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Keywords:

  • fractionation;
  • isotope;
  • photosynthesis;
  • respiration

ABSTRACT

  1. Top of page
  2. ABSTRACT
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. ACKNOWLEDGEMENTS
  8. REFERENCES
  9. Supporting Information

In leaves, although it is accepted that CO2 evolved by dark respiration after illumination is naturally 13C-enriched compared to organic matter or substrate sucrose, much uncertainty remains on whether day respiration produces 13C-depleted or 13C-enriched CO2. Here, we applied equations described previously for mesocosm CO2 exchange to investigate the carbon isotope composition of CO2 respired by autotrophic and heterotrophic tissues of Pelargonium×hortorum leaves, taking advantage of leaf variegation. Day-respired CO2 was slightly 13C-depleted compared to organic matter both under 21% O2 and 2% O2. Furthermore, most, if not all CO2 molecules evolved in the light came from carbon atoms that had been fixed previously before the experiments, in both variegated and green leaves. We conclude that the usual definition of day respiratory fractionation, that assumes carbon fixed by current net photosynthesis is the respiratory substrate, is not valid in Pelargonium leaves under our conditions. In variegated leaves, total organic matter was slightly 13C-depleted in white areas and so were most primary metabolites. This small isotopic difference between white and green areas probably came from the small contribution of photosynthetic CO2 refixation and the specific nitrogen metabolism in white leaf areas.


Abbreviations
GOGAT

glutamine 2-oxoglutarate aminotransferase

IRMS

isotope ratio mass spectrometry

PAR

photosynthetically active radiation

TCA cycle

tricarboxylic acid cycle

INTRODUCTION

  1. Top of page
  2. ABSTRACT
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. ACKNOWLEDGEMENTS
  8. REFERENCES
  9. Supporting Information

Illuminated leaves simultaneously assimilate CO2 by gross photosynthesis and produce CO2 by photorespiration and day respiration. It is now 28 years since the fundamental mathematical background of 12C/13C fractionation associated with these processes, integrated into net photosynthesis, was published by Farquhar, O'Leary & Berry (1982). The mechanisms by which photosynthetic processes per se fractionate between carbon isotopes are now known, with the prevalent effect of CO2 diffusion (4.4‰ in air) and carboxylation by ribulose-1,5-bisphosphate carboxylase/oxygenase (29‰) (for a review, see Farquhar, Ehleringer & Hubick 1989). Still, some uncertainty remains on both the magnitude and the sign of photorespiratory and day respiratory fractionations. This topic has been at the heart of physiological studies (for a review, see Ghashghaie et al. 2003), namely because photorespiratory and day respiratory CO2 losses might have an effect on the carbon isotope composition (δ13C) of leaf organic matter and, furthermore, both photorespiratory and day respiratory fractionations (denoted as f and e, respectively) are critical parameters for internal conductance calculations (Evans et al. 1986; Bickford et al. 2009).

While several pieces of evidence indicate that photorespiration fractionates against 13C by around 11‰ (Tcherkez 2006; Lanigan et al. 2008), the carbon isotope fractionation associated with day respiration is less well-known. The latter is usually defined with respect to carbon fixed by current net photosynthesis, that is:

  • image

where R are 13C/12C isotope ratios. Some authors have suggested that day respiratory CO2 is 13C-depleted by a few per mil compared to net fixed carbon (Gillon & Griffiths 1997). So did theoretical studies that took advantage of intramolecular δ13C values within glucose and triose phosphates, with predicted values of around 5‰ (Tcherkez et al. 2004). Predictions based on metabolic pathways and the δ13C of their substrates are nevertheless impeded by our incomplete knowledge of day respiratory metabolism.

It is clear that respiratory CO2 evolution is inhibited by light in most leaves (for a review, see Atkin et al. 2000) and, furthermore, it is believed that day respiration involves metabolic bypasses due to enzymatic inhibitions and down-regulations by light (for a review, see Nunes-Nesi, Sweetlove & Fernie 2007). Typically, enzymes responsible for decarboxylations (CO2 liberation by day respiration) are down-regulated in the light, as in the case of mitochondrial pyruvate dehydrogenase (Tovar-Mendez, Miernyk & Randall 2003) and decarboxylases associated with the tricarboxylic acid (TCA) cycle (Igamberdiev & Gardeström 2003; Tcherkez et al. 2005) as well as malic enzyme (Hill & Bryce 1992; Igamberdiev, Romanowska & Gardeström 2001). Such decarboxylases are associated with 12C/13C isotope fractionations which are in turn sensitive to metabolic commitments (for a specific discussion, see Schmidt and Kexel 1997 and Tcherkez 2010). In other words, the δ13C value of evolved CO2 depends on both the 13C-abundance in respiratory substrate and metabolic fluxes sustaining respiration.

Since glycolysis (glucose-6-phosphate utilization) is strongly inhibited in the light, the main day respiratory substrates are thought to be triose phosphates produced in the light, that are naturally 13C-depleted (Gleixner et al. 1998; Tcherkez et al. 2004). Nevertheless, radiometric studies have suggested that CO2 evolved in the light may partly originate from stored compounds such as starch (Pärnik et al. 2002), that are 13C-enriched. Accordingly, 13C-tracing studies have shown that the TCA cycle is fed by organic acids like citrate and malate, accumulated during the night (Tcherkez et al. 2009; Gauthier et al. 2010). The decarboxylated C-atom positions (–COOH groups) of such organic acids are 13C-enriched (Melzer & O'Leary 1987; Schmidt 2003) due to the input of 13C-enriched carbon by phosphoenolpyruvate carboxylase. Therefore, there is no clear picture on whether the isotopic signal of day respiration is 13C-depleted (dominated by the degradation of triose phosphates) or 13C-enriched (dominated by the degradation of reserves).

Recently we used gas-exchange and isotopic data at the mesocosm level to show that CO2 evolved in the light by the whole mesocosm is slightly 13C-depleted, with an isotopic contribution of day respiration to the net photosynthetic fractionation of around –0.5‰ (Tcherkez et al. 2010). Nevertheless, at the mesocosm-scale, day respiration results from the combination of both heterotrophic (roots) and autotrophic (leaves) respiratory fluxes, which remain difficult to deconvolute.

