C 4 photosynthetic isotope exchange in NAD-ME- and NADP-ME-type grasses

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1 Journal of Experimental Botany, Vol. 59, No. 7, pp , 2008 doi: /jxb/ern001 Advance Access publication 28 March, 2008 SPECIAL ISSUE RESEARCH PAPER C 4 photosynthetic isotope exchange in NAD-ME- and NADP-ME-type grasses Asaph B. Cousins 1,2, *, Murray R. Badger 1,2 and Susanne von Caemmerer 1 1 Molecular Plant Physiology Group, Research School of Biological Sciences, Australian National University, Canberra, Australian Capital Territory, 2601 Australia 2 ARC CoE, Plant Energy Biology, Research School of Biological Sciences, Australian National University, Canberra, Australian Capital Territory, 2601 Australia Received 27 September 2007; Revised 19 December 2007; Accepted 21 December 2007 Abstract 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. C 4 grasses play an important role in the global carbon cycle, but their contribution to the isotopic composition of atmospheric CO 2 is not well understood. Instantaneous measurements of 13 CO 2 (D 13 C) and C 18 OO (D 18 O) isotope exchange in five NAD-ME and seven NADP-ME C 4 grasses have been conducted to investigate the difference in photosynthetic CO 2 isotopic fractionation in these subgroups. As previously reported, the isotope composition of the leaf material (d 13 C) was depleted in 13 C in the NAD-ME compared with the NADP-ME grasses. However, D 13 C was not different between subtypes at high light, and, although D 13 C increased at low light, it did so similarly in both subtypes. This suggests that differences in leaf d 13 C between the C 4 subtypes are not caused by photosynthetic isotope fractionation and leaf d 13 Cis not a good indicator of bundle sheath leakiness. Additionally, low carbonic anhydrase (CA) in C 4 grasses may influences D 13 C and should be considered when estimating the contribution of C 4 grasses to the global isotopic signature of atmospheric CO 2. It was found that measured D 18 O values were lower than those predicted from leaf CA activities and D 18 O was similar in all species measured. The D 18 O in these C 4 grasses is similar to low D 18 O previously measured in C 4 dicots which contain 2.5 times the leaf CA activity, suggesting that leaf CA activity is not a predictor of D 18 OinC 4 plants. Key words: C 4 grasses, carbonic anhydrase, isotope discrimination, leakiness. Introduction Isotope analysis of atmospheric carbon dioxide (CO 2 )is an important tool for monitoring changes in the global exchange of CO 2 (Flanagan and Ehleringer, 1998; Yakir and Sternberg, 2000). Leaf-level models of carbon isotope exchange (D 13 C) and oxygen isotope exchange (D 18 O) in C 4 plants are used to help interpret the response of C 4 plants to changing environmental conditions as well as predict the contribution of C 4 -dominated ecosystems to the global carbon cycle. However, to interpret the atmospheric CO 2 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 C 4 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 C 4 plants utilize a compartmentalized CO 2 - concentrating mechanism between the mesophyll and bundle sheath cells (BSCs) to increase the CO 2 partial pressure (pco 2 ) around the site of ribulose-bisphosphate carboxylase/oxygenase (Rubisco) in the BSC. The specialized biochemistry and leaf anatomy of C 4 plants result in a pco 2 around the site of Rubisco several fold higher than current atmospheric levels, significantly reducing the * Present address and to whom correspondence should be sent: School of Biological Sciences, Washington State University, Pullman, WA , USA. a.cousins@wsu.edu ª The Author [2008]. Published by Oxford University Press [on behalf of the Society for Experimental Biology]. All rights reserved. For Permissions, please journals.permissions@oxfordjournals.org

