Carbon Isotopes in Photosynthesis

Carbon Isotopesin Photosynthesis Fractionation techniques may reveal new aspects of carbon dynamics in plants

Marion H. O'Leary

he efficiencyof photosynthesis Other materials must be converted to continues to interest biochem- Currentstudies include CO2prior to analysis.Plants are ordi- ists, biologists, and plant narily converted to CO2 by combus- physiologists. Scientists interested in finely tuned, carefully tion. Individualcompounds isolated CO2 uptake are concerned about the from plants are sometimesconverted extent to which the uptake rate is controlled isotope to CO2 by chemical or enzymatic limited by such factors as stomatal fractionationsunder degradation. diffusion and the chemistry of the For naturalmaterials (plants, ani- CO2 absorption process. The frac- defined environmental mals, and minerals), R is approxi- tionation of carbon isotopes that oc- mately0.0112, andonly the lastdigit curs during photosynthesis is one of conditions in this ratio varies. For convenience, the most useful techniques for investi- R valuesare generallyconverted to gating the efficiency of CO2 uptake. valuesof 813C, Atmospheric carbon dioxide con- used to study mechanisms of chemical tains approximately 1.1% of the non- (Melander and Saunders 1980) and 1000 813C 3C[R(sample) R ] radioactive isotope carbon-13 and biochemical (Cleland 1982) process- = (standard)- 1 x 1000 98.9% of carbon-12. During photo- es. Isotopes are used in ecology to synthesis, plants discriminate against establish food chains and biological The standard is carbon dioxide ob- C because of small differences in pathways (Fritz and Fontes 1980, tained from a limestone, called PDB, chemical and physical properties im- 1986, Rounick and Winterbourn from the Pee Dee formation in South parted by the difference in mass. This 1986), and isotope studies of tree Carolina (Craig 1957). The units of discrimination can be used to assign are used to recreate cli- 813C are called mil," or A rings past "per 0/0o. plants to various photosynthetic mates (Hughes et al. 1982). Isotope more negative 8 C means more groups. The isotope fractionation studies of plants are related to all or lighter in mass; a more positive also reflects limitations on photosyn- these areas, because their basis is in 813C means more 13C, or heavier. thetic efficiency imposed by the vari- fundamental chemical processes, and Most natural materials have negative ous diffusional and chemical compo- many of their applications are in the 813C values because they contain less nents of CO2 uptake. When analyzed area of ecology (O'Leary 1981, 13C than the standard. The precision in detail, this fractionation provides Troughton 1979, Vogel 1980). Re- of modern mass spectrometers is at information .about water use efficien- cently developed methods are allow- least ?i0.02 %o0, but sample prepara- cy and indicates that different strate- ing biologists to examine in greater tion errors may bring the total repro- gies are needed for improving water- detail the carbon flow in plants. ducibility of measurements on plant use efficiency in different kinds of materials to 10.2 0/0o. Thus, interpre- plants. Measurement of carbon tations based on differences smaller fractionation in than 1 should be made with Isotope simple isotopes o/ physical and chemical processes is caution. well understood and is commonly The "3C content of carbon dioxide is In the absence of industrial activity, usually determined with a mass spec- the 813C value of atmospheric CO2 is This value for the atmo- Marion H. is a in the trometer specially designed for high- -8 0/oo. O'Leary professor measurement of the ratio is more of Chemistryand Biochem- precision R, sphere slowly becoming nega- Departments combustion of fossil istry at the University of Wisconsin in defined by tive due to the Madison 53706. ? 1988 AmericanInsti- fuel (813C for fossil fuel is approxi- R = -30 tute of BiologicalSciences. 3CO2/12CO2 mately %/oo) (Hoefs 1980).

