Leaf respiration and alternative oxidase in field-grown alpine grasses respond to natural changes in temperature and light

Authors


Author for correspondence:
Stephanie Y. Searle
Tel: + 64 3 348 5996
Email: sys15@student.canterbury.ac.nz

Summary

  • We report the first investigation of changes in electron partitioning via the alternative respiratory pathway (AP) and alternative oxidase (AOX) protein abundance in field-grown plants and their role in seasonal acclimation of respiration.
  • We sampled two alpine grasses native to New Zealand, Chionochloa rubra and Chionochloa pallens, from field sites of different altitudes, over 1 yr and also intensively over a 2-wk period.
  • In both species, respiration acclimated to seasonal changes in temperature through changes in basal capacity (R10) but not temperature sensitivity (E0). In C. pallens, acclimation of respiration may be associated with a higher AOX : cytochrome c oxidase (COX) protein abundance ratio. Oxygen isotope discrimination (D), which reflects relative changes in AP electron partitioning, correlated positively with daily integrated photosynthetically active radiation (PAR) in both species over seasonal timescales. Respiratory parameters, the AOX : COX protein ratio and D were stable over a 2-wk period, during which significant temperature changes were experienced in the field.
  • We conclude that respiration in Chionochloa spp. acclimates strongly to seasonal, but not to short-term, temperature variation. Alternative oxidase appears to be involved in the plant response to both seasonal changes in temperature and daily changes in light, highlighting the complexity of the function of AOX in the field.

Introduction

Plant respiration (R) is a vital process that regulates the exchange of carbon between the atmosphere and the biosphere, resulting in the loss of 30–80% of daily photosynthetic carbon fixation (Poorter et al., 1990; Amthor, 2000; Loveys et al., 2002). As the terrestrial biosphere absorbs more than one-third of anthropogenic CO2 emissions (Schimel et al., 2001; Houghton, 2003; Canadell et al., 2007) small perturbations to R caused by climate change will have large impacts on the global carbon balance. Plant respiration is highly temperature sensitive; therefore, an improved understanding of the temperature response of plant respiration and how it is regulated is needed to accurately predict future plant carbon exchange and cascading effects to the earth climate system.

It is now understood that R acclimates to changes in growth temperature in many species (Larigauderie & Korner, 1995; Tjoelker et al., 1999, 2008; Atkin et al., 2000; Atkin & Tjoelker, 2003; Ow et al., 2008a). Acclimation is typically defined as a reduction in R at a set-point temperature in warm vs cold conditions (Larigauderie & Korner, 1995; Atkin & Tjoelker, 2003; Tjoelker et al., 2008). Full acclimation, or ‘thermal homeostasis’, is achieved in plants grown at different temperatures when R at the growth temperature (Rg) occurs at the same rate in each plant. However, not all plants acclimate and the degree of acclimation varies widely between species (Larigauderie & Korner, 1995; Tjoelker et al., 1999; Loveys et al., 2003).

The physiological mechanisms leading to acclimation are still not fully understood. Concentrations of nonstructural carbohydrates (starches and sugars) are often correlated with changes in respiration and its temperature reponse (Azcòn-Bieto et al., 1983; Tjoelker et al., 1999, 2008; Lee et al., 2005), as is adenylate status (Hoefnagel et al., 1998; Atkin et al., 2002; Covey-Crump et al., 2007). Respiratory enzymes have been shown to have regulatory control over the temperature response of plant respiration; of particular interest is the cyanide-insensitive alternative oxidase (AOX), which catalyzes the reduction of O2 via an alternative pathway and produces, at most, one-third of the ATP generated by respiration via the cytochrome c oxidase (COX) catalyzed pathway (Vanlerberghe & McIntosh, 1997; Millenaar & Lambers, 2003). Electron partitioning through the alternative pathway (AP) is measured using the oxygen isotope fractionation technique, which takes advantage of the fact that AOX discriminates against 18O more strongly than does COX (Guy et al., 1989). Thus, the isotopic signature of the residual oxygen from plant respiration reflects the plant’s ‘discrimination’ against 18O and indicates the relative electron partitioning through each pathway, with higher discrimination values indicating greater AP partitioning, and lower values indicating greater electron partitioning through the cytochrome pathway (CP).

Why would plants utilize an apparently ‘energy-wasteful’ respiratory pathway? The AOX has been proposed to be involved in the stress response of plants to cold (Gonzalez-Meler et al., 1999; Fiorani et al., 2005; Armstrong et al., 2008), heat (Rachmilevitch et al., 2007; Murakami & Toriyama, 2008), drought (Ribas-Carbo et al., 2005b), nutrient deficiency (Gonzalez-Meler et al., 2001; Sieger et al., 2005) and excess light (Noguchi et al., 2001; Yoshida et al., 2007). With regard to temperature, in vivo electron partitioning through the AP has been shown to increase under cold (Gonzalez-Meler et al., 1999; Ribas-Carbo et al., 2000a; Armstrong et al., 2008), as have AOX protein (Vanlerberghe & McIntosh, 1992a; Mizuno et al., 2008) and transcript abundance (Ito et al., 1997; Sugie et al., 2006), although whether or not there is an instantaneous temperature dependency of discrimination in plants is debated (Gonzalez-Meler et al., 1999; Guy & Vanlerberghe, 2005; Armstrong et al., 2008; Macfarlane et al., 2009). A possible mechanism of how the AP acts to reduce stress is through prevention of excess reactive oxygen species (ROS). Reactive oxygen species accumulate in plant mitochondria in response to cold temperatures (Purvis & Shewfelt, 1993; Fiorani et al., 2005; Sugie et al., 2006) and other environmental stresses (Juszczuk et al., 2001; Bartoli et al., 2004), and can cause severe harm to mitochondria (Rich & Bonner, 1978). Using Arabidopsis thaliana mutants over-expressing AOX genes, ROS production (Sugie et al., 2006) and oxidative stress (Fiorani et al., 2005) were found to be lower in response to cold temperatures relative to the wild-type plants, showing that AOX functions to reduce mitochondrial oxidative stress. Because use of the AP necessarily has a negative impact on the efficiency of respiratory energy production, a plant with relatively high electron partitioning via the AP would need to respire at a greater overall rate in order to meet the same energy demands as a plant with a relatively greater flux of electrons through the CP. However, few studies have actually investigated the relationship between changes in the AP and changes in total R, and those that have provide contradictory results (Gonzalez-Meler et al., 1999; Ribas-Carbo et al., 2000a; Armstrong et al., 2008).