Here, we used equations developed for the same sunflower mesocosm and applied them to Pelargonium leaves. We took advantage of variegation (Fig. S1), that is, of the occurrence of white (heterotrophic) and green (autotrophic) areas within single leaves. In the periclinal chimera Pelargonium variety of interest (Pelargonium × hortorum var. Panaché Sud), white areas are caused by the lack of functional photosynthetic chloroplasts in both L2 (hypodermis) and L3 (mesophyll) cell layers (Metzlaf et al. 1982; Gallard 2008). White areas are at the centre of the leaf while green areas are along the border and, ordinarily, are wide enough for gas-exchange measurements on pure green surface areas. Furthermore, this allowed us to calculate CO2-fixation (influx) by green areas and CO2-evolution (efflux) by white areas in the light and the comparison of the photosynthetic isotope fractionation by variegated and green leaves was used to investigate the specific contribution of heterotrophic tissues to day respiration.

Gas-exchange experiments were carried out in both 2 and 21% O2 in order to investigate the specific contribution of day respiration and photorespiration to the isotopic fractionation in the light. Furthermore, we used fossil-derived 13C-depleted CO2 (nearly 40‰-depleted compared to atmospheric, greenhouse CO2) as inlet-CO2 so as to make decarboxylation of pre-labelling (so-called ‘old’) carbon clearly detectable in the light. We also conducted metabolic analyses and compound-specific isotopic analyses to investigate the 13C-distribution in respiratory intermediates and major amino acids. Our experiments show that day respiratory CO2 was strongly 13C-enriched compared to inlet CO2 and net fixed CO2, indicating that reserves are the major source of carbon atoms liberated by day respiration, in both white and green leaf areas. However, when compared to organic matter formed in the greenhouse, day-respired CO2 was slightly depleted by ca. 3‰, showing that day respiration was associated with an isotope fractionation against 13C.

MATERIALS AND METHODS

  1. Top of page
  2. ABSTRACT
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. ACKNOWLEDGEMENTS
  8. REFERENCES
  9. Supporting Information

Plant material

The cultivar used in the present study was Pelargonium × hortorum var. Panaché Sud. Two morphotypes were grown here: a variegated morphotype (the original one, Fig. 1) that produces variegated and, occasionally, totally white leaves, and a green morphotype (obtained by the spontaneous loss of the chimera structure) that only produces green leaves. Plantlets were generated from cuttings planted in potting mix for rooting. Plants were then grown in the greenhouse under 22/18 °C, 60/55% relative humidity, 16/8 h photoperiod (day/night). Plants were automatically watered three times a day with nutritive solution (Hydrokani C2, Yara, Chambourcy, France). Carbon dioxide in air was at natural 13C-abundance (δ13C = –8.9 ± 0.6‰). All gas-exchange and on-line isotopic measurements were carried out on detached leaves, excised in the morning after 3 h illumination in the greenhouse.

image

Figure 1. Photosynthetic response curve of variegated leaves of P. × hortorum between 100 and 600 µmol mol−1 CO2. Data for green areas were either measured directly (closed circles) or recalculated from variegated areas, with Eqns 15 and 16 (semi-filled circles). Measurements were carried out in 21% O2 at 25 °C, under 60% relative humidity and 300 µmol m−2 s−1 PAR. Solid line, hyperbolic trend of the curve.

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Gas-exchange and isotopic measurements

In the following, we describe the procedure used to investigate the isotope fractionation associated with day respiration. The on-line photosynthetic isotope fractionation was measured in either 2% and 21% O2, with both green and variegated leaves. The parameters (e.g. day respiratory rate Rd, compensation point Γ*) that are required thereafter for the calculations were measured with gas exchange experiments. The last section (Calculations) explains how the day respiratory signal was computed from on-line isotopic data.

On-line isotope fractionation

The photosynthesis system used for on-line analyses was similar to that described by Tcherkez et al. (2005). A purpose-built assimilation chamber was connected in parallel to the sample air hose of the gas-exchange system Li-6400 (Li-Cor Inc., Lincoln, NE, USA). Leaf temperature was controlled at 25 °C with a water bath and was measured with a copper-constantan thermocouple plugged into the thermocouple connector of the Li-6400 chamber/IRGA. Inlet air humidity was adjusted using a Li-610 dew point generator, so that outlet air humidity was always at 60–65%. The flow rate was 29 L h−1. Light (fixed at 300 µmol m−2 s−1) was supplied by a halogen lamp (Massive NV, Kontich, Belgium) under which a water bath was placed so as to avoid heating. Inlet CO2 was obtained from a gas cylinder (CO2 Alphagaz N48, Air Liquide, Grigny, France) with δ13C of –45.3 ± 0.2‰. Air at 2% O2 was supplied by a cylinder (Crystal Gas, Air Liquide) connected to the inlet of the Li-6400. Outlet air of the chamber was regularly sent to a sampling-loop (instead of the infra-red analyser of the Li-6400) to measure the δ13C of CO2, as already described by Tcherkez et al. (2005). The isotopic analysis was carried out with an elemental analyser (NA1500, Carlo-Erba, Milano, Italy) coupled to the mass spectrometer (Optima, GV Instruments, Villeurbanne, France). The on-line isotope fractionation (Δobs) was calculated according to Evans et al. (1986).

Respired CO2

The isotope composition of dark-respired CO2 was measured by an incubation method. Leaves were detached from the plant (petiole cut under water) and left in the dark for 20 min. Leaf blades were then excised from the petiole and placed in a Plexiglas tube (volume 0.14 L). The air inside the tube was renewed for 5–6 min with CO2-free air (with a relative humidity of 50%), with a flow rate of 60 L h−1. Then the tube was closed and CO2 was accumulated for 3 min at 25 °C. The air was then sampled with a syringe (Magnum syringe for trace gas, Alltech, Epernon, France), injected into the sampling loop of the elemental analyser (previously cleaned with N2 to remove any CO2 contamination) and the isotopic analysis was carried out. The isotope composition of respired CO2 in the light by white area of variegated leaves (with green areas removed) was measured using the open gas-exchange system described previously, with a low flow rate of 10 L h−1 so as to increase the isotopic difference between outlet and inlet. Measurements were carried out on naturally completely white leaves but also on variegated leaves from which the green area had been removed. No significant difference was observed between the two.

Rd and Γ*i measurements

A/ci curves were carried out at different light levels (300, 200 and 100 µmol m−2 s−1) under either 2% or 21% O2 at 25 °C, using the Li-6400 gas-exchange system with the 6 cm2 red-blue chamber. Air humidity was controlled at 60% with the Li-610 dew point generator. The surface area of 6 cm2 was adapted (i.e. small enough) for individual measurements on pure green parts of variegated leaves. Rd, Γ*i and Γi (ci-based CO2 compensation points) were determined with the method of Peisker & Apel (2001).

