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Keywords:

  • acclimation;
  • arbuscular mycorrhizal (AM) fungi;
  • protein abundance;
  • Q10;
  • root respiration (R);
  • temperature

Summary

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  • • 
    The arbuscular mycorrhizal (AM) symbiosis is ubiquitous, and the fungus represents a major pathway for carbon movement in the soil–plant system. Here, we investigated the impacts of AM colonization of Plantago lanceolata and temperature on the regulation of root respiration (R).
  • • 
    Warm-grown AM plants exhibited higher rates of R than did nonAM plants, irrespective of root mass. AM plants exhibited higher maximal rates of R (RmaxR measured in the presence of an uncoupler and exogenous substrate) and greater proportional use of Rmax as a result of increased energy demand and/or substrate supply. The higher R values exhibited by AM plants were not associated with higher maximal rates of cytochrome c oxidase (COX) or protein abundance of either the COX or the alternative oxidase.
  • • 
    Arbuscular mycorrhizal colonization had no effect on the short-term temperature dependence (Q10) of R. Cold-acclimated nonAM plants exhibited higher rates of R than their warm-grown nonAM counterparts. By contrast, chilling had a negligible effect on R of AM-plants. Thus, AM plants exhibited less cold acclimation than their nonAM counterparts.
  • • 
    Overall, these results highlight the way in which AM colonization alters the underlying components of respiratory metabolism and the response of root R to sustained changes in growth temperature.

Introduction

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Plant respiration (R) is an integral component of the terrestrial global carbon cycle, with 30–70% of the CO2 fixed by daily photosynthesis being released by plant R (Poorter et al., 1990; Loveys et al., 2002). Globally, plant R releases c. 60 Gt C yr−1 (Schlesinger, 1997; Field, 2001); in comparison, anthropogenic CO2 releases are only c. 8 Gt C yr−1. Roots contribute 30–50% of the CO2 released by whole plant R (Poorter et al., 1990) and up to 60% of total soil CO2 efflux (Hanson et al., 2000). Alterations in rates of root R can, therefore, have profound effects on the carbon economy of individual plants, and influence CO2 exchange rates at ecosystem and global scales (Hanson et al., 2000; Gifford, 2003; Schulze, 2006).

One factor that is likely to play an important role in determining variations in specific rates of root R is a plant's mycorrhizal status. For example, formation of the arbuscular mycorrhizal (AM) symbiosis, which is characteristic of the majority of land plant species (Smith & Read, 2008), has been associated with higher rates of root R compared with nonAM plants (Baas et al., 1989; Valentine & Kleinert, 2007) and increased soil CO2 release (Langley et al., 2005). Several factors could contribute to higher rates of root R in AM-colonized plants, including increased substrate availability associated with enhanced nutrient uptake and increased demand for respiratory products (i.e. ATP, reducing equivalents and TCA cycle intermediates) (Hughes et al., 2008). For example, respiratory ATP is probably required for each of the four stages of nutrient uptake by an AM plant (i.e. ion uptake by the external fungal hyphae, ion transport within the fungus, ion export by the internal hyphae, and ion uptake by plant root cells) (Hughes et al., 2008). Increased demand for ATP is likely to decrease adenylate restriction of flux through phosphofructokinase, pyruvate kinase, the pyruvate dehydrogenase complex and the proton-translocating steps of the mitochondrial electron transport chain (Wiskich & Dry, 1985; Loef et al., 2001). It may also explain why mitochondria concentrate around the arbuscules in root cortex cells of Medicago truncatula colonized by the AM fungus Glomus intraradices (Lohse et al., 2005). However, no study has investigated whether formation of the AM symbiosis is associated with a decline in adenylate restriction, a change in the capacity of individual steps of the respiratory system associated with ATP synthesis (e.g. cytochrome c oxidase, COX) and/or overall respiratory capacity.

Temperature contributes to variations in specific rates of root R. Understanding how AM colonization impacts on the temperature response of root R is vital if global circulation models (GCMs) are to predict future rates of CO2 release by soils into the atmosphere. In most GCMs, root R is assumed to increase in a simple exponential manner in response to temperature, with a Q10 (temperature sensitivity of R; the proportional increase in respiration per 10°C increase in temperature) of 2.0 (Cox, 2001). In reality, however, the Q10 of root R is highly dynamic, with Q10 values of 1.1 to 4.6 reported (Boone et al., 1998; Tjoelker et al., 1999; Loveys et al., 2003). Variations in Q10 likely reflect shifts in the control exerted by maximum enzyme activity, substrates and/or adenylate limitations (Atkin & Tjoelker, 2003). Covey-Crump et al. (2002) found that Q10 values of root R in nonAM plants increased in response to increased substrate supply or reduced adenylate restriction. Given that formation of the AM symbiosis might increase ATP turnover (and thus reduce adenylate restriction and/or substrate supply) (Hughes et al., 2008), the short-term temperature dependence of root R may be greater in plants when colonized by AM fungi compared with those that are uncolonized. There is evidence that ectomycorrhizal (ECM) colonization alters the short-term temperature dependence of root R (Koch et al., 2007). Boone et al. (1998) concluded that the Q10 values for mycorrhizas and rhizosphere heterotrophs must be substantially greater than those of roots per se in a mixed temperate forest. By contrast, there was no evidence that Q10 values differ among mycorrhizal roots, extraradical mycelium (ERM; the external fraction of mycorrhizal hyphae in soil) and soil lacking both roots and ERM in ECM seedlings of Pinus muricata (Bååth & Wallander, 2003). Moreover, Langley et al. (2005) found that the Q10 of soil CO2 efflux was similar in AM and nonAM sunflower plants growing in pots. Thus, there is currently no consensus about whether mycorrhizal colonization alters the Q10 of root R.