The timescale on which acclimation and its underlying physiological and biochemical mechanisms occur is also unclear. Acclimation of dark respiration (Rd) to temperature has also been found to occur quickly (2–10 d) in some species under controlled-environment conditions (Rook, 1969; Rychter et al., 1988; Atkin et al., 2000; Bolstad et al., 2003; Armstrong et al., 2008) and even under field conditions (Bolstad et al., 2003), but this contrasts with the findings of Ow et al. (2008a,b). However, whereas changes in respiration brought about through acclimation are generally found to be sustained over long periods of constant temperature (Larigauderie & Korner, 1995; Arnone & Korner, 1997; Armstrong et al., 2008), changes in electron partitioning through the AP (Armstrong et al., 2008), AOX protein (Vanlerberghe & McIntosh, 1992b; Mizuno et al., 2008) and transcript abundance (Ito et al., 1997; Sugie et al., 2006) are sometimes found to be transitory on the timescale of days. Thus, it is unclear how AOX functions to alter metabolic function over multiple timescales, and how this is related to changes in respiration in variable temperature environments. Importantly, to the best of our knowledge, all studies on electron partitioning through the AP and the CP have been conducted on laboratory-grown plants. It is therefore unknown whether AP respiration changes with fluctuating environmental conditions in the field, how quickly this may occur and how it is related to changes in total R.

Alpine ecosystems provide a unique environment in which to study the natural response of plants to environmental stress. Alpine plants are regularly exposed to harsh conditions, including cold temperature, wind, high sunlight and drought. Day-to-day fluctuations in temperature may be rapid and extreme. Additionally, altitudinal gradients can be used as natural environmental gradients of temperature and other variables. Lastly, alpine areas are of high ecological importance, covering c. one-fifth of the total terrestrial area, featuring high plant diversity and being particularly vulnerable to climate change (Korner, 2004).

Here, we investigated the responses of Rd and AP engagement in field-grown Chionochloa pallens and Chionochloa rubra (evergreen perennial tussock grasses) to daily and seasonal changes in ambient temperature. We utilized four sites along an altitudinal gradient on Mt Hutt, New Zealand, in order to extend the range of natural temperature variability experienced by these grasses. We addressed the following questions. On what timescale does acclimation of leaf respiration occur in field-grown alpine grasses? Does the oxygen isotope discrimination of respiration change in response to cold stress in the field? Is this mediated through changes in AOX protein abundance? Does AOX contribute towards the seasonal acclimation of R in these species?

Materials and Methods

Sampling

All grasses studied were found growing in situ on Mt Hutt, Canterbury, New Zealand (43°32′S, 171°33′E). Chionochloa rubra Zotov was sampled at low-altitude (450 m above sea level) and mid-altitude (1070 m) sites; Chionochloa pallens Zotov was sampled at mid-altitude (1070 m) and high-altitude (1600 m) sites. Measurements of ambient temperature at each altitude were taken at 1.4 m above the ground (thermistors: YSI, Yellow Springs, OH, USA) every 30 s, with daily mean, minimum and maximum stored in a datalogger (CR21X, Campbell Scientific, Logan, UT, USA). Measurements of photosynthetically active radiation (PAR) were made using a LiCor 190SB quantum sensor (LiCor, Lincoln, NE, USA) taking measurements every 5 s. Precipitation was measured at the low- and mid-altitude stations using 35-mm tipping buckets (Environmental Measurements, Osney Mead, UK).

Two field studies were conducted in order to determine the timescale of physiological changes in Chionochloa spp.: a seasonal study with approximately monthly measurements at all altitudes and a short-term study on Chionochloa at only the mid-altitude site (1070 m). Measurements for the seasonal study were made at all altitudes, seven times: in December 2007 and in January–April, October and November 2008 (respiration was also measured in November 2007 and is presented here). Measurements were not taken between April and October 2008, and C. pallens (upper site) was not sampled in October 2008 because of deep snow cover. All results from the seasonal experiment were pooled between altitudinal sites for each species (i.e. data from C. rubra at 450 m and 1070 m have been pooled, and data from C. pallens at 1070 m and 1600 m have been pooled), as we did not detect an altitudinal effect in the results. Measurements for the short-term experiment took place on seven occasions between 27 December 2008 and 9 January 2009 at the middle site only. Different plants were sampled each day of both experiments. At mid-morning on each sampling day, grasses were cut at the base of the shoot and transported back to the laboratory for measurements (= 6 for all measurements).

Respiration measurements

Dark-respiration measurements were conducted using a portable infrared gas analysis system (Li-Cor 6400; Li-Cor, Lincoln, NE, USA). One-yr-old leaves were measured from excised shoots that were wrapped in damp paper towels and kept in the dark during transport (< 1 h) and placed in growth cabinets at the University of Canterbury to manipulate instantaneous temperature. Measurements were made at 10, 20, 25 and 30°C in the seasonal study, and at 10, 14, 18, 22 and 26°C in the short-term study. The temperature response of respiration was modelled using a modified Arrhenius equation (Ryan, 1991; Turnbull et al., 2003; Kruse & Adams, 2008):

image(Eqn 1)

where R is the respiration rate, R10 is the respiration rate at a reference temperature (T0) of 10°C, Ta is the measurement temperature of R, g is the ideal gas constant (8.314 J mol−1 K−1) and E0 is the temperature sensitivity of respiration, which is related to the overall activation energy. Nonlinear curve fitting was performed using the Marquardt–Levenberg algorithm (Sigma Plot v8.0; SPSS Inc., Chicago, IL, USA). Respiration at the prevailing field growth temperature (Rg) was calculated by substituting calculated R10 and E0 values into Eqn 1 and using either growth temperature or the minimum temperature the night before sampling.

