JXB Advance Access originally published online on March 28, 2008
Journal of Experimental Botany 2008 59(7):1695-1703; doi:10.1093/jxb/ern001
RESEARCH PAPER |
C4 photosynthetic isotope exchange in NAD-ME- and NADP-ME-type grasses
1Molecular Plant Physiology Group, Research School of Biological Sciences, Australian National University, Canberra, Australian Capital Territory, 2601 Australia
2ARC CoE, Plant Energy Biology, Research School of Biological Sciences, Australian National University, Canberra, Australian Capital Territory, 2601 Australia
* Present address and to whom correspondence should be sent: School of Biological Sciences, Washington State University, Pullman, WA 99164-4236, USA. E-mail: a.cousins{at}wsu.edu
Received 27 September 2007; Revised 19 December 2007 Accepted 21 December 2007
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Abstract |
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Monitoring photosynthetic isotope exchange is an important tool for predicting the influence of plant communities on the global carbon cycle in response to climate change. C4 grasses play an important role in the global carbon cycle, but their contribution to the isotopic composition of atmospheric CO2 is not well understood. Instantaneous measurements of 13CO2 (
13C) and C18OO (18O) isotope exchange in five NAD-ME and seven NADP-ME C4 grasses have been conducted to investigate the difference in photosynthetic CO2 isotopic fractionation in these subgroups. As previously reported, the isotope composition of the leaf material (
13C) was depleted in 13C in the NAD-ME compared with the NADP-ME grasses. However, 13C was not different between subtypes at high light, and, although 13C increased at low light, it did so similarly in both subtypes. This suggests that differences in leaf 13C between the C4 subtypes are not caused by photosynthetic isotope fractionation and leaf 13C is not a good indicator of bundle sheath leakiness. Additionally, low carbonic anhydrase (CA) in C4 grasses may influences 13C and should be considered when estimating the contribution of C4 grasses to the global isotopic signature of atmospheric CO2. It was found that measured 18O values were lower than those predicted from leaf CA activities and 18O was similar in all species measured. The 18O in these C4 grasses is similar to low 18O previously measured in C4 dicots which contain 2.5 times the leaf CA activity, suggesting that leaf CA activity is not a predictor of 18O in C4 plants. Key words: C4 grasses, carbonic anhydrase, isotope discrimination, leakiness
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Introduction |
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Isotope analysis of atmospheric carbon dioxide (CO2) is an important tool for monitoring changes in the global exchange of CO2 (Flanagan and Ehleringer, 1998; Yakir and Sternberg, 2000). Leaf-level models of carbon isotope exchange (
13C) and oxygen isotope exchange (18O) in C4 plants are used to help interpret the response of C4 plants to changing environmental conditions as well as predict the contribution of C4-dominated ecosystems to the global carbon cycle. However, to interpret the atmospheric CO2 isotopic signature requires an understanding of the isotopic fractionation steps associated with specific processes during leaf gas exchange (Yakir and Sternberg, 2000). Additionally, it remains unclear how photosynthetic isotope discrimination differs between the two major C4 biochemical subtypes; the NAD malic enzyme (NAD-ME) and the NADP malic enzyme (NADP-ME) which differ in their biochemistry, leaf anatomy, and geographic distribution (Hattersley, 1983; Hatch, 1987).
Most C4 plants utilize a compartmentalized CO2-concentrating mechanism between the mesophyll and bundle sheath cells (BSCs) to increase the CO2 partial pressure (pCO2) around the site of ribulose-bisphosphate carboxylase/oxygenase (Rubisco) in the BSC. The specialized biochemistry and leaf anatomy of C4 plants result in a pCO2 around the site of Rubisco several fold higher than current atmospheric levels, significantly reducing the rates of photorespiration and consequently increasing their photosynthetic efficiency, particularly under high light and temperature (Hatch, 1987; Kanai and Edwards, 1999). The dominance of C4 plants in open, hot, and arid environments is largely related to the enhanced photosynthetic efficiency of C4 plants compared with C3 plants under these environmental conditions (Osmond et al., 1982; Pearcy and Ehleringer, 1984; Long, 1999; Ghannoum et al., 2001). Within the C4 species, the geographic distribution of the subtypes differ with the occurrence of NADP-ME, increasing with increases in the average annual rainfall and the NAD-ME decreasing as a percentage of total C4 grasses (Teeri and Stowe, 1976; Hattersley, 1983; Sage, 2001). Traditionally, the difference in geographic distribution between the two C4 subtypes has been linked to differences in physiology, primarily the difference in bundle sheath leakiness [
: defined as the fraction of CO2 fixed by phosphoenolpyruvate carboxylase (PEPC) that subsequently leaks out of the BSCs] as estimated from the carbon isotope composition of leaf material (13C) (Hattersley, 1982; Farquhar, 1983). However, there are numerous factors which influence 13C in C4 leaves, including the availability of intercellular CO2, temperature, light intensity, and post-photosynthetic fractionation, and Henderson et al. (1992) found no correlation between 13C of leaf material and the short-term measurements of 13C in 11 C4 species.
