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Keywords:

  • allocation;
  • δ13C;
  • diurnal variation;
  • functional groups;
  • isotope fractionation;
  • pyruvate positional labelling;
  • respiration;
  • respired CO2

Summary

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Supporting Information
  • • 
    The first broad species survey of diurnal variation in carbon (C) isotope signatures of leaf dark-respired CO213Cres) is presented here and functional differences and diurnal dynamics are linked to fractionation in different respiratory pathways, based on 13C-labelling experiments.
  • • 
    δ13Cres was analysed with a rapid in-tube incubation technique in 16 species.
  • • 
    A large diurnal increase in δ13Cres (4–8‰) occurred in evergreen, slow-growing and aromatic species and correlated significantly with cumulative photosynthesis, whereas no variation occurred in herbaceous, fast-growing plants or temperate trees. The diurnal increase in δ13Cres declined almost proportionally to reductions in cumulative light and was reduced in growing compared with mature leaves.
  • • 
    Pyruvate positional labelling provided direct evidence that functional groups differ in C allocation between respiratory pathways owing to different metabolic demands for growth, maintenance and secondary metabolism. Diurnal increase in C flux through pyruvate dehydrogenase (for investment in, for example, isoprene or aromatic compounds) combined with consistently low Krebs cycle activity resulted in pronounced increase in δ13Cres in evergreen and aromatic species. By contrast, fast growing herbs with high respiratory demand exhibited no diurnal changes since C was fully respired. Hence, diurnal δ13Cres pattern may provide information for C allocation in plants.

Introduction

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Supporting Information

Increasing atmospheric carbon dioxide concentrations and corresponding climate change have increased the demand for a better process-based understanding of carbon (C) exchange processes (i.e. photosynthesis and respiration) at individual plant and ecosystem scales. Stable C isotopes are a sensitive tool for disentangling C fluxes from the leaf to the ecosystem level (Yakir & Sternberg, 2000; Bowling et al., 2008) and for analysing biophysical and biochemical processes in photosynthetic pathways. The marked discrimination against the heavier isotope (13C) during photosynthesis has been well characterized (Farquhar et al., 1982, 1989). Similarly, fractionation during photorespiration is thought to be significant (Sharkey, 1988; Gillon & Griffiths, 1997), while apparent fractionation during dark respiration has long been considered negligible (Lin & Ehleringer, 1997). However, there is now evidence for substantial apparent fractionation leading to differences between the C isotope composition of leaf dark-respired CO213Cres) and its putative substrates in many C3 species (Ghashghaie et al., 2003). Therefore, δ13Cres has become the subject of several recent studies (Schnyder et al., 2003; Tcherkez et al., 2003; Nogués et al., 2004; Hymus et al., 2005; Klumpp et al., 2005; Prater et al., 2006; Bathellier et al., 2008). In general, foliar δ13Cres has been found to be 13C-enriched compared with a wide variety of metabolites, for example, an enrichment of 9‰ in dark-respired CO2 relative to plant organic material was found in Nicotiana sylvestris (Ghashghaie et al., 2001). This apparent fractionation effect is highly variable, changing with species and environmental factors (for a review, see Ghashghaie et al., 2003). Based on these laboratory experiments there is now increasing knowledge on the mechanisms accounting for fractionation occurring during dark respiration at the leaf level (Tcherkez et al., 2003; for recent reviews see Ghashghaie et al., 2003; Werner et al., 2007a). Indeed, based on the heterogeneous C isotope distribution in hexose molecules (DeNiro & Epstein, 1977; Rossmann et al., 1991; Gleixner et al., 1998), Ghashghaie et al. (2001) indicated two metabolic origins for the respired CO2 (oxidation of pyruvate releases 13C-enriched CO2 relative to substrate while the acetyl-CoA decarboxylated through the Krebs cycle is depleted) possibly accounting for the 13C-enrichment of the overall respired CO2 compared with respiratory substrates. Recent work has also shown that CO2 respired by tree trunks is in general 13C-enriched, while that of roots is 13C-depleted compared with their respective bulk organic matter or carbohydrates (Badeck et al., 2005; Klumpp et al., 2005; Gessler et al., 2007; Maunoury et al., 2007; Bathellier et al., 2008).

However, only a few studies have focused on potential diurnal short-term variations of δ13C of dark-respired CO2. Indeed, assuming that the pool of fresh assimilates carries C of variable isotope composition resulting from changes in photosynthetic discrimination, it can be expected that the isotope ratio of dark-respired CO2 may also change during the light period, even without involving any fractionation by the process of respiration itself. In field studies, Hymus et al. (2005) and Prater et al. (2006) found a pronounced enrichment of respired CO2 along a light period up to 5–10‰ when compared with the respired δ13CO2 measured during the dark period. This 13C enrichment was correlated with the concomitant cumulative CO2 assimilation (Prater et al., 2006). Similarly, rapid dynamics, though with smaller magnitudes, have been shown in other ecosystem compartments, for example, at the trunk and soil levels (Maunoury et al., 2007; Kodama et al., 2008).

The isotopic signature of ecosystem-respired CO213CR) is a complex response of different respiratory sources, including respiration by autotrophic and heterotrophic organisms. Ecosystem respiration is still poorly understood even though it is a major component of the global C balance (Valentini et al., 2000; Reichstein et al., 2002; Davidson et al., 2006). Understanding the driving environmental factors of δ13CR is therefore important for applications of isotope-based models of the global C budget. So far, the short-term dynamics of the C isotopic composition of respired CO2 have been disregarded in most studies despite their potential implications, for example, for the sampling protocols used to collect nocturnal Keeling plots. There is increasing evidence of rapid dynamics (minutes to hours) of δ13CR (i.e. 4‰ and 6‰ during one night; Bowling et al., 2003; Werner et al., 2006, respectively). However, data on this topic are scarce and the understanding and identification of the isotopic effects during dark-respiration defined by Tcherkez et al. (2003) are far from being resolved (Tcherkez & Farquhar, 2005). Taking advantage of the rapid in-tube incubation method (Werner et al., 2007b), this paper aims to investigate the different metabolic processes influencing the isotope composition of respired CO2 by analysing diurnal variation in dark-respired δ13CO2 in a wide range of ecotypes and species.

