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Maintenance of Growth Rate at Low Temperature in Rice and Wheat Cultivars with a High Degree of Respiratory Homeostasis is Associated with a High Efficiency of Respiratory ATP Production

Maintenance of Growth Rate at Low Temperature in Rice and Wheat Cultivars with a High Degree of... Abstract Some plants have the ability to maintain similar respiratory rates (measured at the growth temperature) when grown at different temperatures. This phenomenon is referred to as respiratory homeostasis. Using wheat and rice cultivars with different degrees of respiratory homeostasis (H), we previously demonstrated that high-H cultivars maintained shoot and root growth at low temperature [Kurimoto et al. (2004)Plant Cell Environ., 27: 853]. Here, we assess the relationship between respiratory homeostasis and the efficiency of respiratory ATP production, by measuring the levels of alternative oxidase (AOX) and uncoupling protein (UCP), which have the potential to decrease respiratory ATP production per unit of oxygen consumed. We also measured SHAM- and CN-resistant respiration of intact roots, and the capacity of the cytochrome pathway (CP) and AOX in isolated mitochondria. Irrespective of H, SHAM-resistant respiration of intact roots and CP capacity of isolated root mitochondria were larger when plants were grown at low temperature, and the maximal activity and relative amounts of cytochrome c oxidase showed a similar trend. In contrast, CN-resistant respiration of intact roots and relative amounts of AOX protein in mitochondria isolated from those roots, were lower in high-H plants grown at low temperature. In the roots of low-H cultivars, relative amounts of AOX protein were higher at low growth temperature. Relative amounts of UCP protein showed similar trends to AOX. We conclude that maintenance of growth rate in high-H plants grown at low temperature is associated with both respiratory homeostasis and a high efficiency of respiratory ATP production. (Received February 28, 2004; Accepted May 7, 2004) Introduction Plant mitochondria have two ubiquinol-oxidizing pathways, the cytochrome pathway (CP) and the alternative pathway. The latter consists of one enzyme, the alternative oxidase (AOX). AOX is not coupled to H+ translocation and, therefore, ATP production, and has the potential to catalyze wasteful respiration in higher plant mitochondria. The precise role of AOX has not been defined in plants, but it is thought to prevent production of reactive oxygen species (ROS) in the respiratory chain, especially under stress conditions, by helping to prevent over-reduction of the ubiquinone pool (Purvis and Schewfelt 1993, Wagner 1995, Maxwell et al. 1999, Vanlerberghe and Ordog 2002, Millenaar and Lambers 2003). Many abiotic stresses, including sudden exposure to low temperature, induce synthesis of AOX (Wagner and Krab 1995). The increase in AOX capacity at low temperature (Elthon et al. 1986, McNulty and Cummins 1987) is often associated with de novo synthesis of AOX protein (Stewart et al. 1990, Vanlerberghe and McIntosh 1992, Gonzàlez-Meler et al. 1999). In hypocotyls and leaves of mung bean grown at low temperature (Gonzàlez-Meler et al. 1999) and chilling-sensitive maize leaves (Ribas-Carbo et al. 2000), the in vivo activity of AOX also increased. However, such increases in AOX activity and capacity were not observed in other tissues or species, such as soybean cotyledons (Gonzàlez-Meler et al. 1999) and a chilling-insensitive maize cultivar (Ribas-Carbo et al. 2000) when grown at low temperatures. Thus, there is still some confusion about the responses of AOX to low growth temperature and the role of AOX at different temperatures. Some plants have the ability to maintain their respiratory rates (measured at the growth temperature), even when grown at different temperatures, a phenomenon referred to as respiratory homeostasis (Atkin and Tjoelker 2003). We investigated root respiration and plant growth in two wheat cultivars with a high degree of homeostasis (H), and in one wheat cultivar and one rice cultivar with a low H (Kurimoto et al. 2004). The plants with high H showed a tendency to maintain their relative growth rate (RGR), irrespective of growth temperature, whereas the plants with low H grown at 15°C showed lower RGR than those grown at 25°C. We suggested that respiratory homeostasis would help to maintain RGR at lower growth temperatures. However, the contribution of AOX to respiration may influence the rate of ATP production to a major extent. We therefore asked the question: is there any difference in response of AOX to low growth temperature between high-H and low-H plants? The uncoupling