined elsewhere (19); aliquots In control experiments, the supernatant solutions of the ammonium sulfate residuies which were stored from the 80 % ammonium sulfate fraction were CHEI'NG, ET Al,. SERTNE METABOTISM IN HIGHER PLANTS 1(81 5i assayed for dehydrogenase and transalm inase activi- after a 60 mmit incutbation at 37' by the addlition of ties. None were detected. All enzymle assays were 0.1 ml of 4.5 N perchloric acid. performned on the same dlay that the plants were The enzyimatic activities of all tissuies werer> ron- halrvested. -Protein conicelntrations were (letermllin(d tinel\ (leterminie(l at 4 levels of enzymie and(1 the 1w the method of Lowry et ail., ( 13) uising l)bovine linear relationship betwveen enzylmie concentration an(l ser-umiii all)umin as the standard. pyruvate produce(d wvas used in the calculation of Standard Assays for GDH aiid PGDH. The specific activity. A complete reactioni niiixtur-e tlhit standard assay conditions (lescribed previously were wvas stopled(at zero tinie w\as used to miiollitor for- uised for GDH (29) and PGDTI (31). Ilitial the presence of pvruvate in the enzyme solutioni. velocities were measured uisinig a Gilford 2000 re- Additional controls includ(le( comiiplete reaction inix- cording spectrophotometer. Assavs were carried out tures except for the followving: A) enzyme, to de- in triplicate on eaclh saample and the average valtue terumiine possible nonenzymatic transamination a.l well reported. as nonenzvmiatic loss of hydroxypvruvate: B) ala- Standard Assay for- P-Hydroxvpv,it-natc :i.-Gluta - nine, to nieasuire enzvnatic loss of hvdroxvpvrnvate: nate Transamiiinase. Detailed conditions for the and C) hydroxyp) ruvate, to (leternmine production determiniationi of this transaminase have been pub- of pyruvate from alanine by other possible reactions. lished previously (24). The present stuidies were Finally, a complete reaction mixture, in \hich both car-ried out essentially in the same manner except substrates wAere omitted and 1.0 pumole of P!yruvat- that the com)plete reaction mixture contained: so- wvas added, was used as an internald standard to dium bor,ate butffer, pH 8.2, 100 pumoles; pyridoxal moniitor for pyruvate recovery. phosiphate. 2.5 X 10-2 ',moles; EDTA, 50 ,imoles: Following deproteinization, 0.25 nml of 1 N KOHI L-glutamate, 10 tmoles: 2-mercaptoethanol. 0.o0 was added, with vigorous stirring, to 1.0( ml aliquots ,umoles; and P-hydroxypyruvate. 5 tmoles. in a total of the supernatant solutions. After chilling, insol volume of 1.4 ml. The same controls as used pre- uble potassium perchlorate was renmoved bv centrifiu- viouslv, i.c. zero time control, internal a-ketoglu- gation. Aliquots of the resulting supernatant solu- tarate standard, and complete reaction mixtulres in tioIns were diluted 1 :1 (v/v) withl 1.0( M phosphate which either P-hydroxypvruivate or L-glutamate was buffer, pH 7.4, prior to the spectropllotometric de- omitted (cf. 24). were carried out under the above termination of unr-eacted hvdroxypyruvate and of thle condition,s with water added where substrates wer-e pyruvate produced bhy transamination. omitted. The reaction was stopped by tlle addition Aliquots (0.5 ml) of the above diluited soluitiolis of 0.1 ml of 4.5 N perchloric acid and precipitated were transferred to cuvettes wlhiclh contaille(d 10 protein was remove(l bv centrifugation. .Aliquots of Mmoles of