Plant Physiol. (1983) 72, 37430032-0889/83/72/0037/07/$00.50/0

Development and Characterization of Ribulose-1,5-bisphosphateCarboxylase'EVIDENCE INDICATING A LACK OF CARBOXYLASE FUNCTION IN CO2 FIXATION IN ENDOSPERMOF GERMINATING CASTOR BEAN SEEDLINGS

Received for publication August 30, 1982 and in revised form December 12, 1982

JOSHUA H. WONG2 AND CHAUNCEY R. BENEDICTDepartment ofPlant Sciences, Texas A & M University, College Station, Texas 77843

ABSTRACT

Ribulose-1,5-bisphosphate carboxylase activty was found In endospermof germinating castor bean seed Ricmu cemmais and was localized inproplastids. The endospenn carboxylase has been extensively purified andis composed of two different subunits. Tlhe molcular weights of the nativecarboxylase and its subunits were 560,000, 55,000, and 15,000 daltons,respectively. The Mlchaells-Menten constants, K.,, for the endospermcarboxylase with respect to ribulose 1,5-bisphosphate, bicarbonate, C02,and magnesium In millimolar are 0.54, 13.60, 0.92, and 0.57, respectively.The endosperm carboxylase was activated by Mge' and HCO3-. Thepreincubation of the carboxylase with 1 millmolar HCOs- and 5 milllarMgCa resulted in activation by low ad inhibitio by high concentratoiosof 6-p_osphogluconate.

In studies of dark 14C0 fixation by endosperm slices, I14CImalate and114CIcitrate were the predominantly labeled products after 30 seconds ofexposure of the tissue to H4CO3-. In pule-chase experiments, 87% of thelabel is malate, and citrate was transferred to sugars after a 60-minutechase with a small amount of the label appearing in the incubation medimas "CO2. The minimal incorporation of the label from 4CO2 into phos-phoglyceric acid indicated a lack of the endosperm ribulose-1,5-bispbos-phate carboxylase participation in the endosperm's CO2 fixation system.The activities of key Calin cycle enzymes were examined In the endo-sperms and cotyledons ofdark-grown castor beans . Many of theseautotrophic enzymes develop In the dark In these tissues. The synthesis ofribulse-1,5-bisphosphate carboxyase in the nonphotosynthetic endo-sperms is not repressed in the dark, and high leels of enzymic activityappear with germination. AM of the Calvin cycle enzymes are present In thecastor bean endosperm except NADP-lnked glyceraldehyde 3-P dehydro-genase, and the absence of this dehydrogenase probably prevents thefunctioning of these series of reactions in dark CO2 fixaton

We have reported significant levels of RuBPCase3 and other

'Supported by the Texas Agricultural Experiment Station and theRobert A. Welch Foundation under Grant A-482. Portion of this workwas taken from a thesis submitted by J. H. W. to Texas A & M Universityin partial fulfillment of the requirements for the Degree of Doctor ofPhilosophy.

2J. H. W. is presently a postdoctoral fellow in the Biochemistry De-partment, University of Missouri, Columbia, MO 65211.

3 Abbreviations: RuBPCase, ribulose-l,5-bisphosphate carboxylase;PEP, phosphoenolpyruvate; Gald3P, glyceraldehyde 3-phosphate; PGA,3-phosphoglycerate; RuBP, ribulose 1,5-bisphosphate; 6-PGluA, 6-phos-phogluconate; FBP, fructose 1,6-bisphosphate; Ru5P, ribulose 5-phos-phate; R5P, ribose 5-phosphate; FBPase, fructose 1,6-bisphosphatase;OAA, oxaloacetate.

enzymes of the Calvin cycle in nonphotosynthetic endosperms ofgerminating castor beans (2, 12). Dark 14CO2 fixation in theendosperm slices labels the 3,4 position of the glucose moiety ofsucrose (27). The accepted model of this labeling pattern is CO2incorporation via C4 acid intermediates through the activity ofPEP carboxylase. This labeling pattern also could be derived viaconversion of CO2 to sugars by a Calvin cycle functioning with aPGA-triose-P shuttle (15) to bypass the missing NADP-linkedGald3P dehydrogenase step. In endosperm, this cycle would pro-vide an additional mechanism for the incorporation of CO2 intosugars along with the activity of PEP carboxylase and subsequenttransformation of C-4 acids into glucose through malate andreversal glycolysis (1, 3).

In this paper, we report several characteristics of Calvin cycleactivity in the germinating castor bean endosperm, includingphysical and catalytic properties of the RuBPCase, quantitationof Calvin cycle enzyme activities, and CO2 fixation by endosperm.