2 1696 Cousins et al. rates of photorespiration and consequently increasing their photosynthetic efficiency, particularly under high light and temperature (Hatch, 1987; Kanai and Edwards, 1999). The dominance of C 4 plants in open, hot, and arid environments is largely related to the enhanced photosynthetic efficiency of C 4 plants compared with C 3 plants under these environmental conditions (Osmond et al., 1982; Pearcy and Ehleringer, 1984; Long, 1999; Ghannoum et al., 2001). Within the C 4 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 C 4 grasses (Teeri and Stowe, 1976; Hattersley, 1983; Sage, 2001). Traditionally, the difference in geographic distribution between the two C 4 subtypes has been linked to differences in physiology, primarily the difference in bundle sheath leakiness [/: defined as the fraction of CO 2 fixed by phosphoenolpyruvate carboxylase (PEPC) that subsequently leaks out of the BSCs] as estimated from the carbon isotope composition of leaf material (d 13 C) (Hattersley, 1982; Farquhar, 1983). However, there are numerous factors which influence d 13 Cin C 4 leaves, including the availability of intercellular CO 2, temperature, light intensity, and post-photosynthetic fractionation, and Henderson et al. (1992) found no correlation between d 13 C of leaf material and the short-term measurements of D 13 Cin11C 4 species. The differences in D 18 O between photosynthetic types may help distinguish the contribution of the influence of C 3 versus C 4 on the global CO 2 cycle. The d 18 O of the H 2 O at the site of CO 2 H 2 O oxygen exchange within a leaf (d ex ) is the primary factor influencing the d 18 Oof CO 2 diffusing out of a leaf (Farquhar et al., 1993). The proportion of CO 2 in isotopic equilibrium with the H 2 Oat the site of oxygen exchange (h) will also influence the d 18 O of the CO 2. The value of h is determined by the balance between the gross flux of CO 2 into the leaf and the activity of CA at the site of CO 2 H 2 O oxygen isotope exchange as this influences the residence time of CO 2 within a leaf and thus the number of hydration reactions per CO 2 molecule (Gillon and Yakir, 2000a, b). Measurements of D 18 O during leaf gas exchange have shown D 18 O to be much lower in two NADP-ME C 4 monocots (Zea mays and Sorghum bicolor) than what is commonly observed for C 3 species (Gillon and Yakir, 2000a, b). A survey of leaf CA activity in species belonging to different photosynthetic functional types found that the C 4 monocots in general had relatively low CA content (Gillon and Yakir, 2001). It has also been shown that D 18 O is sensitive to relatively small changes in CA activity due to an antisense silencing in the C 4 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 C 4 grass species, grown under common growth conditions, were used to address three distinct questions: (i) whether short-term online measurements of D 13 C were different between the two subtypes as generally seen in the leaf d 13 C signature; (ii) whether D 18 O was different between the two subtypes; and (iii) whether CA activity was different between the subtypes and accurately predicted the measured isotopic equilibrium of CO 2 and leaf H 2 O. Materials and methods 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 C 4 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 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. Online 13 CO 2 and C 18 OO discrimination The uppermost fully expanded leaves were placed into the 6 cm 2 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 N 2 and 48 mbar O 2 which contained no water vapour. A portion of the nitrogen/oxygen air was used to zero the mass spectrometer to correct for N 2 O and other contaminants contributing to the 44, 45, and 46 peaks. Pure CO 2 (d 13 C VPDB ¼ 29&, and d 18 O VSMOW ¼24&) was added to the remaining air stream to obtain aco 2 partial pressure of ;531 lbar. Low oxygen (48 mbar) was used to minimize contamination of the 46 (m/z) signal caused by the interaction of O 2 and N 2 to produce NO 2 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 13 CO 2 discrimination The model of C 4 carbon isotope discrimination (D 13 C) from Farquhar (1983) was used to determine which factors in the model would influence D 13 C consistent with the experimental data. The simplified model predicts that D 13 C ¼ a þðb 4 þðb 3 sþ 3 / aþ 3 