Global atmospheric carbon dioxide concentration ([CO2]) has increased since the industrial revolution (IPCC 2001, IPCC 2007). Atmospheric [CO2] in recent past was approximately 350 µmol mol-1 (IPCC 2001, IPCC 2007) and the current amount may even be higher (Tans 2010). By the year 2100, it is predicted that atmospheric [CO2] would be between 730 and 1020 µmol mol-1 (IPCC 2007). Increased [CO2] can affect plant ecosystem (Ceulemans and Mousseau 1994, Körner 2006), species richness and composition (Wang 2007, Langley and Megonigal 2010). At the individual plant level, increased [CO2] generally enhance leaf level photosynthesis, lower stomatal conductance to water vapour (gs) and transpiration rate (E), increase water and nutrient use efficiencies (Dang and Cheng 2004, Ainsworth and Rogers 2007, Cao et al. 2007, Huang et al. 2007, Ambebe et al. 2009) at least in the short term. The effects of increased [CO2] on plant growth and physiological processes may however, be limited by other factors such as soil temperature and nutrient status of the soil (Lukac et al. 2010).
Increased in atmospheric [CO2] is expected to be accompanied by a 1.4-5.8 oC increase in global air temperatures (IPCC 2001). Any increase in air temperature may result in increased soil temperature (Domisch et al. 2002) because both are determined by the energy balance at the ground surface and positively well correlates (Zheng et al. 1993). Soil temperature can affect plant's physiological processes such as carbon fixation, stomatal conductance, transpiration rate, nutrient uptake and nutrient re-translocation as well as carbon dioxide uptake (Cai and Dang 2002, Dang and Song 2004, Pregitzer and King 2005). The effects of increased atmospheric [CO2] and increased temperatures are expected to be highest in the boreal forest (IPCC 2007), where plant growth is limited by soil temperature and nutrient supply (Jarvis and Linder 2000).
In the boreal forest, the three most important factors that limit tree growth are soil temperature and the trees' ability to capture of CO2 and nutrients (Jarvis and Linder 2000). At present climatic conditions, the boreal forest is characterised by low soil temperatures (Domisch et al. 2002). However, there is evidence that the region has been warming up faster than other regions on earth (Serreze et al. 2000). Increased atmospheric [CO2] coupled with warmer temperatures may change species composition, competitive ability and resource use (Stewart et al. 1998). Two congeneric conifer species in the region that may be impacted greatly by these predicted changes are black spruce (Picea mariana (Mill). B.S.P) and white spruce (Picea glauca (Moench) Voss). These two species are the most widely distributed conifers in the boreal forest of North America (Nienstaedt and Zasada 1990, Sims et al. 1990, Viereck and Johnston 1990, Haavisto and Jeglum 1995). The Arctic Climate Impact Assessment (ACIA) climate models suggest that rapid warming of the boreal forest may not allow the growth of commercially valuable white spruce while the spread of black spruce in western Canada's boreal forest may be reduced on poor sites (Juday et al. 2005). On the other hand however, the increased in soil temperature and [CO2] can extend the species growing season and increase their photosynthetic rate and biomass production as long as there is enough nutrients and moisture (Strömgren and Linder 2002).