Isotope composition of metabolites

Sucrose was extracted and purified by high-performance liquid chromatography (HPLC) as already described by Duranceau et al. (1999). The first soluble anionic fraction (first elution peak of the HPLC) was collected, vacuum-dried and then analysed. Amino acids were analysed for isotopic composition using gas chromatography-combustion-isotope ratio mass spectrometry (GC-C-IRMS, combustion module and Isoprime mass spectrometer, GV Instruments, Villeurbanne, France), with a procedure similar to that described in Mauve et al. (2009). Briefly, 100 mg of freeze-dried leaf powder were extracted with 1 mL cold water (0 °C) and deproteinated by heat treatment at 100 °C for 1 min. The extract was then dried with a speed-vac and redissolved in dimethylformamide (DMF). TBDMSTFA-reagent (Sigma-Aldrich, Saint Quentin Fallavier, France) was used to yield t-butyldimethylsilyl derivatives. An internal standard [C18-alkane, –31.4‰, calibrated against the International Atomic Energy Agency (IAEA) standard, glutamic acid] was poured in the sample for correcting any isotopic offset. Detected metabolites were malate, fumarate, threonine, serine, glutamate, glutamine + tartarate and aspartate. A mixture of these compounds (from Sigma-Aldrich), the isotope composition of which was determined by elemental analysis-mass spectrometry, was also derived and analysed by GC-C-IRMS and used as a calibration sample to determine the δ13C of the added carbon load associated with chemical derivation (for more details, see Mauve et al. 2009). It should be noted that arginine could not be analysed with this method, although being very abundant in white areas of variegated leaves. In fact, arginine was degraded by the chemical derivation. The derivation procedure used here did not fractionate between isotopes, as it involved complete reactions (Meier-Augenstein 1999); however, all metabolites were not eventually detected with the mass spectrometer with the same yield because of other (non-fractionating) steps (e.g. solubility in DMF). In the present case, under our conditions, aspartate was hardly detected when below 10 000 nmol g−1 DW and so could not be analysed in green areas. Glutamine co-eluted with tartaric acid and so the δ13C value indicated in Table 4 is the carbon isotope composition of the sum of the two compounds. The compound-specific analyses were checked for accuracy and repeatability with the regular injection of a standard sample (mixture of amino acids). The standard deviation on δ13C values of amino acids and the internal standard (C18-alkane) was 0.20 and 0.10‰, respectively.

Table 4.  Carbon isotope composition (δ13C, in‰) of different components and most abundant metabolites in green and white areas of variegated leaves of P. ×hortorum
CompoundGreen areaWhite area
  1. The carbon percentage of the SAF is also indicated between brackets. Sucrose and the SAF were purified by HPLC, vacuum-dried and the δ13C value was then obtained with elemental analysis-IRMS analyses. The δ13C value of organic acids and amino acids was obtained with chemical derivation and gas chromatography-combustion-IRMS analyses (see Material and Methods). nd, not detected with the IRMS (insufficient current signal for isotope analysis). Bottom, total amount in amino acids, in mmol m-2 and calculated concentration-weighted δ13C average (‰) of detected amino acids. Mean ± SD (n = 3). Significant differences (P < 0.01) between green and white areas are indicated with an asterisk.

  2. SAF, soluble anionic fraction.

Total organic matter–30.8 ± 0.3–31.2 ± 0.2
Sucrose–29.3 ± 0.9–30.2 ± 0.5
SAF–27.9 ± 0.8* (23.1 ± 0.9*)–30.1 ± 0.5* (16.1 ± 0.6*)
Fumarate–18.3 ± 1.2–18.6 ± 1.1
Malate–18.3 ± 2.0–18.2 ± 2.1
Serine–12.6 ± 0.8–13.4 ± 1.7
Aspartatend–22.4 ± 1.2
Threonine–24.3 ± 0.5nd
Glutamate–18.0 ± 1.2–21.3 ± 0.3
Glutamine + Tartarate–32.6 ± 0.4–32.2 ± 0.5
Total amino acid content142.3 ± 10.8*380.9 ± 14.5*
Average δ13C–21.5–25.2
Isotopic analyses of solid samples

The isotope composition of organic matter, soluble anionic fraction and sucrose was measured using elemental analysis and mass spectrometry on samples weighted in tin capsules. Glutamic acid of known isotope composition (USGS-40, –26.39‰, IAEA) was used to calibrate isotopic analyses. All isotope compositions δ13C are given with respect to V-PDB.

Metabolic analyses

The amino acid composition was analysed on freeze-dried leaf samples, using the o-phtalaldehyde derivation followed by HPLC and fluorometric quantification, as already described in Noctor et al. (2007).

Calculations

The mathematical background has already been given previously (Tcherkez et al. 2010). The principles of calculations are recalled briefly as follows. Symbols used thereafter are listed in Table 1.

Table 1.  List of symbols used in the present paper. dl, dimensionless
SymbolMeaningUnits
aIsotope fractionation associated with CO2 diffusion in air
aiIsotope fractionation associated with internal CO2 dissolution and diffusion
ALeaf net CO2 assimilationµmol m−2 s−1
AgRecalculated net CO2 assimilation of green areas (Eqn 15)µmol m−2 s−1
bIsotope fractionation associated with carboxylation
caCO2 mole fraction in the atmosphereµmol mol−1
ccInternal CO2 mole fraction at the carboxylation sites (chloroplasts)µmol mol−1
ciLeaf intercellular CO2 mole fractionµmol mol−1
cigRecalculated intercellular CO2 mole fraction in green areas of variegated leaves (Eqn 16)µmol mol−1
d*Unscaled isotopic signal of (photo)respiratory decarboxylations in the light (Eqn 3)µmol mol−1
δ13CavfAverage δ13C value of net fixed CO2 over the whole experiment time (Eqn 10)
δ13Cdayδ13C value of day-respired CO2 (Eqn 11)
δ13Cnightδ13C value of night-respired CO2
δ13Coutδ13C value of CO2 in outlet air
δ13Cwδ13C value of CO2 evolved by white areas in the light
ΔgIsotope fractionation associated with net photosynthesis of leaf green areas (Eqn 12)
ΔobsOn-line isotope fractionation associated with net photosynthesis
eIsotope fractionation associated with respiratory CO2 evolution in the light, with respect to net fixed CO2 (Eqn 9)
ewIsotope fractionation associated with respiratory CO2 evolution by white leaf areas in the light (Eqn 13)
fIsotope fractionation associated with photorespiratory CO2 evolution
gmInternal CO2 conductancemol m−2 s−1
gsStomatal CO2 conductancemol m−2 s−1
Γici-based CO2 compensation point (Eqn 8)µmol mol−1
Γ*ici-based CO2 compensation point in the absence of day respiration (Eqn 5)µmol mol−1
kCarboxylation efficiency defined as vc/ccmol m−2 s−1
kappApparent, ci-based carboxylation efficiency (Eqn 6)mol m−2 s−1
pProportion of ‘old’ carbon in day-respired CO2 (Eqn 14)%
RdLeaf respiration rate in the lightµmol m−2 s−1
RdwRespiration rate of white areas in the lightµmol m−2 s−1
σProportion of green surface area in variegated leaves (Eqn 15)dl
vcCarboxylation velocityµmol m−2 s−1
Isotopic contribution of (photo)respiration d*

