Single-cell C 4 photosynthesis: efficiency and acclimation of Bienertia sinuspersici to growth under low light

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1 Research Single-cell C 4 photosynthesis: efficiency and acclimation of Bienertia sinuspersici to growth under low light Samantha S. Stutz 1,2, Gerald E. Edwards 1 and Asaph B. Cousins 1 1 School of Biological Sciences, Washington State University, Pullman, WA 99164, USA; 2 Present address: Department of Biology University of Mexico, Albuquerque, NM 87131, USA Author for correspondence: Asaph B. Cousins Tel: acousins@wsu.edu Received: 22 August 2013 Accepted: 13 November 2013 doi: /nph Key words: Bienertia sinuspersici, carbon isotope discrimination, CO 2 leakiness, photosynthetic efficiency, single-cell C 4 photosynthesis, tunable diode laser absorption spectroscopy. Summary Traditionally, it was believed that C 4 photosynthesis required two types of chlorenchyma cells to concentrate CO 2 within the leaf. However, several species have been identified that perform C 4 photosynthesis using dimorphic chloroplasts within an individual cell. The goal of this research was to determine how growth under limited light affects leaf structure, biochemistry and efficiency of the single-cell CO 2 -concentrating mechanism in Bienertia sinuspersici. Measurements of rates of CO 2 assimilation and CO 2 isotope exchange in response to light intensity and O 2 were used to determine the efficiency of the CO 2 -concentrating mechanism in plants grown under moderate and low light. In addition, enzyme assays, chlorophyll content and light microscopy of leaves were used to characterize acclimation to light-limited growth conditions. There was acclimation to growth under low light with a decrease in capacity for photosynthesis when exposed to high light. This was associated with a decreased investment in biochemistry for carbon assimilation with only subtle changes in leaf structure and anatomy. The capture and assimilation of CO 2 delivered by the C 4 cycle was lower in low-light-grown plants. Low-light-grown plants were able to acclimate to maintain structural and functional features for the performance of efficient single-cell C 4 photosynthesis. Introduction In C 4 plants, CO 2 is concentrated around the enzyme ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco), which reduces the rate of photorespiration and increases the rate of photosynthesis (Hatch, 1987; von Caemmerer & Furbank, 1999). In Kranz-type C 4 plants, this occurs by diffusive resistance of CO 2 from the site of decarboxylation of C 4 acids in bundle sheath (BS) to the mesophyll cells (MC). This resistance is dependent on several factors, including the site of decarboxylation in different C 4 subtypes, liquid phase resistance to CO 2 diffusion in the cytosol, the position of organelles and the BS cell wall (von Caemmerer & Furbank, 2003). However, some CO 2 diffuses out of the BS cells, reducing photosynthetic efficiency, which increases the number of absorbed quanta per CO 2 assimilated (Skillman, 2008; Ubierna et al., 2013). Therefore, at a given rate of delivery of CO 2 to the BS cells via the C 4 cycle, the photosynthetic efficiency depends on the fraction of the CO 2 fixed by the Calvin Benson cycle relative to the fraction lost by conductance of CO 2 out of the BS cells. Thus, the efficiency of the CO 2 - concentrating mechanism can be estimated as leakiness (/), defined as the fraction of CO 2 fixed by phosphoenolpyruvate carboxylase (PEPC) in the C 4 cycle that subsequently diffuses out of the BS cells following decarboxylation of C 4 acids (Farquhar, 1983; von Caemmerer, 2003). Leakiness results in a decrease in photosynthetic efficiency, as the C 4 cycle consumes energy; in malic enzyme-type C 4 species, two ATP molecules are required per turn of the cycle for the regeneration of phosphoenolpyruvate (PEP) (Hatch & Osmond, 1976; Pengelly et al., 2010). Unfortunately, / is not directly measurable, but can be estimated by comparing measurements of photosynthetic discrimination against 13 CO 2 (D obs ) with the theoretical model of 13 CO 2 discrimination (D modeled ; Evans et al., 1986; von Caemmerer et al., 1997; Ubierna et al., 2013). Numerous studies have shown that D obs increases with decreasing photosynthetically active radiation (PAR), suggesting a decrease in photosynthetic efficiency of C 4 plants under low PAR (Henderson et al., 1992; Cousins et al., 2006, 2008; Kromdijk et al., 2008, 2010; Tazoe et al., 2008; Pengelly et al., 2010; Ubierna et al., 2013). This increase in D obs has been attributed to an increase in / caused by a disruption in the balance between the C 3 and C 4 cycles, with decreased CO 2 concentrations in the BS cells (C s ) and increased rates of oxygen fixation v o (Henderson et al., 1992; Cousins et al., 2006; Tazoe et al., 2006, 2008; Kromdijk et al., 2008, 2010; Pengelly et al., 2010). However, Ubierna et al. (2011, 2013) demonstrated that factors other than /, such as carbon isotope fractionation associated with respiration and miscalculations of C s, can also explain some of the reported increase in D obs under low PAR. Traditionally, it was thought that dual-cell Kranz-type anatomy was required for efficient C 4 photosynthesis, with features of 220

2 Phytologist Research 221 BS cells providing resistance to CO 2 leakage (von Caemmerer, 2003; von Caemmerer & Furbank, 2003). However, it has been demonstrated in four terrestrial plants within the Chenopodiaceae family (Bienertia sinuspersici, B. cycloptera, B. kavirense and Suaeda aralocaspica) that Kranz-type anatomy is not required for C 4 photosynthesis (Voznesenskaya et al., 2001; Sage, 2002; Edwards et al., 2004; Akhani et al., 2005, 2012). Instead, these halophytic species from the Arabian Peninsula utilize dimorphic chloroplasts within a single cell to perform NAD-malic enzyme-type C 4 photosynthesis (Voznesenskaya et al., 2001; Akhani et al., 2005, 2009; Offermann et al., 2011). Bienertia sinuspersici has bienertioid anatomy, in which peripheral chloroplasts, oppressed to the plasma membrane, support the C 4 CO 2 -concentrating mechanism, and a group of centrally located chloroplasts and mitochondria is the site for C 4 acid decarboxylation and Rubisco fixation (Freitag & Stichler, 2002; Voznesenskaya et al., 2002; Edwards et al., 2004; Offermann et al., 2011; Sharpe & Offermann, 2013). It has been suggested that singlecell C 4 plants maintain the resistance to CO 2 diffusion by manipulating cell size and the distance between the chloroplasts involved in the C 3 and C 4 cycles (Leisner et al., 2010; King et al., 2012). As CO 2 diffuses times more slowly through water than air, single-cell C 4 plants may gain increased resistance to CO 2 diffusion by increasing cell size (von Caemmerer, 2003). King et al. (2012) found that