Oxygen and Hydrogen Isotope Fractionation - Plant Physiology

Plant Physiol. (1990) 93, 325-3320032-0889/90/93/0325/08/$01 .00/0

Received for publication September 25, 1989and in revised form January 16, 1990

Oxygen and Hydrogen Isotope Fractionation duringCellulose Metabolism in Lemna gibba L."

Dan Yakir*2 and Michael J. DeNiro3Department of Earth and Space Science, University of California Los Angeles, Los Angeles, California 90024

ABSTRACT

Lemna gibba L. B3 was grown under heterotrophic, photohet-erotrophic, and autotrophic conditions in water having a varietyof hydrogen and oxygen isotopic compositions. The slopes of thelinear regression lines between the isotopic composition of waterand leaf cellulose indicated that under the three growth conditionsabout 40, 70, and 100% of oxygens and carbon-bound hydrogensof cellulose exchanged with those of water prior to celluloseformation. Using the equations of the linear relationships, weestimated the overall fractionation factors between water and theexchanged oxygen and carbon bound-hydrogen of cellulose. Atleast two very different isotope effects must determine the hy-drogen isotopic composition of Lemna cellulose. One reflects thephotosynthetic reduction of NADP, while the second reflectsexchange reactions that occur subsequent to NADP reduction.Oxygen isotopic composition of cellulose apparently is deter-mined by a single type of exchange reaction with water. Underdifferent growth conditions, variations in metabolic fluxes affectthe hydrogen isotopic composition of cellulose by influencing theextent to which the two isotope effects mentioned above arerecorded. The oxygen isotopic composition of cellulose is notaffected by such changes in growth conditions.

large isotopic fractionations in going from leaf water to or-ganic matter (10, 14, 28). It is essential that the relationshipsbetween the isotopic composition of leaf water and organicmatter be thoroughly understood to interpret isotopic ratiosof organic matter. To date, only a limited number of studiesdirectly addressed this question (14, 28). In these studies eitheroxygen or hydrogen isotopes were investigated. A combinedisotopic study of the two elements would be very useful. Thisis because oxygen and hydrogen isotopic ratios of water, theultimate source of plant isotopic signals, are highly correlatedwith each other (12, 26), while the additional oxygen andhydrogen isotope effects associated with biochemistry are not(14, 28). Thus, changes in oxygen and hydrogen isotopiccomposition of organic matter that are correlated may indi-cate source effects while noncorrelated changes would indicateadditional biochemical effects.

Isotopic fractionations between water and cellulose, theorganic fraction that is typically analyzed, can be expressed:

acellulose - water = Rcellulose/Rwater (1)where R denotes the ratio between the heavy and the lightisotopes, i.e. 180/160 or D/H. These ratios, in cellulose orwater samples, are usually reported with reference to a stand-ard, in this case SMOW4, using the 6 notation,

The use of natural variations in the ratios of stable isotopesin plant biology has grown in recent years (24). Althoughcarbon isotopes have been the main focus of attention (22),oxygen and hydrogen isotopes have much to offer. The dif-ferences in isotopic composition between ground water andleaf water, caused by the effects of transpiration on leaf water,are the basis for the potential use of stable isotopes in plantwater relations (15, 29). Moreover, the isotopic compositionof leaf water is recorded in leaf organic matter (5, 12, 16, 18,20). An integrated isotopic signal that reflects the averagegrowing conditions of the plant, with respect to temperature,humidity and water availability, may therefore be obtained.Leaf water is the source for all organically bound hydrogen

and is also the major factor influencing the oxygen isotopiccomposition of organic matter (9). In both cases there are

'Supported by National Science Foundation grants DMB 84-05003 and DMB 88-96201, U.S. Department of Energy grant DE-87ER60615, and United States-Israel Binational Agricultural Re-search and Development Fund grant SI-0024-85.

2 Present address: Botany Department, Duke University, Durham,NC 27706.

3Present address: Department of Earth Sciences, University ofCalifornia, Santa Barbara, CA 93106.