Over longer time periods, the response of root R to temperature will depend on the extent of thermal acclimation. Acclimation can result in cold- and warm-grown plants exhibiting similar rates of R when measured at their respective growth temperatures (i.e. respiratory homeostasis; Larigauderie & Körner, 1995). Cold-acclimated plants also exhibit higher rates of R than their warm-grown counterparts, when R is measured at a single moderate temperature (e.g. 20–25°C). There is growing evidence that thermal acclimation of root R is common in nonAM plants (Atkin et al., 2005a and references cited therein) and that respiratory acclimation occurs in soils (reflecting CO2 release by roots, AM fungi and other heterotrophs), as shown by the response of a tallgrass prairie system to artificial warming (Luo et al., 2001). Moreover, there is some indication that some species of ECM fungi grown in axenic cultures (Malcolm et al., 2008) can acclimate to temperature. Similarly, the ERM of the AM fungus Glomus mosseae exhibited near-complete respiratory homeostasis in a soil-warming experiment (Heinemeyer et al., 2006). Although it is not known if formation of the AM symbiosis alters the degree of acclimation exhibited by root R, differences between AM and nonAM plants might be expected if the acclimation potential of the plant partner differs from that of the intraradical mycelial (IRM; i.e. AM hyphae inside the root) of the AM symbiosis. Maximal respiratory homoeostasis in roots of nonmycorrhizal plants requires the production of new tissue (Loveys et al., 2003). As the lifespan of AM fungal tissue is short (Staddon et al., 2003), rapid turnover of IRM in roots experiencing a change in growth temperature could potentially alter the extent of respiratory homeostasis.

Our study assessed the interactive effects of colonization by AM fungi and temperature on rates of root R in Plantago lanceolata. The following hypotheses were tested: (i) rates of root R are higher in AM plants than in their nonAM counterparts (i.e. same plant species with or without AM inoculum); (ii) higher rates of root R in AM plants reflect increases in substrate supply and/or demand for respiratory ATP, combined with an increase in respiratory capacity (particularly of enzymes associated with ATP production); and (iii) the Q10 and degree of thermal acclimation of R are both greater in roots of AM plants than in their nonAM counterparts.

Materials and Methods

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Plant material and growth conditions

Experiments were conducted using Plantago lanceolata L. (ribwort plantain). Seed was obtained from Emorsgate Seed Ltd (Norfolk, UK). Surface-sterilized seeds that had been soaked overnight in deionized water were planted into 10-cm-wide pots (fitted with 20 µm screen discs at their base) containing a growth medium consisting of a 50 : 50 mix of sand and Terra-Green (a calcined attapulgite clay soil conditioner, Oil-Dri, Wisbech, UK). Thinning of germinated seedlings ensured that each pot contained one plant. The growth medium also contained 0.5 g l−1 of sterilized bonemeal (Vitax, Coalville, UK). Arbuscular mycorrhizal treatments received 50 g wet weight of Glomus hoi (Berch & Trappe) isolate UY 110 inoculum added to the sand : Terra-green medium. The nonAM controls received 50 g wet weight of the AM inoculum, which had been autoclaved (121°C; 30 min). The AM inoculum consisted of P. lanceolata root medium colonized with G. hoi and included the sand and Terra-Green growth medium. The inoculum was checked to confirm the presence of both root colonization and spores before addition to the pots. In addition, nonAM pots received 10 ml of filtered washings of the AM inoculum, passed through a 20 µm mesh and No.42 Whatman filter paper (Whatman International Ltd, Maidstone, UK) to remove AM propagules, to limit initial differences among pots in starter microbial communities (Hodge, 2001, 2003).

Randomized pots were placed in a growth cabinet (Snijders Microclima 1750, Snijders Scientific BV, the Netherlands) provided with a constant temperature of 21°C (16 h day, 60% RH) with 300 µmol m−2 s−1 PPFD (daily quanta input of 17.3 mol m−2 d−1) provided by fluorescent tubes. Pots were kept at near 20% moisture (a percentage of total mass) throughout the experiment by weighing the pots every few days and adjusting the water content as required. NonAM plants were fed once a week with 10 ml of Rorison's nutrient solution (1.0 mm MgSO4·7H2O, 2.0 mm Ca(NO3)2·4H2O, 1.0 mm K2HPO4·3H2O, 68 µm Fe-EDTA, 10 µm MnSO4·4H2O, 46.3 µm H3BO3, 0.15 µm (NH4)6Mo7O24·4H2O, 1.5 µm ZnSO4·7H2O, 1.6 µm CuSO4·5H2O). For AM plants, the same nutrient solution was used, with the exception that K2HPO4·3H2O was omitted and replaced with KCl. Bonemeal was therefore the AM plants’ only source of P and was added to provide a largely insoluble source of phosphate that would be accessible to, and promote colonization by, AM fungi but would provide little P to the nonAM plants.

In preliminary experiments, it was found that removal of AM extraradical mycelium (ERM) (using tweezers) had little effect on measured rates of O2 uptake by roots colonized by G. hoi. Similarly, bleaching of colonized roots for 5 s (using 2% NaOCl) had no effect on respiration rates. Thus, respiration rates exhibited by AM plants were assumed to reflect O2 uptake by the combination of roots and IRM, without contribution by the ERM. Preliminary experiments also suggested that the impact of G. hoi colonization on respiration rates differed between young/small and older/larger roots. Because of this, we decided to sample plants at several time points after pots were placed into the 21°C maintained growth cabinet (22, 29, 31, 42, 50, 57, 70 and 80 d after sowing of seeds). To assess the effect of 10 d cold treatment on nonAM and AM treatments, a subset of plants was shifted on three occasions (32, 50 and 70 d after sowing of seeds) from the 21°C cabinet to an identical cabinet set to constant 7°C for a further 10 d (i.e. plants were harvested 42, 60 and 80 d after sowing).