Black spruce grows on sites ranging from dry sands, gravels and shallow soils on bedrock through deep nutrient rich mineral soils on uplands to waterlogged nutrient deficient peatlands (Haavisto and Jeglum 1995). In Ontario's boreal forest, 54% of black spruce stands is on upland mineral soils with 46% on peatlands (Haavisto and Jeglum 1995). On the other hand, white spruce grows best on moist upland sites and on well aerated and well drained floodplains (Nienstaedt and Zasada 1990). On moist upland mineral soils, the species co-occur with or without other species such as jack pine (Pinus banksiana Lamb.) (Nienstaedt and Zasada 1990, Sims et al. 1990, Haavisto and Jeglum 1995). On peatlands and floodplains however, black spruce grows in association with white spruce only if aeration is adequate (Nienstaedt and Zasada 1990, Viereck and Johnston 1990, Haavisto and Jeglum 1995). The wide natural range of the two species makes them ideal to study in the context of increased [CO2] as temperature and nutrient status of the soils are also varied within the species range. Root growth of both species increase rapidly at soil temperatures above 10 oC (Grossnickle 2000). Soil temperature for optimum growth of black and white spruce seedlings under field conditions is between 19 and 21 oC (Heninger and White 1974, Tyron and Chapin 1983, Grossnickle and Blake 1985, Odlum and Ng 1995). Under controlled conditions, soil temperature of 15 oC may be optimum both for species root growth (Peng and Dang 2003). A soil temperature of 22 (black spruce) and 21 oC (white spruce) has also been reported as the optimum for maximum the species net photosynthetic rate (Dang and Cheng 2004). Maximum nutrient uptake in spruce occurs at soil temperatures of 20 oC under current CO2 levels (Gessler et al. 1998). Soil temperatures above this cause decline in root uptake of nitrate and ammonium (Gessler et al. 1998).
In this paper, we report on the physiological responses of boreal black and white spruce seedlings to the interactive effects of soil temperature and nutrient supply under current and projected atmospheric [CO2]. The specific objectives were to examine the: (i) photosynthetic rate of both species under mono and mixed growth conditions (ii) in vivo activities of Rubisco under mono and mixed growth conditions and (iii) photosynthetic nutrient use efficiency of the two species at mono and mixed growth conditions. Numerous studies (Grossnickle and Blake 1985, Grossnicke 2000, Cai and Dang 2002, Domisch et al. 2002, Dang and Cheng 2004, Ambebe et al. 2009, Cheng 2009) have reported on the effects of low soil temperature on boreal tree seedlings physiology. A concern that remains to be answered is whether growth at elevated [CO2] would cause an upward shift in the species current optimum soil temperature in response to increase warming associated with higher [CO2]. We attempt to address this by growing the seedlings in their current optimum soil temperature and at a projected warmer temperature. It was expected that the species current optimum soil temperature would shift upwards in response to increased [CO2] and result in greater photosynthetic rate in both species, especially when nutrients are adequate.
If any increase in boreal forest productivity in response to warmer temperatures and increased [CO2] is to be maintained, it will have to be accompanied by an increase in soil nutrient (Lukac et al. 2010). Studies on plant nutrition mostly focus on nitrogen (N) because it is the nutrient required in the largest quantity and most likely to limit carbon gain (Chapin et al. 1987). However, deficiencies of other nutrient elements have been reported (Reich et al. 2009). The rate of photosynthesis is dependent on leaf phosphorus concentration ([P]) (Herold 1980) and its deficiency inhibits photosynthesis (Lambers et al. 2008, Reich et al. 2009). It has also been shown that P supply influences partitioning of N, including to Rubisco (Warren and Adams 2002, Warren et al. 2005). Leaf potassium ([K]) affects photosynthesis through regulation of stomatal aperture (Pallardy 2007) and any imbalance may result in decreased photosynthetic rate (Pallardy 2007). Specifically, P and potassium (K) deficiencies have been observed in black spruce (Wells 1994, Teng et al. 2003) and in white spruce (Truong and Gagnon 1975). To address such limitation, we maintained a constant ratio of the three major nutrient elements (N, P and K). Low nutrient supply usually limit plant's photosynthetic rate (Aerts and Chapin 1999, Ainsworth and Long 2005). However, increased [CO2] can partially compensate for low nutrient supply in the short term, by decreasing the amount of Rubisco required to fix a given amount of carbon (Percy and Bjorkman 1983). While we expected that photosynthetic rate would be higher at elevated [CO2] and high nutrient supply, it was also hypothesised that the negative effects of low nutrient supply on photosynthesis would be partly compensated for by elevated [CO2], especially at the warmer soil temperature.