phosphate buffer, pH 7.4, and 1.22 tmoles the deproteinized solutionis (1.0 ml) were diluted of DPNH in a filial volumile of 28 niil. The ab- witlh 0.25 ml of water before the addition, witlh sorbancy at 340 rmpt was measured wvith a Gilford vigorous stirring, of 0.25 ml of 1 N KOH. The 2000 slpectrophotometer usinlg a water blank before soltitionis were chilled and( insoluble potassiuim per- aand after the addition of 0.1 ml of spinach GDH chlorate wvas removed by centrifugation. Aliquots (40 X l103 units). Due to the favorable eqiuilibriumiii of the resulting supernatant solutions were assaved of this reaction, Keq -- 0.33 X 1012 (8 ), any bly- for a-ketoglutarate. The determinationi of this droxypvruvate present is reduced immediately with a a-keto acid and all other coneditions wer-e identical concomitant decrease in DPNH concentration. A to those ouitlined earlier (24). control assay mixture containing only buffer, wvater, Stanidard Assay for- HvidroxAvpvrz'ltatc : -.-4lan ince DPNH, and sipinachl GD()H \was used to monitor for Transaminiase. The method usede for the determina- any iionspecific oxidation of DPNH. After correc- tioni of this transaminiase is de-cribed in detail belowv. tion.s for volume chaniges and any changes in al- In gener,al, the procedure involves the measurement sorb)aicy of contr-ol assay mlixtulres, un reacted h\ - of the p1-rodutct of the reactioni, lpyruvate. in the droxypyruvate concentrationi is calculated froml the presence of tinreacted hydroxypyruvate. Both of observed chanige in absorbancy at 340 mni using the these a-keto acids are substrates for lactate (lel- molar extinctionl coefficienit of DPNH. drogenase (14), but only the latter compountd is a Following the above step, 0.1 ml of a soluttioni of -substrate fo- slpinach GDH (8). In the assay pro-- crystalliine lactate dehydrogenase (0.25 nig) was cedure, uii reacted hydroxypvruvate is quantitatively- added to the contents of the control and experimental reduced in the presence of excess DPNI-I and spinach cuvettes. Since excess DPNH G;DH is still present, alnv prior to the spectrophotonietric determinlation pyruvate in the reaction mixtures is reduced to lac- of pyruivate withl lactate dehvdrogenase. tate with the simultaneous oxidation of DPNH In the standard assay for the transaminase, var\r- The changes in absorbancv at 340 m.u with control ing levels of enzyme were preincubated for 10 min and experimental reaction mixtures are used for the at 37° with sodiunm borate buffer, pH 8.2., 100 umoles; calculation of pyruvate concentration as oultlined pyridoxal phosphate, 2 X 10-2 ,nuoles; and L-alanine. above for hydroxypyruvate. 10 The Mkmoles. reaction was started bv the addition Certain points about the assay proce(lllre should of 6 ,nioles of hydroxypyruivate to give a final be noted. The of the of suIml pyruvate pro(luced aindI voltumie 1.4 ml. The reaction wias terminated the unreacted hydroxvpvruvate recovered shotuld be 1816 1 Pl,A91T i4'41SIOLOGY equal to the 6 ,umoles of hydroxypyruvate added at Table II. Specific Activities of 3-PhosphoglyceratC and the start of the reaction. In most experiments, this D-Glycerate Dehydroqtenases in Pea Leaves recovery was 90 %. The controls carried out in the During Development transaminase assay that permitted a measure of Experimental condition.s as described in table I. hydroxypyruvate recovery, i.e. nonenzyme blank, zero mixture minus Age and growth PGDH: time control, and complete reaction GDH PGDH GDH alanine, demonstrated that under the conditions of conditions the assay there was routinelv a small nonenzvmatic nifAsmoles per 1m1g protcinl ratio loss of hydroxypyruvate. In the control in which ber inin substrates were omitted and pyruvate added in the 10 days, light 10..4 4- 1.6' 3.5 + 0.9 0.34 " 23.0 + 1.8 3.3 + 0.3 0.14 transaminase assay, recoveries of pyruvate by the 11 " ± ± than 90 %. 