Most studies on fractionation during dark respiration have been performed on fast-growing herbaceous species under laboratory conditions. Werner et al. (2007b) were the first to show pronounced differences in two different functional plant types under standardized conditions: no significant diurnal variation in δ13Cres occurred in a fast-growing herb, while a pronounced δ13CLight–Dark amplitude of 8‰ occurred in a Mediterranean oak. Here, we explore the hypothesis that the extent of diurnal increase in δ13Cres varies between plant functional types. We present the first species survey to characterize different functional groups in relation to structural and metabolic features such as leaf thickness, C : N ratios and photosynthesis. Further, we use pyruvate positional labelling experiments, which provide the first direct evidence of the importance of apparent fractionation processes in respiratory pathways for the observed functional differences in diurnal variation of plant δ13Cres.

Materials and Methods

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Supporting Information

Plant material – growth and experimental conditions

Controlled conditions  Woody species including trees (4-yr-old Quercus ilex L. seedlings (height approx. 40 cm), 2-yr-old Pinus pinea L.) and shrubs (2-yr-old Arbutus unedo L., Ceratonia siliqua L., Citrus hystrix DC., Ficus benjamina L. and Halimium halimifolium L.) as well as herbaceous plants (Tolpis barbata Gaertn., Oxalis triangularis A. St-Hil.) and aromatic species (Mentha piperita L. and Rosmarinus officinalis L.) were grown under stable controlled climate conditions. Artificial light in a growth chamber was provided from 08 : 00 h to 20 : 00 h (or 09 : 00 h to 21 : 00 h) with 200 µmol m−2 s−1 for all species, up to 350 µmol m−2 s−1 for oak leaves. The air temperature was 25°C and 15°C during the light and dark periods, respectively. The relative air humidity was 60%. Plants received 150 ml of water twice a week and were fertilized once a week with 1/8th strength of Hoagland's Fertilizer Solution.

Field conditions  Two herbaceous species (Trifolium pratense L., Bellis perennis L.) and three deciduous trees (Quercus petraea L., Carpinus betulus L. and Sorbus cashmiriana Hedl.) were sampled near the University campus of Bielefeld, Germany. Fully developed leaves from south-facing canopy were collected in June 2008 during two periods of the day: at the beginning of the light period (between 06 : 15 h and 07 : 00 h) and at the end of the light period (from 21 : 15 h to 22 : 00 h). At the time of collection, the mean temperature was 11°C in the morning and 25°C with a maximum light level of 1145 µmol m−2 s−1 in the afternoon.

Leaf structure parameters

Specific leaf area (SLA) was calculated as the ratio of leaf area, measured with a leaf area meter (Delta-T Scan, Cambridge, UK), to leaf dry weight, measured after drying samples for 48 h at 60°C. The relative leaf water content ((FW – DW)/FW), with FW and DW representing fresh and dry weights, respectively, was determined.

Gas exchange measurements

Net photosynthesis was measured on H. halimifolium and Q. ilex mature leaves five times during the day (after 1, 4, 7, 10 and 11 h 45 min of light) using a WALZ CMS-400 minicuvette system (WALZ, Effeltrich, Germany) equipped with an IRGA (BINOS 100, CO2 and H2O channels, Rosemount, Chanhassen, USA). Consecutive measurements on attached leaves were performed at 25°C under growth light intensity and 380 ppm CO2 with controlled leaf vapour pressure deficit and waiting at least 30 min for acclimatization. Carbon dioxide accumulation along the photoperiod was expressed in mol CO2 m−2 s−1 and calculated by multiplying the averaged net C assimilation by the considered duration of the light period (1, 4, 7, 10 and 11 h 45 min).

Changing incident light conditions

To evaluate the dependence of the diurnal variation in δ13Cres on the daily C gain, growth light intensity was reduced to 50% by increasing the distance between the plants and the light source on H. halimifolium plants, grown from seeds under full light or 80 µmol m−2 s−1, as well as in acclimatizing half of the slow-growing Quercus ilex trees to the low-light conditions (180 µmol m−2 s−1) for 3 wk. Alternatively, H. halimifolium and Q. ilex were subjected to a 3-h dark period in the middle of the diurnal course.

Isotope measurements

δ13C of respired CO2 Twelve sampling times were chosen to reflect the diurnal cycle of the respired CO2 signature: 6 : 00 h, 7 : 45 h (before the light period), 9 : 00 h, 12 : 00 h, 15 : 00 h, 18 : 00 h, 19 : 45 h (during the light period), 20 : 15 h, 20 : 30 h, 21 : 00 h, 22 : 00 h and 23 : 00 h (during dark). Sampling and analysis were performed by the rapid in-tube incubation method as described in Werner et al. (2007b, see below). The diurnal increase in δ13Cres13CLight–Dark, expressed in ‰) was calculated when not otherwise specified as the difference between δ13Cres measured at the end of the light and dark periods.