Respiration measurements

On each measurement day, whole-root systems were carefully removed from individual pots and washed. For 21°C-grown plants, each whole-root system was then divided longitudinally into two equivalent halves using a new razor blade. Respiration rate (nmol O2 (g fresh mass)−1 s−1) of one half of the root system was then measured at 21°C, with the other half being used to measure respiration at 7°C. Temperatures were controlled using two refrigerating Lauda E100 water baths (Lauda, Köningshofen, Germany) and insulation on the tubing and cuvettes. By measuring respiration at 21 and 7°C, we could subsequently assess the impact of AM colonization on the short-term temperature dependence of respiration of the warm-grown plants. For 10-d, 7°C-acclimated plants, measurements were made at 21°C only. Respiration was measured polarographically using Clark-type O2 electrodes (Dual Digital Model 20; Rank Brothers, Cambridge, UK), with roots being sealed in an air-tight cuvette containing 30–50 ml of Rorison's nutrient solution buffered (pH 5.8) with 10 mm morpholine ethane sulphonic acid (MES). Roots were left to equilibrate for 10 min; the rate of R was then measured over the subsequent 5 min. Thereafter, maximal rates of respiration (Rmax) were determined via injection of glucose (to a final concentration of 50 mm) and uncoupler (carbonyl cyanide m-chlorophenylhydrazone (CCCP), to a final concentration of 2 µm) into the cuvette via a canula in the lid of the cuvette, waiting for another 10 min, and then determining the rate of O2 uptake over the subsequent 5 min. To avoid O2 limitations to respiration, we ensured that all measurements were terminated before the O2 concentration in the cuvette fell below 40% of air saturation. For all measurements, corrections were made for the low rate of background O2 uptake by the electrodes themselves. Following measurement of respiration, the fresh mass (FM) of each root sample was determined (after blotting to remove excess moisture) and the sample was stored in 1% KOH to enable subsequent mycorrhizal assessment.

Quantifying AM colonization

For AM root colonization assessment, roots were cleared in KOH (90°C, 10 min), acidified in HCl (room temperature, 1 min) and stained with acid fuchsin (90°C, 20 min) (as Kormanik & McGraw (1982) but without phenol). AM colonization was examined with a Nikon Optiphot-2 microscope using brightfield and epifluorescence (Merryweather & Fitter, 1991) and at ×200 magnification. Mycorrhizal scoring, using 100 intersections, was carried out using the method of McGonigle et al. (1990). The percentage of AM structures (arbuscules, vesicles and total root length colonized (RLC; the percentage of total intercepts where AM structures were present)) were recorded for each intersection.

Enzyme activity and protein abundance

To determine whether colonization by G. hoi altered the capacity of COX in 21°C-grown and 10-d 7°C acclimated plants, extracts of AM-colonized and nonAM plants were prepared from 300 mg (FM) of frozen (−80°C) root material taken from an additional set of plants. For both temperature treatments (21°C grown and 10-d 7°C acclimated), plants were 41 d old when assayed for COX activity. COX activity in the extracts was then determined as described in Millenaar et al. (2001). Frozen roots from the same plants were also used to assess the effects of AM colonization and growth temperature treatment (warm-grown and 10-d cold-treated) on the concentration of COX subunit II) and mitochondrial alternative oxidase (AOX) (Campbell et al., 2007). A commercially available primary antibody (Agrisera, Vännäs, Sweden) was used to detect COX (used at 1 : 1000 primary, 1 : 10 000 secondary anti-rabbit). AOX was detected using a monoclonal antibody raised against Sauromatum guttatum AOX (Elthon et al., 1989), used at 1 : 500 primary, 1 : 500 secondary anti-mouse. Blots were immunostained with polyclonal rabbit or mouse antibodies, as required, and visualized using enhanced chemiluminescence (ECL; Amersham Biosciences Ltd, Amersham, UK, or the Pierce SuperSignal West Dura Extended Duration Substrate; Pierce Biotechnology, Rockford, IL, USA). The X-ray film was developed, scanned, and analysed using Adobe Photoshop 5.5 and ImageJ (US National Institutes of Health, Bethesda, MD, USA).

Calculations and statistical analyses

The short-term temperature sensitivity of R (Q10) of warm-grown plants was calculated over the 7–21°C measurement range, using the following equation:

  • Q10= (R21/R7)[10/(21–7)](Eqn 1)

where R21 and R7 are the respiration rates of roots measured at 21 and 7°C, respectively. Acclimation of respiration was assessed using the ‘set temperature’ and ‘homeostasis’ methods, as described in Loveys et al. (2003). Briefly, for the set-temperature method, we divided rates of R exhibited by cold-acclimated plants by rates exhibited by warm-grown plants; the rates compared were measured at 21°C in each case. The ‘homeostasis’ method requires a comparison of R rates at the respective growth temperatures of two contrasting growth temperature treatments; in our case, we compared rates of R of warm-grown and cold-acclimated plants at 21 and 7°C, respectively (the respective growth temperatures of each treatment).

Statistical analyses were carried out using SPSS v10, Sigmaplot v8.02 (SPSS Science, Birmingham, UK), and Microsoft Excel 2000. In order to normalize the data, log10 transformations were carried out and linear regressions then fitted. The F-ratio method for homogeneity of linear regression slopes was used to compare AM vs nonAM treatments, and warm-grown vs 10-d cold-acclimated treatments. The slopes were then tested for parallelism and the intercepts compared using a t-test.

Results

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Root size, dry matter content and AM colonization

Measurements of root metabolism and mycorrhizal status were conducted on individual whole roots ranging from 0.15 to 7.40 g FM. Over the 0.15–0.55 g FM range where dry mass (DM) values were also recorded, dry matter content (DMC, the ratio of root DM to FM) was slightly higher in colonized roots (0.185 ± 0.006) than in their nonAM counterparts (0.170 ± 0.005). In warm-grown AM plants, the average percentage of RLC was 27.0 ± 2.9%, with average arbuscule frequency being 9.3 ± 2.9%. Vesicle structures were not observed during the AM root colonization assessments. There was no colonization in the nonAM control plants.

