role malate synthase citric acid synthesis maturing ...embryo halves incubated with 114ciglyoxylate...

Plant Physiol. (1981) 67, 875-88 10032-0889/8 1/67/0875/07/$00.50/0

Role of Malate Synthase in Citric Acid Synthesis by MaturingCotton Embryos: A Proposal'

Received for publication June 19, 1980 and in revised form November 18, 1980

JAN A. MIERNYK2 AND RICHARD N. TRELEASE3Department of Botany-Microbiology, Arizona State University, Tempe, Arizona 85281

ABSTRACT

Cotton embryos from 34 to 54 days after anthesis were analyzed fororganic acids, and enzymes associated with organic acid metabolism.During this developmental period, embryos accumulated citrate. Malatesynthase activity appeared at 46 days after anthesis and increased rapidlyto 54 days. Of other enzymes examined, only citrate synthase activityincreased during this period. As isocitrate lyase activity was absent fromcotton embryos during maturation, an alternative source of glyoxylatewould be required for in vivo malate synthase activity. Of several metabolicsources tested, glycine was converted to glyoxylate via a transaminationreaction.

Halves of 50-day (mature) cotton embryos incorporated radioactivityfrom Il-14Clacetate, l-14Clglyoxylate, and Ij_14Cjglycine into organicacids. Embryo halves incubated with 114CIglyoxylate plus 13Hlacetatesynthesized double-labeled malate and citrate. Radioactive citrate isolatedfrom 50-day cotton embryos incubated with 11-14Clacetate was degraded;label was distributed as follows: 55% in C1, 33% in Cs, and 12% in C6.Taken together, these data strongly suggest participation of malate syn-thase in citrate production in vivo.

Separation of organeUes by sucrose density gradient sedimentationrevealed that malate synthase, malate dehydrogenase, and citrate synthasewere compartmentalized together only in the peroxisome fraction (1.24grams per milliliter). Peroxisomes isolated from 50-day embryos, whenincubated with glyoxylate and 13Hiacetyl-CoA, synthesized labeled malateand citrate, but only radioactive citrate accumulated. Incubations withglycine plus a-ketoglutarate, in place of glyoxylate, also resulted in syn-thesis of radioactive citrate.A metabolic scheme ilustrating the participation of cotton embryo

peroxisomes in citrate synthesis is proposed. This scheme suggests afunction for plant peroxisomes not previously elucidated. The ontogeneticand metabolic relationship between these organelles and glyoxysomesactive in gluconeogenesis during postgerminative growth remains to beexamined.

Malate synthase and isocitrate lyase are unique enzymes of theglyoxylate cycle by which germinated oilseeds produce succinatefrom acetyl-CoA (3). These and other enzymes of the cycle arelocalized within specialized peroxisomes called glyoxysomes (3).The only reported occurrence of malate synthase and isocitratelyase activities in a higher plant tissue not involved in lipid tocarbohydrate conversion is in ripening fruits (2, 32) where theseenzymes are thought to be involved with organic acid intercon-

'Supported by National Science Foundation Grant PCM 7823156.2Present address: Department of Biology, Queens University, Kingston,

Ontario K7L 3N6.3 To whom reprint requests should be addressed.

versions (32).The glyoxylate cycle also is operative as an anaplerotic pathway

in microorganisms grown on alkanes or C2 carbon sources (22).There have been several reports of malate synthase without ac-companying isocitrate lyase activity in microorganisms (18, 28).In Rhizobia, Stovall and Cole (31) proposed a role for malatesynthase in organic acid metabolism.

In contrast to the enormous body of knowledge that existsconcerning seed germination, relatively little is known about seeddevelopment (12). Choinski and Trelease (8) reported that devel-oping cotton embryos synthesized catalase-containing peroxi-somes beginning about 22 DAA.4 Malate synthase activity devel-oped just prior to desiccation (46 DAA) and coequilibrated withcatalase activity in the peroxisome region of sucrose-density gra-dients. Isocitrate lyase activity has not been detected at any timeduring cotton embryo development (8, 12, 25, 33). It now appearsthat malate synthase, in the absence of detectable isocitrate lyaseactivity, occurs universally among ungerminated oilseeds (25).The purpose of this investigation was to test for in vivo malatesynthase activity and for the possible involvement of malatesynthase in organic acid metabolism during the late stages ofcotton embryo development.

