Journal of Experimental Botany, Vol. 52, No. 357, pp. 829-838,
April 15, 2001
© 2001 Oxford University Press
Original Papers |
The response of the high altitude C4 grass Muhlenbergia montana (Nutt.) A.S. Hitchc. to long- and short-term chilling
Department of Botany, University of Toronto, 25 Willcocks St., Toronto, Ontario, Canada M5S3B2
Received 22 June 2000; Accepted 8 November 2000
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The acclimation of C4 photosynthesis to low temperature was studied in the montane grass Muhlenbergia montana in order to evaluate inherent limitations in the C4 photosynthetic pathway following chilling. Plants were grown in growth cabinets at 26 °C days, but at night temperatures of either 16 °C (the control treatment), 4 °C for at least 28 nights (the cold-acclimated treatment), or 1 night (the cold-stress treatment). Below a measurement temperature of 25 °C, little difference in the thermal response of the net CO2 assimilation rate (A) was observed between the control and cold-acclimated treatment. By contrast, above 30 °C, A in the cold-acclimated treatment was 10% greater than in the control treatment. The temperature responses of Rubisco activity and net CO2 assimilation rate were similar below 22 °C, indicating high metabolic control of Rubisco over the rate of photosynthesis at cool temperatures. Analysis of the response of A to intercellular CO2 level further supported a major limiting role for Rubisco below 20 °C. As temperature declined, the CO2 saturated plateau of A exhibited large reductions, while the initial slope of the CO2 response was little affected. This type of response is consistent with a Rubisco limitation, rather than limitations in PEP carboxylase capacity. Stomatal limitations at low temperature were not apparent because photosynthesis was CO2 saturated below 23 °C at air levels of CO2. In contrast to the response of photosynthesis to temperature and CO2 in plants acclimated for 4 weeks to low night temperature, plants exposed to 4 °C for one night showed substantial reduction in photosynthetic capacity at temperatures above 20 °C. Because these reductions were at both high and low CO2, enzymes associated with the C4 carbon cycle were implicated as the major mechanisms for the chilling inhibition. These results demonstrate that C4 plants from climates with low temperature during the growing season can fully acclimate to cold stress given sufficient time. This acclimation appears to involve reversal of injury to the C4 cycle following initial exposure to low temperature. By contrast, carbon gain at low temperatures generally appears to be constrained by the carboxylation capacity of Rubisco, regardless of acclimation time. The inability to overcome the Rubisco limitation at low temperature may be an inherent limitation restricting C4 photosynthetic performance in cooler climates.
Key words: Chilling, C4 plants, Muhlenbergia, photosynthesis, temperature response, Rubisco.
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Introduction |
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C4 plants often dominate high light habitats from warm climates, but are uncommon above 50° latitude and 2000 m elevations where minimum growing season temperatures average less than 8 °C (Long, 1983
). Numerous explanations have been proposed for failure of most C4 species to occur in cold climates. On the one hand, the C4 pathway may be inherently limiting at low temperature because chilling damages one of the steps of the C4 pathway. In particular, phosphoenolpyruvate (PEP) regeneration by PEP carboxykinase or pyruvate Pi-dikinase (PPDK) has been implicated as the leading cold-sensitive step that curtails C4 photosynthesis in colder climates (Long, 1983; Leegood and Edwards, 1996). Long-term chilling experiments with Echinochloa crus-galli, for example, show a reduction in the activity of PPDK following a 1–3 d exposure to chilling temperature that is correlated with reduced photosynthetic performance (Potvin et al., 1986; Simon and Hatch, 1994). Similarly, 14 d chilling of the warm-adapted grass Zoysia japonica caused a loss of PEP carboxykinase capacity that was correlated with a 75% decrease in CO2 assimilation rate (Matsuba et al., 1997). Alternatively, photosynthetic pathway may be of relatively little importance in cooler settings, with prior adaptation to tropical conditions minimizing the success of most C4 species in cold climates (Ehleringer and Monson, 1993; Long, 1999). Because the C4 syndrome is putatively of recent evolutionary origin in tropical climates (Ehleringer et al., 1997), there may have been insufficient time for more than a few C4 species to evolve chilling tolerance and invade low temperature habitats. As with tropical C3 species, multiple lesions may follow chilling exposure in most C4 plants, both within the photosynthetic apparatus and throughout the whole plant.