The (photo)respiratory component is denoted here as d* as follows (Farquhar et al. 1982):

  • image(1)

where Δobs is the net photosynthetic (‘observed’) carbon isotope discrimination, and a, ai and b are the fractionations associated with diffusion in air, dissolution and diffusion in water, and carboxylation, respectively. ca, ci and cc are the CO2 mole fractions in the atmosphere, in the intercellular spaces and at the carboxylation sites, respectively. d* is equal to inline image where e and f are the carbon isotope fractionations associated with day respiration (the rate of which is Rd) and photorespiration. Γ* is the CO2 compensation point in the absence of day respiration and k is the carboxylation efficiency.

The value of d* obtained here includes both leaf day respiration of photosynthetic and heterotrophic tissues (green and white areas of variegated leaves, respectively). Using mass balance, Tcherkez et al. (2010) have shown that:

  • image(2)

where A is the net assimilation rate of the whole leaf. ew is the carbon isotope fractionation (in ‰) associated with CO2 evolution from the white leaf area in the light, and Rdw the corresponding respiration rate (in µmol m−2 s−1). Δg is the net photosynthetic fractionation of the green leaf area. In other words, the apparent value of d* obtained from Δobs (as explained below) includes an additional term that represents heterotrophic respiration. That is,

  • image(3)

Under our experimental conditions, k was within the range 0.06–0.11 and A/ca was about 0.15–0.27 so that ewRdwca/A was relatively small compared to eRd/k. Thus, the inclusion of the heterotrophic term due to the white leaf area did not compromise the applicability of the equations given below to the entire leaf.

We used the following relationship (Tcherkez et al. 2010) to measure d*:

  • image(4)

In the present study, A/ca curves were carried out by varying the CO2 level. In fact, the use of Eqn 4 does not require ca to be constant as d* is assumed ca-independent and we use the intercept (and not the slope). It should be noted that the extrapolation of (b – Δobs)ca when A tends to zero is much more reliable than the use of (b – Δ0)ca (where Δ0 is the extrapolated value of Δobs when A tends to zero) because Δobs itself cannot be written as a linear function of A and therefore, a linear extrapolation is not accurate. We thus applied Eqn 4 for A [RIGHTWARDS ARROW] 0. In practice, we did linear regression of (b – Δobs) ca versus A plots, taking into account only the linear part observed for ‘small’A values, that is, below 60% of the A value observed at ambient (400 µmol mol−1) CO2.

Carboxylation efficiency k

Carboxylation efficiency is defined as vc/cc where vc is the carboxylation rate and cc is internal CO2 mole fraction. In other words, the computation of k requires the knowledge of internal conductance gm for any ca value to calculate cc. Here, we did not have any measurement of gm along the A/CO2 response curve and we thus used apparent k-values (denoted as kapp) computed from A/ci curves, using the following:

  • image(5)

where Γ*i is the ci-based CO2 compensation point in the absence of Rd, obtained with A/ci curves (Peisker method). Equation 5 may be rearranged to:

  • image(6)

kapp may be interpreted as the k-value obtained with an infinite internal conductance. As such, kapp is certainly smaller than k. If internal conductance were low, say, 0.1 mol m−2 s−1, kapp would be nearly two times smaller than k. Our results obtained in Table 3 would change, with very low (negative) e-values and p higher than 100%. In addition, the δ13C value of day-evolved CO2 (δ13Cday) would then approach 0‰. Such values are not plausible. We therefore consider that internal conductance was certainly not small under our conditions and at most, the results given in Table 3 may be seen as minimal values of p and δ13Cday.

Table 3.  Day respiratory parameters and apparent carbon isotope fractionation of CO2 evolution in the light (e) by green and variegated leaves of P. ×hortorum
 d*kappe (Eqn 7)e (Eqn 9)δ13Cavfδ13Cdayp
μmol mol−1mol m−2 s−1%
  1. The calculation of e is based on data from Fig. 2 and Eqns 7 or 9, with the value of kapp (apparent carboxylation efficiency) reported here or the ΓiΓ*i-value of Table 2.

In 21% O2
Green morphotype3110.093–31.8–21.3–62.0–30.199
Variegated morphotype       
 Total area, variegated2480.061–27.45–30.5–60.8–33.390
 Green area, recalculated3870.079–16.2–13.8–57.6–40.960
In 2% O2
Green morphotype–320.125–18.8–54.0–35.179
Variegated morphotype       
 Total area, variegated–1370.082–26.1–56.7–30.598
 Green area, recalculated–1090.098–23.9–56.1–32.192
Average     –33.786
Respiratory isotope fractionation e

The isotope fractionation associated with day respiration was calculated using the d* value obtained graphically (see previous discussion). The day respiratory rate Rd and the ci-based compensation point Γ*i were measured independently using the Peisker method (Peisker & Apel 2001). The apparent carboxylation efficiency kapp was computed as described above (previous section). For both variegated and green leaves, we used Eqn 3, with corresponding Rd, Γ*i and kapp values measured directly, as follows:

  • image(7)

where f is the isotope fractionation associated with photorespiratory CO2 release (11‰, Tcherkez 2006; Lanigan et al. 2008). It should be noted that under our conditions (δ13C of inlet CO2 at –45‰), CO2 evolved by day respiration was 13C-enriched relative to current fixation. As a result, the observed value of e was always (very) negative. It does not mean that day respiration fractionated strongly against 12C but rather, this effect reflected the contribution of ‘old’ carbon (at ca. –30‰) to CO2 evolution in the light.

The respiratory fractionation e was also computed using Eqn 6 applied at A = 0, that is:

  • image(8)

where Γi is the ci-based CO2 compensation point. Substituting Eqn 8 into Eqn 7 then gives:

  • image(9)

Equation 9 was applied at 21% O2 only, since reliable values of Γ*i and Γi are more difficult to obtain under 2% O2 (both Γi and Γ*i are very small). Under 2% O2, Eqn 7 was used with inline image.