The differences in 18O between photosynthetic types may help distinguish the contribution of the influence of C3 versus C4 on the global CO2 cycle. The 18O of the H2O at the site of CO2–H2O oxygen exchange within a leaf (ex) is the primary factor influencing the 18O of CO2 diffusing out of a leaf (Farquhar et al., 1993). The proportion of CO2 in isotopic equilibrium with the H2O at the site of oxygen exchange (
) will also influence the 18O of the CO2. The value of is determined by the balance between the gross flux of CO2 into the leaf and the activity of CA at the site of CO2–H2O oxygen isotope exchange as this influences the residence time of CO2 within a leaf and thus the number of hydration reactions per CO2 molecule (Gillon and Yakir, 2000a, b). Measurements of 18O during leaf gas exchange have shown 18O to be much lower in two NADP-ME C4 monocots (Zea mays and Sorghum bicolor) than what is commonly observed for C3 species (Gillon and Yakir, 2000a, b). A survey of leaf CA activity in species belonging to different photosynthetic functional types found that the C4 monocots in general had relatively low CA content (Gillon and Yakir, 2001). It has also been shown that 18O is sensitive to relatively small changes in CA activity due to an antisense silencing in the C4 dicot Flaveria bidentis even when rates of photosynthesis were unaltered (Cousins et al., 2006b).
In this study, five NAD-ME- and seven NADP-ME-type C4 grass species, grown under common growth conditions, were used to address three distinct questions: (i) whether short-term online measurements of 13C were different between the two subtypes as generally seen in the leaf 13C signature; (ii) whether 18O was different between the two subtypes; and (iii) whether CA activity was different between the subtypes and accurately predicted the measured isotopic equilibrium of CO2 and leaf H2O.
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Materials and methods |
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Growth conditions
Plants were grown during the summer months in a glasshouse under natural light conditions (27 °C day and 18 °C night temperatures). Twelve C4 grass species, five from the NAD-ME and seven from the NADP-ME subtypes (Table 1), were grown in 5.0 l pots in garden mix with 2.4–4 g of Osmocote/l of soil (15/4.8/10.8/1.2 N/P/K/Mg+trace elements: B, Cu, Fe, Mn, Mo, Zn; Scotts Australia Pty Ltd, Castle Hill, Australia) and watered daily. There were 3–4 replicate pots per species and each pot contained 2–3 individuals of the same species.
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Online 13CO2 and C18OO discrimination
The uppermost fully expanded leaves were placed into the 6 cm2 leaf chamber of the LI-6400 portable gas-exchange system (Li-Cor, Lincoln, NE, USA) and equilibrated under measurement conditions for a minimum of 1.5 h (Cousins et al., 2006a, b). Air entering the leaf chamber was prepared by using mass flow controllers (MKS instruments, Wilmington, MA, USA) to obtain a gas mix of 909 mbar dry N2 and 48 mbar O2 which contained no water vapour. A portion of the nitrogen/oxygen air was used to zero the mass spectrometer to correct for N2O and other contaminants contributing to the 44, 45, and 46 peaks. Pure CO2 (
13CVPDB = –29
, and 18OVSMOW=24) was added to the remaining air stream to obtain a CO2 partial pressure of 
531 µbar. Low oxygen (48 mbar) was used to minimize contamination of the 46 (m/z) signal caused by the interaction of O2 and N2 to produce NO2 with the mass spectrometer source. Simultaneous measurements of leaf gas exchange and isotope discrimination were determined by linking the LI-6400 gas exchange system to a mass spectrometer through an ethanol/dry ice water trap and a thin, gas-permeable silicone membrane (Cousins et al., 2006a, b, 2007; Griffiths et al., 2007).
