JXB Advance Access first published online on April 9, 2008
This version published online on April 24, 2008
Journal of Experimental Botany, doi:10.1093/jxb/ern062
RESEARCH PAPER |
Low temperature effects on leaf physiology and survivorship in the C3 and C4 subspecies of Alloteropsis semialata
1Department of Animal and Plant Sciences, University of Sheffield, Sheffield S10 2TN, UK
2Botany Department, Rhodes University, PO Box 94, Grahamstown 6140, South Africa
* To whom correspondence should be addressed. E-mail: c.p.osborne{at}sheffield.ac.uk
Received 9 December 2007; Revised 28 January 2008 Accepted 12 February 2008
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Abstract |
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The species richness of C4 grasses is strongly correlated with temperature, with C4 species dominating subtropical ecosystems and C3 types predominating in cooler climates. Here, the effects of low temperatures on C4 and C3 grasses are compared, controlling for phylogenetic effects by using Alloteropsis semialata, a unique species with C4 and C3 subspecies. Controlled environment and common garden experiments tested the hypotheses that: (i) photosynthesis and growth are greater in the C4 than the C3 subspecies at high temperatures, but this advantage is reversed below 20 °C; and (ii) chilling-induced photoinhibition and light-mediated freezing injury of leaves occur at higher temperature thresholds in the C4 than the C3 plants. Measurements of leaf growth and photosynthesis showed the expected advantages of the C4 pathway over the C3 type at high temperatures. These declined with temperature, but were not completely lost until 15 °C, and there was no evidence of a reversal to give a C3 advantage. Chronic chilling (5–15 °C) or acute freezing events induced a comparable degree of photodamage in illuminated leaves of both subspecies. Similarly, freezing caused high rates of mortality in the unhardened leaves of both subtypes. However, a 2-week chilling treatment prior to these freezing events halved injury in the C3 but not the C4 subspecies, suggesting that C4 leaves lacked the capacity for cold acclimation. These results therefore suggest that C3 members of this subtropical species may gain an advantage over their C4 counterparts at low temperatures via protection from freezing injury rather than higher photosynthetic rates.
Key words: Alloteropsis semialata, C3 photosynthesis, C4 photosynthesis, chilling, cold acclimation, freezing, photodamage, quantum yield, temperature
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Introduction |
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Grasses using the C4 photosynthetic pathway dominate the tropical and subtropical savannas, but low temperatures limit their poleward distribution, and C4 species are replaced at higher latitudes and altitudes by plants with the C3 pathway (reviewed by Sage et al., 1999). The primary role of temperature as a constraint on C4 plant distributions is demonstrated by significant positive correlations between, C4 species richness and growing season temperature for grass floras on each of the ice-free continents (Teeri and Stowe, 1976; Vogel et al., 1978; Hattersley, 1983; Collins and Jones, 1986; Cavagnaro, 1988; Pyankov et al., 2000).
The leading explanation for these patterns is based upon experiments showing that quantum yield (maximum photosynthetic efficiency under light-limitation) is greater in C4 than C3 plants at high temperatures, due to the suppression of photorespiration in the C4 pathway (Ehleringer and Björkman, 1977). However, the energetic costs of C4 photosynthesis mean that its quantum yield is lower than that of the C3 pathway when photorespiration is decreased by low temperatures. Since light-limited photosynthesis is a key determinant of canopy photosynthesis, it is therefore proposed that the high C4 efficiency translates into a competitive advantage over grasses with the C3 type in hot climates, and vice versa in cool climates, with a crucial crossover temperature at 21–26 °C (Ehleringer, 1978; Ehleringer et al., 1997).
Since the C4 cycle saturates the carboxylase reaction of Rubisco, the theoretical maximum rate of photosynthesis by a C4 plant under high light conditions is always greater than a C3 plant, for a given amount of Rubisco (Long, 1999). However, this potential is commonly not achieved at low temperatures because of photoinhibition or injury in the chilling (0–12 °C) and freezing (<0 °C) temperature ranges (Long, 1983). These effects are exacerbated by light, and seem to reflect the tropical evolutionary origins of C4 species rather than an intrinsic vulnerability of C4 photosynthesis to low temperatures (Long, 1999).
Previous experimental investigations of these mechanisms have controlled for ecological background by focusing on C3/C4 species pairs from the same cool temperate or high elevation habitats (Long et al., 1975), or for phylogenetic distance by comparing congeneric C3 and C4 eudicot species (Osmond et al., 1980). However, close taxonomic affinity between C3 and C4 species in the ecologically important grass family (Poaceae) is rare because ancient origins of the C4 pathway in this group have led to significant divergence between C3 and C4 clades (Grass Phylogeny Working Group, 2001). Although some studies have compared C3 and C4 species of Panicum (Henning and Brown, 1986), recent molecular phylogenies show that it is a polyphyletic genus with significant divergence between its C3 and C4 species (Aliscioni et al., 2003).
The grass Alloteropsis semialata has C3 and C4 variants (Ellis, 1974) that are classified as subspecies (Gibbs Russell, 1983). No other example of such closely related C3 and C4 species has yet been identified in the Poaceae, and A. semialata therefore provides a unique means of controlling for phylogenetic distance in comparative studies. Sequencing of the chloroplast gene ndhF suggests that the C3 subspecies represents a reversion from a C4 ancestor (Ibrahim, 2007; Ibrahim et al., 2007), and the two subspecies occupy an overlapping range of open grassland habitats in South Africa (Ellis, 1981; Gibbs Russell, 1983). In this region of overlap, both experience mean minimum July (midwinter) temperatures in the chilling range (5–15 °C; New et al., 1999) and a mean frost-free period ranging from nine months to more than two years (Werger and Ellis, 1981).
