Dorsoventral variations in dark chilling effects on photosynthesis and stomatal function in Paspalum dilatatum leaves

Abstract

The effects of dark chilling on the leaf-side-specific regulation of photosynthesis were characterized in the C₄ grass Paspalum dilatatum. CO₂- and light-response curves for photosynthesis and associated parameters were measured on whole leaves and on each leaf side independently under adaxial and abaxial illumination before and after plants were exposed to dark chilling for one or two consecutive nights. The stomata closed on the adaxial sides of the leaves under abaxial illumination and no CO₂ uptake could be detected on this surface. However, high rates of whole leaf photosynthesis were still observed because CO₂ assimilation rates were increased on the abaxial sides of the leaves under abaxial illumination. Under adaxial illumination both leaf surfaces contributed to the inhibition of whole leaf photosynthesis observed after one night of chilling. After two nights of chilling photosynthesis remained inhibited on the abaxial side of the leaf but the adaxial side had recovered, an effect related to increased maximal ribulose-1,5-bisphosphate carboxylation rates (V_cmax) and enhanced maximal electron transport rates (J_max). Under abaxial illumination, whole leaf photosynthesis was decreased only after the second night of chilling. The chilling-dependent inhibition of photosynthesis was located largely on the abaxial side of the leaf and was related to decreased V_cmax and J_max, but not to the maximal phosphoenolpyruvate carboxylase carboxylation rate (V_pmax). Each side of the leaf therefore exhibits a unique sensitivity to stress and recovery. Side-specific responses to stress are related to differences in the control of enzyme and photosynthetic electron transport activities.

Introduction

The understanding of plant responses to environmental stresses is crucial for models of plant behaviour in a changing climate. Plant growth and development are strongly influenced by the perception of signals from the environment. The perception of light quality and day length, for example, influences plant growth and architecture, flowering time, germination, and other processes. Similarly, nutrient availability, temperature, and interactions with other organisms also modify plant development. Exposure to low non-freezing (chilling) temperatures induces structural, developmental, physiological, cellular, and molecular alterations in plants (Lyons, 1973; Smallwood and Bowles, 2002; Sung et al., 2003) that are largely associated with altered membrane function (Lyons, 1973). Chilling also leads to the induction of a network of cold-responsive genes, which are considered to be responsible for the biochemical and physiological changes that allow plants to acclimate to a low temperature growth environment. Cold acclimation involves the regulated expression of effecter genes that are collectively called the COLD-REGULATED (COR) genes, whose function is to protect cell membranes and thereby prevent cellular dehydration. Low-temperature induction of the COR genes is regulated by CRT/CRE BINDING FACTOR (CBF) genes, whose expression is influenced by the plant circadian clock, which regulates the magnitude of the transcriptional activation of the CBF genes by low temperatures. For example, CBF expression is lower when chilling is experienced in the dark than in the light (Maruyama et al., 2004; Fowler et al., 2005). Moreover, chilling disrupts the circadian pattern of activity of some enzymes in leaves such as sucrose phosphate synthase and nitrate reductase, whose activation is delayed following exposure to chilling (Jones et al., 1998).

Persistent inhibition of photosynthesis occurs when plants are exposed to chilling temperatures in a diverse group of plant species. The extent of chilling-induced inhibition depends on the species, the sensitivity to chilling, the duration of the stress, and the light intensity experienced in the photoperiod following the stress. Photosynthesis is decreased following exposure to chilling temperatures even when the stress is applied in the dark in some C₃ (e.g. Flexas et al., 1999; van Heerden et al., 2003a; Feng and Cao, 2005) and C₄ (e.g. Ludlow and Wilson, 1971; Pasternak and Wilson, 1972; Pittermann and Sage, 2001) species. Dark-chilling-induced limitations to stomatal function have been reported in C₃ and C₄ plants, and these contribute to the observed inhibition of photosynthesis rates at least in C₃ species (e.g. Pasternak and Wilson, 1972; Martin et al., 1981; Bauer et al., 1985; van Heerden et al., 2003a; Feng and Cao, 2005). Non-stomatal limitations on whole leaf CO₂ assimilation have also been reported after dark chilling in both C₃ and C₄ plants. For example, direct effects on electron transport (Shen et al., 1990; van Heerden et al., 2003b; Bertamini et al., 2006; Strauss et al., 2007) and the activities of photosynthetic enzymes (Jones et al., 1998; Jun et al., 2001; Pittermann and Sage, 2001; van Heerden et al., 2003a, b; Zhou et al., 2004) have been observed.

The dorsoventral regulation of photosynthesis is well characterized in dicotyledonous C₃ plants and is related to the presence of two types of mesophyll cell and a continuous airspace system that provides gaseous continuity across the leaf blade (Terashima and Inoue, 1985a,b; Nishio et al., 1993; Evans, 1995; Sun et al., 1998; Sun and Nishio, 2001; Evans and Vogelmann, 2003). Unlike dicotyledonous C₃ plants, the adaxial and abaxial sides of C₄ monocotyledonous leaves like maize comprise essentially two separate compartments as there are physical dorsoventral restrictions to airspace continuity (Long et al., 1989). Moreover, the regulation of photosynthesis is different on each side of C₄ monocotyledonous leaves that belong to the NADP-malic enzyme (NADP-ME) subtype such as Paspalum dilatatum (Soares et al., 2008; Soares-Cordeiro et al., 2009) and maize (Long et al., 1989; Driscoll et al., 2006; Soares-Cordeiro et al., 2009). Marked differences in the leaf-side-specific responses of photosynthesis and stomatal closure to light orientation are observed in such C₄ monocotyledonous leaves. This led us to propose that each side of such leaves has independent signalling pathways associated with light perception, photosynthesis, and stomatal closure (Soares-Cordeiro et al., 2009).