MATERIALS AND METHODS

Embryos. Continuously flowering cotton (Gossypium hirsutumL. cv. Deltapine 61) plants were glasshouse grown at a 12-h 35/25C thermoperiod and irrigated with one-fourth strength Hoaglandsolution. Flowers were tagged on the day of anthesis, and devel-oping seeds were selected according to their chronological age,fresh weight, and degree of seed coat sclerification (Fig. 1) (8).

Reagents. Substrates and coupling enzymes were purchasedfrom Sigma. The lithium salt of CoA was purchased from P-LBiochemicals and used to prepare acetyl-CoA as previously de-scribed (26). All other chemicals were of analytical prade. Isotopespurchased from New England Nuclear were: [1- 4C]acetate (60mCi/mmol), [2-'4Clacetate (60 mCi/mmol), [3H]acetate (500 mCi/mmol), [3Hlacetyl-CoA (500 mCi/mmol), and [1_14C]glycine (60mCi/mmol). Amersham supplied [1_14CJglyoxylate (8.33 mCi/mmol), whereas [1,5-'4CJcitrate (10 mCi/mmol) was from ICNPharmaceuticals. All isotopes were chromatographed to checkradiochemical purity before use.

Organic Acid Determinations. Excised embryos were homoge-nized in an equal volume of cold 0.6 M HC104, using a motor-driven Teflon Potter-Elvehjem homogenizer. Homogenates wereclarified by centrifugation (20 min, 27,000g), neutralized with 5M K2CO3, the KC104 removed by centrifugation, and the finalsupernatants assayed for acids. Recoveries, based on addition ofstandards to initial homogenates, were greater than 90%o; therefore,

4Abbreviations: DAA, days after anthesis; DH, dehydrogenase; AT,aminotransferase.

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MIERNYK AND TRELEASE

t is ~~31 4: D.

FIG. 1. Morphology of ovules excised from boils at various ages post-anthesis. Incipient seed coat sclerification is apparent at the chalazal endof ovules at 38 DAA. Sclerification of the seed coat is uniform at 50 DAA.

values presented are uncorrected. Malate, lactate, pyruvate, oxal-oacetate, fumarate, oxalate, isocitrate, glycolate, and glyoxylateconcentrations were determined enzymically as described in Berg-meyer (4). Published methods were used to quantitate citrate andaconitate (27), and succinate (34).Enzyme Activities. Clarified homogenates were prepared as

previously described (8). Published assay procedures were fol-lowed for malate synthase and isocitrate lyase (25), malate DHand citrate synthase (8), aconitase and glycolate oxidase (9), NAD-and NADP-isocitrate DH (11), NAD- and NADP-malic enzyme(10), fumarase (17), ATP-citrate lyase (15), and NAD- and NADP-glyoxylate DH (29). Aspartate: a-ketoglutarate AT (EC 2.6.1.1)was assayed by coupling with malate DH. Complete reactionmixtures contained; 70 umol Mops-KOH, pH 8.2, 3 ,umol aspar-tate, 60 nmol pyridoxal-5'-phosphate, 1.25 ,umol NADH andextract in a final volume of 1.0 ml. After recording Au0 for"NADH oxidase" activity, the reaction was initiated with 0.1 mlof 5 mM a-ketoglutarate. Addition of exogenous malate DH wasunnecessary. Alanine: a-ketoglutarate AT (EC 2.6.1.2) was as-sayed in the same manner, but coupled through 1 unit of pig heartlactate DH.

Concentrations of soluble and insoluble (that which sedimentswhen centrifuged at l0,OOOg for 10 min) protein were determinedby the method of Bradford (5). Lipids were extracted and quan-titated as previously described (26). A detailed report on carbo-hydrate extraction and quantitation is in preparation (35).Enzyme and Metabolite Localization. Homogenization of em-

bryos and zonal-rotor sucrose density gradient centrifugation wereconducted as previously described (26). Nonaqueous homogeni-zation and subsequent protein body isolation were by the methodof Yatsu and Jacks (36). Nonaqueous homogenization and isola-tion of a crude organelie fraction by discontinuous hexane/CCI4gradient centrifugation were conducted as described by Fry andBidwell (16).Metabolism of Isotopically Labeled Compounds. Sixty embryo

halves were incubated (cut portion down) in 9-cm plastic Petridishes containing 2 discs ofWhatman 1 paper and 2 ml of solution(isotope in sterile, deionized H20). Dishes were sealed with siliconegrease and wrapped with parafilm. All incubations were conductedin darkness. A shallow center well contained 1 ml phenethylamineto trap respired CO2. After incubation, embryos were extensivelywashed to remove any surface adsorbed isotope, then extracted asdescribed for organic acids. Neutralized extracts were partitionedagainst H20-saturated CHC13 to obtain a lipid fraction, and theaqueous phase separated into basic (amino acids), neutral (sug-ars), and acidic (organic acids) fractions by ion-exchange chro-matography (6). The organic acid fraction was further examinedby descending chromatography on Whatman 1 paper, using tert-

