photosynthetic characteristics of c3-c4 intermediate species' · photosynthesis of c3-c4...

Plant Physiol. (1983) 71, 944-9480032-0889/83/7 1/0944/05/$00.50/0

Photosynthetic Characteristics of C3-C4 Intermediate FlaveriaSpecies'I. LEAF ANATOMY, PHOTOSYNTHETIC RESPONSES TO02 AND C02, AND ACTIVITIES OF KEYENZYMES IN THE C3 AND C4 PATHWAYS

Received for publication September 8, 1982 and in revised form December 14, 1982

MAURICE S. B. Ku, RUSSELL K. MONSON2, ROBERT 0. LITTLEJOHN, JR., HITOSHI NAKAMOTO,DONALD B. FISHER, AND GERALD E. EDWARDSBotany Department, Washington State University, Pullman, Washington 99164

ABSTRACT

Four species of the genus Flaveria, namely F. anomala, F. liaris, F.pubescens, and F. ramosissima, were identified as intermediate C3-C4 plantsbased on leaf anatomy, photosynthetic CO2 compensation point, 02 inhi-bition of photosynthesis, and activities of C4 enzymes. F. anomala and F.rawosissina exhibit a distinct Kranz-like leaf anatomy, similar to that ofthe C4 species F. trinervia while the other C3-C4 intermediate Flaveriaspecies possess a less differentiated Kranz-like leaf anatomy. Photosyn-thetic CO2 compensation points of these intermediates at 30°C were verylow relative to those of C3 plants, ranging from 7 to 14 microliters per liter.In contrast to C3 plants, net photosynthesis by the intermediates was notsensitive to 02 concentrations below 5% and decreased relatively slowlywith increasing 02 concentration. Under similar conditions, the percentageinhibition of photosynthesis by 21% 02 varied from 20% to 25% in theintermediates compared with 28% in Lycopersicon esculentW, a typical C3species. The inhibition of carboxylation efficiency by 21% 02 varied from17% for F. ramosissima to 46% for F. anomala and were intermediatebetween the C4 (2% for F. trinervia) and C3 (53% for L. escudentun) values.The intermediate Flaveria species, especially F. ramosissima, have substan-tial activities of the C4 enzymes, phosphoenolpyruvate carboxylase, pyru-vate, orthophosphate dikinase, NADP-malic enzyme, and NADP-malatedehydrogenase, indicating potential for C4 photosynthesis. It appears thatthese Flaveria species may be true biochemical C3-C4 intermediates.

All available evidence suggests that C4 plants have evolved fromancestors possessing the C3 pathway of photosynthesis and thishas occurred independently many times in taxonomically diversegroups (3, 21). At present, the precise evolutionary transition, atthe anatomical, physiological, and biochemical levels, from a C3to a C4 plant is not clear. It is generally believed that studies ofC3-C4 intermediate species might provide insight into the evolutionof C4 photosynthesis. In addition, since most of the world'simportant crops are C3 plants, there has been considerable interestin improving their productivity by screening for mutants withreduced rates of photorespiration or by incorporating C4 charac-teristics into C3 plants (3, 19, 20). Thus, the search for naturally

' This research was supported in part by a grant from Washington StateUniversity Graduate School and gift funds from Monsanto AgriculturalProducts Company, St. Louis, MO.

'Present address: Department of Environmental, Population, and Or-ganismic Biology, University of Colorado, Boulder, CO 80309.

occurring CrC4 intermediates and the study of their anatomical,physiological, and biochemical characteristics are of importanceto both theoretical and applied disciplines of plant biology.

Since 1975, naturally occurring species intermediate between C3and C4 plants have been found in the genera Panicum (6), Mollugo(22), and Moricandia (2). The intermediate nature of these speciesis based on Kranz-like leaf anatomy, low photosynthetic CO2compensation point, and a reduced level ofphotorespiration. Mostrecently, two species of Flaveria (F. anomala and F. pubescens)have also been identified as C3-C4 intermediates based on lowCO2 compensation point at 21% 02 (1). In the present study, weexamined the leaf anatomy, photosynthetic response to C02,sensitivity of net photosynthesis to 02, and activity ofkey enzymesin C3 and C4 photosynthesis of several species of Flaveria, a genusapparently having C3, C4 and C3-C4 intermediate species (1, 21).