In-tube incubation measurements  To measure the isotopic composition of respired CO2, collected leaf segments or entire fully developed leaves were inserted into a 12 ml glass vial (Exetainer; Labco, High Wycombe, UK). The vials were flushed in the dark for 1 min with CO2-free air, provided by a 10 l min−1 membrane pump pushing atmospheric air through two Plexiglas columns (height, 29 cm; diameter, 4 cm) of soda lime (Carbosorb Sodalime granules; BDH Laboratory Supplies, Poole, UK), as described in Werner et al. (2007b). Leaves were left to respire in the dark for precisely 3 min to gain sufficient CO2 (> 350 ppm) for analysis in the mass spectrometer and minimize the incubation time. A precise incubation time is required as large isotope effects can occur within minutes upon darkening (Barbour et al., 2007; Werner et al., 2007b), in all functional groups (data not shown). After 3 min incubation the isotope samples were immediately measured with an IRMS (Isotope Ratio Mass Spectrometer, IsoPrime; GV, Manchester, UK) interfaced to an autosampler (Microgas; GV).

Positional 13C-labelling experiments  Based on Tcherkez et al. (2005), who use 13C-labelled pyruvate molecules to quantify the relative respiratory fluxes in illuminated and darkened leaves, mature leaves from H. halimifolium and O. triangularis were fed through the transpiration stream with 13C-labelled pyruvate solutions (5 mm pyruvate labelled either at the C1 or both at the C2 and C3 carbon positions: 99%13C; Cambridge Isotope Laboratories, Andover, MA, USA). Leaves were cut at the petiole, immediately recut under water and incubated in the labelled pyruvate solution in the climate chamber. After a 15-min incubation the δ13Cres of leaves or leaf discs was determined by the in-tube incubation method as described earlier.

δ13C of total leaf organic material  Leaves were collected 1 h before sunrise and 1 h before sunset and immediately oven-dried at 60°C for 48 h. After placing samples in desiccators overnight at room temperature, individual leaves were weighed and milled to fine powder, and 2 mg was used for mass spectrometer analysis.

Sample preparation was performed in an elemental analyser (EuroVector, Hekateck, Germany) interfaces to the IRMS. Samples are automatically combusted and analysed in a continuous-flow isotope ratio mass spectrometer (IsoPrime, GV Instruments, Manchester, UK). Samples were standardized to IAEA-CH-4 and IAEA-CH-6 (International Atomic Energy Agency, Vienna, Austria). A cross-calibrated laboratory gas standard was measured every nine samples to quantify any drift. Values are reported relative to vPDBee, and repeated measurements precision was 0.05‰.

Statistical analyses

If not indicated otherwise, all experiments were repeated independently at least three times and the standard error is given. Analyses of variance and LSD post hoc tests were performed using statistica software (Statsoft Inc., Tulsa, USA) at P < 0.05.

Results

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Supporting Information

Diurnal variation in δ13C of respired CO2

Taking advantage of the in-tube incubation technique (Werner et al., 2007b), diurnal variations of dark-respired δ13CO2 of mature leaves (δ13Cres) were analysed in 16 different species grown under natural or controlled conditions (Fig. 1, Table 1). Overall, δ13Cres ranged widely from −16 to −32‰ 15 min before sunset (δ13CLight) and from −20 to −32‰ 15 min before sunrise (δ13CDark). There was a marked diurnal dynamic in δ13Cres allowing the identification of different functional groups regarding the diurnal increase in δ13Cres13CLight–Dark, i.e. the difference between δ13Cres measured at the end of the light and dark periods). Generally, the functional groups followed two distinct diurnal patterns: (1) a significant increase of δ13Cres during the light period (δ13CLight–Dark) ranging from 1.4 to 7.9‰, Table 1) followed by a continuous decrease in δ13Cres during the dark period; and (2) no significant changes in δ13Cres throughout the light and dark periods (Fig. 1, Table 1). Examples of the characteristic diurnal pattern of δ13Cres are shown in Fig. 1a. The three-slow growing evergreen species (H. halimifolium, Q. ilex and A. unedo) exhibited the largest δ13Cres amplitude with δ13CLight–Dark values of 7.9, 7.3 and 6.9‰, respectively, while the fast-growing herbs Tolpis barbata and Oxalis triangularis showed no pronounced diurnal changes (Fig. 1a). A significant diurnal δ13Cres increase (2.7–6.5‰) was also found in the sclerophyllous P. pinea, F.  benjamina, the Mediterranean evergreen C. siliqua and in the aromatic species R. officinalis, M. piperita and C. hystrix (Table 1). In the diurnal δ13Cres cycle the most enriched signatures were found at the end of the daylight period, whereas the most depleted values occurred during the night (Table 1). These patterns were also observed under natural conditions, with a δ13CLight–Dark of 4.2‰ in C. betulus and 2.9‰ in Q. petraea but no significant variation in the deciduous tree S. cashmiriana or in the herbs T. pratense, B. perennis (Fig. 1b, Table 1).

image

Figure 1. Diurnal variation in the leaf dark-respired δ13CO213Cres) from nine different species. The dark period is indicated by the black bars. (a) Halimium halimifolium (closed diamonds), Arbutus unedo (open diamonds), Quercus ilex (squares), Tolpis barbata (open circles, central point) and Oxalis triangularis (grey circles) grown under controlled conditions (light period from 08 : 00 h to 20 : 00 h; growth light intensity 200–350 µmol m−2 s−1). (b) Quercus petraea (closed squares), Bellis perennis (circles), Sorbus cashmiriana (diamonds) and Carpinus betulus (open squares). Leaf samples were collected in June 2008 (daylight from 07 : 00 h to 22 : 00 h. Three to eleven independent replicates (± SE).

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Table 1.  Carbon isotopic composition of the dark-respired CO213Cres) from fully mature leaves grown either under controlled or natural (*) conditions at the end of the dark and light periods (δ13CDark and δ13CLight, respectively)
Functional groupSpeciesδ13Cres (‰)δ13CLight–Dark (‰)
δ13CDarkδ13CLight
  • *

    Those samples were collected in June 2008 between 06 : 15 h and 07 : 00 h (δ13CDark) and from 21 : 15 h to 22 : 00 h (δ13CLight). The other species were sampled at 07 : 45 h and 19 : 45 h, that is 15 min before the end of the dark and the light period, respectively.