12 " " 33..7 3.2 3.1 0.5 0.09 spectrophotometric assay were greater - 2.2 + 0.5 0.07 Assay for D-Glycerate Kinase. The spectropho- 14 ' " 31..4 1.8 15) 36.. -+- 1.1 2.2 + 0.4 0.06 tometric procedure described bv Lamprecht et al., 17 " 38..9 ± 1.6 2.1 + 0.3 0.05 (11) was used for the assav of this enzyme. 12 days, dark 7'.7 + 0 6.9 + 0 0.90 Specific Activities of Enzyme. One unit of 16 " " 7..4 + 0.3 5.8 + 0 0.78 enzyme activitv for both dehydrogenases and trans- Mean deviation of results from at least 3 separate aminases is defined as that a-mount of enzyme that experiments. catalyzes the formation of 1 mpumole of product per growth min under the standard conditions of assav. Spe- within the time protein. activity remains relatively constant cific activitv is units per mg of period studied. On the other hand, the specific activity of GDH shows a marked increase between Results 10 and 12 days, after which time there is a gradual increase to a relatively constant level. These re- Distribution Studies. The levels of activity of sults confirm observations made by Stafford and PGTDH and GDH found in a variety of seeds and Magaldi (22) who reported similar changes in GDH leaves are summarized in table I. PGDH is widely activity at this stage of development. The over-all distributed in seeds in contrast to GDH which is trend of increasing activities of GDH, as compared either absent or present at very low levels. In those to PGDH, during leaf development is seen more seeds in which both activities were detected, the clearly when the ratios of the respective activities level of PGDH greatly exceeds that of GDH. In are used as the criterion of change. In contrast to contrast to seeds, mature leaves show the opposite the results obtained in the light, a reciprocal trend distribution of the 2 dehydrogenases. Since PGDH is observed in etiolated pea leaves as reflected by an is primarily associated with seeds and GDH with increase in the PGDH:GDH ratios. leaves, the activities of the 2 dehydrogenases in The levels of activity of the 2 dehydrogenases in different tissues were compared in gerninating peas. apical meristem during development (table III) Developmental Studies on GDH and PGDH. differ significantly from those in leaf. PGDH ac- The levels of activity of both dehydrogenases in tivity is several fold higher and GDH is significantly leaves during development of peas are given in table lower in apical meristem than in leaf. However, as TT. When plants are grown in the light, PGDH 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. Lea-t Seed Leaf Specific activities off: Source PIGDH GDH PGDH GDH m,rntoles per mtq protein per min 2.1 40.0 Pea (Pisum sativum) 10.4 <0.3 3.4 0.4 5.8 24.2 Soybean (Glycine mnax) 2.6 9.4 Snap Bean (Phaseolus vulgarus) 17.0 1.2 4.5 0 <0.3 8.9 Mung Bean (Phaseolus aureus) <0.3 18.1 Oat (Avena sativa) 2.5 0 Wheat aestivum) <0.3 <0.3 (Triticutn 7.6 <0.3 Wheat Germ <0.3 Z0.3 Beet (Beta vulgaris) 14.4 Parsley (Petroselinum crispum) 1.5 27.8 Spinach (Spinct' ia ol. racca) <0.3 12.7 Radish (Raphanus sativas) O.. .i...