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Photosynthesis plays a crucial role in ecosystem sustainability (Millennium Ecosystem Assessment, 2005). In order to combat climate change and ensure the sustainability of natural and cultivated ecoystems, it is essential to understand the responses of photosynthesis to fluctuating environmental conditions, particularly atmospheric CO2 enrichment. Surprisingly little attention has been given to the effects of increased atmospheric CO2 on C4 photosynthesis, despite the fact that many of the most productive bio-energy/bio-fuel crops are monocotyledonous C4 species. It is important, therefore, to gain a better understanding of how CO2 enrichment will alter stomatal function and photosynthesis in such species, and particularly to elucidate any interactions with respect to light orientation, which is a crucial regulator of these processes (Poulson & DeLucia, 1993). Structure–function relationships in photosynthesis have been intensely studied in dicotyledonous leaves (Terashima & Saeki, 1983; Cui et al., 1991; Vogelmann, 1993), which exhibit internal gradients in light and photosynthetic capacity (Terashima & Inoue, 1985a,b). The highest photosynthesis rates are not found near the leaf surface where the light intensity is highest, but are observed in the middle and lower palisade layers (Nishio et al., 1993; Evans, 1995; Sun et al., 1998; Sun & Nishio, 2001; Evans & Vogelmann, 2003), which have higher electron transport activities and greater amounts of photosynthetic proteins (Terashima & Inoue, 1985b; Terashima & Evans, 1988; Sun & Nishio, 2001). The adaxial surfaces of dicotyledonous C3 leaves have characteristics that resemble classic ‘sun’ leaves, while the abaxial surfaces have properties that are consistent with ‘shade’ leaves (Oya & Laisk, 1976; Terashima, 1986; Terashima & Evans, 1988; Lambers et al., 1998). Growth at high CO2 alters the regulation of photosynthesis on the adaxial and abaxial leaf surfaces of maize (Zea mays) leaves (Domes, 1971; Driscoll et al., 2006), but very little information is available on the effects of CO2 enrichment on the surface-specific regulation of photosynthesis in other monocotyledonous C4 species.
Atmospheric CO2 availability exerts a strong influence on the dorso-ventral organization of leaf structure, composition and photosynthetic activity (Taylor et al., 1994, 2001; Croxdale, 1998; Masle, 2000; Lake et al., 2001; Poorter & Navas, 2003; Martin & Glover, 2007). Similarly, growth CO2 concentrations exert control over stomatal density and patterning (Larkin et al., 1997; Lake et al., 2002). Atmospheric CO2 enrichment decreases stomatal densities in the leaves of dicotyledonous C3 species (Woodward et al., 2002). However, the degree of this response to CO2 enrichment differs between species and little information is available in the literature on such effects in monocotyledonous C4 species (Woodward & Kelly, 1995; Woodward et al., 2002). The CO2-signalling pathways that orchestrate these changes in leaf structure and composition responses remain poorly characterized (Gray et al., 2000; Ferris et al., 2002), but signals transported from mature to developing leaves are considered to be important regulators of such responses (Coupe et al., 2006; Miyazawa et al., 2006).
The present study was undertaken in order to characterize the relative effects of light orientation and CO2 availability on photosynthesis and stomatal responses in Paspalum dilatatum, which, like maize, is a C4 monocotyledonous species of the NADP-malic enzyme (NADP-ME) subtype. Paspalum dilatatum is a common grass species of the prairies of South America (Pinto da Silva, 1969; Usuda et al., 1984) and the wetter areas of Australia (Pearson et al., 1985; Brown, 1999) and has already been characterized in terms of photosynthetic responses to environmental stress (e.g. Marques da Silva et al., 1991; Bernardes da Silva et al., 1999; Cavaco et al., 2003; Carmo-Silva et al., 2007) and to CO2 enrichment (Greer et al., 1995; von Caemmerer et al., 2001). Here we report the influence of light orientation on the dorso-ventral regulation of photosynthesis and stomatal conductance, and the effect of growth CO2 on the symmetry of the photosynthetic responses on the adaxial and abaxial leaf surfaces to incident light (either adaxial or abaxial), together with acclimatory responses in whole-plant morphology, biomass, whole-leaf photosynthesis, stomatal patterning and leaf composition.