  • Diurnal changes of δ13Cres were calculated as the difference between light and dark values (δ13CLight–Dark, ‰ ± SE).

  • Data are expressed in ‰ (n ≥ 4; ± SE). ns, No significant differences (anova and LSD test, P < 0.05).

Woody
 TreesCarpinus betulus*−25.0 (± 0.1)−20.8 (± 1.3)+4.2
Pinus pinea−28.6 (± 0.4)−25.9 (± 0.2)+2.7
Quercus ilex.−25.9 (± 0.5)−18.6 (± 0.8)+7.3
Quercus petraea*−24.7 (± 0.7)−21.8 (± 1.0)+2.9
Sorbus cashmiriana*−20.3 (± 0.8)−19.9 (± 0.5)ns
 ShrubsArbutus unedo−22.9 (± 0.6)−15.9 (± 0.6)+6.9
Ceratonia siliqua−25.1 (± 0.4)−23.7 (± 0.1)+1.4
Citrus hystrix−30.5 (± 1.1)−26.4 (± 0.7)+4.1
Ficus benjamina−24.4 (± 0.6)−21.1 (± 0.4)+3.4
Halimium halimifolium−28.8 (± 0.2)−20.9 (± 0.8)+7.9
Rosmarinus officinalis−27.4 (± 0.4)−20.9 (± 0.6)+6.5
 HerbaceousBellis perennis*−32.4 (± 1.5)−31.8 (± 0.3)ns
Mentha piperita−30.2 (± 0.5)−24.0 (± 0.8)+6.3
Oxalis triangularis−32.0 (± 0.5)−30.9 (± 0.3)ns
Tolpis barbata−24.6 (± 0.6)−24.9 (± 0.8)ns
Trifolium pratense*−31.3 (± 0.6)−31.6 (± 0.6)ns

δ13C of total leaf organic matter and of soluble sugars

No significant diurnal changes in the C isotopic signature of total leaf organic matter (δ13COM, measured 1 h before sunrise and sunset) was identified in any of the species investigated (Fig. 2a,b). The highest 13COM values were obtained in the Mediterranean species A. unedo, and in S. cashmiriana (−26 to −28‰), and the most negative 13COM values in herbs (−31 to −33‰; see Fig. 2). The δ13COM was 13C-depleted compared with δ13Cres except for the two herbaceous species B. perennis and T. pratense, which exhibited almost similar δ13COM and δ13Cres (compare Table 1 and Fig. 2). The enrichment of δ13Cres relative to δ13COM reached up to 11‰ at the end of the light period for H. halimifolium (Fig. 2). The isotopic composition of soluble sugars, including glucose, fructose and sucrose, and their respective concentrations were determined in four selected species: Q. petraea, Q. ilex, H. halimifolium and T. barbata at two periods of the day, just before sunset and sunrise (data not shown). Those results revealed no significant change in δ13C of the three sugars between the two periods investigated and for all species. In addition, the sucrose concentration slightly decreased during the night while glucose and fructose contents remained constant throughout the day (not shown).

image

Figure 2. Relationship between carbon isotopic signatures of leaf respired CO213Cres) and total leaf organic matter (δ13COM) both measured (a) at the end of the light period and (b) the end of the dark period of species grown either in controlled conditions in the glasshouse or under natural conditions (see the Materials and Methods section). Means (± SE) of at least three replicates from independent leaves are represented.

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Relationship of δ13Cres diurnal course with basic structural parameters

The relationship between leaf characteristics (specific leaf area, SLA), water content and the C : N ratio) with the diurnal δ13C enrichment in leaf respired CO2 allowed the identification of three major functional groups (Fig. 3). Sorted by increasing δ13CLight–Dark amplitude, these are: herbaceous species which had the highest relative water content (> 0.75), SLA (> 0.028 m2 g−1) and the lowest C : N ratio (< 15); deciduous trees, the conifer species and several shrubs (C. hystrix, F. benjamina and C. siliqua) with intermediate values of all leaf parameters; and Mediterranean evergreen trees and shrubs with thick leaves and hence, very low SLA (< 0.015 m2 g−1), high C : N ratio (> 23) and a wide range of water contents as well as aromatic plants exhibiting different leaf structures such as needles, mesophyllic or xerophytic leaves (see Fig. 3).

image

Figure 3. Relationships between the δ13Cres diurnal increase (δ13CLight – Dark, corresponding to the difference between δ13Cres measured at the end of the light period and the end of the dark period) and structural parameters. SLA (specific leaf area), leaf water content (estimated as (FW − DW)/FW, FW and DW representing the fresh and dry weights, respectively) and the total leaf carbon to nitrogen ratio (C : N ratio) of different functional types: herbaceous species (A, circles: Bellis perennis, Oxalis triangularis, Tolpis barbata and Trifolium pratense), trees (B–C, squares) including deciduous temperate trees (Quercus petraea, Carpinus betulus and Sorbus cashmiriana), a conifer species (Pinus pinea) and the Mediterranean oak (Quercus ilex), evergreens or semi-deciduous species (B–C, diamonds; Arbutus unedo, Halimium halimifolium, Ficus benjamina and Ceratonia siliqua) and aromatic species (B–C, triangles; Citrus hystrix, Mentha piperita and Rosmarinus officinalis).

