Carbon Isotopes in Photosynthesis

Carbon Isotopesin Photosyn

Fractionationtechniquesmay reveal new aspects of carbon dynamics in plants

Marion H. O'Leary

heefficiencoyf photosynthesis

continues to interest biochemists, biologists, and plant

Currentstudies include

physiologists. Scientists interested in CO2 uptake are concerned about the extent to which the uptake rate is

finely tuned, carefully controlled isotope

limited by such factors as stomatal diffusion and the chemistry of the CO2 absorption process. The frac-

fractionationsunder defined environmental

tionation of carbon isotopes that occurs during photosynthesis is one of

conditions

the most useful techniques for investi-

gating the efficiency of CO2 uptake.

Atmospheric carbon dioxide con- used to study mechanisms of chemical

tains approximately 1.1% of the non- (Melander and Saunders 1980) and

radioactive isotope carbon-13 and biochemical (Cleland 1982) process-

98.9% of carbon-12. During photo- es. Isotopes are used in ecology to

synthesis, plants discriminate against establish food chains and biological C because of small differences in pathways (Fritz and Fontes 1980,

chemical and physical properties im- 1986, Rounick and Winterbourn

parted by the difference in mass. This 1986), and isotope studies of tree

discrimination can be used to assign rings are used to recreate past cli-

plants to various photosynthetic mates (Hughes et al. 1982). Isotope

groups. The isotope fractionation studies of plants are related to all

also reflects limitations on photosyn- these areas, because their basis is in

thetic efficiency imposed by the vari- fundamental chemical processes, and

ous diffusional and chemical compo- many of their applications are in the

nents of CO2 uptake. When analyzed area of ecology (O'Leary 1981,

in detail, this fractionation provides Troughton 1979, Vogel 1980). Re-

information .about water use efficien- cently developed methods are allowcy and indicates that different strate- ing biologists to examine in greater

gies are needed for improving water- detail the carbon flow in plants.

use efficiency in different kinds of

plants.

Measurement of carbon

Isotope fractionation in simple physical and chemical processes is

isotopes

well understood and is commonly The "3Ccontent of carbon dioxide is

usually determined with a mass spec-

MarionH. O'Learyis a professorin the Departmentsof Chemistryand Biochemistry at the Universityof Wisconsin in

trometer specially designed for highprecision measurement of the ratio R, defined by

tMutaedoisfoBni5o3lo7g0i6c.al?Sc1ie9n8c8eAs.mericanInsti-

R = 3CO2/12CO2

Other materials must be converted to

CnaOril2yprcoionrvteoratendatloysCisOP. 2labnytsacormeobruds-ition. Individuaclompoundsisolated from plants are sometimesconverted

to CO2 by chemical or enzymatic

degradation. Fornaturalmaterials(plants,ani-

mals, and minerals), R is approximately0.0112,andonlythelastdigit in this ratio varies. For convenience, R valuesare generallyconvertedto valuesof 813C,

813C 3=C[RR((ssatmanpdlea)rd)- 1] x 11000000

The standard is carbon dioxide ob-

tained from a limestone, called PDB,

from the Pee Dee formation in South

Carolina (Craig 1957). The units of

813C are called "per mil," more negative 8 C means

or mor0e/0o.

A

or lighter in mass; a more positive

813C means more 13C, or heavier.

Most natural materials have negative 813C values because they contain less 13Cthan the standard. The precision of modern mass spectrometers is at least ?i0.02 %o0, but sample preparation errors may bring the total reproducibility of measurements on plant materials to 10.2 0/0o. Thus, interpretations based on differences smaller

than 1 o/ should be made with caution.

In the absence of industrial activity, the 813C value of atmospheric CO2 is -8 0/oo. This value for the atmosphere is slowly becoming more negative due to the combustion of fossil

fuel (813C for fossil fuel is approxi-

mately -30 %/oo) (Hoefs 1980).

