Diurnal variation in the stable isotope composition of water and dry matter in fruiting Lupinus angustifolius under field conditions
L. A. Cernusak,
Environmental Biology Group and Cooperative Research Center for Greenhouse Accounting, Research School of Biological Sciences, Australian National University, GPO Box 475, Canberra, ACT 2601, Australia and
Environmental Biology Group and Cooperative Research Center for Greenhouse Accounting, Research School of Biological Sciences, Australian National University, GPO Box 475, Canberra, ACT 2601, Australia and
In this paper, we present an integrated account of the diurnal variation in the stable isotopes of water (δD and δ18O) and dry matter (δ15N, δ13C, and δ18O) in the long-distance transport fluids (xylem sap and phloem sap), leaves, pod walls, and seeds of Lupinus angustifolius under field conditions in Western Australia. The δD and δ18O of leaf water showed a pronounced diurnal variation, ranging from early morning minima near 0‰ for both δD and δ18O to early afternoon maxima of 62 and 23‰, respectively. Xylem sap water showed no diurnal variation in isotopic composition and had mean values of −13·2 and −2·3‰ for δD and δ18O. Phloem sap water collected from pod tips was intermediate in isotopic composition between xylem sap and leaf water and exhibited only a moderate diurnal fluctuation. Isotopic compositions of pod wall and seed water were intermediate between those of phloem and xylem sap water. A model of average leaf water enrichment in the steady state (Craig & Gordon, pp. 9–130 in Proceedings of a Conference on Stable Isotopes in Oceanographic Studies and Palaeotemperatures, Lischi and Figli, Pisa, Italy, 1965; Dongmann et al., Radiation and Environmental Biophysics 11, 41–52, 1974; Farquhar & Lloyd, pp. 47–70 in Stable Isotopes and Plant Carbon–Water Relations, Academic Press, San Diego, CA, USA, 1993) agreed closely with observed leaf water enrichment in the morning and early afternoon, but poorly during the night. A modified model taking into account non-steady-state effects (Farquhar and Cernusak, unpublished) gave better predictions of observed leaf water enrichments over a full diurnal cycle. The δ15N, δ13C, and δ18O of dry matter varied appreciably among components. Dry matter δ15N was highest in xylem sap and lowest in leaves, whereas dry matter δ13C was lowest in leaves and highest in phloem sap and seeds, and dry matter δ18O was lowest in leaves and highest in pod walls. Phloem sap, leaf, and fruit dry matter δ18O varied diurnally, as did phloem sap dry matter δ13C. These results demonstrate the importance of considering the non-steady-state when modelling biological fractionation of stable isotopes in the natural environment.
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There is currently major interest in understanding the role of plants in the global carbon cycle (Prentice et al. 2001), and it has already become evident that small variations in the natural abundances of the stable isotopes of C, N, O, and H can provide unique insights into the processes controlling carbon uptake and release from vegetation (Griffiths 1998). However, meaningful interpretation of isotopic data requires a sound understanding of the biological fractionation of stable isotopes over relatively short time periods and between the component parts of a plant.
It is well known, for example, that the water contained in leaves becomes enriched in the heavy isotopes of O and H (18O and D) during transpiration. Theory has been developed to account for such enrichments (Craig & Gordon 1965; Dongmann et al. 1974; Farquhar & Lloyd 1993). The organic matter of plants partly reflects the 18O enrichment of leaf water, essentially by tracking and integrating differences in 18O signals generated under varying evaporative conditions and transpiration rates during the growth of the plant (Barbour & Farquhar 2000). The same oxygen isotope signal of leaf water is also reflected in the portion of gaseous CO2 which, having entered into leaves via stomata, then diffuses back out again without being captured by photosynthetic enzymes. This enrichment in the 18O/16O ratio of atmospheric CO2 by plants has important implications for estimating carbon fluxes between the biosphere and the atmosphere (Farquhar et al. 1993). Finally, the 18O/16O ratio of leaf water is also recorded in the gaseous O2 that is evolved during photosynthesis, and variations in 18O/16O of atmospheric O2 provide a basis for constructing historic estimates of terrestrial and marine productivity (Bender, Sowers & Labeyrie 1994).
The heavier isotope of carbon (13C) is discriminated against during photosynthesis, with the degree of discrimination depending in part on the type of photosynthetic pathway (Bender 1968). In C3 plants, the fractionation correlates positively with the gaseous concentration ratio between intercellular and ambient CO2 (Farquhar, O’Leary & Berry 1982), and thereby negatively with plant water use efficiency (Farquhar & Richards 1984). Extents of discrimination against 13C also differ when CO2 is fixed by terrestrial versus marine organisms (Francey et al. 1995), and to a lesser extent among different classes of terrestrial ecosystems (Lloyd & Farquhar 1994). Consequently, 13C discrimination can be an important parameter in global and ecosystem carbon budgets. Because assimilated carbon can cycle through plants and ecosystems in different ways before returning to the atmosphere, detailed consideration of daily and seasonal fractionation events taking place within plants becomes important when constructing carbon budgets based on 13C/12C ratios.
The availability of nitrogen in an ecosystem plays an important role in determining ecosystem productivity (Vitousek & Howarth 1991), and related natural abundance ratios for 15N/14N in soil and plant components have been shown to comprise potentially useful integrators of the types and turnover rates of nitrogen cycling involved (Robinson 2001). Yet again, understanding is required of how isotopic fractionations of nitrogen are mediated during daily transformation and transport events, from initial assimilation right through to incorporation into specific organic components of different plant organs.
In this investigation, we set out to understand more fully the biological fractionations of these stable isotopes which occur during the functioning of a plant under field conditions, where environmental parameters vary considerably with time of day and plants adjust accordingly in terms of their gaseous exchanges and partitioning processes. We specifically report on daily variation in the stable isotopes of C, N, O and H in the water and dry matter of xylem, phloem, leaves, and fruits of lupin, a legume commonly grown as a grain crop in Western Australia, and already subject to extensive earlier studies on the carbon, nitrogen, and water economies of fruits under both glasshouse and field conditions (Pate, Sharkey & Atkins 1977; Pate, Atkins & Perry 1980; Pate, Williams & Farrington 1985).