To avoid complications associated with adaptation to warm climates, the question of whether there are inherent weaknesses of the C4 pathway at low temperature should be addressed using C4 species originating from the cold-extremes of the C4 geographic range. Numerous C4 species from cold climates have been identified, most often in the genera Bouteloua, Miscanthus, Muhlenbergia, and Spartina (Long, 1999; Sage et al., 1999). These species are noted for stable photosynthesis rates at low temperatures, and do not show chilling injury in the C4 cycle enzymes as observed in species originating in low latitudes. For example, in Spartina anglica, a PCK monocot from cool temperate salt marshes, C4 cycle enzymes are not inhibited and A shows a 43% increase following chilling treatment (Matsuba et al., 1997). Similarly, the montane genus Miscanthus from east Asia has numerous species that exhibit stable photosynthetic capacity at low temperature and are able to produce high yields despite chilling (Long, 1999). From these studies, it is apparent that the photosynthetic apparatus of C4 plants is not necessarily prone to failure in cold conditions, and thus chilling injury may not be the ultimate explanation for the general absence of the C4 syndrome from cold climates. These observations, however, do not address whether overall performance of C4 photosynthesis is poor relative to C3 photosynthesis at low temperature. While the C4 photosynthetic apparatus may be stable at low temperature, it remains possible that there is an inherent limitation preventing C4 plants from matching the performance of C3 species. Inferior quantum yield of C4 relative to C3 plants is an example of a performance limitation that may restrict C4 photosynthesis in cool, shady habitats (Ehleringer et al., 1997). Lower quantum yield, however, should not prevent C4 occurrence in high light habitats of low temperature.
In a recent report, the temperature response of net CO2 fixation was evaluated in Bouteloa gracilis from the Rocky Mountains, USA (Pittermann and Sage, 2000). No evidence was observed for dissociation of C4-cycle enzymes at low temperature, and photoinhibition was minor under the experimental conditions used. Rubisco has been implicated as an important control over C4 photosynthesis at moderate to low temperature (Björkman and Pearcy, 1971; Pearcy, 1977; Caldwell et al., 1977). Consistently, the photosynthetic response to temperature in Bouteloua gracilis was equivalent to the temperature response of the Rubisco Vmax. below 17 °C (Pittermann and Sage, 2000). These results indicate the low Rubisco content of C4 relative to C3 plants may be an important impediment to the success of C4 species in cold climates. Unlike C3 species that have an abundance of Rubisco in all photosynthetic cells, Rubisco in C4 species is restricted to the bundle sheath. On a leaf area basis, Rubisco quantity in a C4 leaf is typically one-quarter to one-third that of a C3 plant with an equivalent photosynthesis rate at 25 °C to 30 °C (Sage and Pearcy, 2000). Because of the high thermal dependence of the Rubisco Vmax, the in vivo capacity of Rubisco below 10–15 °C is low relative to its value above 25 °C. Thus, because C4 species have low quantities of Rubisco, they are prone to also have lower photosynthetic potential at cool temperatures than C3 species (Leegood and Edwards, 1996; Pittermann and Sage, 2000). Also, by directly limiting carbon gain at low temperature, low Rubisco capacity could predispose C4 plants to photoinhibition.
In addition to the genus Bouteloua, the other leading C4 group from high altitude regions of North America is the grass genus Muhlenbergia. A number of Muhlenbergia species occur above 3300 m, making it North America's highest C4 genus. The Rocky Mountain species Muhlenbergia montana (NAD-malic enzyme subtype; Hattersley and Watson, 1992) occurs to at least 3100 m in the sub-alpine zone. To complement the prior work with B. gracilis, the research reported here examined the temperature response of photosynthesis and Rubisco activity in a high elevation ecotype of Muhlenbergia montana. In addition to short-term temperature responses of net CO2 assimilation and Rubisco activity, the acclimation responses of photosynthesis and the PSII redox state to chilling conditions were investigated.
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Plant material
Muhlenbergia montana tillers were obtained in the vicinity of Kenosha Pass, Colorado (39°25' N, 105°45' W; 3100 m above sea level) and planted into 4.0 l pots of 60% Pro-mix (Premier Brands, Redhill, Pennsylvania), 20% perlite and 20% sand. All grasses were initially grown outdoors during August of 1996. They were subsequently transferred to a growth cabinet (Conviron E-15, Conviron Ltd. Winnipeg, Manitoba) where light intensity and temperature were controlled to gradually increase from a 16 °C, 8 h dark period to a 26 °C, 16 h light period at a maximum PPFD of 700 µmol m-2 s-1. All plants were fertilized weekly with a half-strength Hoaglands solution.