Respiration was not significantly different when measured on excised leaves in the laboratory vs attached leaves in the field; and it did not change significantly when measured at the same temperature over a 12-h period in the laboratory (data not shown). This is consistent with previous findings that R is stable in well-hydrated excised tissues over many hours (Turnbull et al., 2005).

Oxygen isotope measurements

Changes in electron partitioning through the two possible terminal oxidase pathways in respiration were assessed in vivo based on the premise that the CP and the AP discriminate against the heavier isotope of oxygen (18O) to different extents (Guy et al., 1989; Ribas-Carbo et al., 2005a). We adopted an ‘offline’ method, modified from that of Nagel et al. (2001), which allows measurements of oxygen isotope discrimination in field-grown plants. Leaves were cut into 7-cm sections and placed in 12-ml Exetainers (Labco, High Wycombe, UK) containing a half pellet of KOH to absorb excess CO2, and placed in a water bath to control temperature. Samples were incubated in the dark at 24°C for 1 h for the seasonal study and at 20°C for 1.5 h for the short-term study. Three to four Exetainers were filled with varying amounts of leaf material from each sample to achieve a range of oxygen consumption. After the incubation period, two syringes were inserted into a 12-ml Exetainer; one syringe was empty and the other was filled with water to replace the volume in the Exetainer as the gas was displaced into the empty syringe. The gas-containing syringe was then removed and inserted into a pre-evacuated 3.8 ml Exetainer. Pressure was allowed to equilibrate between the syringe and the 3.8 ml Exetainer.

Gas samples were stored before being analyzed on a Delta IV isotope ratio mass spectrometer (IRMS) equipped with GasBench II (Thermo Fisher Scientific, Waltham, MA, USA). Precision gas mixtures were used to calibrate the IRMS and corrections were made for the slight nonlinearity of its response. A small amount of gas (5–15% of the total volume) was present in the evacuated Exetainers before use; at several times during and after the study, Exetainers injected with nitrogen (N) or helium (He) were used to determine the amount and composition of this gas, and its presence was corrected for in samples. Stored samples were found to leak at a rate of 0.7% per month. Because of the availability of the IRMS, samples were stored for 0–10 months. Standard gases of varying O2 : N2 ratios, stored over various periods of time, were used to determine the specific effect of storage on the O2 : N2 ratio of samples and this was corrected for. Leakage depended on both storage time and the original concentration of oxygen present. These effects were found to be strongly linear and were corrected for in stored samples. The specific effect of storage on 18/16O was determined empirically: a large set of samples was collected in May 2009; half were analyzed on the IRMS immediately (following the procedure described above), while half were stored for 10 months. A strong linear relationship between 18/16Ostored and 18/16Ofresh was determined and utilized to correct for changes in 18/16O as a result of storage. After applying these corrections, no systematic effect of storage time on discrimination values was found. While it is possible that leakage occurred during the transfer of gas from 12-ml to 3.8-ml Exetainers, this was not tested. Calculations of oxygen-isotope fractionation were made as described by Guy et al. (1989) and Ribas-Carbo et al. (2005a,b) with modifications to yield a value of discrimination against 18O/16O (D). We used changes in the O2 : N2 ratio to reflect oxygen drawdown, and did not include samples with > 50% oxygen consumption. We analyzed all replicates of each species together; thus, the SE presented represents the combined error of measurement and error of biological variation. Following Guy et al. (1989), we report the SE of the regression, which is more appropriate than the R2 value given the large and variable number (18–24) of points used in each calculation of D.

We have validated this method by producing similar results to those already published in the literature. We grew Phaseolus vulgaris plants under conditions similar to those reported in Noguchi et al. (2001). Our control and inhibited discrimination values for leaves sampled early in the night were in the range of those previously reported: 25.8 ± 0.5, 21.6 ± 0.4 and 20.5 ± 0.4‰ for KCN-inhibited, salicylhydroxamic acid (SHAM)-inhibited and control treatments, respectively, compared with 26.7 ± 1.0, 19.0 ± 2.2 and 22.1 ± 2.7‰ (Noguchi et al., 2001). Additionally, we have measured KCN-inhibited D in cotyledons of Glycine max to be 30.5‰, similar to that reported in Gonzalez-Meler et al. (1999) and Ribas-Carbo et al. (1997).

We were unable to determine the end points of oxygen isotope fractionation for AOX and COX because we were unable to sufficiently inhibit C. rubra and C. pallens with SHAM and KCN, despite exhaustive efforts. The methods attempted include various concentrations of inhibitors (2–100 mM) and incubation times (10–180 min), vacuum infiltration, gaseous infiltration of KCN, slicing leaf tissues and stripping of the cuticle; none of these methods resulted in a residual respiration rate of < 30% when both inhibitors were applied. Therefore, changes in D are used here to indicate relative changes in electron partitioning through the CP and the AP.

Immunoblotting

Sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS-PAGE) was performed using 4–12% gradient polyacrylamide gels. Freeze-dried ground leaf samples were centrifuged at 10 000 g in a buffer solution and the supernatant was discarded. The remaining pellet, assumed to contain cell membranes and cell walls, was prepared in a buffer solution containing 42% 2-mercaptoethanol and loaded onto the gels. Proteins were transferred to a nitrocellulose membrane using the iBLOT system (Invitrogen, San Diego, CA, USA), and immunoblotting was performed using the Snap i.d. system (Millipore, Billerica, MA, USA) using a monoclonal antibody raised against AOX (Elthon et al., 1989) at a dilution of 1 : 300. Antibodies were detected using an anti-mouse horseradish peroxidase (HRP) conjugate and noncommercial enhanced chemiluminescence (ECL) solution (Haan & Behrmann, 2007). The blots were photographed using a cryo-cooled digital camera (Chemigenius, Syngene, Cambridge, UK). The blots were then incubated in a 15% solution of H2O2 to deactivate the anti-mouse HRP conjugate before application of an anti-COX serum at a dilution of 1 : 600 for C. rubra and a dilution of 1 : 1200 for C. pallens (Agrisera, Vannas, Sweden). An anti-rabbit HRP conjugate was used to detect COX. A representative blot is shown in Supporting Information Fig. S1.