Is the respiration rate affected by AM colonization?

To assess the effect of mycorrhizal colonization on rates of respiration, we measured whole-root O2 uptake at several stages of plant development. Irrespective of mycorrhizal status, rates of in vivo respiration (R) exhibited by warm-grown plants (measured at 21°C) declined exponentially with increasing root FM according to a power-function relationship. Given this, we used log-log plots to compare rates of R in nonAM and AM plants (Fig. 1). Rates of R were consistently higher in AM plants as demonstrated by the fact that a single equation did not describe the log-log relationship between rates of root R and FM in both nonAM and AM plants (Fig. 1, P < 0.001). There was no difference in the slope of the regression lines between the AM and nonAM plants (Table 1; P > 0.05); importantly, however, the intercept was significantly higher in AM plants than in their nonAM counterparts. Therefore, for any given root FM, the AM plants exhibited a higher rate of root R than the nonAM plants; P < 0.001 (Table 1, Fig. 1). Although absolute differences in rates of R between the treatments were greatest in young, small roots (Fig. 1), differences in rates of R were consistent through development when assessed on a proportional basis (as evidenced by the lack of significant difference in slopes of the log-log rate of R–FM plots; Table 1), with AM plants exhibiting, on average, nearly 50% higher rates of R than their nonAM counterparts. Clearly, therefore, rates of root R were affected by AM colonization.

image

Figure 1. Rate of respiration (R) in roots colonized by arbuscular mycorrhizal (AM) fungi (open circles) and nonAM roots (closed circles) in relation to the fresh mass (FM) of whole roots, with the x- and y-axes shown on a log10 scale. Values are for individual Plantago lanceolata plants harvested at different time points (22–80 d after sowing) for measurements made at 21°C for warm-grown plants. On each sampling day, three to eight replicate plants were measured, with a total of 43 and 47 replicate measurements being recorded for nonAM and AM plants, respectively. Respiration rates were measured in the absence of exogenous substrate or uncoupler. First-order regression lines are show to illustrate differences between the mycorrhizal treatments. Values of r2, y-axis intercept and slope are shown in Table 1.

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Table 1.  Regression analysis of log10-transformed data for nonarbuscular mycorrhizal (AM) and AM Plantago lanceolata plants (warm-grown)
y-axis parameterMycorrhizal statusr2y-axis interceptSlopeF-ratio comparison of regressions (nonAM vs AM)P (y-axis intercepts)P (slopes)
  1. x = log10(of FM in g), y = log10(of respiration in nmol O2 (g FM)−1 s−1), where FM is fresh mass. Rates of respiration were measured either in the absence of effectors (i.e. R) or in the presence of exogenous substrate plus uncoupler (i.e. maximal rates of R, (Rmax)). See Fig. 1 for data on log R vs log FM.

  2. ns, not significant.

RNonAM0.160.137−0.169F2,86 = 17.61 (P < 0.001)< 0.0001ns
AM0.440.313−0.237   
RmaxNonAM0.300.369−0.207F2,86 = 3.10 (P < 0.05)< 0.0001ns
AM0.300.430−0.183   

To assess the impact of mycorrhizal status on respiratory capacity (Rmax), we also measured rates of O2 uptake in the presence of exogenous substrate and uncoupler. As was the case in Fig. 1, rates of Rmax measured at 21°C exhibited an ontogenetic decline (data not shown), with rates of Rmax being consistently higher in AM plants than in their nonAM counterparts (Table 1, P < 0.05). Moreover, the slope of log-log Rmax–FM plots did not differ between nonAM and AM plants (Table 1; P > 0.05), whereas the intercepts did differ (Table 1, P < 0.0001). In both treatments, the proportion of respiratory capacity used (R/Rmax) remained constant with increasing root mass in both treatments (P > 0.05; data not shown), with the average R/Rmax ratio being greater in AM plants than in their nonAM counterparts (Table 2). Thus, the faster-respiring AM plants used a greater proportion of their respiratory capacity than the slower-respiring nonAM plants. At a measuring temperature of 7°C, average R/Rmax values were higher in both treatments (Table 2). Taken together, these results suggest that the higher rates of R exhibited by AM plants (Fig. 1) are underpinned by a higher Rmax combined with greater use of that higher Rmax, and that in vivo rates of R become constrained by Rmax at low measuring temperatures, particularly in nonAM plants.

Table 2.  Comparison of respiratory parameters for warm-grown arbuscular mycorrhizal (AM) and nonAM Plantago lanceolata plants
Mycorrhizal statusR/RmaxQ10
21°C7°CRRmax
  1. R/Rmax values represent the proportion of respiratory capacity of roots used under warm (21°C) and cold (7°C) measuring conditions. R/Rmax represents the ratio of rates measured in the absence of effectors (R) divided by rates exhibited in the presence of an uncoupler (2 µm CCCP) and exogenous substrate (50 mm glucose) (Rmax). Also shown are the Q10 values (i.e. proportional decline in respiration per 10°C drop in temperature) of R and Rmax for nonAM and AM 21°C-grown plants. Values are the mean of all measured replicates, irrespective of root mass (for both parameters, regression analysis showed that there was no systematic change with increasing mass). For each column of data, P values (from a one-way ANOVA) compare nonAM with AM plants (ns, not significant at P > 0.05). Values in brackets are the number of individual replicates.