< ... ~ <0.31.3 5.4 Endive (Cichoriun en(liva) . . . . . CHE,N(G LE-SERINT MIETABOLISM IN- HIGHER PLANTS11 18A17 'Fable 111. Spccific Activities of 3-Plhosphoglyccrate (tia days. Etiolation appears to have little or no effect )-Glveerate Delhxdrogenascs in7 Apical MeristeJii on PGI)H whereas GDH activity decreases to values During Pea Dezelopment lower thlla those correspoIldinig to the samile tilime Experimental conditions as described in table 1. p)eriod wVhen pl)laIts are grown in the lighit. In epicotyls durin,g dlevelopment (table I-V). \ge and growth PGDH: (GDH remains constant the conditions G(DH P(DH GDH throughout timie )erio(l studied. HoNwever, PGDH shows a ldecrease ill in,pamoics Per iug proteinl ratio activity after the eleventh day, problal)ly reflecting per litinl the cessatioin of growth in this region of the plant 10 days, light 2.8 ± 0.21 12.9 ± 0 4.6 as development proceeds. Tlle last tisstue studied 11 " " 8.1 ± 1.7 14.4 ± 1.6 1.8 with to the 13 respect activities of the 2 (elev(ll-ogeialses 8.3 ± 1.1 1.5.4 + 1.3 1.9 was the 14 6.4 + 1.0 16.0 ± 0.9 2.; during growth cotyledon (table V). The .; ± 0.9 1.5.4 + 0.6 2.8 levels of GDH activity remlain essentialily constanit 16 " 4.5 -f- 0.5 14.5 -4- 0.3 3.2 until the seventeenth day of growth after which tinie 17 II 4.3 ± 0 16.0 ± 0.1 3.7 activity of this enzyme could not be detected. The 12 days. dark 2.7 ± 0.1 14.6 ± 0.4 5.4 level of PGDHI activity deecreased withi timle until 14 " 2.4 ± 0.3 10.4 ± 0.4 4.3 tulndetectable after 15 days of growth. 16 " " 3.2 - 0.1 11.4 ± 0.5 3.6 The levels of both (lehydrogenases observed ill Mean deviation of results from at least 3 separate el)icotyl and cotyledn(lo drinigu development reflect growth experimetnts. the physiological role of these tisstues. The epicotyl is not a 1)art of tlle l)lant's storage systemii and would not be exp)ected to show a rapid turnover- of starch. fIn accordance, both enzyimies were low in this tissue tlhroughout the timiie period investigated. The coty- Table IV. Spccifit Activities of 3-Phosphoglycerate (old D-Glycerate Dchydrogenatises in Pea Fpicotls ledons, which supply reserve food to the growing Durinig Developentt plant, age anId die after an iniitial period of metabolic Experimental conditions as described in table 1. activity. Accordingly, the enizyme levels remained relatively coinstant or decreased, and after the seven- Age and growth l'GDH: teenith dav neither activitv could be detecte(l. conditions GDH PGDH GDH hl!AIIl(eS per lllfJ prtetin r(ti0 Table Vt. Specific Activities of Hydroxypyruzvate:L- per Jllinj Vmlaninc an1d Phosphzohvdro.rvpvrniva(te:L-Gllltahllatc 10 days, ligh.1 0.7 + 0.11 1.3 + 0.5 1.9 Ti7lrmsawitiases I)nring Pea Dcvelopnienet I. . 11 1.0 + 0.1 3.3 ± 0.6 3.3 Experimental cotnditions as described in table I. 13 0.8 ± 0.2 2.8 + 0.4 3.5 15 g,.. 0.8 + 0.1 1.9 ± 0.2 2.4 16 + Hydroxy- P-Hydroxy- 0.7 0.1 1.2 ± 0.3 1.7 p)yruvate: pyruvate: MMean deviation of results from at least 3 separate L-alanine i.