Amphistomatous leaves from dicotyledonous and monocotyledonous C₃ and C₄ species can exhibit differential regulation of stomatal closure on the adaxial and abaxial surfaces. Generally, the stomata on the adaxial surface are considered to be less sensitive to light intensity (Turner, 1970; Pemadasa, 1979; Travis and Mansfield, 1981; Goh et al., 1995; Soares et al., 2008), as well as to water stress conditions (Turner and Singh, 1984; Lu, 1988; Wang et al., 1998). However, stomatal conductance was shown to be more affected on the adaxial surface in the dicotyledonous C₃ species Rumex obtusifolius grown under CO₂ enrichment (Pearson et al., 1995). Side-specific differences in stomatal movement have been related to variations in calcium ion concentration in the guard cells (De Silva et al., 1986), which are in turn regulated by the metabolic and signalling activities of the adjacent mesophyll cells in response to changes in CO₂ or light availability (Mott et al., 2008). Recent evidence suggests, however, that photosynthesis does not contribute directly to the signalling network that regulates stomatal opening (von Caemmerer et al., 2004; Baroli et al., 2008).

The presence of a two-compartment structure in C₄ monocotyledonous leaves with little or no apparent airspace continuity between the upper and lower sides, would allow separate dorsoventral systemic stress signalling pathways. The hypothesis that was tested in the present study was that the leaves of C₄ monocotyledonous species such as P. dilatatum (Soares et al., 2008; Soares-Cordeiro et al., 2009) might exhibit differential responses and acclimation to chilling on the adaxial and abaxial sides of the leaf. The two-airspace system may thus be useful not only in the optimization of whole leaf photosynthesis but also in the regulation of surface-specific stress defence-signalling cascades. In the following experiments therefore we have explored the possibility that each side of P. dilatatum leaves responds independently to the stress imposed by dark chilling.

To date, relatively few studies that focus on enhancing our current understanding of how the side-specific regulation of photosynthesis and stomatal movements is altered in response to environmental stress have been performed. Studies on CO₂ enrichment in maize (Driscoll et al., 2006) and P. dilatatum (Soares et al., 2008) revealed that each surface of the leaf responded differently to altered CO₂ availability. While photosynthesis on the abaxial surfaces was largely unchanged in the leaves of these two C₄ NADP-ME species when plants were acclimated to elevated growth CO₂ levels, CO₂ assimilation rates were decreased on the adaxial surfaces. No information is available in the literature on the effects of chilling on the surface-specific regulation of photosynthesis and stomatal conductance values in monocotyledonous C₄ species. The present study was therefore undertaken to characterize the effects of dark chilling on photosynthesis and stomatal function on each side of P. dilatatum leaves and to determine whether chilling stress results in side-specific regulation in this C₄ grass species, which is partially tolerant to the stress imposed by dark chilling (Cavaco, 2000).

Materials and methods

Plant material

Paspalum dilatatum seeds were germinated and grown for 6 weeks in controlled environment cabinets (Sanyo SGC228.CFX.J; Sanyo, Osaka, Japan) as described previously (Driscoll et al., 2006; Soares et al., 2008; Soares-Cordeiro et al., 2009). The controlled environment cabinets provide a constant CO₂ concentration of 350±20 μl l⁻¹ (Eurotherm Ltd, Worthing, UK), 80% relative humidity, a temperature of 25 °C in the day and 19 °C at night, with a 16 h photoperiod at an irradiance of 600–650 μmol m⁻² s⁻¹ at pot height (400–700 nm). Irradiance was provided by Phillips Master TL5 HO 49w/830 fluorescent lamps. Plants were watered daily throughout the period of the experiments.

Dark chilling treatments and sampling

For the dark chilling treatments plants were transferred to a controlled growth chamber for one or two consecutive nights. The conditions in the controlled growth chamber were as above, except that the night temperatures were maintained at 4 °C. Leaf pigment and soluble protein determinations and gas-exchange measurements were performed on the middle (widest) part of the youngest fully expanded leaves in the subsequent light periods after one and two consecutive nights of dark chilling. Leaf harvest and measurements were performed between 1 and 5 h after the beginning of the light period.

Determination of leaf pigments and soluble protein

Leaf pigments [chlorophyll a (chla), chlb, total chlorophyll, and carotenoids] and soluble protein content were determined in the middle sections of the youngest fully expanded leaves of plants that had been either maintained at optimal growth temperatures or subjected to one or two nights of dark chilling, as above. All methods used were essentially as described previously (Soares et al., 2008; Soares-Cordeiro et al., 2009). Leaf samples were ground in liquid nitrogen and quartz sand. Pigments were determined according to the method of Lichtenhaler and Wellburn (1983) after extraction in 96% ethanol (v/v). Soluble proteins were extracted in sodium phosphate buffer as described by Soares et al. (2008). Total soluble protein content was determined according to the method of Bradford (1976).

Gas-exchange measurements

Photosynthetic gas-exchange measurements were performed using an infrared gas analyser (model wa-225-mk3; ADC, Hoddesdon, UK) either on whole leaves using custom-made standard Parkinson-type whole leaf chambers designed for CO₂ and water vapour analysis (Novitskaya et al., 2002) or on each leaf surface separately using custom-made dual Parkinson-type chambers designed for CO₂ and water vapour analysis (Soares et al., 2008; Soares-Cordeiro et al., 2009).

Gas-exchange measurements were performed on the middle sections of the youngest fully expanded attached leaves of plants that had been either maintained at optimal growth temperatures or subjected to one or two nights of dark chilling. Light was oriented either to the adaxial surface or to the abaxial surface, by inverting the leaves in the chambers. Since P. dilatatum leaves are not large enough to fully separate the two sides of the dual chamber, the leaves were expanded with gas-tight tape to seal each half of the chamber, preventing any flux of gas between the two sides as previously described by Soares et al. (2008) and Soares-Cordeiro et al. (2009).

