Photosynthetic Carbon Metabolism in Isolated …2 mMNa2EDTA, 1 mMMnCl2, 0.25 mmKH2PO4, 5 mMD-iSo ascorbate, 2 mm thioglycolate, and 2% (w/v) polyvinylpyrroli- done 40 …

Plant Physiol. (1973) 51, 787-792

Photosynthetic Carbon Metabolism in Isolated Maize BundleSheath Strands12

Received for publication November 3, 197

RAYMOND CHOLLET3Department of Agronomy, University of Illinois, Urbana, Illinois 61801WILLIAM L. OGRENUnited States Regional Soybean Laboratory, North Central Region, Agricultural Research Service, UnitedStates Department of Agriculture, Urbana, Illinois 61801

ABSTRACT

Photosynthetically active bundle sheath strands capable ofassimilating up to 8 micromoles C02 per milligram chloro-phyll per hour have been isolated from fully expanded leavesof Zea mays L. Mesophyll cell contamination of the prepara-tions was negligible, as evidenced by light and electron micros-copy and by a high ratio of chlorophyll a to chlorophyll b inthe strands. Ribose 5-phosphate markedly stimulated the rateof photosynthetic "4CO2 fixation by the isolated strands. In con-trast, both pyruvate and phosphoenolpyruvate had a compara-tively small stimulatory effect on bundle sheath '4CO2 fixation.After 5 minutes of photosynthesis in "4C-bicarbonate, 95% ofthe incorporated "4C was found in compounds other than C4-dicarboxylic acids, most notably in 3-phosphoglycerate andsugar phosphates. A similar distribution of 14C was observedin the presence of exogenous ribose 5-phosphate. Extracts ofbundle sheath strands contained high specific activities of"malic" enzyme, phosphoglycolate phosphatase, hydroxypyru-vate reductase, and ribulose 1, 5-diphosphate carboxylase,whereas the specific activities of NADP+-malate dehydrogenaseand phosphopyruvate carboxylase were extremely low. Theseresults indicate that the Calvin cycle occurs in the bundlesheath cells of maize.

Higher plants can be divided into two major groups, C3 andC4 species, based on the initial products of photosyntheticCO2 fixation. In C3 plants such as soybean and tobacco, CO2is initially fixed into 3-PGA4 by RuDP carboxylase. In C4species, atmospheric CO2 is initially fixed into oxaloacetateby PEP carboxylase (20). Another characteristic of all C.

1 This study was supported in part by Agricultural ResearchService, United States Department of Agriculture CooperativeAgreement No. 12-14-100-11,159(34), administered by the Agri-cultural Research Service, Beltsville, Md.

2Publication No. 763 of the United States Regional SoybeanLaboratory, Urbana, Ill.

' Present address: E. I. du Pont de Nemours & Company, CentralResearch Department, Experimental Station, Wilmington, Del.19898.

4 Abbreviations: 3-PGA: 3-phosphoglycerate; RuDP: ribulose1. 5-diphosphate; PEP: phosphoenolpyruvate.

plants which distinguishes them from C3 species is the presenceof two major chloroplast-containing leaf cell types: the bundlesheath cells which tightly surround the vascular tissue, andthe mesophyll cells which in turn surround the bundle sheathlayer (24).

It has been suggested that in the C, panicoid grasses such asmaize, sugarcane, and crabgrass, atmospheric CO2 is initiallyfixed by PEP carboxylase in the mesophyll, with the resultingoxaloacetate being predominantly reduced to malate by anNADP+-specific malate dehydrogenase. The malate is trans-ported to the bundle sheath chloroplasts and decarboxylatedto pyruvate and CO, by "malic" enzyme. The CO2 is refixedby RuDP carboxylase and further metabolized through theCalvin cycle (4, 7, 8, 16, 21, 32). In marked contrast to thiscompartmentation scheme is that recently proposed by Coombsand co-workers (6, 9, 14, 15) and supported by Laetsch andKortschak (25). These authors propose an identical reactionsequence for C, photosynthesis, but suggest that all the en-zymes for photosynthetic carbon metabolism are compart-mented between the mesophyll cytoplasm or nongreen leafcells and the mesophyll chloroplasts, relegating the bundlesheath to a mere amyloplast-like function. A third scheme ofC4 photosynthesis, suggesting that photosynthetic carbon me-tabolism is similar in both cell types, has also been proposed(29).The purpose of our studies has been to re-examine the role