Materials and methods
The study was conducted on a uniform stand of Lupinus angustifolius L. var. Tangil grown as part of a breeding trial at the Mount Barker Research Station (34°38′02′′ S, 117°32′00′′ E). This facility is operated by Agriculture Western Australia and is located approximately 20 km from the township of Mount Barker, Western Australia. The crop was sown on 6 June 2000. All measurements and sap and biomass collections were undertaken during 1 and 2 November 2000 when fruits were well-developed and estimated to be approximately 1 month away from harvest maturity.
Gas exchanges of source leaves and filling fruits on main stems were monitored at 1 to 2 h intervals during the 26 h study period, except during the night when dew was present on leaves and atmospheric vapour pressure approached saturation (Fig. 1). Fluxes of CO2 and water vapour during daylight were measured using an LCA 4 Portable Gas Exchange System (ADC BioScientific Ltd, Hoddesdon, Hertfordshire, UK). Approximately 5 cm2 of leaf or pod surface (two to three leaflets or one to two fruits) were detached and immediately placed inside the gas exchange cuvette and measurements were repeated on eight to ten such sets of leaflets or fruits at each sampling time. Measurements were completed within approximately 5 min of detachment. No decline in gas exchange rates following detachment was observed within this time period.
Ambient air temperature and relative humidity were measured periodically throughout the experiment using a Vaisala temperature and humidity probe (Vaisala Inc., Helsinki, Finland). Leaf and pod temperatures were measured concurrently on three to six leaflets or pods using a 0·13-mm-diameter chromel–constantan thermocouple (Omega Engineering, Stamford, CT, USA).
Xylem sap and phloem sap were sampled at 1 to 2 h intervals throughout the 26 h study period commencing at 0900 h. Xylem sap was collected from the lower main stems of 10 individuals at each sampling time using the mild-vacuum extraction technique developed by Jeschke & Pate (1995) and Pate, Jeschke & Aylward (1995). Phloem sap was obtained by cutting the distal tips of 30–50 fruits and immediately collecting the exudate (Pate, Sharkey & Lewis 1974). Sap samples from individual plants were pooled to form bulk samples (5–10 mL for xylem sap, 30–50 µL for phloem sap) from each sampling time, and samples were immediately decanted into sealable tubes and frozen at −20 °C. Sucrose concentrations of phloem sap were measured at the time of collection using a hand-held refractometer (Pate et al. 1998). Refractometer readings were corrected for the presence of amino acids and amides by measuring the nitrogen concentration of each phloem sap dry matter sample and by assuming amino acids and amides were present in similar profiles to those recorded previously for phloem sap of lupins (Pate et al. 1974; Sharkey & Pate 1976). Combined amino acid and amide concentration estimates ranged from 19 to 28 g L−1, which were similar to those values previously recorded (Pate et al. 1974; Sharkey & Pate 1976).
Leaves, pod walls, and seeds were collected for isotopic analysis of tissue water and dry matter at the same time when xylem sap and phloem sap were being collected. Ten leaflets were randomly selected at each time of sampling and placed in gas-tight collection vials before being frozen. Four to six fruits were similarly collected and their pod walls quickly separated from seeds. Sets of samples were collected in duplicate; one randomly allocated for tissue water extraction and δD and δ18O analyses, the other for analyses of δ13C, δ18O, and δ15N in dry matter. The relative water content (RWC) of the leaves collected for dry matter analyses was determined by dividing the difference between fresh and dry weights by fresh weight. Leaf area was determined with a Li-Cor leaf area meter (Li-Cor Inc, Lincoln, NE, USA), thus enabling data to be expressed in terms of areal concentrations of water within the leaf samples.
We are aware of the substantial alterations in isotope signals of water which can occur during exposure of liquid interfaces to the atmosphere and of the errors which such alterations might introduce when interpreting data. For xylem sap samples, the mild vacuum extraction technique involved an entirely sealed system which siphoned tracheal fluid progressively into a tube of 5–10 mL volume over a period of a few seconds. Samples were then removed from the collection apparatus and immediately decanted into sealable vials. For phloem sap samples, evaporation from exuding droplets was minimized by immediately attracting them (within 5 s) into microcapillary tubes that have extremely small exposed-liquid-surface : volume ratios. The contents of the capillaries were then expelled into small-volume vials (0·1 mL) that were sealed except in the few seconds when additional samples were introduced. In the case of plant tissue samples, transfer within seconds from plants into sealed tubes would be expected to virtually eliminate the opportunity for evaporative enrichment of water isotope ratios during harvest. All samples were immediately frozen at the field site following collection and remained frozen until isotopic analyses were conducted.
To test the possible extent to which evaporative enrichment of water isotope ratios might have occurred during the brief exposure of samples to the atmosphere during sap collection, we performed regression analyses of the δD and δ18O of xylem sap samples against the vapour pressure deficit at the time of sap collection. Vapour pressure deficits ranged from 0 to 28 mbar over the course of the study. If the isotopic composition of the xylem sap samples had been markedly altered by evaporative enrichment during sap collection, one would expect the isotopic composition of the samples to correlate with the vapour pressure deficit (and therefore the evaporation rate) at the time of collection. We observed no relationship between xylem sap isotopic composition and vapour pressure deficit (P = 0·22 for δ18O; P = 0·65 for δD), suggesting that the sap samples were not affected by evaporative enrichment during sap collection.
Tissue water was extracted from leaves, pod walls, and seeds by azeotropic distillation with toluene (Revesz & Woods 1990). Traces of toluene remaining in the water following the distillation were removed by adding wax to the water and warming the water to the melting point of the wax. The δ18O of the water samples extracted from leaves, pod walls, and seeds was measured by a continuous-flow isotope ratio mass spectrometer (CF-IRMS) (Micromass Isochrom-EA, Manchester, UK) following pyrolysis in a Carlo Erba elemental analyser (CE Instruments, Milan, Italy) (Farquhar, Henry & Styles 1997). Xylem water δ18O was similarly measured by this technique. The oxygen isotope ratio of phloem sap water was determined using a method recently described by Gan, Wong & Farquhar (in press) for the determination of the δ18O of the water component of a homogeneous mixture of water and dry matter. In this procedure, the δ18O of one aliquot of phloem sap solution was measured by pyrolysis. A second aliquot of sap was then evaporated overnight at 60 °C, and the resulting dry matter component assayed for δ18O. Isotope composition of the water in the original sap sample was then estimated by mass balance.