MATERIALS AND METHODS

Plants. Two cultivars (Hale and Cimarron) of castor bean seeds(Ricinus communis) were obtained from McNair Seed Company,Plainview, TX. 'Hale' was used for the developmental studies andenzyme purification, while 'Cimarron' was used for 14CO2 fixationexperiments. After dusting with Arasan, seeds were imbibed for24 h and germinated in moist vermiculite at 35°C in the dark. Fordevelopmental studies during germination, seedlings were har-vested from zero (after 24 h imbibition) to 8 d. For the estimationof enzyme activities in developing castor bean seeds followinganthesis, seeds at different stages of development were collectedfrom field-grown plants. The age of developing seeds was esti-mated by changes in the endosperm fresh weight between anthesisand fully developed dry seed.

Etiolated seedlings of Hale were grown in a light-proof box for15 d at 30°C (9). Illuminated or light-treated seedlings were grownin a light-proof box for 10 d and then transferred to light (200 uEm-2 s-) for 5 d at 30°C.

Soluble Extract. Four-d-old endosperms or 15-d-old cotyledonsfrom germinating castor bean seedlings were ground with mortarand pestle on ice bath in 0.1 M Tris buffer (pH 7.5) containing 0.1mM GSH. The homogenate was squeezed through four layers ofcheesecloth and centrifuged at 27,000g for 30 min. An aliquot ofthe supernatant fraction was used as the source of enzymes.Locization of RuBPCase Activity by Discontinuous Sucrose

Density Gradient Centrifugation. Organelles were extracted fromendosperm tissue (10-15 g) by chopping (no blending) with arazor blade in 10 ml 150 mm Tricine buffer (pH 7.5) containing 10mM EDTA and 0.5 M sucrose. The crude homogenate was filteredthrough glass wool and the filtrate was centrifuged at 120g for 10

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WONG AND BENEDICT

min in a Sorval RC-5 centrifuge to remove debris. The supernatantwas centrifuged at 14,500g for 15 min to prepare crude organellepreparation and the resultant pellet was resuspended in 4.0 mlgrinding buer. One to two ml of the crude organelle suspensionwas layered onto a 33-ml discontinuous sucrose gradient containedin 2.8- x 7.8-cm cellulose nitrate tubes. The gradients were com-posed of 5.0 ml of60% sucrose, 7.0 ml of 51% sucrose plus 1.0 mmMgSO4, 7.0 ml each of 43 and 35% sucrose plus 0.5 mM MgSO4,and then 5.0 ml of 25% sucrose plus 0.5 mM MgSO4. All sucrosesolutions (w/v) were made in 0.1 M Tris buffer (pH 7.5). Gradientswere used immediately and placed in a SW 25.1 rotor andcentrifuged in a Beckman model L refrigerated centrifuge at17,500g for 3 h. Fractions of 1.5 ml were collected with an ISCOmodel D Density Gradient Fractionator and an ISCO model 328Fraction Collector equipped with a UV monitor, and absorbancewas measured at 280 nm.RuBPCase Purificaton. RuBPCase was purified from endo-

sperms of 4- to 5-d-old seedlings. Endosperms were separatedfrom testa, hypocotyL epicotyL and cotyledons and rinsed withdeionized H20, blotted dry, and weighed. Endosperms were ho-mogenized for 1 min in a prechilled Sears Insta Blend (60 s at'liquefy' setting) in 0.1 M Tris buffer (pH 7.5) containing 0.1 mmGSH at a ratio of 1 ml buffer to I g fresh weight. The homogenatewas filtered through three layers of cheesecloth and centrifuged at16,300g for 30 min. The crude extracts was filtered through glasswool to remove fat. The supernatant was fractionated with(NH)2SO4 and proteins precipitating in the 45 to 55% (NH4)2SO4fraction were collected by centrifugation. The pellet dissolved in40 to 50 ml of grinding medium and subsequently dialyzed for 18to 24 h with two changes of buffer with an Amicon Diafloultrafiltration cell equipped with XM300 membrane. The firstbuffer was the same as the grinding buffer, the second buffer was10 mm Tris (pH 7.5). The dialysate was centrifuged at 27,000g for15 min to remove insoluble material, and the clarified solutionwas applied to a DEAE-ellulose (Sigma, medium mesh) columnwith a dimension of 2.3 x 28 cm previously equilibrated with 10mm Tris buffer (pH 7.5). Proteins were stepwise eluted from thecolumn with 300 ml of buffer and followed by 300 ml stepgradients of 0.1, 0.15, 0.2, 0.25, and 0.5 M NaCl contained in thesame buffer. Fractions of approximately 8 ml were collected.RuBPCase activity eluted with 0.2 M NaCl and this fraction was

*4-

4-04U

0~I&)

iI-90-CP

I

c

0

C.,

.5

-a

concentrated by ultrafiltration (Amicron) and applied to a BioRadBio-Gel A-SM column (I x 40 cm) equilibrated with 10 mm Trisbuffer (pH 7.5). Proteins were eluted with the same buffer andcollected in 1.2-ml fractions. Carboxylase activity eluted with themajor protein peak and this fraction was concentrated by ultrafil-tration. All steps were carried out at 4°C.