MATERIALS AND METHODS

Plant Material and Growth Conditions. Plants of Flaveriaanomala Robinson, F linearis Lag., F pubescens Rydb., F. ramo-sissima Klatt, F trinervia Mohr, and Lycopersicon esculentum Mill(C3) were obtained by germinating the seeds on top of fime soil inpeat pots which were placed in trays and watered by absorptionor on moist filter paper in Petri dishes. After seedlings reached Ito 3 cm in height, they were transplanted into larger pots filledwith a mixture of peat and sand, and maintained in a growthchamber under a daily regime of 14 h of light at 27°C and 8 h ofdarkness at 22°C. Light was provided by a combination of fluo-rescent and incandescent lamps, giving a photosynthetic photonflux density of 80 nE/cm2 .s at plant height. Plants were wateredwith dilute nutrient solution three times a week. Young and newlyexpanded leaves from 2- to 4-month-old plants were used forexperiments.

Leaf Anatomy. Samples (approximately 4 mm2) of tissue werecut from young, fully expanded leaves and vacuum infiltratedwith cold fixative (2% depolymerized paraformaldehyde and 3%glutaraldehyde in 0.1 M phosphate, pH 7.0). After 2 h, the tissuewas washed with buffer, dehydrated in a graded ethanol series,and embedded in 'L.R. White' embedding medium according tothe supplier's instructions (Polysciences, Inc.). Sections were cutat 2.5-,pm thickness and stained with the periodic acid-Schiffreaction for insoluble carbohydrate (12).Gas Exchange Measurements. CO2 and water vapor exchange

of intact individual leaves were measured with an open IR gasanalysis system as described in a previous paper (18). Leaf tem-peratures were maintained at 30 + 0.5°C using a peltier-cooledheat exchanger. A photosynthetic photon flux density of 180 nE/

2cm . s within the leaf chamber was provided by a combination of944

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PHOTOSYNTHESIS OF C3-C4 INTERMEDIATE FLA VERIA SPECIES

a l-kw multivapor lamp and three 300-w medium flood lamps(Sylvania) after filtration through 20 cm of water. In the CO2response experiments, various gas mixtures were provided bymixing gases from cylinders containing 21% 02 in N2 or 2% 02 inN2 with pure CO2 using a pair of Wosthoff mixing pumps. In the02 response experiments, the gas mixtures were generated bymixing gases from cylinders containing pure O2, pure N2, and 1%CO2 in N2 using a series of three Wosthoff mixing pumps. Therates of photosynthesis were measured after reaching steady state(usually within 30 min).The photosynthetic CO2 compensation points were determined

by extrapolating the initial slope of the CO2 response curvethrough the abscissa. Carboxylation efficiency was determinedfrom the initial slope of the CO2 response curve.Enzyme Extraction. For the assay of PEP3 carboxylase, pyru-

vate,Pi dikinase, and NADP-malic enzyme, leaf extracts wereobtained at 4VC immediately after harvesting the tissue in 4volumes of grinding medium containing 10 mm Tris-HCl, pH 7.5,20 mm MgCl2, 1 mm EDTA, 2.5 mm pyruvate, 100 mm DTE, and2.5% (w/v) insoluble PVP. The crude extract was passed throughone layer of Miracloth and the filtrate was rapidly desalted bypassage through a small Sephadex G-25 column (0.8 cm indiameter and 5 cm in length). The column was pre-equilibratedwith a buffer solution containing 50 mm Tris-HCl, pH 7.0, 5 mmMgCl2, 0.1 mm EDTA, 2.5 mm pyruvate, 0.2% BSA, and 10 mmDTE. About 0.4 ml of the crude filtrate was applied to the columnand desalted by centrifugation of the column at 1400g for 3 minat room temperature (10). The eluate obtained by this techniquewas not diluted and the protein yield was over 90%o of the originalsample. An aliquot was taken for Chl determination prior toapplying the filtrate to the Sephadex column. For the assay ofRuBP carboxylase, PEP carboxykinase, NAD-malic enzyme, andNADP-malate dehydrogenase, a buffer solution containing 50 mMHepes-KOH, pH 7.5, 1 mM MgCl2, 1 mm MnCl2, 5 mm DTE, and2.5% (w/v) insoluble PVP was used for enzyme extraction. Thesame buffer solution without PVP was used for equilibration ofthe Sephadex column.Enzyme Assays and Chi Determination. The following enzymes