Assessments of leaf gas exchange, fluorescence and Rubisco activity
In alpine environments, plants may be exposed to high temperatures during the day, but may be subject to overnight lows near freezing (Jordan and Smith, 1995). The acclimation potential of M. montana was examined by subjecting a subset of the 26/16 °C-grown tillers to 4 °C overnight (8–10 h) conditions for 4 weeks, at which time gas exchange comparisons of photosynthetic responses to temperature and intercellular partial pressure of CO2 commenced. Leaf gas exchange was measured as described earlier (Pittermann and Sage, 2000), using a null balance, thermally-controlled gas exchange system modelled after that described previously (Sharkey, 1985; Field et al., 1989). In all gas exchange measurements, photosynthetic photon flux density was controlled to be slightly above the photosynthetic light saturation point in order to allow for improved thermal control at lower measurement temperatures. Because the light saturation point declined with temperature (Fig. 1), this meant that the measurement PPFD was reduced from above 1800 µmol photons m-2 s-1 at the warmer temperatures to near 800 µmol m-2 s-1 at the cooler temperatures. Vapour pressure difference between leaf and air was set between 8–12 mbar except above 30 °C where it was allowed to rise to near 20 mbar.
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The response of A to temperature was determined by first equilibrating leaves at the daytime growth temperature of 26 °C and saturating light levels for 30–60 min. After this, temperature was increased to near 40 °C and then decreased in steps of approximately 4 °C, with measurements at each step. The exposure to temperature near 40 °C was not observed to depress A subsequently measured at 26 °C, indicating the measurement procedure was sound. The response of A to intercellular CO2 was determined by first equilibrating leaves at 26 °C, light saturation, and air levels of CO2, and then stepping the measurement temperature up to 33±1 °C. After equilibration, the ambient partial pressure of CO2 was increased to approximately 450 µbar, and then reduced in a series of steps to near the CO2 compensation point. Gas exchange parameters were determined at each step after a 15–30 min equilibration period. After completion of the response, the ambient CO2 level was returned to approximately 360 µbar, allowed to stabilize, and A was determined again. If A was within 10% of the original value, the temperature was reduced to 26 °C and the A versus Ci response was again measured as done at 33 °C. After completion of this curve, the procedure was repeated at 13 °C.
During measurements of the temperature response of leaf gas exchange, fluorescence parameters were assessed with an OptiScience OS-500 (Haverhill, MA) modulated fluorimeter. Coefficients of photochemical and non-photochemical quenching (qp and qnp, respectively), the electron-transport rate, and photochemical yield were measured for the 26/16 °C treatment only. Photochemical quenching equalled (Fms –Fs)/Fms-Fod, and qnp equalled Fm-Fms/Fm–Fod, where Fms is maximum fluorescence in the presence of actinic light, Fs is steady-state fluorescence in actinic light, Fm is the maximum fluorescence of dark-acclimated leaves, and Fod is the minimal fluorescence of darkened leaves in the presence of post-actinic far red illumination (Schreiber et al., 1995, as modified in the OS-500 manual).
To determine the effect of short-term chilling on photosynthetic parameters, plants grown at 26/16 °C were transferred to 4 °C for 1 night. The following morning, the grasses were placed in a growth cabinet at 23 °C and 150 µmol photons m-2 s-1 for 1–2 h. The temperature and CO2 responses of A were then determined as described above. To evaluate photoinhibition following the start of night chilling, dark-adjusted Fv/Fm was measured over a 12 d period after transfer of M. montana plants from 16 °C to 4 °C nights. Fv/Fm was measured 30 min after dark exposure on five tillers that were sampled at 4 °C just before morning (time 0 measurement), and 4, 6 and 8 h into the 26 °C day cycle.
Rubisco activity was measured as described previously (Sage et al., 1993; Pittermann and Sage, 2000). Briefly, multiple leaves from at least three fully illuminated plants grown at 26/16 °C were sampled by clipping and rapidly immersing in liquid N2. Samples were then stored in liquid N2 until assay. Activity assays consisted of determining the rate of 14C-CO2 incorporation into acid stable products following a 5–20 min incubation in 15 mM MgCl2 and 10 mM NaHCO3 to activate the extracted Rubisco fully. This procedure produced kcat values of 38 mol CO2 mol-1 Rubisco s-1 at 28 °C for M. montana Rubisco. These are similar to the high kcat values reported for a range of C4 species (Seemann et al., 1984; Sage and Seemann, 1993), indicting our assay procedures were sound.