amyl alcohol:formic acid:H20 (3:1:1, v/v) as the mobile phase.Acids were localized by spraying papers with 0.05% bromphenolblue (w/v) in ethanol. Spots corresponding to authentic standardswere cut out and monitored for radioactivity using a BeckmanLS-8000 liquid scintillation spectrometer. Counting fluid was 16%(v/v) Triton X-100 in toluene containing 4.5 g PPO and 0.1 gPOPOP/1. All results were converted to dpm using the externalstandard method.

[I4C]malate and [14C]citrate were extracted and purified from50 DAA-embryos following a 1-h incubation with 20 ,uCi [1-14C]acetate. Malate (1.65 ,umol, 116,000 dpm) was decarboxylatedwith NADP-malic enzyme, as described by Dittrich (10). Theresulting pyruvate was similarly decarboxylated using bakers yeastpyruvate decarboxylase. Citrate (3.16 ,imol, 273,400 dpm) wassubjected to total stereochemical degradation by the method ofKent (19).

Statistical Analysis. Unless otherwise indicated, all enzyme andmetabolite data are means ± standard error of the means, foreight separate experiments. All isotope experiments were done atleast twice and data presented are means.

RESULTS

Physical and Chemical Parameters. A detailed examination ofcotton embryo development from 34 to 54 DAA was undertaken.Fresh weight increased from 34 to 50 DAA, followed by a declineas bolls opened and seeds desiccated (Table I). Dry weight in-creased from 34 to 46 DAA and leveled off. Soluble proteincontent increased throughout embryogenesis while the insolubleprotein increased from 34 to 42 DAA and remained nearly con-stant thereafter (Table I). Total sugars increased from 34 to 50DAA while starch decreased from 46 to 54 DAA. Levels ofdextrins and reducing sugars were comparatively low and re-mained relatively constant during development.

Organic Acid Analysis. Extraction of mature 54 DAA embryosand paper chromatography of the acidic fraction revealed thepresence of two major and one minor spots corresponding tooxalate, citrate/isocitrate/aconitate, and malate, respectively.Quantitative analysis of organic acids revealed that citrate was amajor acid in developing embryos (Table II).

Oxalate level was high at 34 DAA, decreased at 38-42 DAA,increased at 46-50 DAA then decreased to its lowest level inmature desiccated seeds (Table II). Aconitate increased from 34to 38 DAA, then decreased steadily through 54 DAA (Table II).In contrast, citrate levels increased from 34 to 54 DAA. Malatelevels remained constant through 42 DAA, increased to a peak at46 DAA, then decreased sharply to the lowest level in matureseeds. Lactate, fumarate, and succinate comprised only a smallportion of the total organic acid fraction, and remained relativelyconstant throughout development (Table II). Although recoveriesof standards were <90%o, levels of pyruvate, oxaloacetate, isocit-rate, glycolate, and glyoxylate in extracts were undetectable by theassays employed.Enzyme Activities. Activities of selected enzymes involved with

organic acid metabolism were measured (Table III). Malate syn-thase activity was absent prior to 46 DAA, then increased rapidlyto a maximum at 54 DAA. In contrast, activities of all otherenzymes were measurable throughout the period studied. Citratesynthase activity increased steadily from 34 to 54 DAA. Aconitaseactivity increased from 38 to 42 DAA, then remained constantthrough 50 DAA, before decreasing as embryos desiccated. MalateDH, NADP-isocitrate DH, fumarase, aspartate AT, and alanineAT activities remained relatively constant throughout the devel-opmental period examined (Table III). Aspartate AT used onlya-ketoglutarate as amino-acceptor (pyruvate and glyoxylate werealso tested). Similarly, alanine AT did not utilize either oxaloac-etate or glyoxylate as an amino acceptor. At no stage wereactivities of isocitrate lyase, NAD-isocitrate DH, NAD- or NADP-

876 Plant Physiol. Vol. 67, 1981

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MALATE SYNTHASE AND CITRATE PRODUCTION

Table I. Changes in Weight and Chemical Composition during Late Stages of Embryogenesis and Desiccation ofCotton

Values are mg embryo-' and are means ± standard errors of eight separate experiments.

