JXB Advance Access originally published online on September 24, 2004
Journal of Experimental Botany 2004 55(408):2599-2605; doi:10.1093/jxb/erh263
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RESEARCH PAPER |
Apparent respiratory discrimination is correlated with growth rate in the shoot apex of sunflower (Helianthus annuus)
College of Natural Resources, University of Idaho, Moscow, Idaho, ID 83844-1133 USA
* Present address and to whom correspondence should be sent: Department of Forest Science, Oregon State University, 321 Richardson Hall, Corvallis, OR 97331, USA. E-mail: troy.ocheltree{at}oregonstate.edu
Received 7 January 2004; Accepted 23 July 2004
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Abstract |
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The literature offers no consensus as to whether the
13C of respired CO2 is identical to that of the respiratory substrate, perhaps because of differences in measurement technique and growth conditions. To address this issue, the 13C of respired CO2 from growing sunflower shoot apices was measured and compared with that of soluble carbohydrates extracted from the respiring tissues. Shoot apices were studied because any influence of growth and biosynthesis was expected to be maximally expressed in these rapidly growing tissues. The two most probable substrates, starch and soluble sugars, were similar in 13C (P=0.46). The 13C of respired CO2 was enriched in 13C compared with these putative substrates (P<0.0001). This apparent enrichment ranged from 2.2
–5.7, and decreased with relative growth rate (P<0.0001). The respiratory enrichment was counterbalanced by a depletion in the tissue constructed from the residual carbohydrates. The depletion varied from 2.2 to 3.0 relative to soluble carbohydrates (P<0.05), as predicted from mass-balance arguments. These results support the idea that respired CO2 is enriched relative to its substrates. Variation in growth rates may help to explain the variable amounts of respiratory discrimination described in the literature. Key words: Discrimination, Helianthus annuus, respiration, stable carbon isotopes
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Introduction |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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Plants discriminate against 13C as they assimilate carbon dioxide from the atmosphere (O'Leary, 1981
; Farquhar et al., 1982). Less is known about the isotopic discrimination associated with events following assimilation, in particular, during respiration. Even a small respiratory discrimination could have a significant effect on the isotopic composition of plant tissue because a substantial proportion of assimilated carbon is lost by respiration (O'Leary, 1981; Gillon et al., 1998). Further, the analysis of 13C of respired CO2 has recently been used as a tool for partitioning ecosystem fluxes (Andrews et al., 1999; Bowling et al., 2001). Any discriminatory steps must be accounted for if the 13C of ecosystem respiration is to be interpreted.
The early literature concerning respiratory discrimination was equivocal. For example, respired CO2 from Lycopersicon esculentum Mill. was enriched in 13C by 2.4–8.1 relative to plant tissue (Park and Epstein, 1960), but depleted by 1 in a later report (Park and Epstein, 1961). Respired CO2 was enriched in 13C by 4.9 in Triticum aestivum, but depleted by 0.2–3.7 in four other species (Troughton et al., 1974). These inconsistent results led modellers to treat this potential source of discrimination as negligible (O'Leary, 1981; Farquhar et al., 1982), while recognizing its potential importance.
A recent study measured respiratory discrimination in isolated protoplasts (Lin and Ehleringer, 1997). Protoplasts from bean and corn were incubated in vials and fed with substrate of known 13C. Respired CO2 was collected from the protoplasts and compared with the substrate provided. Under these conditions, respired CO2 had the same 13C as the substrate, thus there was no respiratory discrimination. However, it is uncertain whether the respiratory metabolism of protoplasts changes when isolated from plant tissue. For example, isolating protoplasts would eliminate respiration associated with phloem loading and transport, and might modify any respiration associated with biosynthetic processes. Therefore, this study did not conclusively solve the problem of respiratory discrimination in intact plants.