A. semialata has been developed as a model system for investigating the ecological significance of the photosynthetic pathway for subtropical grasses, by using a common garden experiment to test the interactions between temperature and water availability (Ibrahim, 2007; Ibrahim et al., 2007). Results from the first year of this study showed that leaf photosynthesis was significantly higher in the C4 than the C3 subspecies under high temperatures, moist atmospheric and soil conditions, and clear-skies (Ripley et al., 2007). However, during winter, the pattern was reversed because all of the C4 leaves died, whereas a significant proportion of the C3 canopy survived (Ibrahim, 2007; Ibrahim et al., 2007). The physiological basis for these key findings is explored here, using common garden, greenhouse, and controlled environment studies to test two hypotheses about the differential effects of temperature on the A. semialata subspecies: first, that photosynthesis and growth are greater in the C4 than the C3 subspecies at high temperatures, but this advantage is reversed at temperatures <20 °C; and, secondly, that chilling-induced photoinhibition and light-mediated freezing injury of leaves occur at a higher temperature threshold in the C4 than C3 plants.
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Plant materials
Alloteropsis semialata is a member of the tribe Paniceae and sub-family Panicoideae. Physiology of the C4 subspecies is classified as the ‘PCK-type’ (Hattersley et al., 1977), although high levels of NADP-ME have been measured in the bundle sheath of South African plants (Ueno and Sentoku, 2006). Kranz anatomy is of the ‘Neurachne-type’ (Hattersley and Watson, 1992).
Established plants of the C4 subspecies A. semialata (R.Br.) Hitchc. subsp. semialata and C3 subspecies A. semialata (R.Br.) Hitchc. subsp. eckloniana (Nees) Gibbs Russ. were collected from grasslands near Middelburg (25°50’ S, 29°24 E) (Mpumalanga, South Africa) and Grahamstown (33°18’ S, 26°31’ E) (Eastern Cape, South Africa), respectively. Voucher specimens of each accession are deposited at the University of Sheffield herbarium (C4, Ibrahim 20) and the Schonland Herbarium at Rhodes University (C3, Ibrahim 15). The carbon isotope ratio of leaf tissues and CO2-compensation point for photosynthesis were –11.6
and 1.2 Pa for the C4 and –26.0 and 4.6 Pa for the C3 subspecies, typical for each photosynthetic type (Ripley et al., 2007). Tillers from each were used to establish a common garden experiment at Rhodes University, South Africa (Ripley et al., 2007), or transported to the University of Sheffield, UK, and planted in cylindrical pots (100 mm diameter, 250 mm length) using a 3:1 (v/v) mixture of topsoil (BandQ, Eastleigh, Hants, UK) and perlite (LBS Horticulture, Colne, Lancs, UK).
The latter plants were grown with a slow-release fertilizer (Osmocote, Scotts Miracle-Gro Company, Marysville, OH, USA) under either controlled environment or greenhouse conditions. The controlled environment consisted of a cabinet (BDR-16, Conviron Controlled Environments Ltd, Winnipeg, Manitoba, Canada) imposing a 14 h photoperiod, day/night temperature regime of 25/18 °C, relative humidity (RH) of 70%, and PPFD of 
600 µmol m–2 s–1 at plant height. The greenhouse was heated to give a maximum/minimum temperature range of 30/20 °C, and used supplementary lighting to provide a photoperiod of 14 h and a PPFD of >500 µmol m–2 s–1 at plant height.
Experiment 1: temperature dependency of photosynthesis
The maximum quantum yield was measured on an absorbed light basis (
CO2) to quantify the energetic efficiency of net leaf photosynthesis (A) under light-limited conditions. The temperature dependency of CO2 was determined using a portable open gas exchange system (LI-6400, Li-Cor, Inc., Lincoln, NE, USA) equipped with a red-blue LED light source (LI-6400–02B). A was induced at PPFD >100 µmol m–2 s–1, with pCO2=38 Pa and VPD <1.5 kPa. Once A and stomatal conductance had reached a steady-state (30 min), PPFD was decreased in steps to darkness, and A was recorded when stable (>3 min). Measurements were made at leaf temperatures of 15, 20, 25, and 31 °C for both subspecies, and in addition at 35 °C for the C4 subspecies. Although this species experiences temperatures <15 °C in the field during the night and on cold mornings, these are rare from late morning onwards, and were not therefore used for steady-state gas exchange measurements. CO2 was calculated as the slope of the relationship between A and incident PPFD, using at least five PPFD levels from the linear portion of the response, and had R2 >0.95 (Singsaas et al., 2001). The reflection and scattering of light from the cuvette floor was accounted for in these calculations because the leaves did not completely fill the cuvette. PPFD values <10 µmol m–2 s–1 were excluded from the analysis due to the Kok effect (Singsaas et al., 2001), and the CO2 was expressed on an absorbed light basis using previous measurements of leaf absorptance (0.85 in both subspecies; Ripley et al., 2007).
The temperature response of light-saturated net leaf photosynthesis (Asat) was measured for a different set of plants, grown under controlled environment conditions. Asat was measured using a portable open gas exchange system (HCM-1000, Heinz Walz GmbH, Effeltrich, Germany) under a saturating PPFD of 1250–1350 µmol m–2 s–1 for the youngest fully expanded leaf on a tiller. The gas exchange system was placed in a temperature-controlled cabinet (600H, Gallenkamp Industrial, Loughborough, Leics, UK) to allow measurements at leaf temperatures of 15, 21, 25, 30, 35, and 40 °C. The pCO2 in the leaf cuvette was 38 Pa and VPD maintained at <2.0 kPa or, for measurements at the highest leaf temperatures, <3.0 kPa. The ratio of Ci/Ca did not change at the higher VPD, indicating that photosynthesis was not affected by reduced stomatal aperture at high temperatures. Data were recorded once stomatal conductance and A were stable, usually 20–30 min after the leaf was inserted into the cuvette.