of bundle sheath cells in C4 photosynthesis by using a highlypurified preparation of bundle sheath strands isolated fromfully expanded leaves of maize. We have previously reportedon the effects of oxygen on maize bundle sheath photosynthesis(12, 13). A more complete description of the photosyntheticcarbon metabolism of these bundle sheath strand preparationsis the subject of this report.

MATERIALS AND METHODS

Plant Materials. Zea mays L., var. W23/L317, seeds weregerminated and grown in a soil-sand-peat moss mixture in agrowth chamber with a 12-hr day at 25 C and a 12-hr night at20 C. Light of about 2000 ft-c at the leaf surface was providedby incandescent and cool white fluorescent lamps. Only fullyexpanded primary and secondary leaves from 11- to 14-day-oldseedlings were used. For some of the enzyme assays, crudeleaf extracts from the C3 plant soybean (Gb'cine max [L.]Merrill, vars. Kent and Waseda) were also prepared. Leafmaterial was harvested from the youngest, fully expandedtrifoliolate of 3-week-old plants (grown hydroponically at4500 ft-c) and homogenized as described below.

787

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Plant Physiol. Vol. 51, 1973

8 g OF PRIMARY AND SECONDARY MAIZE LEAVES

SECTION INTO 0.5-1 cm WIDE TRANSVERSE SEGMENTS WITH RAZOR

PACK INTO SEMI-MICRO MONEL HOMOGENIZING VESSEL1 OF WARING BLENDOR

BLEND FOR 1 min. AT FULL SPEED IN 50mi OF SOLUTION I

FILTER THROUGH TWO

F LTRATE( DISCARD )

FILTRATEDISCARD

FILTRATEI DISCARD

FILTRATEI BUNDLE SHEATH STRNDS AND

LOOSE CHLOROPLASTS )

PASS FILTRATE THROUGH AN 80M NYLON NET3 INA FILTER UNIT4 WITH MAGNETIC STIRRING,WASHING MATERIAL ON NET WITH 150 ml OFSOLUTION I

FILTRATE( LOOSE CHLOROPLASTS -DISCARD)

LAYERS OF MIRACLOTH

RESIDUEI

RESUSPEND IN 50 ml OF SOLUTION 1,REBLEND FORI min AT FULL SPEED AND REFILTER

IRESIDUE

IRESUSPEND IN 50ml OF SOLUTION I, REBLEND FORI min. AT FULL SPEED, AND REFILTERI

RESIDUE(EPIDERMIS, BUNDLE SHEATH STRANDS,LOOSE CHLOROPLASTS

IRESUSPEND IN 50ml OF SOLUTION I AND FILTER THROUGH20-AND 35-MESH SIEVES2 WASHING DEBRIS ON SIEVES WITH400 ml OF SOWTION I

RESIDUE( MOSTLY EPIDERMIS-DISCARD )

RESIDUE( BUNDLE SHEATH STRANDS )

The stainless steel semi-micro Monel homogenizing vessel was purchasedfrom VWR Scientific, Baltimore,Md.

2The 20-mesh (840,") and 35-mesh (420P) brass sieves( 3 inch diameter Xlinch deep) were purchased from Sargent-Welch Scientific Co.

3The nylon net used was Nitex' nylon monofilament bolting cloth purchasedfrom Tobler, Ernst a Traber, Inc., New York, N.Y.

4Falcon filter unit, Model 7102, was purchased from Scientific Glass Apparatusand adopted to hold the 80L nylon net.

FIG. 1. Flow chart of technique for isolating bundle sheath-strands from Zea mnays L. leaves (adapted from Edwards and Black [17]).