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Effect of diurnal CO2 assimilation on δ13Cres

The effects of cumulative C gain on the diurnal course of δ13Cres were investigated in plants grown under different light conditions by first, acclimatizing Q. ilex saplings to a 50% reduction in incident light intensity (from 350 to 180 µmol m−2 s−1, Fig. 4a) and second, by decreasing the growth light level of H. halimifolium from 200 to 80 µmol m−2 s−1 (Fig. 4b). In both cases, the diurnal increase in δ13Cres decreased almost proportionally (from 7.3 to 3.7‰ in Q. ilex and from 7.9 to 4.5‰ in H. halimifolium, Fig. 4c,d). To assess the effect of growth light intensity or, more specifically, the cumulative incident light per day on the observed diurnal variations in δ13Cres, both H. halimifolium and Q. ilex were subjected to a 3-h dark period in the middle of the diurnal course. The observed decrease in diurnal δ13Clight-dark amplitude was proportional to the decrease in the daily photoperiod length with a 2.1‰ and 2.7‰ decrease in δ13CLight–Dark in H. halimifolium and Q. ilex subjected to the 3 h-dark period, respectively, compared with plants exposed to a full-time photoperiod of 12 h of light (see the Supporting Information, Fig. S1).

image

Figure 4. Diurnal course of (mature) leaf dark-respired δ13CO2 (a,b) and difference between the diurnal δ13Cres increase obtained at five periods of the day (after 1 h, 4 h, 7 h, 10 h and 11 h and 45 min of light) relative to δ13Cres measured at the end of the dark period (δ13CLight–Dark, expressed in ‰) plotted against corresponding cumulative net CO2 uptake (c,d): (a,c) Quercus ilex grown either under 350 µmol m−2 s−1 (Q. ilex 350, closed squares) or after 3 wk of acclimatization under 180 µmol m−2 s−1 (Q. ilex 180, open squares); (b,d) Halimium halimifolium plants grown under 200 or 80 µmol m−2 s−1 (H. halimifolium 200, closed squares; and H. halimifolium 80, open squares, respectively). Closed bars indicate the dark period. Symbols represent means of at least three independent replicates (± SE); r2 of the linear relationship are given.

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Cumulative CO2 fixation during the light period was calculated from diurnal courses of net C assimilation rates and plotted against the observed diurnal δ13Cres variations (Fig. 4c,d). A highly significant linear correlation was obtained for Q. ilex and H. halimifolium leaves under full (P < 0.05; r2 ≥ 0.93) and reduced light intensities (P < 0.05; r2 ≥ 0.68; Fig. 4c,d). Furthermore, the slopes of these relations were not markedly different, particularly for Q. ilex leaves (Fig. 4c).

Measurements on developing leaves

To evaluate whether the respiratory energy demand of a growing leaf does influence the isotopic composition of leaf-respired CO2, growing versus fully mature leaves of H. halimifolium and A. unedo were compared (Fig. 5). The extent of δ13Cres diurnal increase was markedly decreased in growing compared with mature leaves (δ13CLight–Dark of 6‰ and 7.9‰, respectively, Fig. 5a). Similar results were obtained for A. unedo (with δ13CLight–Dark of 2‰ and 7‰ in growing and mature leaves, respectively, Fig. 5b).

image

Figure 5. Diurnal course of the dark-respired δ13CO2 from fully mature (Mature, closed circles) and growing (Growing, open circles) (a) Halimium halimifolium and (b) Arbutus unedo leaves. The dark period is indicated by closed bars. Symbols represent means of three to six independent measurements (± SE) and statistically significant differences between growing and mature leaves are indicated by an asterisk (P < 0.05).

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Positional 13C-labelling experiments

The diurnal course of δ13Cres of mature leaves from two selected species, with and without diurnal increase of leaf respired CO2 (H. halimifolium and O. triangularis respectively), was investigated after addition of 13C-enriched pyruvate into their transpiration stream (either enriched at the first, or second and third carbon atom positions, 13C1 and 13C2-3-enriched, respectively). When 13C1-enriched pyruvate was supplied, δ13Cres continuously increased during the light period in H. halimifolium leaves and decreased very rapidly after 1 h of darkness (Fig. 6c), following the diurnal pattern of unlabelled δ13Cres (Fig. 1). In O. triangularis, δ13Cres of 13C1-labelled pyruvate was slightly higher in the light than in the dark but remained constant during the light (Fig. 6d). The addition of 13C2-3-enriched pyruvate did not reveal any significant variations in any of the species (Fig. 6c,d).

image

Figure 6. Major expected fluxes of respiratory substrates (grey arrows) explaining δ13C of dark-respired CO2 depending on the respiratory energy demand (a,b) and diurnal course of δ13Cres of two selected species with and without diurnal increase in δ13Cres (Mediterranean shrub Halimium halimifolium (c) and a fast-growing herb Oxalis triangularis (d), respectively) fed with 13C1 or 13C2-3 labelled pyruvate. (a,b) Carbon atoms C3 and C4 of glucose (Glc) and thus C1 of pyruvate (Pyr) which is decarboxylated during pyruvate dehydrogenase (PDH) reaction are 13C-enriched (*C in bold type) while depleted carbons which form acetyl-CoA (Ac. CoA) enter the Krebs cycle (KC). Tinted arrows indicate the major carbon flow. (F.A.) represents fatty acids and (IIº Met.) secondary metabolites. Adapted from Werner et al. (2007a) based on Tcherkez et al. (2003). Lower panels show the diurnal course of the amount of CO2 decarboxylated by the PDH (13C1 pyruvate, closed circles) and in the Krebs cycle (13C2-3 pyruvate, open circles), n = 4–8 ± SE.

