A Review on 13 C Dynamics and Mechanisms Printer-friendly Version Interactive Discussion Diel Variations in the Carbon Isotope Composition of Respired Co 2 and Associated Carbon Sources: a Review of Dynamics and Mechanisms Bgd a Review on 13 C Dynamics and Mechanisms Printer-friendly Version Interac

Introduction Conclusions References Tables Figures Back Close Full Screen / Esc Biogeosciences Discussions This discussion paper is/has been under review for the journal Biogeosciences (BG). Please refer to the corresponding final paper in BG if available. Abstract Introduction Conclusions References Tables Figures Back Close Full Screen / Esc Abstract Recent advances have improved our methodological approaches and theoretical understanding of post-photosynthetic carbon isotope fractionation. Nevertheless we still lack a clear picture of the origin of short-term variability in δ 13 C of respired CO 2 (δ 13 C res) and organic carbon fractions on a diel basis. However, closing this knowledge 5 gap is essential for the application of stable isotope approaches for partitioning ecosystem respiration, tracing carbon flow through plants and ecosystems and disentangling key physiological processes in carbon metabolism of plants. In this review we examine the short-term dynamics in δ 13 C res and putative substrate pools at the plant, soil and ecosystem scales and develop mechanistic explanations for diel δ 13 C res dynamics 10 at each scale. Maximum reported variation in diel δ 13 C res is 4.0, 5.4 and 14.8‰ in trunks, roots and leaves of different species and 12.5 and 8.1‰ at the soil and ecosystem scale in different biomes. Temporal variation in post-photosynthetic fractionation related to changes in carbon allocation to different metabolic pathways is the most plausible mechanistic explanation for observed diel dynamics in δ 13 C res. In addition, 15 mixing of component fluxes with different temporal dynamics and isotopic compositions add to the δ 13 C res variation on the soil and ecosystem level. Understanding short-term variations in δ 13 C res is particularly important for ecosystem studies, since δ 13 C res contains information on the fate of respiratory substrates, and may, therefore, provide a non-intrusive way to identify changes in carbon allocation patterns.


Introduction
Stable carbon isotopes have become an important tool to advance our understanding in carbon cycle processes on different temporal and spatial scales.As carbon travels from the atmosphere through plants and is respired back to the atmosphere by leaf, stems, roots and soil there are many processes, which alter the carbon isotope ratio (generally expressed in the δ-notation (δ 13 C) in ‰ as the relative deviation of the 13 C/ 12 C ratio of a sample from the 13 C/ 12 C ratio of an international standard).Thus, the δ 13 C isotope signature of dark-respired CO 2 (δ 13 C res ) is widely used for tracing carbon flow through plants and ecosystems (e.g.Knohl et al., 2005;Kodama et al., 2008), partitioning ecosystem respiration (e.g.Bowling et al., 2001;Unger et al., 2010a), and disentangling key physiological processes on the plant and stand levels (e.g.Yakir and Sternberg, 2000;Gessler et al., 2009a).Photosynthetic carbon assimilation in C 3 -plants heavily discriminates against 13 C, with the δ 13 C ratio of assimilated carbon being related to the ratio of leaf intercellular and ambient CO 2 concentration (Farquhar et al., 1982).Photosynthetic discrimination leaves an imprint on δ 13 C of newly produced assimilates and respired CO 2 , which are widely used to characterize environmental effects on the physiology of photosynthesis.In addition, post-photosynthetic isotope fractionation processes in enzyme reactions of metabolic pathways downstream of photosynthetic carbon fixation can alter the isotopic signature of the organic matter among organs and chemical compound classes and also affect δ 13 C of respired CO 2 .Thus, driven by the work of Jaleh Ghashghaie's group and others increasing knowledge on isotope fractionation during dark-respiration has been acquired during the last Published by Copernicus Publications on behalf of the European Geosciences Union.
decade (for reviews see Ghashghaie et al., 2003;Badeck et al., 2005;Bowling et al., 2008).However, marked diel variations of dark-respired δ 13 C res , which occur within minutes to hours over the 24 h cycle, have only lately gained scientific attention (e.g.Barbour et al., 2007;Werner et al., 2007;Wingate et al., 2010).Ignoring these short-term variations in δ 13 C res might weaken the power of isotope approaches for disentangling plant and ecosystem processes.
In spite of recent insights into the origin of δ 13 C of different carbon pools (see reviews of Badeck et al., 2005;Bowling et al., 2008;Cernusak et al., 2009), we still lack a clear picture of the physiological mechanisms resulting in isotopic fractionation in metabolic processes downstream of photosynthesis and their implication for diel variation in δ 13 C of different organic carbon fractions and respired CO 2 .
Here, we provide a survey of marked short-term dynamics in respired δ 13 C res and putative substrate pools at the plant, soil and ecosystem scale.We have limited this review to exclusive cover publications evaluating diel (24 h) dynamics in δ 13 C res and providing mechanistic explanations.The mechanistic understanding is a prerequisite for disentangling physiological and environmental information encoded in short-term variations of δ 13 C in both plant organic matter and respired CO 2 .
Compared to our progress in understanding isotope fractionation in general and dark respiratory isotope fractionation in particular, the recognition of diurnal dynamics in δ 13 C res was slow.This was largely attributed to methodological constrains hindering high-time resolved analysis of δ 13 C res .Recently technological advances opened new frontiers to assess the isotopic signature of respired CO 2 at time scales from minutes to hours over the day course, which will be shortly surveyed in the next section (for detailed methodological descriptions see Sect. 3 in companion paper by Werner et al., 2011a).