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Photosynthetic acclimation of M. montana to low night temperature
In Muhlenbergia montana plants grown at 26/16 °C, the saturating light intensity was approximately 1700 µmol m-2 s-1 at 23 °C and 33 °C, but declined at lower temperature such that at 13 °C, it was near 1000 µmol m-2 s-1 (Fig. 1
). Subsequent gas exchange analysis was conducted at 1800 µmol photons m-2 s-1 above 20 °C, and 800–1500 µmol m-2 s-1 below 20 °C.
Initially, the gas exchange and fluorescence responses of plants grown at 26/16 °C day/night temperature (the control treatment) were compared with responses of plants grown at 26/4 °C for over 28 d (the chilled treatment). The temperature response of CO2 assimilation in control and chilled plants was unaffected by growth temperature below a measurement temperature of 25 °C, but diverged above a measurement temperature of 30 °C (P<0.05; Fig. 2). Grasses grown at 26/4 °C reached a maximum photosynthetic rate of 36 µmol m-2 s-1 at a thermal optimum of 36 °C, which is approximately 10% higher than A observed in the 26/16 °C plants. In both chilled and control plants, A and Rubisco activity respond similarly to temperature below 22 °C. Above 25 °C, the temperature response of Rubisco activity diverged from the photosynthetic response, with Rubisco exhibiting a maximum activity that was nearly three times the rate of net CO2 assimilation at 39 °C (Fig. 2).
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Growth at either 26/16 °C or 26/4 °C had no obvious effect on stomatal responses to temperature below 30 °C (Fig. 3
). Stomatal conductance in both cold-hardened and control grasses increased from approximately 0.1 mol m-2 s-1 at 7 °C to 0.3 mol m-2 s-1 at 30 °C. Relative to the warm-grown plants, the conductance was higher in the 26/4 °C tillers at 33–40 °C (Fig. 3A). The ratio of intercellular to ambient CO2 remained constant across a range of temperatures in both chilled and non-chilled M. montana tillers, despite an increase of VPD above 28 °C (Fig. 3B). As a result, Ci corresponding to air levels of CO2 was over 150 µbar for grasses from both growth regimes, a level that was above the CO2 saturation point of A (Fig. 4).
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A reduction in temperature from 33 °C to 13 °C had little effect on the initial slope of the CO2 response in either treatment; in contrast, the CO2-saturated plateau of A declined dramatically with temperature reduction from 33 °C to 13 °C (Fig. 4
). Consequently, the CO2 saturation point of A declined with temperature reduction in plants from each growth regime, from approximately 100 µbar at 33 °C to 75 µbar at 23 °C, and then to below 50 µbar at 13 °C.
The short-term fluorescence response to temperature in M. montana
The fluorescence response to decreasing temperature in M. montana grown at 26/16 °C showed that photochemical quenching (qp) declined from 0.93 at 30 °C to 0.22 at 7 °C (Fig. 5B). By contrast, non-photochemical quenching (qn) varied little over the thermal spectrum, so that the change in fluorescence yield with temperature largely reflected the change in photochemical quenching. The low variation observed in qn reflects the modulation of PPFD to maintain it near the light saturation point of A. Using the yield value, an assumed leaf absorbance of 0.84 and the prevailing PPFD during a measurement, the in vivo temperature response of electron transport rate was expressed relative to the rate at 11 °C to facilitate comparison with A and Rubisco activity (Fig. 5C). Electron transport rate showed a similar relative response to temperature as Rubisco and A below 20 °C, but diverged from the two above this temperature.