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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 Isotopevalues of plants In the 1950s, Craig (1953, 1954) and Baertschi(1953) measured813C values of a variety of natural materials, including plants (reviewed by O'Leary1981). They found that most plants had 813Cvalues in the range -25 to -35 0o/0. They failed to find large species or environmentaleffects on thesevalues. The plants in these initial studies were C3 plants, which fix principally .. !!i CO2 by the action of the enzyme ...... ii . ! .iiiii~•iiii:i...... ".. i i ?iY :ii•: . ribulose bisphosphate carboxylase. ii iil! iii iii.. :...... The C4 photosynthetic pathway, in ..... xt.::::I : . .i ??'?'':ii ::: :ii: ?::::N which CO2 is initially taken up N ...... i~•?••,•.,,=•iiiiN ii. .i.:. -? .. through carboxylation of phospho- }.ii= X:': ...... was discovered in the ...... enolpyruvate, i??? ? ....: .. ,~i;:;~,:l~...il..i..i...... =i 1960s. Following this discovery, .. ?. Bender see also Smith (1968, 1971; -0 -!1_ -14 -16 -18 -20 -i2 -2.4 -26 -2•8 -30 -32• -34 and Epstein1971) discoveredthat C4 AE13r plants are isotopically distinct from C3 plants. C3 plants have 813Cvalues Figure1. Histogramshowing the distributionof 813Cvalues of plantmaterials. This of approximately -28 0/0o, whereas figureis basedon about1000 analysesperformed in fivedifferent laboratories. C4 plants are approximately -14 0/o In subsequentyears, a number of laboratoriesaround the world made fractionationhas a positive sign when 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 (Deleens et al. 1983, Farquharet al. clear distinction between C3 and C4 and chemicalprocesses).1 1982, O'Leary 1981, Peisker 1982, plants (Figure 1), 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 componentfractionations giv- fore, 13Canalysis has become a stan- isotope fractionations(Cleland 1982, en in Table 1. The overall fraction- dardtest for determiningthe pathway 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, chemicalsource of this difference? dependent processes (chemical reac- but it is not simply the sum of a series tions and transport) and in of individualfractionations-instead, Fractionationsin chemical and equilibriumprocesses (chemical equi- the fractionation mostly reflects the libria, dissolution, and phase rate-limitingstep or steps (i.e., those physicalprocesses changes), and both are importantin with the highest resistivity).As a step Plantscontain less '3Cthan the atmo- plants. Table 1 shows isotope frac- becomes more limiting, the observed sphere because the physical and tionations for processes of impor- fractionation approaches the frac- chemical processes involved in CO2 tance in photosynthesis. tionation for that step. uptakediscriminate against 13C. This The importantsteps in CO2 uptake discriminationoccurs because '3C is Theory of isotope in C3plants are shown in Figure2. In heavier than 12C and forms slightly fractionationin the first step, external CO2 is trans- strongerchemical bonds. In addition, plants ported through the boundary layer diffusionof '3CO2 is slower than that The principalfactor affectingthe iso- and the stomata into the internalgas of 12CO2because of this difference in topic compositions of leaves is the space. This process is always to some mass. For the conversion of com- isotope fractionation accompanying extent reversible.Internal CO2 then pound A into compound B, the iso CO2uptake. Followinginitial sugges- dissolvesin the cell sap and diffusesto tope fractionation is defined by tions of Craig (1953), Smith and Ep- the chloroplast,where carboxylation stein (1971), and others occurs. Because the - (O'Leary carboxylation [813C(A) 8'3C(B)] 1981), models for plant isotope frac- step is irreversible,steps subsequent 1 + 813C(A)/1000 tionation have focused on the physi- to carboxylationare not importantin cal and chemicalprocesses accompa- determiningthe isotope fractionation. This fractionation has units of o. nying CO2 uptake, including Both dissolving and diffusion show To avoid confusion with ordinary diffusion, dissolution, and the car- small isotope fractionations(Table 1), 813C values (which represent isotopic but the largest fractionation is that compositions, rather than fraction- 'However,note that some workersin the field connectedwith carboxylation (29 o/o). ations), we call this value A8. The use the opposite sign convention. It is generallyassumed that dissolu-

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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 breeda that would take up CO2 Reference plant Process A, 0/00oo* more rapidly without sacrificingwa- ter-useefficiency. The alternativepos- Equilibria diffusive resist- Solubilityof CO2 in water 1.1 O'Leary1984 sibility, decreasing Hydrationof CO2 -9.0 Mook et al. 1974 ance, has only a very limitedpotential for increasing CO2 uptake, and this Transportprocessest increasewould come at a substantial CO2diffusion in air 4.4 O'Leary1981 cost in water-use As we CO2diffusion in aqueous solution 0.7 O'Leary1984 efficiency. will see below, the situation in C4 Chemicalprocesses plants is different. Spontaneoushydration of CO2 6.9 Marlierand O'Leary1984 The C4 pathway involves sequen- Carbonicanhydrase catalyzed 1.1 Panethand 1985 tial operationof two carboxylasesys- hydrationof CO2 O'Leary tems enters Phosphoenolpyruvatecarboxylase- (Figure3). CO2 initially catalyzedreaction of HC03- the leaf through the stomata and is with phosphoenolpyruvate 2.0 O'Learyet al. 1981 taken up by phosphoenolpyruvate Ribulosebisphosphate carboxylase- carboxylase in the mesophyll cells. catalyzedreaction of CO2 The of this is with ribulosebisphosphate 29.0 Roeske and O'Leary1984 product carboxylation converted to either malate or aspar- *Positivevalues in this table indicatethat the productis depletedin 13Ccompared with the starting tate and is transportedto the bundle state;negative values indicateenrichment. sheath cells, where it is cleaved to tPredictedvalue. This numberhas not been measured. CO2 and some other compound.The CO2 thus