New methodological developments in high time-resolved measurements of δ 13 C res
First attempts to measure δ 13 C res were made with gasexchange systems coupled to isotope ratio mass spectrometers (IRMS), allowing δ 13 C analysis of CO 2 respired by leaves, roots or whole plants in an enclosure (normally a cuvette or phytotron).Alternatively, detached leaves, roots or soil have been incubated in small vials (e.g.exetainer).If flushed with CO 2 -free air the δ 13 C res can be measured directly within 3 min on a gas bench-IRMS (in-tube incubation technique, Werner et al., 2007).High precision IRMS enables "on-line"-measurements, where an open gas-exchange system is directly coupled to the IRMS e.g.via an open-split and a GC-column for CO 2 separation, yielding a time resolution of ca. 5 min (e.g.Schnyder et al., 2003;Klumpp et al., 2005;Werner et al., 2007).Fully continuous monitoring of δ 13 C res can be achieved with new optical laser spectroscopy, e.g.tuneable diode laser spectroscopy (TDLS; e.g.Bowling et al., 2003) or cavity ring down spectroscopy (CRDS; e.g.Wahl et al., 2006), which continuously measure 12 CO 2 and 13 CO 2 concentrations in the gas stream (e.g.Barbour et al., 2007) in e.g.gas exchange chambers (e.g.Kodama et al., 2011) or in ecosystem height profiles (e.g.Wingate et al., 2010).The temporal resolution and precision depends on the integration-time and instrument (e.g.0.25 ‰ at 1 s and about 0.08 ‰ at 30 min integration time for δ 13 C and δ 18 O in CO 2 with a TDLS; Barthel et al., 2011b).
High temporal resolution measurements of δ 13 C res determined in non-equilibrated closed chambers (e.g.Maunoury et al., 2007;Kodama et al., 2008) might, however, be affected by changes in transport isotope fractionation as the CO 2 concentration in the chamber increases and could thus introduce errors under particular conditions (Ubierna et al., 2009) which has created particular concern for δ 13 C measurements of soil respiration (e.g.Nickerson and Risk, 2009).Open dynamic chamber techniques, which can be applied with optical laser spectroscopy (e.g.Bahn et al., 2009;Barthel et al., 2011a) and continuous measurements of δ 13 C in CO 2 over soil profiles (cf.Kayler et al., 2008Kayler et al., , 2010) ) can, however, overcome these potential problems.Thus there are currently at least three independent techniques, which yield accurate measurement of diel dynamics in δ 13 C res , when specific instrument precautions are taking into account.Given the fact that observed ranges in δ 13 C res exceed by far the variation, which may be caused by instrumental noise or nonequilibrium conditions, we have now gained a solid piece of data on short-term (minutes to day) variation in respired δ 13 C res .
Determination of respiratory substrate δ 13 C signatures, which are needed to understand the origins of variation, is not possible at the same high temporal resolution as measurements of δ 13 C res .Even though hyphenated gas chromatographic (GC) and liquid chromatographic (LC) IRMS techniques have enabled us to assess compound specific δ 13 C in organic substrates, destructive sampling and extraction prevents continuous measurements.Moreover, when interpreting data of the isotopic composition of soluble and storage carbohydrates and other fast-turnover compounds, potential artefacts related to the extraction procedures have to be taken into account (Richter et al., 2009).In spite of these problems, more and more data for δ 13 C of respiratory substrates is now available at a temporal resolution of a few hours.This information is a first step towards understanding the mechanisms of variations in diel dynamics in δ 13 C res of different plant organs and ecosystem compound, which are summarized in the next section.
Plant stems and tree trunks (see Fig. 1a) also exhibited marked diel variations in emitted δ 13 CO 2 (up to 4 ‰), sometimes associated with marked seasonal differences (e.g. in Quercus petraea; Maunoury et al., 2007).In contrast to leaves, where highest δ 13 C res values were often observed at the end of the light period, trunk δ 13 C res was most enriched at night (e.g. in Pinus sylvestris; Kodama et al., 2008 and Ricinus communis;Gessler et al., 2009b).
There is limited information on diel dynamics in root δ 13 C res lending a non-uniform picture: only slight variations in δ 13 C res (<2 ‰) occurred in herbaceous and shrubby species under controlled conditions (Gessler et al., 2009b;Wegener et al., 2010).Under natural conditions, however, root δ 13 C res showed a clear diel cycle in wheat (5.4 ‰, Kodama et al., 2011;Fig. 1b) and in a Mediterranean herb, diel δ 13 C res variations increased from 2.4 to 4.6 ‰ during increasing drought (Unger et al., 2010a).In Acacia longifolia and Pinus pinaster a slight increase at the end of the light period of ca. 2 ‰ was observed in the field also under drought conditions (Rascher et al., 2010).
Both ecosystem and soil respiration derive from multiple sources the latter comprising heterotrophic and autotrophic rhizosphere respiration.To stress this origin from multiple sources we term the isotopic composition of CO 2 emitted from the soil or whole ecosystems δ 13 C R .Diel variations in soil δ 13 C R (0.5-5.8 ‰, Table 2) have been reported in Table 2. Survey on nocturnal, diurnal and 24 h-variations in respired δ 13 C R (minimum, maximum and total range) and the variation of the putative substrate (bulk soil OM) of composite fluxes of soil and ecosystem respiration.The ecosystem, environmental conditions and references are given.When several diel courses were available, the variation in the minimum, maximum and range over the measured period was given.ns -not significant; -not determined.grasslands (Dudziak and Halas, 1996;Bahn et al., 2009), forests (Kodama et al., 2008;Fig. 1a, Marron et al., 2009), Mediterranean woodlands (Maseyk et al., 2009;Unger et al., 2010a, b; K. P. Tu and T. E. Dawson, unpublished data), and agricultural systems (Kodama et al., 2011), while nonsignificant diel variations were detected in a boreal forest (Betson et al., 2007) (Table 2).A highly variable range in soil δ 13 C R of 0.3-12.5 ‰ occurred in an experimental garden with deciduous trees (Moyes et al., 2010).
The literature overview in this section clearly demonstrates that the short-term variations in δ 13 C of respired CO 2 do not follow a straightforward pattern and differ between organs, species, ecosystem compartments and ecosystems.This indicates the necessity to understand the processes responsible for the observed patterns and differences among systems.Accordingly we will now focus on the potential mechanisms driving these short-term dynamics.

Mechanisms
The potential mechanisms, which may drive the diel variations in δ 13 C res /δ 13 C R on the plant, soil and ecosystem level can be summarised in three main groups: M1: Substrate driven variations: short-term variations in the carbon isotopic signature of the major respiratory substrate (i.e.sugars or water soluble organic matter) and/or switches between substrates with different carbon isotope composition drive plant δ 13 C res .
M2: Isotope fractionation driven variations: changes in respiratory isotope fractionation in different metabolic pathways over the diel course determine plant δ 13 C res .
M3: Flux ratio driven variations: temporal variability in the contribution of component fluxes with distinct isotopic signatures to composite fluxes (e.g.soil and ecosystem respiration) drive variations in δ 13 C R .
These three mechanisms are not mutually exclusive and a combination of these can and most likely does occur.In the following synthesis we will explore step by step whether the  (Kodama et al., 2011).Gessler et al. (2009b) and Brandes et al. (2006) showed that the δ 13 C of water soluble organic matter is a reasonably good proxy for δ 13 C of the neutral sugar fraction and thus the major respiratory substrate.
three main mechanisms alone or in combination can explain the observed short-term variability in δ 13 C res and δ 13 C R .The complexity of the different processes on the plant level is indicated in Fig. 2 and summarized in Table 3.