Days After Anthesis

34 38 42 46 50 54

Freshweight 71.4±2 80.4±2 82.3 ± 6 90.1±3 91.6±4 52.1±3Dry weight 38.8 ± 1 45.8 ± 2 47.8 ± 7 51.1 ± 2 51.9 ± 1 51.6 ± ISoluble protein 6.4 ± 0.2 8.1 ± 0.6 7.1 ± 0.7 8.5 ± 0.6 9.4 ± 0.4 9.7 ± IInsoluble protein 13.1 ± 0.4 17.1 ± 2 18.3 ± 1 17.8 ± 0.9 17.9 ± 0.8 17.1 ± INeutral lipid 11.1 ± 1 13.3 ± 2 15.1 ± 1 16.1 ± 1 16.0 ± 1 16.0 ± IStarcha 2.1 NDb ND 2.3 1.2 0.4Dextrinc 0.3 ND ND 0.4 0.4 0.4Total sugarsc 2.2 3.8 ND 4.9 5.6 5.9Reducing sugarsc 0.1 ND ND 0.1 0.2 0.2

a Carbohydrate values are means of two determinations.b Not determined.c mg/embryo, glucose equivalents.

Table II. Developmental Changes in Organic Acid Concentration during Cotton Embryo MaturationEmbryos were homogenized in 0.6 M perchloric acid and the homogenates neutralized with 5 M K2CO3,

clarified by centrifugation, and assayed for acids. Values are -means ± standard errors of eight separateexperiments.

Organic Days After Anthesis

Acid 34 38 42 46 50 54

nmol embryo-'Oxalate 1,524 ± 74 1,041 ± 141 1,079 ± 107 1,327 ± 381 1,388 ± 424 932 ± 175Citrate 465 ± 123 636 ± 123 816 ± 123 1,020 ± 117 1,035 ± 277 1,190± 210Aconitate 739 ± 207 1,430 ± 176 1,351 ± 94 1,135 ± 129 930 ± 203 868 ± 114Malate 140 ± 19 143 ± 63 137 ± 34 180 ± 56 128 ± 49 52 ± 18Lactate 44±2 65±3 76±4 66±1 75±2 40±2Fumarate 30 ± 2 34 ± 1 16 ± 4 20 ± 1 49 ± 2 41 ± 2Succinate 20±1 20±1 12±3 18±3 13± 1 18±2

Table III. Developmental Changes in Enzyme Activity during Cotton Embryo MaturationFrench-pressed, clarified homogenates were used directly for assays. Values are means ± standard errors of eight separate experiments.

Days After AnthesisEnzyme

34 38 42 46 50 54

nmol min ' embryo -'

Malate synthase 0 0 43 ± 44 115 ± 38 208 ± 45 319 ± 24Malate DH 8,310 ± 2,004 7,770 ± 2,005 8,330 ± 2,263 7,960 ± 2,736 8,230 ± 2,419 8,390 ± 2,628Citrate synthase 83 ± 28 142 ± 30 150 ± 49 167 ± 47 205 ± 55 279 ± 42Aconitase 40±8 48±8 75±21 79±16 79±12 57±7NADP-Isocitrate DH 55 ± 24 53 ± 10 74± 12 63 ± 19 66 ± 19 62 ± 17Fumarase 103±47 148± 11 134± 1 131±42 141±27 146±64Aspartate AT 327 ± 47 314 ± 40 348 ± 54 323 ± 48 338 ± 15 358 ± 34Alanine AT 327 ± 68 341 ± 18 367 ± 32 333 ± 85 325 ± 28 322 ± 21

malic enzyme, ATP-citrate lyase, glycolate oxidase, or glyoxylateDH found in clarified homogenates from cotton embryos. Theinstability of plant isocitrate DH in homogenates has been previ-ously addressed (11). NAD-isocitrate DH activity, however, wasmeasured in mitochondrial fractions isolated on sucrose density-gradients (unpublished observation).

Organelle Isolation. Organelles were isolated by zonal-rotorsucrose density gradient centrifugation from mature 54 DAAembryos which had been hydrated for 3 h. The mitochondrialpeak (1.21 g ml-', marked by Cyt oxidase) was separated from themicrobody (peroxisome) region (1.24 g ml-', marked by catalase)(Fig. 2). Fifty-four per cent of malate synthase activity applied tothe gradient was recovered in the peroxisome region (Fig. 2).