p i =p a ð1þ where p a and p i represent the pco 2 of the air surrounding the leaf and in the intercellular air spaces, respectively; a (4.4&) is the fractionation during diffusion of CO 2 in air; s (1.8&) is the fractionation during CO 2 leakage from the BSCs; and the fractionation by Rubisco is b 3 ¼30& (Roeske and Oleary, 1984). The combined fractionation of PEPC and the isotopic equilibrium during dissolution of CO 2 and conversion to bicarbonate (b 4 ) was calculated as (Farquhar, 1983) b 4 ¼ 5:7þ7:9V p =V h ð2þ indicating that the fractionation when CO 2 and HCO 3 are not at equilibrium depends on the rate of CO 2 hydration (V h ) and the rate

3 Table 1. Net CO 2 assimilation and carbon isotope discrimination Isotope discrimination in C 4 grasses 1697 List of NAD-ME and NADP-ME C 4 species used in this study, net CO 2 assimilation rates, the ratio of intercellular to atmospheric CO 2 partial pressure (p i /p a ), online carbon isotope discrimination (D 13 C), and the isotopic composition of leaf material (d 13 C) measured in the youngest fully expanded leaves. Gas exchange and D 13 C measurements were determined at both high light (HL; 2000 lmol quanta m 2 s 1 ) and low light (LL; 150 lmol quanta m 2 s 1 ). Leaf material for d 13 C was collected from a similar aged leaf not used for gas exchange. Measurements were made on 3 5 leaves from separate plants for each species and shown are the means 6SE. Different letters indicate significant differences (P < 0.05) between subtypes and measurement conditions. Plant type Net CO 2 assimilation rate (lmol m 2 s 1 ) p i /p a D 13 C& d 13 C& HL LL HL LL HL LL Tribe: Andropogoneae (NADP-ME) Sorghum bicolor Themeda triandra Zea mays Subfamily: Chloridoideae Tribe: Main Chloroid assemblage (NAD-ME) Astrebla lappacea Astrebla pectinata Nd Nd Nd Eragrostis superba Tribe: Paniceae (NADP-ME) Cenchrus ciliaris Chrysopogon fallax Paspalum dilatatum Pennisetum clandestinum Tribe: Paniceae (NAD-ME) Panicum coloratum Panicum miliecium Andropogoneae (NADP-ME) a a a a a a a Chloroideae (NAD-ME) b a a a a a ab Paniceae (NADP-ME) c a a a a a a Paniceae (NAD-ME) a a a a a a b NADP-ME a a a a a a a NAD-ME a a a a a a b of PEP carboxylation (V p ). Values of leakiness (/) were estimated by rearranging Equation 1 and solving for /. Calculations of C 18 OO isotope discrimination Discrimination against C 18 OO (D 18 O) when H 2 O and CO 2 at the site of exchange are at full isotopic equilibrium (h¼1) can be predicted as (Farquhar and Lloyd, 1993) D 18 O ¼ a# þ ed ea ð3þ 1 ed ea where a# is the diffusional discrimination, calculated for each species as described by Farquhar and Lloyd (1993), and e is calculated as p i /(p a p i ). There was little variation in a as the average value for all species was &. The 18 O enrichment of CO 2 compared with the atmosphere at the site of exchange in full oxygen isotope equilibrium with the water was calculated as (Cernusak et al., 2004) D ea ¼ d eð1 þ e w Þþe w d a ð4þ 1 d a where the equilibrium fractionation between water and CO 2 (e 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 d 18 OofH 2 O at the sites of evaporation within a leaf (d e ) can be estimated from the Craig and Gordon model of evaporative enrichment (Craig and Gordon, 1965; Farquhar and Lloyd, 1993) d e ¼ d s þ e k þ e þ þ ðd a d s e k Þ e a ð5þ e i where e a and e i are the vapour pressures in the atmosphere and the leaf intercellular spaces. d a and d s are the isotopic composition of water vapour in the air and source water, and the water taken up by the plant (d s ¼ &), respectively. The kinetic fractionation during diffusion of H 2 O from leaf intercellular air spaces to the atmosphere (e k ) and the equilibrium fractionation between liquid water and water vapour (e + ) 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 d s is equal to the isotopic composition of water transpired by the leaf (d t ) (Harwood et al., 1998). The air flowing into the leaf chamber was dry but well mixed with transpired H 2 O within the chamber such that the average relative humidity within the leaf chamber was %. The proportion of CO 2 in isotopic equilibrium with water at the site of oxygen exchange (h) can be estimated from h ¼ D ca þ a#=ðe þ 1Þ ð6þ D ea þ a#=ðe þ 1Þ where D ca is the oxygen isotope composition of CO 2 at the site of exchange during photosynthesis determined by