Substrate driven variations (M1)
It is well established that different mechanisms and processes can induce diel variations in δ 13 C of primary assimilates in leaves and during transport to heterotrophic plant tissues; thereby potentially inducing short-term variation in δ 13 C res in leaves, stems and root.When we, as a first approximation, assume that respiration is fed by only one major respiratory substrate pool (i.e.new soluble sugars of current photosynthesis) with a homogenous δ 13 C (i.e.all substrate molecules share a comparable δ 13 C at a given time) the following   M1.1:Photosynthetic carbon isotope discrimination (Farquhar et al., 1982), which determines the δ 13 C of primary respiratory substrate, varies over the diurnal course (e.g.Gessler et al., 2007;Wingate et al., 2010) as a result of changes in light intensity, air temperature, vapour pressure deficit (VPD) and other environmental factors, which affect assimilation, stomatal (g s ) and mesophyll conductance (g m ) as well as photorespiration and dark respiration (see Fig. 2 (M1.1), reviewed by Brugnoli and Farquhar, 2000).While we are able to precisely predict changes in carbon discrimination and variations in δ 13 C of fresh assimilates in response to changes in VPD, light and temperature, much less is known on the isotopic effects of mesophyll CO 2 conductance (g m ), photorespiration and dark respiration throughout the day (e.g. Warren and Adams, 2006;Wingate et al., 2007;Lanigan et al., 2008;Tcherkez et al., 2010).There have been recent insights that there is active regulation of internal CO 2 conductance through aquaporins, which are transporting CO 2 across plasma membranes (Hanba et al., 2004;Flexas et al., 2008).This might allow fast diel adjustment of mesophyll conductance to meet photosynthetic requirements (Flexas et al., 2007), but so far measurements of diurnal dynamics in g m are often constrained by methodological issues (Pons et al., 2009).
Overall, photosynthetic discrimination alone cannot explain the strong day-night variations in δ 13 C res (and respiratory substrate) as it is active only during daylight.In addition, sugar δ 13 C values at night are far more positive than predicted by photosynthetic discrimination alone (Tcherkez et al., 2004;Gessler et al., 2008) and thus post-photosynthetic processes must be taken into account in order to fully explain observed diel variations of δ 13 C res .
M1.2: Post-photosynthetic carbon isotope fractionation related to transitory starch metabolism.Starch accumulation during daylight and remobilization at night alter the isotope signal of leaf and phloem-exported sugars on the diel scale (Tcherkez et al., 2004;Gessler et al., 2008; see Fig. 2; M1.2).During the day, the synthesis of transitory starch is either under plant internal control to adapt the storage C supply to environmental conditions (Zeeman et al., 2007) or occurs mainly when the utilisation of newly produced triose-phosphates from the chloroplast becomes rate limiting to carbon assimilation (Beck and Ziegler, 1989).Gleixner et al. (1998) suggested that transitory starch is 13 C enriched relative to soluble sugars because of the isotope effects on the "aldolase-reaction" determined originally  Gleixner and Schmidt (1997).As a consequence, 13 Cdepleted triose phosphates are exported from the chloroplast, which are used for sucrose production during the light period and thus influence δ 13 C res .It has also to be mentioned that the 13 C enriched transitory starch does not provide substrates for respiratory and photorespiratory decarboxylation in irradiated photosynthesizing leaves (Ivanova et al., 2008).During the night the 13 C-enriched transitory starch is used for sucrose synthesis.As a result, a ∼4 ‰ δ 13 C oscillation between light-and dark-exported sucrose has been predicted and observed (Ghashghaie et al., 2001;Tcherkez et al., 2004;Gessler et al., 2008Gessler et al., , 2009a)).
However, these variations in the fast-turnover organic matter pool in leaves had a much lower day-night amplitude than the observed diel changes in respired δ 13 CO 2 (Brandes et al., 2006(Brandes et al., , 2007;;Gessler et al., 2007Gessler et al., , 2008;;Kodama et al., 2008;Werner et al., 2009, see Table 1) and were also phase-shifted compared to δ 13 C res (Kodama et al., 2008, see also Fig. 1).Furthermore, opposing trends in diel variation of δ 13 C res and δ 13 C of the leaf sugars and phloem sugars (Gessler et al., 2007(Gessler et al., , 2009b) ) occurred as shown for leaves of R. communis in Fig. 3a.Others found no significant diel variations in leaf soluble sugars or water soluble organic matter (WSOM) (Hymus et al., 2005;Sun et al., 2009;Werner et al., 2009;Wegener et al., 2010;Rascher et al., 2010) but still strong variations in δ 13 C res (Fig. 3b-c), indicating that diel variations in leaf δ 13 C res cannot be solely explained by changes in the isotopic signature of the substrate.

M1.3: Isotope fractionation during carbon transport:
during phloem transport sugars are continuously released from the phloem but a major part is retrieved again into the sieve tubes (Van Bel, 2003).This process might be responsible for the intermixing of sucrose molecules with different metabolic histories and residence times (Brandes et al., 2006).As a result, the diel variations in δ 13 C originating from starch accumulation and breakdown are dampened with increasing transport distance along the stem in basipetal direction (Fig. 2; M1.3).Consequently, the diel cycle of δ 13 C in organic matter in trunks and stems of trees is mainly dependent on the position along the trunk (Gessler et al., 2007) and dampening as well as time lags have been observed (Keitel et al., 2003;Brandes et al., 2006Brandes et al., , 2007)).At the tree trunk base often no diel variation in phloem sugar δ 13 C was present (Gessler et al., 2007;Kodama et al., 2008;Betson et al., 2007;Rascher et al., 2010) whereas strong diel variations in δ 13 C res are generally observed (Kodama et al., 2008;Maunoury et al., 2007).They thus cannot be explained by variation in substrate δ 13 C (Fig. 3b for trunks P. sylvestris).
In roots there are generally only very low or nonsignificant short-term variations in δ 13 C of sugars or WSOM (Göttlicher et al., 2006;Wegener et al., 2010;Kodama et al., 2011).The lack of short-term variations in roots (Fig. 3d) is highly plausible, given the mixing of sugars with different residence times during phloem transport into the roots.The only exception we are aware of is Ricinus communis grown under controlled conditions, where δ 13 C of root sugars varied by approx 3.7 ‰ within 24 h (Gessler et al., 2009b), which also explained 72 % (p < 0.01) of the diel variation of root δ 13 C res .In contrast, in field-grown plants, root δ 13 C res showed a clear diel variation (Table 1, Unger et al., 2010a), even without significant variations in the respiratory substrate (Kodama et al., 2011;Fig. 1b).
In conclusion, there is a conceptual framework for explaining the observed short-term variations in δ 13 C of sugars and other fast turn-over carbon compounds (Fig. 2).However, δ 13 C variations of new assimilates are too small or uncorrelated to explain δ 13 C res dynamics (Fig. 3), and thus cannot be solely responsible for the diel variations in δ 13 C res , when one major respiratory pool consisting of one compound class is assumed to fuel respiration.Another aspect of substrate-induced variations (M1) might be related to the use of different respiratory substrates: M1.4:A switch between respiratory sources of different storage pools or substrate types including, soluble sugar, starch, lipids or amino acids, or stored and fresh assimilates with different isotopic signature could account for variation in δ 13 C res (Tcherkez et al., 2003;Nogués et al., 2004;Fig. 2;M1.4).
Leaf respiration uses several carbon sources with different isotopic characteristics and residence times (Schnyder et al., 2003;Lehmeier et al., 2008Lehmeier et al., , 2010)).However, in spite of differences in δ 13 C between glucose, fructose and sucrose, mass-balance calculations taking into consideration measured diel changes in pool sizes and δ 13 C signatures could only explain 1.1 ‰ variation in Halimium halimifolium even though observed diel δ 13 C res variation was 8.9 ‰.The amount of explainable variation was even less in four other species (Werner et al., 2009).
The effect of switches between substrate classes (e.g. from sugars to lipids) on δ 13 C res has been shown experimentally during plant starvation under continuous dark (up to 10 ‰ shift, Tcherkez et al., 2003) and may play a role under natural conditions in the case of severe stress, like wilting or senescence (Unger et al., 2010a).However, a complete shift between different respiratory substrates during the day seems rather unlikely for healthy plants under ambient conditions (Hymus et al., 2005).One exception might be a transient shift in utilization of organic acid pools, which accumulated in the light and are rapidly decarboxylated upon darkening.