NonAM0.63 ± 0.03 (43)0.91 ± 0.08 (33)1.83 ± 0.14 (34)2.65 ± 0.11 (34)
AM0.82 ± 0.03 (47)0.87 ± 0.07 (33)1.86 ± 0.14 (39)2.37 ± 0.12 (39)
P valueP < 0.001nsnsns

To assess whether the degree of colonization is positively associated with higher rates of R, we plotted rates of R (measured at 21°C) of warm-grown plants against the percentage of root cross-sectional area occupied by arbuscules (% arbuscules), and percentage root length colonization (% RLC) using data from mycorrhizas where both colonization and rates of R were measured on the same root system. Although there was considerable scatter in the data of R plotted against % arbuscules (Fig. 2a), a significant positive relationship was found (P < 0.05, r2 = 0.15), suggesting that rates of R increase in proportion to the frequency of arbuscule formation, with arbuscules being the putative site of nutrient exchange between symbionts. The relationship between rates of R and % RLC was not significant (P = 0.06, r2 = 0.13; Fig. 2b).

image

Figure 2. Rate of respiration (R) in arbuscular mycorrhizal (AM) Plantago lanceolata plants in relation to the degree of colonization, as measured by percentage of arbuscules (a) or percentage total root length colonization (%RLC) (b). Respiration rates were measured in the absence of exogenous substrate or uncoupler. For (a), the relationship was significant (root R = 1.60 + 0.027 *×% arb; P = 0.041, r2 = 0.15. In (b), the relationship was not significant (P = 0.06, r2 = 0.13). Values are for individual roots (n = 28).

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Formation of the AM symbiosis: impacts on the temperature dependence of R

The effect of mycorrhizal status on the short-term temperature dependence of O2 uptake (Q10) was investigated via measurement of R and Rmax rates at two temperatures (21 and 7°C) and calculation of Q10 values using Eqn 1. There was no significant relationship between Q10 of R and root FM for either nonAM or AM plants (Fig. 3); similarly, the relationship between Q10 of R and root FM was not significantly different between the two treatments (F2,71 = 0.112, P > 0.05, Fig. 3). The overall average value of Q10 of R was 1.85 (Table 2). As provision of exogenous substrate and uncoupler resulted in a greater proportional increase in O2 uptake measured at 21°C than at 7°C, calculated Q10 values were higher for Rmax than for R (Table 2). Importantly, however, there was no significant difference in Q10 values of Rmax exhibited by nonAM and AM plants (Table 2). Thus, while colonization by AM fungi increases in vivo rates of O2 uptake (and Rmax; Table 1), it has no effect on the short-term temperature dependence of respiration rates.

image

Figure 3. Short-term proportional change in respiration (R) per 10°C change in temperature (Q10) values of respiration in relation to the fresh mass (FM) of whole roots for Plantago lanceolata roots colonized by arbuscular mycorrhizal (AM) fungi (open circles) and nonAM roots (closed circles). Q10 values were calculated over the 7–21°C measurement temperature range using the equation provided in the text. Values are for warm-grown individual plants harvested at different time points (22–80 d after sowing). Respiration rates were measured in the absence of exogenous substrate or uncoupler. On each sampling day, three to eight replicate plants were measured, with a total of 35 and 40 replicate measurements being recorded for nonAM and AM plants, respectively. Note: because of technical problems with some 7°C measurements, it was not possible to calculate Q10 values on all days (i.e. no Q10 values are available on day 31 for both treatments, and day 57 for nonAM plants only). As there was no significant change in Q10 values with increasing FM in either mycorrhizal treatment (either in the absence or presence of glucose or CCCP), overall average Q10 values for both mycorrhizal treatments were calculated (Table 2).

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To assess the acclimation of R rates to sustained low temperature on respiration rates, we shifted warm-grown plants from 21 to 7°C for 10 d. In nonAM plants, rates of R measured at 21°C were consistently higher in cold-acclimated plants than in their warm-grown counterparts (Fig. 4a; P < 0.01); the slope of log-log R vs FM plots was similar for warm-grown and cold-acclimated plants. By contrast, no significant differences were found among rates of R exhibited by warm-grown and cold-acclimated AM plants (Fig. 4b). Thus, in contrast to nonAM plants, AM plants did not appear to acclimate to cold; Table 3 shows that acclimation ratios were consistently higher in nonAM plants than in their AM counterparts, as measured using the homeostasis and set-temperature methods. The latter is further illustrated by Fig. 5, where average rates of R measured at 21°C were consistently higher in 10-d, cold-acclimated plants than in their warm-grown counterparts for nonAM plants, but less so for their AM counterparts.

image

Figure 4. Impact of growth temperature treatment (warm-grown, closed circles) and 10-d cold-acclimated (open circles) on rates of respiration (R) in nonarbuscular mycorrhizal (nonAM) Plantago lanceolata roots (a), and arbuscular mycorrhizal (AM) roots (b). In both (a) and (b), symbols represent respiration measured at 21°C in relation to the fresh mass (FM) of whole roots, with the x- and y-axes shown on a log10 scale. Values are for individual plants harvested at different time points (42–80 d after sowing) for measurements made at 21°C, irrespective of the growth temperature treatment. Respiration rates were measured in the absence of exogenous substrate or uncoupler. As plants grew at different rates under the warm and cold growth conditions, we limited the analysis to a common range of root fresh masses in both treatments (0.5–5.5 g). The numbers of replicates for each treatment combination were as follows: nonAM/warm-grown, 38; AM/warm-grown, 38; nonAM/cold-acclimated, 25; AM/cold-acclimated, 23). In (a) F-ratio tests showed data for nonAM plants were best fitted by two separate lines (F2,59 = 5.83; P < 0.01), with the equations log10 R = 0.136 – 0.165 log10 FM and log10 R = 0.286 – 0.230 log10 FM; by contrast, data for AM plants (b) were best fitted by a single regression (comparison of separate lines: F2,57 = 2.99; P > 0.05) with the equation log10 R = 0.295 – 0.157 log10 FM.