Isolation Procedures. Bundle sheath strands (bundle sheathcells attached to vascular tissue) were isolated at 5 C using thecombined mechanical maceration-filtration technique outlinedin Figure 1. The medium used throughout the isolation proce-dure, solution I, consisted of 0.33 M d-sorbitol, 50 mM tris-HCl or Tricine-NaOH (pH 8.0), 5 mM MgCI2, 2 mm NaNO,,2 mM Na2EDTA, 1 mM MnCl2, 0.25 mm KH2PO4, 5 mM D-iSo-ascorbate, 2 mm thioglycolate, and 2% (w/v) polyvinylpyrroli-done 40 (Sigma Chemical Co.).` The isolated strands were re-suspended in 10 ml of solution I, collected by centrifugation at1000g for 1 min, suspended in approximately 5 ml of solutionII containing 0.33 M d-sorbitol, 50 mm tris-HCl or Tricine-NaOH (pH 8.0), 1 mm MgCl2, 2 mm NaNO3, 1 mm Na2EDTA,

5 Mention of a trademark name or a proprietary product does notconstitute a guarantee or warranty of the product by the UnitedStates Department of Agriculture, and does not imply its approvalto the exclusion of other products that may also be suitable.

1 mM MnCl2, 1 mM K2HPO4, and 5 mm dithiothreitol, andstored in ice. All assays were performed within 90 min afterisolation.

Mesophyll chloroplasts were prepared by initially blendingthe leaf material for 5 sec at half speed in 50 ml of solutionI. The resulting brei was filtered through two layers of Mira-cloth (Calbiochem), centrifuged at 2000g for 1 min, and thepellet was resuspended in 1 ml of solution II.

Microscopy. A Microstar Series 10 phase contrast micro-scope (American Optical Co.) equipped with a 35-mm camerawas used to routinely monitor and photograph the isolatedbundle sheath strand preparations.

For electron microscopy, the bundle sheath strands werefixed in buffered glutaraldehyde containing 0.33 M d-sorbitol,thoroughly washed in buffered sorbitol, and postfixed in os-mium tetroxide. The specimens were dehydrated, embedded inEpon 812, sectioned, stained with 2% (w/v) aqueous uranylacetate and basic lead citrate, and examined in an RCAEMU-4 electron microscope.

788 CHOLLET AND OGREN

I

I



MAIZE BUNDLE SHEATH PHOTOSYNTHESIS

Assays. Chlorophyll was determined by thorough extractioninto aqueous 80% (v/v) acetone. Following centrifugation, theconcentration of total chlorophyll and the chlorophyll a-chlo-rophyll b ratio in the acetone extracts were determined aspreviously described (10).

Isolated bundle sheath strands and leaves of maize and soy-bean were homogenized and assayed for NADP+-malate dehy-drogenase, "malic" enzyme (L-malate: NADP+ oxidoreductase[decarboxylating]) (EC 1 .1 .1. 40), and NAD+-hydroxypyru-vate reductase (EC 1. 1. 1. 29) activity as described elsewhere(11). P-glycolate phosphatase (EC 3.1.3.18) activity was as-sayed by the release of Pi from P-glycolate. Reaction vesselscontained 40 mm sodium cacodylate (pH 6.3), 1 mm MgC12,5 mM P-glycolate, and uncentrifuged extract in a total volumeof 1.0 ml (30). The reactions were run for 10 min at 30 C,stopped with 10% (w/v) trichloroacetic acid, the precipitateremoved by centrifugation, and the supernatant assayed forPi (31).