produced is taken up by ribulose bisphosphate carboxylase. tion and liquid-phase diffusion are limited extreme. More quantitative Although the latter enzyme shows a rapid, but good evidence for this is analysis indicates that the carboxyl- large isotope fractionation(Table 1), lacking. If stomatal diffusion is rapid ation resistance is higher than the the effectsof this fractionationare not (stomatal resistance is low) and car- diffusional resistance by up to a factor seen in C4 plants because this step is boxylation is limiting, the predicted of two; diffusion of internal CO2 preceded by an irreversiblestep, the isotope fractionation is 28 %o/,and the back to the outside is faster than carboxylation of phosphoenolpyru- predicted leaf 813Cvalue is -36 o/o. If carboxylation by up to a factor of vate. diffusion is slow (stomatal resistance is two. As in C3 carboxylation,dissolution high), the predicted isotope fraction- Thus, CO2 uptake in C3 plants is and liquid-phasediffusion of CO2are ation is 4 %o/ and the predicted leaf limited more by the rate of carboxyl- assumed to be fast. Carbonic anhy- 813C value is -12 0o/0. To the extent ation of ribulose bisphosphate than drase is present in C4 plants (Reed that diffusion and carboxylation by diffusion. This finding has impor- and Graham 1981); thus, CO2 and jointly limit the rate, the 813C value tant implications for plant breeding. HC03- are expected to be in equilib- will 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 car- for isotope fractionationare stomatal for C3 plants cluster near -28 0/0o, boxylase (either because of increased diffusion and carboxylationof phos- which is nearer to the carboxylation- enzyme activity or because of reduced phoenolpyruvate.If diffusionis facile and carboxylation is limiting, then the predictedleaf 813C is -1 o/oo.On the other hand, if diffusionis limiting Atmosphere Epidermis Internal Phloem and carboxylation is facile, the pre- air space dicted 813C is -12 Mesophy 0/o. 00 Observed813C values for 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 steady-state photosynthesis, and the diffusion is more than car- kq RuBO OG 0 limiting leaf boxylation. Unlike the situation in C3 organic O plants, further improvements in the matter 0 efficiency of C4 plants cannot come about through increases in carboxylation capacity. The 8 C values that are observed Figure 2. Important steps in CO2 fixation during C3 photosynthesis. Sizes of arrows indicate the relative fluxes through the various steps (includingthe reverse steps) in C4 plants are slightly outside the accordingto the best models available.Sizes of symbolsreflect relative concentrations range allowed by this model, and it is of CO2 at various stages. clear that some additional factor is at

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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 Epidermis Phloem Internal Mesophyll made that CO2 is lost from the bundle OBundle sheathduring CO2 uptake by ribulose bisphosphatecarboxylase (Deleens et al. 1983, O'Leary 1981, Peisker ai s cC2 PGAo 1982). Becauseof the large isotope SC3 with ribu- discriminationassociated leaf 00 lose the Co I bisphosphatecarboxylase, e organic 0 CO2thus lost would be enrichedin leaf matter OO in organic 13C,leading to a shift leaf 813C c,matter acidsc o (0O towardmore negative values. In order for this mechanism to Figure3. Importantsteps in CO2 fixation during C4 photosynthesis.Sizes of arrows work, however, the "lost" CO2 must indicate the relative fluxes through the various steps (includingthe reverse steps) escapethe leaf completely-itcannot accordingto the best models available.Sizes of symbolsreflect relative concentrations be recaptured by PEP carboxylase in of CO2 at various stages. the mesophyllcells. Giventhe archi- tecture of C4 leaves and the high efficiencyof CO2 captureby phosphoenolpyruvatecarboxylase, it is their stomates and engage in direct C3 flected in 813C values (Figure 4), and not clearthat this is possible,especial- photosynthesis using ribulose bis- one of the common uses of isotopic has to ly because the CO2 loss must total phosphate carboxylase (Kluge and studies in CAM plants been 20%-40% of CO2fixed. Otherim- Ting 1978, Osmond 1978). determine the proportions of the two portant factors may include respira- When CAM plants absorb CO2 CO2 fixation pathways and the variation, translocation,and developmen- only at night, they have 813C values of tion in proportions with changes in conditions tal effects.Evidence in favor of the approximately -11 0/0o (Nalborczyk environmental (Osmond CO2 loss hypothesishas been ob- et al. 1975, O'Leary 1981). When et al. 1976). Such isotopic data can with measurements tained by Hattersley (1982), who CAM plants engage in only daytime also be correlated showedthat 813C values of C4plants photosynthesis, they have 8 3C values of titratable acidity and gas exchange. character- The leaf succulent Sedum varywith bundle sheath permeability, of approximately -28 0o/0, wrightii with the morenegative values being istic of C3 plants (Nalborczyk et al. grows in a variety of environments in observedfor plantsin whichperme- 1975). the southwestern United States, and ability (and therefore,loss of CO2)is Most often 813C values for CAM study of herbarium specimens reveals expectedto be highest. plants are in the range -10 to -20 oo. that this species shows a greater vari- Thelimiting predictions for C3and Thus, their 813C values serve to distin- ation in leaf thickness than most oth- C4 plants, along with the observed guish them from C3 plants. Distinc- er species in the family. Kalisz and 813C values, are shown in Table 2. tion from C4 plants can generally be Teeri (1986) have shown that in vari- Thesevalues remind us that whereas made on physiological grounds (par- ous populations of S. wrightii, 813C chemical processes are principally ticularly succulence) and on the basis values become more positive, leaves limitingin C3plants, diffusion is prin- of diurnal variations in malic acid become thicker, and growth rates de- cipallylimiting in