Materials and Methods
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- Materials and Methods
All the following experiments were conducted in the laboratories at Rothamsted Research, Harpenden, UK except for the electron microscopy, which was performed at CEBAS-CSIC, Murcia, Spain on samples prepared at Rothamsted Research. Each experiment was repeated at least three times and all experiments involved a minimum of three plants.
Seeds of Paspalum dilatatum Poiret cv. Raki were obtained from the Margot Forage Germplasm Centre, Palmerston North, New Zealand. They were germinated on compost containing slow-release fertilizer in pots (20 cm diameter) and then grown for 6 wk in two controlled-environment cabinets (Sanyo SGC228.CFX.J; Sanyo, Osaka, Japan) in which all the growth conditions were identical except for the atmospheric CO2 concentration, which was maintained at either 350 or 700 µl l−1 throughout the duration of the experiments. CO2 was supplied from a bulk container via a Vaisala GMT220 CO2 transmitter (Vaisala Oyj, Helsinki, Finland). The CO2 contents within the chambers were maintained at 350 ± 20 or 700 ± 20 µl l−1 using a Eurotherm 2704 controller (Eurotherm Ltd, Worthing, UK). The plants were grown under a 16-h photoperiod with a 25°C (day): 19°C (night) temperature regime and 80% relative humidity. Irradiance (600–650 mmol µmol m−2 s−1 at 400–700 nm, at pot height) was provided by Philips Master TL5 HO 49w/830 fluorescent lamps (Philips Lighting UK, Guildford, UK). All plants were well watered daily and the root growth in the pots was checked periodically to ensure that an appropriate pot size was used throughout the growth period. At the harvest point the roots were growing freely.
Whole-plant growth parameters and tissue biomass
In each experiment, between eight and 14 plants were harvested at 6 wk from each CO2 chamber, a time-point that was before anthesis in both air- and high CO2-grown plants. Harvested plants were separated into leaves, stems and roots. The following measurements were performed: root, stem and leaf biomass, specific leaf area (SLA), leaf area ratio (LAR), leaf weight ratio (LWR), leaf density (% leaf dry weight (DW)) and leaf thickness (leaf fresh weight to leaf area ratio).
Photosynthetic gas-exchange measurements were performed using infrared gas analysis (model wa-225-mk3; ADC, Hoddesdon, UK). Two different types of chamber system were used in these studies. One apparatus involves a series of standard Parkinson chambers for whole-leaf measurements (Novitskaya et al., 2002). The second apparatus uses modified Parkinson chambers, as illustrated in Fig. 1. This system enables separate and simultaneous measurements of gas exchange on each leaf surface (Driscoll et al., 2006).
Figure 1. The modified Parkinson leaf chamber used for separate and simultaneous gas exchange measurements on each leaf surface. Different views of the chamber are given to illustrate the following features: 1, light sensor; 2, connecting tubes to the infrared gas analyser; 3, upper side of the modified chamber with light source; 4, lower side of the modified chamber; 5, air supply in to both sides of the chamber; 6, air supply out of both sides of the chamber; 7, humidity and temperature sensors on both sides of the chamber; 8, fan on both sides of the chamber; 9, water jackets on both sides of the chamber; 10, gas-tight tape; 11, Paspalum dilatatum leaf.
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To check the degree of CO2 transport across the leaf, a gas stream containing 10% CO2 was applied to each surface independently in the chamber and the concentration on the other surface was measured. In this system, we could detect no passage of CO2 from one surface to the other through the leaf blade. While the stomata close under these conditions, this simple experiment illustrates that there is no passage of gases from one side of the chamber to the other. Each half of this dual chamber operates essentially as a whole leaf chamber as each half has a distinct gas supply and analysis unit with separate humidity, leaf temperature and individual fan controls (Fig. 1a,b,c). Flow rates were optimized on each side of the chamber to match the pressure on both leaf surfaces. Because the P. dilatatum leaf was not large enough to fully separate the two sides of the chamber, the leaf was expanded with gas-tight tape to seal each half of the chamber, preventing any flux of gas between the two sides (Fig. 1d,e). Irradiance was supplied only from the top of the chamber as described previously (Novitskaya et al., 2002). Leaf temperatures were monitored via thermocouples attached to each surface separately (Fig. 1b) and were maintained by water jackets at 20°C on both the adaxial and abaxial surfaces to ensure that the illuminated surface had exactly the same temperature as the unilluminated surface. All experiments were conducted at 50% relative humidity. The gas composition was controlled by gas mixers supplying CO2 at concentrations as indicated in the figures with 21% O2 and balance N2.