M1.5: Light enhanced dark respiration (LEDR)
is the transient increase in respiration upon darkening in a photosynthesis-dependent manner (Azcon-Bieto and Osmond, 1983;Atkin et al., 1998).Light-acclimated leaves released strongly 13 C-enriched CO 2 as compared to potential substrates in the first 5-10 min after darkening followed by a rapid decline in δ 13 C res (Barbour et al., 2007;Werner et al., 2007).Both the extent of enrichment and the subsequent 13 C-depletion augment during the light period (Fig. 4, Werner et al., 2009) and have been shown to be linearly related to cumulative carbon gain during the light period (Hymus et al., 2005) even under different growth-light conditions (Priault et al., 2009).In Ricinus communis LEDR-dependent 13 C-enrichment was fully explained with the accumulation of 13 C-enriched malate in the light and rapid malate decarboxylation just after darkening (Gessler et al., 2009b, see Fig. 2; M1.5).In the light, both glycolysis and particularly the Krebs cycle (KC) are strongly inhibited (Tcherkez et al., 2005;Nunes-Nesi et al., 2007).Moreover, during illumination probably only a non-cyclic Krebs "cycle" operates in autotrophic tissues (Tcherkez et al., 2009;Sweetlove et al., 2010) because three key enzymes, i.e. the mitochondrial isocitrate dehydrogenase (Igamberdiev and Gardeström, 2003), the succinate dehydrogenase (Popov et al., 2009) and the 2-oxoglutarate dehydrogenase (Gessler et al., 2009b) are inhibited.As a consequence, malate fixed via phosphenolpyruvatecarboxylase (PEPc) can accumulate (see Fig. 5a).However, the malic enzyme is most certainly associated with an isotope effect.If we assume a dynamic Rayleigh process (see Gessler et al., 2009b) δ 13 C res would be more depleted immediately after darkening while getting more enriched as the malate pool declines (Werner et al., 2009).Indeed, such a transient increase is sometimes observed during the first 5-10 min upon darkening (Fig. 4, red arrows); however, a rapid decline from higher δ 13 C res values is generally observed during light-dark transitions (Fig. 4, Barbour et al., 2007;Werner et al., 2007Werner et al., , 2009)), which could indicate several overlaying processes such as a partitioning of malate between the malic enzyme and mitochondrial malate dehydrogenase (Werner et al., 2011b).LEDR is a transient effect and under natural conditions it has been shown to occur at dusk after sunny days (Barbour et al., 2011).Based on current knowledge, the short duration of LEDR cannot explain continuous nocturnal dynamics in δ 13 C res (e.g.Sun et al., 2009Sun et al., , 2010;;Unger et al., 2010a).Furthermore, a diurnal increase in δ 13 C res can also be observed after the first transient LEDR effect (e.g.Fig. 4, grey arrow) indicating processes in addition to LEDR are influencing δ 13 C res .
In conclusion, it is unlikely that diel variations of respired δ 13 CO 2 can be entirely explained by δ 13 C variation in a single substrate or by a switch between substrates.In autotrophic tissues at least part of the day-night differences in δ 13 C res might be attributed to LEDR (Kodama et al., 2011;Barbour et al., 2011) but these do not apply for nonphotosynthetic tissues.As a consequence isotope fractionation driven variations (M2) should be taken into account.