Photosynthetic CO2 fixation by the isolated strands was de-termined by "4CO2 incorporation at 24 C and 2000 ft-c underan atmosphere of 2% oxygen. Unless noted otherwise, thereaction vessels contained bundle sheath strands (6-9 ,tg ofchl), solution II, and 5 mm NaH`4CO3 (1-4 ,uc/,umole) in afinal volume of 1.0 ml. Vessels containing strands were pre-illuminated and gassed with vigorous shaking for 5 min andsealed. The reactions were initiated by injecting NaH"4CO3and terminated after 5 min by injecting 0.1 ml of 6 N aceticacid. Contents of the reaction vessels were centrifuged, ali-quots of the supernatant removed and dried at 90 C, and dpmdetermined by scintillation spectroscopy. For the determina-tion of photosynthetic products, contents of the reaction ves-sels were pooled, centrifuged, and the pelleted strands washedtwice by centrifugation with water. The combined supernatantwas placed on 1 X 13 cm columns of Dowex-1-X8-acetate(200-400 mesh). Nonacidic compounds were eluted with 50ml of water, and aliquots were dried and counted. Acidic com-pounds, including organic acids and sugar phosphates, werethen eluted with 60 ml of 3 N HCl (5), concentrated, and sepa-rated by one-dimensional descending paper chromatographyin liquefied phenol (about 90%)-water-acetic acid-I M EDTA(840:160:10:1 by volume) as described elsewhere (11). Theradioactive areas were located with a radiochromatogramscanner, eluted from the paper, and dpm determined by scintil-lation spectroscopy. The identity of labeled compounds wasdetermined by cochromatography and coincidence with au-thentic nonlabeled compounds as previously described (11). Inall experiments the washed bundle sheath strand pellets con-tained less than 2% of the total 14C incorporated.

RESULTS

The bundle sheath strands isolated by the maceration-filtra-tion technique outlined in Figure 1 characteristically showeda lack of contamination by either intact mesophyll cells orloose chloroplasts, as determined by light and electron micros-copy (Fig. 2). The plastids in the isolated strands were rela-tively agranal, as is typical of mature maize bundle sheathchloroplasts (10, 33), and contained numerous starch grains.The stroma and inner membranes of these plastids appearedto be structurally intact, whereas regions of the chloroplastenvelope were sometimes distorted or ruptured. A typicalyield of isolated strands was about 2% on a total leaf chloro-phyll basis.

In addition to the microscopic observations, the high ratioof chlorophyll a to chlorophyll b in the bundle sheath strandscompared to that in whole maize leaves and isolated mesophyllchloroplasts (Table I) was also indicative of a high degree of

purity (33). From the chlorophyll a to chlorophyll b ratios ofthe various maize leaf fractions, it was calculated that approxi-mately 40% of the total leaf chlorophyll was present in thebundle sheath cell layer (Table I).

Typical maximal rates of photosynthetic "4CO2 fixation bythe isolated bundle sheath strands were 6 to 8 ,moles/mgchl hr. There was no lag in the fixation of "4CO2 following the5-min preillumination period, and the reaction was linear forat least 10 min (data not shown). The concentration of "4C-bicarbonate giving maximal rates of "4CO2 fixation was about30 mm (Fig. 3). The '4C-bicarbonate concentration for 50%maximal velocity (apparent Km) was approximately 10 mMunder 2% oxygen.

Pyruvate and PEP stimulated the rate of light-dependent4CO2 fixation by the isolated strands by less than 3-fold,whereas 2 mm ribose-5-P increased the endogenous rate by anorder of magnitude (Table II). Following 5 min of photosyn-thesis under 2% oxygen in the absence of exogenous sub-strates, approximately 95% of the radiocarbon was found incompounds other than the C,-dicarboxylic acids, malate andaspartate, most notably in 3-PGA and sugar phosphates (TableIII). A similar distribution of "4C was observed when 2 mMribose-5-P was added to the reaction mixtures. In the presenceof exogenous PEP, the amount of label in the C,-acids in-creased, due mainly to increased malate synthesis (Table III).On a chlorophyll basis the specific activities of P-glycolate

phosph,tase and "malic" enzyme in bundle sheath extracts were2.5- and 3.1-fold higher, respectively, than the activities ofthese enzymes in maize whole leaf extracts (Table IV). Incontrast, the specific activity of NADP+-malate dehydrogenasein bundle sheath extracts was extremely low (Table IV). Wehave previously shown that maize bundle sheath strands arenearly devoid of PEP carboxylase activity and enriched inRuDP carboxylase activity when compared to the whole leaf(12). Bundle sheath extracts also showed substantial NAD+-hydroxypyruvate reductase activity, although the specific ac-tivity was less than double that in maize whole leaf extracts(Table IV).