protein (UCP) may also influence the efficiency of ATP synthesis, because protons leak from the intermembrane space to the mitochondrial matrix through UCP, and the proton gradient is dissipated (Sluse and Jarmuszkiewicz 2002). Low temperature induces UCP1 transcript abundance in Arabidopsis seedlings (Maia et al. 1998) and UCP protein abundance in potato tuber (Nantes et al. 1999) and tomato fruit (Holtzapffel et al. 2002). On the other hand, expressions of UCP2 in Arabidopsis seedling and UCP1 in wheat seedlings were insensitive to low temperature (Watanabe et al. 1999, Murayama and Handa 2000). More information on response of UCP to growth temperature is also needed. To investigate a possible relationship between components of the respiratory chain and the homeostasis of respiration, we selected two wheat cultivars with high H and one wheat and one rice cultivar with low H. We grew them at 15 and 25°C, and measured root oxygen uptake rates with and without respiratory inhibitors, KCN and SHAM, which inhibit the cytochrome and alternative paths, respectively. Using isolated mitochondria from the roots of these plants, NADH- and succinate-dependent rates of oxygen uptake via CP and AOX, and the maximal activity of COX, were measured. Respiratory protein abundance was examined by immunoblots for subunit II of COX, AOX and UCP. We discuss the effects of growth temperature on root electron transport system and plant growth. Results and Discussion Respiratory rates in intact roots Root respiratory rates of plants grown at 15°C were faster than those of plants grown at 25°C (Fig. 1A–D). According to Atkin et al. (2004) and Kurimoto et al. (2004), the degree of homeostasis (H) was calculated as: where Rn(m) denotes a respiratory rate of roots of n°C-grown plants, which were measured at m°C. This value occurs between 0 (no acclimation) and 1 (full acclimation). The values of H for Stiletto and Patterson were 0.65 and 0.69, respectively, which was higher than those for Brookton and Amaroo: 0.36 and 0.28, respectively. The average residual respiration (oxygen uptake in the presence of inhibitors of both CP and AOX, KCN and SHAM, respectively) was 21.2±5.7% of total control respiratory rate (data not shown). SHAM-resistant respiration of roots grown at low temperature was also faster than that in plants grown at high temperature, except for cv. Patterson (Fig. 1E–H), which showed similar rates measured at 15 and 25°C. SHAM-resistant respiration of intact tissues can be considered as the potential flux via the CP, but it should be noted that this rate may underestimate the actual capacity of the electron transport pathway (Day et al. 1996). A similar response of SHAM-resistant respiration has been observed in other species. SHAM-resistant respiration was higher in leaves of an Arctic plant grown at lower temperature (McNulty and Cummins 1987). SHAM-resistant respiration also increased in maize leaves grown at 5°C for 5 d (Ribas-Carbo et al. 2000), and cucumber leaves exposed to 8°C for 8 h (Ordentlich et al. 1991). In vivo activity of CP in soybean cotyledons grown at 14°C was faster than that at 28°C (Gonzàlez-Meler et al. 1999). In contrast to the CP, CN-resistant respiration showed different responses to growth temperature between high-H and low-H cultivars (Fig. 1I–L). Again, while CN-resistant respiration is an estimate of the potential flux via AOX, it may not accurately reflect AOX capacity of intact tissues. In roots of cv. Amaroo with low H, the CN-resistant respiration of roots grown at the lower temperature was significantly greater that in those grown at the higher temperature (Fig. 1L). This increase in CN-resistant respiration at low temperature was also reported in other studies (Elthon et al. 1986, McNulty and Cummins 1987, McCaig and Hill 1977). However, the CN-resistant respiration of roots in high-H cultivars showed a different response to growth temperature, being higher in roots grown at 25°C, except at 15°C in the roots of Patterson (Fig. 1I, J). This raised the question of whether these different responses of high-H and low-H plants were due to a difference in mitochondrial electron transport capacities. Respiratory rates in isolated mitochondria The maximal COX activity (cytochrome c-dependent and KCN-sensitive oxygen consumption in detergent solubilised mitochondria) was consistently higher in mitochondria isolated from roots grown at the lower temperature than those at the higher temperature (Fig. 2A–D). Analysis of the results by two-way ANOVA showed that the results were significant, with the probabilities of two factors (growth condition and cultivar) being 0.037 and less than 0.0001, respectively, whereas that of interaction