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Table 3.  Acclimation ratios of respiration for arbuscular mycorrhizal (AM) and nonAM Plantago lanceolata plants
ParameterMycorrhizal statusSampling day
41526080Average
  1. Values were calculated using the ‘set temperature’ (AcclimSetTemp) and ‘homeostasis’ (AcclimHomeo) methods. Acclimation ratios were calculated by comparing rates of respiration exhibited by 10-d, 7°C-acclimated plants with those exhibited by plants kept in the warm (21°C). Ratios are shown for plants sampled on four sampling days (d after potting), with the overall average within a treatment being shown in bold text. High ratios indicate a high degree of cold acclimation.

AcclimSetTempNonAM1.681.731.701.061.54
AM0.801.211.230.991.05
AcclimHomeoNonAMNo data0.560.820.850.74
AMNo data0.420.520.510.48
image

Figure 5. One-to-one plot of respiration rates (R) measured at 21°C of 10-d cold-acclimated Plantago lanceolata plants vs warm-grown plants for arbuscular mycorrhizal (AM; open circles) and nonAM (closed circles) plants. Individual values are the mean of four to eight replicates (± SE) for plants sampled after 41, 52, 60 and 80 d after sowing.

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Enzymatic capacity of COX and protein abundance of the terminal oxidases

Colonization by AM fungi had no effect on the maximal activity of COX (Table 4). Similarly, maximal COX activity was similar in warm- and cold-acclimated roots (Table 4). Growth temperature treatment also had no effect on the relative abundance of COX protein (Table 4); however, in contrast to maximal COX activity, significant differences were found in the relative abundance of COX between nonAM and AM plants, with COX protein abundance being two to three times higher in nonAM plants than in their AM counterparts (Table 4). A contrasting picture is seen when assessing the impacts of mycorrhizal status and growth temperature on the relative abundance of AOX. In warm-grown plants, no differences were found between nonAM and AM plants (Table 4). Exposure to low temperature for 10 d was associated with an increase in AOX protein abundance in nonAM plants, but not in their AM counterparts (Table 4).

Table 4.  Cytochrome c oxidase (COX) activity and relative abundance of COX subunit II and alternative oxidase (AOX) protein for roots of arbuscular mycorrhizal (AM) and nonAM Plantago lanceolata plants
Growth temperatureMycorrhizal statusParameter
COX activity (min g−1 FM)COXII proteinAOX protein
  1. Values are shown for warm-grown (WG; 21°C) and 10-d cold-acclimated (CA; 7°C) plants. Protein abundance is expressed on a fresh mass basis, relative to a standard (Plantago lanceolata leaf samples; Campbell et al., 2007). Values are the mean of eight replicate samples (± SE).

  2. ns, not significant.

Warm-grown (WG)NonAM12.2 ± 0.71.34 ± 0.27 0.16 ± 0.03
AM13.6 ± 2.10.58 ± 0.070.16 ± 0.04
P value (nonAM vs AM)nsP < 0.001ns
10-d cold-acclimated (CA)NonAM13.0 ± 3.01.03 ± 0.090.30 ± 0.05
AM12.3 ± 1.90.59 ± 0.080.12 ± 0.02
P value (nonAM vs AM)nsP < 0.001P < 0.05
Comparison of WG vs CA (P value)NonAMnsnsP <  0.05
AMnsnsns

Discussion

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Do AM plants exhibit consistently higher rates of root R than nonAM plants?

The results shown in Fig. 1 support those of previous studies (Snellgrove et al., 1982; Baas et al., 1989; Valentine & Kleinert, 2007) that reported increased rates of in vivo root R in plants colonized by AM fungi. However, they contrast with those of Silsbury et al. (1983), who found that there was no significant difference in rates of R in older roots of AM and nonAM Trifolium subterraneum. In Silsbury et al. (1983), no attempt was made to account for ontogenetic changes in rates of root R, raising the possibility that AM and nonAM roots were not at a comparable developmental stage at the time of sampling. Moreover, although the proportional stimulation of root R by AM colonization was consistent in young and old roots, the absolute differences between AM and nonAM plants were smaller in older/larger roots than in their younger/smaller counterparts (Fig. 1). Thus, reliance on comparisons of only older root systems could result in nonsignificant differences between the treatments, particularly given the substantial variability in measured rates of root R. Accounting for ontogenetic changes and variability in measured rates of root R is thus essential when comparing respiratory metabolism of AM and nonAM plants. The small difference (< 10%) in dry matter content between AM and nonAM roots may have led to an overestimate in the difference in R expressed on a fresh weight basis, but not sufficiently to remove the effect.

Physiological factors underpinning higher rates of root R in AM plants

The increase in R was underpinned by an increase in the capacity for O2 uptake (Rmax, Table 1) and greater use of that capacity (63 and 82% in nonAM and AM plants, respectively; Table 2). What underlying factors might explain the higher Rmax and R/Rmax values exhibited by AM plants? In the presence of CCCP and glucose, Rmax values of the symbiosis will reflect the capacities for O2 uptake of the root cell and IRM components. For each component, higher Rmax values might be underpinned by increased abundance of glycolytic proteins, higher potential rates of mitochondrial O2 uptake per unit mitochondrial protein (e.g. via increased abundance of rate-limiting proteins and/or changes in the ultrastructure of mitochondria) and/or increased density of mitochondria per unit cell volume. Although we lack data on each of these factors for the plant and fungal partners, colonization of Medicago truncalula roots by the AM fungus Glomus intraradices increases the density of mitochondria in root cells adjoining the arbuscules, and increases activity of the TCA cycle (Lohse et al., 2005). Addition of fungal mitochondria to that of root cell mitochondria may also contribute to the greater rates of Rmax exhibited by AM plants compared with their nonAM counterparts. The higher Rmax values are unlikely associated with increased capacity via the cytochrome pathway of the whole AM system, given the similar maximal COX activity values of AM and nonAM plants (Table 4). Moreover, the low abundance of AOX protein (Table 4) suggests that increases in AOX activity were also unlikely to be responsible for the higher Rmax values exhibited by AM plants. Rather, the greater Rmax values in AM plants likely result from increases in the capacity of enzymatic steps upstream of the terminal oxidases (e.g. the UQ-reducing pathways, TCA cycle and/or glycolysis).