Disc Gel Elecbophoreds. Homogeneity of purified carboxylasewas determined using three different concentrations of polyacryl-amide gel (5, 7, and 10%) according to the method of Davis (8).Mol Wt Detendnation by Disc Gel Electophoesis. The mol

wt of purified endosperm RuBPCase was determined by the discgel electrophoresis method ofHedrick and Smith (14). The relativemobilities (Rm) of the RuBPCase and other protein standardswere measured at gel concentrations of 5, 6, 7, and 8% acrylamide.The mol wt of the purified endosperm carboxylase was estimatedfrom known protein standards. The standard proteins were aldo-lase (158,000) and apoferritin (450,000) from Sigma; L-glutamatedehydrogenase (300,000-350,000) from Calbiochem; and purifiedspinach RuBPCase (550,000) from our laboratory (30).

SDS-Polyacrylamilde Gel Electrphoresis. Analysis of Ru-BPCase subunits content and mol wt by SDS was carried out asdescribed by Weber and Osborne (29) using 10% polyacrylamidegel. The protein standards included BSA (68,000), ovalbumin(dimer) (86,000), ovalbumin (monomer) (43,000), pyruvic kinase(57,000), catalase (60,000), aldolase (40,000), trypsin (23,300), andlysozyme (14,300). All proteins were purchased from Sigma.Dark 4CO2 Fixation by Castor Bean E Slices. About

100 mg of 3-d-old endosperm slices from cv Cimarron werehandcut by straight-edge razor into thin slices. Slices were incu-bated in 5 ml deionized H20 in a 125-mi Erlenmeyer flask at 350Cfor 5 min. At the end of the preincubation period, 0.1 mlNAH"4CO3 (100 ,uCi contained in 1.98 umol) was applied to eachsample. Each sample was incubated for the following times; 5 s,30 s, 1 min, 5 min. Samples was killed by adding boiling 80%ethanol. Samples were subsequently extracted eight times byboiling 80% ethanol. Combined extracts were acidified by 10 mlconcentrated formic acid and then degassed by flushing with N2and dried in vacuo at 40°C. The alcohol-soluble residue wasdefatted by 50 ml diethyl ether and the residue was dissolved in50 ml of deionized H20. The water soluble fraction was taken todryness in vacuo at 40°C and redissolved in 10 ml of H20. An

I Wt * Age, doys VW

F1o. 1. Development of RuBPCase activity in endospems of developing and germinating castor bean seedings. Developing seeds were obtainedfrom field-grown plants and incease in fresh weight of endosperm was used as a measure of seed development. The exact age from anthesis was notknown. Mature seeds were germinated in moist vermiculite in the dark at 35°C, and dayO was 24 h after inhibition.

Plant Physiol. Vol. 72,198338

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RICINUS ENDOSPERM RuBP CARBOXYLASE

0C

m0

:0

a-

%.O

0U,0

h.

-

C.)0nen

Tube NumberFIG. 2. Distribution ofcastor bean endosperm RuBPCase activity among organelle fractions separated from 4- to 5-d-old endosperm by discontinuous

sucrose density gradient centrifugation. Relative units: cpm x 106/tube; specific activity was 2.2 x 105 cpm/pmol. Sol prot, soluble proteins; Mito,mitochondria; Proplast, proplastids; Glyoxy, glyoxysomes.

Table I. Purif4cation of Castor Endosperm RuBPCase

Purfictin Sop Total Total Specific Purifi- ilProtein Activity Activity caton

siLMol C02pmol fixed min-'

mg min M -fold %protein

Crude extract 8,389 167.78 0.02 100.045-55% (NH4)2SO4 880 61.6 0.07 3.5 36.7Dialyzate 750 52.50 0.07 3.5 31.3DEAE-Cellulose 38 22.42 0.59 29.5 13.4Agarose (Bio-Gel A-5m) 3 2.46 0.82 41.0 1.5

aliquot was measured into 10 ml of scintillation cocktail (whichwas prepared by dissolving 5 g of PPO, 100 g of naphthalene, 10ml of H20 and then added 1,4-dioxane to make 1 L) for radioac-tivity assay to a ±0.2% error using a Beckman LS-200B scintilla-tion spectrometer system. The remaining water-soluble fractionswere either dried in vacuo and stored at -15'C or used immedi-ately for analyses.Dowex Resin Chromatography. The water-soluble fraction was

separated into acidic, basic, neutral fractions and the acidic frac-tion was further fractionated into its component organic acids andphosphate esters as described previously (4).