AX = [(Rsample/Rstandard) - 1] * 1000%o (2)where X is 80 or D. Combining the "a" and "6" notationsone can obtain:

6cellulose = acellulose-water * 6water +

[(acellulose-water - 1) * 1000%o] (3)

Because a values are close to unity for the processes involvedin cellulose synthesis (e.g. typically 1.00 ± 0.10), Equation 3can be simplified, for a first approximation, to:

6cellulose = 6 water + F (4)

where F, the difference between the isotopic composition ofcellulose and water, is an approximation of the deviation ofa from unity in %o. F and a can be related by the approxi-mation F= 1000 ln a.

If there is an additional source besides water whose oxygenor hydrogen is incorporated directly into cellulose without

'Abbreviations: SMOW, standard mean ocean water; F6P, fructose6-phosphate; G6P, glucose 6-phosphate; RPP, reductive pentosephosphate; TP, triose phosphates.

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Plant Physiol. Vol. 93, 1990

exchanging with water, Equation 3 must be modified to:

6cellulose = n(6water + F) + (1 - n)by (5)

where n is the fraction of hydrogen or oxygen that is influ-enced by water and Y is the additional source. It is importantto emphasize that an intramolecular isotopic homogeneity foroxygen and hydrogen in Y, as well as in cellulose, must beassumed because of present limitations imposed by the ana-lytical procedures; i.e. we cannot measure isotope ratios forindividual positions within molecules.

Equation 5 can be rearranged to obtain:

6cellulose = n * 5water + [n * F + (1 - n)by] (6)

In this study we grew Lemna in water with a variety ofisotopic compositions and determined the corresponding iso-topic ratios of leaf cellulose and that of any external sourcefor oxygen or hydrogen. This enabled us to use the relation-ships described in Equation 6 to obtain information on theoxygen and hydrogen fractionation factors (F) between waterand cellulose associated with biochemical processes. By grow-ing the Lemna autotrophically, heterotrophically, or photo-heterotrophically we could relate the fractionation factorsobtained in each case to the metabolic pathways active underthe specific growth conditions.

MATERIALS AND METHODS

Plant Materials

Lemna gibba L. B3 was grown at 27°C. For each of theexperiments described here, 9 to 15 Lemna plants at the two-frond stage were transferred to a 3 L flask containing 500 mLof E media (7), E media without sucrose, or E media pluskinetin (2.5 lsM) for photoheterotrophic, autotrophic, andheterotrophic growth conditions, respectively. The media wasprepared with tap water to which various amounts (1-3 mL)of water enriched in '80 and deuterium (Isotec Inc. Miamis-burg, OH) were added to obtain ranges in the oxygen andhydrogen isotopic composition of the water.

Heterotrophically grown plants were kept in complete dark-ness except for 2 min of weak red light every 8 h. For theother growth conditions, continuous light from a mixture ofwhite fluorescent and incandescent light (about 200 ,uE m-2s-') was used. For the photoheterotrophic conditions the flaskswere tightly closed, whereas for the autotrophic conditions astream of dry compressed air was introduced into the flasksthrough a 9 mm Pyrex tube inserted in the rubber stopper.The air flow rate was increased gradually during the first 2weeks of each experiment from 10 to a maximum of 40 mLmin-'. This was found to supply the minimum flow ratenecessary to not limit growth. The level of moisture and theCO2 concentration ofthe input air were tested using a portablegas exchange system (LiCor 6000, LiCor, Lincoln, NE). Con-centration of CO2 for the autotrophic experiments was 330,uL L-', except for one experiment in which a special mixtureof air containing 1500 ,L L` CO2 was used. A 40 cm longPyrex tube served as an air cooled condenser on the outlet ofevery flask. High relative humidity, assumed to be close to100%, was indicated by the heavy condensation inside theflasks and along the first 1 to 2 cm of the condenser. The level

of the media in each flask was marked in the beginning of theexperiment; the change at the end of the experiment wasnegligible. The experiments were terminated (after about 2weeks when sucrose was supplied in the media and about 3weeks with no sucrose) when the media surface was fullycovered with Lemna leaves (several thousands fronds). It isan important feature ofthe experimental setup that at the endof each experiment the contribution of the small plant inoc-ulum to the harvested biomass was negligible. Upon termi-nation of each experiment, the Lemna plants were quicklyfiltered and dried with a paper towel before a sample of 2 gwas sealed in a test tube for vacuum distillation of leaf water(11). The rest of the plants were used for cellulose (10) orstarch (2) extractions.