C4plants. content. crease as an increasing proportion of The balance between night and day CO2 is absorbed at night. fixation in CAM is re- Environmental effects have also CAMplants CO2 plants Desert plants and other succulents absorb CO2 by the pathway known Table 2. Predictedand observed813C values for C3 and C4 plants. as Crassulaceanacid metabolism (CAM; Kluge and Ting 1978, Os- Predicted8"3C mond 1978). At these plants night, C4 opentheir stomates and absorbCO2 Model C3 plants plants malic acid in order to synthesize by diffusionlimiting, use of carbox- phosphoenolpyruvate carboxylationfast -12 0/00 -12 0/o and malate in a ylase dehydrogenase ([COz(i)]approaches zero) process similar to that seen in C4 These plants accumulate high carboxylationlimiting, plants. diffusion -38 /o -1 levels of malic acid overnight. During fast o0/ ([C02(i)] approaches[C02(ext)]) the following morning, stomates close and this malic acid is decarboxylated. carboxylationand diffusion -25 o0/o -6.5 0/oo The CO2 thus formed is taken up by equallylimiting = 1/2 ribulose bisphosphate carboxylase in ([co2(i)] [CO(ext)] a process akin to that in the bundle of the -25 to -29 o/oo -12 to -16 sheath cells C4 plants. During observed 813C 0/oo afternoon, many CAM plants open

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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 been studiedin detail for the Mexican to CO2 for the isotopic analysis. For Kalanchoe daigremontiana,the perennialsucculents Cremnophila lin- This isotope fractionation reflects isotope fractionationassociated with guifolia and Sedum greggii and their only the CO2 fixation process, and malatesynthesis changes from -4 o/oo F1 hybridin an attemptto determine the resultingisotopic signal is free of at 17 oC to 0 oo at 27 oC becauseof environmentalversus genetic determi- complications due to postcarboxyla- an increasein carboxylationcapacity nants of CAM (Teeriand Gurevitch tion events, import and export pro- coupled to a decrease in stomatal 1984). Largevariations in b13Ccould cesses, and contributions from day- aperture(Deleens et al. 1985). be seen in all three populations, re- time CO2 fixation. The isotopic There is an interestingdiscrepancy flecting variations in the proportion compositionso obtainedmust be cor- between these results and results of of carbon taken up by the CAM rected for contributions of respired combustion studies. As noted above, pathway, as expected from 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 Figure 4 is only over from the previousday. The final at night, the leaf "13Cvalue is approx- qualitatively correct. The limiting 13C value for newly fixed carbon imately -11 o0/. However, studiesof Vl3C values for pure C3 and pure was -4 to -7 o/. for various species new carbon incorporatedinto malate CAM are probablyvariable with en- (Deleens et al. 1985, Holtum et al. give approximately-7 0/o. This dif- vironmentalconditions, and this vari- 1983, O'Leary and Osmond 1980), ferencemay be due to CO2 loss dur- ation has not been taken into account both for growth-chamberplants and ing the morning;during malate decar- in studies to date. for field-grownplants. boxylation and CO2 reabsorptionby The combustion studies of 613C Comparison with models devel- ribulose bisphosphate carboxylase, values of CAM plants reflect the in- oped in connectionwith C4 photosyn- the internal CO2 concentration be- trinsicisotope fractionationsassociat- thesis (Figure3) reveals that noctur- comes quite high (Cockburn et al. ed with the two CO2 fixation path- nal CO2 uptake is controlled jointly 1979), and a small amount of CO2 ways, as well as the proportions of by diffusion and carboxylation to escapes from the leaf. Becauseof the carbonfixed by each of the two path- provideoptimum CO2 absorption per large isotope discriminationassociat- ways. The first attempt to measure amount of water lost, and this bal- ed with ribulose bisphosphate car- the two intrinsicfractionations sepa- ance is maintained(by adjustmentof boxylase, this lost CO2 is very heavy, rately was that of Nalborczyk et al. stomatal aperture)even in the face of with a 813Cvalue of approximately (1975), who exposed one set of CAM varying CO2 concentrations(Holtum +20 0/.2 Lossof thisheavy CO2 is a 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. Detailed studies of the isotope CO2 between carboxylation and re- fractionationassociated with noctur- turn to the atmosphere is approxi- Respiration nialCO2 fixation have been made by mately 1:1. This balance is different O'Leary and Osmond (1980), who from that in C4 plants. Gas-exchange The 8"13Cvalue of a leaf reflectsprin- purifiedmalic acid, the initialproduct studies confirmthis conclusion (Hol- cipally the isotope fractionationasso- of CO2 fixation, and degraded it to tum et al. 1983). Temperatureeffects, ciated with photosynthetic carbon convert carbon-4 of this material which are not visible in combustion fixation and thus provides a useful (which came from atmosphericC02) studies, can be seen in these studies. indicationof the operationof the C3, C4, and CAM photosynthetic pathways. However, other effects may % Daytime COa Fixation also contributeto the overall isotopic picture, In addition to the possible 100 80 60 40 20 0 F contribution of CO2 loss from the I I I I 1 I I bundle sheath cells 28 during C4 photo- synthesisand CO2loss duringdeacid- ification in CAM plants, other losses 24 of carbon from leaves may also contribute. All plants respire,and in so doing, 20 they may lose significantamounts of CO2. If this CO2 has the same 13C 16 - value as the leaf froin which it is lost, then this loss is of no consequencefor the content of the leaf. How- - isotope 12 ever, if respiredcarbon is depletedin 1 I I i I I l I 13Ccompared to the leaf, then the leaf 0 20 40 60 80 100 will become 13Cenriched as a result % Nocturnal COs Fixation 2M. H. O'Learyand I. W. Treichel,1987. Un- Figure4. Predicted8 3Cvalue for CAMplants as a functionof theproportions of CO2 published data, University of Wisconsin, fixedat nightand during the day. Madison.