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Exposure of non-hardened M. montana plants to low temperature
To test the short-term effect of a reduction in temperature on M. montana plants acclimated to 26/16 °C, several tillers were placed in a 4 °C chamber overnight and the temperature and CO2 response of A was determined the following day. Below 20 °C, there was little difference in the response of A to temperature between the tillers exposed to cold for 1 night versus 28 (Fig. 6A). Above 20 °C, however, the maximum CO2-assimilation rate was 35% less in the non-acclimated, stressed plants relative to the cold-acclimated plants, and the thermal optimum of A was reduced 7 °C after the first night at 4 °C. The stress treatment also reduced both the initial slope and the CO2-saturated rate of A by a large amount (Fig. 6B). For example, after the initial 4 °C exposure, A in the non-acclimated plants was reduced by over 60% at a Ci of 50 µbar compared to the cold-acclimated grasses. After the first night at 4 °C, dark-adapted Fv/Fm declined from 0.77 in the early morning to 0.70 8 h later (Fig. 7). The Fv /Fm ratios were near 0.78 after 7 and 12 d of 4 °C night treatment, indicating full recovery. Chlorosis and other leaf discoloration were not observed in any tillers during the chilling exposure.
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The temperature response of A in plants grown at 26/16 °C and 26/4 °C
Muhlenbergia montana is tolerant of low temperatures given sufficient time to acclimate. Although there was a substantial depression of photosynthetic activity the day after initial exposure of non-acclimated plants to chilling temperatures, this depression was gone after 4 weeks of growth at low night temperature. Because M. montana plants at higher elevation will routinely experience chilling during the growing season, it is probable that they generally exist in a state of chilling tolerance. Therefore, long-term chilling does not induce distinctive lesions within the CO2-concentrating mechanism that would prevent C4 success in cold climates. Instead, performance limitations associated with Rubisco capacity may be more important in determining C4 distribution in cold climates.
In C4 plants, Rubisco operates near CO2 saturation, in contrast to C3 species where it operates well below CO2 saturation except at low temperature (Leegood and Edwards, 1996; Pittermann and Sage, 2000). As a result, in vitro Rubisco capacity should reflect photosynthetic capacity in C4 plants under conditions where Rubisco is limiting A (von Caemmerer and Furbank, 1999). In M. montana (Fig. 2) and Bouteloua gracilis (Pittermann and Sage, 2000), Rubisco capacity is very similar to photosynthetic capacity below 18 °C, indicating high Rubisco control of A. Above 20 °C, Rubisco capacity rises well above A, indicating little metabolic control. This interpretation is also supported by analysis of the CO2 response of A. In M. montana, the occurrence of the operational Ci above the CO2-saturation point at all measurement temperatures demonstrates that stomatal limitations have no effect on the temperature response of A below the thermal optimum. Instead, the temperature response of A depends upon the biochemical processes controlling the CO2-saturated rate of A. In C4 species, the CO2-saturated rate of A primarily reflects either the capacity of Rubisco, the capacity of electron transport to regenerate RuBP, or pyruvate-Pi dikinase activity (von Caemmerer and Furbank, 1999). Reductions in Rubisco content by antisense procedures reduce the CO2-saturated plateau of A, but have little effect on the initial slope of the CO2 response of A (Furbank et al., 1996, 1997; von Caemmerer et al., 1997). Consistent with a Rubisco limitation, low temperature had a large effect on the CO2-saturated rate of A, but had little effect on the initial slope of the A versus Ci response.
In C4 plants. PEPCase is a major limitation in the initial slope region of the A/Ci response at low CO2 (von Caemmerer and Furbank, 1999). For example, in Amaranthus edulis, PEPCase accounts for nearly 70% of the metabolic control of A at a Ci of 30 µbar, but only 20–30% of the control in air at 25 °C (Dever et al., 1997). When A is reduced by reductions in PEPCase activity, the initial slope is reduced, as shown by transformation using PEPCase-deficient mutants (Dever et al., 1997) and PEPCase inhibitors (Brown and Byrd, 1993). In M. montana, the initial slope of the A/Ci response is little affected by low measurement temperature, indicating little controlling role of PEPCase at air levels of CO2 and cooler temperatures.
The response of A versus Ci in C4 species cannot readily be used to differentiate between limitations in electron transport versus Rubisco capacity, as reductions in each can have similar qualitative effects on the A/Ci response (von Caemmerer and Furbank, 1999). The fluorescence data, however, does evaluate whether electron transport capacity is a principal control at low measurement temperature. When the relative response of Rubisco capacity, A and the electron transport rate are compared, similar relative responses below 20 °C were observed, indicating the low temperature response of electron transport rate in M. montana is largely a function of the temperature response of photochemical quenching. Changes in photochemical quenching often reflect limitations in carbon metabolism enzymes, for example, limitations in Rubisco capacity (Labate et al., 1990; Furbank et al., 1996). Because the Vmax of Rubisco appears to establish a maximum ceiling for CO2 fixation, it may also determine the rate of photochemical quenching. The rate of electron transport in vivo could, in turn, be regulated to match this limitation.