Determination of Isotopic Composition

A 0.5 mL sample of the vacuum distilled leaf water wasequilibrated with CO2 at 25°C. An aliquot of the CO2 wascryogenically purified under vacuum and was used for themass spectrometer analysis as described before (1 1). A secondsubsample of 5 uL was used to determine the hydrogenisotopic composition. The sample was sealed in a capillarywhich was broken under vacuum and the water was passedover uranium at 750°C to form, quantitatively, uranium oxideand release molecular hydrogen (4). The hydrogen was ex-panded into a sample tube and used for the mass spectromet-ric analysis.The carbon isotopic composition of organic samples (cel-

lulose, sucrose, etc.) was determined by heating them inevacuated sealed quartz tubes in the presence of copper,copper oxide, and silver at 875°C, then purifying the CO2formed during combustion cryogenically prior to analyzing itmass spectrometrically. The oxygen isotopic composition oforganic samples was analyzed after heating (525°C) about 6mg in an evacuated tube with HgCl2 to obtain a mixture ofC02, CO, and HCI. The HCI was removed by passing the gasover liquid isoquiniline. The CO was disproportionated toCO2 and C by high voltage discharge and the combined CO2fractions were collected for mass spectrometer analysis asdescribed before (5, 23). Approximately 50 mg of each cellu-lose sample was nitrated by the nitric acid:acetic anhydridemethod (8) to remove hydroxyl hydrogen that may exchangewith water during the preparative procedure. A sample (about10 mg) of the cellulose nitrate, which contained only carbon-bound nonexchangeable hydrogen, was sealed under vacuumwith copper, copper oxide and silver and was heated at 520°C.The water formed was passed over uranium as describedabove to produce molecular hydrogen, which was used forthe mass spectrometer analysis.Oxygen isotopic composition of air 02 was determined by

quantitatively converting it to CO2. About 15 mL of airsample was introduced into an evacuated analysis system.After freezing out water and CO2 the air was circulated, bymeans of a mercury Toepler pump, over red hot graphite(about 2 g) in a small platinum vessel for 25 min. The CO2thus produced was collected cryogenically and used for themass spectrometer analysis.

All mass spectrometer analysis was performed on a MAT-250 mass spectrometer and 6 values (see introduction for

326 YAKIR AND DENIRO

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OXYGEN AND HYDROGEN STABLE ISOTOPES IN LEMNA LEAF CELLULOSE

definition) reported relative to the SMOW standard for oxy-gen and hydrogen and the PDB standard for carbon. Theprecisions of the isotopic analyses were ±0.2 and ±1 %o foroxygen and hydrogen in water, +0.5%o for oxygen in atmos-pheric 02 and organic samples, ±0.1 %o for carbon in organicsamples and ±Y%o for hydrogen in cellulose nitrate.

RESULTS AND DISCUSSION

Heterotrophic Growth Conditions

Sucrose in the media is the only carbon source when plantsare grown in complete darkness. Under these completelyheterotrophic growth conditions, the observed slope of thelinear regression line between the isotope ratios of celluloseand water was about 0.4 for both oxygen and hydrogen (Fig.1, A and D). These linear relationships can be interpretedwith Equation 6 when we use for by the isotopic compositionofsucrose (380 = +33.0%o, AD = -41 %o). The slope indicatesthat about 40% of oxygen and of carbon-bound hydrogenhave exchanged with and have had their isotopic compositioninfluenced by water prior to cellulose formation. This estimatefor oxygen is in agreement with that previously made withcarrot cell culture (28).From Equation 6 and using the y-intercepts obtained from

Figure 1, A and D and the isotopic composition of sucrose

reported above, we calculated the overall fractionation factors,F1, between water and the exchanged oxygen or carbon-bound hydrogen in cellulose formed in the dark. The valuesobtained for Fl were +26.1%o and +158%o for oxygen andhydrogen respectively. The value for oxygen is about 10%o

+80.0 L Y=O.38X+30.4

+40.0

+80.0

f-

+40.0

0

0co

0

+90.0

+50.0

+10.0 r'-20.0

A

I I

+20.0 +60.0 +100.0 -150 -50 +50

8180 of leaf water 8D of leaf water

higher than that reported previously for heterotrophicallygrown carrot tissue culture (28), but identical to that obtainedin the same study when glycerol, rather than sucrose, was

used as the carbon source. The relationship between hydrogenisotopic composition of water and that of the total organicmatter of algae grown in the dark has been studied (13, 14).It was suggested that under these conditions, the organicallybound hydrogen assumed the isotopic composition of theexogenously supplied sugar, i.e. no exchange with water oc-curred. This was clearly not the case in the present study.