Malate DH activity (20%o recovered on gradients) was localizedmostly in the mitochondrial region, but a distinct peak was foundin the peroxisome region (Fig. 2). These data are consistent withour unpublished observation of at least three electrophoreticallydistinct isozymes of malate DH in clarified homogenates fromdeveloping cotton embryos. Citrate synthase activity (54% re-covered on gradients) showed a similar distribution in both mi-tochondrial and peroxisome regions (Fig. 2).

Approximately 10%1o of citrate present in embryos was associatedwith the crude organelle fraction isolated by nonaqueous proce-dures (data not shown), but no citrate was associated with proteinbodies isolated by centrifugation through anhydrous glycerol.

In Vitro Metabolism of Labeled Compounds. An experiment

Plant Physiol. Vol. 67, 1981 877

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o 12ca)0

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11

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was deindt eemnewehrprxsme,ioae orm5

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FIG. 2. Isopycnaicgradient centrifugation of aclarified homogenate of

54 DAA embryos. 150 embryos (hydrated for 3 h) were chopped with

razor blades in a modified electric knife and centrifuged at 270g for 10

min. The supernatant was injected onto a dynamically loaded sucrose

gradient (30-60%, w/w) in a Beckman JCF-Z zonal rotor and centrifugedfor 3 h at 25,000g. Malate synthase activity in tubes 12 to 15 represents

38% of that applied. Catalase activity is Luck units (8), malate DH is 2molmin-' fraction-', all others are omol min-' fraction-'. Buoyant density is

g/ml.

was designed to determine whether peroxisomes, isolated from 50

DAA embryos, were capable ofsynthesizing malate and/or citrate.

The peroxisome fraction selected (1.25 g ml-') was free of mito-

chondrial contamination, judged by lack of fumarase and Cytoxidase activities, but contained malate synthase, malate DH, and

citrate synthase activities. Portions of the fraction were incubated

with [3HJacetyl-CoA plus various substrates. Short incubations (2

min) with glyoxylate resulted in synthesis of labeled malate andcitrate, while longer incubations (20 min) resulted in only labeledcitrate (Table IV). Incubations with isocitrate, glycolate, or glyox-ylurea did not yield any labeled organic acids. Incubations (20min) with glycine plus a-ketoglutarate resulted in as much incor-poration into citrate as incubations with glyoxylate (Table IV).

In Vivo Metabolism of Radioactive Compounds. Experimentswith various radioactive metabolites were carried out to test certainaspects of organic acid metabolism (Table V). Embryos harvested38 DAA incorporated [1-'4Clacetate into lipids, whereas 50 DAAembryos did not. Nearly all of the [l-14CJglyoxylate was decar-boxylated in 38 DAA embryos, whereas a majority of label in 50DAA embryos was recovered in the organic acid fraction. [1,5-"Cjcitrate incorporation into 38 DAA embryos resulted in ageneral distribution among the various fractions. In contrast, morethan two-thirds of ['4C]citrate taken up by 50 DAA embryosremained as citrate after a 6-h incubation. Radioactivity from [1-14C]glycine was incorporated into the organic acid fraction (al-though most dpms were recovered with amino acids). Organicacid incorporation was inhibited by simultaneous incubation with10 mm unlabeled glyoxylate, without any effect on total uptake.A 4-h incubation with [1-'4Clacetate resulted in most of the labelin the organic acid fraction, and a subsequent 6-h incubationperiod (after removal of labeled acetate) resulted in decreasedlabeling of organic acids and an increase in amino acids. Ofparticular interest was the apparent "chasing" of label out ofmalate into citrate (see changes in malate and citrate labeling,Table V). A double-label experiment, where 50 DAA embryoswere incubated for 6 h with [1-_4Cjglyoxylate plus [3H]acetate,resulted in quantitatively similar double labeling of total organicacids, and of citrate and malate. The majority of radioactivityfrom both glyoxylate and acetate was found in the sugar fraction.Incubations with [2-14C]acetate gave a different result than with[1_-4C]acetate incorporation studies; most of the radioactivity ap-peared in the sugar fraction with [2-14Clacetate.Inasmuch as the above results suggested that malate synthase

was active in vivo, this was tested directly by incubating 50 DAAembryos with [l-14Clacetate, isolating and purifying labeled mal-ate and citrate, and degrading the acids to determine the positionof incorporated label. Malate was equally labeled in C1 and C4,with no label in internal carbons (Fig. 3). Fifty-five per cent oflabel in citrate was located in C1, with 33% in C5 and 12% in C6.Internal carbons were not labeled (Fig. 3).As malate synthase appeared active in vivo and involved in