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Discussion

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Supporting Information

The primary objective of this study was to investigate species-specific differences in the magnitude of diurnal variation in δ13C of leaf dark-respired CO213Cres). The diurnal amplitude in δ13Cres varied from 0 to 8‰ among the species examined. Such differences could be attributed to distinct functional plant groups: all evergreen, slow-growing or aromatic species studied exhibited large variation in δ13Cres with a common diel pattern. The δ13Cres continuously increased during the light period compared with the morning values, followed by a decrease during the dark period. Conversely, herbaceous and fast-growing plants did not exhibit marked temporal variations. These results precisely match previous data obtained for T. barbata (Werner et al., 2007b), for one Pinus species (Pinus elliottii; Prater et al., 2006) and for Q. ilex (Hymus et al., 2005; Werner et al., 2007b) in natural conditions, being a strong indication that measured δ13Cres on glasshouse-grown species were fully representative. Nevertheless, changes in δ13Cres diurnal patterns could occur under natural conditions with, for example, drought or temperature.

There has long been evidence for enriched δ13C signals in leaf-respired CO2 relative to leaf organic matter or respiratory substrates (Park & Epstein, 1961; Duranceau et al., 1999; Ghashghaie et al., 2001; Tcherkez et al., 2003; Klumpp et al., 2005), which has been explained by apparent fractionation processes in the respiratory pathways (Ghashghaie et al., 2001, 2003; Tcherkez et al., 2003). Our positional labelling experiments (Fig. 6) provide first evidence, that these processes are also involved in the diurnal changes in δ13Cres. In short, enrichment in dark leaf-respired δ13CO2 is mainly attributed to the heterogeneous 13C-distribution within hexose molecules (Fig. 6), where C3 and C4 are 13C-enriched compared with other positions because of fractionation in the aldolase reaction (Rossmann et al., 1991; Gleixner & Schmidt, 1997). During glycolysis, C1 of pyruvate derived from enriched C3 and C4 of glucose molecules is decarboxylated by pyruvate dehydrogenase (PDH), releasing 13C-enriched CO2, while the lighter carbon atoms are incorporated in acetyl-CoA and decarboxylated in the Krebs cycle. Acetyl-CoA molecules are partially deviated to the biosynthesis of metabolites, for example, fatty acids and secondary compounds, well known to be 13C-depleted compared with carbohydrates (Park & Epstein, 1961). Accordingly, Ghashghaie et al. (2001) proposed that if the carbohydrate molecule is fully consumed during dark respiration no apparent fractionation will be observed (i.e. the overall CO2 released by dark respiration carries the isotopic signature of the substrate). By contrast, in the case of a deviation of light carbon (acetyl-CoA) into biosynthetic pathways, the overall respired CO2 is 13C-enriched.

The pyruvate positional 13C-labelling provides direct evidence that changes in the relative activity of the PDH-reaction (decarboxylation of 13C1-labelled pyruvate) and Krebs cycle (decarboxylation of 13C2-3-labelled pyruvate) do occur (Fig. 6). Moreover, it indicates the importance of changes in relative C flux rates through both respiratory pathways: the diurnal increase in δ13Cres was caused by a marked increase in the C flux through PDH into secondary metabolism relative to the Krebs cycle activity, which remained constant throughout the day (for H. halimifolium, Fig. 6c). Hence, diurnal variations in δ13Cres are related to an increased metabolic activity of the PDH, which exceeded the C flow into Krebs cycle by several times. By contrast, a stable low activity of both pathways was observed in the herb O. triangularis (Fig. 6d), which is consistent with the lack of diurnal variation in δ13Cres in herbaceous, fast-growing species. Nevertheless, the amount of CO2 released from C1 by PDH exceeded the CO2 released in the Krebs cycle (13C2-3-labelled pyruvate, Fig. 6d) even in this species. This indicates that not all the glucose molecules are fully respired even in fast-growing species, but that some acetyl-CoA molecules and/or intermediates of the Krebs cycle, which are precursors for multiple anabolic and catabolic reactions, may always be allocated into other pathways.

Effect on the balance between carbon supply and demand on δ13Cres

Differences between functional plant groups may be attributed to marked differences in magnitude of C supply through photosynthesis (‘supply function’) relative to the respiratory demand for growth and maintenance respiration (‘demand function’).

The demand function is sustained by distinct respiratory energy demand of the functional groups: slow-growing, evergreen woody species, with high diel variation in δ13Cres, generally exhibit a low demand in respiratory substrates in contrast to actively growing species with a high respiratory energy demand (e.g. herbs). The respiratory demand of a species can be broken up into growth and maintenance respiration. The latter contributes to a larger proportion of total plant respired CO2 in slow-growing as opposed to fast-growing plants (Amthor, 1984). Given the differences in leaf structure such as higher lignin content in woody compared with herbaceous plants, Bowling et al. (2008) estimated that growth respiration would produce twice as enriched δ13Cres in woody than in herbaceous plants.

The demand function is further supported by the reduced diurnal δ13Cres variation in growing leaves, with a higher respiratory energy demand, compared with mature leaves from the same evergreen plants (Fig. 5). Most evergreen species exhibit a flush-like growth during a short period (Werner et al., 1999) and woody species can sustain new growth from C reserves of the previous year (Damesin & Lelarge, 2003). By contrast, in fast-growing species, the growing shoots may provide a strong C sink for the whole plant. This is further supported by Ocheltree & Marshall (2004) who found that the enrichment in δ13Cres of Helianthus annuus relative to soluble sugars was negatively correlated to its relative growth rate. It supports the importance of maintenance vs growth respiration to explain isotopic increase in δ13Cres.

However, differences in respiratory energy demand between functional groups are not expected to change on a diurnal time-scale. Hence, what seems to be of greater importance is the balance between the respiratory energy demand and the C supply rate, which can indeed exhibit marked diurnal changes. The supply function is reinforced by the fact that the increase in δ13Cres is linearly related to the cumulative CO2 fixation during the light period (Fig. 4), as already reported for Q. ilex leaves (Hymus et al., 2005). Moreover, by impairing the potential CO2 accumulation through a decreased light intensity or by interrupting the light period, the diurnal increase in δ13Cres declined almost proportionally. The positive linear relationship between the increase in δ13Cres and the cumulative CO2 uptake may indicate that with the diurnal accumulation of metabolites in excess of the respiratory demand a larger proportion can be diverted into secondary metabolism, as shown through the pyruvate-labelling experiments (Fig. 6c). This is supported by findings of Prater et al. (2006) who induced less enriched δ13Cres on P. elliottii needles by artificial shading of leaves. Thus, the diurnal increase in δ13Cres can be attributed to the increasing flux into secondary metabolism with increasing C supply during the day when the sugar pools are filled and the respiratory demand is met.