Photoheterotrophic Growth Conditions

When Lemna plants were grown in closed vessels withsucrose in the media as before but under continuous light,the slope of the cellulose/water line was significantly higherthan that observed in the dark (Fig. 1, B and E). Under theseconditions the leaves were green and had a functional pho-tosynthetic apparatus that was, most likely, operating at thecompensation point (1). At least part of the sucrose taken inby the plants was metabolized through the RPP pathwayunder these conditions, i.e. while refixing respired CO2. Ascan be expected, this leads to a further exchange with waterof both oxygen and hydrogen originating from sucrose. Theslopes of about 0.7 for the linear regression lines between theisotope ratios of cellulose and water (Fig. 1, B and E) indicatethat now about 70% of the oxygen and carbon-bound hydro-gen of cellulose underwent exchange with water prior tocellulose formation. Because there was no external source forC02, or hydrogen, the remaining 30% must have come di-rectly from sucrose with no exchange with water. (The lack

+125

+25

-75

+125 cXC

0

+25c

0

co

-75 000

1+50

Figure 1. Relations between leaf water and cel-lulose oxygen (A, B, C) and hydrogen (D, E, F)isotopic compositions in Lemna gibba grownunder heterotrophic (A, D) and photohetero-trophic (B, E) conditions with sucrose in themedia as carbon source and under autotrophicconditions with normal dry air (C, F). The corre-lation coefficients for the linear regression linesare better than 0.9 in all cases.

-50

-150

Y=0.45X+48.6

_~~~~~~~~~~~

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of external CO2 contribution was confirmed by the 6'3C valueof cellulose, -10.6%o, similar to that of the cane sugar,-I 1.4%o, provided in the media. In contrast, cellulose fromautotrophically grown Lemna had a 5'3C value of -23.6%o).Under photoheterotrophic growth conditions, some ex-

change occurs in the light that did not occur when Lemnawas grown heterotrophically in the dark. The oxygen orhydrogen isotopic composition of cellulose grown photohet-erotrophically can be described as follows:

3cellulose = P(6water + F2) +

q(6water + Fl) + [1 - (p + q)]by (7)

where p is the fraction of oxygen or carbon-bound hydrogenthat exchanged with or derived from water respectively duringmetabolic steps taking part in the chloroplast that are engagedonly in the light, q is the fraction that exchanged with orderived from water during metabolic steps independent ofchloroplast activity that are engaged both in the dark and inthe light, and byis the isotopic composition ofthe exogenouslysupplied sucrose. This treatment involves a few assumptions.First, the pathways operating in the dark, and the correspond-ing exchange reactions with water, also operate in the lightand to the same extent. Second, the fractionation factor (Fl)between cellulose and water for these sections of the pathwayremains the same in the dark and in the light. Third, anyintermediate metabolized via the RPP pathway, the pathwayengaged in the light, had the opportunity to exchange all itsoxygen and hydrogen with water.

Equation 7 can again be rearranged to describe the linearrelationship observed between the isotopic ratios ofwater andcellulose:

3cellulose = (P + q)6water +

tp * F2 + q * Fl + [1-(p + q)]byl (8)

Equation 8 reduces to Equation 6 when p = 0 (q = n). Weassume that the proportion of oxygen, or hydrogen, that wasincorporated directly from sucrose into cellulose and theproportion of oxygen or hydrogen that originated from su-crose but underwent exchange with water via the 'dark reac-tions' was the same in the dark and in the light (i.e. q/[ 1 -(p + q)] = constant). We can, therefore, estimate q by com-paring the ratios n/( 1 - n) from the heterotrophic experiment(i.e. Eq. 6) and q/[ 1 - (p + q)] from the photoheterotrophicexperiment (i.e. Eq. 8). Doing so, we obtain 0.24 and 0.21 forq (and consequently 0.47 and 0.44 for p) based on the resultsfor oxygen or hydrogen isotopic composition respectively (Fig.1, A and B and 1, D and E, respectively). Using these estimatesof p and q in Equation 8 to interpret the linear regressionlines shown in Figure 1, B and E, we obtain F2 values of+33%o and +27.2%o for hydrogen and oxygen respectively.As indicated above, we assumed that the intermediates of