-glutamate growth experiments. Tissue Age transaminase tran saminase davs inpAn10oles per m1i.l pr-oteinl pernin Seed <0.1 1.8 01 Leaf 10 8.2 -+ 0.5 4.6 0.1 Table V. Specific Activities of 3-Phlosphoglvccrate (anid 11 8.5 + 1.6 4.7 + 0.4 D-Glvcerate Dehydrogeniases in Pea Cotyledons 13 8.8 1.6 4.2 + 0.2 During Dezvelopmiewn 15; 13.1 0.2 4.5 + 0.6 16 11.1 + 0.7 4.0 0.6 Experimental conditions as described in table I. Apical ineristem 10 9.4 + 0.6 8.7 + 0.7 Age and growtth PGDH: 11 9.5 1.1 11.1 0.4 conditions GDH PGDH GDH .. 13 8.6 + 1.1 10.7 + 0.4 .. 15 8.5 + 0.6 12.4 + 0.8 1m,umoles per mytg protein. r-atio ~,, 16 8.3 + 0.9 11.7 pernlin 0.3 10 days, light 1.A4 ± 0.31 4.7 0.7 3.4 Meani deviation of results from 2 separate growtli 11 " "1 .6 ±+ 0.2 2.9 + 0.5 1.8 experiments. 12 1.:7 0.3 1.8 -+- 0.4 1.1 13 " 1.,3 0.3 1.2 4- 0.4 0.9 14 5 0.3 0.5 -+: 0.1 0.3 Developmental Studies oni Transamninases. The 15 "~ " 1. -+- 0.3 *.. levels of activity of hydroxypyruvate :L-alanine and 16 " " 1 .5 + 0.3 *P-hydroxvpyruvate :L-glutamate transaminases found 17 L" 1 .5 0.3 during development of the pea plant are given in Mean deviation of rresults from at leastI 3 separate table VI. Because of the amounts of enzyme prep- growth experiments. aration required for the transaminase assavs. only 1818 1818PLAN'T PHYSIOLOGY Table VII. . Issaj for D-Glyceratc Kiiiase in plhohylated pathway plays a predominant role in Pea Leaves g-reen leaves. The functional role of both pathways Pea leaves from plants grown in the field w-ere must be closely associated with carbohydrate metabo- treated and assayed as described under 'Materials and lism in view of the intermediates involved. Mlethods. In general, the routes for carbohydrate utilizationi or formation in plants vary not only with the stage Comnponients of Phosphoglycerate of development but also with different types of plants. assay system formation Since the pea is a starch-storing seed, upon germi- in,uiolses per nig protein per nin nation there is a rapid turnover of this material as Complete 15.0 evidenced by respiratory quotients of approximately Complete 15.5 one. Although starch is the major reserve in the Minus D-glycerate 0 endosperm, fat and protein also play an important Minus ATP 0 role in seed development. As germination proceeds. Minus enzyme 0 the plant begins to replenish its sugars concomitant with the onset of photosynthesis. In green tissues. seed, leaf and a-pical meristemn were investigated. hexose phosphates are the net prodticts of carbonl II contrast to the dehydrogenases, the activities of aIssimilation by the photosynthetic carbon cycle. both transaminases remained relatively constant in Another route for sugar formationl involves the both leaf and apical meristem over the time period glyoxylate cycle in which a net conversion of carbon studied. However, the levels of activity of a given from fatty acids to carbohydrate is effected. This transaminase are generally comparable to those of Pathway is known to be of importance in fat-storing the dehydrogenase of the corresponding pathwav in seeds during germination whenl massive breakdown the tissues investigated. In seeds, for example. hy- of stored fat and its conversion to carbohvdrate droxypyruvate :L-alanine transaminase is barely de- occurs (2). The key enzymes of this pathway, i.e. tectable, as is GDH, whereas the 2 enzymes of the isocitritase and malate svnthetase, are knowin to be phosphorylatecd pathway are readily demonstrable. absent from seed until germination when de novo I-n leaf, the activity of hydroxypyruvate :L-alanine synthesis occurs (12, 17). A third route