Pulse and Chase Labeing Experhnent. About 100 mg of 3-d-old castor bean endosperm slices (Cimarron) were handout witha straight-edge razor into thin slices. After the 5 min preincubationin 5 ml of H20 at 350C, 0.1 ml NaH"4CO3 (100 1&Ci contained in1.98 pmol) was added and the sample was allowed to fix 14CO2 for30 s. The mixture was poured over two layers of cheesecloth, andthe collected slices were washed for 30 s with deionized H20.Slices were then transferred to another 5 ml of I mm NaHCO3solution for the chase periods of 30 s, 1, 5, 10, 30, and 60 min.Slices were killed by boiling 80% ethanol at the end of thedesignated incubation period, after an aliquot of the incubationmedium was removed for radioactivity assay for respiratory 14CO2.Extraction and ion-exchange chromatographic separation of con-stitutes were carried out as described above.Enzym Assays. RuBPCase was assayed by a modification of

the method of Benedict (2, 18). The reaction mixture contained inpmol: 100 Tris buffer (pH 7.5); 2.5 GSH; 5 MgCl2; I RuBPtetrasodium salt; 25 KH C05 which contained 5 #Ci radioactivity;

..

5% 7% Io%

Fio. 3. A photograph showing the agarose-purified, endosperm Ru-BPCase on three different percentages of poly-acrylamide gels.

100 to 200 pg enzyme contained in a final volume of 0.5 ml.Activation and inhibition by 6-PGluA was examined in the stan-dard assay mixture of RuBPCase with a final volume of 0.5 mL

39

umum

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WONG AND BENEDICT

6L w

6.01.

00

Co4.0-

2.0[

A1

1.0 2.0 3.0 4.0 5.0 6.0Mol Wt, x 105

FIG. 4. Detcrmination of mol wt of native castor bean endospermRuBPCase using four different concentrations of polyacrylamide gel and50 to 75 ,g of protein. Protein standards were: Aid, aldolase; Cat, catalase;GDH, glutamate dehydrogenase; Apofer, appoferritin; Castor and Spin-ach, castor and spinach RuBPCase.

5.0

4.81.

qo

50-j

4.6Io

4.4.

4.21r

0.2 0.4 0.6 08Relative Mobiliy

FIG. 5. Determination ofmol wt ofcastor bean endosperm RuBPCase'stwo subunits by 1% SDS and 10% polyacrylamide gel. Protein standardswere: Lys, lysozme; SS, small subunit; Try, trypsin; Aid, aldolase;Ova(mono), ovalbumin-monomer, LS, large subunit; PK, pyruvic kinase;Cat, catalase; BSA; Ova(di), ovalbumin-dimer.

The reaction blank contained all of the reagents except RuBP.Two methods of preincubation were used in this report: (a) theassay mixture excluding RuBP was preincubated for 10 min, theninitiated the reaction by RuBP; (b) enzyme was preincubated withonly Mg2+ for 10 min and this mixture was used to initiate thereaction. Method a was used routinely as standard procedure,whereas method b in combination with method a was used for theactivation and inhibition studies by 6-PGluA. Both reaction mix-tures were incubated at 350C for another 15 min and were stoppedwith 1.0 ml concentrated HC1. After stopping the reaction, thevolume was brought to 10 ml with water and the sample bubbledwith N2 for 15 min to remove unreacted 14CO2. Enyme activitywas determined by liquid scintillation spectrometry of an aliquotof the reaction mixture as described previously.

Table I. Summary ofKm and VK Values ofRuBPCase EndospermRuBPCase was preincubated in reaction mixture for 10 min and initi-

ated by addition of RuBP as described in "Materials and Methods."

Endosperm Spinach (25)mM

KmHCO3- 13.6 10-20CO2 (calculated) 0.92Mg2+ 0.57 1RuBP 0.54 0.14.25

,umol CO2fixed min-'mg' protein

Vine1HC03- 0.99Mg2+ 0.83RuBP 0.30

-c

0._

46

0

C)4-0

m

0 0.5 1.0 1.5 2.0(6-PGuA), mM

FIG. 6. The effect of varying 6-PGluA on RuBPCase activity in thepresence of low (A) and high (B) concentration of NaH'4CO3. Enzymewas preincubated with Mg2", HCO3- at varying concentrations of6-PGluAfor 10 min (0); the reaction was initiated by RuBP. Enzyme was prein-cubated with only Mg2e for 10 min (0); this mixture was used to initiatethe reaction. Relative units: cpm x 104. Specific activity: 1.8 x 10' cpm/pmol.