Calculations of 13CO2 discrimination
The model of C4 carbon isotope discrimination (13C) from Farquhar (1983) was used to determine which factors in the model would influence 13C consistent with the experimental data. The simplified model predicts that
 | (1) |
) is the fractionation during diffusion of CO2 in air; s (1.8) is the fractionation during CO2 leakage from the BSCs; and the fractionation by Rubisco is b3=30 (Roeske and Oleary, 1984). The combined fractionation of PEPC and the isotopic equilibrium during dissolution of CO2 and conversion to bicarbonate (b4) was calculated as (Farquhar, 1983)
 | (2) |
) were estimated by rearranging Equation 1 and solving for .
Calculations of C18OO isotope discrimination
Discrimination against C18OO (18O) when H2O and CO2 at the site of exchange are at full isotopic equilibrium (=1) can be predicted as (Farquhar and Lloyd, 1993)
 | (3) |
is calculated as pi/(pa–pi). There was little variation in a’ as the average value for all species was 7.9±0.1. The 18O enrichment of CO2 compared with the atmosphere at the site of exchange in full oxygen isotope equilibrium with the water was calculated as (Cernusak et al., 2004)
 | (4) |
w) was determined at the LI-6400 measured leaf temperature (30 °C) as described by Cernusak et al. (2004) and Cousins et al. (2006b).
The 18O of H2O at the sites of evaporation within a leaf (e) can be estimated from the Craig and Gordon model of evaporative enrichment (Craig and Gordon, 1965; Farquhar and Lloyd, 1993)
 | (5) |
a and s are the isotopic composition of water vapour in the air and source water, and the water taken up by the plant (s = –5.3±0.3), respectively. The kinetic fractionation during diffusion of H2O from leaf intercellular air spaces to the atmosphere (k) and the equilibrium fractionation between liquid water and water vapour (+) was calculated according to Cernusak et al. (2004) and Cousins et al. (2006b). During the online gas exchange measurements, the leaves remained in steady-state conditions for a minimum of 1.5 h and under those conditions the value of s is equal to the isotopic composition of water transpired by the leaf (t) (Harwood et al., 1998). The air flowing into the leaf chamber was dry but well mixed with transpired H2O within the chamber such that the average relative humidity within the leaf chamber was 39.4±0.9%.
The proportion of CO2 in isotopic equilibrium with water at the site of oxygen exchange () can be estimated from
 | (6) |
ca is the oxygen isotope composition of CO2 at the site of exchange during photosynthesis determined by
 | (7) |
It has been suggested that the extent of in a leaf can also be calculated from in vitro CA assays coupled with the unidirectional flux of CO2 into the leaf (Gillon and Yakir, 2000a, b, 2001) from the equation initially developed by Mills and Urey (1940):
 | (8) |
) (Gillon and Yakir, 2001). Leaf CA activity (CAleaf) is determined as the product of the CA hydration rate constant (kCA, mol m–2 s–1 bar–1) and the mesophyll pCO2 (pm). The rate constant kCA is calculated from in vitro measurements of CA activity in leaf extracts (see below). The gross influx of CO2 into a leaf [Fin=gt pa; where gt is the total conductance of CO2 from the atmosphere to the site of CO2–H2O oxygen exchange (Gillon and Yakir, 2000a)] as well as pm determine the residence time (=pm/Fin) of CO2 within the leaf.
Enzyme activities
CA activity was determined on 1 cm2 section taken from the same leaves used for gas exchange. Leaf samples were collected after the gas exchange measurements and subsequently frozen in liquid nitrogen and stored at –80 °C. Tissue was ground on ice in 600 µl of extraction buffer [50 mM HEPES-KOH, pH 7.4, 10 mM dithiothreitol (DTT), 1% polyvinlypolypyrrolidone (PVPP), 1 mM EDTA, 0.1% Triton] with 20 µl of protease inhibitor cocktail (Sigma, St Louis, MO, USA) and briefly centrifuged. CA activity was measured on leaf extracts using mass spectrometry to measure the rates of 18O2 exchange from labelled 13C18O2 to H216O (Badger and Price, 1989; von Caemmerer et al., 2004). Measurements of leaf extracts were made at 25 °C with a subsaturating total carbon concentration of 1 mM. The hydration rates were calculated from the enhancement in the rate of 18O loss over the uncatalysed rate, and the non-enzymatic first-order rate constant was applied (pH 7.4, appropriate for the mesophyll cytosol). The CA activity was reported as a first-order rate constant kCA (mol m–2 s–1 bar–1), and kCApm gives the in vivo CA activity at that particular cytosolic pCO2.