citrate production, it seemed possible that accumulated citrateserved as an early metabolic substrate during germination and/orpostgerminative growth. Citrate levels showed a sharp drop be-tween 4 and 8 h following imbibition, then again between 24 and28 h (Fig. 4). The level of citrate 28 h after soaking was approxi-mately one-half that in dry seeds. The majority of [14C]citratetaken up between 4 and 8 h remained in the organic acid fraction,nearly 10%1o was evolved as CO2 and incorporated into lipids (Fig.4). Most citrate taken up between 24 and 28 h remained in theorganic acid fraction, although there was some incorporation intoamino acids and sugars. Nearly one-fourth of incorporated labelwas evolved as CO2 and 13% was recovered in lipids (Fig. 4).

DISCUSSION

Fresh and dry weight, lipid, and starch contents of cottonembryos grown under our conditions were similar to previousreports for field grown cotton (1). Insoluble protein levels, whichpresumably represent storage protein were similar to measure-ments of purified storage proteins by Kisla (20). Comparative dataon soluble protein, dextrins, total, and reducing sugars appearlacking.Mauney et al. (24) observed the presence of malate (>7 mg

ml-') in extracts of 12 to 14 DAA cotton embryos. Ergle and


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Table IV. Incorporation of[3H]acetyl-CoA, in the Presence of Selected Substrates, into Malate and/or Citrate byPeroxisomes Isolatedfrom 125 SO-DAA Cotton Embryos

Each incubation consisted of 1.7 ml of the 1.25 g ml1' gradient fraction (0.048 mg protein), 1.0 ml 200 mmTris-HCl, pH 8.0, 3 umol substrate, 3 pmol NAD, 4 pmol MgCl2, and 0.22 ,umol [3Hlacetyl-CoA (50 ,uCi jmnol-)in a final volume of 3.0 ml. Reaction was stopped by addition of 1.0 ml of 10%1o (w/v) trichloroacetic acid.Products were isolated by ion-exchange and paper chromatography and radioactivity determined by liquidscintillation spectrometry.

Selected Substrate

Glyoxylate Glyoxylate Isocitrate Glycolate Glyoxylurea Glycine plus a-(2 min) (20 min) (20 min) (20 min) (20 min) (20kmin)

dpmMalate 30,469 0 0 0 0 0

Citrate 223,439 252,102 0 0 0 261,661

Table V. Metabolism of Radio-labeled Metabolites by Cotton EmbryosMalate and citrate were separated from total organic acids by descending paper chromatography using tert-amyl alcohol:formic acid:H20 (3:1:1, v/

v). Values are percent of total dpm taken up, except for citrate and malate which are percent of total organic acids. Values are means oftwo experiments.

Embryo Incubation dpm Taken Total Or- AminoAge Isotope Time Up CO2 Lipids ganic Malate Citrate Acids Sugars

Acids Ais Sgr

DAA h % of total dpm taken up38 [1_14C]Acetate 6 644,000 80.038 [1-'4C]Glyoxylate 6 444,316 87.8 1.1 0 0 10.138 [l,5-'4C]Citrate 6 271,200 9.5 17.9 28.6 7.1 9.9 5.7 38.350 [l-'4C]Glyoxylate 6 466,580 0 60.7 26.9 7.250 [1,5-'4CjCitrate 6 207,320 10.2 0 89.8 6.4 68.2 0 050 [11-'4C]Glycine 6 721,460 12.9 0 15.3 61.7 10.150 [1-'4C]Glycine + 10 mm glyoxyl- 6 690,940 19.1 0 2.6 76.0 2.4

ate50 [11-'4C]Acetate 4 939,880 7.2 0 57.7 1.6 11.5 18.6 16.5

Acetate chase 6 939,880 11.0 0 36.8 0.3 12.8 37.0 15.150 [11-'4C]Glyoxylate 6 352,000 2.6 0 27.9 2.1 1.3 24.2 45.2

+ [3H]acetate 6 1,013,935 0 0 25.5 3.2 2.5 20.2 54.350 [2-'4CJAcetate 6 297,924 1.3 0 20.0 2.1 14.4 15.7 49.0

54.9 0

H COOHHOOC-C 4OH

H H, 2COOH

CITRATE

11.50

0

33.1

FIG. 3. Stereospecific distribution (%) of "4C in malate and citrateisolated from 50 DAA embryos incubated with [l-'4Clacetate for 1 h.Carbons are numbered as described by Kent (19).