DISCUSSIONThe rates of endogenous photosynthetic CO2 fixation by

isolated maize bundle sheath strands are considerably lowerthan those reported for leaf cells isolated from tobacco (23).This may be due, in part, to the comparatively harsh macera-tion procedure (Fig. 1) required to isolate the strands freefrom whole mesophyll cell contamination. Some indication ofdeleterious isolation effects is suggested by the lack of com-plete structural integrity of the bundle sheath chloroplast en-velope (Fig. 2B). In addition, the extremely high Kin of thestrands for bicarbonate (Fig. 3) may also suggest a deleteriouseffect of the isolation procedure. The observed Km is at least10 times that reported for isolated chloroplasts (22, 28) andC3 leaf cells (23), and almost identical to that reported forpartially purified maize RuDP carboxylase (3). Other possibleexplanations for the relatively low rates of endogenous photo-synthetic CO2 fixation by maize bundle sheath strands may bea reduced capacity of mature agranal bundle sheath chloro-plasts to generate NADPH through noncyclic electron flowfrom water (1, 2, 27) or a leaching of carbon cycle intermedi-ates.The increased specific activities of "malic" enzyme. P-glyco-

late phosphatase, NAD+-hydroxypyruvate reductase (Table IV),and RuDP carboxylase (12) in bundle sheath extracts, as com-pared to maize whole leaf extracts, indicates that the Calvincycle is present in these preparations. This contention is sup-ported by the findings that: (a) the predominant products of

Plant Physiol. Vol. 51, 1973 789



CHOLLET AND OGREN

.~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~.

.,7, .':'f.,FP,,a a t o 4 A,

;t ew * w:w_A o a E r1

._ ,. .v.0-' -f.e

_;

k;

', 'iftSX A/'tX fl //549/ 7 0K

t

//'A

' .

/ .R- ...,1# :s \ Ce. to' .,>. * tb s .: _

;r ,; _X;§r>:.

-f.ff * o z ;

.f S- F; ; i nSt F . A... :. .. .f ' f i.£; ;' _ .:.^

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y., +;

.: eS

<.f : {.

,, + .!

.: :.::.. J,;, F -,j,,Jr.

.S.... Ki.?. .:W S t2

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FIG. 2. A: Phase contrast light micrograph of a representative field of isolated maize bundle sheath strands showing the characteristic lackof intact mesophyll cells. The arrows indicate remnants of mesophyll cell walls. X 560. B: Electron micrograph of a section through an isolatedmaize bundle sheath strand showing the relatively agranal nature of the bundle sheath chloroplasts. X 22,500.

790 Plant Physiol. Vol. 51, 1973

e.Y .



MAIZE BUNDLE SHEATH PHOTOSYNTHESIS

Table I. Chlorophyll a-Chlorophyll b Ratios and theDistributtioni of Chlorophyll int Various Maize

Leaf Fractions

Leaf Fraction Chl a-Chl b Chl Distribution'

ratio So

Whole leaves 4.03Mesophyll chloroplasts 3.23 61Bundle sheath strands 6.11 39

Calculated algebraically by the method of Woo et al. (33).

6

5

E4a

w

x3

csJ

0

vo2

-j

0

I

0 10 20 30mM NaH'4c03

40 50

FIG. 3. Dependence of the rate of photosynthetic "CO2 fixationby maize bundle sheath strands on bicarbonate concentration. Ratesof "CO2 fixation were calculated from the amount of "C in-corporated during 5 min photosynthesis in 2% 02.

5-min bundle sheath photosynthesis are 3-PGA, sugar phos-phates, and glycerate (Table III), (b) the addition of ribose-5-P, a Calvin cycle intermediate, greatly stimulates photosyn-thetic CO2 uptake (Table II), and (c) the Warburg effect, a

phenomenon of Calvin cycle photosynthesis, occurs in maizebundle sheath photosynthesis (12, 13). These observationsprovide conclusive evidence that schemes of maize photosyn-thesis suggesting a lack of photosynthetic CO2 fixation in thebundle sheath (9, 25) are not tenable. The bundle sheath cellsof other C4 species likely are similarly active, since Calvincycle enzymes or photosynthetic CO2 fixation or both havebeen found in all cases where either intact bundle sheath cells(16, 18, 19. 26, 27) or nonaqueous bundle sheath chloroplasts(32) have been isolated and examined. A functional Calvincycle has also been reported in an unspecified chloroplast-typeisolated from young, unexpanded leaves of maize (28).