were assayed spectrophotometrically at 340 nm in a total volumeof 1 ml at 25 to 27°C as previously described: PEP carboxylase(24), pyruvate,Pi dikinase (23), NADP-malic enzyme (13), NADP-malate dehydrogenase (13), and NAD-malic enzyme (9). RuBPcarboxylase and PEP carboxykinase were assayed radiometricallyin a total volume of 0.15 ml at 30°C using NaH"CO3 accordingto (17) and (7), respectively. Chl concentration and Chl a/b ratioswere determined according to Wintermans and De Mots (26) afterextraction in 96% (v/v) ethanol.

RESULTS AND DISCUSSION

Leaf Anatomy. The leaf anatomy of F. trinervia, as shown inFigure IA, is characteristic of a C4 or Kranz leaf. A layer of welldeveloped bundle sheath cells surrounds the vascular tissue. Thebundle sheath cells contain numerous chloroplasts in a centripetalposition. Surrounding the bundle sheath cells are palisade paren-chyma beneath the adaxial epidermis and spongy parenchymaabove the abaxial epidermis, typical of a C4 dicotyledonous leaf.The bundle sheath chloroplasts were considerably larger, appear-ing more elongated than those of the palisade and spongy paren-chyma cells.

3 Abbreviations: DTE, dithioerythritol; PEP, phosphoenolpyruvate; T,photosynthetic CO2 compensation point; CE, carboxylation efficiency;RuBP, ribulose 1,5-bisphosphate.

FIG. 1. Leaf transactions of F. trinervia (upper), F. ramosissima (mid-dle), and F. linearis (lower). BS, bundle sheath cell; PP, palisade paren-chyma cell; SP, spongy parenchyma cell. Bar = 100 jum.

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Plant Physiol. Vol. 71, 1983

Leaf transections of F. ramosissima (Fig. 1) and F. anomala (notshown) exhibited Kranz-like anatomy with an obvious chloro-plast-containing layer of bundle sheath cells. However, in contrastto F. trinervia, in these two species the Kranz cells were lessdistinctive; the mesophyll and bundle sheath chloroplasts ap-peared similar in size, not all of the bundle sheath chloroplastswere in a centripetal position and a smaller proportion of the leafchloroplasts were in the bundle sheath tissue. In comparison to F.ramosissima and F. anomala, F linearis (Fig. 1) and F. pubescens(not shown) have a similar, but less developed, Kranz-like leafanatomy.

In a previous study (21), F trinervia was classified as a C4 plantand F anomala, F. linearis, F. pubescens, and F ramosissimaclassified as C3 plants based on leaf anatomy. However, W. V.Brown had observed that certain species including F. oppositifolia,F linearis, and F. floridana were intermediate between non-Kranzand Kranz leaf ahatomy (cited as a personal communication inRef. 21). In the present study, we have clearly shown that Ftrinervia possesses a typical C4 or Kranz leaf anatomy, while otherFlaveria species studied display a varying degree of intermediacybetween non-Kranz and Kranz leaf anatomy.

Photosynthetic Responses to CO2 and 02. Whole leaf netphotosynthesis as a function of intercellular CO2 concentrationwas measured in the various Flaveria species for comparing theircarbon assimilation efficiencies. Photosynthesis by F. trinerviashowed a steep initial slope in response to varying intercellularCO2 concentration and saturated at low CO2 concentrations(around 150,d/l) (Fig. 2A), which is typical of a C4 plant. Therewas no apparent inhibition of photosynthesis by 21% 02. Incontrast, photosynthesis at 21% 02 by L. esculentum, a typical C3plant, responded slowly to CO2 and was not saturated until theintercellular CO2 concentration reached 330 ,ul/l (Fig. 2B). Therates of photosynthesis in L. esculentum were greatly enhanced at2% 02 at all CO2 levels. These results are similar to those of earlierstudies (4, 14) which show that in C4 plants CO2 is fixed moreefficiently at low CO2 concentrations in the intercellular spaces(a steeper initial slope) and photosynthesis saturates at relativelylower (subatmospheric) levels of CO2 than in C3 plants. Thephotosynthetic CO2 response curves of F. pubescens and Fanomala were intermediate between those of F trinervia and L.esculentum (Fig. 2, A and B). Similar responses of photosynthesisto CO2 were also observed with F linearis and F ramosissima(data not shown).The photosynthetic CO2 compensation point (Tr) is another