Three types of measurement were made on the attached leaves of plants that had been either maintained at optimal growth temperatures or subjected to one or two nights of dark chilling: (i) single measurements of gas-exchange parameters on the whole leaves and on each leaf surface separately at an ambient CO₂ concentration of 350 μl l⁻¹ and at an irradiance of between 900 and 1000 μmol m⁻² s⁻¹; (ii) CO₂-response curves for photosynthesis were performed on whole leaves and on each side of the leaf separately at an irradiance between 900 and 1000 μmol m⁻² s⁻¹. In these experiments, the CO₂ concentration within the chambers was increased in a stepwise manner from 50 to 1000 μl l⁻¹, and (iii) light-response curves for photosynthesis were performed on whole leaves and on each side of the leaf separately at an ambient CO₂ concentration of 350 μl l⁻¹. In these experiments, the light intensity was increased stepwise from darkness to 1500 μmol m⁻² s⁻¹.

The whole leaf response curves for photosynthesis allow us to model some of the key parameters of C₄ photosynthesis, using the C₄ photosynthetic models described by von Caemmerer (2000). Such C₄ photosynthetic models have only been described for whole leaf photosynthesis. It was therefore not possible to use the leaf-surface-specific data obtained from the CO₂- and light-response curves for photosynthesis to determine the various C₄ photosynthetic parameters on each side of the leaf. In addition, we consider that the interpretation of the maximal electron transport rate and maximal photosynthetic rate values may not be trivial using values obtained for each leaf surface (S. von Caemmerer, personal communication) because we have not determined the exact amount of light that reaches the side of the leaf that is not under direct actinic illumination.

Steady-state rates for CO₂ assimilation and stomatal conductance were determined at a leaf temperature of 20 °C. This temperature was maintained constant for both whole leaf and side-specific measurements of parameters on the adaxial and abaxial surfaces, by water jackets as previously described by Soares et al. (2008). Measurements were conducted at 50% relative humidity and the irradiance was provided by overhead broad-spectrum lamps and supplied only from the top of the chamber both for the whole leaf measurements and for each leaf surface measurement (Soares et al., 2008). Data presented for whole leaf and leaf-surface-specific parameters are the mean values obtained from leaves of nine plants per measurement that were analysed on different batches of plants grown over the whole experimental period.

Measurements of the internal resistance to gas diffusion

Ambient CO₂ concentration, intercellular CO₂ concentration, and stomatal conductance values were determined on the adaxial and abaxial surfaces of P. dilatatum leaves as follows. The gas flow and pressure were maintained equal and constant on each side of the leaf but a differential ambient CO₂ concentration was applied across the leaves such that one side was maintained at 2500 μl l⁻¹ CO₂ while the other received 0 μl l⁻¹ CO₂. Light was oriented to the adaxial leaf surface in all measurements at an intensity of 550 μmol m⁻² s⁻¹. The leaf temperature was maintained at 20 °C with a relative humidity of 50%.

C₄ photosynthetic model predictions for whole leaf photosynthesis

CO₂-response curves:

Whole leaf CO₂-response curves for photosynthesis were used to calculate C₄ photosynthetic parameters, according to the whole leaf C₄ photosynthetic model of von Caemmerer and Furbank (1999) and as described in detail by von Caemmerer (2000). These models are based on the earlier models of Berry and Farquhar (1978) and Peisker (1979). This model allows the prediction of the maximal phosphoenolpyruvate carboxylase (PEPC) carboxylation rate (V_pmax) and maximal ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) carboxylation rate (V_cmax) using enzyme-limited photosynthesis equations and the maximal electron transport rate (J_max) using light- and electron-transport-limited photosynthesis equations. The model requires that a saturating light intensity is used to perform the measurements. We thus had to achieve a compromise with regard to the light intensity in order to avoid photoinhibition in the light period following the treatments when the plants were dark-chilled. While the light intensity (900–1000 μmol m⁻² s⁻¹) used in the present experiments was not fully saturating for photosynthesis according to the data shown in Fig. 4, it is nearly saturating while low enough to prevent photoinhibition in plants that had experienced dark chilling. Nevertheless, it is important to note that while the values obtained through the application of the C₄ photosynthetic model in this study cannot be considered to be the absolute maximal values, they can accurately be used to compare the effects of dark chilling and light orientation on the leaf.

Fig. 4.

The effect of light orientation and dark chilling on the light-response curves for photosynthesis in whole P. dilatatum leaves. Plants were either maintained at optimal growth temperatures (black circles) or subjected to dark chilling for either one (light grey inverted triangles) or two (dark grey squares) nights. Light intensity was increased in a stepwise manner from 0 to 1500 μmol m⁻² s⁻¹ with orientation to either the adaxial (A) or the abaxial (B) leaf surface. Data are the mean values±SE of nine plants per treatment.

In addition, the C₄ photosynthetic model requires the use of some parameters measured at 25 °C (von Caemmerer, 2000). Some parameters are dependent on temperature and thus it was important to calculate the values appropriate to the temperatures at which the present curves were performed (20 °C). This is the case for the Michaelis–Menten constants of Rubisco for CO₂ (K_c) and for O₂ (K_o), and the Michaelis–Menten constant of PEPC for CO₂ (K_p). An Arrhenius function was used to transform the K_c, K_o, and K_p values to 20 °C, (Badger and Collatz, 1977):

where Q₁₀ is the temperature coefficient that represents the factor by which the rate of a reaction increases for every 10 °C rise in the temperature. The tabled values of K_c and K_o at 25 °C (650 μbar and 450 mbar, respectively) and the Q₁₀ values for K_c and K_o (2.24 and 1.63, respectively) (von Caemmerer, 2000) were used to calculate the K_c and K_o at 20 °C. The K_p value tabled at 25 °C (80 μbar) (von Caemmerer, 2000) and the Q₁₀ value for K_p of 1.603, calculated from the PEPC activation energy value (34.8 kJ mol⁻¹) obtained for P. dilatatum plants at temperatures >20 °C (Cavaco, 2000), were used to calculate the K_p value at 20 °C. The parameters and the values used to apply the photosynthetic model are described in Table 1.

Table 1.