the photosynthetic reactions that occurred only in the light-grown plants have exchanged or derived all their oxygens orhydrogens, respectively, with water. It is clear, however, thatsome of these must have been reexchanged as they passedthrough the 'dark reactions,' i.e. the same section of thepathway reflected also in the heterotrophic conditions. F2,therefore, represents a composite of Fl, for the reexchange

via the 'dark reactions,' and F3, the fractionation factorassociated only with that section of the pathway that wasactive in the light. Thus:

F2 = r * Fl + (I - r)F3 (9)

We now treat the sugars formed autotrophically in the sameway as we did the exogenously supplied sucrose under theheterotrophic conditions, as sugars from both sources areincorporated into cellulose in a similar way. Therefore, wecan obtain an estimate of F3 by substituting the fraction ofoxygen or hydrogen exchanged with water under hetero-trophic growth conditions for r in Equation 9. Doing thiscalculation, we obtain F3 values of -69%o and +27.9%oo forhydrogen and oxygen, respectively.

Undoubtedly, the fractionation factors given above do notreflect isotope effects during a single reaction (Fig. 2). In fact,each reflects the integration of isotope effects of completesections of pathways. In the dark, Fl may reflect exchangethat occurs as glucose is polymerized directly into cellulose,i.e. due to F6P-G6P isomerization, together with the ex-change that occurs as some sugars are completely metabolizedas they enter the respiratory pathway. In the light, the situationis more complex. The high starch content observed in theleaves from the photoheterotrophic conditions indicated thatsubstantial amounts of the exogenously supplied sugar wasmetabolized to TP. These compounds could enter the chlo-roplast, the site of starch accumulation, where part of themcould be involved in various steps of the RPP pathway,including that of hydrogen donation by NADPH, beforeeither exiting the chloroplast or being converted to glucoseand polymerized to starch. There are several steps along thisroute that allow both hydrogen and oxygen exchange withwater. Interestingly, both in the dark and in the light almost

CO2

CO2

0-C02-

1TtTP -*-o- starch

~-TCA -gyoyipentose phosphate )gluconeogenesis J

G6P *- sucrose

+

cellulose

F3

F1

F2

Figure 2. A simplified representation of the pathways involved in themetabolism leading to the synthesis of cellulose in C3 plants underautotrophic or heterotrophic conditions or a combination of the two.The parts of the pathways that correspond to the different overallfractionation factors, Fl, F2, and F3, discussed in the text, areindicated. The values corresponding to these factors are reported inTable II.


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all the steps allowing hydrogen (19) and oxygen exchangewith water involve ketose-aldose isomerization.The fractionation factors obtained for oxygen, Fl, F2, and

F3 were all within the range +26%o to +28%o. These valuesare consistent with the direct observation of oxygen isotopefractionation occurring during carbonyl hydration (fraction-ation factor +27%o, ref. 27) and also support the hypothesisthat under physiological conditions this reaction is the onlyway oxygen may exchange with water (27; see also 25). Theonly difference among the calculated fractionation factors,with regard to oxygen, is that each represents different sectionsof pathways, or metabolic fluxes, that allow different propor-tions of oxygens to exchange with water.With regard to hydrogen, the results indicate that a strong

discrimination against D is associated with photosyntheticactivity. This is in agreement with an observation previouslymade in algae (13, 14). The reactions during which NADP isreduced by protons from water and, consequently, hydrogenis donated by NADPH during photosynthesis, are the onlylikely candidates for this effect. This is because all otherreactions that could allow hydrogen exchange with water canoccur under heterotrophic conditions as well. However, sinceNADPH formation involves an irreversible step and, in ad-dition, practically all NADPH is consumed during well bal-anced photosynthetic activity, it is most likely that the nega-tive isotope effect is associated only with the reduction ofNADP with protons from water.

Clearly, the isotope effect associated with NADPH was onlypartially expressed in F3 because F3 represents various addi-tional steps that allow the exchange of hydrogen with water,i.e. in the RPP pathway. Little is known about the proportionsof the various metabolic fluxes involved in carbohydratemetabolism under photoheterotrophic conditions. Hydrogenstable isotope analysis, however, may prove to be an idealtool to study these fluxes, once the specific fractionationfactors involved are determined.