single-cell C 4 plants had similar photosynthetic efficiencies to their Kranz-type counterparts under high PAR, but / increased more under low PAR relative to / of Kranz-type C 4 plants, even when taking into account changes in C s, photorespiration via ribose-1,5- bisphosphate (RuBP) oxygenation and the contribution of mitochondrial day respiration to 13 CO 2 exchange. However, measurements of / presented in King et al. (2012) were made under low O 2 levels (4.6 kpa), reducing rates of photorespiration and complicating estimates of C s under low PAR (Ubierna et al., 2013). In addition, King et al. (2012) assumed that the substrate for day respiration was the same as the substrate for photosynthesis (i.e. the isotopic signature of CO 2 was the same for photosynthesis and respiration, Wingate et al., 2007). However, this assumption might not be valid when the isotopic signature of the growth CO 2 is different from that of the measurement CO 2. Indeed, the manipulation of differences in the isotopic composition of growth and measurement CO 2 has been elegantly used to demonstrate the contribution of photorespiration and day respiration to leaf CO 2 exchange (Gillon & Griffiths, 1997; Gillon et al., 1998). In B. sinuspersici, the carbon isotope composition (d 13 C) of leaf dry matter, which is largely determined by fractionation during photosynthesis, varies considerably. For example, plants grown in chambers ranged from 19.3& in King et al. (2012) to a characteristic C 4 signature of 14.1& in Smith et al. (2009). However, analyses of Bienertia species in natural habitats consistently show C 4 -type isotope signatures (Akhani et al., 2005, 2009, 2012). Some of the variation in chamber-grown plants is attributed to developmental differences in young and mature leaf tissue (Voznesenskaya et al., 2002; King et al., 2012), but it has also been suggested that differences in growth conditions affect d 13 C (Leisner et al., 2010; King et al., 2012).However,ithasnot been demonstrated how dry matter d 13 C and cellular leaf structure relate to / in response to growth under different light conditions. Therefore, the goal of this study was to test the relationship among leaf structure, capacity of carboxylases and photosynthetic efficiency in the single-cell C 4 plant B. sinuspersici grown under moderate and low light (ML and LL, respectively). The objectives were as follows: to determine how the photosynthetic efficiency of the CO 2 -concentrating mechanism responds to light-limited growth conditions; to see how leaf cell structure and biochemistry are affected by growth light conditions; and to use differences in growth and measurement CO 2 isotope signatures to determine the influence of day respiration on measurements of D obs and / in response to changes in PAR. This was accomplished by measuring leaf carbon isotope discrimination with a tunable diode laser absorption spectroscope (TDLAS), short-term labeling of recent photoassimilates with 7 and 58& CO 2, spectrometer assays of Rubisco and PEPC activities, and light microscopy to compare leaf cell structure in plants grown under ML vs LL. Materials and Methods Growth conditions and plant propagation Bienertia sinuspersici Akhani was grown in an environmental chamber (Econair Ecological Chambers Inc., Winnipeg, MB, Canada) under ambient CO 2 (isotopic signature 10.7&) at 32 C: 18 C, 20% : 40% relative humidity and 16 h : 8 h day : night. PAR was lmol quanta m 2 s 1 for ML and lmol quanta m 2 s 1 for LL at pot level. As reported previously (King et al., 2012), all plants were top watered with tap water daily and once a week with 50 mm NaCl and Peters fertilizer (Scotts Miracle-Gro, Marysville, OH, USA). Plants were grown side by side with low light provided by a shade structure made of 0.25-in PVC pipe covered with a black shade cloth (Polysack Plastic Industries, Nir Yitzhak, Negev, Israel), which reduced light quantity by 60%, but had no effect on light quality. Plants grown under LL were rotated once a week to ensure uniform treatment, and ML plants were randomly positioned in the center of the chamber. Plants were propagated from both cuttings and seeds (for details on plant propagation, see King et al., 2012). Once the propagated plants showed substantial root and vegetative growth (c. 6 8wk), they were transferred to 7.6-l pots, one plant per pot, in a mixture of c. one-half potting soil (Sun Gro Horticulture, Seba Beach, AB, Canada) and one-half Turface (Profile Products LLC, Buffalo Grove, IL, USA). The ML plants were placed in the center of the chamber (PAR = lmol quanta m 2 s 1 ) and the LL plants were initially placed under a slanted shade screen (PAR = 300 lmol quanta m 2 s 1 ) for 1.5 2wk before being placed under the shade structure (PAR = lmol quanta m 2 s 1 ). Plants grown in ML were analyzed 3 4wk after transplanting; to compensate for their slower growth, LL plants were analyzed 5 6 wk after being placed under the shade structure. The LL plants were measured when the above-ground crown was of a similar size to that of the ML plants at the time of

3 222 Research Phytologist measurements. As a result of the differences in growth rate, there were two separate cohorts of ML plants, whereas a single cohort of LL plants was used during the measurements to ensure similar-sized plants. Measurements of gas exchange and leaf carbon isotope exchange A LI-6400XT (LI-COR Biosciences, Lincoln, NE, USA) was coupled to a TDLAS (model TGA100; Campbell Scientific, Inc., Logan, UT, USA) to measure online carbon isotope discrimination for both light- and O 2 -response curves (Sun et al., 2012; Ubierna et al., 2013). Isotope calibration consisted of a zero CO 2 tank, three mixing tanks from 3.3 to 10 Pa CO 2 with the same isotopic signature, a calibration tank (Liquid Technology Corporation, Apopka, FL, USA), followed by the LI-COR reference and sample (Ubierna et al., 2013). Boundary layer conductance depending on leaf area and flow rate was calculated according to Ubierna et al. (2013). Average values ranged from to mol m 2 s 1, which had an insignificant impact on the calculated parameters. Photosynthetic discrimination D 13 C (D obs ) was calculated according to Evans et al. (1986): D obs ¼ where ξ is: nðd o d e Þ 1 þ d o nðd o d e Þ P e n ¼ ðp e P o Þ Eqn 1 Eqn 2 where d o and d e are the d 13 Cvaluesofairentering(e)andleaving (o) the chamber, respectively, and P e and P o are the CO 2 partial pressures entering and leaving the chamber, respectively. Measured values are reported in Supporting Information Figs S1 and S2. Light- and O 2 -response curves Medium-aged branches, having few minor branches, were selected for analysis. The branch was placed in an opaque conifer chamber (LI-COR Biosciences) with an RGB LED light source (LI-COR Biosciences) attached to a LI-6400XT. Before sealing the chamber, the