Parameters used for modelling C₄ photosynthesis based on the CO₂-response curves for photosynthesis (von Caemmerer, 2000)

Parameter	Value	Description
Kc	0.973 mbar	Michaelis–Menten constant of Rubisco for CO2 (variable with temperature, corrected to 20 °C)
Ko	575 mbar	Michaelis–Menten constant of Rubisco for O2 (variable with temperature, corrected to 20 °C)
Kp	101 μmol mol−1	Michaelis–Menten constant of PEPC for CO2 (variable with temperature, corrected to 20 °C)
O	21%	O2 partial pressure in the bundle sheath and mesophyll cells
gbs	3 mmol m−2 s−1	Bundle-sheath conductance to CO2
gi	2 mol m−2 s−1	Mesophyll conductance to CO2
Rd	0.01*Vcmax	Mitochondrial respiration
Rm	0.5*Rd	Mesophyll mitochondrial respiration
γ*	0.000193	Half the reciprocal of Rubisco speficity
X	0.4	Partitioning factor of electron transport rate
θ	0.7	Empirical curvature factor
F	0.15	Factor that corrects for spectral quality of light
abs	0.85	Absorptance of leaves
I	1000 μmol m−2 s−1	Irradiance used
Ia	361.2 μmol m−2 s−1	Total absorbed irradiance

A similar approach to that described by Massad et al. (2007) was used to estimate V_pmax, V_cmax, and J_max. First, an asymptotic exponential model was selected as it provides the best description of the variation in CO₂ assimilation rates with intercellular CO₂ concentration (C_i) for each individual plant according to the least squares method using the program Statistical Package for Social Sciences (SPSS) (version 15.0, 2006; SPSS Inc., Chicago, IL, USA).

The equations that were used to determine enzyme-limited photosynthesis and light- and electron-transport-limited photosynthesis (as shown in Table 2) were applied using the Mathematica version 6.0 (2007) software (Wolfram Research Inc., Champaign, IL, USA). Data obtained from each individual plant were analysed in the range 0–500 μl l⁻¹, in a stepwise manner (i.e. per 5 μl l⁻¹ C_i increase) in order to determine V_pmax, V_cmax, and J_max values at each C_i. The estimated values of V_pmax, V_cmax, and J_max were then plotted against C_i. The mean values were then determined for each individual plant in the ranges 50–80 μl l⁻¹ C_i for V_pmax and 300–500 μl l⁻¹ C_i for V_cmax and J_max. The values obtained for each plant were then used to determine the mean values for V_pmax, V_cmax, and J_max for each treatment. Values for mitochondrial respiration and mesophyll mitochondrial respiration were then calculated from the V_pmax and V_cmax data according to von Caemmerer (2000).

Table 2.

Equations for enzyme-limited photosynthesis and for light- and electron-transport-limited photosynthesis used to predict the maximal PEPC carboxylation rate (V_pmax), maximal Rubisco carboxylation rate (V_cmax), and the maximal electron transport rate (J_max) from the CO₂-response curves of whole leaf photosynthesis according to von Caemmerer (2000)

Parameter	Equations
Vpmax and Vcmax	Equations for enzyme-limited photosynthesis:
Jmax	Equations for light- and electron-transported-limited photosynthesis:
	Equation characterizing the relationship between electron transport rates and absorbed irradiance:

See Table 1 for all abbreviations except A (CO₂ assimilation rate), C_i (intercellular CO₂ concentration), C_m (mesophyll cells CO₂ concentration), C_s (bundle sheath cells CO₂ concentration), and J_t (predicted electron transport rate).

Light-response curves:

Light-response curves for photosynthesis in whole leaves were analysed (Statistica 8.0, 2007; StatSoft, Inc., OK, USA) to determine the apparent quantum yield (ϕ), curvature degree (θ), maximal rate of photosynthesis (A_max), and mitochondrial respiration (R_d), using the equations below as described by von Caemmerer (2000) and Lambers et al. (1998):

where, A is the CO₂ assimilation rate, and I the irradiance used.

Each parameter was calculated for each individual curve and then the mean values were obtained for each dark chilling and light orientation treatment. The C₄ photosynthetic light-response curve model requires that analysis is performed under conditions of saturating CO₂ concentrations. In the present study, the light-response curves for photosynthesis were performed at 350 μl l⁻¹ CO₂ (corresponding to a C_i value of ∼120–200 μl l⁻¹), which is a saturating CO₂ concentration (according to Fig. 3). The predicted ϕ, θ, A_max, and R_d values determined from the model are therefore considered to be accurate.

Fig. 3.

The effect of light orientation and dark chilling on the CO₂-response curves for photosynthesis in whole P. dilatatum leaves. The light-response curves for photosynthesis were performed at 350 μl l⁻¹ CO₂ (corresponding to a C_i value of ∼120–200 μl l⁻¹), which is a saturating CO₂ concentration. Plants were either maintained at optimal growth temperatures (black circles), or subjected to dark chilling for either one (light grey inverted triangles) or two (dark grey squares) nights. The light intensity was orientated either to the adaxial (A) or the abaxial (B) leaf surface. Data are the mean±SE of nine plants per treatment.

Statistical analysis

Statistical analysis of the data was performed using parametric tests at a stringency of P<0.05. The significance of variation for mean values regarding pigment, protein, single gas-exchange measurements obtained for whole leaves and for each leaf side separately and for whole leaf photosynthetic model determinations was analysed using an ANOVA followed by a Tukey HSD test. All the analyses were performed separately for each parameter.

Results

Effects of dark chilling stress on leaf pigments and protein

The leaves of plants that had been subjected to a single night of dark chilling showed a decrease in the content of both chla and chlb. However, the chla/chlb ratio was increased because the chilling-induced decrease (19%) in chlb was higher than that (9%) in chla (Table 3). After a second night of chilling the decrease in chla and chlb was of a similar magnitude (13% and 14%, respectively). Thus, the ratio chla/chlb was not significantly different from that of the control plants that had been maintained under optimal temperatures. Leaf carotenoid content decreased significantly after the second consecutive night of chilling (Table 3) but the ratio of chla plus chlb to carotenoids was unaffected by dark chilling. The amount of soluble protein in the leaves was not affected by the chilling treatments (Table 3).