Attached last fully expanded leaves of the primary tiller of air- and CO2-grown plants were used to obtain CO2- and light-response curves for whole-leaf photosynthesis and photosynthesis on each leaf surface separately. Light was oriented directly to the adaxial surface or to the abaxial surface (by inverting the leaf in the chamber). For CO2-response curves, CO2 was increased step-wise from 50 to 1000 µl l−1 at an irradiance of between 900 and 1000 µmol m−2 s−1. Light-response curves for photosynthesis were obtained via step-wise increases in irradiance from darkness to 1500 µmol m−2 s−1 at 360 µl l−1 CO2. Steady-state gas-exchange measurements were obtained for each treatment after at least 10 min of incubation in each light and CO2 condition. Vapour water deficit was kept constant throughout the assay in both types of analysis.
Leaf optical properties
Leaf reflectance and transmittance measurements were performed on each surface of P. dilatatum plants grown at 350 µl l−1. Attached leaves of the same age and stage of development as those used for gas exchange were measured across the spectrum from 400 to 800 nm at 1-nm intervals. Five measurements were made on both sides of each leaf parallel to the middle vein and three leaves were assayed per experiment. Reflectance and transmittance were measured using reflection and irradiance integration spheres (Avasphere-30 reflection and irradiance integration spheres; Avantes, Eerbeek, the Netherlands) connected to a USB-2000 spectroradiometer (Ocean Optics, Dunedin, FL, USA). The measuring light was provided by a laboratory-built stabilized quartz-halogen source connected to a 0.6-mm optical fibre (type QP600-2-VIS-BX; Ocean Optics). In both cases the angle of incidence of the measuring beam was 8°. The 100% reflectance signal was obtained using a white reflectance standard (Spectralon; Labsphere, North Sutton, NH, USA). This was calibrated against a sheet of glass microfibre paper (Type GF/A; Whatman, Brentford, UK), which was used as a second standard. Leaf absorptance was calculated for each wavelength, as 1 – (reflectance + transmittance).
Determination of pigments and protein content
Leaf pigments (chlorophyll a (chla), chlb, total chlorophyll and carotenoids) and soluble protein contents were determined in the leaves of air- and high CO2-grown plants. Leaf sections (4 cm), excised from both sides of each leaf parallel to the middle vein, were ground in liquid nitrogen and quartz sand. Pigments were determined according to the method of Lichtenhaler & Wellburn (1983) in ethanol (96%) extracts. Total leaf soluble proteins were extracted in 1 mm sodium phosphate buffer (pH 7.0) containing 10 mm dithiothreitol (DTT), 1 mm phenylmethanesulphonylfluoride or phenylmethylsulphonyl fluoride (PMSF), 5 mm 2-mercaptoethanol and 1% (weight/volume (w/v)) Polyclar AT (Sigma-Aldrich, Saint Louis, MO, USA). Protein was determined according to the method of Bradford (1976).
Analysis of epidermal structure
The structure of the epidermis was examined in the leaves of air- and high CO2-grown plants using comparable sections to those used for photosynthesis measurements. Leaf sections were painted on the adaxial and abaxial surfaces with clear nail varnish. Epidermal imprints were then stripped from both surfaces and examined by optical microscopy (Olympus BH-2; Olympus Optical Co. Ltd, Tokyo, Japan). The number of stomata was counted on 24 randomly selected digitized images from the adaxial or the abaxial epidermal imprints of eight plants. To calculate epidermal and stomatal cell areas and densities, the dimensions of at least 100 cells from the adaxial or the abaxial epidermal layers were measured per experiment using Sigma ScanPro photographic analysis software, version 5 (Sigma Chemical Co., St Louis, MO, USA). The stomatal index was calculated as the number of stomata/(number of epidermal cells + number of stomata) × 100, as defined by Salisbury (1927). The ratio of stomata on each surface was calculated from the number of stomata counted on the adaxial and abaxial surfaces.