More recent studies have consistently found that CO2 respired from intact leaves was enriched in 13C compared with respiratory substrate (Duranceau et al., 1999, 2001; Ghashghaie et al., 2001, 2003; Tcherkez et al., 2003). These studies have isolated respiratory substrate from tissues and compared it directly with the CO2 respired. This approach uses the following equation:
 | (1) |
s and p are the 13C values of the source and product (CO2), respectively. In the older literature, s was assigned the value of plant tissue; these recent papers have used the isotopic composition of probable respiratory substrates to estimate respiratory discrimination. The latter approach yields a better estimate of respiratory discrimination because it more directly reflects sources and substrates. These studies have found respiratory CO2 enriched in 13C by 2–7 relative to the respiratory substrate (Duranceau et al., 1999, 2001; Ghashghaie et al., 2001, 2003; Tcherkez et al., 2003). Physiological mechanisms that would lead to fractionation based on shifts in catabolic and anabolic processes have been proposed (Duranceau et al., 1999; Tcherkez et al., 2003). These metabolic shifts might be especially pronounced if the growth rate was varied experimentally; growth rate differences would vary the proportional allocation of photosynthate among biosynthetic pathways (Waring et al., 1985; Tcherkez et al., 2003; Ghashghaie et al., 2003). The objective of this study was to help resolve the question of respiratory discrimination and to work toward a general explanation of its magnitude. CO2 respired from shoot apices of intact sunflowers growing at different rates was collected and compared with their putative respiratory substrate to calculate respiratory discrimination. The magnitude of respiratory discrimination was correlated with the relative growth rate.
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Materials and methods |
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Sunflowers (Helianthus annuus) were grown at the University of Idaho Research Greenhouse. Seeds were sown in 5'' pots containing a Peat-lite soil mixture consisting of 70–80% sphagnum moss (SunGro Sunshine Mix no. 1, SunGro Horticulture Inc., Bellevue, WA). Plants were watered to field capacity every 2 d and fertilized every third watering with a 16:16:17 (N:P:K by vol.) fertilizer (Scotts-Sierra Horticultural Products Company, Marysville, OH). Average growth conditions throughout the experiment were day and night-time temperatures of 20.5±3.5 °C and 16.6 ±1.6 °C, respectively; and day and night-time relative humidities were 40.4±8.9% and 36.3±4.3%, respectively. Plants were grown under natural light that averaged 1066 µmol m–2 s–1 PPFD for the growth period.
Individual plants were randomly assigned to one of four light treatments: 10, 30, 60, or 100% of incident photosynthetically active radiation (PAR). Except for the 100% light treatment, the treatments used different types of shade cloth. Measured PAR transmission averaged 9, 31, and 58% of incident PAR for the treatments above, as measured using a Sunfleck ceptometer (Decagon, Pullman, Wa) on three occasions. There were nine replicates of each light treatment and six plants were randomly assigned to each replicate. The replicates were shade structures constructed of 0.75" PVC pipe covered with the varying types of shade cloth (Dewitt Company, Sikeston, MO). The replicates measured 46x61 cm in area and were spaced 15 cm apart. The height of the shade structures was adjusted to exceed the heights of the growing plants. Shade cloth completely covered the top of the treatment area and two opposing sides. On the remaining two sides only the upper 20 cm were covered with shade cloth. The open sides were in the direction of airflow, which increased the mixing of air around the plants.
Plants were grown for 10 d under ambient conditions in the growth room before random assignment to a treatment. Treatments were applied for 14 d before sampling began. Subjects were sampled in random order, which required 15 d to complete. Although some plants had begun to form flower buds by the end of the experiment, none had progressed as far as forming the disc that would later support florets. Furthermore, plants with signs of flower buds were not included in the sampling.
Relative growth rates
Relative growth rates were calculated for the plant tissue from which respired CO2 was collected. To obtain maximal RGR, only the distal portion of the plant was used. The initial biomass was estimated using a regression model instead of directly measuring biomass from a subsample of each treatment. There was enough variation in the size of the plants within treatments that measuring only a subsample, even by direct measurement, would have caused greater error than modelling each plant.