Table 3.

Effects of dark chilling on leaf pigment and protein contents. Plants were either maintained at optimal growth temperatures (controls) or were subjected to dark chilling for either one or two consecutive nights

Data represent the mean values ± SE of seven leaves per treatment. Statistical analysis was performed separately for each parameter. The different letters represent statistical differences at P<0.05. Chl, chlorophyll.

	Control	1 Night Chilling	2 Nights Chilling
Chla (mg m-2)	572 ± 15.7 a	518 ± 14.7 b	499 ± 4.9 b
Chlb (mg m-2)	156 ± 4.9 a	127 ± 4.3 b	135 ± 2.0 b
Chla+b (mg m-2)	727 ± 2.0 a	645 ± 18.9 b	634 ± 5.7 b
Carotenoids (mg m-2)	114 ± 3.3 a	108 ± 2.6 b,a	100 ± 1.4 b
Chla/Chl b ratio	3.7 ± 0.06 a	4.1 ± 0.04 b	3.7 ± 0.05 a,b
Chla+b/Carotenoid ratio	6.4 ± 0.07 a,b	6.0 ± 0.06 a	6.3 ± 0.10 a,b
Soluble proteins (mg m-2)	3084 ± 148.9 a	2773 ± 65.3 a	2797 ± 78.8 a

Effects of dark chilling on whole leaf photosynthesis and stomatal conductance

Whole leaf photosynthesis rates at ambient CO₂ and a light intensity of 900–1000 μmol m⁻² s⁻¹ were 14% lower when CO₂ assimilation was measured with light oriented to the abaxial surface (Fig. 1A). Whole leaf CO₂ assimilation rates measured under adaxial illumination were 32% and 14% lower after, respectively, one and two nights exposure to dark chilling compared with controls that had been maintained under optimal temperatures. Photosynthesis was only decreased under abaxial illumination after two nights of chilling and then only by 20%. Stomatal conductance values were higher in control plants under abaxial illumination than under adaxial illumination (Fig. 1B). No effects of dark chilling on the stomatal conductance were detected either under adaxial illumination after one or two nights of dark chilling or under abaxial illumination after one night of chilling. However, a 40% decrease in stomatal conductance was observed under abaxial illumination after two nights of stress (Fig. 1B).

Fig. 1.

The effect of light orientation and dark chilling on whole leaf photosynthesis (A) and stomatal conductance (B) in P. dilatatum leaves. Plants were either maintained at optimal growth temperature (controls), or subjected to dark chilling for either one or two nights. Light was oriented to either the adaxial (closed bars) or the abaxial (open bars) leaf surface. Data are the mean±SE of nine plants per treatment with each gas-exchange parameter measured separately. The different letters represent significant differences (P<0.05).

Effects of dark chilling on photosynthesis and stomatal conductance on each leaf surface

The dark chilling treatments affected CO₂ assimilation differently on each leaf surface under adaxial illumination. The CO₂ assimilation rates on each leaf surface were similar under adaxial illumination in control plants (Fig. 2A). Under the same light condition, a single night of exposure to the chilling stress was sufficient to decrease the photosynthetic rates by half. However, photosynthesis rates recovered on the adaxial surface, but not on the abaxial surface after two nights of chilling in these conditions.

Fig. 2.

The effect of light orientation and dark chilling on leaf-side-specific rates of photosynthesis (A) and stomatal conductance (B) in P. dilatatum leaves. Plants were either maintained at optimal growth temperatures (controls), or subjected to dark chilling for either one or two nights. CO₂-assimilation rates (A, C) and stomatal conductance (B, D) were determined on the adaxial (closed bars) and the abaxial (open bars) leaf surfaces. Light was oriented to either the adaxial (A, B) or the abaxial (C, D) leaf surface. Data are the mean values±SE of nine plants per treatment with each gas-exchange parameter measured separately. The different letters represent significant differences (P<0.05) and * indicates values below the levels of detection.

Photosynthesis on the adaxial surface was below the level of detection under abaxial illumination. However, the abaxial surface had increased CO₂ assimilation rates compared with the values obtained under adaxial illumination (Fig. 2A, C). In contrast to photosynthesis under adaxial illumination, CO₂ assimilation rates on the abaxial surface were unaffected by one night of chilling, but they were decreased (21%) after two consecutive nights of chilling (Fig. 2C). Dark chilling had similar effects on stomatal conductance under adaxial (Fig. 2B) and abaxial illumination (Fig. 2D).

Effects of dark chilling on the whole leaf CO₂-response curves for photosynthesis

Whole leaf CO₂ assimilation rates increased with C_i under all conditions irrespective of the light orientation (Fig. 3). The steady-state rates of photosynthesis were similar after one night of chilling when light was oriented either to the adaxial or the abaxial surface of the leaves. However, steady-state rates of photosynthesis in the control leaves maintained under optimal growth temperatures and those of plants that had experienced two nights of dark chilling were highest when light was oriented to the adaxial surface (Fig. 3A). The initial slopes of the CO₂-response curves for photosynthesis were similar in control and chilled leaves regardless of light orientation. However, chilling had different effects on the steady-state rates of photosynthesis measured under adaxial or abaxial illumination. Photosynthesis rates were decreased under adaxial illumination after the plants had been chilled for one night (Fig. 3A). However, a partial recovery was observed after the second chilling treatment (Fig. 3A). In contrast, a second night of chilling led to a greater inhibition of photosynthesis when measured under abaxial illumination (Fig. 3B). These data indicate that the adaxial sides of the leaves are better able to recover from chilling-induced inhibition of photosynthesis than the abaxial sides.

Effects of dark chilling on the whole leaf light-response curves for photosynthesis

Under saturating light intensities (900–1000 μmol m⁻² s⁻¹) photosynthesis rates were significantly lower when light was oriented to the abaxial surface (Fig. 1). However, this inhibition was not observed under lower light intensities (Fig. 4). Thus, the kinetics of the light-response curves for photosynthesis were largely unaffected by dark chilling under either adaxial or abaxial illumination (Fig. 4). Only the light-saturated rate was decreased (Figs 1, 4).