The greater R/Rmax ratios exhibited by the AM plants could have resulted from an increase in respiratory substrate availability and/or increased respiratory product demand (ATP, NADH, and/or carbon skeletons) across the whole AM system. Our data do not allow us to determine whether one or both of these factors were responsible for the rise in R/Rmax. However, because mycorrhizal colonization increases the proportion of plant carbon allocated below ground (Douds et al., 1988), Hughes et al. (2008) hypothesized that carbon availability might be less limiting to root R in AM plants. Moreover, the exchange of carbon and nutrients between the root and the fungus could lead to an increased demand for respiratory products in the plant component of the symbiosis (Hughes et al., 2008), as may the energy and carbon skeleton costs associated with fungal growth and tissue maintenance. For the latter, it seems likely that formation of arbuscules (which were positively correlated with rates of R in AM plants; Fig. 2) would increase the demands for respiratory products. The formation of arbuscules necessitates the production of new plasma membrane material in the plant cell, which may lead to an increase in the specific respiratory product demands associated with plant tissue maintenance (Hughes et al., 2008). Individual arbuscules also turn over rapidly (Alexander et al., 1989), thus making this additional demand for respiratory products a potentially persistent feature of roots colonized by AM fungi (Hughes et al., 2008). If correct, this may explain why the density of mitochondria in root cells is greatest in the regions adjoining individual arbuscules (Lohse et al., 2005).

Our use of CCCP and glucose to estimate colonization-mediated changes in Rmax is similar to that of Noguchi et al. (2001) and Covey-Crump et al. (2002), who used them to estimate changes in respiratory capacity during acclimation to contrasting growth irradiances and temperature, respectively. Addition of CCCP dissipates the proton-motive force across plant membranes, including the inner mitochondrial membrane, resulting in ADP availability increasing; it can also increase flux through glycolysis as a result of removal of adenylate restriction of phosphofructokinase and pyruvate kinase. Addition of exogenous glucose increases substrate supply and can result in a slight decrease in adenylate restriction as a result of ATP consumption during the transport of glucose into the cell (Hatzfeld & Stitt, 1991). Thus, in the presence of both effectors, respiratory rates are thought to be near-maximal.

Cytochrome and alternative oxidases in AM and nonAM plants

At the outset of our study, we hypothesized that in addition to exhibiting higher rates of root R, plants colonized by AM fungi would also exhibit increased capacity for O2 uptake by pathways associated with ATP synthesis. In contrast with the nonphosphorylating AOX pathway, electron flux via the COX pathway results in proton translocation and subsequent ATP synthesis. Thus, it was expected that maximal COX activity would be higher in AM plants (compared with their nonAM counterparts). However, the absence of any differences in maximal COX activity (Table 4) strongly refutes this hypothesis. Thus, if colonization by AM fungi does increase the demand for ATP, as recently suggested (Hughes et al., 2008), the most parsimonious explanation is that this increase in ATP demand can be met by the COX capacity already present in roots before colonization by the AM fungi. Indeed, the capacity of the COX is often far greater than the measured rates of in vivo respiration (Millar et al., 1995), particularly when measured at moderate temperatures in young roots (Millar et al., 1998).

An intriguing finding in our study was the apparently lower abundance of COX subunit II protein in the AM plants compared with their nonAM counterparts, both in warm-grown and cold-acclimated plants (Table 4). This result contrasts with the near homeostatic rates of maximal COX activity irrespective of mycorrhizal status or growth temperature (Table 4). One explanation for this apparent discrepancy is that the COXII primary antibody that has been used successfully in plants (Campbell et al., 2007) does not react with the COX subunit II protein in the fungal component of the symbiosis. The target sequence used, though highly conserved in plants, is only partly conserved in fungal COX sequences. Because of this, it is possible that the fungal protein may have been detected only weakly, if at all, by this antibody. If correct, then this might suggest that the fungal component may represent a large proportion of overall respiratory capacity in the symbiosis fungus, given the near-twofold difference in COXII protein concentrations between the nonAM and AM plants (Table 4).

Although cold has been found to increase COXII protein/activity levels in roots of one of the four genotypes used by Kurimoto et al. (2004), growth temperature had no significant effect on either parameter in the other three genotypes used in that study. Similarly, we found that COX activity and COXII protein concentrations were similar in warm-grown and cold-acclimated plants, irrespective of the mycorrhizal status of the plants (Table 4). What is lacking, however, are estimates of electron partitioning between the AOX and COX pathways in warm- and cold-grown plants (e.g. using the 18O-fractionation technique; Guy et al., 1989). Similarly, estimates of AOX and COX pathway activity in AM and nonAM plants are needed.

In contrast to COXII, AM and nonAM plants exhibited the same AOX protein concentrations in warm-grown plants. We know that AOX is found in most plants (Vanlerberghe & Mcintosh, 1997), that AOX is found in some fungi (Lambowitz et al., 1989; Sakajo et al., 1991), and that root AOX protein concentrations can increase in response to sustained cold treatment (Kurimoto et al., 2004). However, it is not known if AOX is found in AM fungi. Although our study provides some insight into AOX abundance in roots colonized by an AM fungus, further work is needed to determine the extent to which the plant and fungal partners differ in AOX abundance, their relative contribution to AOX abundance in the whole AM system, and the extent to which the symbiotic partners differ in temperature-induced changes in AOX protein concentrations.