Physiological limitations at sub-optimal temperatures were further investigated by using a portable open gas exchange system (CIRAS-1, PP-Systems International Ltd, Hitchin, Herts, UK) to measure the responses of A to Ci following the protocol of Long and Bernacchi (2003). Measurements were made at leaf temperatures of either 25 °C or 15 °C, a saturating PPFD of 1200 µmol m–2 s–1, VPD <1.5 kPa, and 8–10 values of pCO2, equally distributed either side of the inflexion of the curve. Biochemical models of C3 and C4 photosynthesis (Farquhar et al., 1980; von Caemmerer, 2000) were fitted to the initial slope or saturated portion of the individual curves after Wullschleger (1993), accounting for the temperature-dependence of Rubisco kinetics in the C3 model (Bernacchi et al., 2001). A bundle sheath conductance to CO2 of 3 mmol m–2 s–1 and a saturating supply of phosphoenolpyruvate (PEP) were assumed in the C4 model (von Caemmerer, 2000). Effects of temperature on the model parameters were investigated using a paired Student's t test for the C3 leaves, and an unpaired t test for the C4 leaves, where one value was missing.
Experiment 2: temperature dependency of leaf growth
The temperature response of the linear leaf extension rate (LER) was measured for plants grown under controlled environment conditions, sampling duplicate leaves for five replicate plants of each subspecies. At the end of the photoperiod, a steel ruler was used to measure the length of an elongating leaf from ligule to tip, to the nearest 0.5 mm. Plants were then watered to drip-point and placed in a dark, humid, controlled temperature cabinet (600H, Gallenkamp Industrial) for 16 h. Measurements of leaf length were repeated, and the overnight growth increment used to calculate LER.
The measurements were repeated for temperatures of 5, 10, 13, 15, 17, 20, 24, 28, 32, and 35 °C, each set using a calibrated max–min thermometer (CIS Calibration Laboratories, Coalville, Leics, UK) and fluctuating overnight by <1 °C. The temperatures were applied in a random order, and plants returned to the controlled environment growth environment for >36 h between each measurement. To investigate the influence of light on LER, an additional set of measurements were made at a PPFD of 600 µmol m–2 s–1 and a temperature of 25 °C.
The temperature dependency of LER was also investigated in the common garden experiment during a vegetative period of growth prior to flowering in spring 2005. An elongating leaf was randomly selected for six individuals of each subspecies, each in a different block of the experiment. The tip of each leaf was attached, via a crocodile clip and 0.264 mm inelastic nylon braid (Gudebrod Inc., Pottstown, PA, USA) passed over a pulley, to a small brass weight. The pulley and line were arranged so that the weight hung in front of a plastic ruler, and the whole apparatus was welded or wired onto a steel fencing stake. The LER was estimated to the nearest 1 mm at 17.00 h each day over a 9 d period, using the position of the weight against the ruler. Air temperature was recorded every 30 min at 3.5 m (Vantage Pro Weather Station, Davis Instruments, Hayward, CA, USA). Although leaf temperatures may exceed these values during the middle of the day, they do not differ between the subspecies (Ibrahim, 2007).
Experiment 3: low temperature thresholds for photoinhibition and leaf injury
Temperature thresholds for photoinhibition and leaf injury were investigated by exposing plants from the greenhouse to acute low temperature events. The events were designed to mimic natural diurnal cycles of light and temperature at the common garden field site in South Africa, and were applied using a controlled environment growth room (Conviron BDW 40, Conviron Controlled Environments Ltd). Three plants of each subspecies were subjected to minimum air temperatures (Tmin) of either –5, 0, 5, or 15 °C. Different plants were used for each of the temperature treatments, which were applied in a random order and repeated, to give six replicates for each Tmin. To ensure that temperature treatments were applied to leaves but not the roots, each plant pot was wrapped in polythene and immersed in an insulated waterbath held at 15 °C throughout the experiment. The effect of protecting the roots from low temperature was assessed by repeating the Tmin= –5 °C treatment without the waterbaths, and allowing the soil to freeze.
Plants were first exposed to 24 h at 15 °C, with an 8 h photoperiod and a PPFD of 900 µmol m–2 s–1 at plant height, chosen to approximate a sunny day in the greenhouse. The value of 15 °C was chosen as a compromise; pilot studies showed that this ‘control’ temperature was associated with some inhibition of photosynthesis, but higher temperatures would have necessitated faster cooling rates to achieve Tmin. Since leaf injury was the focus of this experiment, and is correlated with the rate of cooling, the minor inhibitory effects of the control treatment were accepted as a cost of achieving night-time cooling rates similar to those measured in South Africa.
After the initial 24 h period, rates of net leaf photosynthesis were measured at 14.00 h using a portable open gas exchange system (CIRAS-1, PP-Systems International, Ltd) on duplicate marked leaves for each plant. The maximum quantum efficiency of photosystem 2 (Fv/Fm) was determined for the same leaves after 15 min dark adaptation, using a portable, pulse-modulated, chlorophyll fluorimeter (FMS-2, Hansatech Instruments Ltd, King's Lynn, Norfolk, UK). At the end of the photoperiod (17.00 h), temperature in the growth room was lowered to Tmin+5 °C at a constant rate of 4 °C h–1, after which the cooling rate was slowed to reach Tmin at 08.00 h. From 09.00 h, PPFD was ramped upwards in three equal 300 µmol m–2 s–1 steps to reach a maximum at 12.00 h, while temperature was increased to 15 °C at a rate of 4 °C h–1. Measurements with a hand-held infrared thermometer (Cyclops 330S, Minolta/Land Infrared, Bristol, PA, USA) at ‘dawn’ verified that leaf temperature was within 3 °C of the air temperature.