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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 of respiration.Unfortunately, it is not Aquaticplants and algae Water-useefficiency possibleto measurethe isotopicconsequences of respiration during Aquaticplant 813Cvalues are more With the realizationthat isotope frac- steady-statephotosynthesis, because difficultto understandthan those of tionation in plants is a reflectionof respiration-derivedCO2 cannot be terrestrialplants becauseof the im- the balance between diffusional and separatedfrom other CO2. Measure- portanceof diffusionin photosynthe- chemical processes, Farquharet al. mentmade under nonphotosynthetic sis of aquaticplants. Diffusion of CO2 (1982) suggested that isotopic com- conditionsmay not give an accurate dissolvedin wateris ordersof magni- positions of plants should correlate indicationof eitherthe quantityor tudeslower than diffusion of CO2in with internal CO2 concentration, the isotopicnature of normalrespira- air. Not surprisingly,in aquatic [CO2(i)],according to the equation tion, especiallyfor photorespiration,plants diffusion is often limiting,so whichis associatedwith CO2uptake the isotope fractionationis small, A5 = a + [b - a][CO2(i)]/[C02(ext)] by ribulosebisphosphate carboxylase even for C3plants. Althoughthe 813C in C3plants. value of CO2 in air is relativelycon- wherea is theisotope fractionation in data to date do not the value of dissolved Experimental stant, 813C CO2 CO2diffusion (4.4 %0) and b is the providea clearanswer to thequestion is variable, and dissolved quite CO2 carboxylationfractionation (29 o0/0 of whether respirationfractionates differsfrom dissolved HCO3- by ap- for C3plants), [C02(i)] is the internal carbon isotopes (O'Leary 1981), proximately 9 %/o. gas-phaseCO2 concentration,and muchless the questionof whetherthe Recent studies have attempted to [CO2(ext)]is the externalCO2 con- isotopicconsequences of respiration account quantitativelyfor these fac- centration.Because gas exchange also may changewith speciesor environ- tors. Osmondet al. (1981) surveyeda provides a value of [C02(i)], this ment.The lack of informationon this varietyof aquaticplants and simulta- equation provides an important issue becomesparticularly trouble- neously made measurementsof the bridgebetween the two methodsand some in such quantitativestudies as isotopic composition of dissolved in- helpsto validatethe theoreticalmod- water-useefficiency. organic carbon. In rapidly flowing els for isotopefractionation. streams in which mixing was good Severalstudies have now shown Longer-termeffects and carbon was readily available, thatthe isotopicmethod and the gas- plants often showed isotope fraction- exchangemethod give consistentval- Accordinto thispicture of CO2fixa- ations similar to those of terrestrial ues of [CO2(i)].Isotopic analysis of tion,the C contentof leavesreflects C3plants, indicatingthat neithermix- malateand independent gas-exchange the isotope fractionationassociated ing nor diffusionwas rate limiting.In measurementsboth show that in K. with CO2uptake, with perhaps small sluggishwater, isotope fractionations daigremontianachanging the external modificationsas a resultof respira- were small, becausediffusion is limit- CO2 concentrationover the range toryprocesses. This snapshot view of ing in CO2 uptake. Similar results from 100 to 1000 ppm does not CO2fixation is not correct. have been Raven et al. entirely reported by change the ratio [CO2(i)]/[CO2(ext)], The isotopiccontent of a leaf pro- (1982). in spite of a substantialchange in vides an integratedview of carbon Recent identificationof the aquatic CO2 uptakerate. Instead,stomatal gain and loss over the whole history plants Isoetes lacustrisL. and Isoetes apertureis adjustedto keepthis ratio of theleaf. Although most leaf carbon howellii as CAM species is based on constantand optimizewater-use effi- appearsto be introduceddirectly by criteriaother than isotopic composi- ciency(Holtum et al. 1983). photosynthesiswithin the leaf, total tion (Keeley and Busch 1984, Rich- Isotopic compositions of halo- carbonalso includescarbon that was ardson et al. 1984). The isotope frac- phytesbecome more positive by up to fromelsewhere in the imported plant tionation is small, because of limited 10 0%/0 with increasingsalinity (Guy duringearly stages of leaf develop- availability of CO2. The occurrence et al. 1980, Nealeset al. 1983). The ment and excludescarbon that has of CAM in these cases appearsto be a aboveequation suggests that thisob- beenlost fromthe leaf by respiratory responseto limited CO2 availability. servationmight be due to a decrease processesand by export to the re- Likewise,algae, which take up CO2 in [CO2(i)], and gas-exchangestudies mainderof the plant. An accurate by means of ribulose bisphosphate are consistentwith that suggestion model of the isotopic compositionof carboxylase, show isotope fraction- (Guy and Reid 1986). Most of the a leaf must includeall these processes. ations that vary with environmental change in photosynthetic rate with Import and export processes are CO2 concentration (Kerby and Raven salinityis due to a changein stomatal poorly understood and are often 1985). In laboratory experiments, conductance. In spinach, increasing in slighted developmentof quantita- small isotope fractionations (some- salinityto 200 mM NaCI shifted813C tive isotopic models. These processes times approaching 0 %ooo) are ob- to more positive values by 5 can be to the extent that 0/0, ignoredonly served when CO2 is limiting, and indicatinga decreasein [CO2(i)],con- the carbon gained and lost is isotopi- fractionations of 20 %o or more are sistent with results of gas exchange cally the same as whole leaf carbon. observed when CO2 concentration is studies (Downton et al. 1985). Simi- Studies of the changes in isotopic high. In field studies, isotope fraction- larly, the flaccamutant of tomato has content during development are in- ations may vary over this entire a high stomatal conductance,a high conclusivewith regardto the question range, with most of the variation pre- [CO2(i)],and an isotope fractionation of whether these effects are sumably being due to variations in 1-2 %0/ larger than wild type (Brad- significant. CO2 availability. ford et al. 1983).