Approximately 40% of the total leaf chlorophyll in themaize plants used in the present experiments is located in thebundle sheath cells (Table I). Thus if a particular enzyme wereexclusively localized in the bundle sheath, the specific activityof that enzyme (on a chlorophyll basis) in bundle sheath ex-tracts would be approximately 2.5-fold greater than the enzymeactivity in extracts of the whole leaf. The specific activities of' malic" enzyme, P-glycolate phosphatase (Table IV) and RuDPcarboxylase (12) in bundle sheath extracts are 2.3- to 3.1-foldgreater than the specific activities of these enzymes in maize

whole leaf extracts. Conversely, the specific activities ofNADP+-malate dehydrogenase (Table IV) and PEP carboxylase(12) in bundle sheath extracts are only 2% of the total leafactivities. The distribution of these five enzymes fully supportsthe scheme of C. photosynthesis advanced by Hatch and co-workers (4, 32) and by others (7, 8, 16, 18, 19, 21, 27). Whilethe data reported here and elsewhere (12, 13) cannot excludethe possibility that some Calvin cycle activity occurs in themesophyll, it clearly indicates that the enzymes of maize photo-synthesis are not evenly distributed between cell types. Of theenzymes assayed, "malic" enzyme and the enzymes involved inC3 photosynthesis are preferentially, if not exclusively, locatedin the bundle sheath, whereas the enzymes of the C, cycle arelocated outside the bundle sheath, presumably in the mesophyll.Alternative schemes of C4 photosynthesis are primarily basedon differential enzyme extraction, but the results obtained withthis technique are highly variable (7, 9, 21, 29).

Table II. Effect of Exogenious Substrates onz "CO2 Fixationby Maize Bunidle Sheath Stranids

Fixation was assayed at 5 mm NaH'4C03, 27 02, and 42000ft-c for 5 min.

Exogenous Substrate Light Dark

;mmoles "C02 fixed/mg cht hr

No addition 2.7 0.082 mM pyruvate 3.5 0.102 mM PEP 7.8 0.282 mm ribose-5-P 27.2 0.77

Table III. Effect of Exogenous Substrates onz the Productsof Maize Bunzdle Sheath Photosynzthesis

The data are given as percentage of "4C eluted from Dowex-1-X8-acetate columns.

Distribution of 14C in the Presence ofProducts'

No Addition 12 mm ribose-5-P 2 mms PEP

1 ~ ~~~~14CNonacidic compounds 8.8 1.1 1.6Sugar phosphates (in- 52.6 I 69.3 31.8cluding 3-PGA)

Glycerate 15.0 8.0 2.0Malate and aspartate 5.2 4.2 47.62Others 18.4 17.4 17.0

1 The strands had photosynthesized for 5 min in 5 mm NaHl4C03and 2c,% 02 before stopping the reaction with acetic acid. Theproducts were separated and identified as described in "Materialsand Methods."

2 Mostly malate (over 90%c).

Table IV. Enzyme Aclivities in Leaf anid BunzdleSheath Extracts

Enzyme Maize Maize Bun- SoybeanW5hole Leaf dle Sheath Whole Leaf

Amoles/mg chi-hr

"Malic" enzyme 611 1919 5P-glycolate phosphatase 143 353NADP+-malate dehydrogenase 328 4 18NAD+-hydroxypyruvate reduc- 153 232

tase

Plant Physiol. Vol. 51, 1973 791



792 CHOLLET i

LITERATURE CITED

1. ANDERSEN, K. S., J. NM. BAIN, D. G. BISHOP, AN'D R. 'M. SMILLIE. 1972. Photo-system II activity in agranal bundle sheath chloroplasts from Zea mays.Plant Physiol. 49: 461-466.