4 1698 Cousins et al. D ca ¼ D18 O a# 1 þ D 18 O e It has been suggested that the extent of h in a leaf can also be calculated from in vitro CA assays coupled with the unidirectional flux of CO 2 into the leaf (Gillon and Yakir, 2000a, b, 2001) from the equation initially developed by Mills and Urey (1940): ðð CAleaf h ¼ 1 e =FinÞ=3Þ ð8þ where CA leaf /F in represents the mean number of hydration reactions for each CO 2 molecule inside the leaf (k s ) (Gillon and Yakir, 2001). Leaf CA activity (CA leaf ) is determined as the product of the CA hydration rate constant (k CA, mol m 2 s 1 bar 1 ) and the mesophyll pco 2 (p m ). The rate constant k CA is calculated from in vitro measurements of CA activity in leaf extracts (see below). The gross influx of CO 2 into a leaf [F in ¼g t p a ; where g t is the total conductance of CO 2 from the atmosphere to the site of CO 2 H 2 O oxygen exchange (Gillon and Yakir, 2000a)] as well as p m determine the residence time (s¼p m /F in )ofco 2 within the leaf. Enzyme activities CA activity was determined on ;1 cm 2 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 ll 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 ll 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 18 O 2 exchange from labelled 13 C 18 O 2 to H 2 16 O (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 18 O 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 k CA (mol m 2 s 1 bar 1 ), and k CA p m gives the in vivo CA activity at that particular cytosolic pco 2. Dry matter d 13 C 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 CO 2 was analysed by mass spectrometry. The d was calculated as [(R sample R standard )/R standard ]31000, where R sample and R standard are the 13/12 C of the sample and the standard V-Pee Dee Belemnite, respectively. Results Net CO 2 assimilation rates When plant species were grouped into photosynthetic subtypes (NAD-ME versus NADP-ME), the rates of net CO 2 assimilation were similar between the two groups at both high (2000 lmol quanta m 2 s 1 ) and low (150 lmol quanta m 2 s 1 ) measuring light conditions (Table 1). However, if net CO 2 assimilation was analysed ð7þ 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 CO 2 partial pressures (p i /p a ) was similar between the NAD-ME and NADP-ME subtypes, and there was no difference in p i /p a between species of different tribes (Table 1). Isotope discrimination There was no difference in photosynthetic carbon isotope discrimination (D 13 C) between photosynthetic subtypes, nor was there any difference in D 13 C between tribes at either light level (Table 1). In all species measured, except for Themeda traiandra, D 13 C was higher at low light compared with high light (Table 1). The carbon isotope composition of leaf material (d 13 C) determined on a similar aged leaf as used for the gas exchange measurements was more depleted in the heaver isotope ( 13 C) 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 (D 18 O), 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 (k CA, 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 CO 2 hydration (CA leaf ), which takes into account the internal CO 2 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). The ratio of the rate of PEPC carboxylation, estimated from the rates of net CO 2 assimilation, to the rate of CO 2 hydration estimated from CA activity (V p /V h ) 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 CO 2 leakage across the BSCs (/: defined as the fraction of CO 2 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 V p /V h were lower in all species than when V p /V h was not considered in the / calculations (Table 3). Isotope discrimination versus p i /p a and the extent of isotopic equilibrium The majority of the measured values of D 13 C fall within the theoretical relationship of D 13 Ctop i /p a as predicted from the model of C 4 carbon isotope discrimination