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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 Following the correlation of 813C al measurements is approximately light intensity increasedfrom 100 to with [C02(i)] came the realization +0.2 %0/o.The remainderof the vari- 500 the observed that for C3plants, the isotopiccom- abilitymust be due to individualdif- 813ClEinsteins'm-2"s-1,value became more positive by positionshould correlate with water- ferencesamong speciesor environ- 2 %o. No such trend is apparentin use efficiency.More water-efficientmental effects. similarstudies with C4 plants.8These plantsshould have more positive 813C The observedvariation becomes studies provide an importantcontrol values. Recent studies from Far- smallerwhen measurementsare re- for attemptsto use 8•1C of tree rings quhar'sgroup demonstrate that this strictedto a singlespecies and a single to constructpast climates (Hugheset correlationcan be usedto screencul- environment.For soybean (a C3 al. 1982). tivarsfor water-useefficiency (Con- plant),combustion analysis of 25 cul- don et al. 1987, Farquharand Rich- tivars grown side-by-sideshowed a Short-term measurements ards 1984). In wheat, various standarddeviation of +0.36 0/o.5 A cultivarsdiffering in water-useeffi- similarexperiment with 120 cultivars As noted above, combustion studies ciency from 2.0 to 3.7 mmol C/mol of Zea mays(a C4plant) grown side- integrateover the entire life of a leaf, H20 variedin isotope fractionation by-side showed 813C = -11.6 and it is likely that useful short-term from22 to 19 o0/.3 This methodis +0.4 0/00 in a system in which the informationis lost in this integration. fasterand cheaper than other screen- experimentalerror for multiplemea- Methods are currently being devel- ingmethods and thus has tremendous surements of a single sample was oped that permitmeasurement of iso- a short economic implications for plant -0.3 o0/.6 tope fractionationduring peri- breeding.However, it shouldbe not- Interestingly,differences in ploidy od (1-12 hr) in the life of a plant. ed that the method is probablypracti- level do not seem to translate into These methods may provide a new cal only when comparinga single differencesin 813C in either C3 or C4 window on environmentaland other species grown in a single environ- plants. In Z. mays, a tetraploidstrain short-termeffects. ment. It is unlikely that the same had a 813C value only 0.3 0/%omore The first short-term method methodcan be used for C4 plants negativethan the mean value for 120 (O'Leary and Osmond 1980) made because of the low [C02(i)] and the other strains of the same species. In use of malate accumulatedovernight smallerrange of 813C values that alfalfa, genetically identical diploid, in CAM plants. Studies of environ- would result from changes in tetraploid, and octaploid strains dif- mental and species variations have [C02(i)]. fered by less than 0.3 %0/o.7Thus, the demonstratedthat the method gives ratio [C02(i)]/[CO2(ext)]seems to be useful indications of environmental Fine the measurements preservedacross ploidy levels, in spite effects on stomatal apertureand car- tuning of significant biochemical and ana- boxylation capacity. The use of isotopicmethods for dis- tomical differences. Other short-term methods make tinguishingamong C3, C4, and CAM In addition, there are small organ use of the changein isotopic composi- plantsis now well established.This and biochemicaldifferences within a tion of atmosphericCO2 duringpho- proceduredepends on large differ- plant: Nonphotosynthetic tissues tosynthesis in a closed system. This encesin 813Cvalues, which are easily (e.g., stem and roots) are generally2- approach can be applied either in a measured and easily interpreted. 4 0/%o more positive than leaves flowing system like that used in gas However,there is currentlyconsider- (O'Leary 1981). Individualchemical exchange (Evanset al. 1986) or in a ableinterest in the questionof wheth- components may also differ. Lipids, closed, nonflowing system (O'Leary er smallvariations in 813Cvalues (of in particular,are severalper mil more et al. 1986). The advantage of the theorder of 1-4 o/oo)may be of usein negativethan other materials,appar- flowing system is that measurements biology. The water-use efficiency ently as a result of the isotope frac- are made at steady-state, and the studiescited aboveare one example tionation associated with the decar- propertiesof the plant are likely to be of this approach. boxylation of pyruvic acid (O'Leary quite reproducible.The disadvantage Combustionanalyses