328

BioScience Vol. 38 No. 5

This content downloaded from 129.236.31.165 on Wed, 09 Sep 2015 19:33:03 UTC All use subject to JSTOR Terms and Conditions

Isotopevaluesof plants

In the 1950s, Craig(1953, 1954) and

Bueaserotfsachvi(a1r9ie5t3y)omf neaastuurraedlm81a3teCrviaalls-,

including plants (reviewed by

O'Leary1981). Theyfoundthatmost

plants had 813Cvalues in the range l-a2r5getsope-3ci5eso0or/0e. nTvihreoynfmaielendtatleofffeincdts on thesevalues.

The plants in these initial studies were principallyC3plants, which fix CO2 by the action of the enzyme ribulose bisphosphate carboxylase. The C4 photosynthetic pathway, in which CO2 is initially taken up through carboxylation of phosphoenolpyruvate,was discoveredin the 1960s. Following this discovery,

}.=.i.i......i..

i??? ..

!i

i

i! i

??'?':'ii ::: :ii:

.X. :':

.. ? ,~?i..;i:l.;.~i.,.i:.l.~..........:....=i

. ..

.

:I::: .ii..........".. iii!i.liii?~iiii?Yii.ii.i...i.:i::.i.i.?..:. .

. .

xt. :

.

.-?.. i~?N???,?.,,=i?i.i.i.?.i:.i.:N:N..:..:....

....... ..

Bender (1968, 1971; see also Smith and Epstein1971) discoveredthat C4

-0 -!1_ -14 -16 -18 -20 -i2 AE13r

-2.4 -26 -2?8 -30 -32? -34

plants are isotopically distinct from

C3plants.C3plantshave 813Cvalues Figure1. Histogramshowingthe distributioonf 813Cvaluesof plantmaterialsT. his of approximately -28 0/0o, whereas figureis basedon about1000analyseps erformeidn fivedifferenltaboratories. C4Ipnlasnutbs saerqeuaepnptryoexaimrsa,taelynu-1m4be0/roof

laboratoriesaround the world made fractionationhas a positivesignwhen boxylation step itself. Severalmathe-

similar measurementson thousands 13Cis transformedmore slowly than matical models have been suggested of plants species and established a 12C(as is the case in most physical (Deleenset al. 1983, Farquharet al.

clear distinctionbetween C3 and C4 and chemicalprocesses).1

1982, O'Leary 1981, Peisker 1982,

plants (Figure1), with little overlap Many physical,chemical,and bio- 1984, 1985), all of which are based

betweenthe two distributions.There- chemical processes have significant on the componentfractionationsgiv-

fore, 13Canalysishas becomea stan- isotope fractionations(Cleland1982, en in Table 1. The overall fraction-

dardtestfordeterminingthepathway Melanderand Saunders1980). Frac- ation in such a complex system is a

of CO2 fixation. What is the bio- tionations can occur both in time- combination of these components,

chemicalsourceof this difference? dependentprocesses (chemicalreac- but it is not simplythe sumof a series

tions and transport) and in of individualfractionations-instead,

Fractionationsin chemicaland equilibriumprocesses(chemicalequi- the fractionationmostly reflectsthe

physicalprocesses

libria, dissolution, and phase rate-limitingstep or steps (i.e., those changes),and both are importantin with the highestresistivity).As a step

Plantscontainless '3Cthanthe atmo- plants. Table 1 shows isotope frac- becomesmore limiting,the observed

sphere because the physical and tionations for processes of impor- fractionation approaches the frac-

chemicalprocesses involved in CO2 tance in photosynthesis.

tionation for that step.

uptakediscriminateagainst13C.This

The importantstepsin CO2uptake

discriminationoccurs because '3C is heavierthan 12Cand forms slightly strongerchemicalbonds. In addition,

Theory of isotope fractionationin plants

in C3plantsareshown in Figure2. In the first step, external CO2 is transported through the boundary layer

diffusionof '3CO2 is slowerthanthat of 12CO2because of this difference in mass. For the conversion of com-

The principalfactoraffectingthe iso- and the stomatainto the internalgas topic compositions of leaves is the space.This processis alwaysto some isotope fractionation accompanying extent reversible.InternalCO2 then

pound A into compound B, the iso CO2uptake.Followinginitialsugges- dissolvesin the cell sapanddiffusesto

tope fractionation is defined by

tions of Craig(1953), Smithand Ep- the chloroplast,where carboxylation

[813C(A) - 8'3C(B)] 1 + 813C(A)/1000

stein (1971), and others (O'Leary occurs. Because the carboxylation 1981), models for plant isotope frac- step is irreversible,steps subsequent tionation have focused on the physi- to carboxylationarenot importantin

cal and chemicalprocessesaccompa- determiningthe isotopefractionation.