Fractionation driven variations (M2)
Since δ 13 C res markedly deviates from substrate δ 13 C, we have to assume that diel variation in δ 13 C res may be mainly affected by carbon isotope fractionation during respiration.The following mechanism might be involved: M2.1: Fragmentation fractionation and enzyme related isotope effects: the often observed 13 C enrichment in respired CO 2 above the organic source is assumed to originate from the fragmentation of the substrate molecule due to heterogeneous isotope distribution (Tcherkez et al., 2003(Tcherkez et al., , 2004;;see Figs. 2f, 5, 6).There is a non-homogeneous distribution of δ 13 C within the glucose molecule where C-3 and C-4 are 13 C-enriched compared to other positions due to isotope fractionation in the aldolase reaction (Fig. 6, Rossmann et al., 1991;Gleixner and Schmidt, 1997;Hobbie and Werner, 2004).During glycolysis, C-1 of pyruvate derived from enriched C-3 and C-4 of glucose is decarboxylated by pyruvate dehydrogenase (PDH) (cf.Tcherkez et al., 2003).Consequently, the PDH reaction releases 13 C-enriched CO 2 , whereas the remaining molecule enters the Krebs Cycle (KC) which releases in turn 13 C-depleted CO 2 -compared with the mean δ 13 C of the original glucose molecule (see Fig. 5, 6).Any change in the relative contribution of CO 2 decarboxylated in the KC versus by PDH to total CO 2 production may thus cause variations in δ 13 C res .Furthermore, kinetic and equilibrium isotope fractionation in glycolysis and KC may also occur.Pyruvate is also the substrate for amino acid synthesis and the PDH reaction has been recognized to imply kinetic isotope effects on all three C atoms of pyruvate (Melzer and Schmidt, 1987).The kinetic isotope effect on the PDH reaction is also responsible for the 13 C depletion of (acetogenic) lipids (DeNiro and Epstein, 1977), which implies a metabolic branching point at the pyruvate stage.In case of a non-quantitative conversion of pyruvate to acetyl-CoA and CO 2 and a metabolic branching point at pyruvate, the kinetic isotope effect on the C-1 of pyruvate will be expressed in vivo and, as a consequence, the released CO 2 will be depleted in 13 C relative to C-3 and C-4 of glucose.In conclusion, it is most likely a mixed influence of fragmentation fractionation and enzymatic isotope effects related to metabolic flux rates (e.g.see Fig. 5) which together drive δ 13 C res variations.
The potential 13 C enrichment of PDH-derived CO 2 above the whole glucose molecule as a result of "fragmentation fractionation" depends on the extent of intramolecular 13 C variation.The maximum deviation of a particular C atom from the average δ 13 C value of the molecule was determined as 6.3 ‰ for glucose in yeast (by stepwise biochemical degradation, Rossmann et al., 1991, see Fig. 6).Recently, NMR data for sucrose showed a somewhat larger enrichment at the C-3 and C-4 position (Fig. 6) and in particular a larger intramolecular deviation between the C-4 to C-6 positions (of 13.3 ‰, Gilbert et al., 2009) compared to the data from Rossmann et al. (1991: 11.2 ‰).Nevertheless, assuming a complete isomerisation reaction between glyceraldehyd-3-phosphate and dehydroxyacetonphosphate, the isotopic signature within the pyruvate molecule would be similar.However, new emerging NMR data indicate that the heterogeneous 13 C distribution in carbohydrates may vary among species and be related to environmental conditions www.biogeosciences.net/8/2437/2011/Biogeosciences, 8, 2437-2459, 2011  Rossmann et al., 1991).Two distinct situations are indicated: (A) full decarboxylation of the carbon molecules in the Krebs cycle (KC) or (B) high investment into secondary metabolism and fatty acid synthesis.Isotope fractionation processes in the KC are exemplified by citrate synthase and α-ketoglutarate (αKG) dehydrogenase (α = 1.023) (see Tcherkez andFarquhar, 2005 andliterature therein, Bulzenlechner et al., 1996;Melzer and Schmidt, 1987;Schmidt, 2003).Further, the potential involvement of an enriched malate pool (−5.1 ‰, Barbour et al., 2007) which is produced during phospoenolpyruvate carboxylase (PEPc) reaction with small kinetic enzyme fractionation against 13 C (α = 1.002) and equilibrium fractionation against 12 C (α = 0.991) during HCO − 3 equilibration (O' Leary 1981, Farquhar et al., 1989) is indicated in (A) (dashed line).The human malic enzyme fractionates in favour of 12 C (α = 1.014,Rishavy et al., 2001, see Gessler et al., 2009b), the reaction following a Rayleigh distillation process after the light-dark transition (see also Barbour et al., 2007).Adapted from Barbour et al. (2007), Werner et al. (2009) and Gessler et al. (2009b).(Gilbert et al., 2011), which may add to species-specific differences in δ 13 C res .
Utilizing the data from Rossmann et al. (1991;Figs. 5, 6), the potential variation in δ 13 C res due to fragmentation fractionation can be calculated: if only pyruvate decarboxylation by PDH is assumed (i.e. when the KC cycle is fully inhibited in the light) δ 13 C res of C-1 of −21 ‰ is released (Fig. 5b), whereas the complete decarboxylation of the glucose molecules in KC produces δ 13 C res of the substrate with −25 ‰ (Fig. 5a).Thus the shift from 0 to 100 % decarboxylation in the KC produces an isotope shift of 4 ‰ (illustrated in Fig. 7 for 0 or 100 % carbon flow into KC decarboxylation).However, the KC is also an important source for amino-acid biosynthesis, providing carbon skeletons for glutamic and aspartic acid (notably amino acids which are strongly enriched in 13 C, Hayes, 2001).If pyruvate is not fully respired, both equilibrium and kinetic isotope effects that occur in the KC (Tcherkez and Farquhar, 2005) could lead to more negative δ 13 C res .The relative decrease in δ 13 C res depends on the carbon partitioning into KC and the effective enzymatic isotope fractionations, which are dependent on the flux rate.
Figure 7 exemplifies on a theoretical basis the potential effects of varying carbon flux rates from 0-100 % through the PDH and KC considering isotope fractionation by (i) citrate synthase (ε CS ∼ 23 ‰) and (ii) α-ketoglutarate dehydrogenase (ε KG ∼ 23 ‰)(fractionation factors taken from Tcherkez and Farquhar, 2005 and references therein, see also legend Fig. 5).
If the carbon flow into the KC is low (e.g. 5 %), isotope fractionation is high and the CO 2 released in KC enzymatic reactions will be strongly depleted in 13 C (−48.9 ‰).However this has little effect on the overall δ 13 C res as it constitutes only a small fraction which is mixing into the enriched δ 13 CO 2 flux released by PDH (−21 ‰).Inversely, if the carbon flow into KC decarboxylation is high (e.g.95 %) the effective isotope fractionation diminishes, and δ 13 C in respired CO 2 approaches −25 ‰.However, Fig. 7  illustrates that the largest decrease (∼9 ‰) occurs at intermediate mixing ratios (at 50 % in the given example), when the CO 2 release from KC decarboxylation is still relatively depleted (38.6 ‰) and constitutes two-thirds of the overall CO 2 evolved (due to two decarboxylation steps in the KC) so that the total δ 13 C res decreases to −29.8 ‰ (Werner, 2010).Potential isotope fractionation can also occur in the PDH reaction as explained above, which would further deplete the Acetyl-CoA at the C-2 position if the reaction was incomplete (Melzer and Schmidt, 1987, effect indicated on the zaxis, Fig. 7).
This example indicates that on a theoretical basis large isotope effects can occur through fractionation effects and metabolic branching points in the respiratory pathways, however to what extend this will be expressed in vivo still remains to be resolved.Only recently, Werner et al. (2011b) postulated that the isotope effects associated with the KC enzyme reactions do most probably not lead to in vivo isotope discrimination.On the one hand, the inner mitochondrial membrane is impermeable for acetyl-CoA (Voet and Voet, 1995) and thus acetyl-CoA will react quantitatively with oxaloacetate to citrate.As a consequence, the citrate synthase reaction should not lead to any isotope fractionation in the acetyl-part of citrate (Werner et al., 2011b).On the other hand, the whole KC is assumed to work as an organised enzyme complex (Srere et al., 1996).The proposed channelling of the KC substrates at reduced concentrations would avoid metabolic branching to other enzymatic reactions (Srere et al., 1996).As a consequence, theoretically possible kinetic isotope effects on KC enzyme reactions would not be expressed in vivo (Werner et al., 2011b).We need at that time clear experimental evidence if kinetic isotope fractionation occurs in the KC under physiological conditions as postu- with f 1-3 being the carbon flux (where f 2 equals f 3 as both carbon atoms enter the KC as Acetyl-CoA moiety), and δ 13 C-1-3 the isotopic composition of the carbon molecules at the C-1 to C-3 positions of pyruvate and ε denotes the enzymatic isotope effect.The effective enzyme isotope fractionation (ε eff ) in the KC of the citrate synthase (ε CS ) and the α-ketoglutarate dehydrogenase (ε KG ) is dependent on the carbon flow in the KC (see Hayes, 2001) by: ε eff = α 1+ε•f − 1. Potential fractionation effects were calculated by varying the carbon flow rates into KC (f 2,3 ) from 0-100 %, assuming fractionation factor of ε CS and ε KG of −23 ‰ (see Tcherkez and Farquhar, 2005 and Fig. 5 for details).PDH could also potentially fractionate if the reaction is incomplete (Melzer and Schmidt, 1987) which would further deplete δ 13 C res , which was tested assuming f 1 of 50-100 % (z-axis), but occurrence of the latter processes in vivo is unknown.lated by Tcherkez (2010) or not as concluded by Werner et al. (2011b).
In general, it has to be considered that, both the KC and to a lesser extend also the mitochondrial PDH are down-regulated in the light (Budde and Randall, 1990;Tcherkez et al., 2009;Werner et al., 2009) and that only part of the KC reactions may function in a non-cyclic manner (e.g.Sweetlove et al., 2010).In the dark when the KC is reorganized again, the impact of fragmentation fractionation together with potential kinetic isotope discrimination in vivo will depend on how much of the respiratory substrate is oxidized to CO 2 and which portion is used for biosyntheses.Large fractionation effect within the mitochondria may probably only be relevant during transitory stages (e.g. during up-regulation upon darkening).There are, however, other metabolic branching points within the cell, which could www.biogeosciences.net/8/2437/2011/Biogeosciences, 8, 2437-2459, 2011 C. Werner and A. Gessler: Diel variations in the carbon isotope composition lead to fractionation effects and carbon partitioning along different pathways has to be considered: M2.2: Variations of fluxes in the metabolic pathways: the relative carbon fluxes involved in metabolic pathways may change depending on the metabolic status of cells, tissues or plants.Day-night variations with relatively higher allocation of carbon to isoprenoids (Loivamäki et al., 2007) and various other secondary compounds (Ayan et al., 2006) as well as the non-cyclic nature of the KC in the light (Tcherkez et al., 2009) are thus highly plausible triggers for changes in the relative contribution of PDH to KC derived CO 2 as described above (see Fig. 2; M2.2).Increased activity of the oxidative pentose phosphate pathway (PPP), which decarboxylates the 13 C depleted C-1 position of glucose, can be significant in roots (Dieuaide-Noubhani et al., 1995;Bathellier et al., 2008Bathellier et al., , 2009)).Thus, temporal changes in the partitioning of carbon originating from glucose-6-phosphate between glycolysis and PPP might also be responsible for diel variations in δ 13 C res .
Marked differences between carbon allocation into different metabolic pathways, i.e. an increasing secondary metabolism when carbon accumulated throughout the day, could be related to differences between plant functional groups (Priault et al., 2009).A variety of secondary compounds including volatile isoprenoids, oxygenated VOCs, aromatics, and fatty acid oxidation products can be emitted by plants (e.g.Jardine et al., 2010a).The biosynthesis evolves the decarboxylation of the 13 C-enriched C-1 from pyruvate (or phosphenolpyruvate), leading to the biosynthesis of VOCs from the PDH bypass, 2-C-methy.l-D-erythritol 4-phosphate, and mevalonic, shikimic, and fatty acid pathways (e.g.Jardine et al., 2010b).In contrast to the mitochondrial PDH, the plastidial PDH is activated by lightinduced changes in the stroma (Tovar-Méndez et al., 2003) as it fuels Acetyl-CoA for fatty acid synthesis and secondary metabolism.Fatty acid biosynthesis involves plastidic PDH.In the light, CO 2 originating from the 13 C-enriched C-1 of pyruvate will be released and -most probably -re-fixed during photosynthesis.Chain elongation of fatty acids occurs partially in the cytosol and involves acetyl-CoA originating (via the citrate shuttle) from the Krebs-Cycle (Bowher et al., 2008; see Fig. 1 in Werner et al., 2011b).Starting from PEP, the C-1 of pyruvate is released as CO 2 by the mitochondrial PDH for the acetyl-CoA supply in this reaction.While the 13 C-enriched C-1 from pyruvate will be released as CO 2 , pyruvate positional labelling showed that the 13 C depleted C-2 and C-3 carbon atoms of the acetyl-moiety are emitted as a variety of volatile isoprenoids and oxygenated VOCs (such as isoprene, acetaldehyde, ethanol, or acetic acid) (Jardine et al., 2010b).VOC emissions generally range about 2-5 % (e.g.Guenther et al., 1995) of mostly recently fixed carbon (Ghirardo et al., 2011), but can increase at least by an order of magnitude during stress conditions (e.g.Kesselmeier et al., 2002), which may potentially cause a large effect on the emitted δ 13 CO 2 .In plants, two different pathways are responsible for the biosynthesis of isopentenyl diphosphate, which is the central intermediate for isoprenoids (Bick and Lange, 2003).In the plastidic deoxyxylulose 5-phosphate (DXP) pathway CO 2 originating from the 13 C enriched C-1 of pyruvate is released in the DXP synthase reaction.To our knowledge no investigations have been performed whether there is an in vivo kinetic isotope effect on this reaction.Since the isoprene synthesis is strongly photosynthesis and thus light dependent (Niinemets et al., 1999) re-fixation of the CO 2 released in the chloroplast is likely.For the cytosolic mevalonate pathway acetyl-CoA is the starting point.It originates from Krebs cycle citrate and thus its supply involves the release of CO 2 originating from the C-1 of pyruvate by the mitochondrial PDH (see Fig. 2a in Werner et al., 2011b).
Moreover, acetaldehyde is emitted from a variety of plant species (Nguyen et at., 2009) and one potential mechanism involved in its production is the decarboxylation of pyruvate by leaf pyruvate decarboxylase (PDC) (Karl et al., 2002).As in the PDH reaction, the PDC reaction releases the C-1 of pyruvate as CO 2 and a kinetic isotope effect on this reaction might cause a further 13 C depletion of the acetaldehyde produced (DeNiro and Epstein, 1977).
A pyruvate positional 13 C-labelling experiment provided further direct evidence that diel changes in the relative activity of the PDH-reaction (measured after 5 min darkening) occurred in species with marked increase in δ 13 C res (Priault et al., 2009).Diel variations in δ 13 C res were related to an increased metabolic activity of the PDH probably due to an increase in carbon allocation to secondary metabolism, while carbon flow into KC remained at a constant low level (Priault et al., 2009;Wegener et al., 2010).In contrast, an herb without significant diel variation in δ 13 C res and presumably low secondary metabolism had a stable, low activity of both PDH and KC activity throughout the day (Priault et al., 2009;Wegener et al., 2010).
Considering mass-balance requirements, the release of highly enriched δ 13 C res could be counterbalanced by the emission of 13 C-depleted VOCs; otherwise a compensating effect on the δ 13 C of leaf organic matter would have to occur.Interestingly, Wegener et al. (2010) observed that strong 13 C enrichment of leaf respired CO 2 above substrate was highly correlated with differences in autotrophic vs. heterotrophic tissue 13 C, i.e. species with high diel leaf δ 13 C res enrichment had larger 13 C-differences between leaf and root WSOM than species with lower diel leaf δ 13 C res .Nevertheless, most leaves (particularly evergreen or longer-lived leaves) do not exhibit a progressive 13 C depletion once the leaf has matured (Eglin et al., 2009;Werner and Máguas, 2010).Thus, a counterbalancing effect from the emission of VOCs depleted in 13 C might be a plausible explanation.
Moreover, a close positive correlation between respiration rate and respiratory isotope fractionation over the diel course was observed for trunks of P. sylvestris and for shoots of Triticum aestivum (Kodama et al., 2008;2011): with more positive δ 13 C res at low compared to high respiration rates.Comparable results were observed by Ghashghaie et al. (2003) for three herbaceous species.Furthermore, as observed by Kodama et al. (2008Kodama et al. ( , 2011) ) respiration rates increased linearly with temperature.One reason for the decreasing 13 C enrichment of respired CO 2 with increasing temperature might be attributed to the temperature-dependent kinetic isotope effect of the PDH reaction (Tcherkez et al., 2003).Moreover, as glycolysis (and thus decarboxylation of pyruvate) are less temperaturedependent than mitochondrial oxidation capacity and thus KC mediated CO 2 flux (cf.Berry and Raison, 1981;Atkin et al., 2000), the relative contribution of CO 2 from glycolysis to total respiration might also increase at lower temperatures (e.g. in the night) explaining the higher apparent isotope fractionation at lower respiration rates.Thus, the fractionation hypothesis based on temporal variations in fragmentation fractionation during respiration (M2.1) due to changes in carbon fluxes through different metabolic pathways (M2.2) might offer a conclusive explanation for day-night variations of δ 13 C res .
A particular case is the variation of carbon fluxes, which are directed in opposite directions: M2.3: Re-fixation of CO 2 by PEPc causes a CO 2 flux in the direction opposite to the respiratory flux (Fig. 2; M2.3).PEPc discriminates against 13 C by ca.2.2 ‰.Equilibrium dissolution of CO 2 into water concentrates 13 CO 2 in the gas phase by 1.1 ‰, while the hydration equilibrium favours 13 C by 9 ‰ (O' Leary, 1991), resulting in an overall discrimination of 5.7 ‰ against 12 C (Farquhar et al., 1989;Brugnoli and Farquhar, 2000).Thus PEPc activity causes the produced organic matter to be 13 C enriched whereas the remaining (non-fixed) CO 2 is relatively 13 C depleted.Thus (re)-fixation by PEPc can also alter the δ 13 C of CO 2 emitted from a plant.Since both processes and the effective isotope fractionations cannot be separated the isotopic difference between putative substrate and respired CO 2 is often referred to as apparent isotope fractionation (e.g.Gessler et al., 2009b).
It is known that differences in PEPc activity among organs can cause differences in apparent respiratory isotope fractionation and thus in δ 13 C of respired CO 2 along the plant axis (Badeck et al., 2005).PEPc activity has been found in all plant organs (e.g.Hibberd and Quick, 2002;Berveiller and Damesin, 2008) and thus PEPc activity may also be involved in diel variations in δ 13 C res .The anaplerotic PEPc reaction in leaves of C 3 -plants is activated in the light (Duff and Chollet, 1995) to replenish the carbon skeletons of the TCA used for biosynthesis.Theoretically, the increased PEPc activity during day might thus be directly responsible for 13 C enriched CO 2 emitted from light acclimated leaves.In roots and stems, however, Gessler et al. (2009b) did not find any relation between PEPc activity and δ 13 C res .It is consequently unlikely that PEPc mediated re-fixation of CO 2 played a large role in observed diel variations in δ 13 C. We, however, need more information on diel variations in PEPc activity with simultaneous assessments of δ 13 C res from different species to draw more reliable conclusions.
In summary M2.1 and M2.3 can explain part of the variation in δ 13 C over the diel course.However, they give no explanation for δ 13 C res values more positive than the δ 13 C of the enriched position in glucose (−21 ‰ in our example calculation Fig. 7, or 4 ‰ above the mean glucose δ 13 C, according to the values of Rossmann et al., 1991) and thus other co-occurring processes such as LEDR in leaves must also occur.
At the soil and ecosystem scale, mixing of different respiratory fluxes varying over the diel course might also be involved in the diel pattern of δ 13 C of emitted CO 2 and might even enhance the short-term variations.