Fixation, embedding and sectioning for optical and electron microscopy
Leaf blade samples (1 mm2) were sectioned from the middle of the last fully expanded leaf of P. dilatatum plants grown at 350 µl l−1 CO2. Sections were fixed at ambient temperature in 4% (v/v) paraformaldehyde and 1% (v/v) glutaraldehyde in 0.2 m sodium phosphate buffer (pH 7.2) for 3.5 h. The samples were then washed in the same buffer three times for 15 min. They were then dehydrated at ambient temperatures using a graded ethanol series (35, 50, 70, 96 and twice at 100% (v/v) ethanol) with 30 min exposure at each step. The embedding was carried out in London Resin White acrylic resin (LRWhite; Electron Microscopy Sciences, Fort Washington, PA, USA) according to the following schedule: 75% (v/v) ethanol + 25% (v/v) LRWhite, 50% (v/v) ethanol + 50% (v/v) LRWhite, 25% (v/v) ethanol + 75% (v/v) LRWhite, 100% (v/v) LRWhite (1 h each series), and 100% (v/v) LRWhite overnight. The samples were transferred to tubes filled with resin and polymerized under nitrogen ambient at 55% for 24 h. Transverse semithin leaf sections (0.5 µm) for optical microscopy and transverse ultrathin leaf sections for electron microscopy (50–60 nm) were prepared using a Leica EM UC6 ultramicrotome (Leica Microsystems, Wetzlar, Germany). Optical microscopy sections were stained with toluidine blue. Electron microscopy sections were mounted on nickel grids and stained with lead citrate (Reynolds, 1963) and 2% (w/v) uranyl acetate. Optical microscopy sections were observed using a Leica DMR light microscopy (Leica Microsystems, Wetzlar, Germany).
In situ immunolocalization of ribulose 1,5-bisphosphate carboxylase/oxygenase and phosphoenolpyruvate carboxylase
The nickel grids were incubated at ambient temperature for 30 min in phosphate-buffered saline (PBS) containing 5% (w/v) bovine serum albumin (BSA). They were then incubated at room temperature for 3 h with either rabbit preimmune serum (dilution 1 : 500), rabbit anti-ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco) large subunit (Agrisera AB, Vännäs, Sweden) (dilution 1 : 250) or rabbit anti-phosphoenolpyruvate carboxylase (PEPC) (courtesy of Jean Vidal, Institut de Biotechnologie des Plantes, Centre National de la Recherche Scientifique, Université de Paris-Sud, France) (dilution 1 : 500), in the above PBS/BSA mixture. After washing twice with PBS (5 min each wash), the sections were incubated at ambient temperature for 1.5 h with goat anti-rabbit antibodies labelled with gold 10 nm (British Biocell International, Cardiff, UK) diluted in PBS containing 1% (w/v) BSA (dilution 1 : 50). The sections were washed sequentially with PBS containing 1% (w/v) BSA and then twice with PBS alone followed by five washes with filtered (0.2 µm) ultra-pure water (5 min each wash). The grids were dried at ambient temperature. Immunogold electron microscopy images were collected using a Philips Tecnai 12 electron microscope (Philips, The Hague, the Netherlands) operated at 80 kV. The number of gold particles on the adaxial and abaxial sides of the leaves and the parameters of ultrastructural morphology were quantified using the 3.2 image analysis software (Soft Imaging System, Münster, Germany). Quantification of gold labelling was performed on three leaf samples per experiment with a total of 45 different bundle sheath (BS) and mesophyll (M) cells measured in each analysis.
The data were statistically analysed using parametric tests at a stringency of P < 0.05. The significance of variation in mean values for growth parameters and pigment and protein determinations was determined using a t-test. The significance of the data for epidermal structure and immunological measurements was analysed using ANOVA and Tukey HSD tests.