by which transaminase is higher than that of P-hydroxvpyru- l)Iants can form precursors of hexose is the glycolate, vate :L-glutamate transaminase in agreement with or glyoxvlate-serine, pathway. -GGlycerate has beeni the relative levels of activity of the 2 dehydrogenases (leimonstrated as an interme(liate in the pathway fromil in this tissue. Although there is no decrease in the glvcine or serine to sucrose in pea leaves (15). level of activity of hydroxypyruvate :L-alanine trans- Trhe einzymes of the noniplhosphorvlated plathway, aminase in apical meristem as compared to leaf (as GTDH and hydroxypyrtluvate :1.-alaninie transaiminase. was observed with GDH), the activity of P-hydroxy- mllust function in the glycolate pathway and thus pro- pyruvate :L-glutanmate transaminase is higher in this vide the enzymatic link between serine and D-glVc- tissue than it is in leaf which is consistent with the errate. The role of these 2 enzymes in this gluco- changes observed for PGDH in these 2 tissues. neogenic route is further sulpported by the present a In view of the relationship of D-glycerate to demonstration of D-glycerate kinase in pea leaves. gl-colytic intermediates and of the high levels of This activity wvas shown to require D-glvcerate and activity of the enzymes of the nonphosphorvlated to be dependent upon ATP. The presence of this pathwway in leaves, studies wvere carried out to de- kinase completes the circuit -of enzymies necessary termine if glvcerate kinase is present in pea leaves. for the conversion of serine to triose-P precursors The results (table VII) clearly establish the presence of sugars. That these enzymes are inivolved in a of this enzvme in this tissue. gluconeogenic role in leaf is further indlicated by the results of the light vs. dark growth experimiients. Discussion \When peas -were grown in the dark, leaves were foulnd to cotntain reduiced levels of GDH over those 'lihe miiaini plupose of this sttidv was to gain in- )resent in light-grow6,n plants; a sinmilar resuilt was .>i;g,ht into the relativ e sigilificainice of the phos- obtaiined with apical meristem. These observations correlate well with the findings of Miflin et hlorylated anid nonphosphorylated lathways for atl. ( 15) serinie mnetabolism during development. Since germi- who showed that in pea leaves the conversion of nia,ting see(l consists of an organized collection of ,erine to sucrose is light dependent. miany tissues which differ widely in ftunction and The present studies do not rule ouit the ilossi- fate. it was necessary to investigate the individual b)ilitv that in certain tissues, e.g. leaf, apical meristem. parts of the developing plant if meanlingful results the nonphosphorylated pathway mnay also serve as a were to be obtained. On the basis of the relative route for serine biosynthesis from carbohvdrate in- levels of activity of dte individual enzymes, the termediates. In fact, the labeling patterns in D-glyc- present studies indicate that the phosphorylated path- erate, formed in tobacco leaves during 14CO0 photo- way is of major importance for serine metabolism in synthesis, suggest that this compound can be formed tissues associated with rapid cell proliferation, e.g. int vivo directly from intermediates in the reductive seed and apical meristem, and that the nonphos- pentose phosphate cycle as well as via the glycolate CHF,UNG, ET AL.