5 Isotope discrimination in C 4 grasses 1699 Table 2. Oxygen isotope discrimination and leaf carbonic anhydrase List of NAD-ME and NADP-ME C 4 species used in this study, online oxygen isotope discrimination (D 18 O), the rate constant of leaf CA (k CA ), and leaf CA activity (CA leaf ) measured in the youngest fully expanded leaves. Measurements were made on 3 5 leaves from separate plants for each species and shown are the means 6SE. Different letters indicate significant differences (P < 0.05) between subtypes and measurement conditions. Plant type D 18 O k CA CA leaf Tribe: Andropogoneae (NADP-ME) Sorghum bicolor Themeda triandra Zea mays Subfamily: Chloridoideae Tribe: Main Chloroid assemblage (NAD-ME) Astrebla lappacea Astrebla pectinata Eragrostis superba Tribe: Paniceae (NADP-ME) Cenchrus ciliaris Chrysopogon fallax Paspalum dilatatum Pennisetum clandestinum Tribe: Paniceae (NAD-ME) Panicum coloratum Panicum miliecium Andropogoneae (NADP-ME) a a a Chloroideae (NAD-ME) a a a Paniceae (NADP-ME) a a b Paniceae (NAD-ME) a a 23069b NADP-ME a a a NAD-ME a a a developed by Farquhar (1983) (Fig. 1). The theoretical relationship of D 13 C and p i /p a was calculated with a / value of 0.25, and the initial CO 2 carboxylation reaction catalysed by PEPC relative to the CO 2 hydration by CA (V p /V h ) was assumed to be either zero (solid line) or 0.4 (dashed line; Fig. 1). The 0.4 value for V p /V h was taken as the maximum value estimated in Table 3. Values of D 13 C 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 D 18 O was higher in the C 4 dicots compared with the C 4 monocots as would be predicted from Equation 3 based on the higher p i /p a values in the dicots (Fig. 2). However, D 18 O showed little relationship with p i /p a within the dicots and monocots, unlike the model predictions, and D 18 O values were substantially lower at each p i /p a than predicted from Equation 3 (Fig. 2). Additionally, the proportion of CO 2 in isotopic equilibrium with water at the site of oxygen exchange (h) predicted from the number of hydration reactions per CO 2 molecule (k s ) was substantially higher than the h values determined from D 18 O measurements in all species including the C 4 dicots Table 3. The ratio of V p /V h and leakiness List of NAD-ME and NADP-ME C 4 species used in this study, the ratio of PEPC and CA activity (V p /V h ), and bundle sheath leakiness estimated without and with factoring CA activity. Measurements were made on 3 5 leaves from separate plants for each species and shown are the means 6SE. Different letters indicate significant differences (P < 0.05) between subtypes and measurement conditions. Plant type V p /V h / / with CA Tribe: Andropogoneae (NADP-ME) Sorghum bicolor Themeda triandra Zea mays Subfamily: Chloridoideae Tribe: Main Chloroid assemblage (NAD-ME) Astrebla lappacea Astrebla pectinata Eragrostis superba Tribe: Paniceae (NADP-ME) Cenchrus ciliaris Chrysopogon fallax Paspalum dilatatum Pennisetum clandestinum Tribe: Paniceae (NAD-ME) Panicum coloratum Panicum miliecium Andropogoneae (NADP-ME) a a a Chloroideae (NAD-ME) a a a Paniceae (NADP-ME) b a a Paniceae (NAD-ME) b a a NADP-ME a a a NAD-ME a a a which have higher k s values compared with the C 4 monocots (Fig. 3). The value of h in the C 3 dicot was close to the theoretical value of h estimated from k s values. Discussion Leaf 13 C 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 13 C compared with the NADP-ME species (Table 1). The depletion in 13 Cin 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 CO 2 across the BSCs. However, the present measurements of D 13 C under high light conditions indicated that leakiness does not vary between the subtypes and does not support the notion that d 13 C differs between the subtypes because of differences in leakiness (Tables 1, 3; Fig. 1). This is similar to