Effects of dark chilling on calculated photosynthetic response curve parameters

The orientation of the leaf to light has effects on whole leaf V_pmax, V_cmax, and J_max values. In plants maintained at optimal growth temperatures, these parameters were 48%, 19%, and 22% lower, respectively, under abaxial relative to adaxial illumination (Table 4). However, the estimated values for the whole leaf A_max, ϕ, θ, and R_d were not affected by light orientation at optimal growth temperatures (Table 5).

Table 4.

The effects of light orientation and dark chilling on whole leaf maximal phosphoenolpyruvate carboxylase (PEPC) carboxylation rates (Vpmax), maximal ribulose-1,5-bisphosphate (Rubisco) carboxylation rates (Vcmax) and maximal electron transport rates (Jmax). Plants were either maintained at optimal growth temperatures (controls) or subjected to dark chilling for either one or two consecutive nights

Data represent the mean values ± SE of nine plants per treatment and were calculated following the application of the C₄ photosynthetic model of von Caemmerer (2000) to the whole leaf CO₂-response curves (See Fig. 3). Statistical analysis was performed separately for each parameter. The different letters represent statistical differences at P<0.05.

	Vpmax(μmol m-2 s-1)	Vcmax(μmol m-2 s-1)	Jmax(μmol m-2 s-1)
Light Adaxial
Control	111 ± 4.3 a	32 ± 1.0 a	214 ± 15.6 a
1 Night Chilling	52 ± 1.4 c	22 ± 0.7 c	144 ± 11.3 b
2 Nights Chilling	63 ± 1.0 b,c	28 ± 1.1 a,b	183 ± 10.2 a,b
Light Abaxial
Control	58 ± 2.9 b,c	26 ± 0.5 b	167 ± 1.6 b
1 Night Chilling	67 ± 4.8 b	24 ± 0.8 b,c	152 ± 6.4 b,c
2 Nights Chilling	66 ± 2.5 b,c	22 ± 1.1 c	148 ± 3.5 c

Table 5.

The effect of light orientation and dark chilling on whole leaf maximal rate of photosynthesis (Amax), apparent quantum yield (φ), curvature degree (θ) and mitochondrial respiration (R_d) in plants that had been maintained at optimal growth temperatures (controls) or subjected to dark chilling for either one or two consecutive nights

Data represent the mean values ± SE of nine plants per treatment and was calculated accordingly to von Caemmerer (2000) and Lambers et al. (1998) from the whole leaf light-response curves (See Fig. 4). Statistical analysis was performed separately for each parameter. The different letters represent statistical differences at P<0.05.

	Amax(μmol m-2 s-1)	ϕ(*102 μmol μmol-1)	θ(relative units)	Rd(μmol m-2 s-1)
Light Adaxial
Control	41 ± 2.4 a	5.7 ± 0.58 a	0.64 ± 0.045 a	1.9 ± 0.06 a
1 Night Chilling	26 ± 0.6 b	4.6 ± 0.66 a	0.82 ± 0.044 a	1.8 ± 0.08 a
2 Nights Chilling	39 ± 2.7 a	6.5 ± 0.23 a	0.69 ± 0.090 a	1.9 ± 0.13 a
Light Abaxial
Control	43 ± 1.7 a	4.2 ± 0.40 a	0.64 ± 0.073 a	1.6 ± 0.05 a
1 Night Chilling	37 ± 1.0 a	5.4 ± 0.12 a	0.61 ± 0.035 a	1.6 ± 0.03 a
2 Nights Chilling	37 ± 1.0 a	4.6 ± 0.50 a	0.64 ± 0.073 a	1.7 ± 0.07 a

One night of chilling decreased the V_pmax, V_cmax, J_max, and A_max values by between 30 and 50% when light was oriented to the adaxial leaf surface (Tables 4, 5). These parameters largely recovered to control values after the second night of chilling, except for V_pmax, which showed values similar to those measured after one night of dark chilling (∼45% decrease) (Table 4). The V_pmax and A_max values were similar in control and dark-chilled plants when the light was oriented to the abaxial surface (Tables 4, 5). Under abaxial illumination the estimated values for V_cmax and J_max decreased by 15% and 11%, respectively, but only after two nights of chilling (Table 4). Dark chilling and light orientation had no effects on ϕ, θ, and R_d (Table 5).

CO₂ diffusion between the adaxial and abaxial sides of the leaf

The diffusion of CO₂ between the adaxial and abaxial sides of the P. dilatatum leaves was measured in plants maintained at optimal growth temperatures (Fig. 5). When the adaxial side of the leaf was exposed to high atmospheric CO₂ levels with zero amounts on the abaxial side of the leaf (Fig. 5A), C_i values were below the level of detection on the abaxial surface (Fig. 5B) despite the stomata being fully open on that surface (Fig. 5C). Conversely, when the abaxial side of the leaf was exposed to high atmospheric CO₂ levels with zero amounts on the adaxial side of the leaf (Fig. 5D), C_i values were below the level of detection on the adaxial side (Fig. 5E) despite the stoma being fully open on that surface (Fig. 5F).

Fig. 5.

Internal resistance to CO₂ diffusion across P. dilatatum leaves. Ambient CO₂ concentrations (C_a; A, D), intercellular CO₂ concentrations (C_i; B, E), and stomatal conductance values (C, F) were determined on the adaxial (dark bars) and abaxial (white bars) surfaces of attached P. dilatatum leaves, on plants that were maintained at optimal growth temperatures. The ambient CO₂ concentration was maintained either at 2500 μl l⁻¹ on the adaxial surface and 0 μl l⁻¹ on the abaxial surface (A–C) or 2500 μl l⁻¹ on the abaxial surface and 0 μl l⁻¹ on the adaxial surface (D–F). Light was oriented to the adaxial leaf surface. Data are the mean values±SE of four plants per treatment. The different letters represent significant differences (P<0.05) and * indicates values below the level of detection.