Hydrogen isotope ratios of water samples were measured in Perth using a CF-IRMS system (Delta Plus XL; Finnigan Mat, Bremen, Germany) incorporating an elemental analyser with pyrolysis reactor (Thermoquest PYRO/EA; Finnigan Mat). For phloem sap water δD, the phloem sap remaining after the δ18O analyses was distilled cryogenically under vacuum to collect water separately from dry matter. The δD of the phloem water was then determined as for the other water samples.
The δ15N and δ13C of xylem sap and phloem sap dry matter were determined by CF-IRMS using the Perth-based ANCA combustion system (PDZ Europa Ltd, Northwich, Cheshire, UK) coupled to a dual-inlet mass spectrometer (SIRA9; VG Isogas, Wythenshawe, Cheshire, UK). The δ15N and δ13C of leaf, pod wall, and seed dry matter were determined by CF-IRMS with an Isochrom mass spectrometer (Micromass, Manchester, UK) coupled to a Carlo Erba elemental analyser (CE Instruments) housed at the Australian National University.
The δ18O of leaf, pod wall, and seed dry matter was determined using the method described by Farquhar et al. (1997). Xylem sap dry matter δ18O was similarly determined after evaporation of sap water at 60 °C. Isotope ratios were expressed using delta (δ) notation with respect to the standards of air for nitrogen, Pee Dee Belemnite for carbon, and Vienna Standard Mean Ocean Water for oxygen and hydrogen. Carbon isotope ratios were converted to discrimination values (Δ) (Farquhar, Ehleringer & Hubick 1989a) by assuming an isotopic composition for atmospheric CO2 of −7·8‰.
We tested for differences between isotope ratios of various dry matter components using analysis of variance. If significant differences were detected, Tukey's method was used for pair-wise comparisons. Statistical analyses were conducted in systat 9·0 (SPSS Inc, Chicago, IL, USA).
Modelling leaf water isotopic enrichment
Observations of the isotopic enrichments of leaf water over that of source water (Δ18O or ΔD for oxygen or hydrogen, respectively) were compared to the predictions of steady-state and non-steady-state models. Steady-state isotopic enrichment at leaf evaporative sites (Δes) was modelled as described previously (Craig & Gordon 1965; Dongmann et al. 1974; Farquhar et al. 1989b) using the expression
where ɛ* is the equilibrium fractionation factor between liquid and vapour, ɛk the kinetic fractionation that occurs during diffusion from the leaf to the atmosphere, Δv the isotopic discrimination between atmospheric vapour and source water (i.e. Δv = Rv/Rs − 1; Rv = 18O/16O or D/H of vapour and Rs = 18O/16O or D/H of source water), and ea/ei the ratio of ambient to intercellular vapour pressures. The ɛ* was estimated using the regression equations of Bottinga & Craig (1969) for oxygen and Majoube (1971) for hydrogen. The ek was calculated for oxygen according to the equation (Farquhar et al. 1989b)
where rs and rb are the stomatal and boundary layer resistances for diffusion of water vapour in air. The coefficients 28 and 19 in the above equation represent discrimination factors (scaled to per mil) for the respective diffusions of H218O through stomata (Merlivat 1978) and leaf boundary layer (Farquhar et al. 1989b). The corresponding coefficients used for calculating ɛk for HDO were 25 and 17.
We predicted the average steady-state leaf water enrichment from the estimated enrichment at the evaporative sites using the following formula (Farquhar & Lloyd 1993):
The ρ is a dimensionless number termed the Péclet number which is defined as EL/(CD), where E is transpiration rate, L a scaled effective path length, C the molar concentration of water, and D the diffusivity of H218O or HDO in water.
The steady-state model of isotopic enrichment at the evaporative sites can then be further modified to take into account non-steady-state effects (Farquhar and Cernusak, unpublished) as follows:
where Δen is the non-steady-state enrichment (‰), W the leaf water concentration (mol m−2), t time (s), g total conductance of stomata plus boundary layer (mol m−2 s−1), and wi the mole fraction of water vapour in the leaf intercellular spaces (mol mol−1). By analogy, the average leaf water isotopic enrichment in the non-steady-state (ΔLn) can be modelled as
Equations 1 to 5 were parameterized to develop steady-state and non-steady-state predictions of evaporative site and average leaf water isotopic enrichment. For Eqn 2, ɛk was estimated using gas exchange data; estimates of boundary layer conductance were based on measurements of average leaf dimensions and wind speed at the adjacent weather station (Mount Barker, Western Australia). We estimated Δv by assuming that atmospheric water vapour was in isotopic equilibrium at the mean daily temperature with source water as inferred from measurements of the isotopic composition of xylem sap water. Values for ea/ei were estimated from measurements of relative humidity, air temperature, and leaf temperature. Transpiration rates from the gas exchange measurements conducted during daylight hours were used in Eqn 3. In the same equation, we assumed a scaled effective path length of 8 mm (Flanagan et al. 1994). Equations 4 and 5 were solved iteratively using the Solver function in Microsoft Excel and assuming starting values for Δen and ΔLn of 15 and 40‰ for oxygen and hydrogen at 0930 h on day 1 (the start of the study). Values for W and g were measured during the day. At times in the night when gas exchange measurements were not possible, we assumed a g of 75 mmol m−2 s−1. The predicted enrichment values were compared to observed enrichment values (Δo), calculated as
where δo is the observed δD or δ18O of leaf water, and δs is the δD or δ18O of source water.