branch was photographed and the projected leaf area was estimated using ImageJ (US National Institutes of Health, Bethesda, MD, USA). The branch was acclimated for at least 1 h in the LI-COR chamber at 25 C (leaf temperature), 2000 lmol quanta m 2 s 1, 3.8 Pa CO 2 and 18.4 kpa O 2.Energy balance calculations of leaf temperature and boundary layer conductance were determined from the LI-COR software. The relative humidity within the chamber was between 50% and 70% with a vapor pressure deficit of 1 2 kpa. Ambient CO 2 was scrubbed from the air entering the LI-6400 and added back from a & CO 2 cartridge (isi GmbH, Vienna, Austria). To test the contribution of day respiration to leaf CO 2 isotope exchange, light-response curves were additionally measured with a & CO 2 tank. CompoundQ (Apiezon Products, M&I Materials Ltd, Manchester, UK) was placed around the conifer chamber gaskets and the plant stem to minimize leaks. The lightresponse curves were measured in the following order: 2000, 1500, 1000, 800, 600, 500, 400 and 2000 lmol quanta m 2 s 1.Eight measurements of photosynthesis over c. 30 min were taken before PAR was changed and after the LI-COR infrared gas analyzers (IR- GAs) were matched. The respiration rate was measured at 3.8 Pa CO 2 and 18.4 kpa O 2 after 30 min of dark adaptation following each light- or O 2 -response curve. After the light-response curves, six mature leaves towards the base of the branch, nine medium leaves and nine young leaves towards the apex were harvested for dry matter d 13 C and total leaf nitrogen. Mature leaves were defined as those closest to developing axillary branches with few salt glands, whereas medium leaves were fully expanded with many salt glands, and young leaves were not fully expanded with numerous salt glands (Edwards et al., 2004). The O 2 -response curves of photosynthesis and isotopic exchange were measured at 4.6, 13.8, 18.4, 27.6 and 36.9 kpa O 2 (5%, 15%, 20%, 30% and 40% O 2 ) at 2000 lmol quanta m 2 s 1 and 3.8 Pa CO 2. Plants were acclimated for at least 1 h under the first O 2 concentration with the order of the oxygen concentrations randomly selected. Oxygen concentration was controlled using mass flow controls (Aalborg Instruments & Controls Inc., Orangeburg, NY, USA) connected to compressed nitrogen and oxygen tanks and a 2-l mixing flask. Photosynthetic efficiency (leakiness) Leakiness (/) for LL plants was estimated under high PAR (2000 and 1500 lmol quanta m 2 s 1 ) using the enzyme-limited model of C 4 photosynthesis (von Caemmerer, 2000; Ubierna et al., 2013), and under low PAR (1000, 800, 600, 500 and 400 lmol quanta m 2 s 1 ) using the light-limited model of C 4 photosynthesis (von Caemmerer, 2000; Ubierna et al., 2013). The light-limited model of C 4 photosynthesis was used under all PAR levels for ML plants, because ML plants did not reach saturation under measurement PAR. The enzyme-limited model of C 4 photosynthesis was used to estimate leakiness for all O 2 partial pressures for LL plants. However, the light-limited model of C 4 photosynthesis was used to estimate leakiness for all O 2 partial pressures for ML plants, because these plants did not reach saturation under 2000 lmol quanta m 2 s 1. Leakiness, including the ternary effect (Farquhar & Cernusak, 2012; Ubierna et al., 2013), under high PAR was estimated as (see Table 1): / HL ffi ð1 t b 0 3 ÞD obs P a a ðp a P i Þ 1þt ð ÞP i b4 0 þ e0 Rm A þ 0:5Rd s þ e0 Rm A þ 0:5Rd Rd Eqn 3 A þrd where t is the ternary effect (see Farquhar & Cernusak, 2012; Ubierna et al., 2013), P i is the CO 2 partial pressure in the intercellular air space, P a is the atmospheric partial pressure of CO 2, a is the weighted fractionation across the boundary layer and stomata in series (4.4&), b 0 4 ( 5.7&) is the effect of CO 2

4 Phytologist Research 223 Table 1 Definitions and units for symbols in the text Symbol Definition Equation/notes Units A Net rate of CO 2 assimilation lmol m 2 s 1 a s 13 C fractionation during diffusion 4.4& through the stomata b 3 13 C fractionation including carboxylation, day Eqn 8 & respiration and photorespiration b C fractionation during carboxylation by 30& Rubisco b 4 Fractionation against 13 CinCO 2 by PEPC including Eqn 9 respiratory activity b 0 4 Fractionation by CO 2 dissolution, hydration and 5.7& PEPC activity C a CO 2 mole fraction in the atmosphere 380 lmol mol 1 C i CO 2 mole fraction in the intercellular air lmol mol 1 spaces C m CO 2 mole fraction in the mesophyll cells C m = C i lmol mol 1 C s CO 2 mole fraction in the bundle sheath cells lmol mol 1 e 13 C fractionation during decarboxylation 6 in this study & e 13 C fraction during decarboxylation including Eqn 4 & measurement artifacts e* Difference in 13 C discrimination by respiration Eqns 5 and 6 & due to day respiration utilizing previous assimilates E Rate of transpiration mmol m 2 s 1 f 13 C fractionation during photorespiration , 11.6 in this study & g ac Conductance to diffusion of air in CO 2 and CO 2 in air mol m 2 s 1 g bs Bundle sheath conductance to CO 2 mol m 2 s 1 g m Mesophyll conductance Assumed infinite C i = C m mol m 2 s 1 R d Leaf mitochondrial respiration in the light lmol m 2 s 1 R m Mesophyll mitochondrial respiration rate in the light R d lmol m 2 s 1 s Fractionation during leakage of CO 2 out of the 1.8 & bundle sheath cells t Ternary correction coefficient t = (a ac E)/(2g ac ) & v c Rubisco carboxylation rate lmol m 2 s 1 v o Oxygenation rate lmol m 2 s 1 v p PEP carboxylation rate lmol m 2 s 1 a ac Fractionation factor for the isotopologies of CO 2 =1 + a diffusing in air D Photosynthetic discrimination against 13 C & D obs Observed 13 C photosynthetic discrimination calculated Eqn 1 & with the tunable diode laser absorption spectroscope measurement / Leakiness Eqns 3 and 7 Unitless ξ Ratio of rate of CO 2 entering the chamber to rate of net CO 2 fixation by the leaf Eqn 2 Unitless PEP, phosphoenolpyruvate; PEPC, phosphoenolpyruvate carboxylase; Rubisco, ribulose 1,5-bisphosphate carboxylase/oxygenase. dissolution and PEPC activity at 25 C (Farquhar, 1983), R d is an estimate of leaf mitochondrial respiration occurring during the day (based on the rate measured in the dark), R m is the rate of mitochondrial day respiration in the MCs, calculated as R m = 0.5R d, A is the net photosynthetic rate, b3 0 is Rubisco fractionation (30&)ands(1.8&) is the fractionation of CO 2 leaving the BS cells (Roeske & O Leary, 1984; Henderson et al., 1992). The value e 0 was calculated as in Wingate et al. (2007): e 0 ¼ e e Eqn 4 where e 0 is the 13 C fractionation during decarboxylation, including measurement artifacts, e is the respiratory fractionation during decarboxylation, 6& (Wingate et al., 2007), and e* is the difference between the use of recent photoassimilate and the use of other substrates for day respiration as in Wingate et al. (2007). To model /, assuming all respiratory consumption of substrates occurs using recent photoassimilates, the following equation for e* was used: e recent ¼ d 13 C sample d 13 C sample D obs Dobs Eqn 5 where d 13 C sample and D obs are the isotopic signature of the LI-COR sample line and the photosynthetic discrimination during the measurement, respectively. However, the influence of day respiration on fractionation with the use of old photoassimilates e* was modeled as:

5 224 Research e old ¼ d 13 C sample d 13 C growth d 13 C growth d 13 C dry Dobs Eqn6 where d 13 C growth is the isotopic signature of the air in which the plants were grown ( 10.7& in this study) and d 13 C dry is the isotopic signature of the plant dry matter ( 21.3 and 17.9& for LL and ML plants, respectively). Leakiness under low PAR was modeled according to Ubierna et al. (2013) incorporating the ternary effect (Farquhar & Cernusak, 2012; Ubierna et al., 2013): / LL ¼ P s P i P i D obs ð1 t ÞP a a ðp a P i Þ ð1 þ tþp i b 4 ð1 þ t Þðb 3 sþ The terms b 3 and b 4 are defined as (Farquhar, 1983): Eqn 7 b 3 ¼ b3 0 e0 Rd f 0:5v o Eqn 8 v c v c and b 4 ¼ b 0 4 e0 Rm v p Eqn 9 where f is the fraction during photorespiration, 11.6& (Lanigan et al., 2008), and v o and v c are the rates of oxygenation and carboxylation respectively, by Rubisco. The parameters v p, v o, v c and total electron flux (J t ) were estimated under light-limited conditions using models described previously (von Caemmerer, 2000; Ubierna et al., 2011, 2013; eqns 3 18). Dry matter N content and d 13 C Frozen leaf tissue, consisting of two mature leaves, three medium leaves and three young leaves, was freeze dried for 48 h and ground to a fine powder. Total N was measured by combustion in an elemental analyzer (ECS 4010; Costech Analytical, Valencia, CA, USA). Dry matter d 13 C was determined by mass spectroscopy (Delta PlusXP; Thermofinnigan, Bremen, Germany) and calculated as [(R sample R standard )/R standard ] , where R sample is the ratio of 13 C/ 12 C in the sample and R standard is the ratio of 13 C/ 12 C in the standard, V-Pee Dee Belemnite. Specific leaf area (SLA) To estimate SLA, mature, medium and young leaves were cut from different branches and placed on wet filter paper before obtaining fresh weight and leaf area. Subsequently, leaves were dried in an oven at 60 C for 1 wk and then weighed. Six replicate plants were measured per treatment. Enzyme assays and chlorophyll content Enzyme activity was measured for both LL and ML plants according to Cousins et al. (2007). Three leaves (mature, Phytologist medium and young) from several different branches were taken from a single plant. Leaf area was determined from photographs of leaves using ImageJ and leaves were ground together in a cold room at 4 C in 1000 ll of extraction buffer (50 mm Hepes KOH, ph 7.8, 1% polyvinylpyrrolidone (PVPP), 1 mm EDTA, 10 mm dithiothreitol, 0.1% Triton X), 5 ll of protease inhibitor cocktail (Sigma) and fine sand. After grinding, the leaf extract was briefly centrifuged and 500 ll of supernatant was incubated with 15 mm MgCl 2 and 15 mm NaHCO 3 to fully activate Rubisco. Rubisco activity was determined in an assay buffer of 100 mm EPPS NaOH, ph 8.0, 20 mm MgCl 2, 1 mm EDTA, 10 mm ATP, 50 mm creatine phosphate, 20 mm NaHCO 3, 0.2mM NADH, 12.5 U ml 1 creatine phosphokinase, 250 U ml 1 carbonic anhydrase, 22.5 U ml 1 phosphoglycerate kinase, 20 U ml 1 glyceraldehyde-3-phosphate dehydrogenase, 56 U ml 1 triose-phosphate isomerase and 20 U ml 1 glycerol-3-phosphate dehydrogenase, and the reaction was initiated with 20 ll of 20.4 mm RuBP. PEPC activity was determined in an assay buffer of 100 mm EPPS NaOH, ph 8.0, 20 mm MgCl 2, 1mM EDTA, 0.2 mm NADH, 5 mm glucose-6 phosphate, 1 mm NaHCO 3 and 12 U ml 1 malate dehydrogenase, and the reaction was initiated with 10 ll of 4 mm PEP. Enzyme activity for both assays was determined spectrophotometrically by following the decrease in NADH absorbance over time at 340 nm, correcting for the non-specific decrease in absorbance at 400 nm (Thermo Fisher Scientific Inc., Houston, TX, USA). Chlorophyll was extracted in 95% ethanol for 48 h in the dark on a shaker at 4 C, and the absorbance of chlorophyll was measured at 649 and 665 nm (any non-specific absorbance measured at 700 nm was subtracted from these values) with a spectrometer. Chlorophyll concentration was calculated according to Ritchie (2006) with five replicates per treatment, consisting of pooled mature, medium and young leaves. Anatomical measurements Two square millimeters of medium-aged leaves from the middle of the branch were fixed in 2% paraformaldehyde and 2% glutaraldehyde in 0.1 M phosphate buffer. Subsequently, the samples were transferred to 2.5% glutaraldehyde, 3.5% formaldehyde, 0.1 M sodium cacodylate, 0.12 M sucrose, 10 mm ethylene glycol tetra-acetic acid (EGTA) and 2 mm magnesium chloride overnight at 4 C. The samples were rinsed in a 0.1 M sodium cacodylate buffer and post-fixed in 2% OsO 4 for 2 h at room temperature, dehydrated in an ethanol series and embedded in Spurr s resin. Cross-sections (800 nm thick) were made and stained with toluidine blue in 1% sodium borohydrate, and digital images were collected with a camera (Jenoptik ProgRes Camera; Optik, Systeme, Jena, Germany) attached to a compound light microscope (Olympus BH-2; Olympus Optical Co. Ltd, Tokyo, Japan). Images of five replicate plants from each treatment were analyzed with ImageJ (US National Institutes of Health) to measure the distance between the central chloroplastic compartment (CCC) and the intercellular airspace (IAS) (lm), the total length

6 Phytologist Research 225 of MC wall exposed to IAS (lm) and the width of the leaf section analyzed (lm). The corresponding total MC surface area exposed to IAS per (one side) leaf surface area (A mes, lm 2 lm 2 ) was inferred (Evans et al., 1994) as: (a) A mes ¼ Total length of MC appressed to ISA Section width 1:43 Eqn 10 where 1.43 is the curvature correction factor taken from Evans et al. (1994). The cross-sectional area of MC and their CCC was measured by light microscopy on cells isolated by gentle maceration of leaves as described by Leisner (2009). Leaf thickness, length and width were measured with digital calipers on medium-aged leaves from the middle of the branch. (b) Statistical analysis Statistical analysis was performed using Statistix software (Analytical Software, Tallahassee, FL, USA). Three-way repeatedmeasures ANOVAs were used for A, g s, D 13 C and leakiness by PAR, treatment and tank for light-response curves. For O 2 - response curves, two-way repeated-measure ANOVAs were used to compare ML and LL plants for A, g s, D 13 C and leakiness by O 2 and treatment. The branch was the repeated measure for all analyses. Results were deemed to