The leading mechanism proposed for failure of C4 species to occur in cold climates has centred around cold-lability of PPDK (Long, 1983; Simon and Hatch, 1994). PPDK from warm-adapted species dissociates from its tetramer state upon exposure to temperatures less than 11 °C, although PPDK forms from cold-adapted species appear more stable at low temperature (Edwards et al., 1985; Leegood and Edwards, 1996). Modelled responses of A to Ci in C4 plants indicate that a limitation in PEP regeneration via PPDK decreases the CO2-saturated plateau of A in a manner similar to that observed in M. montana at lower temperature (von Caemmerer and Furbank, 1999; Fig. 4). Because of this, the results presented here do not rule out a PPDK limitation at low temperature. In cold-acclimated M. montana plants, however, there is little evidence that PPDK dominates metabolic control over A at low temperature. Should cold-lability cause PPDK activity to become limiting at low temperature, A in the grasses grown at 26/4 °C would be less between 7 °C and 36 °C relative its value in the grasses grown at 26/16 °C. This response was not observed—the CO2 assimilation rate was equal in both chilled and warm-acclimated M. montana below 20 °C (Fig. 1A). Furthermore, an enhancement of photosynthesis occurred at the thermal optimum of the cold-hardened plants, indicating acclimation to chilling increases the capacity of the step that controls A at the thermal optimum. Should PPDK share in the metabolic control of A at low temperature, its level of control would at most be close to that of Rubisco, given the similarity between the Rubisco Vmax response and A below 20 °C.
While results with M. montana show no evidence for a photosynthetic lesion in cold-acclimated plants, there is significant inhibition in the rate of photosynthesis in non-acclimated plants the day after initial exposure to 4 °C nights. This inhibition is associated with marked reduction in both the initial slope of the CO2-response of A and the CO2-saturated rate of A above 20 °C. Notably, there is little change in A below 18 °C where Rubisco control is hypothesized to be high. Photoinhibition is slight as indicated by a modest reduction in Fv/Fm in the dark. These results indicate that the cause of the reduction in A the day after initial night chilling is not Rubisco capacity nor a photoinhibitory lesion, but may be more likely a problem with electron transport capacity or one of the C4 cycle reactions. Electron transport probably does not become limiting in these conditions, based on gas exchange responses of C4 plants in which electron transport capacity has been selectively reduced. In C4 species, reduction in light intensity disproportionately reduces the rate of electron transport, and this in turn reduces A (von Caemmerer and Furbank, 1999). Light reduction affects only the CO2-saturated plateau of the A/Ci response in C4 plants, not the initial slope (Sage, 1986; Leegood and von Caemmerer, 1989; Peisker and Diez, 1990; Furbank et al., 1996), thus indicating that a disproportionate reduction in the rate of electron transport would have little effect on the initial slope, contrary to what was observed in the cold-stressed plants. The reduction in A at both high and low Ci is consistent with a marked reduction in either the capacity of PEPCase and PPDK (von Caemmerer and Furbank, 1999). The observation that the reduction in A occurs at warmer rather than cool temperatures is also consistent with a reduction in one of the C4 cycle enzymes. At warmer temperatures, the km of Rubisco for CO2 rises substantially, while the specificity for CO2 declines (Leegood and Edwards, 1996). Because of these changes in Rubisco kinetics, any lesion in the ability of the C4 cycle to concentrate CO2 in the bundle sheath would have a greater effect at warmer, rather than cooler temperatures.
The possible lesion in the C4 cycle of M. montana the day after initiating cold treatment is consistent with numerous studies observing cold-induced injury to PEPCase or PPDK in chilled C4 plants. In Echinochloa crus-galli, a reduction in both the initial slope and the CO2-saturated portion of the A/Ci curve occurs following transfer of plants to 14/8 °C (Potvin et al., 1986). This reduction is associated with a concurrent decrease in PPDK activity (Potvin et al., 1986; Simon, 1987). Loss of PEPCase and PEP carboxykinase activity accounted for a severe reduction in photosynthesis at 25 °C in Zoysia japonica following growth at 10/7 °C (Matsuba et al., 1997). Numerous other reports for PEPCase and PPDK decline in response to cold have been reported in a number of C4 species such as Panicum maximum and Digitaria sanguinalis (Leegood and Edwards, 1996). Notably, most of the species where cold-induced injury to C4 cycle enzymes occurred either originated in warm climates, or were associated with short-term responses where little time for acclimation was allowed (Leegood and Edwards, 1996; Pittermann and Sage, 2000).