The relationship between the enrichments of δD and δ18O provides an additional means of evaluating predictions made from the models. Theoretical predictions of δD–δ18O slopes in leaf water have been compared to measured values in earlier studies (Allison, Gat & Leaney 1985; Walker et al. 1989; Yakir, DeNiro & Gat 1990; Walker & Lance 1991), but by using Eqns 1 to 5, this approach can be extended to include kinetic fractionation of the boundary layer outside the stomata, the differential diffusivities of HDO and H218O in water, and non-steady-state effects. For a series of measurements under varying conditions, a regression line can be drawn through the points yielding a slope and intercept in ΔD–Δ18O space. The slope of such a line is sensitive to changes or errors in the estimate of atmospheric vapour isotopic composition, and so should provide a means of validating assumptions relating to isotopic equilibria between source water and atmospheric vapour. We tested for discrepancies between regression slopes stemming from modelled versus measured data using analysis of covariance in systat 9·0 (SPSS Inc).
As expected during the approach to summer in a Mediterranean environment, large diurnal variations in ambient air temperature and relative humidity occurred (Fig. 1a & b). Air temperature varied markedly, peaking near 27 °C in mid-afternoon and then falling below 8 °C at night. Relative humidity decreased during the day to near 20% by afternoon, but then recovered fully to 100% early in the night.
The highest photosynthetic rates of source leaves occurred in the early morning (Fig. 1c) followed by a progressive decrease during the rest of the morning through to mid-afternoon. As leaf-to-air vapour pressure deficits moderated in the late afternoon a second, albeit smaller, peak in photosynthesis occurred. A negative linear correlation existed between mean leaf photosynthetic rates and vapour pressure deficit (R = −0·75, P = 0·01, n = 10), presumably reflecting increasingly reduced stomatal conductance under increasing water stress. Intact whole fruits exhibited slight net gaseous gains of carbon early in the mornings, but incurred continuous net losses of CO2 for the remainder of the day. However, because these losses were consistently less during the day than after dark, pod wall photosynthesis appeared capable of significant refixation of CO2 respired to the fruit interior by the seeds. Transpiration losses of leaves and fruits tended to be greater in the morning and early afternoon than in late afternoon, again indicating progressive decreases in stomatal conductances during the day (Fig. 1d).
As to be expected, leaf RWC decreased during the morning hours when transpiration rates were highest, reaching a minimum in the early afternoon (Fig. 2a). It then increased until the following morning as the water balance of leaves was progressively restored. The sucrose concentration of phloem sap increased throughout the morning with a sharp peak at 0·38 mol L−1 in the early afternoon (Fig. 2b) preceding a steady decline until the following morning. Although apparently diminishing through the night, phloem sap sucrose concentration did not fall below 0·30 mol L−1, despite photosynthesis having ceased many hours earlier. A negative relationship was observed between daytime phloem sap sucrose concentration and leaf photosynthetic rates (R = −0·72, P = 0·02, n = 10), namely the opposite of what one might expect if the current rate of sugar production in photosynthesis were directly determining sap concentration. Phloem sap sugar concentration was also negatively correlated with leaf RWC (R = −0·85, P < 0·0001, n = 15) and positively correlated with atmospheric vapour pressure deficit (R = 0·73, P = 0·002, n = 15).
Isotopic composition of dry matter components
The δ15N values of bulk dry matter of transport fluids, leaves, and fruits changed only marginally during the day. But as seen from the mean values given in Fig. 3a, the organic matter of fruits and especially leaves was consistently more depleted in 15N than that of either xylem sap or phloem sap. Of the transport fluids, phloem sap was depleted in 15N by more than 1‰ compared to xylem sap.
The corresponding mean δ13C values for dry matter components showed phloem sap to be less negative than xylem sap (Fig. 3b). As to be expected from earlier studies showing phloem dominated import of organic solutes by lupin fruits, seed dry matter showed a carbon isotope ratio conforming more closely to that of phloem sap dry matter than xylem sap dry matter. The δ13C of pod-wall dry matter was about 1‰ more negative than that of seed dry matter, whereas δ13C of source leaves was 3‰ more negative than that of seeds. The δ13C values for samples of phloem sap dry matter collected through the study were positively correlated with sucrose concentration for samples taken during daylight hours (Fig. 4). However, δ13C values did not show a significant correlation with sucrose concentration for those few sap collections made at night (Fig. 4).
Mean oxygen isotope ratios differed amongst all dry matter components with the exception of the similar values recorded for phloem sap and seeds (Fig. 3c). The values ranked somewhat similarly in relative magnitude to those mentioned above for δ13C, with leaf dry matter least enriched, pod wall dry matter most enriched, and mean values for phloem sap, xylem sap, and seeds intermediate between leaves and pod walls. Marked fluctuations were observed in time courses for δ18O in dry matter for both phloem sap (Fig. 5a) and leaves (Fig. 5b). Thus, dry matter of phloem sap was least enriched in the morning, increased in the afternoon, and then remained enriched throughout the night before decreasing again the following morning. The δ18O values for leaf dry matter also increased in the morning, but then decreased throughout the night before increasing the following morning. Diurnal changes in δ18O of pod-wall and seed dry matter (Fig. 5c) were similar to, but less pronounced, than those for leaves.
Isotopic composition of water components within the system
A strong diurnal variation was observed in the oxygen and hydrogen isotope ratios of leaf water (Fig. 6a & b), encompassing maxima of 23 and 62‰, respectively, in the early afternoon, followed by almost linear decreases in both entities to well-defined minima near 0‰ just before sunrise. By contrast, xylem water, presumably recently accessed from the soil by roots, showed no appreciable diurnal variation in either δ18O or δD with values fluctuating only marginally around means of −2·3 and −13·2‰, respectively.
Phloem sap water generally showed δ18O and δD signatures intermediate between current values for leaf water and xylem sap water (Fig. 6a & b), and the moderate diurnal variations shown for phloem sap water lagged slightly behind those for bulk leaf water. Maximum phloem sap water isotope ratios were 15·2‰ for δ18O and 48‰ for δD, with minimum values of 7·6 and 19·8‰, respectively.
Isotopic compositions (δ18O and δD) differed only slightly in water of pod walls and seeds, showing relatively slight diurnal fluctuations encompassing a range of values intermediate between those for water in phloem sap and xylem sap (Fig. 6a & b). Mean values of δ18O were 6·6‰ for pod-wall water and 6·9‰ for seed water, whereas mean values of δD were 12·3‰ for the same components.