be significant at P<0.05 and Tukey s test was used for post hoc comparisons. A paired twotailed Student s t-test was used for enzyme assays, microscopy measurements, SLA, chlorophyll content, dry matter d 13 C and dark respiration rate between treatments. (c) Results Oxygen-response curves The net rate of CO 2 assimilation was higher for ML relative to LL plants under all O 2 partial pressures (Fig. 1a). In addition, the net rate of CO 2 assimilation declined with increasing partial pressure of O 2 in plants from both treatments; however, the O 2 sensitivity was greater in LL (1.8% decrease (kpa O 2 ) 1 ) relative to ML (1.0% decrease (kpa O 2 ) 1 ) plants (Fig. 1a, Table S1). Stomatal conductance (g s ) was higher in ML plants across all partial pressures of O 2, but was constant with O 2 for both treatments (Fig. 1b, Table S1). There was no difference in P i /P a between treatments or in response to O 2 (Fig. 1c, Table S1). Photosynthetic discrimination (D obs )increasedwitho 2 partial pressure in both ML and LL plants, but D obs responded more to O 2 in LL plants (Fig. 2a, Table S1). In ML plants, there was a gradual increase in / with increasing O 2,whereas,inLLplants, therewasanimmediaterisein/ at 13.8 kpa O 2, which leveled outafterthispoint(fig.2b,tables1).leakinessinllplantswas higher than in ML plants under all O 2 conditions (Fig. 2b). Light-response curves In ML plants, there was a strong response of photosynthesis up to full sunlight (PAR = 2000 lmol quanta m 2 s 1 ), whereas the Fig. 1 Oxygen-response curves for low-light-grown (LL, open squares) and moderate-light-grown (ML, closed circles) Bienertia sinuspersici plants measured with a CO 2 isotopic signature of 7&. (a) CO 2 assimilation rate (A), (b) stomatal conductance (g s ) and (c) ratio of intercellular CO 2 to ambient CO 2 partial pressure (P i /P a ). Measurements were made with photosynthetically active radiation (PAR) of 2000 lmol m 2 s 1 and 3.8 Pa CO 2 at 25 C. Measurements represent averages SE of six replicates. response in LL plants was more hyperbolic, increasing gradually at higher PAR (Fig. 3a). Rates of net CO 2 assimilation (A) were higher in ML relative to LL plants at PAR > 800 lmol m 2 s 1, but similar at PAR < 800 lmol m 2 s 1 (Fig. 3a, Table S2). Rates of assimilation at PAR = 2000 lmol quanta m 2 s 1 (equivalent to full sunlight) in ML plants (c. 40 lmol m 2 s 1 ) were c. two-fold higher than in LL plants (< 20 lmol m 2 s 1 ). In addition, in ML plants, A was higher when the source was 58& rather than 7& CO 2 (P < 0.01), but not for LL plants (Fig. 3a, Table S2). Stomatal conductance (g s ) was higher in ML relative to LL plants across all PAR levels (Fig. 3b, Table S2), regardless of the isotopic signature of the measurement CO 2.In ML plants, g s was significantly higher in 58& than in 7&

7 226 Research Phytologist (a) (a) (b) (b) (c) Fig. 2 Oxygen-response curves for low-light-grown (LL, open squares) and moderate-light-grown (ML, closed circles) Bienertia sinuspersici plants with a CO 2 isotopic signature of 7&. (a) Leaf discrimination (D 13 C) for LL- and ML-grown plants. (b) Bundle sheath leakiness (/) was modeled assuming plants were using recent photoassimilates in respiration. Measurements were made with photosynthetically active radiation (PAR) of 2000 lmol m 2 s 1 and 3.8 Pa CO 2 at 25 C. Measurements represent averages SE of six replicates for 7& for both light treatments. CO 2 (P < 0.01) under high PAR, but, in general, g s increased in ML plants with increasing PAR (Fig. 3b, Table S2). The ratio of P i /P a was higher in ML relative to LL plants (P < 0.01) and decreased with increasing PAR for both treatments (Fig. 3c, Table S2; P < 0.01). Values of P i /P a were significantly different between CO 2 isotopic signatures for both growth conditions and all PAR levels (P < 0.01). Measured D obs increased more with decreasing PAR in ML relative to LL plants, regardless of the CO 2 isotopic signature (Fig. 4a,b). In ML plants, D obs increased with decreasing PAR more under 58& relative to 7& CO 2 (Fig. 4b); however, there was no significant difference in D obs between the two measurement CO 2 isotopic signatures in LL plants (Fig. 4a, Table S2). Leakiness (/) was calculated assuming the substrate for day respiration was either from recent photosynthate (recent) fixed during the measurements or from previous photosynthate (old) fixed in the growth chamber. Leakiness generally did not respond to changes in PAR in LL plants, except when measurements were made with the 58& CO 2 source and it was assumed that old photosynthate was the substrate for day respiration (Fig. 4c). Under these conditions, / decreased with Fig. 3 Light-response curves for low-light-grown (LL) Bienertia sinuspersici plants with CO 2 isotopic signatures of 7& (open squares) and 58& (closed squares), and moderate-light-grown (ML) plants with CO 2 isotopic signatures of 7& (open circles) and 58& (closed circles). (a) CO 2 assimilation rate (A), (b) stomatal conductance (g s ) and (c) ratio of intercellular CO 2 to ambient CO 2 (P i /P a ). Measurements were made under 3.8 Pa CO 2 at 25 C. Measurements represent averages SE of six replicates for 7& and three replicates for 58& measurements for both light treatments. decreasing PAR and was significantly different from the other estimates of / below 1000 lmol quanta m 2 s 1. However, / did not change with PAR in LL plants measured under 7& CO 2, whether the substrate for R d was assumed to be recent or old photoassimilate (Fig. 4c). In plants grown under ML, / increased with decreasing PAR measured under 58& CO 2 and assuming recently fixed photoassimilate was used as the substrate for day respiration (Fig. 4d). However, / modeled assuming that plants used old photoassimilate did not increase under decreasing PAR, and was significantly lower at low PAR than / estimated under 58& CO 2, assuming that recent photoassimilate was the substrate for day respiration. Leakiness in ML plants measured