The main stem of each plant was marked with a horizontal line 3 cm from the apex of the stem, which hereafter is referred to as the sampling-line. The stem diameter was measured at the sampling-line, height of the stem above the sampling line, and the length and width of all leaves attached above the line. Measurements from 10 plants harvested the following day were fitted to a model to estimate plant biomass above the sampling-line. Because plants were not harvested until the day following the initial measurements, the height above the sampling-line was often greater than 3 cm. Therefore, the height above the sampling-line had to be included as a parameter in the model. The model was as follows:
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Final biomass, b2, was measured directly as the dry weight of all plant tissue above the sampling-line. These two biomass values were used to calculate the relative growth rate of the plant tissue above the sampling-line using the following equation:
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Collection and analysis of respired CO2
Respired carbon dioxide was collected for stable isotope analysis using glass flasks plumbed into a closed gas-exchange system. The gas-exchange system was a Li-Cor 6200 portable photosynthesis system with a 4.0 l sample chamber (Model no. 6000-10, Li-Cor Inc., Lincoln, NE). The flasks contained 
350 ml and were sealed at either end with manual valves. They were placed immediately downstream of the sample chamber. The chamber was loaded with a plant shoot after the shoot had been in darkness for at least 1 h; the delay was intended to avoid any post-illumination burst of respiration (Atkins et al., 1998). The plants were inserted so that the sampling line lay along the centre of the chamber gasket.
CO2 was then scrubbed from the system using a soda-lime trap. At the end of the scrubbing, CO2 concentrations were generally around 40 µmol mol–1. The concentrations never reached zero because the gas analyser lay between the chamber and the soda-lime trap; therefore the shoot was able to release additional CO2 into the scrubbed air-stream as the gas made its circuit around the system. The effectiveness of the soda-lime trap was tested by re-plumbing the system so that the trap was placed between the chamber and the infrared gas analyser; in this configuration, it was statistically not possible to distinguish CO2 concentrations from zero. This suggests that the scrubber was highly effective; the complete scrubbing led to the suggestion that the scrubber was unlikely to contribute to isotopic fractionation.
After scrubbing, CO2 was allowed to build up inside the chamber until its concentration reached 350 µmol mol–1. The time required to reach this concentration ranged between 10 min and 1 h, depending on the size of the shoot and its growth rate. The valves were then closed and the flask returned to the laboratory for isotopic analysis. The 13C of this sample was used as an estimate of p in equation 1. Thus equation 1 was used to estimate fractionation focusing on CO2, rather than biomass, as a product. The samples were analysed at the Idaho Stable Isotopes Laboratory at the University of Idaho on a Finnigan Mat Delta-Plus Isotope Ratio Mass Spectrometer (Finnigan MAT Bremen, Germany) coupled with a Pre-Con (Finnigan MAT, Bremen, Germany), which concentrated the carbon dioxide and separated it from N2O. The samples were calibrated against a CO2 standard from Oztech (Dallas, TX, USA). The standard deviation of repeated analyses of an injected standard was 0.25.
Isotopic composition of the respired CO2 was corrected for minor leakage into the system during sampling. Leakage was estimated from the conductance of the empty system to CO2. The following equation was used to calculate system conductance:
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 | (5) |
13CT is the isotopic composition of collected CO2, T is rate of CO2 collection, 13Ca is the isotopic composition of ambient CO2, 13CR is the isotopic composition of respired CO2, and R is the respiration rate. Equation 5 was rearranged to solve for 13CR:
 | (6) |
13Ca was estimated twice each night and averaged. The leakage rates were so low that they had relatively little influence on the 13CR estimates. The correction reduced the values by 0.04–0.87 and averaged 0.26, similar to the error due to repeated analyses of a standard in the mass spectrometer.
Harvesting and carbohydrate extraction
After CO2 was collected from each plant, all plant material above the sampling-line was removed, plunged into liquid nitrogen, and placed on dry ice until it could be stored in an ultra-cold freezer (–79±1 °C). In the laboratory, samples were removed from the freezer and immediately placed on a bench-top freeze-dry system (LABCONCO, Kansas City, Missouri). Freeze-dried samples were ground to a fine powder in a glass jar with two stainless steel balls on a shaker (Eberback Co. Ann Arbor, Michigan). Plant tissue (2 mg) was then packed into tin cups and analysed for carbon stable isotopic composition.