The stimulation of tree growth and function by enhanced [CO2] and warmer temperatures are species-specific (Engel et al. 2005, Lukac et al. 2010). This implies the potential to alter species composition, competitive ability and natural distribution of tree species in the future (Saxe et al. 2001). Questions that arise from such potential changes include whether congeneric black spruce and white spruce would respond differently to the interactive effects of soil warming and fertilisation under current and future [CO2] and whether the response would different when the species are grown isolated and together in mixture with one another. Black spruce can grow well on both rich upland sites and nutrient-poor peatlands while white spruce only grown well on fertile upland sites. Black spruce therefore appears physiologically more plastic than white spruce (Patterson et al. 1997). In response to nutrient demand, black spruce appears to have lower nutrient requirement and wider temperature range than white spruce (Viereck and Johnston 1990, Nienstaedt and Zasada 1990, Patterson et al. 1997, Way and Sage 2008). While black spruce may grow on wide of range sites, responses of plants to increased atmospheric [CO2] vary with the nutrient plasticity of the species (Brown and Higginbotham 1986). And species occurring on nutrient poor soils such as black spruce may have slower response to increased [CO2] and increased nutrient supply (higher NUE) than white spruce which usually grows on richer soils. Moreover, on fertile sites, fast growing species have high nutrient uptake kinetics (Jackson et al. 1990) and are able to deplete rich nutrient patches before slow growing species with low uptake kinetics are able to extract enough nutrients (Jackson et al. 1990, Caldwell et al. 1996, Lambers et al. 2008). Plants with high uptake kinetics may however be disadvantaged when nutrient supply is low because of greater energy requirements (Jackson et al. 1990). As such, species adapted to low soil fertility with lower uptake kinetics can out-compete species adapted to high nutrient supply, on poor sites (Aerts and Chapin 1999). It is therefore expected that white spruce will benefit more from the increased [CO2] and higher nutrient supply than black spruce. At the low nutrient treatment however, black spruce may have higher photosynthetic gains than white spruce. Because the two species have different nutrient requirements, nutrient utilisation would be maximised when the two species are grown together. As such, photosynthetic rate in the mixed-species experiment would be higher than in the mono experiments.
Materials and Methods
Plant Materials
The experiment was conducted at Lakehead University's Thunder Bay, Ontario Campus, from December 2009-April 2010. One year old black spruce (Picea mariana [Mill.] B.S.P.) and white spruce (Picea glauca [Moench] Voss.) seedlings raised in a commercial nursery were used for the study. All the seedlings were of uniform size and form at the beginning of treatment. Seedlings of each species were either grown isolated in a single pot (mono experiment) or combined together in one larger pot (mixed-species experiment). Soil volume of each seedling in the mono experiment was 1767.38 cm3. For seedlings in the mixed-species experiment, each plant was allocated a soil volume 10% less than each plant received in the mono experiment. This was to ensure that, there is below-ground plant-plant interaction but minimal above ground interaction. The growing medium was premium grade vermiculite-peat moss mixture (50:50, v/v).
Experimental Design and Growth Conditions
Two experiments were carried out simultaneously. In experiment one (mono-grown), 8 individual seedlings of each species were each grown in a single pot. In the second experiment (mixed-species experiment), 8 seedlings of each species were grown together in the same pot (total of 16 in one pot). Apart from the number of plants per pot, all other conditions were the same for the mono-grown and mixed-species experiments. For each experiment, the design was a split-split-plot with CO2 concentration as the whole plot, soil temperature as the sub-plot and nutrient supply as the sub-sub plot. There were two CO2 (ambient, 360 and elevated, 720 µmol mol-1) levels with two replications, two soil temperature regimes within each CO2 treatment. There were two nutrient levels within each soil temperature treatment. The last factor, species, was completely randomised in the design. Arrangement of each temperature control box, pot and plant within each plot was randomised.