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An accurate knowledge of the impacts of the environment on photosynthesis in pasture grasses such as P. dilatatum is crucial in any prediction of ecosystem viability and sustainability in the face of climate change. Here we show that P. dilatatum plants grew better and had double the total biomass under the high CO2 growth conditions. Paspalum dilatatum benefits from CO2 enrichment in terms of growth and biomass production, as observed in other C4 species (e.g. Ghannoum et al., 1997, 2001; Wand et al., 1999, 2001). These results are perhaps surprising given the widely accepted view that C4 plants might benefit less than their C3 counterparts from growth in a high CO2 world because of their intrinsic CO2-concentrating mechanisms (Bowes, 1993). However, the presence of the ATP-dependent CO2-concentrating systems in C4 species has probably already resulted in a substantial degree of acclimation of photosynthesis to a CO2-enriched state, at least in the BS cells. C4 species may therefore accommodate the large increases in carbon gain that result from CO2 enrichment better than C3 species. In the present study, the P. dilatatum plants grown at the higher CO2 concentration had decreased shoot:root ratios and showed other changes in whole-plant morphology parameters that are consistent with the acclimation of source–sink processes to CO2 enrichment and increased carbon gain (Ghannoum et al., 1997, 2001; Walting & Press, 1997).
Acclimation to CO2 enrichment often involves changes in leaf morphology, stomatal patterning and the structure of the leaf epidermis (Lake et al., 2001; Martin & Glover, 2007). Growth at the higher CO2 concentration increased the density of stomata in P. dilatatum leaves, particularly on the abaxial surface, as has been reported in Panicum antidotale (Tipping & Murray, 1999). However, the changes observed here in P. dilatatum were somewhat different from those observed previously in maize, which is also a C4 monocotyledonous NADP-malic enzyme species (Driscoll et al., 2006). Maize leaves grown with CO2 enrichment had fewer, much larger cells on both surfaces. Paspalum dilatatum leaves grown under similar conditions had a greater number of smaller stomata on both surfaces with a significant increase in epidermal cell numbers only on the abaxial surface. The nature of the responses of epidermal structure to CO2 enrichment is therefore species-specific. This is perhaps not surprising given the complex repertoire of environmental and developmental signals that influence cell size and cell number (Taylor et al., 1994, 2001; Masle, 2000; Ferris et al., 2001, 2002) as well as stomatal density and patterning (Larkin et al., 1997; Lake et al., 2002). However, stomatal densities were increased by high CO2 in the present study with P. dilatatum as in P. antidotale (Tipping & Murray, 1999), suggesting that CO2 is a less powerful negative regulator of stomatal density in Paspalum and Panicum than in Arabidopsis (Gray et al., 2000).
Like maize leaves (Domes, 1971; Driscoll et al., 2006), P. dilatatum leaves display a pronounced dorso-ventral asymmetry in the regulation of photosynthesis, each surface showing unique characteristic responses to available CO2 and light. The specific dorso-ventral regulation of photosynthesis with respect to light orientation to the adaxial or abaxial surface was observed at both growth CO2 concentrations. This regulation is not related to surface-dependent differences in light absorption, reflectance or transmission as the two leaf surfaces have the same optical properties. Light-harvesting complexes and photosystems are therefore equally distributed on the adaxial and abaxial sides of the leaf. While it is logical to assume that gradients in CO2 fixation rates that have been documented in dicotyledonous C3 leaves (Oya & Laisk, 1976; Terashima, 1986; DeLucia et al., 1991; Sun & Nishio, 2001; Evans & Vogelmann, 2003) are also present in monocotyledonous C4 leaves, there have been no comparative studies on C4 leaves that enable deductions to be made regarding the nature of possible mechanisms of acclimation to growth at high CO2. However, it is clear that the dorso-ventral gradients in photosynthetic machinery that determine adaxial/abaxial light absorption and CO2 fixation characteristics in C3 leaves must be entrained early in leaf development (Smith & Ullberg, 1989; Evans et al., 1993; Poulson & DeLucia, 1993; James & Bell, 2000; Ustin et al., 2001) as they can only be changed by inversion of the leaves at the onset of development (Terashima et al., 1986; Smith et al., 1997). Similar to the leaves of other monocotyledonous species, P. dilatatum leaves grow vertically from the sheath and then bend over so that the adaxial surface tends to be uppermost with the abaxial surface beneath. However, the leaves tend to curl as they expand so that the adaxial and abaxial surfaces can both experience orientation to the light in different sections of the same leaf.