Bands were quantified using densitometry (ImageJ; NIH, Bethesda, MD, USA). We and our collaborators have previously determined the density of AOX protein bands to respond linearly to changes in the amount of protein over the range of our analyses in several species; this approach was used in Campbell et al. (2007). Thus, we assumed a linear response of band density to protein abundance in Chionochloa spp. In order to control for differences in the brightness between western blots, a standard plant sample was run on each blot (a sample of C. pallens from November 2008 with an average amount of protein was used to standardize all C. pallens samples, and a sample of C. rubra from January 2009 was used to standardize all C. rubra samples). All other samples were normalized to this standard (i.e. the quantity of protein detected in any particular sample was divided by the quantity of protein detected in the standard plant sample analyzed on the same blot). The protein abundance for each sample was then divided by the mean abundance of all analyzed samples for each protein and species in order to equalize the distribution for AOX and COX. Relative AOX protein abundance was then divided by relative COX abundance for each sample in order to calculate the AOX : COX ratio. Thus, an AOX : COX ratio of 1 represents the mean AOX : COX ratio for each species in this study.

Leaf characteristics

For analysis of both carbohydrates and N, leaves were dried and ground in a ball mill. Soluble sugar and starch were analyzed following the method of Tissue & Wright (1995). Soluble sugars were extracted from leaf powder in a solution of methanol, chloroform and water. The remaining pellet was treated with 35% perchloric acid to hydrolyse the starch. Soluble sugars and starch were determined colorimetrically using phenol and sulphuric acid. The percentage of starch and the percentage of sugar were summed to calculate total nonstructural carbohydrates (TNC).

Leaf N was analyzed on a Europa Scientific 20/20 isotope analyzer at the Waikato University stable isotope unit (Hamilton, New Zealand). Specific leaf area (SLA) was measured on 15-cm-long sections cut from the middle of the leaves. Leaf area was determined by measuring the width of the section at both ends using calipers, and treating the leaf as a trapezoid in calculations. Leaf sections were then dried and weighed. Specific leaf area was calculated as fresh area divided by DW. Specific leaf area was used to convert respiration measurements to mass basis, except in November 2007, when SLA measurements were not taken; measurements from December 2007 were used for calculations in this month.

Chlorophyll fluorescence

Measurements of chlorophyll fluorescence yield (FV/FM) were made in the field using a Mini-PAM (Pulse Amplitude Modulator; Walz, Effeltrich, Germany). Leaves were dark adapted for 20 min before measurements were made.

Statistical analysis

Statistical analyses were performed using R 2.4.0 (R Development Core Team). Simple linear regressions were used to test correlations between variables. One-way ANOVA was used to test changes in the short-term study. A Chow test was used to test differences in the slope of discrimination between measurement days in the short-term study. All results were considered significant if < 0.05.

Results

Seasonal study

Temperature and precipitation at the middle field site (1070 m) over the course of the study are shown in Fig. 1. Precipitation did not differ significantly between the lower and middle field sites (= 0.553), or between months at the lower (= 0.426) or middle (= 0.667) sites.

Figure 1.

 (a) Daily mean temperature and (b) daily precipitation at the middle field site weather station (1070 m above sea level, Mt. Hutt, Canterbury, New Zealand) from 29 November 2007 to 12 November 2008 (the course of the seasonal field experiment). Precipitation did not differ significantly between months.

The appropriate time window explaining the extent and rate of acclimation of respiration to changes in ambient temperature was determined by evaluating the strength (R2 value) of the relationship between R10 and ambient temperature averaged over a range of time-periods (Fig. 2). The relationship was similar between E0 and ambient temperature (data not shown). The R2 value increased to a transient peak at 3–5 d, after which it decreased and then increased to a maximum for each species and site at c. 120 d; therefore, 120 d was chosen as the time window for calculating the ambient temperature to which R in C. rubra and C. pallens acclimated.

Figure 2.

 Correlation (R2 value) for the linear regression between the respiration rate at a reference temperature (T0) of 10°C (R10) and temperature averaged over an increasing time window preceding the day of measurements (in days) for Chionochloa rubra (solid line) and Chionochloa pallens (dotted line) in the seasonal study.

R10 was significantly and negatively correlated with 120 d averaged temperature in both species (Fig. 3a,b), while E0 was significantly and positively correlated with 120 d averaged temperature in C. pallens only. Rates of respiration at 10°C (R10) ranged from 0.07 to 0.41 μmol CO2 m−2 s−1. As a result of a high leaf mass per area (128–236 g m−2; data not shown), R10 on a mass basis was relatively low, in the range of 0.3–2.9 μmol COkg−1 s−1 (Fig. 3). Respiration at the field growth temperature (Rg) was calculated using the R10 and E0 values measured on each sampling date. The Rg value calculated using 120 d averaged growth temperature negatively correlated with growth temperature in C. pallens and showed no response in C. rubra (Fig. 3e,f). When calculated using the minimum temperature the night before measurement, Rg showed a scattered response to the previous night’s minimum temperature in both species, but there was no evidence that (acclimated) Rg increases with temperature (Fig. 3g,h).

Figure 3.

 Respiratory parameters for Chionochloa rubra and Chionochloa pallens in the seasonal study. Values shown are mean ± SEM. (a, b) R10 (respiration at a set-point temperature of 10°C) plotted against growth temperature (field temperature averaged over 120 d before measurement). (c, d) E0 (the instantaneous temperature sensitivity of respiration) plotted against growth temperature. (e, f) Rg (respiration at the growth temperature (averaged over 120 d before measurement)) plotted against growth temperature. (g, h) Rg (calculated using the minimum temperature the night before sampling) plotted against the previous night’s minimum temperature.