physiological trait that distinguishes C3 from C4 plants: C4 plantsexhibit a near-zero T (0-5 ul/l1), whereas C3 plants usually have Tabout 50 ful/l under atmospheric conditions. The T values ofvarious Flaveria species and L. esculentum, obtained by extrapo-lation of the photosynthetic CO2 response curves to zero CO2 (Fig.2), are shown in Table I. F. trinervia showed a T less than 1 ,ul/l atboth 2% and 21% 02, typical of C4 plants. On the other hand, thevalue for L. esculentum was 54 l/l and was greatly reduced at 2%02. The T values of the intermediate Flaveria species compared tothat of the C3 plant are very low, ranging from 7 to 14 ftl/l at 21%02. The T of F. ramosissima of 7 pd/l approaches that obtained forC4 plants. The low T in these species at 21% 02 suggests that theyhave a reduced rate of photorespiration. In contrast to L. esculen-tum, there was no significant influence of lowering the 02 concen-tration from 21% to 2% on the T of these Flaveria species. The T ofthe intermediate Flaveria species are lower than or comparablewith those typically reported for the C3-C4 intermediate species inPanicum (6, 14), Moricandia (2, 11, 25), and Mollugo (22). Re-cently, Apel and Maass (1) also reported that F. anomala and F.pubescens possess an intermediate T between the C3 and C4 Flaveriaspecies at 21% 02. Thus, F. anomala, F. linearis, F pubescens, andF. ramosissima can be classified as C3-C4 intermediates based onleaf anatomy and photosynthetic CO2 compensation point.

E 2000

CPE

0) 150

E-1

C,n

c

00&-50

c

-

0:

V)

0

E

0

a.0

0~

I.

(A)

v / /

0

,/I -1( /l

vf 1"

100 200 300 400Intercellular C02 concentration (,ul/l)

100 200 300 400Intercellular C02 concentration (pi/1)

500

FIG. 2. The response of net photosynthesis to varying intercellular CO2concentration for various Flaveria species and L. esculentum (C3) at 2%(open symbols) and 21% 02 (solid symbols). A, F trinervia (A, A; note, ateach CO2 level, rates of photosynthesis at the two 02 levels coincided,giving essentially no effect of 02), F. pubescens (0, 0); B, F. anomala (O,), L esculentum (0, U). The assay conditions were 180 nE/cm2.sphotosynthetic photon flux density, 340 pd/l C02, and 30 ± 0.5°C leaftemperature. Note different scales for ordinates ofA and B. Data presentedrepresent one replication although two measurements were made for eachspecies.

The intermediate species are also less sensitive to 02 inhibitionof photosynthesis, relative to C3 species. CE, derived from theinitial slope of the photosynthetic CO2 response curve, measuresthe efficiency of different species in utilizing low levels of CO2.The data of CE for various Flaveria species and L. esculentum,measured at 21% and 2% 02, are presented in Table I. The C4species F. trinervia had the highest CE and exhibited essentiallyno inhibition of CE by 21% 02, presumably due to the CO2concentration mechanism of the C4 pathway of photosynthesis. L.esculentum had slightly higher CE than the intermediate Flaveria

11)aI n

946 KU ET AL.



PHOTOSYNTHESIS OF C3-C4 INTERMEDIATE FLAVERIA SPECIES

Table I. Photosynthetic C02 Compensation Point and CarboxylationEffciency at 2% and 21% 02 of Various Flaveria Species and Lycopersicon

esculentum

C02Compensation Carboxylation Efficiencyb

PointSpecies

Inhibition21%02 2%02 21%02 2%02 by

21% 02J.L/l

F. anomala 12 11 1.02 1.89 46F. linearis 8 6 0.94 1.63 42F. pubescens 14 11 0.83 1.21 31F ramosissima 7 5 1.07 1.29 17F. trinervia

KU ET AL. Plant Physiol. Vol. 71, 1983

Table II. Activity of Several Key Enzymes of C4 Photosynthesis in Leaf Extracts of Various Flaveria Species and Panicum milioidesSee "Materials and Methods" for the assay of enzymes. RuBPC, ribulose 1,5-bisphosphate carboxylase; PEPC, PEP carboxylase; PPDK, pyruvate,Pi

dikinase; NADP-ME, NADP-malic enzyme; NAD-ME, NAD-malic enzyme; PEP-CK, PEP carboxykinase; NADP-MDH, NADP-malate dehydroge-nase.