8 Phytologist Research 227 (a) (b) Fig. 4 Light-response curves for low-lightgrown (LL) Bienertia sinuspersici plants with CO 2 isotopic signatures of 7& (open squares) and 58& (closed squares) and moderate-light-grown (ML) plants with CO 2 isotopic signatures of 7& (closed circles) and 58& (open circles). Photosynthetic discrimination (D 13 C) for LL (a) and ML (b) plants. Bundle-sheath leakiness (/) was modeled for LL (c) and ML (d) plants assuming that plants were using either old photoassimilate (LL 7&, closed circles; LL 58&, closed squares; ML 7&, closed hexagons; ML 58&, closed down triangles) or recent photoassimilate (LL 7&, open circles; LL 58&, open squares; ML 7&, open hexagons; ML 58&, open down triangles). Measurements were made under 3.8 Pa CO 2 at 25 C. Measurements represent averages SE of six replicates for 7& and three replicates for 58& for both treatments. (c) (d) under 7& CO 2 did not change with declining PAR, assuming either recent or old photoassimilate. Leaf biochemical characteristics Rubisco activity was c. two-fold higher and PEPC activity was c.three-fold higher per unit leaf area in ML relative to LL plants (P < 0.01; Table 2), although the ratio of PEPC to Rubisco was not significantly different between treatments. In addition, total chlorophyll, chlorophyll a/b and total leaf N were not significantly different between treatments (Table 2). However, the isotopic signature of leaf dry matter (d 13 C) was significantly more depleted in 13 CinLL( &) relative to ML ( &; P < 0.01; Table 2) plants. Rates of dark respiration were significantly higher (c. two-fold) in ML ( lmol CO 2 m 2 s 1 ) relative to LL ( lmol CO 2 m 2 s 1 ; P < 0.01; Table 2) plants. Anatomical changes The SLA, the leaf area to dry mass ratio, was significantly higher in LL relative to ML plants (P < 0.01; Table 3). In addition, the ratio of the individual MC area to the area of the CCC was significantly higher in LL relative to ML plants (P < 0.05; Table 3). However, leaf length and thickness, the path length from the CCC to the IAS, and the distance of the MCs exposed to the IAS were not significantly different between treatments (Table 3). The planar area per MC was greater in LL relative to ML plants; Table 2 Leaf biochemical properties of Bienertia sinuspersici grown under low-light (LL, PAR = lmol m 2 s 1 ) and moderate-light (ML, PAR = lmol m 2 s 1 ) conditions Parameter LL ML Rubisco (lmol CO 2 m 2 s 1 ) ** ** PEPC (lmol CO 2 m 2 s 1 ) ** ** PEPC/Rubisco Chlorophyll a + b (mg m 2 ) Chlorophyll a/b Dry matter d 13 C(&) ** ** Leaf nitrogen (mmol m 2 ) Dark-type respiration (lmol CO 2 m 2 s 1 ) ** ** PEPC, phosphoenolpyruvate carboxylase; Rubisco, ribulose 1,5-bisphosphate carboxylase/oxygenase. Measurements represent averages SE of four to six replications from pooled mature, medium and young leaves. Similar to the measurements of photosynthesis, the rates of dark-type respiration were made on branches and leaves, and the rates are expressed on a projected leaf area. Significant differences between treatments determined with Student s t-test: **, P < however, this difference was not significant. The planar area of the CCC was the same between treatments (Table 3). Discussion Our results demonstrate the capacity for photosynthesis and the CO 2 -concentrating mechanism in the single-cell C 4 plant

9 228 Research Phytologist Table 3 Leaf properties of Bienertia sinuspersici grown under low-light (LL, PAR = lmol m 2 s 1 ) and moderate-light (ML, PAR = lmol m 2 s 1 ) conditions Parameter LL ML SLA, leaf area/leaf dry mass (cm 2 g 1 ) ** ** Planar mesophyll cell area (lm) Planar CCC area (lm) Mesophyll cell area/ccc area * * Leaf thickness (mm) Leaf length (mm) Path length to intercellular airspace (lm) A mes, mesophyll cell length exposed to IAS per leaf area (lm 2 lm 2 ) CCC, central cytoplasmic compartment in mesophyll cells; IAS, intercellular air space. Measurements show the means and standard errors of five to six replicates from medium-aged leaves for all parameters except SLA. The estimates of SLA were pooled from mature, medium and young leaves. Significant differences between treatments determined with Student s t-test: *, P < 0.05; **, P < B. sinuspersici is influenced by the growth light condition. ML plants had c. two-fold higher rates of photosynthesis under high PAR, c. two-fold higher Rubisco and c. three-fold higher PEPC activities per unit leaf area, and an estimated c. 20% lower leakiness under current atmospheric levels of O 2 (18.4 kpa O 2, Fig. 4). This shows that the single-cell C 4 plant has a characteristic acclimation potential: when light is limiting during growth, there is less investment in biochemistry for carbon assimilation. However, growth light conditions caused only subtle changes in leaf structure and anatomy, except that the SLA was higher in LL plants. In species with non-succulent planar leaves grown under LL, the leaves are often thinner, cells smaller and, in some cases, layers of cells in the leaf are reduced (Terashima et al., 2006; Pengelly et al., 2010). Below, we discuss how biochemical and structural acclimation under light-limited growth conditions affects photosynthesis in B. sinuspersici. Oxygen-response curves The CO 2 -concentrating mechanism in Kranz-type C 4 plants reduces the rate of photorespiration by concentrating CO 2 around Rubisco (Edwards & Walker, 1983; Hatch, 1987; Keeley & Rundel, 2003; Sage, 2004). Therefore, C 4 plants are typically not as sensitive as C 3 plants to high O 2 concentrations. However, the efficiency of the single-cell CO 2 -concentrating mechanism to changes in O 2 partial pressure has not been studied. King et al. (2012) estimated the photosynthetic efficiency in two single-cell C 4 plants, B. sinuspersici and S. aralocaspica, under low O 2 (4.6 kpa), and showed that, under these conditions, / in singlecell C 4 plants was similar to that of Kranz-type plants. However, the low O 2 during these previously published estimates of / in the single-cell C 4 plants may have masked an inefficient CO 2 - concentrating mechanism. If the single-cell C 4 plants have an inefficient CO 2 -concentrating mechanism, D obs and / should be more sensitive to changes in O 2 than expected for C 4 plants. Furthermore, differences in leaf anatomy caused by the light-limited growth condition could also affect the O 2 sensitivity of the CO 2 -concentrating mechanism in single-cell C 4 plants. In our measurements, as the O 2 partial pressure increased, D obs increased by 3& and 2& in LL and ML plants, respectively (Fig. 2a,b), suggesting a slightly greater sensitivity to increasing O 2 in LL plants. Rates of net CO 2 assimilation (A) were higher in ML relative to LL plants regardless of O 2 partial pressure (Fig. 1a). In addition, at ambient CO 2 concentrations (3.8 Pa), A decreased with increasing O 2 in plants from both growth conditions. However, it should be noted that the O 2 sensitivity of A seen in B. sinuspersici has also been demonstrated in several Kranz-type C 4 plants (Dai et al., 1993; Maroco et al., 1997, 2000). Some sensitivity of A to O 2 can occur in C 4 plants as a result of photorespiration, but it is expected to be rather low relative to C 3 plants because of the CO 2 -concentrating mechanism. In B. sinuspersici, the slightly higher sensitivity of A to O 2 in LL plants suggests that the acclimation of the single-cell C 4 system to LL altered the capacity and efficiency of the CO 2 - concentrating mechanism. This is further supported by measurements of leaf CO 2 isotope exchange in response to O 2, which showed that D obs increased more in LL relative to ML plants from low to high O 2. Furthermore, / increased