of largecol- 1981). is that the isotopic change in the CO2 lectionsof C3 plantsgrown under a Recent studies indicate that there streamdue to photosynthesisis quite varietyof conditionsand analyzedin are small environmental effects on small, and thus the calculatedisotope a number of different laboratories C13Cvalues in C3 plants. When toma- fractionation (which may be 5-10 give a rangeof 513Cvalues. Although to plants were grown in a controlled- times larger than the observed an adequatestatistical analysis of all environment chamber at varying light change) is subject to a large uncer- the data in the literaturehas not been levels and temperatures, systematic tainty. Larger isotopic changes are done, comparison of large data sets trends were found in 513C values. As obtained in the second method, but from a numberof laboratoriesgives a growth temperature was increased results are rendered uncertain by to the in CO2 in the mean for all C3 plants of -27.1+2.0 ooo from 17 C 32? C, 513C value changes concentration and for C4 plants of -13.1+ 1.2 0/,.4 became more negative by 3 oo. As atmosphere (and consequently The reproducibility of individu- changes in stomatal aperture) that sJ. Teeri and J. Gurevich, 1986. Unpublished occur during the experiment. Both data, University of Chicago, Chicago. methods give results consistent with 3Note that the numbers given here are isotope 6J. Holtum, D. Weber, and M. H. O'Leary, data from the combustion method. fractionations, not isotopic compositions. 1983. Unpublished data, University of Wiscon- 4M. H. O'Leary, 1987. Unpublished data, Uni- sin, Madison. versity of Wisconsin, Madison. 7See footnote 2. 8Seefootnote 2.

334 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 These methods will be useful for Farquhar. 1987. Carbon isotope discrimina- and growth in Sedum wrightii. Ecology 67: studying a variety of environmental tion is positively correlated with grain yield 20-26. and effects. and dry matter production in field-grown Keeley, J. E., and G. Busch. 1984. Carbon species wheat. Crop Sci. 27: 996-1001. assimilation characteristics of the aquatic Craig, H. 1953. The geochemistry of the stable CAM plants, Isoetes howellii. Plant Physiol. carbon isotopes. Geochim. Cosmochim. 76: 525-530. Conclusions Acta 3: 53-92. Kerby, N. W., and J. A. Raven. 1985. Trans- The first of studies of carbon 1954. Carbon-13 in plants and the port and fixation of inorganic carbon by phase relationships between carbon-13 and car- marine algae. Adv. Bot. Res. 11: 71-123. isotope fractionation in plants took bon-14 variations in nature. J. Geol. 62: Kluge, M., and I. Ting. 1978. Crassulacean advantage of the large differences 115-149. Acid Metabolism. Springer-Verlag, New among C3, C4, and CAM plants. Dur- 1957. Isotopic standards for carbon York. this the method and oxygen and correction factors for mass Marlier, J. F., and M. H. O'Leary. 1984. Car- ing period isotopic spectrometric analysis of carbon dioxide. bon kinetic isotope effects on the hydration became a standardmethod by which Geochim.Cosmochim. Acta 12: 133-149. of carbon dioxide and the dehydration of new species could be placed in these Deleens, E., A. Ferhi, and 0. Queiroz. 1983. bicarbonateion. J. Am. Chem. Soc. 106: categories.The currentphase involves Carbon isotope fractionation by plants using 5054-5057. finely tuned, carefully controlled the C4 pathway. Physiol. Veg. 21: 897-905. Melander, L., and W. H. Saunders. 1980. Reac- studiesof fractionationunder Deleens, E., I. Treichel, and M. H. O'Leary. tion Rates of Isotopic Molecules. Wiley isotope 1985. Temperature dependence of carbon Interscience, New York. defined environmental conditions. isotope fractionation in CAM plants. Plant Mook, W. G., J. C. Bommerson, and W.H. These studies are providingdetails on Physiol. 79: 202-206. Staverman. 1974. Carbon isotope fraction- carbon flow Downton, W. J. S., W. J. R. Grant, and S. P. ation between dissolved bicarbonate and during photosynthesis Robinson. and stoma- carbon dioxide. Earth Plan. Sci. and on how various steps contribute 1985. Photosynthetic gaseous in tal response of spinach leaves to salt stress. Lett. 22: 169-175. to the overall rate of CO2 uptake Plant Physiol. 77: 85-88. Nalborczyk, I., L. J. LaCrois, and R. D. Hill. plants. Wecan anticipatethat studies Evans, J. R., T. D. Sharkey, J. A. Berry, and 1975. Environmental influences on light and in the future will reveal many new G. D. Farquhar. 