Flux ratio driven variations (M3)
On the soil and ecosystem level the net respiration flux consists of several component fluxes and mixing between these fluxes with potentially different isotopic signatures and associated diel variation in both δ 13 C res and flux rates has to be considered for the explanation of temporal variations of δ 13 C R .We have discussed above that the diel patterns (i.e. the timing of maxima and minima) in δ 13 C res differ among respiratory fluxes from different plant tissues.Furthermore δ 13 C of soil and plant respiration are not synchronous (e.g.Kodama et al., 2008;Unger et al., 2010a), and even soil and ecosystem respiration fluxes are partially phase-shifted with distinct diel patterns (e.g.Unger et al., 2009), so that strong temporal dynamics in the component fluxes and consequently δ 13 C R of the total flux have to be expected.There are several processes, which drive the diel variation in different component fluxes.
M3.1: Effect of diel changes in abiotic drivers and physical factors on component fluxes: on the one hand different respiratory components (e.g.above and belowground respiratory sources) experience different amplitudes and phase-shifted diel variations due to changes in abiotic environmental factors (such as temperature, moisture and PPFD).On the other hand, respiratory sources differ in their responsiveness to these abiotic drivers, thus resulting in changes in the mixing-ratios of respiratory fluxes.
At the soil scale it is often reported that temperature and moisture are the main drivers for CO 2 flux (e.g.Davidson et al., 1998;Carbone et al., 2008;Paterson et al., 2009), which are both characterized by a marked diurnal cycle.Moreover, diurnal temperature changes are buffered and phaseshifted compared to air temperature with increasing soil depth.As a consequence, the resulting soil and ecosystem respiration flux consists of a temporally variable mixture of different component fluxes with different isotope signatures www.biogeosciences.net/8/2437/2011/Biogeosciences, 8, 2437-2459, 2011 (e.g.Werner et al., 2006;Unger et al., 2010a).Many soils show a marked gradient in δ 13 C of soil organic matter (SOM) within the soil profile (Ehleringer et al., 2000).Thus, diel changes in the contribution of CO 2 originating from different soil layers and thus from organic substrates with different δ 13 C can induce diel variations of the net soil efflux δ 13 C R .Maseyk et al. (2009) estimated that the depth-related increase in SOM and respired δ 13 C could contribute to ∼0.5 ‰ of the observed diel-scale variability in soil δ 13 C R through temperature driven shifts in the relative contribution of δ 13 C res from different soil depths.Soil δ 13 C R can also be influenced by physical effects on soil CO 2 diffusivity (Stoy et al., 2007).The diffusive velocity of CO 2 through the soil pores is altered by the physical environment, such as porosity and soil moisture (Stoy et al., 2007), and thus diurnal changes during drying and wetting of upper soil layers may alter mixing ratios from different soil depths.Transient diffusive isotope fractionation during non-steady state conditions could induce diel variation in soil δ 13 C R , which seemed particularly large when soil respiratory fluxes were low but the variability (fluctuation) was high (Moyes et al., 2011).
A further aspect is the different responsiveness of respiratory components to variations in abiotic drivers.At the soil scale, CO 2 flux derives from two major components with different isotopic signatures: autotrophic and heterotrophic soil respiration, which are two fully distinct processes, controlled by different underlying factors (see Brüggemann et al., 2011 and literature therein for details), particularly regarding their temperature sensitivity.To date, published results yield a non-uniform picture: in some ecosystems autotrophic soil respiration was found to have a higher temperature sensitivity than heterotrophic soil respiration (e.g.Boone et al., 1998;Bhupinderpal-Singh et al., 2003).In these systems the proportional contribution of autotrophic respiration may therefore increase from the morning to the afternoon, thus producing diel variations in soil δ 13 C res (Carbone et al., 2008;Marron et al., 2009).In contrast, others (e.g.Bol et al., 2003;Hartley and Ineson, 2008;Vanhala et al., 2007) suggested that heterotrophic respiration with recalcitrant soil organic material as substrate was highly temperature sensitive.
Additionally, it has been suggested that growth respiration might be temperature insensitive while maintenance respirations might exhibit large temperature sensitivity (Kuzyakov and Gavrichkova, 2010).If growth and maintenance respiration differ in δ 13 C res due to differences in respiratory substrates (see M1) or respiratory isotope fractionation (see M2) any change in temperature will lead to changes in δ 13 C R .
Moreover, recently it has been questioned whether soil respiration is mainly driven by environmental factors such as soil temperature and moisture (Liu et al., 2006;Vargas and Allen, 2008;Kuzyakov and Gavrichkova, 2010) as opposed to biotic factors.There is evidence that soil respiration can be partially decoupled from soil temperature, probably because of the impact of recent photosynthates as substrates for root and (myco)rhizosphere respiration.Thus substrate-driven changes through the input of labile carbon compounds needs to be considered as a driving factor causing short-term variations in δ 13 C R .