Photosynthesis and chlorophyll fluorescence measurements of the marked leaves were repeated at 14. 00 h. Plants were then returned to the greenhouse for 10 d, and injury was scored for every leaf in the canopy, classifying leaves as dead (>80% necrotic), damaged (<80%, but >10% necrotic), or undamaged (<10% necrotic). The interacting effects of temperature and subspecies on photosynthesis, Fv/Fm, damage, and mortality were tested using two-way ANOVA after arcsine transformation of the data. For the plants in which roots were allowed to freeze, Student's t test was used to evaluate the differing responses of these same variables between the subspecies.
Experiment 4: induction of cold acclimation and photoinhibition by chronic chilling
A further experiment determined whether cold acclimation during a chilling pretreatment could protect leaves from injury during a subsequent freezing event. Photoinhibition during this chilling pretreatment was also quantified using chlorophyll fluorescence measurements.
Four plants of each subspecies were transferred from the greenhouse to controlled growth environments (BDR-16, Conviron Controlled Environments Ltd) applying day/night temperature regimes of either 25/20 °C (control) or 15/10 °C (chilling) for 1 week. The relative humidity (RH) was 70%, photoperiod 14 h, and PPFD at plant height 600 µmol m–2 s–1. Temperatures in the chilling treatment were then lowered to 10/5 °C for a further week, while the control was held at 25/20 °C, giving a pretreatment duration of 2 weeks. To avoid confounding the effects of treatment and cabinet, the plants and treatments were exchanged between the cabinets at the midpoint of each week.
Chlorophyll fluorescence quenching analyses were carried out at intervals during the pretreatment period using a portable fluorimeter (FMS-2, Hansatech Instruments Ltd). The operating efficiency of photosystem 2 (PS2) was first measured under growth conditions, and Fo' estimated following Maxwell and Johnson (2000). Fv/Fm was subsequently measured after 15 min dark adaptation, and used to calculate fast-relaxing non-photochemical quenching (NPQ), and photochemical quenching (qP), the fraction of Fv/Fm realized under growth conditions (Maxwell and Johnson, 2000; Baker et al., 2007).
On the final day of pretreatments, plants were transferred at 14.00 h to a controlled environment room (Conviron BDW 40, Conviron Controlled Environments Ltd). Roots were kept at 15 °C using water baths, and leaves exposed that night to the –5 °C freezing treatment described for Experiment 3. Leaf injury was scored as before. The effects of pretreatment and subspecies were tested using arcsine-transformation followed by two-way ANOVA, for fluorescence parameters on the final day of treatments, leaf mortality and damage.
Experiment 5: changes in leaf water relations during cold acclimation in the field
The cold acclimation process was investigated further using measurements of leaf water potential in plants from the irrigated treatment of the common garden experiment. Measurements were made at weekly intervals before the first ground frost on 30 May 2006, and repeated immediately after the frost. Minimum air temperatures during May were in the chilling range, varying from 1–12 °C.
Leaves were cut from up to seven replicate plants of each subspecies, sealed in polythene bags with wet tissue paper and allowed to hydrate overnight at room temperature. Moisture release curves were constructed using a custom-built Scholander pressure chamber (Turner, 1981), and leaves were then oven-dried to obtain relative water content (RWC) and the dry matter content of saturated leaves (DMC). Pressure–volume (P–V) curves were derived from these data and analysed via a stepwise method, first fitting a straight line to values below the turgor loss point (TLP) to estimate the response of osmotic potential (
) to RWC. Estimated values of were then subtracted from measured leaf water potential (L) values to obtain pressure potential (P). A modified exponential equation (‘PVC’ from Schulte and Hinckley, 1985) was fitted to values of P and RWC, and its zero intercept used to estimate the TLP. Young's modulus of elasticity (
) was calculated as the slope of the moisture release curve between saturation and the TLP (Lenz et al., 2006). The interacting effects of subspecies and date (for the first two sampling dates only) were tested on L,sat, ,sat, , TLP, Smax, and DMC using two-way ANOVA, since missing values (i.e. a non-orthogonal design) precluded the use of repeated measures ANOVA.
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Experiment 1: temperature dependency of photosynthesis
Values of
CO2 in the C4 subspecies were invariant with temperature, and had an average of 0.074 mol mol–1 between 15 °C and 35 °C (Fig. 1a). By contrast, CO2 in the C3 subspecies showed a strong, negative, linear relationship with leaf temperature, declining from a value of 0.072 mol mol–1 at 15 °C to 0.042 mol mol–1 at 31 °C (Fig. 1a). The crossover temperature range for CO2 between these subspecies was therefore 
17 °C (Fig. 1a).
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The maximum value of Asat and its temperature optimum were both greater in the C4 than in the C3 subspecies (Fig. 1b). Differentiation of the fitted curves in Fig. 1b showed that the C4 leaves achieved a maximum Asat of 30 µmol m–2 s–1 at 34 °C, while the C3 leaves reached a maximum of 17 µmol m–2 s–1 at 28 °C. However, Asat was more strongly limited by sub-optimal temperatures in the C4 than C3 subspecies, and the advantage of C4 photosynthesis was completely lost at 15 °C (Fig. 1b).