Interestingly, the oxygen isotopic composition of cellulosewas similar to that obtained for starch from the same leaves(Table I). As mentioned above, under photoheterotrophicconditions, starch was formed in the chloroplasts from exog-enous carbohydrates ( 17). The results, therefore, indicate thatboth starch and cellulose are metabolized from a common,well mixed, pool ofTP and there are no oxygen isotope effectsassociated with the specific steps leading thereafter to eitherproduct.

Table I. 6180 Values of Water, Cellulose, and Starch from Leaves ofL. gibba Grown Photoheterotrophically with Sucrose in the Media

Lemna gibba

Water Cellulose Starch

6180

-14.5 +20.7 +22.7+25.5 +45.6 +47.3+42.2 +56.8 +55.7+62.0 +69.7 +67.2+81.8 +83.3 +81.5

Autotrophic Growth Conditions

When Lemna plants were grown under continuous light inmedia containing only mineral salts with a flow of dry air,the slope of the linear regression line between the isotopiccomposition of cellulose and water was approximately 0.8 forboth oxygen and hydrogen (Fig. 1, C and F).

It is important to distinguish at this point between theisotope effects associated with the biochemical processes,which are the main subject of this study, and source effectson the isotopic ratios of cellulose. As discussed above, water,under autotrophic conditions, determines both the hydrogenand oxygen isotopic composition of plant organic matter.Biochemical isotope effects must remain constant as plantsare grown in water with different isotopic compositions butunder otherwise identical conditions. Under these circum-stances the slope of the relationship between the isotopicratios of cellulose and water should be one. If this is the case,direct estimate of F could be obtained from Equation 4. Theresults presented in Figure 1, C and F, with slopes of about0.8, did not produce the expected relationships. In the nextsegment, we argue that these lower slopes are due to effectsof atmospheric moisture that entered the experimental sys-tem. We then make the appropriate corrections and proceedto obtain an estimate of the fractionation factor, F, betweencellulose and water as mentioned above.

Initially, we tested and rejected two possible hypothesesthat could explain a slope of less than one for the relationshipbetween the oxygen isotopic ratios of cellulose and water. Thefirst possibility was the incorporation of an isotopic signalfrom air 02. This possibility, which is all but ruled out by theexperiments reported by Berry et al. (3), was eliminated inthe present study. When Lemna plants were grown in aircontaining oxygen with a 6180 value of 377 %o, the 6180 ofleaf cellulose was +21.5%o, similar to the value of +20.9%oobtained for control plants, for which the 6180 of the air 02was +23.6%o. Although atmospheric oxygen must be fixedinto glycolate during photorespiration (3), this isotopic signalis lost due to partial release as CO2 and isotopic exchange ofthe oxygens derived from 02 with water before the productsof photorespiration are incorporated into sugars. We alsotested the possibility that some oxygen from air CO2 is incor-porated into cellulose without exchanging with water. If thiswere the case, growing Lemna with higher concentrations ofCO2 in the air should increase the proportion of nonex-changed oxygen incorporated into cellulose, thereby loweringthe slope of the cellulose-water 6180 relationship. GrowingLemna with air containing 1500 ,uL L-' C02, compared with330 uL L` measured in the normal dry air, produced acellulose/water slope of 0.71 (Fig. 3, compare with a slope of0.77 in Fig. IC). This is not a substantial shift for a CO2concentration five times higher than the control. We thereforeconcluded that isotopic signals from neither atmospheric 02nor atmospheric CO2 were incorporated into cellulose.The most striking point in the results shown in Fig. 1, C

and F is the similarity in the slopes obtained for oxygen andfor hydrogen. Water is not only the sole source for all organ-ically bound hydrogen but also the only factor that couldaffect both the oxygen and hydrogen isotopic ratios simulta-

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+80.0

+40.0 -

0-20

-20.0 +20.0 +60.0

8180 of leaf water

Figure 3. Relation between the oxygen isotopic composition of leafwater and that of cellulose in L. gibba grown with an air mixturecontaining 1500 ,uL L-' C02, under continuous light at 270C. Corre-lation coefficient of the linear regression line is 0.995.