Isotopic analysis was also performed on the starch and water-soluble fractions, which were extracted from the harvested tissues using procedures described by Wanek et al. (2001). Briefly, 100 mg of plant sample was boiled in a methanol:chloroform:water (5:3:1, by vol.) mixture and then centrifuged. The supernatant was treated with chloroform and water to separate any remaining non-soluble compounds. The water-soluble fraction was placed on a cation-exchange column and then an anion-exchange column and rinsed with deionized water. The effluent, which was assumed to be dominated by the soluble carbohydrate fraction, was dried in a tin cup for isotopic analysis (Wanek et al., 2001). The pellet was resuspended in the M:C:W mixture, centrifuged, and rinsed three times with deionized water. The insoluble fraction was then incubated with 
-amylase, which hydrolysed the starch to glucose molecules. The soluble carbohydrates released by the enzyme were separated from the remaining non-soluble fraction by treatment with chloroform and water. The water-soluble fraction was then dried in tin cups for isotopic analysis of the starch. A test of this method using purified starch and glucose found no fractionation during the extraction (T Ocheltree, JD Marshall, unpublished data). Plant tissue, starch, and soluble carbohydrates were analysed for their respective stable carbon isotopic compositions by combustion in an NC 2500 Elemental Analyser (Thermo Quest Italia, S.P.A., Rodanao, Milan, Italy) on-line with a Finnigan MAT Delta-Plus Isotope Ratio Mass Spectrometer (Finnigan MAT Bremen, Germany). These samples were also run at the Idaho Stable Isotope Laboratory with an average standard deviation of 0.13 for repeated measurements of a solid standard.
System checks
The system was tested for fractionation of 13C caused by the pump in the Li-Cor 6200. With the sample chamber empty, the air was passed through a soda-lime trap for 10 min to remove all CO2 from the system. The system was run without plant material inserted. Pure CO2 with a known 13C was injected into the system to raise the CO2 concentration to 450 µmol mol–1 CO2. Samples (1.0 ml) were removed from the system at the beginning and end of a 60 min period (equivalent to the longest sample collection time) for isotopic analysis. The difference between the initial and final 13C of the system air was used to estimate the impact of leakage on the measurements. The Li-Cor 6200 caused a change in the isotopic composition that averaged 0.1±0.4.
The system was also tested for the discrimination associated with passing air through soda-lime to remove CO2. Air was passed through a soda-lime trap for 10 min to remove all the CO2 from the system. As above, pure CO2 at a known 13C value was injected into the system. Samples (1.0 ml) were taken at the beginning and end of 30 s, 40 s, and 60 s of exposure to soda-lime. The air remaining inside the collection system after the scrubbing procedure was 1.5 lighter than the injected CO2. This fractionation due to scrubbing might be of concern were it not for the completeness of CO2 removal under experimental conditions, as noted earlier.
This set of system checks revealed that potential sources of fractionation during the collection of CO2 were small compared with the differences measured between respired CO2 and soluble carbohydrates. The fractionation associated with the soda-lime trap (1.5) would have affected a negligible proportion of the final CO2 in the sample. Running the Li-Cor 6200 for 60 min caused an average fractionation of 0.1±0.4. Finally, the calculations were corrected for any ambient greenhouse air that leaked into the collection system, but the average value for this correction was approximately equal to the experimental error, which was small relative to measured discrimination from carbohydrate to respired CO2.
All data were analysed using SAS 8.0 (SAS Institute Inc., Cary, NC). A mixed-effects model was used for the analysis. Variation among individual plants was treated as a random variable, which removed the plant-to-plant variation from the analysis of the treatments (Littell et al., 1996). The light treatments were analysed as fixed effects. All pairwise contrasts, both between and within treatments, were tested using a contrast test in the ‘proc mixed’ procedure in SAS 8.0. The effect of RGR on respiratory discrimination and tissue 13C was tested using two-way Analysis of Variance (ANOVA). Unless otherwise specified, significant differences were determined at the P <0.05 level.
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Results |
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Relative growth rates and cumulative biomass differed among shading treatments (P=0.008 and P=0.0008, respectively, Fig. 1). Relative growth rates were 50 (SE=20) mg g–1 d–1 on the growing tips of plants under 10% of incident light. This was approximately one-fifth the 240 (SE=80) mg g–1 d–1 observed on plants growing under 60% light. As a consequence of these RGR differences, the amount of biomass accumulated above the sampling-line was also lower in the 10% light (0.3 g, SE=0.08 g) and 30% light treatments (0.7 g, SE=0.16 g) than in the 60% and 100% light treatment (2.0 g, SE=0.57 g).