Assay procedures for Gald3P dehydrogenase (NAD- andNADP-linked), FBP aldolase, PGA kinase, triose-P isomerase,transaldolase, transketolase, Ru5P kina, R5P isomerase, FBPase,PEP carboxylase, PEP carboxykinase, and malic dehydrogenasewere carried out as described by Benedict (2).

Protein Determination. Protein was determined by the methodof Lowry et al. (20). Protein content of sucrose gradient fractions

GDH

* Cat

Try

SstLyS

40 Plant Physiol. Vol. 72, 1983

A ^I

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41RICINUS ENDOSPERM RuBP CARBOXYLASE

Table III. Relative Percentage of Radioactivity Incorporated in Various Organic Acids

Total Relative Count Incorporated in Various AcidsTime dpm x

10-3 L-Aspartic Unknown Glyceric Suyoxyat Malic Citric Fumaric PGAglyoxyate

30 s 65.0 6.6 3.5 31.8 54.7 3.41 min 202.6 7.5 4.1 2.6 33.5 47.6 1.0 3.65 min 1,048.9 3.9 2.4 1.7 3.2 40.3 44.7 1.1 2.6

30 40Time, min

FIG. 7. Changes in the distribution of "4C among various metabolitegroups in castor bean endosperm during an extended chase in H12CO3-.After a period of 30 s in NaH'4CO3 and 30 s wash, endosperm slices weretransferred to a 5-ml solution of 1 mM NaH'2CC0 for designated chaseperiods. AA (amino acids), OA (organic acids), and sugars were separatedby Dowex resin chromatography.

was determined by Coomassie Brilliant Blue G-250 of Bradford(6). BSA (Sigma crystallized and lyophilized) was used as thestandard.

RESULTS

RuBPCase: Development and Compartmentation in Enido-sperm. The data in Figure 1 show that almost no RuBPCaseactivity is detected during seed development, which is consistentwith earlier studies (26). Endosperm carboxylase activity devel-oped during the first 4 d of germination reaching a maximum of1.3 pmol min-' g-' fresh weight, subsequently declining as storagematerial was being depleted (Fig. 1).

Figure 2 shows the distribution of RuBPCase activity amongthe various organeile fractions of castor bean endosperm extract.Ninety % of the RuBPCase activity applied to the top of thegradient was recovered in the proplastid/glyoxysome fraction.RuBPCase: Purification and Characterizations. Purification of

RuBPCase from endosperm is shown in Table I. The specificactivity of purified enzyme was over 0.8 pmol CO2 fixed minmg-1 protein which was about 50% of the specific activity ofpurified spinach leaves enzyme (7, 25). The purified enzyme wasseen as a single electrophoretic band at three different concentra-

Table IV. Enzymic Activities of the Reductive Pentose Phosphate Cycleand Related Enzymes in Castor Endosperm Extract (4- to 5-Day-Old)

Enzymes Activity

,umol g 'fresh wt h-Ru5P kinse 0.01RuBPCase 0.04PGA kinase 16.00Gald3P dehydrogenaseNAD- (reversible) 0.60NADP- (reversible) Not detectableNADP- (nonreversible)' 0.03

Triose-P isomerase 3.80FBP aldolase 0.10FBPasepH 8.8 0.04pH 7.5 0.04pH 6.8 0.05pH 6.6 0.03

R5P isomerase 0.02Transkeltolase 0.01Transaldolase 0.01PEP carboxykinase 0.03PEP carboxylase 0.01Malic dehydrogenase 16.00

a Kelly and Gibbs (16).

Table V. Enzymatic Activities of the Reductive Pentose Phosphate Cyclein Dark- and Light-Grown Castor Bean Cotyledons (15-Day-old)

Dark-Growna Light-

,umol g'fresh wt h-Gald3P dehydrogenaseNAD 0.10 0.55NADP- Not detectable 0.2

FBP aldolase 0.08 0.07FBPasepH 8.8 Not detectable 0.01pH 7.5 0.03 0.02pH 7.0 0.05 0.02pH 5.5 0.04 0.01

PGA kinase 1.90 1.60Transketolase 0.17 0.13Transaldolase 0.02 0.01Triose-P isomerase 6.30 8.90RuBPCase 0.13 0.10R5P isomerase 0.28 0.38Ru5P kinase 0.18 0.24

* Seedlings were grown in a light-proof box for 15 d at 30°C.b Seedlings were grown in a light-proof box for 10 d and then were

transferred into light (200 1tE m-2 s-1) for 5 d at 30°C.

.-

00._0.-

0

c0

4-

.0

0~

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WONG AND BENEDICT

tions of polyacrylamide gel (Fig. 3).The mol wt of the native enzyme and SDS-dissociated Ru-

BPCase was determined by polyacrylamide disc gel electropho-resis (14, 29) and estimated to be 560,000, which was similar tothe mol wt of RuBPCase in green leaves (Fig. 4). Subunit mol wtwere 55,000 and 15,000, respectively (Fig. 5).