Species Chl a/b RuBPC PEPC PPDK NADP-ME NAD-ME PEP-CK NADP-MDH

,urmol/mg Chl.hF. anomala 2.82 564 123 23 25 22 0.6 133F. linearis 2.92 561 123 3.7 66 50 0.6 23F. pubescens 2.78 497 207 27 90 18 1.7 54F. ramosissima 3.40 412 162 37 153 34 0.4 135F. trinervia 3.87 309 908 290 1091 47 4.3 758P. milioides 3.01 76 5.5 14 4.1 N.D.a

a N.D., not detectable.

about one-tenth of those of the C4 species. In P. milioides, the C4enzymes, particularly pyruvate,Pi dikinase and the C4 acid decar-boxylases, were very low in activity, which is consistent with therecent report that this species fixes CO2 solely by the C3 pathway(8). These results suggest that the C3-C4 intermediate Flaveriaspecies, in contrast to Moricandia arvensis (11, 25) and P. milioides,may be capable of fixing some CO2 through the C4 pathway.The C4 pathway in the genus Flaveria occurs mostly in the

advanced annual species and is proposed to have arisen from C3species relatively recently under arid, tropical conditions (21). Thegenus appears to contain a number of species which exhibit C3-C4intermediate characteristics (1, 21). In the present study, F.anomala, F. linearis, F pubescens, and F. ramosissima are identifiedas intermediate species between C3 and C4 plants based on leafanatomy, photosynthetic CO2 compensation point, sensitivity ofphotosynthesis to 02, and activities of C4 enzymes. Whereas themechanism of reduced photorespiration in M. arvensis and P.milioides remains unknown, it appears that a limited degree of C4photosynthesis may be responsible for the lower CO2 compensa-tion points and reduced rates of photorespiration in the C3-C4intermediate Flaveria species. Thus, some of the Flaveria speciesmay be in the process of evolution from C3 to C4 photosynthesisat both the anatomical and biochemical levels.

Acknowledgments-We wish to thank Dr. A. M. Powell for providing seeds ofFlaveria species and Gail Schmitt for preparing the leaf cross-sections.

LITERATURE CITED

1. APEL P, I MAASS 1981 Photosynthesis in species of Flaveria CO2 compensationconcentration, 02 influence on photosynthetic gas exchange and 93C values inspecies of Flaveria (Asteraceae). Biochem Physiol Pflanzen 197: 396-399

2. APEL P, H OHLE 1979 C02-compensation point and leaf anatomy in species ofthe genus Moricandia DC (Cruciferae). Biochem Physiol Pflanzen 174: 68-75

3. BJORKMAN 0 1976 Adaptive and genetic aspects of C4 photosynthesis. In RHBurris, CC Black, eds, C02 Metabolism and Plant Productivity. UniversityPark Press, Baltimore, pp 287-310

4. BJORKMAN 0, HA MooNEY, J EHLERINGER 1975 Photosynthetic response ofplants from habitats with contrasting thermal environments. Comparison ofphotosynthetic characteristics of intact plants. Carnegie Inst Year Book 74:743-751

5. BROWN RH 1980 Photosynthesis of grass species differing in carbon dioxidefixation pathways. IV. Analysis of reduced oxygen response in Panicummilioides and Panicum schenckii Plant Physiol 65: 346-349

6. BROWN RH, WV BROWN 1975 Photosynthetic characteristics of Panicum mil-ioides, a species with reduced photorespiration. Crop Sci 15: 681-685