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The
13C of starch was not significantly different from that of the water-soluble carbohydrate fraction (P=0.46; Fig. 2). The regression between these carbon fractions is significant (P <0.0001) with a slope of 0.9, but the lack of a significant difference between starch and sugar indicates that the slope cannot be distinguished from one. Therefore, these fractions were treated collectively when being discussed as respiratory substrates.
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Soluble carbohydrates, bulk tissue, and respiration varied in
13C, but the magnitude of variation differed among treatments (Fig. 3; Table 1). Compared with the soluble carbohydrates, respired CO2 was always 13C-enriched (Fig. 3). The mean difference between the 13C of respired CO2 and carbohydrates ranged from 2.2 to 5.7 across treatments (Interaction, P <0.0001; Table 1). By contrast, the residual carbon in plant tissue was always depleted in 13C (by 2.2–3.0) relative to carbohydrate (Fig. 3).
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Isotopic composition also varied among the light treatments (Fig. 3). For example, the
13C of soluble carbohydrate and whole-plant tissue were both more negative in the 10% light treatment than in plants grown in 100% light. These treatment differences were best resolved in the soluble carbohydrate fraction. It was not possible to detect differences in the 13C of respired CO2 among treatments, presumably because the standard errors of 13C of respired CO2 were greater than those of the other sample types (Fig. 3). Nonetheless, when respiratory discrimination was calculated, treatment differences were detected (Fig. 4). Plants grown at 10% light discriminated in favour of 13C (i.e. negative discrimination in the production of CO2) by almost 6 relative to carbohydrate, while those grown in the 60% and 100% light treatments discriminated in favour of 13C by 2.2 and 2.5, respectively (Fig. 4).
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RGR was positively correlated with respiratory discrimination, although the Pearson's correlation was only 0.43 and the standard error of the residuals was large (1.8
). An ANOVA showed a positive relationship between RGR and respiratory discrimination when both variables were treated as continuous and not averaged by treatment (P <0.0001). As in the analysis of treatments effects (Fig. 4), the slowly growing plants showed the highest respiratory discrimination. |
Discussion |
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These data generally agree with published reports that whole tissue is depleted in 13C by 2.4–3.0
(Fig. 2) relative to starch and sucrose pools (Deleens and Garnier-Dardart, 1977; O'Leary, 1981). Similarly, these results support earlier reports of a 2–7 fractionation during respiration (Duranceau et al., 1999, 2001; Ghashghaie et al., 2001; Tcherkez et al., 2003). The range of respiratory discrimination values found for Helianthus annuus (2.2–5.7) is similar to the range previously reported for this species (3–6) (Ghashghaie et al., 2001). These studies, like ours, compared respired CO2 directly to a presumed respiratory substrate. It was possible to correlate discrimination with relative growth rates (Fig. 4). This ability to detect this phenomenon was enhanced because the growing shoot apex of the plant was measured, magnifying the effect of growth rate on differences in discrimination. Because the apices were growing at different rates among treatments, it was possible to account for the extent to which respiratory discrimination is associated with biosynthetic processes.
The RGR of the 100% light treatment was lower than that under 90% and 60% light treatments. The easiest explanation is that there were differences in the initial biomass of the plants in these treatments. However, all plants were grown under ambient conditions for 10 d before beginning the treatments and plants were randomly assigned to the treatment levels, which removed any potential bias in initial biomass. There are two other likely explanations: water stress and maturity of plant tissue. The lack of difference in RGR between the 100% and 60% light treatments could have been due to soil drying. The plants were watered more often as they grew until the plants grown in full sunlight were being watered every day. Even so, they often had dry soil. If the RGR values are recalculated, excluding the plants harvested in the last 6 d of sampling (which probably experienced the greatest water stress), the mean RGR for the 100% light treatment increases to 247 mg m–1 d–1, 12 mg m–1 d–1 greater than the plants grown in 60% light. The maturity of the plants should not have caused difficulties because all the plants were harvested before they began to form the floral disc.