Neither R10 nor E0 was significantly correlated with percentage sugar or percentage starch (see Table 1 for data). Leaf N was positively correlated with R10 and with growth temperature in C. pallens (R2 = 0.373; P = 0.032) but not in C. rubra (Table 1). Leaf N was not correlated with E0.

Table 1.   Mean values ± SEM for R10, E0, Rg, D, AOX : COX protein abundance ratio, soluble sugar, starch, nitrogen and FV/FM during the course of the seasonal study
 R10 (μmol CO2 kg−1 s−1)E0 (kJ)Rg (μmol CO2 m−2 kg−1)Discrimination (ml−1)AOX : COX proteinSugar (%)Starch (%)Leaf nitrogen (%)FV/FM
  1. AOX, alternative oxidase; COX, cytochrome c oxidase; D, oxygen isotope discrimination; E0, the temperature sensitivity of respiration; FV/FM, chlorophyll fluorescence yield; R10, the respiration rate at a reference temperature (T0) of 10°C; Rg, respiration rate at the growth temperature.

Lower siteNovember 20071.00 ± 0.0950.1 ± 5.10.60 ± 0.08      
Chionochloa rubraDecember 20070.95 ± 0.0443.4 ± 0.70.71 ± 0.0321.68 ± 0.050.80 ± 0.234.58 ± 0.2411.64 ± 0.841.15 ± 0.050.863 ± 6
January 20081.14 ± 0.1440.2 ± 4.01.06 ± 0.1317.77 ± 0.970.68 ± 0.305.67 ± 0.7511.37 ± 0.661.05 ± 0.050.829 ± 7
February 20080.70 ± 0.1060.6 ± 8.80.46 ± 0.0816.85 ± 0.640.97 ± 0.434.75 ± 0.4020.40 ± 1.381.00 ± 0.060.823 ± 4
March 20080.63 ± 0.1364.3 ± 9.20.36 ± 0.1019.56 ± 0.91 6.15 ± 0.9411.93 ± 0.461.16 ± 0.030.821 ± 5
April 20080.36 ± 0.0659.7 ± 1.50.33 ± 0.0520.73 ± 0.970.81 ± 0.269.22 ± 1.0115.28 ± 1.301.16 ± 0.070.834 ± 1
October 20081.92 ± 0.3042.0 ± 5.51.01 ± 0.2319.73 ± 0.500.98 ± 0.1211.30 ± 1.2112.75 ± 0.341.36 ± 0.040.837 ± 2
November 20081.48 ± 0.1145.6 ± 4.01.42 ± 0.1121.01 ± 1.341.24 ± 0.166.57 ± 0.9212.11 ± 0.971.12 ± 0.060.840 ± 3
Middle siteNovember 20070.97 ± 0.1246.0 ± 5.20.59 ± 0.10      
C. rubraDecember 20071.16 ± 0.1432.4 ± 6.10.91 ± 0.1523.03 ± 0.571.76 ± 0.265.12 ± 0.3213.38 ± 0.701.17 ± 0.050.856 ± 3
January 20081.07 ± 0.1733.2 ± 5.00.94 ± 0.1721.70 ± 1.190.72 ± 0.125.47 ± 0.8613.56 ± 0.691.20 ± 0.060.834 ± 6
February 20080.91 ± 0.0731.0 ± 3.00.56 ± 0.0620.27 ± 0.552.28 ± 0.055.33 ± 0.2723.55 ± 1.581.07 ± 0.100.823 ± 5
March 20080.50 ± 0.0762.1 ± 6.60.29 ± 0.0521.63 ± 1.13 6.36 ± 0.4712.96 ± 0.501.00 ± 0.080.806 ± 7
April 20080.30 ± 0.1074.6 ± 19.30.22 ± 0.0821.06 ± 0.721.27 ± 2.0613.94 ± 2.2519.62 ± 1.690.89 ± 0.050.828 ± 2
October 20081.78 ± 0.2340.1 ± 5.20.89 ± 0.1720.53 ± 0.450.91 ± 0.2810.3 ± 0.7916.46 ± 0.741.00 ± 0.110.825 ± 4
November 20081.32 ± 0.0842.1 ± 3.70.97 ± 0.0822.01 ± 0.711.38 ± 0.3010.38 ± 1.5911.53 ± 0.840.93 ± 0.070.813 ± 4
Middle siteNovember 20071.80 ± 0.1838.0 ± 5.61.17 ± 0.15      
Chionochloa pallensDecember 20071.40 ± 0.1650.2 ± 6.20.94 ± 0.1423.42 ± 0.930.79 ± 0.177.86 ± 1.0013.03 ± 1.180.97 ± 0.060.840 ± 5
January 20080.92 ± 0.1560.0 ± 5.10.72 ± 0.1322.85 ± 0.870.84 ± 0.066.76 ± 0.4714.54 ± 0.960.94 ± 0.050.846 ± 4
February 20081.00 ± 0.1061.0 ± 2.60.38 ± 0.0321.22 ± 0.992.17 ± 0.226.97 ± 0.3420.67 ± 1.220.93 ± 0.040.824 ± 3
March 20080.67 ± 0.0573.7 ± 7.50.35 ± 0.0520.16 ± 1.000.80 ± 0.048.21 ± 1.6618.97 ± 0.590.95 ± 0.060.830 ± 4
April 20080.52 ± 0.0875.8 ± 8.50.33 ± 0.0720.21 ± 0.750.76 ± 0.1113.18 ± 0.4822.27 ± 2.430.96 ± 0.040.827 ± 2
October 20082.88 ± 0.2843.9 ± 4.81.33 ± 0.2520.42 ± 0.882.58 ± 0.3914.2 ± 0.8917.40 ± 1.411.05 ± 0.040.811 ± 5
November 20081.73 ± 0.1344.3 ± 2.11.24 ± 0.1020.63 ± 1.441.41 ± 0.0817.94 ± 3.2612.43 ± 1.000.94 ± 0.070.829 ± 4
Upper siteNovember 20071.13 ± 0.2760.2 ± 9.90.62 ± 0.19      
C. pallensDecember 20071.43 ± 0.1860.9 ± 4.60.81 ± 0.1323.80 ± 1.261.44 ± 0.128.41 ± 0.4315.77 ± 0.921.09 ± 0.030.846 ± 8
January 20080.86 ± 0.1063.9 ± 3.50.59 ± 0.08 1.08 ± 0.118.52 ± 0.6018.46 ± 1.031.00 ± 0.040.833 ± 3
February 20081.06 ± 0.0557.2 ± 2.70.38 ± 0.0322.16 ± 0.430.81 ± 0.198.60 ± 0.6423.36 ± 2.100.90 ± 0.050.810 ± 2
March 20080.80 ± 0.0468.0 ± 3.90.33 ± 0.0322.08 ± 0.831.38 ± 0.269.01 ± 0.3218.76 ± 1.090.95 ± 0.050.816 ± 5
April 20080.47 ± 0.0774.2 ± 7.50.28 ± 0.0521.05 ± 0.540.57 ± 0.0415.53 ± 1.0635.05 ± 4.460.83 ± 0.050.809 ± 3
November 20081.97 ± 0.1740.0 ± 2.61.36 ± 0.1221.77 ± 0.515.16 ± 0.678.94 ± 0.6415.93 ± 1.601.02 ± 0.040.804 ± 3

