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30 - 4 ANGERT ET AL.: BIOLOGICAL EFFECTS ON THREE O 2 ISOTOPES[23] According to equation (1), the 17 D of the photosyntheticallyproduced oxygen is17 R W17 R W17 D W ¼ ln C ln : ð11Þ17R17 ref R ref[24] Subtracting equation (11) from equation (10), andusing equation (5) yields17 D 17 BSS D W ¼ ln18 a C q: ð12ÞFigure 2. Schematic plot (not to scale) of Ln 17 O versusLn 18 O for a closed system in production-uptake steadystate. Point ‘‘W’’ represents oxygen that is produced byphotosynthesis in photosystem 2 from water, and ‘‘BSS1’’and ‘‘BSS2’’ represent biological steady states. The slope ofthe line that connects ‘‘BSS1’’ and ‘‘W’’ is equal to q 1 andthe slope of the line connecting ‘‘BSS2’’ and ‘‘W’’ is equalto q 2 (q =ln( 17 a)/ln( 18 a)). When q of the system equals q 1the system reaches the steady state condition indicated by‘‘BSS1’’ with an 17 O excess of 17 D BSS1 . Since C equals toq 1 , 17 D BSS1 equals to the 17 D W . When q of the system equalsq 2 the system reaches steady state indicated by ‘‘BSS2’’with 17 O excess of 17 D BSS2 that is lower than 17 D BSS1 . Thehorizontal distance between ‘‘BSS’’ and ‘‘W’’ is thesystem’s equivalent of the global Dole Effect (in Ln 18 Oterms). The difference between 17 D BSS1 and 17 D BSS2 isgiven by the system’s ‘‘Dole Effect’’ times the differencebetween q 1 and q 2 .[20] Rearranging equation (8) and substituting it intoequation (4) givesl BSS ¼ ln ð17 aÞln 18 ¼ q: ð9Þð aÞ [21] Hence, the slope of the line connecting the O 2 of abiological system in production-uptake steady state, and theO 2 produced from the substrate water, is equal to the valueof q. However, as we will show in section 2.3 below, thevalue of l is not always equal to the value of q.[22] The 17 D in production-uptake steady state can befound by substituting equations (7) and (8) into equation (1),[25] When q equals C, the 17 D value of the air in steadystate equals to that of the oxygen that is produced from thesubstrate water. However, if q is different from C, then17 D BSS is controlled both by q and 18 a.[26] Currently, the value of 17 D W cannot be measureddirectly with sufficient accuracy. However, this value canbe estimated by conducting a terrarium experiment inwhich the q value of the uptake process is known (thisknown q will be noted as q 1 ). In such an experiment, thevalue of 17 D in steady state ( 17 D BSS1 ) will be identical tothat of 17 D W , if we choose C = q 1 . By conducting anadditional experiment in which the q value of the uptakeprocess is unknown (this unknown q will be noted as q 2 ),we can calculate the value of q 2 from the 17 O excess insteady state of this experiment ( 17 D BSS2 ), by rearrangingequation (12),ð17 D 17 BSS 2 D W Þq 2 ¼ q 1ln 18 : ð13Þð aÞ[27] The value of ln( 18 a) for the system can be found byrearranging equation (8),ln 18 a ¼ ln 18 R BSS = 18 R W : ð14Þ[28] The right-hand side of equation (14) is the Ln 18 Ovalue of ‘‘BSS’’ versus ‘‘W,’’ which is equivalent to theterrarium Dole Effect in Ln 18 O terms (the value of the DoleEffect in d 18 O terms is greater by 0.3% if SMOW is thereference and lower by 0.3% if atmospheric O 2 is thereference). Hence, the calculation of q 2 from equation (13)is identical to the solution presented graphically in Figure 2.[29] To estimate q values by the method describe above,the q value of at least one process (q 1 ) must be knownindependently. This value can be found by conductingexperiments in systems in which there is O 2 uptake butno production.2.3. O 2 Removal Only[30] In our dark respiration and binary diffusion experiments,O 2 is only removed from the system. The change inisotopic composition in such experiments follows theRaleigh distillation equation,1717 R W = 17 a18 R W = 18 aD BSS ¼ ln C ln : ð10Þ17R18 ref R refx e ¼ ln x R= x R 0; ð15Þln f
ANGERT ET AL.: BIOLOGICAL EFFECTS ON THREE O 2 ISOTOPES 30 - 5where x R 0 is the initial isotope ratio x O 16 O/ 16 O 2 and f is theremaining 16 O 2 fraction. Substituting equation (15) intoequation (4) yieldsl R ¼17 e¼ g; ð16Þ18ewhere the subscript R stands for Raleigh distillation.Equation (16) shows that in a system where there is onlyuptake of oxygen, the value of l is different from thevalue of q. The value of q in ‘‘removal only’’ experimentscan be calculated by substituting equation (16) intoequation (5),q ¼ ln ð 1 þ l R 18 eÞlnð1 þ 18 eÞ : ð17Þ[31] The range of 18 e in biological uptake is 15 to32% and the value of q in mass-dependent fractionationis about 0.5. Hence, l R will be larger by 0.002–0.004 than qin most systems. For O 2 samples close in composition to thestandard O 2 , using slightly different C values will notsignificantly affect the calculated 17 D. However, for thepurpose of the present study where comparisons betweensteady