-SERINE METABOLISM IN IIIGHER PLANTS1 18,19 pathway (6). By analogy to animal s)ystems, D-glyc- 7. HESS, J. L. AND N. E. TOLBERT. 1967. Glycolate erate cotuld arise either from D-2-P-glycerate bv the pathwaNa in algae. Pant Phvsiol. 42: 371-79. actioIl of a specific phosphatase (4) or more in- 8. HOLZER, H. AND A. HOILLDORF. 1957. Isolierung directly from fructose by the following conversions: Von D--Glycerat-dehydrogenase, einige Eigenschaften des- zur fructose fructose-4-P -4 D-glyceraldehyde > D- Enzymes unid seiine Verrx enidung enzy- matischoptischen Bestimmung v-on1 Hydroxypyrnivat glycerate (10). In plant tissues, sucrose would nebeni Pvruvat. Bioclheml. Z. 329: 292-319. provide a reads' soturce of fructose bv the action of 9. ICHIHARA, A. AND D. -.M. GREENBERG. 1957. Fur- invertase (ssucrase). ther stuidies on the pa,thw ay of serine formation The fact that ungerminiated seeds contaiin signifi- from carbohydrate. J. Biol. Chem. 224: 331-40. cant levels of PGDH anld P-hydroxypyruvate:i.-glu- 10. L.%AMPRECHT, W. AND F. :HEINZ. 1958. IsolierUng tamate transaminase, while those of GDH and hy- xvon Glycerinaldehyddehvd(lrocgenase aus Rattenleber droxvpyruvate :iL-alanine transaminase are either zur Biochemie des Fructosestoffwechsels. Z. absent or very low, is indicative that the Naturforschl. 13B: 464-65. phosphoryl- 11. T. F. AND aited is titilized for serine formation in LA-MPRECHT, W., DI.\M[ANSTEIN, HEINZ, pathvay this P. BALDE. 1959. Phosphorylierung von D-Gly- tissule. In addition, the results suggest that in those cerinsaure zu 2-Phospho-D-glycerinsdure mit Gly- tissues associated with rapid cell proliferation, such ceratkinase in Leher, 1. Z. Phvsiol. Chem. 316: as apical imieristem, the same pathway is of greater 97-112. importance. Since D-3-P-glycerate is one of the 12. LONGO, C. P. 1968. Evidence for de novo syn- early products of CO., fixation in the photosynthetic thcsis of isccitritase and malate synthetase in ger- carbon cycle. the first substrate for the minating peanut cotyledons. Plant Physiol. 43: phosphoryl- 660-64. aited pathway is readily available in tissues carrying otut photosvntlhesis. 13. LOWRY, 0. H., N. J. ROSEBROUGH, A. L. FARR, AND Although the levels of activity R. J. RANDALL. 1951. Protein measurement with of GDH anid hydroxypyruvate :i-alanine transami- the folin-phenol reagent. J. Biol. Chem. 193: 265- niase exceed those of PGDH and P'-hydroxypyruvate: 75. L.-glutamnate transaminase in leaf, the latter enlzymes 14. MEISTER, A. 1952. Enzymatic preparation of a- alre readily (lemonstrable in this tissue. These re- keto acids. J. Biol. Chemn. 197: 309-17. sulIts are consistent \\-ith those from in vivo 14CO., 15. M11LIN, B. J., A. F. H. MfARKER, AND C. P. WHIT- studies whiclh incdicate that both cytoplasmic (glylco- TINGHA-M. 1965. The miietabolism of glycine and late pathway) and chloroplastic glycolate by pea leaves in relation to photosyn- (phosphorvlated thesis. Biochim. l)patway) routes funct.ion for biosynthesis Biophvs. Acta 120: 266-73. ser-inie in 16. RABSON, R., N. E. TOLBERT, AND P. C. KEARNEY. lplant leavres (cf. 6). 1962. Formatioin of serine and glyceric acid by the glycolate pathway. Arch. Biochem. Biophys. Acknowledgments 98: 154-63. 17. RvcHITER, G. A. AND J. H. CHERRY. 1968. De The authors are indebted to Professor F. K. Skoog, iiovo synthesis of isocitritase in peanut (Arachis lepartmnent of Botany, for the generous use of the green- hypogaea L.) cotyledons. Plant Physiol. 