This fractionation has units of o. To avoid confusion with ordinary

813Cvalues (which represent isotopic

compositions, rather than fractionations), we call this value A8. The

nying CO2 uptake, including Both dissolving and diffusion show

diffusion, dissolution, and the car- smallisotopefractionations(Table1),

but the largest fractionationis that

'However,note that some workersin the field use the oppositesign convention.

coIntnisegcetenwdeirtahlclyaarssbuomxyeldathtiao(t2nd9issoo/olu).-

May 1988

329

This content downloaded from 129.236.31.165 on Wed, 09 Sep 2015 19:33:03 UTC All use subject to JSTOR Terms and Conditions

Table 1. Carbon isotope fractions associated with photosynthesis.

photorespiration), then we could

Process

A, 0/00oo*

Reference

breeda plantthatwould takeup CO2 more rapidlywithout sacrificingwa-

Equilibria Solubilityof CO2in water Hydrationof CO2

1.1

O'Leary1984

-9.0

Mook et al. 1974

ter-useefficiency.The alternativepossibility, decreasing diffusive resistance,has only a verylimitedpotential for increasingCO2 uptake, and this

Transporpt rocessest CO2diffusionin air CO2diffusionin aqueoussolution

4.4

O'Leary1981

0.7

O'Leary1984

increasewould come at a substantial cost in water-use efficiency. As we will see below, the situation in C4

Chemicalprocesses Spontaneoushydrationof CO2 Carbonicanhydrasecatalyzed hydrationof CO2 Phosphoenolpyruvatcearboxylasecatalyzedreactionof HC03with phosphoenolpyruvate Ribulosebisphosphatecarboxylasecatalyzedreactionof CO2 with ribulosebisphosphate

plants is different.

6.9

Marlierand O'Leary1984 The C4 pathway involves sequen-

1.1

Panethand O'Leary1985

tial operationof two carboxylasesystems (Figure3). CO2 initially enters

the leaf through the stomata and is

2.0

O'Learyet al. 1981

taken up by phosphoenolpyruvate

carboxylase in the mesophyll cells.

29.0

Roeskeand O'Leary1984

The productof this carboxylationis convertedto either malate or aspar-

*Positivvealuesin thistableindicatethattheproductis depletedin 13Ccomparedwiththestarting state;negativevaluesindicateenrichment.

tate and is transportedto the bundle sheath cells, where it is cleaved to

tPredictedvalue.This numberhas not beenmeasured.

CO2and some other compound.The

CO2 thus produced is taken up by

tion and liquid-phase diffusion are rapid, but good evidence for this is lacking. If stomatal diffusion is rapid (stomatal resistance is low) and carboxylation is limiting, the predicted isotope fractionationis 28 %o/,and the predictedleaf 813Cvalue is -36 o/o. If diffusion is slow (stomatal resistanceis

limited extreme. More quantitative analysis indicates that the carboxylation resistance is higher than the diffusional resistance by up to a factor of two; diffusion of internal CO2 back to the outside is faster than carboxylation by up to a factor of two.

ribulose bisphosphate carboxylase. Although the latter enzyme shows a large isotope fractionation(Table1), the effectsof this fractionationarenot seen in C4plants becausethis step is precededby an irreversiblestep, the carboxylation of phosphoenolpyruvate.