Kinetic properties of the purified endosperm RuBPCase aresummarized in Table II. The Km was V. values for HC03, C02,Mg2+, and RuBP were similar to those values for purified spinachleaf RuBPCase (25) and other endosperm preparations (2, 12, 24).In separate experments, 10 min preincubation of RuBPCase withMg2+ and HC03- resulted in 10% greater specific activity (datanot shown).6-PGluA is known to modify RuBPCase activity (7, 28), and

the endosperm enzyme is regulated in the same fashion (Fig. 6, Aand B). When the enzyme was preincubated for 10 min at 350Cwith various concentrations of 6-PGluA along with MgCl2 and 1mm NaHCO3, maximal activation obtained at 0.05 to 0.1 mm 6-PGluA was 56 to 61% (Fig. 6A, upper curve). Less activation wasobserved when the enzyme was preincubated with MgCl2 alone(Fig. 6A, lower curve). As 6-PGluA concentration increased be-yond 0.1 mm, an inhibitory effect was obtained. At 50 mM,NaHCO3 inhibition ofthe endosperm RuBPCase by 6-PGluA wasobtained by either preincubating the enzyme with 6-PGluA,MgCl2, and NaHCO3 at the same time or with MgCl2 alone (Fig.6B). The activation by 6-PGluA at low concentrations of HCO3-is typical of RuBPCase with a mol wt larger than 500,000 consist-ing of two types of subunits (7, 28).CO, Fixatio by Endosperm Tissue. The initial products of

'4CO2 fixation in endosperm slices were analyzed to determinewhether RuBPCase significantly contributed to dark CO2 fixation.Exposure of the endosperm slices to H"4CO3 for 30 s resulted inincorporation of 59% of label in the organic acid fraction and 24%of label into the amino acid fraction (data not shown). After 30sof exposure to H4CO3, the percent distribution of label intoorganic acids was as follows: malate, citrate, and PGA accumu-lated, respectively, 31.8, 54.7, and 3.4%, of the label (Table III).Exposure of endosperm slices to H14C03 for 5 min did notincrease partitioning of label into PGA (Table III). Principleproducts of CO2 fixation were malate and citrate.

In the pulse-chase experiment (30 s - 30 s), the distribution oflabel incorporated into the organic acids, sugars, and amino acidswas 45, 36, and 8%, respectively (Fig. 7). During the 30-s washperiod, it was shown that 11% of the label was lost from organicacids and accumulated in the neutral fraction. Exposure of theslices to 60 min of (Fig. 7) cold HCO3 resulted in a transfer of87% of the label in the organic acids to the sugars with a smallfraction (14%) appearing as 14CO2 in the incubation medium (Fig.7). In separate experiments, 70% of the label in malate and citratewas converted to sugars (data not shown).

Proffie of Enzyme Activitsn Enspe and Cotyledons.Activities of various Calvin cycle and related enzymes in castorbean endosperm (Table IV) and in dark- and light-grown castorbean cotyledons (Table V) were measured to compare the amountsof autotrophic enzymes in dark-maintained tissue relative to illu-minated tissue. Cotyledons become functional photosynthetic tis-sue, but like endosperm, developed high levels of RuBPCaseactivity (on a fresh weight basis) in darkness (9). Autotrophicenzymes RuBPCase, RuSP kinase, and alkaline FBPase are pre-sent in the endosperm of germinating seed. RuBPCase, Ru5Pkinase, but not alkaline FBPase are present in etiolated castorbean cotyledons. NADP-linked Gald3P dehydrogenase was miss-ing in both endosperms and cotyledons of dark-grown seedlings.In response to light, measurable quantities of alkaline FBPaseactivity and greatly increased levels of NADP-linked Gald3Pdehydrogenase activity were detected in the castor bean cotyledons(Table V). Separate experiments showed NADP-linked Gald3P

dehydrogenase was not synthesized in endosperm germinatedunder light (unpublished observation).

DISCUSSION

The developmental pattern of endosperm RuBPCase parallelsgluconeogenic activity development in germinating castor beanseeds (1, 24). The development of carboxylase activity over a 4-dperiod is suggestive of a protein synthetic event rather thandevelopment by activation of a precursor from the developingseed. This observation is consistent with earlier study (12) thatdevelopment of carboxylase activity in endosperm of castor beanis inhibited by antibiotics such as cycloheximide and chloram-phenicol which inhibit protein synthesis on 80S and 70S ribo-somes, respectively. The amount of RuBPCase activity in endo-sperm on a g fresh weight basis approaches levels found in greenleaves, and this development is independent of light (12). Theseobservations are taken as evidences that the development ofRuBPCase activity in endosperm of germinating castor bean inthe dark requires a cooperation of synthetic events between thecytoplasm and proplastids.Among various physical and kinetic properties examined, (mol