The relationships between R10 and the AOX : COX protein ratio and between the AOX : COX protein ratio and 120 d averaged temperature are shown in Fig. 4. Neither R10 nor 120 d averaged temperature in C. rubra correlated with the AOX : COX protein ratio (Fig. 4a,c; = 0.892). In C. pallens, the AOX : COX protein ratio showed a statistically insignificant negative relationship with the 120 d averaged temperature (Fig 4b; R2 = 0.262; = 0.089) and was significantly and positively correlated with R10 (Fig 4d; R2 = 0.538, = 0.006). Inclusion of an outlier in the regression analyses, shown in grey in Fig. 4(b,d), resulted in the statistics R2 = 0.362 and = 0.030 for Fig. 4(b) and R2 = 0.393 and = 0.022 for Fig. 4(d).

Figure 4.

 The ratio of alternative oxidase (AOX) and cytochrome c oxidase (COX) protein abundances in the seasonal study plotted against the respiration rate at a reference temperature (T0) of 10°C (R10) in Chionochloa rubra and Chionochloa pallens (c, d), and against growth temperature (field temperature averaged over 120 d before measurement) (a, b). (b, d) Grey-coloured symbols show outliers that were not included in the regression analysis. Values shown are mean ± SEM. An AOX : COX ratio of 1 represents the mean AOX : COX ratio of each data set.

Discrimination (D), as measured by oxygen isotope fractionation, did not correlate with ambient temperature, R10, E0, the AOX : COX protein ratio, or the contents of soluble sugars or starch. D correlated positively with the previous day’s integrated PAR in both species (Fig. 5; C. rubra: R2 = 0.322, = 0.034; C. pallens: R2 = 0.525, = 0.008). Owing to the difficulty in obtaining end points of discrimination for AOX and COX, we cannot estimate absolute values of electron partitioning through the AP, but interpret our raw discrimination numbers as a measure of relative changes in the electron partitioning between the two oxidases. A higher D indicates increased electron partitioning through AOX, which discriminates more heavily against 18O than does COX. Thus, we infer greater AP relative to CP engagement with high ambient light in these species.

Figure 5.

 Oxygen isotope discrimination of respiration (D) of Chionochloa rubra (a) and Chionochloa pallens (b) in the seasonal study plotted against the previous day’s integrated photosynthetically active radiation (PAR). Values shown are mean ± SEM.

Chlorophyll fluorescence yield (FV/FM) ranged from 0.803 to 0.863 (Table 1) and did not correlate with any measured parameters or environmental variables.

Short-term study

Although a large variation in daily average temperature was observed at the middle site over the course of the short-term experiment (e.g. a drop from 19 to 7°C; Fig. 6a), there were no significant changes in R10 (= 0.667; 0.526), E0 (= 0.177; 0.883), discrimination (= 0.263, df = 43; Fig 6b–d), or AOX or COX protein abundance (Table 2) in either C. rubra or C. pallens. There were significant changes in the percentage of TNC over the course of the experiment, but no clear trend was evident (Table 2; < 0.001 in both species).

Figure 6.

 Short-term study. (a) Mean daily field temperature for each day of the experiment. (b–d) Respiration rate at a reference temperature (T0) of 10°C (R10; basal capacity), temperature sensitivity (E0) and oxygen isotope discrimination (D), respectively. Chionochloa rubra (closed circles), Chionochloa pallens (open circles). Values shown are mean ± SEM.

Table 2.   Percentage total nonstructural carbohydrates (TNC), and alternative oxidase (AOX) and cytochrome c oxidase (COX) protein abundance for each day of the short-term study
DayTNC (%)AOXCOX
  1. Values shown are mean ± SEM. P-values for one-way ANOVA are shown for each variable, with significance: ***, < 0.001.

