Plant material and growth conditions
Two Plantago species (lowland Plantago major L. and alpine Plantago euryphylla Briggs, Carolin & Pulley) were used in the experiments. Seeds of P. major originated from near-sea-level sites in the Netherlands. Plantago euryphylla seeds were collected at a field site in Kosciusko National Park, NSW, Australia at an altitude of 1940 m (G.R. 165687). Seeds of P. major were germinated on moist filter paper and then transferred to sand moistened with a half-strength modified 1/8 Hoagland solution in a growth cabinet (25 : 10°C, 12 : 12 h light:dark; photon flux density (PFD) 50 µmol m−2 s−1). After approx. 14–21 d of establishment, P. major seedlings were transferred to 30-l tanks filled with aerated modified 1/8 Hoagland solution containing 2 mm NO3− as the nitrogen source (Poorter & Remkes, 1990; Atkin et al., 2006). Solutions were changed weekly and the pH was adjusted daily to 5.8. Plants were grown under controlled conditions (12 : 12 h day:night; 300 µmol photons m−2 s−1; 70% relative humidity) in three growth cabinets that differed in temperature (constant 13, 20 or 27°C).
Plantago euryphylla seeds were planted on soil and initially grown in a controlled environment chamber with an irradiance of 190 µmol m2 s−1 and 23 : 14°C day:night temperature. Once the seedlings were large enough, they were transferred into pots containing Cocopeat and transferred to the 13, 20 or 27°C cabinets described in the previous paragraph. The pots were irrigated with nutrient solution identical to that used for the lowland species, with the exception that the solution contained equal concentrations of both NO3− and NH4+. Initial growth of P. euryphylla on organic media and provision of NH4+ were necessary as P. euryphylla produced healthier leaves when grown on organic media than on hydroponic culture and developed nutrient deficiency symptoms when provided with only NO3− nitrogen. Once plants had established on the organic media for 35–40 d, they were transferred to hydroponic culture (containing modified Hoagland solution as above, but with 1 mm NO3− and 1 mm NH4+) for a further 3 wk.
Whole-plant gas exchange was measured on both species. Whole-shoot photosynthesis (P), shoot respiration (shoot R) in darkness, and root respiration (root R) were measured in four randomly selected plants of each species at each growth temperature (13, 20 and 27°C) when the plants had reached a total fresh mass of approximately 3–6 g (Poorter & Welschen, 1993). Measurements of shoot and root CO2 exchange were conducted as described in Loveys et al. (2002), but with the impact of variable measurement temperatures on CO2 exchange also being assessed. Intact plants were placed into cuvettes with the roots and shoots in separate compartments (see Den Hertog et al. (1993) for additional details of the experimental system); as the shoot compartment was considerably larger than the shoot diameter, leaf display was the same in the compartment as in the growth cabinet. Air within the shoot compartment was well mixed using fans positioned on opposite corners of the compartments, with air passed through the shoot compartment at a flow rate of 12–14 l min−1. The root compartment was filled with 800 ml of aerated nutrient solution (see previous section) plus buffer (10 mm (2-N-morpholino)ethanesulfonic acid (MES)), with air passed through the root compartments at a flow rate of 0.9–1.1 l min−1. The irradiance that shoots were exposed to was the same as that in the growth cabinets (provided by HPI-T 400-W high-pressure mercury lamps (Philips Nederland BV, Eindhoven, the Netherlands) in both cases). Root R in buffered nutrient solution was measured immediately after the P measurement. Shoot R was recorded after the plants had been in darkness for approx. 0.5 h. CO2 fluxes from the shoot and root compartments were measured using a Li-Cor 6262 infrared gas analyser (Li-Cor, Lincoln, NE, USA) in an open system (air flowing from the root compartments was dried before passing through the gas analyser). In the first set of measurements assessing the impact of shoot temperature on gas exchange, root temperature was set to that experienced by plants in their respective growth cabinet (13, 20 or 27°C). To assess the impact of air temperature on shoot R and P, we exposed shoots sequentially to 20, 27 and then 34°C. At each shoot temperature, P in the light, root R and then shoot R in darkness were measured; root temperature was kept at the growth temperature throughout. Although root respiration at 13°C was measured, measurements of shoot gas exchange at 13°C were technically not possible (because the heat load from the mercury lamps exceeded our capacity to cool the compartment environment). Thus, for the 13°C-grown plants we did not obtain a measurement of shoot R or P at the growth temperature. Estimates of shoot CO2 exchange at 13°C were made, however, using information on the temperature dependence of leaf R (in darkness and in the light) and leaf P at growth irradiance (Atkin et al., 2006). To establish the short-term temperature dependence of root R over a common measurement temperature range (20–27°C), we conducted an additional set of experiments where roots were first exposed to 20°C and then to 27°C, with root R being measured in each case (with shoots illuminated); in each case, shoots were kept at the growth temperature, with the exception of the 13°C-grown plants, where shoot temperature was set to 20°C.