wt, presence of large and small subunits, mol wt of the twosubunits, Km values, V., values, and 6-PGluA activation), en-dosperm RuBPCase is similar to the RuBPCase of green leaves(7, 25, 28). The localization of RuBPCase activity in proplastidsof germinating castor bean endosperm (Fig. 2) is in agreementwith earlier studies (23, 24).A key biochemical function of castor bean endosperm is its

ability for gluconeogenesis to convert acetate derived from fattyacid catabolism into carbohydrates via malate (1, 3, 23). Highactivities of many gluconeogenic pathway enzymes (NAD-linkedGald3P dehydrogenase, triose-P isomerase, FBPase, PGA kinase,PEP carboxylase, and PEP carboxykinase) are consistent with thiskind of metabolism (Table IV; Ref. 23). As we have shown,endosperm tissue also incorporates and converts CO2 into sugars.Incorporated "4CO2 labels the 3,4 position of glucose (27). Thepresence of most of the enzymes of Calvin cycle in endospermtissue suggested the possibility that CO2 is incorporated intoglucose not only via malate and reversal of glycolysis, but also viaPGA and Calvin cycle action utilizing the NAD-linked Gald3Pdehydrogenase to form a PGA-triose-P shuttle (15, 16) to bypassthe absence of NADP-linked Gald3P dehydrogenase. Studies onCO2 fixation in endosperm show that RuBPCase does not functionin dark CO2 fixation (Table III) and the pathway for CO2 tosugars involves the anaplerotic CO2 fixation intoOAA and malate,after randomization of label between the C4 and C, carboxylgroups of the C4 acids through the action of fumarase and thenfollowed by conversion of OAA to PEP and gluconeogenesis (1,3). A similar conclusion was reached from work with marrowcotyledons (19). The percentage ofCO2 fixed into sugars is greaterthan theoretical considerations (19, 27) and indicates that signifi-cant amounts of CO2 are refixed via PEP carboxylase (Fig. 7).

Reasons for castor bean endosperm developing high activitiesof Calvin cycle enzymes are still not known. It is established thatmany etiolated tissues develop high levels of RuBPCase (5, 9-1 1,13, 17, 18), but few studies document the activities ofthe completeset of autotrophic enzymes in etiolated materials (5, 8). Lightgenerally produces increased levels of Calvin cycle enzymes andalso causes qualitative changes in levels of some enzymes such asNADP-linked Gald3P dehydrogenase (5, 11, 13, 18, 21, 22) andFBPase (5, 13, 18). In the development of Calvin cycle enzymesin castor bean cotyledons, light-induced increases of FBPase andNADP-linked Gald3P dehydrogenase activities seem to be locicontrolling the presence and operation of a complete Calvin cycle(Table V). The cycle is not operable in dark, ie. less than 5% oflabel is incorporated into sugars after 10 min "CO2 fixation byetiolated castor bean cotyledons (unpublished data), although

42 Plant Physiol. Vol. 72, 1983

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RICINUS ENDOSPERM RuBP CARBOXYLASE

most of the enzymes have been developed. In endosperm, thetransaiption and translation of nuclear and plastid genes result innear complete complement of Calvin cycle enzymes, particularlyRuBPCase, which appear to be products of constitutive genes.However, the Calvin cycle is not fimctional in endosperm becauseNADP-linked Gald3P dehydrogenase is missing (Table IV).

Acknowledgment-We are grateful to Dr. Paul Reibach for the preparation ofthe sucrose density graient experiment and to Mrs. Jackie Hall for valuable technicalassistance. Valuable criticism of the manuscript by Dr. T. Kagawa is gratefullyacknowledged.

LITERATURE CITED

1. BRSvm H 1969 Glyoxysomes of castor bean endosperm and their relation togluconeogenesis. Ann NY Acad Sci 168: 313-323

2. BENEDIcr CR 1973 The presence of ribulose-1,5-diphosphate carboxylase in thenonphotosynthetic endosperm of germinating castor beans. Plant Physiol 51:755-759

3. BENwicr CR, H BRvmts 1961 Formation ofsucrose from malate in germinatingcastor beans I. Conversion of malate to phosphoenolpyruvate. Plant Physiol36: 540-544

4. BENDICT CR, WWL WONG, JHH WONG 1980 Fractionation of the stableisotopes of inorganic carbon by Plant Physiol 65: 512-517

5. BRADBEm IW 1971 Plastid development in primary leaves of Phaseolus vulgaris.J Exp Bot 22: 382-390

6. BRADPoRD MM 1976 A rapid and sensitive method for the quantitation ofmicrogram quantities of protein utilizing the principle of protein-dye binding.Anal Biochem 72: 248-254