M3.2: Substrate driven changes in component flux
rates due to different responsiveness to input of recent assimilates: assuming that soil and ecosystem respiration rates are strongly influenced by photosynthetic assimilate supply to the soil (cf.Ekblad and Högberg, 2001, recent reviews by Davidson et al., 2006 andPaterson et al., 2009;Högberg and Read, 2006;Trumbore, 2006;Bahn et al., 2009) photosynthesis should influence δ 13 C R in two ways.First the isotopic signature of the labile carbon transferred from the canopy to roots and rhizosphere should be imprinted on the CO 2 respired from mycorrhizal roots and associated rhizosphere microorganisms.In addition, the ratio of heterotrophic to autotrophic contributions to respiratory fluxes is most likely altered.Soil and ecosystem δ 13 C R are indeed often well correlated with environmental factors driving changes in photosynthetic discrimination during the preceding days (e.g.Ekblad and Högberg, 2001;Werner et al., 2006).The rapid transfer of photosynthates to roots, root exudates and subsequent respiration in the rhizosphere has been demonstrated by 13 C labelling experiments (e.g.Carbone and Trumbore, 2007;Högberg et al., 2008;Bahn et al., 2009;Subke et al., 2009;Barthel et al., 2011a).Bahn et al. (2009) showed that in a grassland recent plant-assimilates were respired in the soil from the late morning hours onwards, whereas previous day assimilates were the substrate during the night and early morning hours.Moreover, there are new indications suggesting a tight and rapid coupling between the onset of photosynthetic activity during the light period and increased C-supply to rhizosphere respiration (Mencuccini and Hölttä, 2010;Kuzyakov and Gavrichkova, 2010), which could be mediated by pressure-gradient waves.This mechanism could enable a tight coupling between phloem sugar loading with new assimilates and root-released exudates which would circumvent the time-lags associated with basipetal transport ways (Mencuccini and Hölttä, 2010).
Thus, the autotrophic soil flux is likely more dynamic over the diel cycle than heterotrophic respiration resulting in diel variations in soil δ 13 C R (e.g.Carbone et al., 2008).
As the soil flux constitutes a large proportion of total ecosystem respiratory flux in many ecosystems (e.g.Davidson et al., 2006) it may markedly contribute to diel variations in ecosystem δ 13 C R .At the ecosystem level, an additional factor is the fact that respiration of leaves is strongly inhibited in the light (Tcherkez et al., 2008) but may exhibit a marked increase with very positive δ 13 C res (LEDR) at the beginning of the dark period (Barbour et al., 2011).A maximum peak in δ 13 C res after sunset (duration of 60-100 min) was correlated with the light intensity on the prevailing day, showing higher 13 C enrichment during sunny compared to cloudy days (Barbour et al., 2011), thus confirming the patterns observed in leaves (Prater et al., 2006;Priault et al., 2009).Indeed, markedly 13 C-enriched CO 2 has been measured in tree crowns at night (Mortazavi et al., 2006).Unger et al. (2010a) have shown in an isotopic-mass balance approach how different ecosystem components can vary in flux rates and δ 13 C res over short-term scales, with marked impacts on ecosystem δ 13 C R .Given the high short-term dynamics of multiple sources in an ecosystem, it needs to be critically reassessed whether a simple two-source mixing model for Keeling-plots can adequately describe processes at the ecosystem scale.K. P. Tu and T. E. Dawson (unpublished data) concluded that the ecosystem mass-balance could be closed only at predawn, when most ecosystem processes became relaxed and changes in fluxes were small.
A further complication may arise if large portions of respired CO 2 are not released to the atmosphere but internally transported between organs as it has been suggested for xylem CO 2 (see Teskey et al., 2008).If a large portion of root respired CO 2 is transported via the xylem water inside the plant and subsequently re-fixed in stem and twigs and/or emitted via the stem to the atmosphere, it would add a further variable source coupled to diel changes in xylem flow delivering depleted δ 13 C res compared to atmospheric δ 13 CO 2 .However, recent studies from Kodama et al. (2008) and Ubierna et al. (2009) showed that the influence of CO 2 from belowground -potentially transported with and stored in the xylem water -had only negligible influence on δ 13 C res of trunk respired CO 2 .Aubrey and Teskey (2009) calculated that on a daily basis, the amount of CO 2 that moved upward from the root system into the stem via the xylem stream in a poplar plantation rivaled that which diffused from the soil surface to the atmosphere.If part of this CO 2 is released via trunk or twigs (or refixed via PEPc or stem photosynthesis), if it deviates in δ 13 C from CO 2 produced in the above-ground tissues and if the contribution to trunk or stem efflux varies over the diel course, temporal variations in δ 13 C of ecosystem respired CO 2 would also result.
In summary, the mechanisms driving composite fluxes such as soil and ecosystem CO 2 fluxes are complex, since changes in the contribution of the relative flux rates of component fluxes with different isotopic signatures have to be taken into account.There is increasing recognition on close feed-backs between plant carbon assimilation and rhizosphere and soil respiration (see Brüggemann et al., 2011), but its impact on diel variations in δ 13 C R remain to be resolved.Variable contributions of different component fluxes, might at least partially explain the strong variations in δ 13 C R observed on the ecosystem level.We certainly need experiments targeted towards assessing the short-term variability of the isotopic fluxes from different ecosystem compartments and how they contribute to δ 13 C of ecosystem respired CO 2 .The emerging laser spectroscopic techniques which allow direct determination of 13 CO 2 and 12 CO 2 fluxes on the ecosystem level (Griffis et al., 2008) and within individual compart-ments (Wingate et al., 2010) will provide a powerful tool for such studies in the future.