Previous work has shown that one control over the isotopic composition of respired CO2 is the substrate utilized for respiration (Tcherkez et al., 2003). They found that the 13C of respired CO2 correlated with the respiratory substrate utilized by the plants, as indicated by the respiratory quotient (RQ). The isotopic composition of respiration became 13C depleted as plants used more fatty acids as substrate (Tcherkez et al., 2003). The respiration rate of these plants also correlated with the RQ and the isotopic composition of respiration. In the current study, no correlation was found between the respiration rate and the 13C of the respiration from plants (data not shown). It is possible that this correlation was not seen, and perhaps there was a respiratory substrate switch, because the respiration rates in this work were not manipulated by temperature or starvation. All plants in this study were grown in the same greenhouse and therefore exposed to the same temperatures. Neither were the plants exposed to long periods of darkness. Plants were able to replenish carbohydrate supplies each day and even the plants in the lowest light treatment showed positive growth rates. This suggests that the plants were not starving. Additional studies investigating the relationship between RGR, respiration rates, and respiratory substrate are needed.
Respiratory discrimination is not only influenced by substrate 13C, but also by the relative rates of the biosynthetic pathways engaged. The C-3 and C-4 positions of glucose are 13C-enriched, which leads to a depletion in acetyl-CoA (Rossman et al., 1991), which is used for fatty acid synthesis. If less acetyl-CoA is being used for fatty acid production and remains in the TCA cycle, respiration would be less 13C-enriched (Tcherkez et al., 2003; Ghashghaie et al., 2003). As plants grow larger, more of the plant respiration is dominated by maintenance respiration (Amthor, 1989). Lipid demand should be lower for maintenance respiration as the need to synthesize new cell membranes decreases. This lower demand for the lipids may allow more 13C-depleted acetyl-CoA to remain in the TCA cycle and eventually be released as CO2. It is hoped that these results will spur further investigations on lipid production, plant growth, and respiration.
Respiration by isolated protoplasts does not fractionate against 13C (Lin and Ehleringer, 1997). The apparent contradiction between the in vitro results and this study requires explanation. It is speculated that protoplasts change their fundamental physiology when isolated from their cellular matrix. Another key difference between the protoplast and recent whole-tissue experiments is in how discrimination was calculated. The in vitro study estimated discrimination starting from a substrate dissolved in the growth medium. This approach would include in the final discrimination value any discrimination due to diffusion and uptake of the substrate. By contrast, this study addressed the discrimination starting from carbohydrates already inside the tissues. However, it is possible that there are distinct pools of carbohydrates even within the shoot apex, and these distinct pools might have different isotopic composition.
The possibility is acknowledged that the apparent respiratory discrimination found in this study could also be influenced by phloem sugar translocation. If sugars exported from the plant tissue above the sampling-line were depleted in 13C relative to the bulk carbohydrate pool, the isotopic composition of respiratory substrates might be more enriched than the bulk carbohydrate pool. Under this scenario, the bulk pool would yield a value for s in equation 1 that would be too low and respiratory enrichment of 13C might be inferred where none in fact had occurred. It is also conceivable, especially in the 10% light treatment, that phloem transport delivered carbohydrates upward across the sample line. Phloem transport would not matter if the transported sugars were well mixed into the bulk carbohydrate pool before leaving the leaf, i.e. if they were isotopically similar to the sugars that remained behind. Additional research comparing phloem contents to bulk soluble carbohydrate pools should answer this question.
The most compelling argument in favour of respiratory discrimination comes from simple mass-balance considerations (Park and Epstein, 1960, 1961; DeNiro and Epstein, 1977). If it is true that the sucrose and starch pools represent the isotopic composition of substrate for both respiration and biosynthesis, then the observed enrichment in 13C in respired CO2 must be offset by an equal depletion. The observed depletion of 13C in tissue relative to substrate is thus a necessary complement to the enrichment. In fact, if respiratory discrimination were known, it might be possible to estimate the proportional carbon losses due to respiration from the difference between tissue and respiratory substrates. It would be useful to conduct a complete mass balance on whole plants. Such an analysis of whole-plant isotopic fluxes will be necessary to support the use of these tools in the analysis of ecosystem-level fluxes.
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