During A/CO2 curve experiments, the isotope composition of outlet CO2 (surrounding air), δ13Cout, was measured and so was the net photosynthetic fractionation (Δobs). It was therefore possible to calculate the average net-fixed CO2 as follows:

  • image(10)

where x stands for the CO2 step number of the experiment (A/CO2 curve) and yx is the contribution of that step to the time-integrated photosynthesis (inline image). The average isotope composition of day-evolved CO2 was then calculated as follows:

  • image(11)

In order to get values associated with pure green areas of variegated leaves (and not whole variegated leaves), similar calculations were carried out, with corresponding Rd, Γ*i and kapp values measured directly on green areas. While δ13Cout was the same in the calculations, the net photosynthetic fractionation Δobs had to be corrected by subtracting the contribution of respiration of the white area, so as to obtain the pure ‘green’ fractionation (denoted as Δg), using Eqn 2:

  • image(12)

where A is net photosynthesis (of the whole leaf), Rdw is the day respiration rate of the white area (measured directly) and ew is the fractionation associated with day respiration of the white area. The latter was simply obtained using the δ13C value of CO2 evolved by illuminated white area (δ13Cw):

  • image(13)
Proportion of ‘old’ carbon in day evolved CO2

The proportion of carbon that had been previously fixed (and not fixed by current assimilation) and liberated by day respiration during our experiment (denoted as p) was estimated with mass balance. The carbon isotope composition of ‘old’ respired carbon was assumed to be equal to that of total organic matter (δ13Ctom), that reflected the average signature of the carbon fixed during growth in the greenhouse. Therefore, p was given by:

  • image(14)

We recognize that there may be an isotope fractionation associated with CO2 liberation from ‘old’ carbon. Nevertheless, as explored in the present paper, the day respiratory fractionation is rather small and this causes negligible errors (typically less than 5%) in P-values computed with Eqn 14.

Back calculation of A and ci for green areas of variegated leaves

When experiments were conducted with variegated leaves, the assimilation value A obtained did account for the whole leaf surface area. To apply Eqn 4 (with Δg-values) and Eqn 6 to ‘pure’ green parts, both A and ci had to be corrected for the contribution of the white area. Using the proportion of green surface area (denoted as σ), we have:

  • image(15)

where Ag is the net assimilation rate of the green area. Similarly, using the relationship A = gs(caci) and assuming that stomatal conductance gs is the same for both green and white areas, we have:

  • image

where ci is the ‘apparent’ value (measured for the whole variegated leaf). This gives:

  • image(16)

where cig is the ‘true’ci value within the green area. The assumption gs remained the same over the whole leaf area was not critical. In fact, transpiration rates did indicate that gs was similar between white and green parts (data not shown). Furthermore, we show here that A/ci curves obtained on green areas and that recalculated from variegated areas (with Eqns 15 & 16) were similar (Fig. 1).

RESULTS

  1. Top of page
  2. ABSTRACT
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. ACKNOWLEDGEMENTS
  8. REFERENCES
  9. Supporting Information

Respiratory properties of leaves

Dark and day respiration rates were measured in both Pelargonium morphotypes: variegated and green (Table 2). In variegated leaves, separate measurements were carried out on white and green areas. Green areas of both morphotypes had similar dark and day respiration rates (no statistically significant difference) of around 2 and 0.6 µmol m−2 s−1. Similarly, the CO2 compensation point in the absence of day respiration, Γ*i, was similar, of around 46 µmol mol−1. White areas respired more in the dark (ca. 1.4 µmol m−2 s−1) than in the light (ca. 0.5 µmol m−2 s−1), demonstrating that a small photosynthetic assimilation occurred although it did not compensate for CO2 evolution. In other words, roughly 60% of respired CO2 was re-fixed by photosynthesis in the light in white areas. Unsurprisingly then, CO2 respired by illuminated white areas was slightly (though not significantly) 13C-enriched (–24.9‰) compared to dark-respired CO2 (–26.2‰) due to photosynthetic fractionation against 13C. Nevertheless, such a 13C enrichment caused by photosynthesis was modest, suggesting that in white areas, respiratory metabolism produced 13C-depleted CO2 in the light (compared to the dark), thereby compensating partly for the enrichment caused by photosynthetic CO2 fixation.

Table 2.  Photosynthetic and respiratory properties of green and variegated leaves of P. ×hortorum
 RnightRdayΓ*iΓiΓ*iδ13Cnightδ13Cday
µmol m−2 s−1µmol m−2 s−1µmol mol−1µmol mol−1
  • Day respiration rates and ci-based Γ* values were measured using the Peisker method. A/ci curves were carried out in 21% O2 at 25 °C, under 60% relative humidity. For photosynthetic green areas, the δ13C value of day-respired CO2 is investigated thereafter in the present paper (see Fig. 2).

  • a

    The day respiration rate of white parts in variegated leaves is a net respiration value (observed CO2 evolution in the light); that is, the gross respiration value is certainly larger. na, not applicable. Mean ± SD (n = 4).

Green morphotype1.88 ± 0.200.54 ± 0.0845.1 ± 0.58.7 ± 1.0–20.5 ± 0.9na
Variegated morphotype      
Green area2.05 ± 0.210.66 ± 0.1047.5 ± 1.59.8 ± 1.5–22.5 ± 1.1na
White area1.38 ± 0.250.54 ± 0.35anana–26.2 ± 0.9–24.9 ± 1.5

Validity of back calculations for variegated leaves

When gas exchange experiments were carried out on variegated leaves, net photosynthesis and photosynthetic fractionation were influenced by respiration of white areas of leaves. In the light, white areas did respire though at a lower rate than in the dark (see above and Table 2). Therefore, the assimilation rate of ‘pure’ green areas had to be recalculated from data obtained for whole leaves, with a known day respiratory rate of the white area (Eqns 15 & 16). Figure 1 shows such recalculated A and ci values and compares the results with A and ci values obtained directly on green areas. Clearly, our calculations gave satisfactory values, very similar to that obtained on green areas. We therefore used such a correction of A and ci thereafter (Fig. 2b, redrawn from Fig. 2a).

image

Figure 2. Relationship between ca(b–Δobs) and net CO2 assimilation in variegated (half-closed symbols) and green leaves (open symbols) of Pelargonium×hortorum. Data were obtained on-line at 25 °C, 300 µmol m−2 s−1 PAR, in 21% O2 (circles) or 2% O2 (squares). Observed data are plotted in A (variegated leaves) and C (green leaves). Panel B represents the relationship associated with green areas of variegated leaves, recalculated from data obtained on whole variegated leaves (panel a). Continuous lines: linear regressions. Intercepts are (21% and 2% O2 in that order): 248 and –137‰µmol mol−1 (A), 292 and –91‰µmol mol−1 (b) and 311 and –32‰µmol mol−1 (c). Linear regressions are carried out over the range A ≤ 0.6 A400 (see Material and Methods for more details) and are all significant (P < 0.01).