2. ANDERSON, J. M., K. C. Woo, AND N. K. BOARDMAN. 1971. Photochemicalsystems in mesophyll and bundle sheath chloroplasts of Cs plants. Biochim.Biophys. Acta 245: 398-408.

3. ANDREWS, T. J. AND M. D. HATCH. 1971. Activity andl properties of ribulose-diphosphate carboxylase from plants with the C4-dicarboxylic acicl pathwayof photosynthesis. Phytochemistry 10: 9-15.

4. ANDREWS, T. J., H. S. JOHNSON, C. R. SLACK, AND M. D. HATCH. 1971. Malicenzyme ancl aminotransferases in relation to 3-phosphoglycerate formationin plants with the C4-dicarboxylic acid pathway of photosynthesis. Phyto-chemistry 10: 2005-2013.

5. ATKINS, C. A. AND D. T. CANVIN. 1971. Analysis of 14C-labeled acidic photo-synthetic products by ion-exchange chromatograplhy. Photosynthetica 5:341-351.

6. BALDRY, C. W.. C. BCCKE, AND J. COONIBS. 1971. Progressive release of car-boxylating enzymes during mechanical grinding of sugar cane leaves. Planta97: 310-319.

7. BERRY, J. A., WV. J. S. DOWNTON, AND F. B. TREGUNNA. 1970. Thle pllotosyn-thetic carbon metabolism of Zea mays and Gomphrena globosa: the loca-tion of the CO2 fixation and the carboxyl transfer reactions. Can. J. Bot.48: 777-786.

8. BOWES, G. AN'D W. L. OGREN. 1972. Oxygen inhibition and other propertiesof soybean ribulose-1,5-diphosphate carboxylase. J. Biol. Chem. 247: 2171-2176.

9. BUCKE, C. AND S. P. LoN`G. 1972. Location of enzymes assimilating and shut-tling carbon dioxide in sugar cane and maize leaf tissue. In: G. Forti, WI.Avron, and A. Melandri, eds., Proceedings of the Second International Con-gress on Photosynthesis Research, Vol. 3. Dr. W. Junk N.V. Publishers, TheHague. pp. 1935-1941.

10. CHOLLET, R. AND D. J. PAOLILLO, JR. 1972. Greening in a virescent mutant ofmaize. I. Pigment, ultrastructural, and gas exchange studies. Z. Pflanzen-physiol. 68: 3044.

11. CHOLLET, R. AND W. L. OGREN. 1972. Greening in a virescent mutant of maize.II. Enzyme studies. Z. Pflanzenphysiol. 68: 45-54.

12. CHOLLET, R. AND W. L. OGREN. 1972. Oxygen inhibits maize bundle sheathphotosynthesis. Biochem. Biophys. Res. Commun. 46: 2062-2066.

13. CHOLLET, R. AND W. L. OGREN. 1972. The Warburg effect in maize bundlesheath photosynthesis. Biochem. Biophys. Res. Commun. 48: 684-688.

14. COOMBS, J. 1971. The potential of higher plants with the phosphopyruvicacid cycle. Proc. R. Soc. Lond. B. 179: 221-235.

15. COOMBS, J. AND C. W. BALDRY. 1972. C-4 pathway in Pennisetf m puirpureum.Nature N\ew Biol. 238: 268-270.

16. EDWARDS, G. E. AND C. C. BLACK. 1971. Photosynthesis in mesophyll cells andbundle sheath cells isolated from Digitaria sanguinalis (L.) Scop. leaves. In:M. D. Hatch, C. B. Osmond, and R. 0. Slatyer, eds., Photosynthesis andPhotorespiration. Wiley-Interscience, New York. pp. 153-168.

ND OGREN Plant Physiol. Vol. 51, 1973

17. EDWARDS, G. E. AN-D C. C. BLACK, JR. 1971. Isolation of mesophyll cells an(Ibundle sheath cells from Digitaria saiigsisalis (L.) Scop. leaves and a scan-ning microscopy study of tile internal leaf cell morphology. Plant Physiol.47: 149-156.

18. EDWARDS, G. E., S. S. LEE, T. LM. CHEN, AN-D C. C. BLACK. 1970. Carhoxyla-tion reactions and photosynthesis of carbon compounds in isolated meso-phyll and bundle sheath cells of Diyitaria sanguinalis (L.) Scop. Biochemn.Biophys. Res. Commun. 39: 389-395.