Influence of fractionation of enzymes and respiratory substrates on δ13Cres

The observed diurnal variation in δ13Cres of up to 8‰ exceeds the variation that can be expected from the heterogeneous intramolecular 13C distribution of a glucose molecule (6‰, Rossmann et al., 1991; Hobbie & Werner, 2004). However, isotope effects of the respiratory decarboxylating enzymes could increase the difference between CO2 evolved via PDH reaction and the Krebs cycle. Tcherkez & Farquhar (2005) assumed that PDH fractionates (Melzer & Schmidt, 1987) but they also suggested that this fractionation would not be evident in case of full decarboxylation of pyruvate molecules (Tcherkez & Farquhar, 2005). Nevertheless, they have shown with quantum chemical calculations, that the enzyme citrate synthase, which catalyses the first step of the Krebs cycle, has an isotope effect of 23‰ (Tcherkez & Farquhar, 2005). Calculations of the overall isotope effects revealed that the Krebs cycle is a source of 13C depletion, both in organic acids that are intermediates in the cycle and in the respired CO2 (Tcherkez & Farquhar, 2005). Hence, this process could increase the difference in δ13Cres above the expected difference originating from heterogeneous distribution within the glucose molecule.

The extent to which respiratory processes fractionate is further dependent on the pool sizes of the substrates (i.e. with increasing pool sizes during the day there is a higher probability for fractionation to occur). Further, the observed increase in δ13Cres could be caused by an increase in δ13C of the respiratory substrates through either (i) a diurnal decrease in photosynthetic discrimination via temporal variation of stomatal and internal conductance and Rubisco activity; or (ii) a shift to respiratory sources with more enriched isotopic signatures or (iii) a change in the relative flux rates from respiratory substrates with different δ13C.

Leaf respiration uses several C sources, including soluble sugars, starch, lipids or amino acids with a rapid turnover and different isotopic characteristics and residence times (Schnyder et al., 2003; Nogués et al., 2004). Further, a change in isotopic signature of stored vs fresh assimilates, that can contribute to up to 50% of respiration, can account for variation in δ13Cres (Schnyder et al., 2003; Nogués et al., 2004). The δ13Cres has been shown to vary over the course of various days in darkness under constant environmental conditions. Those changes were associated with the depletion of different substrate pools and/or shifts in the relative contributions of dark-respiratory pathways (Tcherkez et al., 2003). Could these processes also occur in the light?

We did not observe diurnal changes in either the pool sizes or δ13C of different sugars (data not shown) or leaf organic matter (Fig. 2), in agreement with other recent works that reported little diurnal variation in δ13C of different respiratory substrates, despite marked diurnal variations in δ13Cres (Hymus et al., 2005; Göttlicher et al., 2006). Furthermore, marked diurnal changes in photosynthetic discrimination and thus, in δ13C of fresh assimilates are unlikely under the controlled conditions in the climate chamber. Vapour pressure deficit and temperature were kept constant in the glasshouses, thus, changes in those parameters can also be ruled out as a potential source of variations. Further, as these plants were grown for a prolonged period under constant conditions, there was presumably no substantial difference in the isotopic signature of old and new C reserves. However, there are further potential fractionation processes, such as transitory starch accumulation and remobilization, which have been found to govern the diel rhythm of δ13Cres in short-term turnover pools of soluble sugars in leaves and phloem-transported organic matter (Tcherkez et al., 2004; Gessler et al., 2007).

Light-enhanced dark respiration

Upon darkening of a leaf an immediate increase followed by a subsequent decrease in δ13Cres occurs (Werner et al., 2007b), and hence the time of dark-incubation is important. It has been argued that this transient peak is related to light-enhanced dark respiration (LEDR; Barbour et al., 2007) that can be observed as a post-illuminatory respiration pulse (Atkin et al., 1998). Although the metabolic origin of such an effect is not well known (Atkin et al., 1998), organic acids might be the respiratory substrates during this peak (Cornic, 1973), which might have a different δ13C signature from glucose. Barbour et al. (2007) have suggested that malate could be a substrate for respiration by the NAD+ malic enzyme during the LEDR peak, which would produce enriched CO2. Our on-going work does not provide a strong support for this hypothesis. First, the transient decrease in δ13Cres typically lasts for 30–60 min (up to 120 min, Werner et al., 2007b) which is longer than the time frame of LEDR and second, we observed this transient peak during the decarboxylation of 13C1-labelled pyruvate, which may not be expected if the major source of LEDR is the decarboxylation of (unlabelled) organic acid via the NAD+ malic enzyme. An alternative explanation could be a rapid increase in PDH activity, which is downregulated during the light period (Tcherkez et al., 2005), and subsequent rapid decarboxylation of the available sugar pools, reflecting the pool size and C flow rates. However, more research is needed to clarify the underlying processes. Nevertheless, the diurnal increase in δ13Cres has similar amplitude when measured after 30 min darkening (Q. ilex, data not shown) and the reported values are consistent with data from Hymus et al. (2005) who incubated leaves for approx. 15–30 min. This indicates, that even if the LEDR has an influence on δ13Cres immediately upon darkening, the diurnal pattern observed in this study will be maintained.