The CO2 in the elevated greenhouses were generated from electronic ignition natural gas CO2 generators (model GEN-2E, Custom Automated Products, Inc, Riverside, CA). CO2 levels in each of the four greenhouses were monitored and automatically controlled with Argus CO2 sensors and control system (Argus, Vancouver, BC, Canada). Day-time soil temperatures were 20 and 25 oC. The 20 oC soil temperature (CurrentOT) represents current optimum for both species (Grossnickle 2000) while the 25 oC (ProjectedOT) is based on 5 oC projection. Each day-time soil temperature was lowered by approximately 4-6 oC at night to cater for lower night temperatures and simulate natural phenomenon. Changing of day-time and night-time soil temperature occurred 90 minutes before air temperature was changed as soil temperature change is slower than air temperature change. Each soil temperature was controlled using a separate control system consisting of a large leak-proof box (112 cm wide, 196 cm long, 16 cm deep) filled with water to a suitable level and a circulatory pump (model AC-2CP-MD, March Mfg. Inc., Glenview, Illinois, USA). The CurrentOT system was equipped with a flow-through cooler-heater (model 911), while the ProjectedOT system had a flow-through heater (model 210, PolyScience, Nile, Illinois, USA) to control the water to the desired temperature. The system was insulated to minimise heat exchange with the greenhouse air. The high nutrient regime (150, 60, 150, 80, 40, 60 ppm nitrogen, phosphorus, potassium, calcium, magnesium and sulphur respectively) represents fertile boreal sites and was based on Landis (1989) rapid growth phase nutrient recommendation for conifers. The low nutrient treatment was 10% of each of the high nutrient treatment concentration. Fertilisation was done once a week except.
Environment conditions in the greenhouses were maintained and controlled using an Argus control system (Argus, Vancouver, BC, Canada) as follows: photoperiod of 16 hours, relative humidity of 55 ± 5% and day and night air temperatures of 25 ± 2 oC and 15 ± 2 oC respectively. Natural light in the greenhouse (approximately 700 μmol m-2s-1) was supplemented on cloudy days, early mornings and late evenings with high-pressure sodium lamps. The supplementary light was approximately 220 μmol m-2s-1 at the canopy level. Watering was done twice a week.
Measurements
Gas exchange
Foliar gas exchange was measured 4.5 months into the treatment. Gas exchange was measured on current year needles using PP-Systems Ciras-1 open gas exchange system (Hitchin, Herefordshire, U. K.) with a Parkinson conifer leaf cuvette. Environmental conditions in the cuvette were controlled automatically as follows: temperature 25 oC, relative humidity 50%, photosynthetically active radiation (PAR) 800 µmol m-2 s-1. The light was supplied from the cuvette's built-in tungsten lamp. A-Ci curves (CO2 assimilation rate, A, and sub-stomatal [CO2], Ci) were measured sequentially from 50, 150, 250, 360, 500, 700, 900 to 1200 µmol mol-1 [CO2]. Leaf temperature inside the cuvette averaged 24 oC. Net photosynthetic rate (Pn, µmol CO2 m-2 s-1), stomatal conductance (gs, mmol m-2 s-1) and transpiration rate (E, mmol H2O m-2 s-1) were estimated at growth [CO2] based on Farquhar et al. (1980). Photosynthesis was also measured at a common ambient, 360 µmol mol-1 [CO2] (Pn360).
Photosynthetic water use efficiency (WUE, µmol CO2 mmol H2O-1), amount of carbon gain per unit water was calculated as: WUE = Pn/E, where Pn and E were rates at growth [CO2]. Maximal carboxylation capacity of Rubisco (Vcmax), maximal photosynthetic electron transport capacity (Jmax) and triose phosphate utilisation (TPU) were estimated from the A-Ci curves using an A-Ci based on Sharkey et al. (2007). All gas exchange parameters and Rubisco biochemical activities are expressed on a projected leaf area basis. Leaf area was determined using the Regent Winseedle system (Regent Instruments, Québec City, QC, Canada).