19. EDWARDS, G. E. AND M. GCTIERREZ. 1972. 'Metabolic activities in extracts ofmesophyll and bundle sheath cells of Pas'icusin mni?acelnt (L.) in relation tothe C4 dicarboxylic acid pathway of photosynthesis. Plant Physiol. 50: 728-732.

20. HATCH, MI. D. AN-D C. R. SLACK. 1970. Photosynthetic CO2-fixation pathwa)ays.Annu. Rev. Plant Physiol. 21: 141-162.

21. HUANG, A. H. C. AND H. BEEVERS. 1972. Microbody enzymes and carboxylasesin sequential extracts from Cs and C3 leaves. Plant Physiol. 50: 242-248.

22. JEN-SEN, R. G. AN-D J. A. BASSHANM. 1966. Photosynthesis by isolated chloro-plasts. Proc. Nat. Acad. Sci. U.S.A. 56: 1095-1101.

23. JENSEN, R. G., R. I. B. FRAN-CKI, AN-D 'M. ZAITLIN. 1971. Metabolism of sep-arated leaf cells. I. Preparation of photosynthetically active cells from to-bacco. Plant Physiol. 48: 9-13.

24. LAETSCH, W. M. 1971. Chloroplast structural relationships in leaves of C4plants. In: 'M. D. Hatch, C. B. Osnrond. andI R. 0. Slatyer, eds., Photo-synthesis and Photorespirationi. Wiley-Interscience, New York. pp. 323-349.

25. LAETSCH, W. M. AND H. P. KORTSCHAK 1972. Chloroplast structure andfunction in tissue cultures of a C4 plant. Plant Physiol. 49: 1021-1023.

26. Liu, A. Y. AND C. C. BLACK, JR. 1972. Glycolate metabolism in mesoplhyllcells and bundle sheath cells isolated from crabgrass, Digitaria sanguinalis(L.) Scop., leaves. Arch. Biochem. Biophys. 149: 269-280.

27. MAYNE, B. C., G. E. EDWARDS, AND C. C. BLACK, JR. 1971. Spectral, physical,and electron transport activities in the photosynthetic apparatus of meso-phyll cells and bundle sheath cells of Digitaria sanguinalis (L.) Scop. PlantPhysiol. 47: 600-605.

28. O'NEAL, D., C. S. HEW, E. LATZKO, AND 'M. GIBBS. 1972. Photosynthetic car-bon metabolism of isolated corn chloroplasts. Plant Physiol. 49: 607-614.

29. POINCELOT, R. P. 1972. The distribution of carbonic anhydrase and ribulosediphosphate carboxylase in maize leaves. Plant Physiol. 50: 336-340.

30. REHFELD, D. W., D. D. RANDALL, AN-D N\. E. TOLBERT. 1970. Enzymes of theglycolate pathway in plants Without C02-photorespiration. Can. J. Bot. 48:1219-1226.

31. ROCKSTEIN, AT. AND P. W. HERRON. 1951. Colorimetric determination of in-organic phosphate in microgram quantities. Anal. Chem. 23: 1500-1501.

32. SLACK, C. R., M. D. HATCH, AND D. J. GOODCHILD. 1969. Distribution of en-zymes in mesophyll and parenchyma-sheath chloroplasts of maize leaves inrelation to the Cs-dicarboxylic acid pathway of photosynthesis. Biochem.J. 114: 489-498.

33. Woo, K. C., N. A. PYLIOTIS, AN-D W. J. S. DOWNTON. 1971. Thylakoid aggre-gation and chlorophyll a/chlorophyll b ratio in Cs-plants. Z. Pflanzenphysiol.64: 400-413.

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Photosynthetic Carbon Metabolism in Isolated …2 mMNa2EDTA, 1 mMMnCl2, 0.25 mmKH2PO4, 5 mMD-iSo ascorbate, 2 mm thioglycolate, and 2% (w/v) polyvinylpyrroli- done 40 …