Values of Asat at 25 °C and 15 °C were statistically indistinguishable in the C3 subspecies (Figs 1b, 2b), but the same temperature contrast caused a 2-fold change in Asat for the C4 plants (Figs 1b, 2a). The insensitivity of C3 photosynthesis could be explained by the opposing responses of photosynthetic capacity and photorespiration to temperature. Vc,max and Jmax were both significantly lower at 15 °C than at 25 °C, with the temperature sensitivity (Q10) of Vc,max greater than that of Jmax, leading to a 63% increase in the Jmax:Vc,max ratio at 15 °C compared to 25 °C (Table 1). However, the apparent (in vivo) specificity of Rubisco for CO2 relative to O2 rises by 70% between 25 °C and 15 °C (Long, 1991; Bernacchi et al., 2001), leading to an increase in the ratio of carboxylation to oxygenation reactions, and a decrease in photorespiration. According to our model analysis, the net consequence of these opposing effects was that the A/Ci curves were virtually identical for the C3 leaves at 25 °C and 15 °C (Fig. 2b).
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The strong temperature-limitation of photosynthesis in the C4 leaves was attributable to decreases in both Vp,max and Amax (Table 1). The latter is controlled by either the CO2-saturated value of Vc,max or the capacities for PEP or RuBP regeneration under the light-saturated conditions used for A/Ci measurements (von Caemmerer, 2000). The ratio of Vp,max:Amax was 1.4 at both temperatures because Q10 values for Vp,max and Amax were similar (Table 1). This temperature-mediated decrease in photosynthetic capacity in the C4 leaves translated directly into reduced values of A, because the impact of photorespiration is minimized by the C4 CO2-concentrating mechanism.
Experiment 2: temperature dependency of leaf growth
The C4 and C3 subspecies showed differential growth responses to temperature. Maximum values of LER under controlled environmental conditions were achieved at 35 °C and were 35% greater in the C4 than the C3 plants (Fig. 3a). However, LER was more strongly limited by temperature in the C4 subspecies, and its growth advantage over the C3 was completely lost at 15 °C (Fig. 3a), matching almost exactly the crossover temperature for photosynthesis (Fig. 1). Values of LER were statistically indistinguishable in the C3 and C4 plants at controlled temperatures 15 °C, and the low temperature threshold for leaf elongation occurred at <8 °C in both (Fig. 3a). LER was unaffected by light at 25 °C (Fig. 3a).
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Average daily values of LER in the common garden experiment showed a stronger correlation with the maximum temperature (R2 >0.8), than the minimum or average on each day (data not shown). Values of LER were greater in the C4 than C3 plants at high temperatures, but this growth advantage diminished with decreasing maximum temperature (Fig. 3b). At 35 °C, LER for the C4 subspecies was
3 times that of the C3, but the subspecies had statistically indistinguishable growth rates at 12 °C (Fig. 3b). The patterns observed in the field therefore showed a qualitative match with the controlled growth experiment, but the absolute difference between subspecies was greater, and the crossover temperature lower.
Experiment 3: low temperature thresholds for photoinhibition and leaf injury
Exposure of unhardened plants to a controlled environment light regime and temperature of 15 °C caused photoinhibition, manifested in both subspecies as decreases in A and Fv/Fm to 80% of their initial values (Fig. 4a, b). Photosynthesis was further inhibited by exposure to acute episodes of low temperature under high PPFD, but CO2-assimilation and photochemistry differed in their sensitivity to these treatments. A showed a progressive, and approximately linear, decrease with Tmin in both of the subspecies, reaching 30% of its initial value after exposure to –5 °C in the light (Fig. 4a). By contrast, Fv/Fm showed a threshold response in both of the subspecies, remaining at 80% of its initial value after exposure to +5 °C and 0 °C in the light, but falling sharply to 50% of its initial value after the –5 °C freezing event (Fig. 4b). Although there were highly significant effects of temperature for A (two-way ANOVA: F1,48=9.19, P <0.01) and Fv/Fm (F1,50=8.12, P <0.01), there were no statistically significant effects of subspecies (F1,48=0.09 for A, and F1,50=1.22 for Fv/Fm) or the interaction (F1,48=0.06 for A, and F1,50=0.09 for Fv/Fm).
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A further experiment investigated whether allowing the root system to freeze would induce a differential treatment-effect in the C3 and C4 subspecies at –5 °C. This showed a different trend in the two subspecies, reducing A by 50% in the C3 subspecies, but 100% in the C4 [Fig. 4a; t test: t(df)=1.98(10); P=0.075]. However, the same treatment lowered Fv/Fm by an equal amount in the two subspecies [Fig. 4b; t(df)=0.93(4); ns].
Leaf injury showed a threshold response to acute low temperature/high PPFD events. Levels of damage and mortality were uniformly low in the +15 °C and +5 °C treatments, but damage increased abruptly at 0 °C, and mortality at –5 °C, suggesting greater susceptibility to freezing, rather than chilling injury (Fig. 4c, d). There was a highly significant effect of the temperature treatment on leaf damage (two-way ANOVA: F1,43=13.52, P <0.001) and mortality (F1,43=15.84, P <0.001) but, crucially, there was no differential effect of freezing on leaf mortality in the two subspecies (F1,43=0.10 for subspecies and F1,43=0.48 for the interaction). The experiment investigating the influence of root freezing suggested a trend towards greater mortality and lower damage in the C4 than C3 subspecies (i.e. more of the damaged leaves were killed outright) (Fig. 4c, d), but the effect was not statistically significant [(t(df)=1.12(10), ns)].