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(Photo)respiratory component d* in variegated and green leaves

The (photo)respiratory component of net photosynthetic fractionation was determined as the intercept of the plot showing (b – Δobs)ca against A (see Material and Methods). Such a representation is shown in Fig. 2, in which data were obtained either in leaves from the variegated morphotype (Fig. 2a) or leaves from the green morphotype (Fig. 2c). As expected, there was a positive relationship between (b – Δobs)ca and A, because of lower stomatal conductance at increasing CO2 (see Eqn 4). The relationship was flatter at low CO2 for which stomatal conductance was large and close to a maximum due to low CO2 mole fractions. Noteworthy, the intercept was always positive in 21% O2, showing the substantial effect of photorespiration (*) on d*. By contrast, in 2%, intercepts were negative, showing an apparent day respiratory contribution against 12C (negative apparent e-values, also see further discussion). Data obtained on variegated leaves were recalculated to correct for respiration of white areas (Fig. 2b). Clearly, the correction only introduced subtle differences in the general trend (Fig. 2a, b), although the scale of the x axis (photosynthesis) changed a lot. The intercept in both 21 and 2% O2 showed the same pattern, with a positive d* value under ordinary conditions and negative d* value under low photorespiratory conditions. Interestingly, the shape of the (b – Δobs)ca versus A relationship was different in the two morphotypes, with lower (b – Δobs)ca values in the green morphotype (Fig. 2c). This effect is simply due to larger and less variable stomatal conductance values in the green morphotype (data not shown).

Computations were carried out using intercepts obtained in Fig. 2 and the results are shown in Table 3. d* values are all positive in 21% O2 and negative in 2% O2 because of the production of 13C-depleted CO2 by photorespiration under 21% O2. The pure respiratory component d* – * was always negative and accordingly, the calculated e-value was negative in all instances (Table 3). In other words, day respiration produced 13C-enriched CO2 (compared to net fixed carbon), and our estimated values of day-evolved CO2 were comprised between –30 and –40‰, with an average value of ca. –33‰. In the present data, there was no clear difference between green and variegated morphotypes; both were associated with negative e-values and 13C-enriched evolved CO2. Assuming that the isotopic signal of day respired CO2 was due to the contribution of two carbon sources, namely, ‘old’ carbon fixed in the greenhouse before the experiment (δ13C of organic matter, Table 4) and recent carbon fixed during the experiment (δ13Cavf, Table 3), the proportion of ‘old’ carbon, p, was calculated (Table 3). p was always very large, between 79% (green morphotype, under 2% O2) and 99% (green morphotype, under 21% O2).

Carbon isotope composition of metabolites

Major metabolites were extracted from intact leaves sampled in the greenhouse and their isotope composition was analysed by HPLC and EA-IRMS (sucrose, soluble anionic fraction) or GC-C-IRMS (amino and organic acids). The results are shown in Table 4. Metabolites and total organic matter were generally 13C-depleted in white areas, by ca. 1‰. In both white and green areas, malate and fumarate were 13C-enriched (above –20‰), strongly suggesting the input of 13C by the PEPc-catalysed HCO3 fixation. So was serine (up to –12.6‰) showing the activity of photorespiration, that fractionates against 13C for CO2 release, thereby enriching glycine and serine pools. Aspartate was not abundant enough in green areas to allow isotopic analysis, and a similar situation occurred with threonine in white areas. Glutamine + tartaric acid were relatively 13C-depleted compared to other compounds, and appeared very slightly 13C-enriched in white areas compared to green areas. The concentration-weighted signature of detected amino acids was significantly 13C-depleted in white areas. Nevertheless, the far most abundant amino acid in white areas was arginine (Fig. S2) but its δ13C value could not be measured since it was degraded by the derivation treatment. In general, total amino acid concentration was much larger in white (380 mmol m−2) than in green areas (142 mmol m−2). Likely, white areas also contained more nitrates, as witnessed by the lower carbon percentage of the soluble anionic fraction (Table 4).

DISCUSSION

  1. Top of page
  2. ABSTRACT
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. ACKNOWLEDGEMENTS
  8. REFERENCES
  9. Supporting Information

While there is currently a growing literature on the carbon isotope composition (δ13C) of CO2 evolved in the dark by either leaves, roots or ecosystems, less is known on the isotope composition of day-respired CO2 (for reviews, see Ghashghaie et al. 2003; Bowling, Pataki & Randerson 2008). This lack of knowledge stems from the technical difficulties associated with the measurement of the pure, respiratory isotopic signal, which is indeed caused by a (very) small respiratory rate. Elucidating the day respiratory contribution to net photosynthetic 12C/13C fractionation usually requires assumptions on either its magnitude or its associated fractionation. Here, we took advantage of previously described equations (Tcherkez et al. 2010) and applied them to variegated or green Pelargonium leaves in order to (1) determine whether day evolved CO2 is 13C-depleted or 13C-enriched at the leaf level; and (2) examine the carbon source sustaining day respiration.

Origin of CO2 evolved by day respiration

On-line gas exchange and isotopic measurements were carried out on variegated and green leaves and the results were plotted as (b – Δobs)ca versus A. The intercept of such a representation is d*, the unscaled (photo)respiratory component of net photosynthetic fractionation. Under 21% O2, d* was always positive (roughly about 300‰ µmol mol−1, Fig. 2 and Table 3) due to the contribution of photorespiration, *, of about 11 × 45 = 495‰ µmol mol−1. This suggests a pure respiratory contribution eRd/k of roughly –200‰ µmol mol−1. In fact, when experiments were carried out under 2% O2 (very low photorespiratory conditions), d* declined to negative values (–32 to –137‰ µmol mol−1, Table 3). This clearly demonstrates that e < 0, that is, produced 13C-enriched CO2 compared to net fixed CO2. e calculated with Rd and kapp values was very negative (up to –31‰). Such a large apparent fractionation against 12C cannot have originated from 12C/13C isotope fractionations since enzymatic isotope effects of decarboxylases favour 12C (O'Leary 1980). Rather, this clearly indicates that the carbon source used for CO2 production by day respiration did not come from 13C-depleted, recent carbon fixed by net photosynthesis, but mainly from reserves accumulated previously during growth in the greenhouse. The estimated proportion of pre-labelling, ‘old’ carbon in day respired CO2 averaged 86% (Table 3). Similar results have been obtained by Tcherkez et al. (2010) at the mesocosm level in sunflower (Helianthus annuus) plants subjected to 12C/13C labelling: after 1 d labelling, the contribution of ‘new’ carbon to day respiration was ca. 50% only. The results also agree with measurements of photosynthetic 12C/13C fractionation by spruce needles (Picea sitchensis), that strongly suggested the use of 13C-enriched, ‘old’ carbon substrates in the light, causing artificially high Δobs values (Wingate et al. 2007). In addition, 14C-labeling techniques have shown that about 40% of day evolved CO2 originated from low turn-over compounds (Pärnik & Keerberg 2006) such as starch (Pärnik et al. 2002).