When δ18O and δD values for all water components were matched against each other, the data resolved as a single regression line with a slope of 3·1 (Fig. 7). Slopes and intercepts were not appreciably altered when xylem sap water was included in or excluded from the analyses (P = 0·55 for slope; P = 0·81 for intercept). However, as seen in the itemized data points of Fig. 7, the slope of the regression line relating specifically to leaf water differed significantly (P= 0·005) from that drawn through data for both leaf water and xylem sap water. Figure 7 also shows that δD values for xylem sap water fall slightly below those predicted by the local meteoric water line, δD = 7·3 × δ18O + 11·1 (Townley et al. 1993), for Perth, Western Australia (420 km north of Mount Barker). Based on the observed δ18O of xylem sap water, the predicted δD for precipitation water would be −5·7‰, which can then be compared to the observed value of −13·2‰ for δD of xylem sap water entering shoots from roots of lupin plants.
Predictions from steady-state and non-steady-state leaf water models for evaporative site and average leaf water enrichment (Δ18O and ΔD) are detailed in Fig. 8. For 18O enrichment, the steady-state prediction of water at the site of evaporation (Δes) was generally exceeding that actually observed for bulk leaf water during the course of the day, but was much lower than that shown by leaf water at night (Fig. 8a). The steady-state prediction of average leaf water (ΔLs) for δ18O was close to that observed during the day, particularly in the morning and early afternoon. As transpiration eased off in the late afternoon and evening, the ΔLs prediction converged with the Δes prediction. Non-steady-state predictions of the site of evaporation and average leaf water 18O enrichment were close to observed 18O enrichments through the diurnal cycle. The ΔLn was consistently less than the Δen with the magnitude of difference between the two being noticeably larger at times of high transpiration and lower at times of low transpiration. Patterns of the predictions from the various models for ΔD were qualitatively similar to those for Δ18O, but tended to be shifted down relative to observed enrichments.
The slopes and intercepts of the regression lines drawn in ΔD–Δ18O space are presented in Table 1. The regression slope of the Δes predictions differed slightly from that of the ΔLs predictions (P = 0·03), reflecting the different diffusivities of HDO and H218O in water [i.e. 2·34 × 10−9 m2 s−1 for HDO compared to 2·66 × 10−9 m2 s−1 for H218O (Wang 1954)]. Thus, taking the Péclet effect into account results in a slightly lower regression slope. However, the difference was small, judging from the overlap in the 95% confidence intervals around the two estimates. The inclusion of non-steady-state effects had a negligible influence on the predicted regression slopes for both evaporative site enrichment and average leaf water enrichment (P = 0·73 for Δen; P= 0·82 for ΔLn). The ΔD–Δ18O regression slope of the observed enrichment (Δo) was higher than the predicted slopes for both Δes and ΔLs (P = 0·003 for Δes; P < 0·001 for ΔLs). Results were similar when non-steady-state effects were taken into account (P = 0·02 for Δen; P = 0·009 for ΔLn).
Table 1. Slopes and intercepts for ΔD–Δ18O leaf water isotope enrichment relationships from model predictions and observations, and their respective 95% confidence intervals. Symbols are as follows: predicted steady-state evaporative site enrichment (Δes); predicted non-steady-state evaporative site enrichment (Δen); predicted steady-state average leaf water enrichment (ΔLs); predicted non-steady-state average leaf water enrichment (ΔLn); and observed enrichment (Δo)
95% confidence limits
95% confidence limits
This study represents an integrated account of diurnal variations in stable isotope ratios of O and H in water and C, N, and O in dry matter of translocation streams and source and sink organs of the grain legume Lupinus angustifolius. Sampling fruiting plants over a full diurnal cycle, δ18O and δD of the rapidly exchanging water of transpiring leaves and phloem sap were shown to fluctuate through the day to a much greater extent than the bulk tissue water of less actively transpiring pod walls and essentially non-transpiring seeds. As to be expected, soil-derived source water moving up from the roots, and intercepted as stem xylem sap, showed highly consistent δ18O and δD signals, indicating that diurnal oscillations observed for plant components emanated from fractionation processes within rather than outside the plant. Diurnal variations in isotope composition were also observed in the δ13C and δ18O of phloem sap dry matter, and in the δ18O of leaf and fruit dry matter, but not in the δ13C of bulk dry matter components other than phloem sap. The data collectively indicated complex fractionation and mixing phenomena geared principally around diurnal changes in rates of transpiration and CO2 exchange by leaves and pod walls. Parallel observations of δ15N in transport and source and sink components indicated consistently maintained differences in isotopic composition, probably related to fractionation processes during mixing of fixed atmospheric and soil-derived nitrogen, and therefore only marginally related to the processes alluded to above. In the following sections the principal findings will be discussed in further detail.
Isotopic composition of water components within the system
In those few studies in which both δ18O and δD of leaf water have been assessed simultaneously under field conditions (Allison et al. 1985; Bariac et al. 1989; Bariac, Jusserand & Mariotti 1990; Yakir et al. 1990; Walker & Lance 1991; Flanagan, Marshall & Ehleringer 1993; Bariac et al. 1994), the diurnal patterns of enrichment have typically shown mid-afternoon maxima and early morning minima in composition of both isotopes. Our present data conform to this general pattern. The use of a steady-state model for average leaf water enrichment, represented by Eqns 1 to 3, resulted in reasonably accurate predictions of leaf water δ18O, and to a lesser extent δD, during times when the evaporative flux term g·wi was relatively large. However, as g·wi decreased in the afternoon and on through the night, the discrepancy increased between observed values and those predicted by the steady-state model. We conclude that in modelling exercises in which night-time leaf water enrichment is an important component, such as when evaluating components of dark respiration of ecosystems (Flanagan et al. 1997; Flanagan, Kubien & Ehleringer 1999), it will be necessary to adjust for potential errors related to assuming steady-state isotopic enrichment. Yakir (1998) has addressed this problem, and a recent further extension of the approach by Farquhar and Cernusak (unpublished) indicates that the correction for non-steady-state effects is much more influenced by changes in Δen and ΔLn[see Eqns 4 & 5] than by changes in W. For example, in the present study, proportional changes in W over the diurnal cycle were only about 10% (Fig. 2a), whereas the Δen and ΔLn changed by nearly 80% (Fig. 8).