Eaton (13, 14) reported citrate, malate, oxalate and succinate (12.5,11.2, 7.8, and 2.9 meq/100 g dry weight, respectively) in dry cottonseeds. They were unable to detect isocitrate or cis-aconitate usingenzymic assays. Their failure to detect aconitate is surprising inview of the high levels which we observed (Table II). Our prelim-inary examinations showed the aconitate was approximately 50Y%the trans-isomer (23) which would not be detected by enzymicanalysis.

Thirty-eight DAA cotton embryos were physically, chemically(Table I), and metabolically distinct from 50 DAA embryos. In 38DAA embryos, the citrate level (Table II) and citrate synthaseactivity were about half those values in 54 DAA embryos, andmalate synthase activity was absent (Table III). Synthesis ofstorage lipid was a major event at 38 DAA as evidenced by neutrallipid accumulation (26) and incorporation of [l-14Clacetate intolipid (Table V). In contrast, storage lipid accumulation had ceased

by 50 DAA (26) and [1-'4C]acetate was not incorporated intolipids. Much of ['4CJcitrate taken up by 38 DAA embryos wasincorporated into lipids, whereas citrate taken up by 50 DAAembryos remained metabolically stable (Table V). Thus, a shiftfrom active citrate metabolism to citrate storage was apparent inembryos from 38 to 50 DAA. This shift is coincident with thecessation of lipid synthesis, and the appearance ofmalate synthaseactivity. Finally, most [1-14C]glyoxylate taken up by 38 DAAembryos was decarboxylated and no label appeared in the organicacid fraction, while 50 DAA embryos incorporated 28% of labeltaken up into organic acids and 45% into sugars (Table V).Without considering compartmentation, the intracellular concen-trations of amino acids and the corresponding a-ketoacids areoften considered to be in equilibrium. If this assumption is validfor cotton embryos, then these results suggest that a shift in glycinemetabolism also occurs between 38 and 50 DAA.Appearance at 46 DAA and increase in malate synthase activity

to a peak at 54 DAA are in agreement with the previous report byChoinski and Trelease (8). Of other enzyme activities examined,only citrate synthase increased during this period (Table III).Examination of the organic acid fraction revealed that citratelevels increased from 42 to 54 DAA. An involvement of malateand citrate synthases in synthesis of citrate is consistent with theseobservations. Their involvement is further implicated by com-partmentation ofmalate synthase, malate DH, and citrate synthasetogether only in peroxisomes (Fig. 2), and the capability of this

COOH 47.3H-640H 0HCH 0COOH 52.7

L-MALAT E

Plant Physiol. Vol. 67, 1981 879

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1 200

L 9006)

a)\Cu

' 600C.)

co

E 300c 8 16 24

germination time(h)organic amino

age C02 lipids acids acids sugars6h 9.4 8.3 82.3 0 024h 23.9 134 55.2 2.8 4.7

FIG. 4. Citrate metabolism during cotton seed germinapanel shows the decrease in endogenous citrate as postgerrprogressed. At times indicated by arrows, seeds were incutof [1,5-'4Cjcitrate for 4 h before extraction and fraction.metabolites.

PEROXISOME

CYTOSOL

FIG. 5. Proposed model of carbon flow in peroxisorcotton embryos illustrating their possible role in citrate pi

fraction to incorporate labeled acetyl-CoA (incuboxylate) into malate and citrate (Table IV).When 50 DAA embryos were incubated with

plus [3Hlacetate, there was double labeling of orgcluding malate and citrate (Table V). Data obtaine(halves pulsed with acetate and incubation of isolatewith glyoxylate and acetate (Table IV) suggesterapidly converted to citrate. When malate synthesizembryos from [1l-'Clacetate was degraded, carbonequally labeled (Fig. 3). This was consistent wit]acid cycle equilibrated malate, i.e. no peroxisonmalate accumulated. Degradation of labeled citmore complex pattern (Fig. 3), representative of aintracellular citrate pools. Our interpretation is thalleast three distinct citrate pools: a peroxisome-sy:(50%o each in C, and C5) which can readily diffuse ijand two mitochondrial pools. One is a small,synthesized directly by citrate synthase (100%1o of lathe other being the tricarboxylic acid cycle equilibrin C,, 25% each in C5 and C6). The greater incorpthan C6 of the total extracted citrate supports theactive peroxisome malate to oxaloacetate to citrateAs isocitrate lyase activity was absent, an alter

glyoxylate was necessary for involvement of malate synthase incitric acid synthesis. Higher plants can synthesize glyoxylate fromglycolate and glycine. Additionally, various microorganisms havebeen reported to synthesize glyoxylate from purines, oxalate, 3-hydroxyaspartate, 2 hydroxyglutarate, or acetyl-CoA. We did notdetect glycolic acid, glycolate oxidase activity, or glyoxylate DHactivity in extracts of 50 DAA cotton embryos. Clarified homog-enates of 50 DAA embryos did convert allantoic acid or glyoxy-lurea to glyoxylate, but at relatively low rates (unpublished obser-vations). Incorporation of ['4C]glycine into organic acids (TableV) is consistent with in vivo glyoxylate production via glycine AT.