by 10% in LL plants from 4.6 to 13.8 kpa O 2 and then remained constant; however, in ML plants, / decreased by < 10% across all O 2 partial pressures (Fig. 2c,d). These estimates of / used the full Wingate et al. (2007) equation (Eqns 5 and 6 in this text), taking into account differences between the substrate for day respiration and the substrate for photosynthesis. This has important implications when comparing / values reported here with those of King et al. (2012) for B. sinuspersici. In King et al. (2012), estimates of / were determined in ML-grown plants (the isotopic signature of growth CO 2 was c. 10&) and measured under low O 2 (4.6 kpa) with a CO 2 signature of c. 45&. The apparent influence of day respiration in King et al. (2012) on 13 C fraction during decarboxylation (e ) was estimated using e = e + d 13 C measurement d 13 C growth. This assumes that the substrate for day respiration is recent photoassimilate, which has an isotopic signature dependent on D obs and d 13 C of the CO 2 used during the measurements (Ubierna et al., 2013). However, / will be miscalculated if a plant is using old photoassimilate as a substrate for day respiration and the measurement CO 2 has a different isotopic signature from the growth conditions. Therefore, under low PAR, when day respiration represents a greater proportion of branch net CO 2 exchange, the estimate of / from King et al. (2012) may be incorrect because of the simplification used to estimate e*. Below, we discuss the influence of e* and differences in growth light conditions on / in response to changes in measurement PAR. Light-response curves In plants grown under ML and measured under 58 and 7& CO 2, there was a linear increase in A up to full sunlight. ML

10 Phytologist Research 229 plants measured under 58& CO 2 were a different cohort from those measured under 7& CO 2, which probably contributed to the differences seen in A and g s (Fig. 3a,b). However, in LL plants, A became saturated at light levels above growth conditions. If plants acclimate, this typically occurs by not over-investing in components that cannot be used in light-limited conditions (e.g. high capacity of carboxylases). This has also been observed in some Kranz-type C 4 plants grown under two light levels (Tazoe et al., 2008; Pengelly et al., 2010). The increase in D obs as measurement PAR decreased in both LL and ML plants (Fig. 4) is also in agreement with previous reports (see Tazoe et al., 2008; Pengelly et al., 2010; Ubierna et al., 2013). The increase in D obs at low PAR was more pronounced in ML relative to LL plants, regardless of the isotopic signature of the measurement CO 2 (Fig. 4a). Previous studies (Tazoe et al., 2008; Pengelly et al., 2010) have also shown a greater increase in D obs for plants grown under high light or ML relative to plants grown under LL. It is assumed that day respiration stays relatively constant under all PARs, whereas photosynthesis decreases with decreasing PAR. Therefore, under high PAR, the contribution of respired CO 2 to total leaf CO 2 isotope exchange is smaller than the contribution under low PAR. In the current study, rates of dark-type respiration were 2.1 and 6.1 lmol CO 2 m 2 s 1 for LL and ML plants, respectively (Table 2). Therefore, the contribution of respired CO 2 to net CO 2 exchange in ML plants was greater than in LL plants. If a plant was measured using CO 2 depleted in 13 C, the recent photoassimilates would be depleted in 13 C. In addition, plants with higher day respiration rates would yield a greater contribution to leaf CO 2 exchange, which would influence D obs. The simplified equation of Wingate et al. (2007) for e, where e = e + (d 13 C measurement d 13 C growth ), assumes that the isotopic signature of the substrate for day respiration is the same as the isotopic signature of the substrate for photosynthesis (Ubierna et al., 2013). For this assumption to be met, the plant must use recent photoassimilate as a substrate for day respiration; however, this may not always be the case. To test this, we used Eqns 5 and 6 to account for the differences in substrates used for photosynthesis and day respiration. The estimated / changed from 0.4 to 0.15 with decreasing PAR in LL plants, assuming old photoassimilate (acquired in the growth chambers with CO 2 = 10.7&) and measured with 58& CO 2 (Fig. 4c). However, there was little response of / to PAR with measurement CO 2 of 58& and assuming that recent photoassimilate was the substrate for day respiration (Fig. 4c). In addition, when measurements were conducted under 7& CO 2, there was little change in / in response to PAR (Fig. 4c). This demonstrates that misrepresentation of the isotopic signature of the photoassimilate used for day respiration can misestimate / when there is a significant difference between the isotopic composition of recent and old photoassimilates. Furthermore, in the LL plants, / does not appear to change in response to PAR when correctly accounting for the impact of day respiration on leaf CO 2 isotope exchange. For ML plants measured under 58& CO 2, values of / increased from 0.4 to 1.4 with declining PAR, assuming that plants used recent photoassimilate for day respiration (Fig. 4d). However, / does not increase with decreasing PAR if / is modeled assuming plants are using old photoassimilate as a substrate for day respiration. In addition, when plants were measured under 7& CO 2, estimates of / were not influenced by errors in assuming recent vs old substrate for day respiration. Therefore, measurements made at 7& CO 2 can be used to determine whether plants measured at 58& CO 2 are using recent or old photoassimilate as the substrate for day respiration. For example, in ML plants, there was a sharp increase in D obs when measured under 58& CO 2 (Fig. 4d) relative to plants measured under 7& CO 2. If plants used old photoassimilate as a substrate for day respiration, there would not have been a large increase in D obs or / with declining PAR. However, there was a large increase in D obs under 58& CO 2 with declining PAR, indicating that B. sinuspersici used recent photoassimilate as a substrate for day respiration for both LL and ML plants under low PAR. It should be noted that leaves probably use a mixture of both recent and old photosynthate as substrates for day respiration. Therefore, our assumption of leaves exclusively using recent or old photosynthate is probably an over-simplification. This may explain why ML plants measured under 58& CO 2 assuming recent photosynthate overestimated / and assuming old photosynthate under-estimated / relative to plants measured under 7& CO 2 (Fig. 4). In addition, two separate cohorts of plants were used for the 58& and 7& CO 2 measurements; therefore, some of the difference in / could also be attributed to differences