As in C3carboxylation,dissolution

high), the predicted isotope fractionation is 4 %o/ and the predicted leaf 813C value is -12 0o/0. To the extent that diffusion and carboxylation

Thus, CO2 uptake in C3 plants is limited more by the rate of carboxylation of ribulose bisphosphate than by diffusion. This finding has impor-

and liquid-phasediffusionof CO2are assumedto be fast. Carbonicanhydrase is present in C4 plants (Reed and Graham 1981); thus, CO2 and

jointly limit the rate, the 813C value tant implications for plant breeding. HC03- are expected to be in equilibwill be intermediate between these If we could breed plants with a more rium. The steps that are significant

two extremes. Measured 813C values efficient ribulose bisphosphate carfor C3 plants cluster near -28 0/0o, boxylase (either because of increased which is nearer to the carboxylation- enzyme activity or because of reduced

Atmosphere Epidermis Internal air space

Mesophy

Phloem

00

for isotope fractionationarestomatal diffusionand carboxylationof phosphoenolpyruvate.If diffusionis facile and carboxylation is limiting, then the predictedleaf 813Cis -1 o/ooO. n the otherhand,if diffusionis limiting and carboxylationis facile, the predicOtebdse8r1v3Cedi8s1-31C2va0l/uoe.sfor C4 plants

are approximately-14 0/00. Thus, it

appears that, unlike the case in C3

plants, carboxylation capacity in C4

plants is in excess of that needed for

kq

leaf RuBO OG 0

steady-state photosynthesis, and the diffusion is more limiting than carboxylation. Unlike the situation in C3

omragtatneirc

O 0

plants, further improvements in the efficiency of C4 plants cannot come

about through increases in carboxyl-

ation capacity.

Figure 2. Important steps in CO2 fixation during C3 photosynthesis. Sizes of arrows indicate the relative fluxes through the various steps (includingthe reversesteps)

The 8 C values that are observed in C4 plants are slightly outside the

accordingto the best modelsavailable.Sizesof symbolsreflectrelativeconcentrations of CO2at variousstages.

range allowed by this model, and it is clear that some additional factor is at

330

BioScience Vol. 38 No. 5

This content downloaded from 129.236.31.165 on Wed, 09 Sep 2015 19:33:03 UTC All use subject to JSTOR Terms and Conditions

work. The suggestion has often been made that CO2 is lost from the bundle

Epidermis Internal

Mesophyll

Phloem OBundle

sbhisepahthodsuprhiantgcCeaOrb2uoxpytalakseb(eDyerliebeunloseste

al. 1983, O'Leary 1981, Peisker

1982). Becauseof the largeisotope

SC3

ai s

discriminatioanssociatedwith ribu-

lose bisphosphatecarboxylase,the

CO2thuslost wouldbe enrichedin

13C,leadingto a shift in leaf 813C

towardmorenegativevalues.

cC2

PGAo

c, acidsc leaf organic

Co I leaf organic

e matter

matter

o

0000

0

OO (0O

In order for this mechanism to work, however, the "lost" CO2 must

escapetheleafcompletely-itcannot btheermecaepsotuprhedylbclyelPlsE.GP civarebnotxhyelaasrecihni-

Fiancidgcuiocrradet3ien.tghItmeo ptrheoelratbtaienvsettsmftleuopxdseeisnlstaChvrOaoi2ulafgibhxlateht.SieoinzvedasruoirfoiusnysgmsCtbe4poplshsr(oietnfolcselycuntdrteihnlaegsttiihvse.Secirozeenvsceoerfnstaersratretoipowsn)ss of CO2at variousstages.

tectureof C4 leaves and the high

pefhfoiceinenolcpyoyfruCvOat2cecaarpbtouxryelbaysep,iht oiss- their stomates and engage in direct C3

notclearthatthisispossiblee, special- photosynthesis using ribulose bis-

ly because the

20%-40% of

CO2 loss must total

CO2fixed.Otherim-

phosphate carboxylase (Kluge Ting 1978, Osmond 1978).

and

portant factors may include respira- When CAM plants absorb CO2

tion,translocationa,nddevelopmen- only at night, they have 813C values of

flected in 813C values (Figure 4), and one of the common uses of isotopic studies in CAM plants has been to determine the proportions of the two CO2 fixation pathways and the variation in proportions with changes in

tal effects.Evidencein favorof the CO2 loss hypothesishas been ob-

approximately -11 0/0o et al. 1975, O'Leary

(Nalborczyk 1981). When

tained by Hattersley (1982), who CAM plants engage in only daytime

showedthat813Cvaluesof C4plants photosynthesis, they have 8 3C values

environmental conditions (Osmond et al. 1976). Such isotopic data can also be correlated with measurements of titratable acidity and gas exchange.

varywithbundlesheathpermeability,of approximately -28 0o/0, character-

with the morenegativevaluesbeing istic of C3 plants (Nalborczyk et al.

observedfor plantsin whichperme- 1975).

ability(andtherefore,loss of CO2)is Most often 813C values for CAM

expectedto be highest.

plants are in the range -10 to -20 oo.