Differences in δ13Cres between functional plant groups

Our data cover a broad spectrum of leaf-respired CO2 isotopic signatures, ranging from −16 to −32‰, which may be partially attributed to differences in photosynthetic discrimination between functional groups, reflected in differences in δ13C of leaf organic matter (Fig. 2). Differences in leaf structure, such as SLA, leaf water content and C : N ratios (Fig. 3) may also play a role through its effect on stomatal and mesophyll CO2 conductance. Evergreen species have a lower internal CO2 conductance compared with deciduous trees and herbaceous plants (e.g. 0.1 and 0.24 for Q. ilex and Q. petraea, respectively; Roupsard et al., 1996; for references on mesophyll conductance see Ethier & Livingston, 2004). Mesophyll conductance can vary rapidly with incident light, temperature, CO2 and humidity (Piel et al., 2002; Warren & Dreyer, 2006; Flexas et al., 2007), however, to our knowledge there are no reports on diurnal changes in mesophyll conductance. Stomatal conductance remained constant over the day in species with marked increase in δ13Cres (such as Q. ilex, H. halimifolium and A. unedo; data not shown). Further, a diurnal δ13Cres increase was observed for all leaf types investigated (including needles, sclerophyllous and mesophyllous leaves; see Figs 1 and 3) and in M. piperita, a herbaceous aromatic plant with larger SLA and (most probably) high mesophyll conductance. Thus, while species might be clustered into functional groups by leaf morphology, this is not likely to account for the observed diurnal patterns.

One common function of species with a high diurnal increase in δ13Cres seems to be an increased biosynthesis of secondary metabolites for defence, stress avoidance or aromatic compounds. For 10 species of this group, including the aromatic herb M. piperita (Nogués et al., 2006), we found a literature reference for volatile compound emission, particularly isoprene (no data were available for the remaining species).

In Q. cilex and P. pinea, for example, monoterpene emissions were found to be highly variable at the diurnal timescale, and markedly dependent on the incident light level (Staudt et al., 1997; Sabillon & Cremades, 2001). Isoprene emission is closely linked to photosynthesis and can exhibit a marked diurnal increase (Rapparini et al., 2004). Although several pathways can be involved in isoprene synthesis, Affek & Yakir (2003) showed that 72–91% of emitted isoprene was derived from recently fixed C. An inverse relationship between dark respiration rate and isoprene emission was found in several studies and it has been hypothesized that these two processes compete for the same substrate (Rosenstiel et al., 2003, 2004), which would be in agreement with our results. Loreto et al. (2007) confirmed this relationship for young leaves only, showing that the decrease in respiratory demand when leaves mature is accompanied by a progressive and rapid increase in isoprene emission during leaf development. This is in agreement with the reduced diurnal increase in δ13Cres in young leaves (Fig. 5). Further, several species with marked diurnal variation in δ13Cres are aromatic species (e.g. R. officinalis and M. piperita), which do allocate C into secondary compound synthesis of aromatic volatile molecules.

Hence, the diurnal increase in δ13Cres may be attributed to the increasing flux into secondary metabolism with increasing C supply during the day when the sugar pools are filled and the respiratory demand is met.

It will be important to evaluate the impact of these dynamics in δ13Cres at other spatial scales, from organs (e.g. shoots, stems and roots) to the plant level and for other ecosystem compartments (e.g. soil) to identify, how variations in δ13Cres of different ecosystem compartments will influence the integrated signal of ecosystem respiration (δ13CR). There is now increasing evidence that significant diurnal cycles in δ13Cres also occur in many other respiratory sources in the ecosystem, such as trunk (Maunoury et al., 2007; Kodama et al., 2008), soil (Kodama et al., 2008), roots (S. Unger et al., unpublished) and ecosystem (Bowling et al., 2003; Knohl et al., 2005; Werner et al., 2006; Kodama et al., 2008). Such knowledge is of major importance as it may affect the reliability of our estimates of ecosystem respiration, which is used in many modelling approaches to partition ecosystem C fluxes (Yakir & Wang, 1996; Bowling et al., 2001; Knohl & Buchmann, 2005) and may thus affect our predictions on ecosystem response to environmental changes.

Concluding remarks

This is the first large species survey on short-term δ13Cres variations that could be attributed to marked apparent fractionation processes in the respiratory pathways. Our results support the hypothesis that the diurnal increase in δ13Cres is enhanced during the light period in species with a high investment in secondary metabolism, whereas fast-growing herbs and grasses with a high respiratory energy demand do not show this diurnal pattern. Pyruvate positional 13C-labelling provided the first direct evidence that diurnal variations in δ13Cres are related to increased metabolic activity of PDH at low constant Krebs cycle activity, and point out the importance of changes in relative flux rates between both pathways. Differences between functional groups may be attributed to marked differences in the balance between the C supply through the amount of fresh assimilates during photosynthesis versus the respiratory demand for growth and maintenance respiration. Hence, C isotope composition of plant-respired CO2 contains information on the fate of respiratory substrates, and may, therefore, provide a nonintrusive way to identify changes in C allocation patterns. These short-term variations in δ13Cres have marked implications at larger scales, particularly for isotope partitioning studies at the ecosystem level.

Acknowledgements

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Supporting Information

This work was financed by the ISOFLUX project of the Deutsche Forschungsgemeinschaft (DFG, WE 2681/2-2). We gratefully acknowledge valuable comments of J. Ghashghaie and three anonymous referees and the collaboration of S. Nogués for sugar extraction and analysis, as well as the skilful technical assistance B. Teichner and E. Furlkröger, and the proofreading of K. Grieve.

References

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Supporting Information
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Supporting Information

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Supporting Information

Fig. S1 Characteristic diurnal course of δ13Cres from (a) Quercus ilex and (b) Halimium halimifolium mature leaves for which the 12-h photoperiod was decreased by including a 3-h dark period in the middle of the light period.

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