The impact of growth temperature on the relative growth rate (RGR; mg g−1 d−1) of whole plants was calculated for each species using the following formula:
- (Eqn 1)
(Pnet, the measured specific rate of net photosynthesis (i.e. CO2 uptake by carboxylation minus CO2 release by photorespiration and shoot Rday, the specific shoot R in the light); shoot Rnight, the specific shoot R in darkness; root R, the specific rate of root R, assumed to be constant over light and dark periods as shown by Scheurwater et al. (1998); LMR, StMR and RMR, the leaf, stem and root mass ratios (i.e. proportion of whole-plant biomass allocated to each organ), respectively; CC, the carbon concentration.) Carbon concentrations at each temperature were predicted using the relationship between CC and growth temperature (T) reported previously (P. major, CC = 48.81 − 0.68T; P. euryphylla, CC = 31.01 + 0.03T; Loveys et al., 2002). For RGR values at 13°C, predicted rates of gas exchange at 13°C were used (see previous paragraph).
Whole-plant R/Pgross (i.e. the percentage of daily gross photosynthetic CO2 uptake released by whole-plant respiration) values were calculated, where daily whole-plant R was taken as root R plus shoot R over a 24-h period, and Pgross was the measured rate of Pnet plus shoot R during the day. When calculating rates of shoot R during the day (shoot Rday) for each species, we multiplied the measured rates of shoot R in darkness by the ratio of leaf R in the light to that in darkness (Rlight/Rdark) for each growth temperature/measuring combination reported previously (Atkin et al., 2006). For P. major at 13, 20 and 27°C, mean Rlight/Rdark ratios at each respective growth temperature were 1.25, 0.36 and 0.38, respectively. For P. euryphylla at 13, 20 and 27°C, mean Rlight/Rdark ratios were 0.84, 0.61 and 0.44, respectively. Data from Atkin et al. (2006) were also used to take into account the impacts of short-term changes in measuring temperature on Rlight/Rdark ratios. The equation used to calculate whole-plant R/Pgross according to this approach was:
- (Eqn 2)
(12/24, the relative lengths of both the dark and the light periods.) The resultant daily rates were then weighted according to the number of hours over which each gas exchange parameter occurred on each day. For R/P values at 13°C, predicted rates of shoot gas exchange at 13°C were used, these being obtained from measured rates at 20°C and previously measured temperature responses of leaf gas exchange made at growth irradiance (Atkin et al., 2006). To establish the relative contributions of roots and shoots to whole-plant R/Pgross, we also calculated root and shoot R/Pgross values separately using modified versions of Eqn 2. Similarly, the percentage of whole-plant R taking place in roots and shoots was calculated using modified versions of Eqn 2 (with light impacts on shoot R (Atkin et al., 2006) being taken into account).
To assess the impact of variations in night-time shoot temperature alone on R/Pgross of whole plants, we included predicted rates of shoot R at 6 and 13°C in Eqn 2 (for plants grown at 13 and 20°C), using second-order polynomial equations fitted to log-transformed values of shoot R in darkness plotted against measuring temperature:
- 13°C grown:log shoot R = −0.3923 + (0.0576 × T) + (0.00050 × T2)(Eqn 3)
- 20°C grown:log shoot R = −0.0172 + (0.0289 × T) + (0.00009 × T2)(Eqn 4)
(T, measuring temperature.) Measured rates of Pnet during the day (at 20, 27 and 34°C) and root R at the growth temperature were used in the calculations. We also assessed the impact of daytime shoot temperature (20, 27 and 34°C; with night-time shoot and root temperatures kept constant at 13°C) on R/Pgross; for these calculations, measured rates of shoot and root CO2 exchange were used, taking into account variations in the ratio of Rlight/Rdark (Atkin et al., 2006).
Biomass allocation and statistical analyses
Dry masses of leaves, stems and roots were determined and used to calculate LMR, StMR and RMR values (see previous section). Tissues were freeze-dried in a Virtus, Unitop 600 SL freeze dryer (Gardiner, New York, NY, USA). Leaf area was determined using a Li-Cor 3100 leaf area meter (Li-Cor), with the ratio of leaf dry mass to leaf area being used to calculate leaf mass per unit area (LMA) values. Statistical analyses were carried out using the spss software package version 10 (SPSS Inc., Chicago, IL, USA). Where necessary, proportional data were angular transformed to ensure that data were normally distributed and variances homogeneous before analysis using one- or two-way analysis of variance (ANOVA). In cases where data remained nonparametric (e.g. when comparing P. major root R/Pgross values at each growth temperature), the Kruskal–Wallis test was used followed by pair-wise comparison of growth temperatures using the Mann–Whitney U-test. At each growth temperature, comparisons of R/Pgross between the two species were made using one-way ANOVA. One-way ANOVAs were also used to compare the values of Q10 (i.e. the proportional increase in R for every 10°C rise in measuring temperature) of shoot and root R of the two species.