We believe that the data summarized in Fig. 6 represent the first measurements of the stable isotope composition of water collected from phloem bleeding sap. Adar et al. (1995) reported δ18O values for phloem water of tamarisk using a collection technique involving vacuum distillation of tissues from stems and roots. The latter technique could result in contamination of water samples from components other than translocating cells. Data from the above study suggested that 18O-enriched phloem water could be detected in tamarisk roots up to 12 m below the soil surface. In the present study, based on phloem sap water exuded from cut tips of lupin fruits, variation in δ18O and δD over a diurnal cycle encompassed a range of values intermediate between those of leaf water and xylem water. However, water of the phloem stream appeared slightly more enriched than leaf water in the very early morning, probably reflecting slow translocatory rates from leaf to fruit through the previous night, with phloem sap water accordingly reflecting earlier loading conditions when the leaf water was more enriched. In any event, data for Lupinus and those mentioned above for tamarisk suggest that the fluid contents of the sieve tubes remain sufficiently isolated from surrounding tissue water to transmit enrichment signals from source leaves to sink organs such as fruits or roots to which the translocate is passing.
Judging from earlier models of the economies of water, carbon, and nitrogen of lupin fruits (Pate et al. 1977; Pate et al. 1985), water accumulated in pod walls and seeds should be derived from water entering through both phloem and xylem. Measurements of water isotopes recorded here confirm this suggestion, although the large water content of the fruit tissues appears to effectively buffer fruit water against the relatively large diurnal fluctuations concurrently recorded in δ18O and δD of the leaf water (Fig. 6). Complications due to fractionation during transpiration of pods are to be expected, but were difficult to resolve in the present study.
Plotting of hydrogen and oxygen isotope ratios of different plant water components recorded through the diurnal cycle produced a single mixing line (Fig. 7), just as observed earlier for water in the stem, sheath, and various leaves of barley plants on a single occasion at midday in South Australia (Walker & Lance 1991). The resulting regression line drawn through all water components for lupin intersected almost perfectly that of incoming xylem water, as to be expected if the variations in enrichment among phloem and sink tissues resulted specifically from the mixing of enriched leaf water with un-enriched xylem water (see also Yakir et al. 1990; Yakir, Ting & DeNiro 1994). In our analysis, the regression line based solely on values for leaf water intercepted the meteoric water line slightly above the xylem sap water. Dealing more generally with soil–plant–atmospheric systems, Bariac et al. (1994) found that plots of leaf water δD against δ18O varied in slope according to whether samples were collected during the day or night. However, this effect did not appear as a feature that could be resolved in the present data set.
Theory relating to the enrichment for δD relative to that for δ18O has been discussed for water in lakes by Gat (1971), and for the water in leaves by subsequent authors (Allison et al. 1985; Walker et al. 1989; Yakir et al. 1990; Walker & Lance 1991). In the simplest scenario, where atmospheric vapour remains in equilibrium with source water, the expression denoting the slope (S) of the relative enrichments of δD and δ18O at evaporative sites simplifies from S = [ɛ* + ɛk + (Δv − ɛk)ea/ei]H/[ɛ* + ɛk + (Δv − ɛk)ea/ei]O to S = (ɛ* + ɛk)H/(ɛ* + ɛk)O, where the subscripts H and O denote fractionation factors for δD and δ18O, respectively (Gat 1971; Allison et al. 1985). In this simple case, small variations in S will still apply at different temperatures because the quotient (ɛ*)H/(ɛ*)O decreases with increasing temperature, for example from 9·33 at 10 °C to 8·88 at 20 °C. In contrast, the effect of the partitioning of ɛk between stomata and leaf boundary layer is negligible because the changes are proportionally very similar for HDO and H218O. Thus, for the range of boundary layer and stomatal conductances encountered in the present study, the quotient (ɛk)H/(ɛk)O varied only from 0·8929 to 0·8932. Overall then, S should vary little for water at evaporative sites where atmospheric vapour remains in equilibrium with source water, namely an S-value of 3·20 at 10 °C versus 2·97 at 20 °C. However, as noted previously, when the Péclet effect is taken into account the estimate for average leaf water will differ slightly from that for evaporative sites.
In practice, however, there is likely to be diurnal variation in temperature such that Δv will not equal –ɛ* at all times during the diurnal cycle. In addition, if ea varies, identical values for ea/ei may occur at different leaf temperatures. In such cases the relative changes in δD and δ18O will differ through the day (Walker & Lance 1991). Moreover, although it is generally expected to be the case that atmospheric vapour is close to isotopic equilibrium with source water in natural systems (Jouzel et al. 1991), there may be specific situations that will not conform to this generalization (White & Gedzelman 1984).
The regression slopes drawn in ΔD–Δ18O space and listed in Table 1 differ from the slope (S) as defined by Gat (1971) in that they are drawn through several points collected through a diurnal cycle and plotted as enrichment over source water (i.e. Δ). For the values in Table 1, the modelled regression slope differed slightly from the observed slope, namely with a predicted value for ΔLn of 2·25 versus an observed value of 2·73. This small discrepancy may have resulted from the assumption that isotopic equilibrium existed between atmospheric vapour and source water. Our estimate of δ18O of water vapour, based on the equilibrium fractionation factor at the mean daily temperature, was −11·85‰. This is close to the mean vapour δ18O observed in Perth between July 1996 and January 1998 for measurements conducted on a semi-weekly basis (mean =− 12·26‰, SD = 1·63‰, n= 56; John Rich, Ph.D. dissertation, unpublished). The slight under prediction of ΔD for average leaf water could then have resulted from an error in the estimate of atmospheric vapour δD and this would also account for the discrepancy between the modelled and observed ΔD–Δ18O slope. Our equilibrium estimate for vapour δD was −97·6‰, which differs from the mean value observed in Perth of −85·0‰ (SD = 10·1‰, n= 107; John Rich, Ph.D. dissertation, unpublished). However, use of the mean Perth value in the ΔD models resulted in larger discrepancies between the predicted and observed ΔD–Δ18O slope and larger errors in the diurnal ΔD predictions.