43: house and its facilities durilng the course of these studies. 653-59. The technical assistanice of Miss Julie Appert is grate- 18. SALLACH, H. J. 1955. Evidenice for a specific ft1ilx acknowledged. alanine-hydroxypyruvate transaminase. In: Amino Acid Metabolistmi. W. D. McElroy and B. Glass. Literature Cited eds. The Johns Hopkins Press, Baltimore, Marv- land. p 782-87. 19. SALLACH, H. J. 1966. D-Glycerate dehydrogenases 1. BALLOU, C. E. 1960. Biochem. Prep. 7: 66-68. of liver and 2. D. T. AND H. BEEVERS. 1961. Sucrose spinach. In: Methods in Enzymology. CANVINI, WV. A. Wood, ed. Academic Press, New York. s; nthesis from acetate in the germinating castor Vol. IX: heani: kinetics and pathway. J. B,iol. Chem. 236: 221--28. 988-95. 20. SINHIA, S. K. AND E. A. COSSINS. 1965. The .. (HANG, H. AND importance of glyoxvlate in amino acidbhiosynthesis N. E. TOLBERT. 1965. Distribu- in 96: tion of 14C in serine and glycine after 14CO2 plaInts. !3iochem. J. 254-61. p)hotosynt,hesis by isolated chloroplasts. Modifi- 91. STAFFORDn, H. .\., A. MAGALD1, ANI) B. VENNES_ Cation of serine-'4C degradation. Plant Physiol LAND. 1954. T'he enzymatic reduction of hydroxy- 40: 1048-52. pyruvic acid to D-glyceric acid in higher plants. 4. FALLON, H. J. AND W. L. BYRNE. 1965. 2-Phos- J. Biol. Chem. 207: 621-29. phoglvceric acid phosphatase: identification and 22. STAFFORD, H. A. AND A. MAGALDI. 1954. A (Ie- properties of the beef liver enzyme. Biochim. velopmental study of n-glyceric acid dehydrogenase. Biophys. Acta 105: 43-53. Plant Physiol. 29: 504-08. D. HANFORD, J. AND D. D. DAVIES. 1958. Formation 23. TOLBERT, N. E. 1963. Glycolate pathway. In: of phosphoserine from 3-phosphoglycerate in higher Photosyntlhesis Meclhanisms in Green Plants. Natl. plants. Nature 182: 532-33. Acad. Sci. Natl. Res. Council. Pub]. 1145. p 6. HESS, J. L. AND N. E. TOLBERT. 1966. Glycolate, 648-62. serine, glvcine, and glycerate formation during 24. WVALSH, D. A. AND photosynthesis by tobacco leaves. J. Biol. Chem. H. J. SALLACH. 1966. Comn- 241: 5705-11. parative studies on the pathways for serine bio- synthesis. J. Biol. Chem. 241: 4068-76. 1820 PLA.\NT Il1 \ SiOI.O&;\

25. XXVANG, 1). \Ni) R. H1. BuRR{KS. 1963. Carboni miie- Metabolism of C-labele(i anino aci(ls in \\beiat tabolismii of W-C-labeled amino acids in wheat leaves. 1. A pathway of glyoxylate-serinie imietab- leaves. I 1. Serinie and its role in glvcine me- Olisnm. Plant 1hYvsiol. 37: 826-32.

tabolisimi. Plant Physiol. 38: 430-39. 29. EtI.i. l. \\ H. J. i..\(1. 1962. E-vi 26. VA\N.(; 1). AxN) IR. H. BuRIs. 1965. Car-boni miie- (jen1ce fOI- a mammalian i)-glyceric (deh(yrogenac tabolisiii of 14C-labeled aminiio acids in w heat 1. Biol. Cliem1. 237: 910-15. leaves. fI. FSurther studies oni the role of serine 30. W ILLIS. J. .'X) H. J. S mi.A\ cii. 19)63. Ser-ine1 in glycine metabolisnm. Plant Plhysiol. 40: 415-18. biosynthesis fromii hydroxy r-Uivate in plants. Phy- 27. \VA; I ). ANIG R. H. BURRIS. 1965. Carboni imie- tochemistrv 2: 23-28. tabolisiim of glycivie anid serinie in relationi to thle 31. \WVI l i:. 11. 19( 4. The svnthesis oi organiic acids and(I a guianine (le- occurr-etice of l -3-phosplhaoglycerate (dehydlrogenlase rivative. Planit Phvsiol. 40: 419-24. in animal tissues. Biochim. Biophys. A\ca 81: 28. \ DXG \Ni, E W\V YGOOD. 1962. Cai-bon 39-54.