1986. Isotope discrimina- dark CO2 fixation by Kalanchoe daigremon- aspects of both long-termand short- tion measured concurrently with gas ex- tiana. Can.J. Bot. 53: 1132-1138. term of carbon movements change to investigate CO2 diffusion in leaves Neales, T. F.E,M. S. Fraser, and Z. Roksandic. dynamics of higherplants. Aust. J. Plant Physiol. 13: 1983. Carbon isotope composition of the in living systems. 281-292. halophyte Disphyma clavellatum (Haw.) Farquhar, G. D., M. H. O'Leary, and J. A. chinnock (Aizoaceae), as affected by salinity. Berry. 1982. On the relationship between Aust. J. Plant Physiol. 10: 437-444. Acknowledgments carbon isotope discrimination and the inter- O'Leary, M. H. 1981. Carbon isotope fraction- cellular carbon dioxide concentration in ation in plants. Phytochemistry 20: 553- Work at the Universityof Wisconsin leaves. Aust. J. Plant Physiol. 9: 121-137. 567. was supportedby the US Department Farquhar, G. D., and R. A. Richards. 1984. 1984. Measurement of the isotope of Energy,the National ScienceFoun- Isotopic composition of plant carbon corre- fractionation associated with diffusion of dation, and the US Department of lates with water-use efficiency of wheat ge- carbon dioxide in aqueous solution. J. Phys. to C. notypes. Aust. J. Plant Physiol. 11: 539- Chem. 88: 823-825. Agriculture.I am grateful Barry 552. O'Leary, M. H., and C. B. Osmond. 1980. Osmond, Graham Farquhar, and Fritz, P., and J. C. Fontes. 1980. Handbook of Diffusional contribution to carbon isotope Tom Sharkey for helpful comments EnvironmentalIsotope Geochemistry,vol. fractionation during dark CO2 fixation in and to James Teeri for permissionto 1, Elsevier Scientific Publ., Amsterdam, CAM plants. Plant Physiol. 66: 931-934. results. Netherlands. O'Leary, M. H., J. E. Rife, and J. D. Slater. quote unpublished . 1986. Handbook of Environmental 1981. 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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 discriminationin C4 plants. Photosynthetica 16: 533-541. . 1984. Modellvorstellungenzur kohlen- stoff-isotopendiskriminierungbei der photo- synthesevon C3- und C4-pflanzen.Kulturp- flanze32: 35-65. . 1985. Modellingcarbon metabolism in C3-C4intermediate species 2. Carbonisotope discrimination.Photosynthetica 19: 300- 311. Raven,J., J. Beardall,and H. Griffiths.1982. Inorganic C-sources for Lemanea, Clado- phora and Ranunculus in a fast-flowing stream:measurements of gas exchangeand of carbon isotope ratio and their ecological implications.Oecologia 53: 68-78. Reed, M. L., and D. Graham.1981. Carbonic anhydrasein plants: distribution,properties and possible physiologicalroles. Prog. Phy- tochem. 7: 47-94. Richardson,K., H. Griffiths,M. L. Reed,J. A. Raven,and N. M. Griffiths.1984. Inorganic carbon assimilationin the Isoetids, Isoetes Rememberwhen you said you'ddo something lacustrisL. and Lobeliadortmanna L. Oeco- if had the logia 61: 115-121. only you power? Roeske,C. A., and M. H. O'Leary.1984. Car- bon isotope effectson the enzyme-catalyzed carboxylationof ribulosebisphosphate. Bio- chemistry23: 6275-6284. Rounick,J. S., and M. J. Winterbourn.1986. Stable carbon isotopes and carbon flow in ecosystems.BioScience 36: 171-177. Smith,B. N., and S. Epstein.1971. Two categories of '3C/12Cratios for higherplants. Plant Physiol.47: 380-384. Teeri,J. A., and J. Gurevitch.1984. Environ- mental and genetic control of Crassulacean acid metabolismin two Crassulaceanspecies and an F1 hybridwith differingbiomass b83C values. Plant Cell Environ.7: 589-596. Troughton,J. H. 1979. 8 as an indicator of carboxylationreactions. Pages 140-149 in M. Gibbs and E. Latzko,eds. Encyclopedia of Plant Physiology, New Series, vol. 6. Springer-Verlag,Berlin, FRG. Vogel,J. C. 1980. Fractionationof the carbon isotopes duringphotosynthesis. Sitzungsber. HeidelbergerAkad. Wiss. 3: 111-135. Well,now you do.

Nowthat you're sitting in a bigchair, don't let your convictionsgo flabby.They're still there. And now you're in theposition to makegood on them,by giving to the United NegroCollege Fund. Bykeeping tuitions low, we helpsend thousands of deservingstudents to 42 private,predominantly black colleges.Students who will go on to meetthe ever increasing demandfor well rounded, well educated employees in today'scorporations. Please.Help your corporation give something back to society.Give generously to theUnited Negro College Fund, 500 East62nd Street, New York, N.Y. 10021. It'sthe contribution you've always wanted to make. Give to the United Negro College Fund A mindis a terriblething to waste. W 336 APublicThisPubbesionSwt•,olf BioScience Vol. 38 No. 5

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