Dry matter 13C
A similar leaf to the one used during gas exchange was collected, oven-dried at 70 °C, and ground with a mill ball. A subsample of ground tissue was weighed and the isotopic composition determined by combustion in a Carlo Erba elemental analyser, and the CO2 was analysed by mass spectrometry. The was calculated as [(Rsample–Rstandard)/Rstandard]x1000, where Rsample and Rstandard are the 13/12C of the sample and the standard V-Pee Dee Belemnite, respectively.
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Net CO2 assimilation rates
When plant species were grouped into photosynthetic subtypes (NAD-ME versus NADP-ME), the rates of net CO2 assimilation were similar between the two groups at both high (2000 µmol quanta m–2 s–1) and low (150 µmol quanta m–2 s–1) measuring light conditions (Table 1). However, if net CO2 assimilation was analysed by species within a tribe and photosynthetic subtypes (see Table 1) then the Chloroideae-NAD and the Paniceae-NADP had higher rates compared with the Andropogoneae-NADP and Paniceae-NAD under high light conditions only (Table 1). The ratio of intercellular to ambient CO2 partial pressures (pi/pa) was similar between the NAD-ME and NADP-ME subtypes, and there was no difference in pi/pa between species of different tribes (Table 1).
Isotope discrimination
There was no difference in photosynthetic carbon isotope discrimination (13C) between photosynthetic subtypes, nor was there any difference in 13C between tribes at either light level (Table 1). In all species measured, except for Themeda traiandra, 13C was higher at low light compared with high light (Table 1). The carbon isotope composition of leaf material (13C) determined on a similar aged leaf as used for the gas exchange measurements was more depleted in the heaver isotope (13C) in the NAD-ME compared with the NADP-ME and more depleted in the Paniceae-NAD species compared with those species in the Andropogoneae-NADP and Paniceae-NADP tribes (Table 1).
The measured photosynthetic oxygen isotope discrimination (18O), measured at high light only, was similar between species in the NAD-ME and NADP-ME subtypes as well as in the different tribes (Table 2). The leaf CA content expressed as the rate constant (kCA, mol m–2 s–1 bar–1) was also similar between all the NAD-ME and NADP-ME subtypes (Table 2). When CA was expressed as a rate of CO2 hydration (CAleaf), which takes into account the internal CO2 concentration within the leaf, the rates were similar between the NAD-ME and NADP-ME subtypes but higher in the Paniceae compared with both the Chloroideae and the Andropogoneae species (Table 2).
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The ratio of the rate of PEPC carboxylation, estimated from the rates of net CO2 assimilation, to the rate of CO2 hydration estimated from CA activity (Vp/Vh) was similar in the NAD-ME and NADP-ME subtypes but lower in the Paniceae compared with species in the other two tribes (Table 3). The rate of CO2 leakage across the BSCs (
: defined as the fraction of CO2 fixed by PEPC that subsequently leaks out of the BSCs) was similar between species within the two photosynthetic subtypes as well as between species in different tribes (Table 3). The values of calculated with the estimates of Vp/Vh were lower in all species than when Vp/Vh was not considered in the calculations (Table 3).
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Isotope discrimination versus pi/pa and the extent of isotopic equilibrium
The majority of the measured values of
13C fall within the theoretical relationship of 13C to pi/pa as predicted from the model of C4 carbon isotope discrimination developed by Farquhar (1983) (Fig. 1). The theoretical relationship of 13C and pi/pa was calculated with a value of 0.25, and the initial CO2 carboxylation reaction catalysed by PEPC relative to the CO2 hydration by CA (Vp/Vh) was assumed to be either zero (solid line) or 0.4 (dashed line; Fig. 1). The 0.4 value for Vp/Vh was taken as the maximum value estimated in Table 3. Values of 13C in the Andropogoneae and the Chloroideae are generally higher than in the Paniceae, but this trend was not significantly different (Table 1; Fig. 1). Measured 18O was higher in the C4 dicots compared with the C4 monocots as would be predicted from Equation 3 based on the higher pi/pa values in the dicots (Fig. 2). However, 18O showed little relationship with pi/pa within the dicots and monocots, unlike the model predictions, and 18O values were substantially lower at each pi/pa than predicted from Equation 3 (Fig. 2). Additionally, the proportion of CO2 in isotopic equilibrium with water at the site of oxygen exchange () predicted from the number of hydration reactions per CO2 molecule (k) was substantially higher than the values determined from 18O measurements in all species including the C4 dicots which have higher k values compared with the C4 monocots (Fig. 3). The value of in the C3 dicot was close to the theoretical value of estimated from k values.