7. EDWARDS GE, R KANAI, CC BLACK 1971 Phosphoenolpyruvate carboxykinasein leaves of certain plants which fix C02 by the C4-dicarboxylic acid pathway

of photosynthesis. Biochem Biophys Res Commun 45: 278-2858. EDWARDS GE, MSB Ku, MD HATCH 1982 Photosynthesis in Panicum milioides,

a species with reduced photorespiration. Plant Cell Physiol 23: 1185-11969. HATCH MD, M TsuzuKI, GE EDWARDS 1982 Determination of NAD malic

enzyme in leaves of C4 plants. Effect of malate dehydrogenase and otherfactors. Plant Physiol 69: 483-491

10. HELMERHORST E, GB STOKES 1980 Microcentrifuge desalting: a rapid, quantita-tive method for desalting small amounts of protein. Anal Biochem 104: 130-135

1 1. HOLADAY AS, YJ SHIEH, KW LEE, R CHOLLET 1981 Anatomical, ultrastructuraland enzymic studies of leaves of Moricandia arvensis, a C3-C4 intermediatespecies. Biochim Biophys Acta 637: 334-341

12. JENSEN WA 1962 Botanical Histochemistry. WH Freeman and Co., San Francisco13. KANAI R, GE EDWARDS 1973 Separation of mesophyll protoplasts and bundle

sheath cells from maize leaves for photosynthetic studies. Plant Physiol 51:1133-1137

14. Ku SB, GE EDWARDS 1978 Photosynthetic efficiency of Panicum hians andPanicum milioides in relation to C3 and C4 plants. Plant Cell Physiol 19: 665-675

15. Ku SB, GE EDWARDS 1980 Oxygen inhibition of photosynthesis in the C4 speciesAmaranthus graecizans L. Planta 147: 277-282

16. Ku SB,M GUTIERREZ, R KANAI, GE EDWARDS 1974 Photosynthesis in mesophyllprotoplasts and bundle sheath cells ofvarious types ofC4 plants. II. Chlorophylland Hill reaction studies. Z Pflanzenphysiol 72: 320-337

17. LORIMER GH, MR BADGER, TJ ANDitEws 1977 D-Ribulose-1,5-bisphosphatecarboxylase-oxygenase. Improved method for the activation and assay ofcatalytical activities. Anal Biochem 78: 6675

18. MONSON RK, MA STIDHAM, GJ WILLIAMS III, GE EDWARDS, EG URIBE 1982Temperature dependence of photosynthesis in Agropyron smithii Rydb. I.Factors affecting net CO2 uptake in intact leaves and contribution fromribulose-1,5-bisphosphate carboxylase measured in vivo and in vitro. PlantPhysiol 69: 921-928

19. Moss DN 1976 Studies on increasing photosynthesis in crop plants. In RHBumfis, CC Black, eds, CO2 Metabolism and Plant Productivity. UniversityPark Press, Baltimore, pp 31-42

20. OGREN WL 1976 Search for higher plants with modifications of the reductivepentose phosphate pathway ofCO2 assimilation. In RH Burris, CC Black, eds,CO2 Metabolism and Plant Productivity. University Park Press, Baltimore, pp19-30

21. POWELL AM 1978 Systematics ofFlaveria (Flaverinae-Asteraceae). Ann MissouriBot Garden 65: 590-636

22. SAYE RT, RA KENNEDY 1977 Ecotypic differences in the C3 and C4 photosyn-thetic activity in Mollugo verticillata, a C3-C4 intermediate. Planta 134: 257-262

23. SUGIYAMA T 1973 Purification, molecular, and catalytic properties of pyruvatephosphate dikinase from the maize leaf. Biochemistry 12: 2862-2868

24. UEDAN K, T SUGIYAMA 1976 Purification and characterization of phosphoenol-pyruvate carboxylase from maize leaves. Plant Physiol 57: 906-910

25. WINTER K, H USUDA, M Tsuzusu, MR SCHMIrr, GE EDWARDs, RJ THOMAS,RF EVERT 1982 Influence of nitrate and ammonia on photosynthetic charac-teristics and leaf anatomy of Moncandia arvensis. Plant Physiol 70: 616-625

26. WINTEsANS JFGM, A DE MOTS 1965 Spectrophotometric characteristics ofchlorophylls and their pheophytins in ethanol. Biochim Biophys Acta 109:448453

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