7. CHu DK, JA BAssHm 1973 Activation and inhibition of ribulose-1,5-diphos-phate carboxylase by 6-phosphogluconate. Plant Physiol 52: 373-379

8. DAvis BS 1964 Disc electrophoresis. II. Method and application to human serumproteins. Ann NY Acad Sci 121: 404-427

9. DocKmTv A, IM LoRD, MJ MERRm 1977 Development of ribulose-1,5-di-phosphate carboxylase in castor bean cotyledons. Plant Physiol 59: 1125-1127

10. FEAmaBEND J, A PIRSON 1966 Die wirkung des Lichts auf die Bildung vonPhotosynthese-Enzymen in Roggenkeimlingen. Z Pflanzenphysiol Bd 55S:235-145

11. Fn.Lu B, AO KLEN 1968 Changes in enzymatic activities in etiolated beanseedling leaves after a brief illumination. Plant Physiol 43: 1587-1596

12. GnumE LAF, JHH WONG, CR BENDICT 1976 Development of ribulose-1,5-diphosphate carboxylase in non-photosynthetic endosperms of germinatingcastor beans. Plant Physiol 57: 589-593

13. GRAHAm D, AM GRavE, RM SmLUIE 1971 Phytochrome-mediated plastid

development in etiolated pea stem apices. Phytochemistry 10: 2905-291414. HEDIUCK JL, AJ SiaH 1968 Size and charge isomer separation and estimation

of molecular weights of proteins by disc gel electrophoresis. Arch BiochemBiophys 126: 155-164

15. KELY GJ, M GiBBS 1973 A mechanism of the indirect transfer of photosynthet-ically reduced nicotinamide adenine dinucleotide phosphate from chloroplaststo the cytoplasm. Plant Physiol 52: 674-676

16. KELY GJ, M GIBBS 1973 Nonreversiblen-glyceraldehyde 3-phosphate dehydro-genase of plant tissues. Plant Physiol 52: 111-118

17. KLENKOPF GE, RC HUFFAKER, A MATHESON 1970 Light-induced de novasynthesis of ribulose 1,5-diphosphate carboxylase in greening leaves of barley.Plant Physiol 46: 416-418

18. KOBAYASHI H, S AsAW, T AKAZAWA 1980 Development ofenzymes involved inphotosynthetic carbon assimilation in greening seedlings of maize (Zea mays).Plant Physiol 65: 198-203

19. LEEGOoD RC, T ApRms 1978 Dark fixation of CO2 during gluconeogenesis bythe cotyledons of Cucurbitapepo L. Planta 140: 275-282

20. LowRY OH, NJ ROSEBROUGH, AL FnaR, RJ RANDAL 1951 Protein measure-ment with the Folin phenol reagent. J Biol Chem 193: 265-275

21. MARcus A 1960 Photocontrol of formation of red kidney bean leaf triphospho-pyridine nucleotide-linked triose phosphate dehydrogenase. Plant Physiol 35:126-128

22. MARGULES MM 1965 Relationship between red light-mediated glyceraldehyde-3-phosphate dehydrogenase formation and light-dependent development ofphotosynthesis. Plant Physiol 41: 57-61

23. NisHDiuRA M, H BEEvERs 1979 Subceliular distribution of gluconeogeneticenzymes in germinating castor bean endosperm. Plant Physiol 64: 31-37

24. OsmoND CB, T AKAzAwA, H BEvERs 1975 Localization and properties ofribulose diphosphate carboxylase from castor bean endosperm. Plant Physiol55: 226-230

25. PAuLsEN JM, MD LANE 1966 Spinach ribulose diphosphate carboxylase. I.Purification and properties of the enzyme. Biochemistry 5: 2350-2357

26. Simcox PD, EE REID, DT CANVIN, DT DaENs 1977 Enzymes of the glycolyticand pentose phosphate pathways in proplastids from the developingendospermof Ricuws communs L. Plant Physiol 59: 1128-1132

27. STnlE ML 1959 The mechanism of malate synthesis in Crassulacean leaves.PhD thesis. Purdue University, Lafayene, IN

28. TABITA FR, BA McFADDEN 1972 Regulation of ribulose 1,5-diphosphate car-boxylase by6-phospho-D-gluconate. Biochem Biophys Res Commun48: 1153-1159

29. WaER K, M OSBoRN 1969 The reliability of molecular weight determinationsby dodecyl sulfate-polyacrylamide gel electrophoresis. J Biol Chem 244: 4406-4412

30. WONG WWL, CR BENDICT, RJ Kose 1979 Enzymic fractionation ofthe stablecarbon isotopes of carbon dioxide by ribulose-1,5-bisphosphate carboxylase.Plant Physiol 63: 852-856

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