Conclusions
Our review suggests that direct relations between δ 13 C of recent assimilates as the most probable respiratory substrates and respired CO 2 may not be present on a diel time scale and that other factors lead to short-term variations in δ 13 C res and in δ 13 C R of ecosystem-emitted CO 2 .Temporal variation of respiratory isotope fractionation due to temperature effects and changing allocation of carbon to metabolic pathways are highly plausible mechanisms that can explain diel patterns in δ 13 C res .For leaves and other autotrophic organs, LEDR is an additional mechanism most probably responsible for the observed increase in δ 13 C directly after sunset and upon initial darkening.Component fluxes with different and variable isotopic compositions and flux rates further complicate the interpretation of the respiratory isotope signal at the plant, soil and ecosystem scale.The quantification of component isofluxes at different scales including assessments of e.g. in vitro enzyme activities, transgenic PEPc knock-out and overexpressing lines and combined 13 C-labelling and natural abundance studies might all give deeper insights into the origin of short-term variations of respired CO 2 in future.This is highly important since the carbon isotope composition of plant respired CO 2 contains information on the fate of respiratory substrates, and may, therefore, provide a nonintrusive way to identify changes in carbon allocation patterns over various scale levels.

Figure 3 2 Fig. 3 .
Figure 3 2 Fig. 3. δ 13 C res plotted against δ 13 C of potential respiratory substrates during the diel course.(A) data for leaf emitted CO 2 and leaf soluble sugars in R. communis during a 24 h cycle.Each data point represents one individual plant at one time point.Samples were taken twice during the day (10:00; 15:30) and twice during the dark period (22:30; 03:30).Data are from Gessler et al. (2009b).(B) data for trunks of P. sylvestris taken from Kodama et al. (2008).As substrate for respiration we have chosen trunk phloem exudates from the same position where the CO 2 measurements were made.Data are from diel courses measured every 3 h over 4 days.(C and D) data from Rosmariuns officinalis of leaves and roots, respectively; of dark-respired δ 13 C res and WSOM measured every 2-3 h over the diurnal course in Portugal in May and August from Dubbert et al. (2011).

Fig. 4 .
Figure 4 Fig. 4. Diel (grey dashed arrow) and short-term (red arrows) postillumination changes of leaf dark respired δ 13 CO 2 (δ 13 C res ) during 25 min dark phases over the diurnal course (grey areas indicate the dark period).Black bars at the bottom of the figure represent the time during which the measured leaf was darkened, while the rest of the plant remained under the growth light conditions.Data are mean values of Quercus ilex leaves (n = 3, ± SE), reprinted from Werner et al. (2009).

clearly 55 Figure 6 Fig. 6 .
Figure 6 Fig. 6.Relative intramolecular 13 C distribution for different carbon positions calculated as deviation from the mean δ 13 C of the whole molecule.Examples are given for glucose (*), determined by stepwise fermentation by Rossmann et al. (1991) and for the glucose moiety of sucrose (#) determined by NMR by Gilbert et al. (2009).Glucose enters glycolysis where two CO 2 molecules from the C-3 and C-4 positions are decarboxylated by pyruvatedehydrogenase (PDH) and the remaining 13 C-depleted Acetyl-CoA molecules enter the Krebs cycle (KC).

Table 3 .
Potential mechanisms causing diel variation in δ 13 C res at the plant scale.