In any case, during the acclimation process, the lesion that causes the photosynthetic depression in M. montana the day after first chilling is repaired and A recovers, eventually exceeding the rate of photosynthesis of control plants at the thermal optimum. This pattern is consistent with acclimation responses observed in C4 plants from colder climates such as Spartina anglica (Matsuba et al., 1997), Miscanthus species (Long, 1999), and Bouteloua gracilis from high (but not low) elevation (Bowman and Turner, 1993; Pittermann and Sage, 2000). In contrast to these cold-hardy C4 grasses, C4 species from warm habitats such as Zea mays and Zoysia japonica respond to prolonged chilling with increasing reduction of A (Matsuba et al., 1997; Long, 1999).
Temperature effects on the in vivo fluorescence response in Muhlenbergia montana
Photoinhibition is a characteristic symptom of low temperature stress in moderate to high light conditions (Huner et al., 1993). In warm-adapted grasses such as maize, the length of chilling exposure determines the degree of photodamage (Long, 1983, 1999), indicating that thermophillic species cannot easily acclimate to low temperature/high light conditions. By contrast, in M. montana and other cold-adapted C4 species such as Cyperus longus and Bouteloua gracilis, prolonged exposure to sub-optimal temperatures caused little observable damage to the PSII reaction centres during chilling treatments (Blowers and Baker, 1995; Long, 1999; Pittermann and Sage, 2000). Similarly, in cold-adapted C3 species, leaves are capable of full recovery of PSII quantum yield following exposure to 4 °C (Somersalo and Krause, 1990; Huner et al., 1993). This is because of increased rates of electron transport and carbon assimilation and the prolonged accumulation of carotenoids, notably zeaxanthin (Adams and Demmig-Adams, 1995). In M. montana, the high level of non-photochemical quenching across a range of temperature indicates that zeaxanthin formation may be a major means of dealing with excess excitation energy. Non-photochemical quenching primarily involves antenna-level dissipation of light energy by zeaxanthin (Demmig-Adams et al., 1996).
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In a companion study, photosynthetic responses to low temperature of the Rocky Mountain species Bouteloua gracilis were examined using similar techniques as here (Pittermann and Sage, 2000
). Consistent with results from M. montana, the key constraint in B. gracilis below 20 °C appeared to be Rubisco carboxylation, as its thermal response matched closely that of net CO2 assimilation. Little evidence was observed in either study for high control over A by C4 cycle enzymes of plants either acclimated to low temperature, or adapted to low temperature and grown in warmer conditions. Together, these results indicate that the failure of the C4 syndrome in cold environments is not because of a cold-induced lesion, but may be more the result of inferior performance at low temperature that is related to how C4 plants use Rubisco. The ceiling on A that Rubisco establishes below 18 °C could prevent C4 species from matching the photosynthetic performance of C3 competitors in cold climates. To overcome the Rubisco limitation, C4 plants would need to maintain much higher levels of Rubisco, potentially at considerable ecological cost. Some cold-adapted C4 grasses such as Spartina and Miscanthus have more Rubisco relative to C4 plants from warmer environments (Long, 1999), but whether this influences low temperature performance remains to be determined. To evaluate fully whether Rubisco content is a major inherent limitation over C4 performance in the cold, it will be important to examine the pattern of Rubisco use across a range of temperatures using C4 genotypes of varying Rubisco content.
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Acknowledgments |
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We are grateful to David Kubien for critical reading of the manuscript, and the technical assistance of Sam Puvendran. This study was supported by an NSERC grant No. OGP0154273 to RFS and a Strong-Hull Scholarship to JP.
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Notes |
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1 To whom correspondence should be addressed: Fax: +1 416 978 5878. E-mail: rsage{at}botany.utoronto.ca
2 Current address: Department of Biology, University of Utah, Salt Lake City, Utah 84112, USA.
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Abbreviations |
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A, the rate of net CO2 assimilation; Ci, intercellular partial pressure of CO2; PEPCase, phosphoenolpyruvate carboxylase; PPDK, pyruvate-phosphate-dikinase.
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References |
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