Discussion

Plant responses to environmental stresses are important in determining overall productivity and crop yields. The decreases in leaf protein and chlorophyll following short-term exposure to low temperatures observed in the leaves of P. dilatatum, which is nominally regarded as semi-tolerant to dark chilling (Cavaco et al., 2003), are consistent with previous observations in chilling-sensitive plants (Lyons, 1973; Slack et al., 1974; Forde et al., 1975; Haldimann et al., 1995; Nie et al., 1995; Haldimann, 1996; Cavaco et al., 2003; Naidu et al., 2003; Strauss et al., 2007). More cold-tolerant C₄ species such as Miscanthus show a greater degree of cold acclimation capacity (Naidu et al., 2003). The information provided here on the chilling-dependent inhibition of photosynthesis in P. dilatatum leaves, together with observations concerning the differential sensitivities of each leaf surface to chilling-induced inhibition, advances our current understanding of how exposure to stress influences the dorsoventral regulation of photosynthesis and allows us to draw the following conclusions.

(i) Light orientation modifies whole leaf responses to dark chilling:

Under optimal growth conditions whole leaf photosynthesis was decreased under abaxial illumination compared with adaxial illumination (Fig. 1A). Moreover, differences in the calculated V_pmax, V_cmax, and J_max values (Table 4) are related to the lower photosynthesis rates on the adaxial surface under abaxial illumination (Fig. 2C), in agreement with our earlier observations on this and other monocotyledonous C₄ NADP-ME species (Soares et al., 2008; Soares-Cordeiro et al., 2009). Under abaxial illumination, the stomata on the adaxial surface of the leaf were tightly closed and thus it is not possible to determine CO₂ exchange for the adaxial surface. Under these conditions the abaxial surface alone contributes to detectable CO₂ assimilation rates. One possible reason for these differences is that the abaxial surface is more limited by light absorption under adaxial illumination. Under most circumstances the lower side of the leaf would experience much lower light levels than the upper surface (Soares et al., 2008). While side-specific differences in the capacity for light absorption have not been detected to date (Soares et al., 2008), there may be subtle variations that cannot be determined by simple whole leaf measurements. Moreover, the sensitivities of enzymes such as PEPC might be different on the two leaf surfaces.

When the abaxial surface was directly illuminated, the limitation by light availability was diminished and thus there was a marked increase in CO₂ assimilation rates. The rates of photosynthesis on the abaxial surface under abaxial illumination were much higher than those obtained on the adaxial surface under adaxial illumination. This is presumably because the abaxial surface has a higher capacity to absorb light and to assimilate CO₂.

The results presented here show that the responses of whole leaf photosynthesis to dark chilling were modified by light orientation (Fig. 1A, B). This result is similar to that obtained previously with P. dilatatum plants, in response to another environmental stress, i.e. growth with CO₂ enrichment (Soares et al., 2008). It has long been known that dark chilling decreases photosynthesis under adaxial illumination in chilling-sensitive species (e.g. Ludlow and Wilson, 1971; Ivory and Whiteman, 1978; van Heerden et al., 2003a; Feng and Cao, 2005). Chilling-induced inhibition of photosynthesis observed here in P. dilatatum leaves under adaxial illumination after one night of chilling is thus in agreement with previous observations. However, in photosynthesis recovered following a second chilling treatment (Fig. 1A), reflecting the modest chilling sensitivity of this species. The recovery of photosynthesis after the second night of stress, demonstrated by the values for V_cmax and J_max (Table 4), is not observed in many chilling-sensitive C₃ species (van Heerden et al., 2003a; Feng and Cao, 2005). Moreover, photosynthesis was only decreased after two nights of chilling under abaxial illumination (Fig. 1A). The results presented in this study (Fig. 1A, B; Tables 4, 5) together with the data that we have obtained previously in plants grown with CO₂ enrichment (Soares et al., 2008) demonstrate that these stresses initially exert their greatest effects on photosynthesis under adaxial illumination but the resultant inhibition is followed by a rapid recovery upon a second stress exposure. In contrast, under abaxial illumination the P. dilatatum leaf is initially more resistant to dark chilling. This evidence demonstrates that each side of the leaf has a unique sensitivity to dark chilling.

The absence of effects on stomatal conductance after one night of chilling under adaxial illumination (Fig. 1B) suggests that the decrease in the photosynthetic rate is associated with non-stomatal limitations to photosynthesis, as also indicated by the results in Table 4. Moreover, after one night of chilling, the dorsoventral differences in the C_i values on each side of the leaf disappeared. Under optimal conditions of irradiance and temperature, the mean C_i values were 157±15.2 when light was oriented to the adaxial surface, and 251±22.3 when light was oriented to the abaxial surface. After one night of chilling the mean C_i values were similar on both sides of the leaf, being 230±20.1 and 226±19.8, respectively. However, after the second night of chilling the dorsoventral asymmetry in mean C_i values was partially restored; the mean C_i values were then 209±19.8 when light was oriented to the adaxial surface, and 259±23.5 when light was oriented to the abaxial surface.

Dark chilling is known to decrease the activity of several photosynthetic enzymes in C₃ and C₄ plants (e.g. Jones et al., 1998; Pittermann and Sage, 2001; van Heerden et al., 2003a) and also cause decreased electron transport capacity in C₃ plants (e.g. Bertamini et al., 2005; Strauss et al., 2007). The higher decrease in V_pmax observed in P. dilatatum leaves after one night of chilling compared with V_cmax, J_max, and A_max, together with the failure of this parameter to recover after two nights of chilling stress under adaxial illumination (Tables 4, 5), suggests that the maximal activity of the first carboxylation step in C₄ plants has a higher sensitivity to dark chilling. Moreover, the decrease in V_cmax and J_max values under abaxial illumination together with the absence of an effect on V_pmax after the second night of chilling (Table 4) suggest that the maximal activity of the first carboxylation enzyme of C₄ plants on the abaxial surface is less sensitive to dark chilling. It is important to note that while photosynthesis on both sides of the leaf is determined under adaxial illumination, this is not the case under abaxial illumination where photosynthesis on the abaxial side of the leaf alone can be detected. These results strongly suggest that PEPC activity on each side of the P. dilatatum leaf has a different sensitivity to dark chilling.