ThelimitingpredictionfsorC3and Thus, their 813C values serve to distin-

C4 plants,alongwith the observed guish them from C3 plants. Distinc-

813C values, are shown in Table 2. tion from C4 plants can generally be

Thesevaluesremindus thatwhereas made on physiological grounds (par-

chemical processes are principally ticularly succulence) and on the basis

limitinginC3plants,diffusionisprin- of diurnal variations in malic acid

cipallylimitingin C4plants.

content.

The balance between night and day

CAMplants

CO2 fixation in CAM plants is re-

The leaf succulent Sedum wrightii grows in a variety of environments in the southwestern United States, and study of herbarium specimens reveals that this species shows a greater variation in leaf thickness than most other species in the family. Kalisz and Teeri (1986) have shown that in various populations of S. wrightii, 813C values become more positive, leaves become thicker, and growth rates decrease as an increasing proportion of CO2 is absorbed at night.

Environmental effects have also

Desert plants and other succulents

aabssoCrbraCsOsu2labcyetahneapciadthwmaeytakbnoolwisnm Table2. Predictedand observed813Cvaluesfor C3and C4plants.

(CAM; Kluge and Ting 1978, Os-

Predicted8"3C

mond 1978). At night, these plants

opentheirstomatesandabsorbCO2

Model

C3 plants

C4 plants

in order to synthesize malic acid by use of phosphoenolpyruvate carboxylase and malate dehydrogenase in a process similar to that seen in C4

diffusionlimiting, carboxylationfast ([COz(i)]approacheszero)

-12 0/00

-12 0/o

plants. These plants accumulate high levels of malic acid overnight. During the following morning, stomates close

carboxylationlimiting, diffusionfast ([C02(i)] approaches[C02(ext)])

-38 /o

-1 o0/

and this malic acid is decarboxylated. carboxylationand diffusion

-25 o0/o

-6.5 0/oo

The CO2 thus formed is taken up by ribulose bisphosphate carboxylase in

e([qcuo2a(lil)y]lim=it1i/n2g[CO(ext)]

a process akin to that in the bundle

sheath cells of C4 plants. During the observed 813C afternoon, many CAM plants open

-25 to -29 o/oo

-12 to -16

0/oo

May 1988

331

This content downloaded from 129.236.31.165 on Wed, 09 Sep 2015 19:33:03 UTC All use subject to JSTOR Terms and Conditions

beenstudiedin detailforthe Mexican to CO2for the isotopic analysis.

For Kalanchoe daigremontiana,the

perennialsucculentsCremnophilalin- This isotope fractionationreflects isotope fractionationassociatedwith

guifolia and Sedumgreggii and their only the CO2 fixation process, and malatesynthesischangesfrom -4 o/oo

F1 hybridin an attemptto determine the resultingisotopic signalis free of at 17 oC to 0 oo at 27 oC becauseof

environmentavl ersusgeneticdetermi- complicationsdue to postcarboxyla- an increasein carboxylationcapacity

nants of CAM (Teeriand Gurevitch tion events, import and export pro- coupled to a decrease in stomatal

1984). Largevariationsin b13Ccould cesses, and contributionsfrom day- aperture(Deleenset al. 1985).

be seen in all three populations,re- time CO2 fixation. The isotopic Thereis an interestingdiscrepancy

flectingvariationsin the proportion compositionso obtainedmustbe cor- between these results and results of

of carbon taken up by the CAM rected for contributionsof respired combustionstudies.As noted above,

pathway,as expectedfrom Figure4. carbon, randomizationof malate by combustion studies indicate that

However, it should be noted that fumarase, and residual malate left when CAM plants absorb CO2 only

the curve shown in Figure4 is only over fromthe previousday. The final at night,theleaf "13vCalueis approx-

qualitatively correct. The limiting Vl3C values for pure C3 and pure CAM are probablyvariablewith environmentalconditions,and this vari-

13C value for newly fixed carbon imately-11 o0/. However,studiesof

was -4 to -7 (Deleens et al.

o/. for 1985,

various species Holtum et al.