6 1700 Cousins et al. Fig. 1. Carbon isotope discrimination (D 13 C) as a function of the ratio of intercellular to ambient pco 2 (p i /p a ). The lines represent the theoretical relationship of D 13 C and p i /p a where /¼0.25, and the ratio of the PEPC carboxylation to the CO 2 hydration reaction (V p /V h )is either 0 (solid line) or 0.4 (dashed line); see Equation 1. Each point represents the means 6SE of measurements made on 3 5 leaves from separate plants for each species. Symbols represent the NADP-ME monocots (filled diamonds and stars), NAD-ME monocots (open circles and stars), NADP-ME C 4 dicots (filled triangle), and NAD-ME dicot (open triangle). Data for the dicot C 4 NAD-ME and NADP-ME are adapted from Cousins et al. (2006a, 2007). Fig. 2. Oxygen isotope discrimination (D 18 O) as a function of the ratio of mesophyll cytosolic to ambient CO 2 partial pressure (p i /p a ). The line represents the theoretical relationship of D 18 O and p i /p a at full isotopic equilibrium where a# was ; & and D ea ¼33.7& (Equation 3), and the CO 2 supplied to the leaf had a d 18 O of 24& relative to VSMOW. Each point represents the means 6SE of measurements made on 3 5 leaves from separate plants for each species, and symbols are as in Fig. 1. Data for the dicot C 4 NAD-ME and NADP-ME are adapted from Cousins et al. (2006b, 2007). Fig. 3. The extent of isotopic equilibrium (h) as a function of the number of hydration reactions per CO 2 molecule (k s ). Measured values of h were determined from D 18 O using Equation 6. Each point represents a measurement made on an individual leaf for a single species, and symbols are as in Fig. 1 and a C 3 dicot (asterisk). Data for the C 3 dicot and C 4 NAD-ME and NADP-ME dicots are adapted from Cousins et al. (2006a, 2007). The line represents the theoretical relationship between h and k s (Equation 8). 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 D 13 C (Henderson et al., 1992). The quantum yield of CO 2 assimilation has been reported as a good proxy for estimating leakiness in C 4 plants and that the range of quantum yields measured in numerous C 4 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 CO 2 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 13 C isotopic signature of C 4 leaf material (Buchmann et al., 1996; Kubásek et al., 2007). Additionally, reducing the light level during online measurements of D 13 C causes an increase in the C 4 photosynthetic isotopic fractionation against 13 CO 2 (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 C 4 and C 3, causing the

7 overcycling of CO 2 and increasing leakiness; however, a mechanism has not yet been reported. D 13 C was measured under low light conditions (150 lmol quanta m 2 s 1 ) to investigate whether or not D 13 C responded differently in the two C 4 subtypes (Table 1). As previously reported, D 13 C 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 D 13 C in the C 4 subtypes under low light. This indicates that the low light enhancement of D 13 C is widespread in C 4 plants but does not provide an explanation for the differences in d 13 C between the subtypes. It is interesting to note that the method of determining the quantum yield of CO 2 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 C 4 and C 3 cycles. Photosynthetic discrimination does not readily explain the time-tested differences in d 13 C between the NAD-ME and NADP-ME C 4 subtypes, which suggests that differences in post-photosynthetic discrimination of photoassimilate may be responsible for the d 13 C 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 C 4 species (Henderson et al., 1992). C 18 OO discrimination There was no difference in the photosynthetic C 18 OO discrimination (D 18 O) between the C 4 species measured in this study (Table 2). Values of D 18 O showed little increase with an increase in p i /p a, and the measured D 18 O was significantly less in all species than would be predicted from the theoretical relationship of D 18 O and p i /p a (Equation 3; Fig. 2). The low D 18 O in the C 4 grasses is similar to what was measured in the C 4 dicots A. edulis and F. bidentis (Fig. 2), indicating that D 18 O is low in C 4 plants regardless of their evolutionary origin, and low D 18 O may indeed be inherent to C 4 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 (k CA ), nor leaf activity (CA leaf ) between the NAD-ME and NADP-ME species (Table 2). The values of CA leaf for the C 4 grasses are significantly less then previously reported for C 4 dicots including Amaranthus sp., lmol m 2 s 1 (Gillon and Yakir, 2001); A. edulis, lmol m 2 s 1 (Cousins et al., 2007); and F. bidentis, lmol 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 C 4 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 C 4 Isotope discrimination in C 4 grasses 1701 grasses, CA activity is not consistent as the species within the Andropogoneae and Chloroideae had ;60% of the CA leaf activity compared with species of the Paniceae (Table 2). The ratio of the initial photosynthetic carboxylation reaction catalysed by PEPC to the rate of CO 2 hydration by CA (V p /V h ) averaged in all the grasses and was significantly lower in the Paniceae species compared with the other species (Table 3). Values of V p /V h 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 C 4 plants (Hatch and Burnell, 1990). As noted above, there were no differences in D 13 CorD 18 O 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 C 4 grass species. However, V p /V h is low enough to have a major influence on D 13 C in all the C 4 grasses measured here (Fig. 1; Table 3). For example, variation in D 13 C not explained by p i /p a (Fig. 1) can be explained from estimates of V p /V h. Additionally, taking V p /V h into consideration reduced the estimated values of leakiness by ;20% (Table 3). This has implications for interpreting the contribution of C 4 photosynthesis to regional CO 2 exchange in C 4 -dominated grasslands as the photosynthetic discrimination against 13 CO 2 may be underestimated if CA activity is not taken into consideration. CO 2 and H 2 O isotopic equilibrium The exchange of 18 O between CO 2 and H 2 O is facilitated by CA, which catalyses the interconversion of CO 2 and bicarbonate (HCO 3 ), and high CA activity will increase the proportion of CO 2 in isotopic equilibrium with the H 2 O(h). The extent of h measured from D 18 O (Equation 6) was low in all species compared with h estimated from the number of hydration reactions per CO 2 (k s ) (Equation 8; Fig. 3). This suggests that the total leaf CA activity in C 4 monocots does not represent the CA activity associated with the CO 2 H 2 O oxygen exchange which influences D 18 O. This finding is similar to what was previously reported for C 4 dicots which have 2.5 times the CA activity and values of k s compared with the C 4 grasses (Cousins et al., 2006b, 2007) (Fig. 3). The present study suggests that in C 4 species leaf CA activity cannot readily be used as an indicator of the extent of 18 O equilibration. This might be because not all of the CA located in mesophyll cytosol in C 4 species is available at the site of CO 2 H 2 O exchange compared with C 3 species where CA is located in chloroplasts which appress intercellular airspaces (Poincelot, 1972; Ku and Edwards, 1975). Alternatively, the extent of 18 O equilibration may be miscalculated if the estimated values of d e do not accurately describe the isotopic composition of H 2 O at the site of oxygen exchange between leaf H 2 O and CO 2 (Cousins et al., 2006b).

8 1702 Cousins et al. Conclusions Differences in the carbon isotope composition (d 13 C) between the NAD-ME- and NADP-ME-type C 4 grasses are not explained by photosynthetic carbon isotope discrimination (D 13 C) at either high or low light. This indicates that other processes such as post-photosynthetic discrimination have a major influence on d 13 C in C 4 grasses. The low CA in the C 4 grasses may influence D 13 C, and CA activity should be considered when estimating CO 2 bundle sheath leakiness and the contribution of C 4 photosynthesis to the isotopic signature of atmospheric CO 2 in C 4 -dominated ecosystems. Additionally, leaf CA activity does not appear readily to predict the extent of 18 O equilibration between leaf H 2 O and CO 2. Acknowledgements 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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