The degree of stomatal opening was markedly affected by light orientation in P. dilatatum leaves. Abaxial illumination resulted in the closure of stomata on the adaxial surface, a response that was accompanied by a complete suppression of photosynthesis. The differential sensitivity of adaxial and abaxial stomata to light and the absence of CO2 transport between the two surfaces are important factors governing overall photosynthesis rates (Turner, 1970; Pospíšilová & Solárová, 1980). However, the complete suppression of photosynthesis on the adaxial surface when the stomata are closed is surprising given that this is a C4 species able to fix CO2 even with closed stomata, as a result of the presence of a high-affinity PEPC (Bauwe, 1986). Furthermore, these data show that there is little transfer of CO2 across the leaf as the abaxial stomata were open in these conditions.
Photosynthesis on the adaxial surface was much less responsive to low Ci values than that on the abaxial surface. The initial slopes of the CO2-response curves, which represent the maximal PEPC rate according to von Caemmerer (2000), indicate a higher maximal PEPC activity on the abaxial surface. The immunolocalization studies reported here show that there is a more or less uniform distribution of the carboxylating enzyme (Rubisco and PEPC) proteins across the P. dilatatum leaf. PEPC activation in response to increasing light intensities and other factors occurs via post-translational regulation involving an increase in the phosphorylation state of the enzyme protein (Chollet et al., 1996). Our data therefore strongly suggest that dorso-ventral asymmetric gradients operate in P. dilatatum leaves and that these greatly affect the photosynthetic capacity on each surface. We are drawn to the conclusion that the dorso-ventral gradient in photosynthesis in P. dilatatum leaves results from asymmetric enzyme activation across the leaf, which allows much higher activation on the abaxial surface.
Whole-leaf photosynthesis displayed a higher degree of flexibility with regard to light orientation when the P. dilatatum plants were grown at the higher CO2 concentration. The characteristic decreases in maximal CO2 assimilation rates observed with respect to Ci when the light was oriented to the abaxial surface were absent in plants grown at the higher CO2 concentration. Hence, CO2 enrichment caused a small but important adjustment in the dorso-ventral specification of photosynthesis. Light absorption profiles in Flaveria bidentis and maize have shown that, while green light is absorbed throughout the leaf, blue light is only strongly absorbed near the surface with little light penetrating the BS cells (J.R. Evans, T.C. Vogelmann & S. von Caemmerer, pers. comm.). However, given the similarity of internal structure on the adaxial and abaxial sides of monocotyledonous leaves, which have a bilateral symmetry, one would predict that light orientation would have similar levels of penetration from each surface (Moss, 1964; Syvertsen & Cunningham, 1979). Growth at the higher CO2 concentration had little effect on the overall photosynthetic capacity of P. dilatatum leaves, as observed previously (von Caemmerer et al., 2001). However, other studies using CO2 enrichment have yielded rather mixed results in different C4 species. In some studies, enhancement of photosynthesis was reported (LeCain & Morgan, 1998; Wand et al., 2001) but in others down-regulation of photosynthesis was observed (Greer et al., 1995; Ghannoum et al., 1997; Walting & Press, 1997). Here we show that growth at the higher CO2 concentration caused a small but important adjustment in the dorso-ventral specification of photosynthesis, an effect that was most apparent when light was oriented to the adaxial surface. These results indicate that metabolic and/or structural adjustments to CO2 enrichment have occurred within the leaves and that these facilitate the altered response to light orientation.
We conclude that the dorso-ventral specification in the regulation of photosynthesis and stomatal conductance in P. dilatatum arises from genetically programmed differences in stomatal sensitivity to light orientation coupled to a fixed gradient in enzyme activation, probably PEPC activation state. The results presented here together with those reported previously in maize (Driscoll et al., 2006) suggest that dorso-ventral asymmetry in the regulation of photosynthesis and stomatal conductance may be a common feature of monocotyledonous C4 species. The dorso-ventral asymmetry may have functional significance in situations that favour leaf rolling, for example drought avoidance, where such mechanisms would help to prevent water deficits while allowing high rates of photosynthesis.