between plants. Anatomical and biochemical changes Sage & McKown (2006) suggested that Kranz-type C 4 plants are less phenotypically plastic in their response to light-limited growth conditions because of their unique anatomical requirements. However, Pengelly et al. (2010) demonstrated that the Kranz-type C 4 plant Flaveria bidentis showed substantial plasticity when grown under LL, including decreased leaf thickness, smaller cells and increased SLA. In the single-cell C 4 plant B. sinuspersici, there were more subtle effects on leaf anatomy for plants grown under LL vs ML. The characteristic structure of the C 4 chlorenchyma cells was maintained under LL, and growth light conditions did not influence leaf thickness, which is probably related in part to the thick succulent nature of the leaves. The ratio of leaf area to dry mass (SLA) was higher in plants grown under LL conditions, suggesting higher leaf density (possibly associated with cell wall density and starch accumulation). In Kranz-type C 4 plants, it has been suggested that the mesophyll surface area next to the IAS correlates with C 4 photosynthetic capacity (Evans & von Caemmerer, 1996). In LL plants, the mesophyll surface area exposed to IAS was greater than in ML plants; however, this relationship was not statistically significant. In addition, the distance from the CCC to the IAS was 30% greater in ML relative to LL plants (Table 3), but this relationship was not statistically significant. However, the ratio of the

11 230 Research Phytologist individual cell area to the area of the CCC (CA : CCC) was greater in LL relative to ML plants (Table 3). This increase in CA : CCC suggests a greater distance from the CCC to the IAS in LL plants, which might increase the resistance to leakage of CO 2 from the CCC during C 4 photosynthesis. Greater resistance to CO 2 leakage could increase the photosynthetic efficiency of the single-cell CO 2 -concentrating mechanism (von Caemmerer, 2003); however, / in LL plants was higher than in ML plants under all O 2 and PAR conditions when correctly accounting for the influence of day respiration (Figs 3, 4). This suggests that something other than leaf and cell anatomy is driving the differences in / between LL and ML plants. It has been demonstrated that the cell structure, chlorophyll content and biochemical capacity of the leaf can also influence the efficiency of C 4 photosynthesis. However, total chlorophyll was not significantly different between LL and ML plants. These findings are different from other studies with C 4 plants, where ML plants showed lower total chlorophyll per unit leaf area relative to LL plants (Tazoe et al., 2008; Pengelly et al., 2010). In addition, the ratio of chlorophyll a/b was not significantly different in B. sinuspersici between growth treatments, but the ratio followed the same pattern as in Tazoe et al. (2008) and Pengelly et al. (2010), where high-light or ML plants had higher chlorophyll a/b ratios than LL plants. With respect to biochemistry, the activities of Rubisco and PEPC were two and three times higher, respectively, in ML relative to LL plants (Table 2). Pengelly et al. (2010) also observed higher rates of Rubisco and PEPC activity in ML relative to LL F. bidentis. However, the ratio of PEPC/Rubisco activity in B. sinuspersici was not significantly different between treatments (Table 2). This suggests that changes in the relative capacity of the C 3 and C 4 cycles were not a major factor influencing the efficiency of the single-cell CO 2 -concentrating mechanism. However, modeling simulations have demonstrated that / is highest at low photosynthetic capacity (low V cmax and V pmax ; see von Caemmerer, 2003). Dry matter d 13 C values (Table 2) were within the range of the values reported previously for chenopod species grown in environmental chambers (Voznesenskaya et al., 2002; Akhani et al., 2009; Leisner et al., 2010; King et al., 2012). However, LL plants had more negative dry matter d 13 C relative to ML plants (Table 2). This was also observed in F. bidentis reported in Pengelly et al. (2010) and Amaranthus cruentus (Tazoe et al., 2008), suggesting that LL plants had greater / under growth conditions, which was also observed in the online measurements of carbon isotope discrimination in B. sinuspersici. Conclusions There have been few studies on the effect of growth light levels on structural, biochemical and physiological features associated with photosynthesis. The goal of this research was to determine how growth under limited light affects leaf structure and photosynthetic efficiency in a unique single-cell C 4 system. Similar to Kranz-type C 4 plants, a functional C 4 system was maintained in this single-cell C 4 species. Plants grown under ML were more effective in capturing and assimilating CO 2 delivered by the C 4 cycle than were plants grown under LL, which was linked to biochemical rather than anatomical changes. The photosynthetic efficiency of the single-cell C 4 system is insensitive to changes in measurement PAR when correctly accounting for differences in day respiration and photosynthetic discrimination. The results also indicate that this single-cell C 4 plant uses recent photoassimilate as a substrate for day respiration. Together, these data demonstrate that the fully developed single-cell C 4 system in B. sinuspersici is robust when grown under ML. Although, under natural growth conditions, this species is exposed to high-light environments in semi-arid deserts, it can acclimate to growth under LL conditions (c. 10% full sunlight). Acknowledgements We thank Drs A. Gandin and N. Ubierna for helpful discussions on modeling and estimating leakiness. We are also grateful to Drs E. Voznesenskaya and N. Koteyeva for helpful discussions on microscopy, plant propagation and growth. In addition, thanks are due to C. Cody for growth chamber maintenance. This research was supported in part by instrumentation obtained through a National Science Foundation (NSF) Major Research Instrumentation grant no (A.B.C.) and partly by the Division of Chemical Sciences, Geosciences, and Biosciences, Office of Basic Energy Sciences of the US Department of Energy through grant DE-FG02-09ER16062 (A.B.C.), and NSF grant MCB (G.E.E.). References Akhani H, Barroca J, Koteeva N, Voznesenskaya E, Franceschi V, Edwards GE, Ghaffari SM, Ziegler H Bienertia sinuspersici (Chenopodiaceae): a new species from southwest Asia and discovery of a third terrestrial C 4 plant without Kranz anatomy. Systematic Botany 30: Akhani H, Chatrenoor T, Dehghani M, Khoshravesh M, Mahdavi P, Matinzadeh Z A new species of Bienertia (Chenopodiaceae) from Iranian salt deserts: a third species of the genus and discovery of a fourth terrestrial C 4 plant without Kranz anatomy. 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