state O 2 and its substrate water are necessary, a smalldifference in parameter C becomes significant. To illustratethe importance this point, consider a water sample havingLn 18 O= 23% and Ln 17 O= 11.7% with respect to airO 2 . Using equation (1) and C = 0.52, 17 D of this sample iscalculated as 260 per meg. Similar calculation with C =0.524 yields 17 D of 352 per meg.3. Experimental Methods3.1. Dark Respiration Experiments[32] Axenic cultures of Lemna gibba, a small waterplant, were grown in a nutrient solution. Illumination frommixed fluorescent/incandescent lamps was at an intensityof 200 ± 20 mE m 2 s 1 photosynthetic photon flux (PPF),at the level of the fronds, for 12 h d 1 . Air temperaturewas 24°C and 19 ± 0.5°C in the light and dark periods,respectively.[33] The seeds of Orobanche aegyptica, an obligatoryroot parasite, were placed in empty tea bags and suspendedfor sterilization in 1% solution of NaOCl for 5 min. Theseeds were then rinsed thoroughly for 20 min in steriledistilled H 2 O. After the rinse, the seeds were activated by 3days imbibition in 5 ml of H 2 O in 9-cm-diameter Petridishes with one layer of Whatman paper, to which a mixtureof antibiotics was added. The mixture contained 50 mg eachof streptomycin and penicillin G and 25 mg of chloramphenicol,in order to prevent the development of bacterialinfections during the imbibition period.[34] To determine the q value associated with the COX,we inhibited the AOX in the plant samples with 2 mMSalicylhydroxamic acid (SHAM). To determine the valueassociated with the AOX, we inhibited the COX with 1mM NaCN. The inhibitors were added to a closedaerated chamber containing the plant samples and waterfor at least 90 min before measurements were made.Additional control experiments without inhibitors wereconducted with Lemna and natural soil (the resulting qvalue was expected to lie between that of the AOX andthe COX).[35] For the incubation, about 1 g of plant sample wasinserted into 6 cm 3 blood collection tubes (Vacutainers 1 )that were closed with rubber septa. The tubes wereimmersed in water during incubation in order to preventair leaks. After an incubation period of 1 to 10 hours, the airin the tubes was sampled, via a needle, directly to thevacuum preparation line. In other experiments, plant sampleswere incubated in water and the changes in thedissolved oxygen were monitored. At the end of theseexperiments, 120 cm 3 water were sampled in 250 cm 3pre-evacuated flasks closed with a Lowers Happert 1 valve.Sampling and extraction of the dissolved gases were doneaccording to Luz et al. [2002].3.2. Terrarium Experiments[36] We used an airtight terrarium as described by Luz etal. [1999]. The terrarium contained Philodendron plants,soil and natural water from Lake Kinneret, Israel (d 18 O=0.5% versus SMOW). It was illuminated by a fluorescentlamp (100 mE m 2 s 1 ) for 24 h d 1 ,10hd 1 ,4hd 1and 2 h d 1 in different stages of the experiment. Differentlight conditions were used to manipulate the CO 2concentration in the terrarium, which, in turn, affects therelative rate of photorespiration [Badger, 1985]. Theterrarium was also covered and darkened for dark incubations.The CO 2 concentration in the terrarium air wasdetermined by sampling 6 cm 3 air in pre-evacuated bloodcollection tubes (Vacutainers 1 ), and measuring the airsample with an infrared gas analyzer (LI-COR-6252 1 )by a method similar to Davidson and Trumbore [1995].The relative accuracy for CO 2 concentration measurementwas ±5%.3.3. O 2 -N 2 Diffusion Experiments[37] In the diffusion of O 2 in N 2 experiments, a 4-cm 3flask was filled with pure oxygen. The neck of the flask wasfilled with a diffusive medium (plastic sponge) that preventedadvective mixing, and the oxygen that diffused outof the tube was immediately removed by a flow of N 2 . After1–2 hours, the O 2 concentration in the flasks was considerablylowered and the flasks were closed and transferredfor isotopic analysis.3.4. Sample Preparation and Mass Spectrometry[38] Sampling, sample preparation, and mass spectrometrywere according to Angert and Luz [2001], Luz et al.[2002], and Luz and Barkan [2000]. The preparation of thesample included cryogenic removal of water vapor and CO 2,and chromatographic separation of N 2 , which is needed foraccurate 17 D measurements. The samples were frozen at4°K into stainless steel tubes and transferred for analysisin a Finnigan Delta-Plus mass-spectrometer. Correctionswere applied in order to account for the sensitivity ofionization efficiencies of the three isotopic species tovariations in the O 2 /Ar ratio. The analytical error (absolutedifference from the average) for d 18 O, dO 2 /Ar, and D 17 Owas 0.02%, 0.5% and 5 per meg, respectively. All values
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