The predicted steady-state evaporative site enrichment for δ18O of water in transpiring leaves was larger than the observed leaf water enrichments in the morning and early afternoon (Fig. 8a). Previous authors have reconciled similar discrepancies by assuming that some fraction of the water of leaves comprises un-enriched vein water recently derived from the xylem and not yet subject to evaporation (Allison et al. 1985; Leaney et al. 1985; Walker et al. 1989; Walker & Lance 1991; Yakir 1992; Roden & Ehleringer 1999; Roden, Lin & Ehleringer 2000). In the present study, we took a different approach in that variations between predicted evaporative site enrichment and observed leaf water enrichment were partly reconciled by considering the Péclet and non-steady-state effects.
Isotopic composition of dry matter components
The δ18O of oxygen atoms in carbonyl groups of organic molecules is influenced by exchange with those in surrounding free water, involving an equilibrium fractionation factor estimated to be +27‰ (Sternberg & DeNiro 1983; Sternberg, DeNiro & Savidge 1986). The specific possibilities for such exchanges during synthesis of sucrose have been outlined in detail by Farquhar, Barbour & Henry (1998), who ascribe particular importance to processes whereby triose phosphates are exported from chloroplasts and subsequently consumed in sucrose synthesis in the cytoplasm of mesophyll cells. As suggested by the above authors, exchange rates for oxygen atoms in triose phosphates are expected to be fast, so that under high rates of assimilation the equilibrium of oxygen of triose molecules with cytosolic water should be completed well before their incorporation into sucrose. It is expected that there should be no further major exchanges of oxygen atoms occurring until the translocated sucrose is broken down in the sink tissues of the system. Consistent with this, Barbour et al. (2000) found that phloem sap sucrose exported from castor bean leaves was in close isotopic equilibrium with predicted average leaf water.
In the present study, we observed a relatively large diurnal variation in the δ18O of total leaf dry matter (Fig. 5b), apparently indicating that carbohydrates were accumulated and exported with δ18O values consistently more enriched than that of the bulk structural dry matter of leaves. This would be expected if the δ18O signals of assimilated sugars were in close isotopic equilibrium with the δ18O of leaf water at the time of fixation. Earlier data recorded by Sharkey & Pate (1976) for lupin leaves showed a 15% diurnal increase from dawn to dusk in dry matter concentration, with about half the marked afternoon increase accountable as starch deposited in chloroplasts and half as sugar in the cytosol of leaf tissues. In the present study, we observed a 19% increase (12·3 g m−2) in dry matter concentration of leaves from pre-dawn (64·8 ± 1·5 g m−2) to late afternoon (77·1 ± 3·8 g m−2). This observation is reasonable considering that a rough integration of the photosynthetic rates shown in Fig. 1c suggests a daily fixation of 18·5 g dry matter m−2 d−1.
To illustrate how the accumulation and export of sucrose and starch could potentially alter the δ18O of total dry matter of leaves, we performed the following simplified calculation. We began by assuming that photosynthesis, averaged over the day, occurred at a leaf water value of 20‰ (Fig. 6a). Taking the equilibrium fractionation between medium water and organic molecules into account, the assimilated sugars should then have an average δ18O value of 47‰. The pre-dawn leaves had a total dry matter δ18O value of 28‰ (Fig. 5b). Assuming equivalent oxygen concentrations between the assimilated sugars and the pre-dawn leaf, the addition of 12·3 g m−2 of dry matter having an average δ18O of 47‰ would raise the δ18O of total leaf dry matter from 28 to 31‰ by the end of the day. This additional photosynthate would then be exported through the night and the leaf would return to its initial value of 28‰ by pre-dawn the next day. We speculate that there may additionally be some rapid turnover of oxygen atoms in organic compounds not subject to export from the leaf that would in turn contribute to the diurnally fluctuating δ18O of total leaf dry matter. The accumulation and export of enriched carbohydrates, combined with perturbations of this nature, would then be sufficient to account for the 4‰ diurnal variation in total leaf dry matter δ18O that we observed for Lupinus angustifolius. It is worth noting, however, that such a pronounced diurnal fluctuation in the δ18O of leaf dry matter would probably only occur in crop plants such as lupin that have high photosynthetic rates, low leaf mass per area, and pronounced diurnal cycles of carbohydrate accumulation and export. Long-lived, perennial plants that do not possess these traits would not be expected to show a marked diurnal fluctuation in total leaf dry matter δ18O, and of course no diurnal fluctuation would be expected in δ18O of leaf cellulose for any plant.
Somewhat surprisingly, the δ18O signal of phloem sap sucrose collected from pod tips appeared closer to equilibrium with tissue water in pod walls and seeds than with average leaf water. One explanation would be that the phloem sap dry matter at the tips of fruits does not entirely comprise sucrose exported directly from leaves. Unfortunately, unlike Lupinus albus (Pate 1986), Lupinus angustifolius fails to bleed sap from leaf petioles, so it is not possible to intercept translocate immediately at its point of exit and thereby compare its isotopic composition with that of translocate collected 20–30 cm away in recipient fruits.
The sucrose concentration of phloem sap collected in this study from fruits of Lupinus angustifolius showed a strong diurnal variation, as observed previously for Lupinus albus (Sharkey & Pate 1976), and Lupinus angustifolius (Hocking et al. 1978). Although correlating negatively with leaf photosynthetic rate, variations in sucrose concentration tracked closely with leaf water status, as affected by diurnal changes in leaf-to-air vapour pressure deficit and RWC. A response was reported previously for Ricinus communis in which the onset of water stress was shown to induce a net increase in the solute content of sieve tubes, so that positive turgor pressure was maintained in phloem despite steeply declining water potential in the surrounding tissues (Smith & Milburn 1980).