32 Several lines of evidence support this interpretation: a significantfree glycine pool is present in mature cotton embryos (137 nmol

total cotyledon pair-) (7); incubation of embryo halves with [1-'4C1DPM glycine in the presence of a large amount of unlabeled glyoxylate46868 resulted in decreased incorporation into the organic acid fraction45746 (Table V); incubation of isolated peroxisomes with [3H]acetyl-

CoA, glycine, and a-ketoglutarate resulted in synthesis of citrate(Table IV); and finally, 10 ,U aminooxyacetic acid, an AT inhib-

tion. Theupg r itor, blocked incorporation of glycine into organic acids (unpub-minative growth lished data).)atedwithn 5lCi Accumulation of citrate by cotton embryos is not unique;ation of labeled ungerminated maize and soybean seeds (27) and castor beans (15)

also contain considerable citrate. Rapp and Randall (30) recentlyproposed that ATP-citrate lyase from castor bean endosperm wasactive in generation of acetyl-CoA from citrate, for fatty acidsynthesis during postgerminative growth. Decrease in citrate level

eitrate \ and metabolism of ['4Cjcitrate by germinated cotton seeds (Fig. 4)Cs \ suggest that accumulated citrate may be a metabolic substrate

\ during germination and postgerminative growth.CYCLE Maturing cotton embryos accumulate citrate. It is apparent

DH from our labeling studies with isolated peroxisomes and the deg-radation analyses of radioactive citrate that malate synthase could

rnala te / actively participate in this accumulation in vivo. The major shiftin acetate, glyoxylate, and citrate metabolism in embryos between38 and 50 DAA, coupled with the appearance of substantialmalate synthase activity further implicate peroxisomes in citratesynthesis. In the absence of isocitrate lyase activity, glyoxylatemay be supplied by glycine AT, with the other substrate, acetyl-CoA, coming from fl-oxidation of fatty acids (26). Figure 5 is a

nes of 50 DAA proposed scheme of carbon flow in peroxisomes of 50 DAA

roduction. embryos, which is consistent with the above data and compart-mentation of the enzymes. As only 10% of the citrate extracted by

,ated with gly- nonaqueous procedures could be recovered in particulate frac-tions, it is assumed that a majority of the citrate accumulates in

["Ciglyoxylate the cytosol. Neither glycerol-isolated protein bodies nor peroxi-,anic acids, in- somes contained any citrate, therefore the l1o0 particulate citrated from embryo likely resided in the mitochondria.d microbodies The ontogenetic relationship between these peroxisomes inad malate was mature cotton embryos and glyoxysomes involved with postger-Ced by 50 DAA minative gluconeogenesis remains to be elucidated. Kindl andIs 1 and 4 were associates (21) recently have isolated peroxisomes without isocit-h tricarboxylic rate lyase activity from cucumber embryos and named themne synthesized "incomplete glyoxysomes." This implies they are not functionalrate showed a during seed maturation, but become active after addition (orXmixture of all activation) of isocitrate lyase activity during postgerminativet there exists at growth. We propose that the peroxisomes in maturing cottonnthesized pool embryos have a specific role in seed maturation, ie. citrate pro-nto the cytosol, duction. Whether these organelles also are "incomplete glyoxy-transient pool somes," or are degraded and replaced by new "complete" glyox-ibel in C,) and ysomes, is currently being investigated.*ated pool (50%oration into Csproposal of an-sequence.mnate source of

Acknowledgments-Thanks are due R. Monson for assistance in interpretation ofthe double-label experiments, and H. Reeves for his generous gift of[l-'4Clglyoxylate.Additionally, we would like to acknowledge the cooperation and support of theWestern Cotton Research Laboratory, Science Education Administration, UnitedStates Department of Agriculture, Phoenix, AZ.


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881Plant Physiol. Vol. 67, 1981

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role malate synthase citric acid synthesis maturing ...embryo halves incubated with 114ciglyoxylate...

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