neously. We suspected, that water other than that providedin the media "contaminated" the experimental system. Nomoisture was present in the input air (see "Materials andMethods"). It is possible that an appreciable amount of mois-ture diffused back from the moist air in the growth chamberthrough the outlets leading to the atmosphere. To test thishypothesis, we compared the hydrogen isotope ratios of themedia and the condensed moisture in the inner tip of thecondenser on the outlet of a growth vessel. The AD valuesobtained were +54%o for the media and +44%o for thecondensed moisture in one of the vessels used for autotrophicgrowth. The AD value of the atmospheric moisture in the labwas -95%o.

If the condensed atmospheric moisture is at equilibriumwith the free vapor, as would be expected inside the condenser,its hydrogen isotopic ratio would be -95 + 77 = -18%o, since77%o is the equilibrium fractionation factor for hydrogen at27°C (21).

Using this value we estimated the proportion of the atmos-pheric moisture contamination in the sample that was col-lected from the condenser, as follows:

Z(-18%o) + (1 - Z)(+54%o) = +44%o (10)

where Z represents the fraction of water in the condenseroriginating from atmospheric moisture, and -1 8%o, +54%o,and +44%o are the AD values of the condensed moistureoriginating from the atmosphere, the media, and the vapormoisture at the exit from the growth vessel, respectively. Avalue of 0. 14 is obtained for Z.

Using the value of 0.14 for Z and the isotopic compositionofatmospheric moisture reported above, a corrected value forthe relevant part ofleafwater at equilibrium with the moisturein the growth vessel was calculated. The linear regression linesobtained for these corrected water isotope ratios and thecellulose isotope ratios reported in Figure 1, C and F were:

6 18°cellulose = 0.99 bi80water + 27.1 %o r = 0.998 (1 1)

5Dcellulose = 0.95 bDwater -23%o r = 0.998

The intercepts obtained in Equations 11 and 12 can beinterpreted using Equation 4 to give the overall fractionationfactors between water and the exchanged oxygen or carbon-bound hydrogen in cellulose when all metabolites have passedthrough the RPP pathway. The intercept for oxygen in Equa-tion 1 1, +27.1 %o, is in good agreement with the estimatesmade above as well as with previous reports (Table II; 27).This, again, suggests that the same type of fractionationmechanism with the same fractionation factor is operating inall parts of the pathway leading to cellulose. With regard tohydrogen, the intercept in Equation 12, -23%o, must, like F2above, represent a composite of F3, for the reactions associ-ated with photosynthesis only, and Fl, for the subsequentreexchange, estimated as +158%o, from the heterotrophicgrowth experiment (Table II). The results ofthe heterotrophicexperiment provided us also, as discussed above, with a firstapproximation ofthe proportion ofthe reexchange that occursafter sugars are formed, i.e. about 40 to 45% of the oxygenand carbon-bound hydrogen. We can now calculate again anestimate for F3 using Equation 9, substituting -23%o for F2,0.45 for r and +158%o for Fl. We obtain for autotrophicgrowth F3 = -171 %o (see Table II for a summary of the Fvalues). Again, a very strong negative isotope effect associatedwith photosynthetic activity is indicated. The value obtainedhere, however, is much more negative than that obtainedunder photoheterotrophic conditions (F3 = -69%o). Thisdifference may reflect differences in the proportions of themetabolic fluxes involved in carbohydrate metabolism underthe different growth conditions. For example, under photo-heterotrophic conditions (CO2 at the compensation point,normal 02 and light), the photorespiratory flux would berelatively large (6). Under these conditions the effects ofhydrogen donation from NADPH, the likely source of thelarge negative isotope effect, would be less pronounced sincethe opportunity for hydrogen to reexchange with water duringthe photorespiratory pathway would be greater. Under auto-trophic conditions, on the other hand, the flux through theRPP pathway would increase, with no comparable change inthe photorespiratory flux (6). Thus the NADPH effect will bepreserved in the final product, such as cellulose, to a greaterextent. An alternative explanation follows from the observa-tion that a large starch accumulation was observed underphotoheterotrophic but not under autotrophic conditions.Starch metabolism may increase the opportunity for hydrogen

Table II. Overall Fractionation Factors between Water and Cellulosefor Various Sections of Pathways Leading to the Synthesis ofCellulose under Different Growth Conditions

The values for Fl and F2 were calculated based on the linearregression lines reported in Figure 1. F2 was assumed to be acombination of Fl and F3. Fl was assumed to remain constant underall conditions.