Nevertheless, starch is not a plausible substrate as its degradation is down-regulated in the light (Stitt & Heldt 1981; Preiss 1984; Hendricks et al. 2003). More likely, day respiration is fed by the remobilization of organic acids such as malate and citrate. In fact, isotopic tracing carried out in illuminated leaves have recently shown that the commitment of pyruvate to the TCA pathway is very small in cocklebur (Xanthium strumarium) (Tcherkez et al. 2009). Similarly, in rapeseed (Brassica napus), 2-oxoglutarate synthesized in the light mainly originates from night-stored citrate and malate (Gauthier et al. 2010). We nevertheless recognize that CO2 respired in the light is not only associated with mitochondrial metabolism (TCA pathway) but also with chloroplastic pyruvate decarboxylation by the plastidic pyruvate dehydrogenase, which is not down-regulated in the light by phosphorylation (Tovar-Mendez et al. 2003). Chloroplastic pyruvate is produced by glycolysis from chloroplastic triose phosphates that are, in turn, produced by current photosynthesis. Our results indicate that up to 14% of day respired CO2 was associated with pyruvate dehydrogenation in the chloroplast.

Is day respiratory CO2 naturally 13C-depleted?

The day respiratory fractionation e is ordinarily defined with respect to net fixed CO2, and this definition has been applied here in Table 3. As a result, apparent fractionation values are negative, because of the remobilization of carbon stores (see above). Importantly, such a metabolic uncoupling between photosynthesis and day respiration causes inherent difficulties to manipulate the mathematical expression of photosynthetic fractionation Δobs. In other words, as emphasized by Wingate et al. (2007), another particular term should be used in the general expression of Δobs as a substitute for eRd/k.

Still, one may compare the carbon isotope composition of day respired CO2 (Table 3) with that of organic material accumulated in the greenhouse prior to our experiments (Table 4). Day respired CO2 was 13C-depleted by 2–3‰ compared to organic matter and 3–4‰ compared to sucrose. Nevertheless, when green areas or green leaves only were considered, δ13C of respired CO2 appeared quite variable and comprised between –30 and –40‰, that is, 13C-depleted by 0–10‰ compared to organic matter. Our results are consistent with previous studies (already discussed in the Introduction), either theoretical or experimental, that suggested day-respired CO2 was 13C-depleted compared to photosynthates. Furthermore, in sunflower mesocosm, in which leaf respiration was prevalent within the day respiratory isotopic signal, CO2 evolved in the light was found to be 13C-depleted by ca. 5‰ compared to net fixed CO2 (Tcherkez et al. 2010).

We previously argued that lower metabolic commitments in respiratory metabolism, caused by the inhibition of leaf respiration by light, may be at the origin of such a 13C depletion (Tcherkez et al. 2010). In fact, the observed 12C/13C isotope effects of enzymes (e.g. decarboxylases) are large when the reactions of interest are rate-limited (Hayes 2001). In vitro, enzymes involved in respiratory metabolism are known to fractionate against 13C: pyruvate decarboxylation fractionate by 23‰ (Melzer & Schmidt 1987), citrate synthase fractionates by ca. 20‰ (Tcherkez & Farquhar 2005). Although poorly documented, isocitrate dehydrogenase may fractionate by –1.1 to 5.7‰ (Grissom & Cleland 1988; Lin et al. 2008) and 2-oxoglutarate dehydrogenase may fractionate by around 20‰ (Tcherkez & Farquhar 2005). In addition, the decarboxylation of the pentose phosphate pathway (that may occur in the cytoplasm in the light) fractionates by 16‰ (6-phosphogluconate dehydrogenase, Rendina, Hermes & Cleland 1984). Taken as a whole, decarboxylases favour 12C by up to 20‰ and this might explain the 13C depletion of day respired CO2 since metabolic fluxes involved are small. That said, C-atom positions decarboxylated to CO2 are in the form of –COOH (carboxylic groups) within substrate molecules while it has been reported that such groups are generally 13C-enriched (Rinaldi, Meinschein & Hayes 1974; Schmidt 2003; Hobbie & Werner 2004). In citrate, –COOH group are enriched by up to 4‰ compared to the molecular average (Schmidt 2000). Moreover, malate and fumarate were found to be 13C-enriched here (Table 4), by up to 12‰ compared to sucrose. Such an enrichment could not compensate completely for the effect of enzymatic isotope fractionations and as a result, decarboxylated CO2 remained slightly 13C-depleted by ca. 3‰.

12C/13C isotope fractionations in heterotrophic white leaf area

Several studies have reported a general 13C enrichment in both organic matter and metabolites in heterotrophic organs like roots and stems (Badeck et al. 2005; Cernusak et al. 2009). Furthermore, in artificially heterotrophic leaves obtained with an inhibitor of pigment synthesis, Terwilliger & Huang (1996) have shown that total organic matter was also 13C-enriched. Therefore, it is well accepted that heterotrophic tissues are naturally 13C-enriched, although the reasons of such an enrichment remain uncertain (Cernusak et al. 2009).

This pattern is not in agreement with the results obtained here on naturally heterotrophic white leaf areas. Both total organic matter and several metabolites (serine, sucrose, soluble anionic fraction) in white areas were 13C-depleted by around 1‰ compared to green areas (Table 4). Carbon metabolism of white areas was certainly sustained by metabolites produced by the green area of the same leaf. The white area of the leaf blade was at the centre of the leaf and, therefore, the phloem path carrying photosynthates and assimilated nitrogen from the green area of the same leaf went through the white area. Furthermore, although both photosynthesis and photorespiration represented very low carbon fluxes in white areas (Table 2), serine was substantially 13C-enriched, reflecting the 13C-enriching effect of ordinary photorespiration rates on serine and glycine in the green area (Tcherkez & Hodges 2008). That is, metabolites such as sucrose and some amino acids synthesized in white areas were inherited from green areas. Such a conclusion is consistent with other studies carried out in variegated leaves (Ivanova & Sherstneva 1999). As a result, the isotope composition of the carbon source feeding white areas is that of metabolites (e.g. sucrose, glutamate) exported from the surrounding green area.