One would expect that in any translocatory system, sugar concentrations in the phloem sap should increase to a threshold value at which the osmotic pressure of sieve tubes is sufficient to drive the translocation process. It would then follow that as water potentials in the surrounding tissues continue to fall, higher sugar concentrations would be necessary for continued functioning of the transport system. Pate et al. (1998) reported a strong correlation between values for δ13C of phloem sap sugars and concurrent phloem sap sugar concentrations across a wide range of seasonal and habitat conditions for plantation-grown Eucalyptus globulus. This strong relationship is indicative of close coupling between stress and osmotically mediated parameters, particularly as δ13C of plant photosynthate increases consistently with water stress (Farquhar & Richards 1984). In the present study, we observed a similar positive linear relationship between phloem sap sugar concentration and phloem sap sugar δ13C for samples collected over a relatively narrow range of environmental conditions during daylight hours. The observed uncoupling of the relationship at night might then be resolved if night-time translocate were derived, at least in part, from the mobilization of starch stored in leaves during previous photoperiods (Sharkey & Pate 1976; Hocking et al. 1978).
The regression line relating carbon isotope discrimination to sucrose concentration for the daytime samples was Δ13C = 30 − 38·[sucrose] (Fig. 4). This relationship suggests that Δ13C will decrease to 4·4‰, the value expected when stomata are completely closed, at a sucrose concentration of 0·69 mol L−1. At 20 °C, this sucrose concentration corresponds to an osmotic pressure of approximately 2·1 MPa (Nobel 1999), suggesting that canopy gas exchange should cease at leaf water potentials of −2·1 MPa, assuming no excess turgor in sieve tubes at that point. The analysis is simplified insofar as it ignores the osmotic pressure exerted by molecules other than sucrose in the phloem sap. Furthermore, there is no a priori reason to assume that the relationship between Δ13C and phloem sap sugar concentration should be linear over its full range. More detailed analyses of this relationship could prove useful in future investigations.
Mean differences observed in δ13C values for different dry matter components of the Lupinus angustifolius system spanned a range of more than 3‰, with leaves most negative (−27·7‰) and seeds least negative (−24·5‰). Differences of similar magnitude and direction between δ13C of leaves and fruits have been reported for Glycine max (Yoneyama, Fujiwara & Engelaar 2000), Cicer arietinum (Behboudian et al. 2000), Ricinus communis (Yoneyama, Fujiwara & Wilson 1998), and for a range of legume species (Yoneyama & Ohtani 1983). In the present study, the δ13C of seeds of Lupinus angustifolius matched almost exactly that of the phloem sap carbon, whereas the negative values recorded for dry matter of donor leaves might simply have resulted from the structural dry matter of the foliar canopy having been laid down earlier in the growing season when plants were subject to consistently lower water stress. A similar situation where leaves were reported to contribute carbon to phloem sap with less negative δ13C values than their own dry matter was reported by Pate & Arthur (1998) for a study of Tasmanian Blue Gum (Eucalyptus globulus) carried out under essentially similar seasonal and climatic conditions to those experienced at Mount Barker. The above authors also showed that seasonal variations in carbon isotope ratios of phloem sap sugar were consistently reproduced in δ13C values of the new dry matter laid down in shoot tissue and secondary xylem. It is likely that similar seasonal changes in δ13C of phloem sap carbon of Lupinus angustifolius would be progressively recorded in the isotopic signatures of seed dry matter. However, one cannot for the present exclude the possibility of appreciable, albeit probably minor, fractionation events during export from leaves, translocation to fruits, and eventual conversion of sugars to dry matter.
Pod walls of Lupinus angustifolius showed a δ13C for their dry matter that was almost 1‰ more negative than that of the seeds. A similar differential has also been observed between pod walls and seeds of Cicer arietinum (Behboudian et al. 2000). The gas exchange measurements made here, in conjunction with earlier studies on lupin fruits (Pate et al. 1977), indicate a significant refixation of seed-respired CO2 by the pod. Cernusak et al. (2001) recently developed theory describing carbon isotope discrimination during refixation in photosynthetic bark, a process presumably analogous to refixation in pod walls. Their model suggests that refixation should contribute carbon with a δ13C more negative than that of the respiratory carbon source. A greater proportional contribution of refixed carbon relative to carbon originating from source leaves in the dry matter of pod walls compared to seeds could then explain the difference in δ13C between the two tissues.
Nitrogen isotope ratios varied among dry matter components across a surprisingly large range (3·6‰), with nitrogen accumulated in the dry matter of leaves and fruits more depleted in 15N than that currently flowing in the xylem and phloem. A similar tendency for δ15N of vegetative tissue to be depleted relative to incoming xylem δ15N has also been observed for other species: Ptilotus polystachyus (Pate, Stewart & Unkovich 1993), Triticum aestivum (Yoneyama et al. 1997), and Glycine max (Yoneyama et al. 2000). Between the two transport fluids of Lupinus angustifolius, phloem sap was more depleted in 15N than xylem sap, as similarly recorded for Ricinus communis at different times of plant development (Yoneyama et al. 1998), and for Triticum aestivum during grain filling (Yoneyama et al. 1997). In our study at Mount Barker, root systems were well nodulated and therefore able to feed their shoots with fixed nitrogen (δ15N close to 0‰) while also accessing mineral nitrogen from the soil (δ15N for NO3-N recorded for the site in the range of 5·3–5·5‰; J.S. Pate, unpublished results). Differential partitioning of fixed nitrogen to certain plant parts combined with targeting of soil-derived nitrogen to other parts earlier in the growing season might then explain the observed differences in δ15N between different organs of plants sampled at the fruiting stage.
Of the stable isotope ratios that we examined progressively through a diurnal cycle, some components such as δ18O and δD in leaf water showed a pronounced and distinctly diurnal variation, whereas others such as δ13C and δ18O in phloem sap dry matter showed more complex oscillations, possibly reflecting fractionations associated with the daily sugar/starch storage cycles of the leaves. Further insight into the effects of internal cycling and acquisition processes on isotopic fractionation within the source and sink organs of plants will undoubtedly contribute to the successful interpretation of multiple isotopic data sets in terms of plant performance under field conditions.
We thank David Arthur for excellent assistance with field collections. Sue Wood, Chin Wong, and Qifu Ma provided technical assistance with field and laboratory measurements. Lidia Bednarek of the West Australian Biogeochemistry Center, Perth kindly assisted with hydrogen isotope analysis of water samples. We thank John Rich and Jeff Turner for making available their data for isotopes in atmospheric water vapour in Perth. This research was partly funded by the Grains Research Council of Western Australia.
Received 11 November 2001;received inrevised form 26 February 2002;accepted for publication 27 February 2002