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Leaf 13C isotope composition and photosynthetic discrimination
As previously reported (Hattersley, 1982; Ohsugi et al., 1988), the leaf material of species in the NAD-ME subtype was significantly depleted in 13C compared with the NADP-ME species (Table 1). The depletion in 13C in the NAD-ME species has been attributed to difference in leakiness between the two subtypes (Hattersley, 1982; Farquhar, 1983; Ohsugi et al., 1988; Buchmann et al., 1996), and it has been hypothesized that the presence of a suberized lamella in the BSCs of the NADP-ME species reduces the conductance of CO2 across the BSCs. However, the present measurements of
13C under high light conditions indicated that leakiness does not vary between the subtypes and does not support the notion that 13C differs between the subtypes because of differences in leakiness (Tables 1, 3; Fig. 1). This is similar to previous estimates of leakiness in the NAD-ME and NADP-ME species with various techniques (Krall and Edwards, 1990; Hatch et al., 1995) including online measurements of 13C (Henderson et al., 1992). The quantum yield of CO2 assimilation has been reported as a good proxy for estimating leakiness in C4 plants and that the range of quantum yields measured in numerous C4 species reflects the differences in leakiness in these species (Ehleringer and Pearcy, 1983; Farquhar, 1983). However, the greatest difference in the quantum yield is generally not seen between NAD-ME and NADP-ME subtypes but between monocots and dicots (Ehleringer and Pearcy, 1983; von Caemmerer and Furbank, 2003). Additionally, it is difficult to estimate leakiness using the quantum yield measurement given the uncertainties in the production of ATP from absorbed quanta and how this ATP/quanta ratio varies between species (Furbank et al., 1990; von Caemmerer and Furbank, 2003).
The quantum yield of CO2 fixation is typically determined from the initial slope of a light response curve, and low light growth conditions have previously been shown to decrease the 13C isotopic signature of C4 leaf material (Buchmann et al., 1996; Kubásek et al., 2007). Additionally, reducing the light level during online measurements of 13C causes an increase in the C4 photosynthetic isotopic fractionation against 13CO2 (Henderson et al., 1992; Cousins et al., 2006a; Tazoe et al., 2006). It is possible that low light conditions alter the co-ordination between the C4 and C3, causing the overcycling of CO2 and increasing leakiness; however, a mechanism has not yet been reported. 13C was measured under low light conditions (150 µmol quanta m–2 s–1) to investigate whether or not 13C responded differently in the two C4 subtypes (Table 1). As previously reported, 13C increased at low light (Henderson et al., 1992; Cousins et al., 2006a; Tazoe et al., 2006), but there was no significant difference in the response of 13C in the C4 subtypes under low light. This indicates that the low light enhancement of 13C is widespread in C4 plants but does not provide an explanation for the differences in 13C between the subtypes. It is interesting to note that the method of determining the quantum yield of CO2 assimilation from the initial slope of the photosynthetic light response curve may be difficult to interrupt as low light appears to alter the co-ordination of the C4 and C3 cycles.
Photosynthetic discrimination does not readily explain the time-tested differences in 13C between the NAD-ME and NADP-ME C4 subtypes, which suggests that differences in post-photosynthetic discrimination of photoassimilate may be responsible for the 13C differences. As suggested over a decade ago, further studies should be conducted to investigate the differences in respiratory discrimination in a variety of NAD-ME and NADP-ME C4 species (Henderson et al., 1992).
C18OO discrimination
There was no difference in the photosynthetic C18OO discrimination (18O) between the C4 species measured in this study (Table 2). Values of 18O showed little increase with an increase in pi/pa, and the measured 18O was significantly less in all species than would be predicted from the theoretical relationship of 18O and pi/pa (Equation 3; Fig. 2). The low 18O in the C4 grasses is similar to what was measured in the C4 dicots A. edulis and F. bidentis (Fig. 2), indicating that 18O is low in C4 plants regardless of their evolutionary origin, and low 18O may indeed be inherent to C4 photosynthesis (Gillon and Yakir, 2000b, 2001) but not necessarily related to low CA activity (see below).