(ii) Dark chilling effects on photosynthesis are leaf-side specific, the abaxial side being more sensitive to inhibition:

Dark chilling had a greater impact on photosynthesis on the abaxial sides of the leaves under adaxial illumination. In marked contrast, under abaxial illumination the dark chilling effect on the abaxial side of the leaf was observed only on the second consecutive night of chilling (Fig. 2C). These results demonstrate that the acclimation of photosynthesis to dark chilling is most rapid on the surface that is directly exposed to light. Although the effects of dark chilling on metabolism, physiology, and membrane fluidity are expected to be similar on each side of the leaf irrespective of the light orientation, the higher light intensity arriving on the abaxial surface under abaxial illumination may increase the activities of photosynthetic enzymes in such a way as to overcome any metabolic limitations incurred during the chilling treatment. The activation of photosynthetic activity may be insufficient to overcome metabolic constraints resulting from the second chilling treatment. Taken together, these results suggest that the orientation of light is an important determinant of the ability of each side of the P. dilatatum leaf to respond to the stress imposed by dark chilling.

Growth with CO₂ enrichment decreased the photosynthetic rates only on the adaxial sides of the P. dilatatum leaves under adaxial illumination (Soares et al., 2008). A similar response was observed in maize plants grown with CO₂ enrichment (Driscoll et al., 2006). In both studies, photosynthesis fell below the levels of detection on the adaxial sides of the leaves under abaxial illumination, while the abaxial sides of the leaves have rates similar to controls. These results presented on this study and those presented by Driscoll et al. (2006) and Soares et al. (2008) suggest that the effects of environmental stress are different on each leaf surface, revealing the importance of these studies in our current understanding of the dorsoventral regulation of photosynthesis in monocotyledonous C₄ species.

(iii) Differential sensitivity of stomatal conductance to dark chilling suggests leaf-side-specific stress signalling:

The data presented here demonstrate that the two sides of P. dilatatum leaves are not functionally symmetrical, as the C₄ leaf structure might suggest (Cavaco, 2000). Moreover, we show here that each side of the leaf can respond differently to environmental stress. In agreement with this view, stomatal conductance is less sensitive to the effects of dark chilling on the adaxial side of the leaf under adaxial illumination (Fig. 2B). This observation is similar to that obtained in plants subjected to water deficit, a stress that, like chilling requires cellular osmotic adjustment (Turner and Singh, 1984; Lu, 1988; Wang et al., 1998). The lower sensitivity of the adaxial stomata in leaves exposed to water deficit has been explained by a lower sensitivity of the guard cells on this leaf surface to abscisic acid and to calcium ions (De Silva et al., 1986; Wang et al., 1998). While we have no direct evidence for a specific effect of dark chilling on the stomata per se, the different responses of stomata on the adaxial and abaxial surfaces are intriguing, particularly as it may be a common feature of the response of C₄ cereal leaves to stress situations.

The significance of independent, dorsoventral systemic stress signalling pathways

We have previously described the dorsoventral regulation of photosynthesis with respect to light orientation under optimal growth conditions in two monocotyledonous species that belong to the NADP-ME subtype, P. dilatatum and maize, and we have discussed the practical relevance of the findings at a whole-plant level within the context of the natural diurnal light environment that C₄ plants face every day (Driscoll et al., 2006; Soares et al., 2008; Soares-Cordeiro et al., 2009). The data presented here show that each side of the leaf responds differently in terms of inhibition by dark chilling and the ability to recover from the stress. We consider that these differential responses are possible because of the structure of the P. dilatatum leaves, which restricts gas flow. The data presented here confirm the two-compartment model for monocotyledonous C₄ leaves developed in order to explain the dorso-ventral differnces in the regulation of photosynthesis (Long et al., 1989). The finding that dark chilling, like growth with CO₂ enrichment (Soares et al., 2008), modifies not only the dorsoventral regulation of photosynthesis with respect to light orientation but also the acclimation ability of each side of the leaf to the stress, is intriguing. While more studies are required to unravel the physiological relevance of these findings at the whole plant level, it is apparent that the two-compartment, two-airspace system of C₄ cereal leaves would enable leaf-side-specific CO₂-signalling and defence-signalling pathways. This type of compartmentation would also facilitate the independent regulation of stomatal movements on each leaf surface, in a manner that may not primarily be related to photosynthesis (von Caemmerer et al., 2004; Baroli et al., 2008). Moreover, differential dorsoventral stress signalling pathways would allow independent acclimation processes to occur on each side of the leaf. While the significance of leaf-side-specific stress responses remains to be elucidated, the leaf-side-specific regulation of photosynthesis may be controlled by the developmental genetic programme that governs the adaxial–abaxial patterning of leaves (Chitwood et al., 2009).

Glossary

Abbreviations

ϕ

apparent quantum yield

θ

curvature degree

A

CO₂ assimilation rate

A_max

maximal rate of photosynthesis

CBF

CRT/CRE BINDING FACTOR

chla

chlorophyll a

chlb

chlorophyll b

C_i

intercellular CO₂ concentration

COR

COLD-REGULATED

I

Irradiance

J_max

maximal electron transport rate

K_c

Michaelis–Menten constant of Rubisco for CO₂

K_o

Michaelis–Menten constant of Rubisco for O₂

K_p

Michaelis–Menten constant of PEPC for CO₂

NADP-ME

NADP-malic enzyme

PEPC

phosphoenolpyruvate carboxylase

R_d

mitochondrial respiration

R_m

mesophyll mitochondrial respiration

Rubisco

ribulose-1,5-bisphosphate carboxylase/oxygenase

V_cmax

maximal ribulose-1,5-bisphosphate carboxylation rate

V_pmax

maximal phosphoenolpyruvate carboxylase carboxylation rate