Hydrogen OxygenConditions

Fl F2 F3 Fl F2 F3

%O

Heterotrophic +158 +26.1Photoheterotrophic +33 -69 +27.2 +27.9Autotrophic -23 -171 +27.1 +27.7

0(I)0

*130(U4)

00

cov-

0so


(12)

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to reexchange with water subsequent to the RPP pathway andbefore incorporation into cellulose. In any case, it appearsthat F3 obtained under the autotrophic conditions (-171 %O)provides a better estimate of the net isotope effect associatedwith NADPH formation. Undoubtedly, more work is neededto confirm the hypotheses suggested here. But, at the sametime, the potential in using stable isotopes in studying meta-bolic fluxes under different physiological conditions, once thefractionation factors are known, should be obvious.

Support for our hypothesis that there is a single oxygenisotope effect and two major hydrogen isotope effects in goingfrom water to cellulose comes from a recently advancedargument regarding the relationships between the isotoperatios of oxygen and hydrogen in water and leaf matter (26).Plotting the isotope ratios of hydrogen versus those of oxygenfor either leafwater or leaf cellulose yields linear relationships(Fig. 4), (21, 24, 26). It was demonstrated (26) that theequation of the AD versus 0'80 line of leaf cellulose can berelated to that of leaf water by:

WDcellulose = s(aD/a8) 180water +

[1000 (aD - 1) - c - 1000 * s(aD/aI8)(a18 - 1)] (13)

where s is the slope of the water line, aD and a18 are the

+150

+50

-50

a-150

+100

0

-100

-200 L

-20.0 +20.0 +60.0

8180Figure 4. Relations between the oxygen and hydrogen isotopiccompositions of leaf water (U) or of cellulose (El) in L. gibba grownunder heterotrophic (A) and autotrophic (B) conditions. The isotopiccompositions for leaf water reported here are those calculated aftercorrecting for the assumed effects of atmospheric moisture, asdiscussed in the text. Correlation coefficients of the regressions linesare all better than 0.9.

hydrogen and oxygen fractionation factors, respectively, be-tween cellulose and water and c is the intercept of the waterline. Equation 13 describes a linear relationship in which theslope of the cellulose line, s(aD/al8), is a function of the slopeof the leaf water line, s, and the ratio between the hydrogenand oxygen fractionation factors. Since we propose that as wechange from heterotrophic to autotrophic growth conditions,a18 remains constant but aD changes dramatically, one wouldexpect that the ratio aD/a18, and therefore the slope of thecellulose line relative to that of the water line, would alsochange accordingly. We used the slopes of the water linesreported in Figure 4, together with the values of aD (1.171,0.977) and a18 (1.027, 1.027) for heterotrophic and auto-trophic conditions, respectively (derived from the F valuesreported above, see also introduction), to calculate the pre-dicted slopes of the corresponding cellulose lines. We obtainvalues of 1.69 and 1.51 for the slopes of the cellulose linesunder heterotrophic and autotrophic conditions, respectively(i.e. 1.69 = [1.171/1.027]1.48 and 1.51 = [0.977/1.027]1.59).These values are in agreement with the actual slopes obtained(Fig. 4). Thus, not only can we accurately predict changes inslopes in the cellulose relative to the water lines as growthconditions change, but such changes may serve as an indicatorof the metabolic pathway used by the plant in each case.

Obviously more work is needed to identify the specificbiochemical steps responsible for the observed isotope effectsand to determine the fractionation factors involved. Thisinformation is essential to understanding the variations in thestable oxygen and hydrogen isotopic composition of plantmatter. This understanding, in turn, can help in providingnew insights into changes in metabolic fluxes involved inplant metabolism under different growth conditions.

ACKNOWLEDGMENT

Lemna plants were kindly provided by Professor E. Tobin. Theauthors thank Dr. L. Stemnberg for determining the hydrogen isotopiccomposition of the sucrose and for discussions during the course ofthis study.

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Oxygen and Hydrogen Isotope Fractionation - Plant Physiology

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