1983, O'Leary and Osmond 1980),

new carbonincorporatedinto malate

give approximately-7 ferencemay be due to

C0/Oo. 2lTohssisdduirf--

ationhas not beentakeninto account both for growth-chamberplants and ingthe morning;duringmalatedecar-

in studiesto date.

for field-grownplants.

boxylationand CO2reabsorptionby

The combustion studies of 613C Comparison with models devel- ribulose bisphosphate carboxylase,

values of CAM plants reflectthe in- oped in connectionwith C4photosyn- the internal CO2 concentration be-

trinsicisotopefractionationsassociat- thesis (Figure3) revealsthat noctur- comes quite high (Cockburn et al.

ed with the two CO2 fixation path- nal CO2 uptake is controlledjointly 1979), and a small amount of CO2

ways, as well as the proportionsof by diffusion and carboxylation to escapesfrom the leaf. Becauseof the

carbonfixedby eachof the two path- provideoptimumCO2absorptionper largeisotope discriminationassociat-

ways. The first attempt to measure amount of water lost, and this bal- ed with ribulose bisphosphate car-

the two intrinsicfractionationssepa- ance is maintained(by adjustmentof boxylase,this lost CO2is veryheavy,

ratelywas that of Nalborczyket al. (1975), who exposedone set of CAM

stomatalaperture)even in the face of varyingCO2concentrations(Holtum

with +20

0a/.2813LCovsasolufethoifshapeapvroyCxiOm2aitsealy

plants to CO2 only at night and an- et al. 1983, O'Leary and Osmond principal cause of the shift of 8"3C

other set only during the daylight 1980). The partitioning of internal value.

hours.Detailedstudiesof the isotope CO2 between carboxylationand re-

fractionationassociatedwith nocturnOi'aLlCeaOr2yfainxadtiOonsmhaovnedb(e1e9n8m0)a, dwe hboy purifiedmalicacid,the initialproduct

turn to the atmosphereis approximately 1:1. This balance is different

Respiration

from that in C4 plants. Gas-exchange studiesconfirmthis conclusion(Hol-

Tcihpeal8l"yt1h3eCviaslouteoopfeafrlaecatfiornefalteicotnsapsrsion--

of CO2 fixation, and degradedit to tum et al. 1983). Temperatureffects, ciated with photosynthetic carbon

convert carbon-4 of this material which are not visible in combustion fixation and thus provides a useful

(whichcame from atmosphericC02) studies, can be seen in these studies. indicationof the operationof the C3,

C4, and CAM photosyntheticpath-

ways. However, other effects may

% Daytime COa Fixation

also contributeto the overallisotopic

100 28

80

I

I

60

I

40

I

F

1

20

I

I

0

picture, In addition to the possible contribution of CO2 loss from the bundlesheathcells duringC4photo-

synthesisand CO2loss duringdeacid-

ificationin CAM plants, other losses

24

of carbon from leaves may also

contribute.

20

All plants respire,and in so doing, they may lose significantamountsof

CO2. If this CO2 has the same 13C

16 -

valueas the leaffroinwhich it is lost,

thenthisloss is of no consequencefor

12 -

1

I

I

i

I

I

0

20

40

60

the isotope contentof the leaf. How-

ever,if respiredcarbonis depletedin

l

I

13Ccomparedto the leaf,thentheleaf

80

100 will become 13Cenrichedas a result

% Nocturnal COs Fixation

Figure4. Predicte8d 3CvalueforCAMplantsasa functionoftheproportionosf CO2 fixedat nightandduringtheday.

2M. H. O'Learyand I. W. Treichel,1987. Unpublished data, University of Wisconsin, Madison.

332

BioScienceVol.38 No. 5

This content downloaded from 129.236.31.165 on Wed, 09 Sep 2015 19:33:03 UTC All use subject to JSTOR Terms and Conditions

 ................
................

In order to avoid copyright disputes, this page is only a partial summary.

Google Online Preview   Download