The metabolism of white tissues was probably at the origin of the 13C depletion in organic materials. There was a slight refixation of respired CO2 by photosynthesis in white areas, as evidenced by the smaller respiration rate in the light compared to the dark and the (very slight) 13C enrichment in CO2 in the light (Table 2). That said, the fractionation value associated with photosynthetic CO2 fixation was small: using a mass balance calculation, one obtain a fractionation value of around 1‰ only. Such a low value is surprising, e.g. because of the large fractionation of ribulose-1,5-bisphosphate carboxylase/oxygenase (29‰, Roeske & O'Leary 1984). Thus it remains possible that in white tissues, the photosynthetic fractionation is compensated for by an inhibition of day respiration similar to green tissues, with lower (more negative) δ13C values than in the dark. In addition, the PEPc-catalysed carboxylation (that favours 13C with respect to dissolved CO2) might have contributed in the light (see also below) thereby decreasing the apparent fractionation value of CO2 fixation. Still, the refixation of respired CO2 may have produced 13C-depleted assimilates that might have contributed to organic matter and sucrose production in white areas.

CO2 refixation also originated from carbamyl-phosphate synthesis necessary for arginine production (for a review, see Slocum 2005). In fact, arginine synthesis is believed to occur in the light, as demonstrated by 14C tracing (Holden & Morris 1970; Larsen et al. 1981) and the light-inducible pattern of the gene encoding ornithine transcarbamoylase (Williamson, Lake & Slocum 1996). Carbamyl phosphate production fractionates by –1.7‰ (favours 13C) (Tipton & Cleland 1988) and ornithine transcarbamoylase, that fixes carbamyl-phosphate onto ornithine during arginine biosynthesis, fractionates by 9.5‰ in E. coli (Parmentier & Kristensen 1998). The isotope fractionation of the plant ornithine transcarbamoylase is currently not known but it might be even larger; for example, the similar enzyme aspartate transcarbamoylase fractionates by up to 24‰ (Waldrop et al. 1992). This isotope effect probably contributes to the slightly lower 13C abundance in white areas owing to the high arginine content (up to 10 mmol m−2).

The operation of the arginine-involving nitrogen metabolism is consistent with previous studies that suggested white areas of variegated leaves of Coleus blumei played the role of a transitory ‘nitrogen store’ (Gilbert et al. 1998). There was a substantial accumulation of amino acids in addition to arginine (glutamine, aspartate and serine), with nearly a three-fold increase of total molar concentration compared to green areas (Table 4). While asparate and serine were 13C-enriched, glutamine and tartaric acid were 13C-depleted and glutamate was relatively 13C-enriched (Table 4). The isotope composition of tartaric acid was probably close to that of glucose and sucrose (–31‰), from which it is directly derived in Pelargonium (via ascorbate, involving irreversible and O2-dependent reactions, Wagner & Loewus 1973; Helsper & Loewus 1982). Plausibly then, glutamine was at about –33‰, that is, 13C-depleted as compared to glutamate. Such an inconsistency between glutamate and glutamine would certainly reflect the involvement of particular metabolic reactions. That is, the enzymatic isotope fractionations known so far cannot explain the specific 13C enrichment in glutamate compared to glutamine: (1) the conversion of glutamine into glutamate by the glutamine-oxoglutarate aminotranferase (GOGAT) probably fractionates against 13C in the C-5 atom position of glutamine and 2-oxoglutarate and so depletes glutamate in 13C; (2) arginine synthesis firstly involves N-acetylation of glutamate by N-acetylglutamate synthase and so no primary 12C/13C isotope effect is expected in the glutamate moiety in that reaction; (3) glutamine synthesis from glutamate by glutamine synthetase has no isotope effect at ordinary NH4+ concentration (Stoker 1994); and (4) the isotope effects of documented aminotransferases favour 12C in glutamate (Macko et al. 1987; Rishavy & Cleland 1999, 2000). The 13C enrichment in glutamate may have come from the involvement of the PEPc-catalysed carboxylation, that produced oxaloacetate, in turn converted (via citrate) into 2-oxoglutarate and glutamate. Consistently, fumarate and malate were naturally 13C-enriched (Table 4). However, we found that glutamine was (likely) not 13C-enriched. The origin of the 13C enrichment in glutamate has thus to be further investigated, such as possible isotope effects associated with other glutamate-dependent enzymes of arginine biosynthesis (e.g. N2-acetylornithine aminotransferase).

Conclusions and perspectives

We have shown here that stored compounds, likely in the form of organic acids, are the main carbon source for leaf day respiration and in addition, metabolic restrictions caused by the inhibition of respiration by light are plausible explanations for the 13C depletion in evolved CO2. The present study also illuminates the tight relationship between metabolic dynamics and the observed δ13C in CO2. That is, the latter results from: (1) δ13C value of substrates; (2) metabolic pathways and their associated fractionations; and (3) kinetics of metabolic pools (turn-over rates and day-night cycles). Variegated leaves are exquisite examples involving all of these three aspects and in particular, the influence of nitrogen metabolism on isotopic abundance in white tissues needs more investigation. More information on metabolic fluxes involving glutamate and arginine may be studied using compound-specific 15N natural abundances and this will be addressed in a subsequent paper.

ACKNOWLEDGEMENTS

  1. Top of page
  2. ABSTRACT
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. ACKNOWLEDGEMENTS
  8. REFERENCES
  9. Supporting Information

The authors wish to thank the Agence Nationale de la Recherche for its financial support through a project Jeunes Chercheurs, under contract no. JC08-330055. GT, CM and ML wish to thank the Institut Fédératif de Recherche 87 for supporting the equipments and the Quality Management System of the Plateforme Métabolisme Métabolome. GT wishes to thank Prof Gabriel Cornic for passionate discussions on the work.

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  2. ABSTRACT
  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. ABSTRACT
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. ACKNOWLEDGEMENTS
  8. REFERENCES
  9. Supporting Information

Figure S1. Typical morphology of variegated leaves in P. × hortorum var. Panaché Sud. White bar, 1 cm.

Figure S2. Amino acid composition of green (black bars) and white (white bars) parts of variegated P. × hortorum leaves, determined by HPLC analyses. Values are in nmol g−1 DW. Mean ± SE of 6 replicates. b-Ala, beta-alanine, g-ABA, gamma-aminobutyrate, hSer, homoserine, Orn, ornithine.

FilenameFormatSizeDescription
PCE_2241_sm_f1.TIF569KSupporting info item
PCE_2241_sm_f2.TIF3562KSupporting info item

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