Carbonic anhydrase
There was no significant difference in the CA content, presented as the rate constant (kCA), nor leaf activity (CAleaf) between the NAD-ME and NADP-ME species (Table 2). The values of CAleaf for the C4 grasses are significantly less then previously reported for C4 dicots including Amaranthus sp., 389±119 µmol m–2 s–1 (Gillon and Yakir, 2001); A. edulis, 783±153 µmol m–2 s–1 (Cousins et al., 2007); and F. bidentis, 1321±114 µmol m–2 s–1 (Cousins et al., 2006a, b). Our data support the suggestion that low CA may not be due to or explicitly associated with C4 photosynthesis, but that it may instead be a trait of the clade of grasses to which the species in this current study belong (Edwards et al., 2007). Within the C4 grasses, CA activity is not consistent as the species within the Andropogoneae and Chloroideae had 60% of the CAleaf activity compared with species of the Paniceae (Table 2). The ratio of the initial photosynthetic carboxylation reaction catalysed by PEPC to the rate of CO2 hydration by CA (Vp/Vh) averaged 0.24±0.03 in all the grasses and was significantly lower in the Paniceae species compared with the other species (Table 3). Values of Vp/Vh reported here are similar to values reported in an earlier publication which suggested that CA activity may be just sufficient to support rates of photosynthesis in C4 plants (Hatch and Burnell, 1990). As noted above, there were no differences in 13C or 18O between the photosynthetic subtypes and the tribes, indicating that differences in CA activity do not have a large influence on the photosynthetic isotope exchange between the C4 grass species. However, Vp/Vh is low enough to have a major influence on 13C in all the C4 grasses measured here (Fig. 1; Table 3). For example, variation in 13C not explained by pi/pa (Fig. 1) can be explained from estimates of Vp/Vh. Additionally, taking Vp/Vh into consideration reduced the estimated values of leakiness by 20% (Table 3). This has implications for interpreting the contribution of C4 photosynthesis to regional CO2 exchange in C4-dominated grasslands as the photosynthetic discrimination against 13CO2 may be underestimated if CA activity is not taken into consideration.
CO2 and H2O isotopic equilibrium
The exchange of 18O between CO2 and H2O is facilitated by CA, which catalyses the interconversion of CO2 and bicarbonate (HCO3–), and high CA activity will increase the proportion of CO2 in isotopic equilibrium with the H2O (). The extent of measured from 18O (Equation 6) was low in all species compared with estimated from the number of hydration reactions per CO2 (k) (Equation 8; Fig. 3). This suggests that the total leaf CA activity in C4 monocots does not represent the CA activity associated with the CO2–H2O oxygen exchange which influences 18O. This finding is similar to what was previously reported for C4 dicots which have 2.5 times the CA activity and values of k compared with the C4 grasses (Cousins et al., 2006b, 2007) (Fig. 3).
The present study suggests that in C4 species leaf CA activity cannot readily be used as an indicator of the extent of 18O equilibration. This might be because not all of the CA located in mesophyll cytosol in C4 species is available at the site of CO2–H2O exchange compared with C3 species where CA is located in chloroplasts which appress intercellular airspaces (Poincelot, 1972; Ku and Edwards, 1975). Alternatively, the extent of 18O equilibration may be miscalculated if the estimated values of e do not accurately describe the isotopic composition of H2O at the site of oxygen exchange between leaf H2O and CO2 (Cousins et al., 2006b).
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Differences in the carbon isotope composition (
13C) between the NAD-ME- and NADP-ME-type C4 grasses are not explained by photosynthetic carbon isotope discrimination (13C) at either high or low light. This indicates that other processes such as post-photosynthetic discrimination have a major influence on 13C in C4 grasses. The low CA in the C4 grasses may influence 13C, and CA activity should be considered when estimating CO2 bundle sheath leakiness and the contribution of C4 photosynthesis to the isotopic signature of atmospheric CO2 in C4-dominated ecosystems. Additionally, leaf CA activity does not appear readily to predict the extent of 18O equilibration between leaf H2O and CO2.
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Acknowledgements |
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We thank Sue Wood for the carbon isotope analysis of dry matter samples, and Oula Ghannoum for supplying the seeds used in this study. ABC was supported in part by an NSF international postdoctoral fellowship.
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