Short-term temperature sensitivity of root R

Whereas some studies have suggested that the Q10 of mycorrhizal roots will be higher than that of nonmycorrhizal plants (Boone et al., 1998; Koch et al., 2007), our results strongly suggest that colonization by AM fungi does not alter the temperature dependence of root R (Table 2). A similar conclusion was reached by Bååth & Wallander (2003) and Langley et al. (2005) for ECM- and AM-colonized rhizocosm systems, respectively. We had expected Q10 values to be greater in AM plants, as the Q10 is expected to be higher under conditions of reduced adenylate and/or substrate limitation (Covey-Crump et al., 2002). Indeed, we found that the stimulatory effect of CCCP and glucose on root R was lower in AM plants than in their nonAM counterparts (Table 2), suggesting that adenylate and/or substrate limitations were lower in colonized plants. Given this, why weren't Q10 values greater in AM plants? To answer this question, we need to consider how Q10 values are influenced by the balance between enzymatic capacity, substrate supply and adenylates. Q10 values are likely to be higher whenever respiratory flux is more limited by enzymatic capacity than by substrates and/or adenylates (Atkin & Tjoelker, 2003); this explains why Q10 values of root R were highest in the presence of exogenous glucose and CCCP, irrespective of the mycorrhizal status (Table 2). Conversely, transition from limitations in enzyme capacity to limitations imposed by substrate supply (and/or adenylates) can result in a decline in Q10. In our study, we found that respiratory capacity (Rmax) was greater in AM plants than in their nonAM counterparts (Table 1); alone, this might result in the Q10 of AM plants being lower than that in nonAM plants. However, against this is the fact that adenylates and/or substrates were less limiting in AM plants (which alone would increase the Q10). Taken together, we suggest that Q10 values of in vivo R were similar in AM and nonAM plants because the AM-mediated increase in Rmax (Table 1) was countered by the AM-mediated reduction in adenylate and/or substrate limitations (Table 2).

Acclimation to sustained low-temperature treatment

We investigated the ability of AM and nonAM plants to acclimate to a 10-d, 7°C temperature treatment via comparison of rates of root R measured at a common temperature (i.e. the set-temperature method); and comparison of rates of root R at each respective growth temperature (i.e. the homeostasis method) (Atkin et al., 2005b). Both our past (Covey-Crump et al., 2002; Atkinson et al., 2007) and current work (Table 3, Figs 4, 5) show that Plantago lanceolata exhibits considerable thermal acclimation of root R when nonAM plants are exposed to lower temperatures for several days. Our current results demonstrate that cold acclimation in nonAM plants occurs irrespective of whether roots were small or large when shifted to the cold for 10 d (Fig. 4a). By contrast, 10 d exposure to cold had negligible effect on root R of AM-plants, irrespective of root FM (Fig. 4b). Given the importance of respiratory acclimation to temperature for plant performance (Rachmilevitch et al., 2006) and predicting future rates of CO2 exchange between vegetation and the atmosphere (King et al., 2006; Atkin et al., 2008), and the widespread nature of the AM symbiosis (Smith & Read, 2008), this finding could have important implications for our understanding of how future changes in climate might impact on the carbon balance of individual ecosystems and the global carbon cycle. Importantly, however, we need to determine the impacts of AM colonization on thermal acclimation of root R under natural field conditions (both in pre-existing roots that experience a sustained change in growth temperature, and in roots that develop at different temperatures) if we are to accurately account for AM-mediated changes in thermal acclimation in a future, warmer world.

Our finding that AM plants exhibit minimal cold acclimation of root R contrasts with recent studies that have reported considerable thermal acclimation of the ERM of AM fungi (Heinemeyer et al., 2006) and ECM fungi (Malcolm et al., 2008). Thus, the absence of cold-induced increases in rates of root R in our AM plants (Fig. 4b) does not necessarily imply a lack of acclimation potential of the fungus per se. Rather, rates of root R appear to be running at near-maximal values in AM plants, as illustrated by the high R/Rmax ratio (Table 2). To acclimate to cold, AM plants would need to increase the overall capacity of R (i.e. Rmax values would need to increase). Although we did not measure Rmax in cold-acclimated roots, we have previously found little change in Rmax in nonAM plants exposed to 10°C for several days (Covey-Crump et al., 2002). Moreover, higher degrees of thermal acclimation are possible if root tissues develop under a new thermal regime (Loveys et al., 2003), with increases in Rmax likely to underpin the greater degree of acclimation in roots that develop in the cold (Atkin & Tjoelker, 2003). Thus, depending on the extent to which the development of the AM symbiosis is restricted at low soil temperatures (Gavito et al., 2005), it is possible that root R of AM plants can acclimate to sustained cold.

Concluding statements

Given that two-thirds of all land species are capable of forming the AM symbiosis (Smith & Read, 2008), the potential for AM colonization and/or temperature to alter rates of root R (Pregitzer et al., 2000; Hughes et al., 2008), and the importance of root R in determining ecosystem and Earth system functioning (Hanson et al., 2000; Gifford, 2003; Schulze, 2006), it is imperative that we develop a more thorough, process-based understanding of the factors controlling root respiratory CO2 release in AM plants. It was with these issues in mind that we investigated the impacts of AM colonization and temperature on root R of Plantago lanceolata. Our results show that AM plants exhibit higher rates of root R, both in young and older roots. Underpinning the increase in R is a rise in respiratory capacity, as well as increased utilization of that capacity that results from increased energy demand and/or substrate supply in roots of AM plants. Despite such changes, we found no evidence that AM colonization alters the short-term temperature dependence of root R. However, colonization does reduce the extent to which pre-existing roots can increase respiratory rates in response to a 10 d chilling period. Collectively, these results highlight the potential for AM colonization to alter the underlying components of respiratory metabolism and the response of R to sustained changes in growth temperature.

Acknowledgements

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

This work was funded by a Natural Environment Research Council (NERC), UK, grant (reference NE/D008301/1) to AH, OKA and AHF, and by a Nuffield Foundation Undergraduate Research Bursary to SJ. We thank Hans Lambers and two anonymous referees for their comments on the manuscript.

References

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References