Chionochloa rubra
 134.7 ± 1.2  
 320.5 ± 1.31.07 ± 0.140.88 ± 0.18
 631.1 ± 1.9  
 823.7 ± 1.61.23 ± 0.120.72 ± 0.07
1014.5 ± 1.60.95 ± 0.31.11 ± 0.1
1221.7 ± 0.7  
1429.8 ± 1.61.44 ± 0.251.01 ± 0.18
P-value< 0.001***0.4870.266
Chionochloa pallens
 137 ± 0.5  
 327.2 ± 4.20.76 ± 0.120.99 ± 0.25
 637.5 ± 2.2  
 828.5 ± 1.60.53 ± 0.060.73 ± 0.16
1021 ± 21.34 ± 0.411.25 ± 0.21
1229.3 ± 1.9  
1440.5 ± 1.81.03 ± 0.211 ± 0.07
P-value< 0.001***0.1340.359

Discussion

We observed strong acclimation of respiration in Chionochloa spp. when measured over seasonal timescales: respiration at a basal reference temperature of 10°C (R10) decreased significantly with increases in ambient temperature (Fig. 3a,b; Table 1). As a result, respiration at the growth temperature (120 d averaged temperature; Rg) showed no trend with growth temperature in C. rubra, representing a reduction in long-term temperature sensitivity relative to the instantaneous response, and negatively correlated with growth temperature in C. pallens, indicating over-acclimation of respiration to growth temperature in this species (Fig. 3e,f). The response of Rg to the previous night’s minimum temperature was scattered (Fig. 3g,h); thus, acclimation in Chionochloa spp. was not rapid enough to dampen the response of R to the large daily temperature fluctuations experienced in the field.

In field studies that incorporate an assessment of thermal acclimation of respiration, respiration at the growth temperature is typically calculated using a short time window (2–7 d) of averaged temperature (Atkin et al., 2000; Lee et al., 2005; Xu & Griffin, 2006; Tjoelker et al., 2008; Ow et al., 2010). This has been appropriately based on experimental evidence suggesting that the thermal acclimation of Rd is a relatively rapid process in several previously studied species (Rook, 1969; Atkin et al., 2000; Bolstad et al., 2003; Lee et al., 2005; Armstrong et al., 2008). Although we found a small peak in the correlation between respiratory parameters and field temperature averaged over 3–5 d, the strongest relationship occurred when temperature was averaged over a much longer period (110–120 d; Fig. 2). Moreover, while we found significant acclimation of R to seasonal changes in temperature, we found no clear response of R10 and E0 to temperature variation on the timescale of days (Figs 3, 6). We speculate that seasonal changes in respiration in Chionochloa spp. may respond to long-term temperature changes as well as to other seasonal environmental signals such as daylength. Slow acclimation in these perennial alpine grasses may provide an adaptive physiological advantage in the face of constantly fluctuating ambient temperatures (Fig. 1) – a relatively slow seasonal response would avoid frequent metabolic changes and associated construction and/or maintenance costs.

Acclimation of respiration in pre-existing leaves subjected to new temperatures has been proposed to result primarily from changes in the instantaneous temperature sensitivity (E0 or Q10) and not R10 (‘type I’ acclimation; Atkin & Tjoelker, 2003; Atkin et al., 2005. However, in our seasonal study of two perennial grasses with leaves that live multiple years, acclimation resulted from large changes in R10 (‘type II’ acclimation), with higher R10 in winter-acclimated leaves than in summer-acclimated leaves. Thermal acclimation via changes in R10 have been reported in seasonal field studies (Atkin et al., 2000; Xu & Griffin, 2006; Ow et al., 2010) as well as in plants subjected to long-term temperature manipulations (Tjoelker et al., 1999; Bruhn et al., 2007). Our results, along with these studies, support the idea that acclimation in pre-existing leaves can result from changes in R10 (respiratory capacity). Additionally, whereas other studies have found that seasonal or long-term acclimation is accompanied by either no change in the instantaneous temperature sensitivity (E0) of Rd (Tjoelker et al., 1999; Bruhn et al., 2007) or an increase in E0 with cold (Atkin et al., 2000; Ow et al., 2010), we found a positive correlation between E0 and 120 d averaged temperature, with higher values of E0 occurring in the summer and the lowest E0 values occurring in the winter. Although it may seem counter-intuitive to invoke a decrease in E0 when an up-regulation of Rd is required, low E0 during cold months would tend to reduce Rd primarily at high temperatures. This response would have little impact on in situ Rd at the low prevailing growth temperatures in winter, but would prevent an overcompensation of Rd during short periods of warm temperatures that are not uncommon in the variable climate of the New Zealand alpine zone.

We questioned whether acclimation of Rd to temperature in Chionochloa spp. would be underpinned by changes in nonstructural carbohydrates, leaf N, abundance of AOX and COX, and relative changes in electron partitioning to the AP vs the CP. In contrast to a number of studies that link R to carbohydrate status (Tjoelker et al., 1999, 2008; Whitehead et al., 2004; Lee et al., 2005; Ow et al., 2010), we found no relationship between the concentrations of soluble sugars, starches or TNC and respiratory parameters. We did find a significant relationship between R10 and leaf N in C. pallens in the seasonal study, but not in C. rubra (Table 1). However, the change in N in both species was small (0.89–1.35% in C. rubra; 0.83–1.05% in C. pallens), so it is not clear whether N exerts any real control over seasonal acclimation of Rd. We found a suggestive link between seasonal acclimation of Rd and the abundance of AOX and COX proteins in C. pallens. There was a relationship between the AOX : COX protein balance and 120 d averaged temperature in the seasonal study in C. pallens (Fig. 4b), which, although not statistically significant, supports previous laboratory studies reporting increased AOX protein in response to cold treatment temperatures under controlled conditions (Vanlerberghe & McIntosh, 1992b; Gonzalez-Meler et al., 1999; Ribas-Carbo et al., 2000a; Armstrong et al., 2008; Mizuno et al., 2008). We also found a positive relationship between the AOX : COX protein balance and R10 in C. pallens (Fig. 4d). These results suggest that changes in the AOX : COX protein balance have some role in the response of respiration to seasonal temperature changes in this species. In contrast to C. pallens, the AOX : COX protein ratio did not range as greatly in C. rubra, the lower-altitude species (0.68–2.28 in C. rubra; 0.57–5.16 in C. pallens), and it did not correlate with R10 or temperature. A possible ecological explanation of the differences between these related species could be that the high-altitude species experiences more extreme temperature fluctuations and lower temperatures, and must change both engagement of AOX and protein abundance in order to fully adjust its AP output to respond to cold stress. By contrast, we suggest that the low-altitude species may economize on protein construction costs and rely more heavily on changes in the activation state of existing proteins.