Experiment 4: induction of cold acclimation and photoinhibition by chronic chilling
Patterns of leaf freezing injury were modified substantially by the chilling pretreatment, but the response differed between C3 and C4 subspecies. This led to significant interactions between subspecies and treatment for both leaf mortality (two-way ANOVA: F1,12=9.76, P=0.01) and damage (F1,12=5.72, P=0.034). Leaf mortality in the C3 subspecies was approximately halved in the chilling relative to the control pretreatment (Fig. 5a), indicating the development of frost protection. However, the opposite pattern was observed for leaf damage, which showed a significant increase in the chilling compared with control pretreatment (Fig. 5b). In combination, these results show that the C3 leaves were protected from freezing injury through a cold acclimation process, which reduced the number of leaves that were killed outright, but failed to provide complete protection against damage. In the C4 subspecies, leaf mortality was uniformly high across pretreatments (Fig. 5a), whilst damage was low (Fig. 5b), showing that cold acclimation did not develop in these leaves, and most were therefore killed outright by the freezing event.
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Leaf injury in the control treatment was higher than that measured in Experiment 3. Although the illuminated freezing treatments were identical in these experiments, the PPFD pretreatment was not, with plants in Experiment 4 experiencing lower PPFD in the controlled environment than those in Experiment 3 received in the greenhouse. The difference between experiments may therefore be due to differential light acclimation.
The chilling pretreatment caused significant photodamage in leaves of the C3 and C4 subspecies, which occurred largely in the second week under the 10/5 °C (day/night) temperature regime. The operating efficiency of photosystem 2 (PS2) remained static in this treatment for the first week, and then declined rapidly in the second, with a stronger decrease in the C3 than C4 subspecies (Fig. 6a; two-way ANOVA: interaction F1,12=13.04, P <0.01). In both subspecies, this major decrease in efficiency could be attributed to both Fv/Fm (Fig. 6b), a measure of the number of functional reaction centres, and photochemical quenching (qP), the fraction of reaction centres closed under growth conditions (Fig. 6d). The interaction between treatment and subspecies was significant for both (Fv/Fm: F1,12=4.87, P=0.048. qP: F1,12=9.32, P=0.010.). Fast-relaxing (<15 min) non-photochemical quenching (NPQ) did not increase during this period in either subspecies (Fig. 6c).
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Temperature regimes of the greenhouse and the control treatment were similar, but the transfer of plants to the control environment also caused changes in photochemistry. The value of
PS2 increased to its theoretical maximum during the 2-week treatment (Fig. 6a). Since Fv/Fm remained constant throughout this period (Fig. 6b), the rise in PS2 could be attributed to an increase in qP (Fig. 6d). These effects of the control treatment on chlorophyll fluorescence did not differ between the C3 and C4 subspecies.
Experiment 5: changes in leaf water relations during cold acclimation in the field
P–V curves constructed during a natural period of cold acclimation in the common garden experiment showed significant differences between the moisture release characteristics of C3 and C4 leaves. Before the first frost event, the maximum water content of the symplasm (Smax) in the C3 leaves was approximately half that in the C4 (Table 2). This significantly lower water content of cells was consistent with the significantly higher values of leaf dry matter content (DMC) observed in C3 than C4 leaves (Table 2). The leaf water potential at 100% RWC (L,sat) showed a significant interaction between subspecies and dates, decreasing more rapidly between the first and second sampling dates in the C4 than C3 subspecies (Table 2). However, the other characteristics of the moisture release curve, including osmotic potentials at saturation (,sat), , and TLP, showed no significant differences between the C3 and C4 subspecies (Table 2). The TLP tended to decline between the first and second sampling dates (Table 2).
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The net result of these differences is illustrated by the P–V curves constructed immediately before the first frost (23 May; Table 2). Leaf water potential at saturation was lower in the C4 than C3 leaves, but the TLP was statistically indistinguishable between the subspecies. Below the TLP, the response of
(and therefore L) to RWC was significantly steeper in the C3 than C4 subspecies, with critical implications for the behaviour of leaf water below freezing. The potential of water vapor over ice (ice) is significantly lower than that of liquid water at the same temperature (Jones, 1992), and extracellular ice formation in leaves therefore tends to desiccate cells (frost drought). By assuming temperature insensitivity of the shape of P–V curves, ice was used to model the RWC of leaf tissues that would theoretically be achieved if intracellular water fully equilibrated with extracellular ice at –5 °C; i.e. the desiccation effect of extracellular ice formation. This RWC was 77% in the C3 and 44% in the C4 subspecies and demonstrates that extracellular ice has the potential to cause substantially greater desiccation of cells in the C4 leaves. |
Discussion |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionConclusionsReferences |
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Temperature limitation of photosynthesis and growth
Photosynthesis under light-limited and light-saturated conditions and the rate of leaf growth were all greater in the C4 than the C3 subspecies at high temperatures (Figs 1, 3), supporting our first hypothesis. However, the C4 advantage was maintained at significantly lower temperatures than anticipated, with a crossover temperature range beginning at
17 °C for CO2, and 15 °C for Asat and LER, and no evidence of higher values in the C3 plants at any temperature (Figs 1, 3). On the basis of these data, a photosynthetic advantage for the C3 over the C4 subspecies of A. semialata between 10 °C and 15 °C cannot be excluded. However, given the occurrence of photoinhibition in this temperature range, it seems likely that the distribution of the C3 subspecies to higher latitudes and altitudes than its C4 sister is driven by alternative mechanisms.