Owing to the difficulty in obtaining isotopic end points for the two oxidases, we interpret changes in oxygen isotope discrimination as relative changes in electron partitioning through the AP vs the CP, with higher discrimination values indicating a greater contribution of AOX to respiration. This is based on consistent findings in the literature that AOX discriminates more heavily against 18O than does COX (Ribas-Carbo et al., 2005a). The discrimination values reported here range from 17 to 24‰. While values of < 19‰ may be uncommon (Ribas-Carbo et al., 2005a), discrimination values of 17‰ and lower have been previously reported for various plant tissues (Guy et al., 1989; Millar et al., 1998; Nagel et al., 2001; Armstrong et al., 2008). The range in D (i.e. the difference between AOX and COX end points) for leaves has been reported to be 6–11‰, depending on species and the study (Ribas-Carbo et al., 2005a). Assuming a conservative range of 11‰ for Chionochloa spp., our findings show that C. rubra theoretically varies its contribution of AOX to R by up to 48%, and C. pallens by 33%, over the year. Interestingly, unlike the changes in the AOX : COX protein balance, discrimination does not appear to respond to changes in growth temperature and was not related to changes in total respiration.

Similarly to several previous studies (Guy & Vanlerberghe, 2005; Ribas-Carbo et al., 2005b; Vidal et al., 2007), we found no clear correlation between relative AP engagement and AOX protein abundance or the AOX : COX protein ratio. This is not surprising, as existing AOX protein in plant tissues is thought to be activated or engaged through changes in its redox state (Umbach & Siedow, 1993; Umbach et al., 1994, 2006) or by signalling compounds such as pyruvate (Ribas-Carbo et al., 1995, 1997; Millar et al., 1996; Rhoads et al., 1998), salicylic acid (Rhoads & McIntosh, 1992; Lennon et al., 1997) and ROS (Wagner, 1995; Vanlerberghe & McIntosh, 1996). We can therefore expect AP activity to respond more rapidly than protein abundance to environmental triggers.

Importantly, we found that D correlated positively with the previous day’s integrated PAR in both species in the seasonal study (Fig. 5), but not with daylength. This suggests that in Chionochloa spp., electron partitioning through the AP is up-regulated relative to the CP in response to high ambient light in the field. An increase in AP partitioning has been found in response to moderate increases in light in Glycine max cotyledons (Ribas-Carbo et al., 2000b) and high light in A. thaliana (Noguchi et al., 2001). The abundance of the AOX protein has also been shown to increase in response to moderate light in isolated mitochondria of Nicotiana sylvestris (Dutilleul et al., 2003) and to high light in A. thaliana (Yoshida et al., 2007, 2008). Yoshida et al. (2007) showed that the abundance of the AOX protein increased concomitantly with an increase in reducing equivalents in the chloroplast and with a decrease in FV/FM (indicating chloroplastic stress) during exposure to high light intensity, demonstrating that AOX can dissipate excess reducing equivalents during photo-oxidative stress. As FV/FM remained relatively high (0.803–0.863) in our plants at all times throughout the year, it is unclear if we observed a similar process to that reported in (Yoshida et al., 2007). Providing a different perspective, (Ribas-Carbo et al., 2008) found AP activity to increase and CP activity to decrease in soybean cotyledons exposed to red light under nonstressful conditions, indicating that phytochrome can control mitochondrial electron transport. Thus, the response of D to PAR in our study may simply be a direct result of phytochrome regulation.

It is interesting that D correlated best with the previous day’s integrated PAR in the seasonal study, yet did not change on a timescale of days in the short-term study. However, the range of daily integrated PAR observed during the short-term study (12.8–27.8 mol m−2 d−1 ) was considerably smaller than that observed over the course of the seasonal study (3.03–29.3 mol m−2 d−1). Thus, it is possible that the changes in PAR over the short-term study were not sufficient to produce a significant change in D.

Few studies published to date have specifically compared the short- and long-term responses of AOX to temperature. Some studies have found transient (1–7 d) increases in electron partitioning to the AP (Ribas-Carbo et al., 2000a; Armstrong et al., 2008), in AOX protein abundance (Vanlerberghe & McIntosh, 1992b; Mizuno et al., 2008) and in AOX transcript abundance in wheat and in A. thaliana exposed to cold stress (Ito et al., 1997; Sugie et al., 2006); these observed increases in AOX typically reverted after a few days. For instance, Armstrong et al. (2008) found discrimination to peak at day 4 of cold treatment in A. thaliana and to decrease by day 10. Here, we specifically investigated the timescale on which AP engagement and protein abundance respond to temperature changes in a perennial alpine grass and found that, like total Rd, the abundance of AOX and COX proteins did not respond to daily changes in temperature (Fig. 6; Table 2), but only to long-term changes in temperature, indicating a slow turnover of protein pools as part of the seasonal response of R. By contrast, D responded to daily changes in light, suggesting that the activation state of existing AOX and COX proteins is regulated on shorter timescales.

Our findings, that the AOX : COX protein balance was greater with cold temperatures and that discrimination increased with high light intensity, provide field-based support for previous laboratory findings. However, we observed a complex response in the field, with the AOX : COX protein balance and discrimination responding to different abiotic variables and on different timescales. These findings highlight that an improved understanding of how the AP regulates respiration in the field is critical in our ability to connect carbon balance physiology with the ecology of plants in changing environments.

Acknowledgements

Thanks to Ian Reeves, Jenny Ladley and David Conder for technical assistance, Catherine Campbell for immunoblotting training, and Owen Atkin for valuable comments on the manuscript. We gratefully acknowledge the Marsden Fund (The Royal Society of New Zealand) for supporting this project, as well as the New Zealand Department of Conservation for access to field sites.

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