Values of CO2 at 30 °C were 0.070–0.078 mol mol–1 for the C4 and 0.040–0.048 mol mol–1 for the C3 subspecies (95% confidence intervals). The C3 values are slightly lower, and the C4 marginally higher than ranges previously measured for C3 and C4 grasses (C3 range 0.052–0.056 mol mol–1, C4 range 0.060–0.069 mol mol–1; Ehleringer and Pearcy, 1983). Therefore, although the temperature dependency of quantum yield in each subspecies is typical of C3 and C4 photosynthesis, a difference in absolute values results in a lower crossover temperature than expected (Ehleringer et al., 1997). Errors in CO2 of ±10% may have been introduced by the inaccurate estimation of leaf area. However, there is no reason to expect this factor to introduce a major systematic bias, and it is therefore suggested that the low crossover temperature may be a real effect rather than an artefact.
In theory, the C4 photosynthetic pathway has the potential to achieve higher light-saturated CO2-fixation rates than the C3 type at any temperature, given equal investment in Rubisco and insensitivity of the photosynthetic apparatus to chilling injury (Long, 1999). In practice though, C4 plants typically accumulate significantly less Rubisco than C3 species (Long, 1999), and the calculated values of Amax (for the C4) and Vc,max (for the C3) at 25 °C in our experiment (Table 1) are consistent with a 3-fold lower capacity of the enzyme in the C4 than C3 subspecies. In the absence of other limitations, the amount of Rubisco in C4 leaves can become a major constraint on CO2-fixation at low temperatures (Pittermann and Sage, 2000). However, the observed decreases in Vp,max and Amax for the C4 plants between 25 °C and 15 °C (Table 1) are also consistent with a lower capacity for PEP regeneration and a decline in PEP carboxylase (PEPC) activity at sub-optimal temperatures. Both in vitro (Chinthapalli et al., 2003) and in vivo (Chen et al., 1994) studies of PEPC show temperature-dependency of enzyme activity at 15 °C (reviewed by Sage and Kubien, 2007), but cold-lability of enzymes in the PEP regeneration pathway typically occurs only at temperatures <10 °C (reviewed by Long, 1999).
The limitation of Asat by sub-optimal temperatures was significantly less pronounced in the C3 than C4 subspecies (Fig. 1), and is consistent with the opposing responses of Rubisco specificity and photosynthetic capacity (catalytic rate of Rubisco and RubP regeneration rate) to temperature (Long, 1991; Bernacchi et al., 2001). The 15 °C crossover temperature range for Asat may therefore be explained in terms of four interacting factors: (i) the lower activity of Rubisco in C4 than C3 leaves; (ii) the decreased activity of C4 cycle enzymes at 15 °C relative to 25 °C; (iii) elimination of photorespiration in the C4 leaves, which makes photosynthesis directly proportional to photosynthetic capacity; and (iv) the increase in apparent (in vivo) Rubisco specificity, which offsets decreases in its catalytic rate and the RubP regeneration rate of C3 leaves at low temperatures.
Light- and chilling-mediated photodamage
An 8 h exposure of leaves to a temperature of 15 °C under high PPFD led to inhibition of photosynthetic CO2-assimilation and slow-relaxing (>15 min) quenching of chlorophyll fluorescence in both subspecies (Fig. 4), suggesting damage to photosystem 2 reaction centres. This photodamage was exacerbated by acute exposure to freezing and high PPFD over a period of hours (Fig. 4) or chronic exposure to chilling in the range 5–15 °C and lower PPFD over a period of days (Fig. 6). In the latter case, major decreases in qP and Fv/Fm were associated with declining NPQ (Fig. 6), implying uncoupling of photochemistry from the thylakoid proton gradient and/or temperature-mediated decreases in xanthophyll cycling (Bilger and Björkman, 1991).
The observation of chilling-induced photodamage in both C3 and C4 leaves conflicts with our a priori expectation of higher thresholds for chilling injury in the C4 subspecies. Rather, it supports the alternative hypothesis that susceptibility to chilling injury in A. semialata is related to its tropical ancestry. A tropical origin for A. semialata is suggested by the overlap in Equatorial Africa of the distributions for all five species in the Alloteropsis genus (Ellis, 1981). This idea that chilling injury in C4 plants is a consequence of their ancestry, rather than an inherent weakness in their photosynthetic pathway, is supported by observations of chilling tolerance in C4 species which originate from high latitude and altitude habitats (e.g. Miscanthus, Beale et al., 1996; Bouteloua, Pittermann and Sage, 2000; and Muhlenbergia, Kubien and Sage, 2004).
Freezing injury and cold acclimation
Freezing caused leaf injury in unhardened plants of both subspecies under illuminated conditions, but a 2-week chilling pretreatment allowed cold acclimation of the C3 subspecies (Fig. 5). The C4 plants failed to develop the same protection, and suffered the same levels of injury as unhardened plants (Fig. 5). This result demonstrates that, although the C4 subspecies is not more vulnerable to chilling injury, it is significantly more sensitive to freezing. Since both C3 and C4 subspecies have diverged recently from a (sub)tropical common ancestor, differential freezing sensitivity cannot be attributed to the different climatic histories of C4 and C3 evolutionary lineages. Instead two alternative, but not mutually exclusive, interpretations of these results are offered.
First, they could represent ecotypic differentiation between the subspecies that is not directly related to their photosynthetic pathway, but correlated to their differing geographical distributions. The C4 subspecies of A. semialata occupies a range stretching from tropical Australia, through the Asian and African wet tropics, and into seasonally arid subtropical regions of southern Africa. The C3 subspecies co-occurs with its C4 sister in subtropical South Africa, but extends to higher altitudes and further polewards (Ellis, 1981). Although these contrasting distributions are likely to be linked to the difference in photosynthetic pathway, they may also generate ecotypic differences between the subspecies that are not. In the regions of southern Africa where A. semialata is found, winter is characterized by freezing events, but is typically dry (New et al., 1999), and early spring is therefore the main fire season in this region (Carmona-Moreno et al., 2005). A failure to protect leaves from frost in the C4 subspecies could therefore represent an adaptive response to other features of the environment, such as high soil water deficit or fire risk (Ripley et al., 2008). In contrast, the geographic range of the C3 subspecies is characterized by a lower fire risk (Giglio et al., 2006), and an increased probability of winter rainfall (Vogel et al., 1978), so that leaf retention during winter could bring important fitness benefits.
Secondly, cold acclimation mechanisms could be less effective in C4 than C3 plants, or more costly in metabolic terms, perhaps reflecting a fundamentally lower phenotypic plasticity in C4 species (Sage and McKown, 2006). However, field observations during the growing season and manipulation experiments have demonstrated that the leaves of some C4 grass species can resist freezing injury at temperatures of < –10 °C (Sage and Sage, 2002; Márquez et al., 2006; Sage and Kubien, 2007), and may develop frost protection during exposure to chilling (Rowley et al., 1975; Rowley, 1976; Stair et al., 1998). Vulnerability to freezing is therefore not inevitable in C4 grasses; however, most fail to develop significant cold acclimation, and are highly sensitive to freezing (Rowley et al., 1975; Rowley, 1976; Ivory and Whiteman, 1978). These data suggest that this mechanism may be ecologically relevant, and should be considered when explaining C4 grass distributions in relation to temperature.
Two of our experiments suggested that freezing injury in the C4 subspecies of A. semialata may be mediated by plant hydraulics. Preliminary data from experiment 3 indicated that a frozen soil exacerbated damage to C4 photosynthesis, suggesting that ice in the root system interrupted the water supply to thawing leaves. Experiment 5 demonstrated a radically different pattern of moisture release in C3 and C4 leaves; with the potential for significant cellular desiccation by extracellular ice in the C4 that is avoided by the C3, and a lower L,sat in the C4 leaves (Table 2). In combination, these data suggest that freezing injury may be caused by ‘frost drought’, and are consistent with scanning electron microscopy (SEM) observations in other freezing-sensitive or unhardened species (Ashworth and Pearce, 2002; Ball et al., 2004). However, the mechanism that differentiates C3 and C4 species, and its links (if any) to the photosynthetic pathway, remains unknown. One possibility is that suberization of the bundle sheath causes hydraulic isolation from the mesophyll, and makes bundle sheath cells particularly vulnerable to ice damage (Ashworth and Pearce, 2002), but this idea requires further investigation.
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Conclusions |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionConclusionsReferences |
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These experiments have demonstrated significant advantages of the C4 over the C3 pathway for photosynthesis and growth at high temperatures. These were sustained at lower temperatures than expected, and the C4 advantage was only lost in the temperature range
17 °C. However, there was no evidence of a reversal below this point, since both subspecies suffered photodamage on exposure to chronic chilling or acute freezing events. Unhardened leaves of both subspecies suffered freezing injury, but the C3 leaves developed protection via a cold acclimation mechanism that was absent in the C4 plants. Acclimation to low temperature extremes, rather than differential limitation of photosynthesis may, therefore, represent the primary mechanism that discriminates C3 and C4 members of this subtropical species in cool climates.
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Acknowledgements |
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We thank Steve Long for helpful discussions on aspects of this work. This research was supported by a University Research Fellowship from The Royal Society (CPO), an Early Career Project Grant from the British Ecological Society (EJW, CPO), a postgraduate studentship from the Natural Environment Research Council (NERC) (DGI), and a research grant from the South African National Research Foundation (NRF) (BSR).
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Abbreviations |
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A, net leaf photosynthesis (µmol m–2 s–1);; Asat, the light-saturated value of A (µmol m–2 s–1);; Amax, the CO2-saturated value of Asat (µmol m–2 s–1);; Ca, atmospheric pCO2 (Pa);; Ci, intercellular pCO2 (Pa);; DMC, leaf dry matter content (%);; Fv/Fm, the quantum efficiency of photosystem 2 after 15 min dark adaptation (dimensionless);; Jmax, the apparent maximum rate of photosynthetic electron transport in vivo (µmol m–2 s–1);; LER, linear leaf extension rate (mm h–1);; NPQ, fast-relaxing non-photochemical quenching (dimensionless);; pCO2, partial pressure of CO2 (Pa);; PEP, phosphoenolpyruvate;; PPFD, photosynthetically active photon flux density (µmol m–2 s–1);; qP, photochemical quenching, the fraction of Fv/Fm realized under growth conditions (dimensionless);; RH, relative humidity (%);; RWC, relative water content (%);; Rubisco, RubP carboxylase/oxygenase;; RubP, ribulose-1,5-bisphosphate;; Smax, maximum water content of the symplasm (%);; TLP, turgor loss point on the basis of
or RWC;; Tmin, minimum air temperature (°C);; Vc,max, the apparent maximum carboxylation activity of Rubisco in vivo (µmol m–2 s–1);; Vp,max, the apparent maximum PEP carboxylation rate in vivo (µmol m–2 s–1), assuming that PEP is saturating;; VPD, leaf–air vapour pressure deficit (kPa);; , Young's modulus of elasticity (MPa);; CO2, the maximum quantum yield of CO2-fixation on an absorbed light basis (mol CO2 mol–1 photons);; PS2, the operating efficiency of photosystem 2 under growth conditions (dimensionless);; , water potential (MPa);; L, leaf water potential (MPa);; L,sat, the value of L at 100% RWC (MPa);; p, leaf pressure potential (MPa);; , leaf osmotic potential (MPa);; ,sat, the value of at 100% RWC (MPa). |
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