was 0.114. A similar trend was observed with state 3 respiratory rates via the CP (oxygen uptake in the presence of ADP and n-propylgallate) with NADH plus succinate as substrates (Fig. 2 E–H). Two-way ANOVA analysis showed that these results were significant, with a probability of 0.018 for growth condition and 0.015 for cultivar. The probability of interaction was more than 0.05. In cv. Stiletto grown at 15°C, this rate, which represents the maximal CP activity of isolated mitochondria, was about three times that of mitochondria from plants grown at 25°C (Fig. 2E). Lower growth temperature also increased the maximal CP activity of mitochondria isolated from tobacco suspension cells (Vanlerberghe and McIntosh 1992). In contrasts, maximal AOX activity in the high-H and low-H cultivars seemed to respond differently to growth temperature. In the low-H cultivars (Brookton and Amaroo), maximal AOX activity was higher in mitochondria from roots grown at the lower temperature (Fig. 2K, L), whereas, in mitochondria from the roots of high-H cultivars, Stiletto and Patterson, maximal AOX activity was lower in mitochondria from plants grown at the lower temperature compared with those from plants grown at the higher temperature (Fig. 2I, J). However, two-way ANOVA of maximal AOX activity (Fig. 2I–L) yielded probabilities greater than 0.05 for the two factors and their interaction, and comparisons between conditions were not significant with the Tukey-Kramer multiple comparison test. Further analyses are needed to clarify these different responses of AOX between high- and low-H. Nonetheless, these results appear to agree with the CN-resistant oxygen uptake rates observed with intact roots in the high-H cultivars (Fig. 1 I–L), which are also able to maintain their RGR at the lower growth temperature (Kurimoto et al. 2004). These high-H cultivars might sustain more efficient ATP production via their respiratory system at low growth temperatures (i.e., they show lower maximal AOX activity and enhanced CP activity). It is probable, therefore, that the higher RGR was associated with a higher efficiency of ATP production as well as a faster overall respiratory rates in high-H plants. Respiratory chain protein abundance To extend the activity measurements described above, we estimated relative amounts of some key electron transport chain proteins using specific antibodies and purified mitochondria: subunit II of cytochrome c oxidase (COXII), AOX and UCP (Fig. 3, 4). COXII abundance was evaluated using a polyclonal antibody raised against soybean COXII (Daley et al. 2002), which reacted with a single protein at 30 kDa in all of the cultivars (Fig. 3). The relative amount of COXII on a mitochondrial protein basis showed a similar trend between cultivars as did maximal COX activity (Fig. 2A–D). Roots of plants grown at 15°C possessed a larger amount of COXII protein than those grown at 25°C, irrespective of H (Fig. 4 A–D), although these difference were not significant except for Amaroo (P <0.05). In contrast, the relative amount of AOX showed a different response between high-H and low-H cultivars (Fig. 3, 4E–H). We used the monoclonal AOA antibody raised against voodoo lily AOX (Elthon et al. 1989) and detected two bands (32 and 33 kDa) in the roots of the three wheat cultivars, and a single band of 33 kDa in the roots of rice cv. Amaroo (Fig. 3). In the roots of low-H cultivars (Brookton and Amaroo), the relative amount of AOX was higher in plants grown at the lower growth temperature (P <0.1), whereas, in the roots of high-H cultivars grown at 15°C, the reverse was seen (P <0.05 for Stiletto, Fig. 3E–H). There are several reports showing increases in the amount of AOX protein with a decrease in temperature in higher plants. These include mitochondria isolated from maize mesocotyles (Stewart et al. 1990), tobacco suspension-cultured cells (Vanlerberghe and McIntosh 1992), mung bean hypocotyls and leaves (Gonzàlez-Meler et al. 1999), and tomato fruit (Holtzapffel et al. 2002). In rice, mRNA levels of AOX1a and AOX1b increased at low temperature (Ito et al. 1997). Thus, AOX was thought to prevent the generation of ROS under the condition with a highly reduced ubiquinone pool (Purvis and Schewfelt 1993, Wagner and Krab 1995). However, many of these studies involved the sudden imposition of cold on cells or plants grown at a higher temperature, while here, we have grown plants for several weeks at the specified temperature. Under these conditions, the amount of AOX decreased in the roots of high-H cultivars grown at 15°C. UCP abundance showed a similar trend to that of AOX protein (Fig. 3, 4 I–L). The amount of UCP was estimated using a polyclonal antibody raised against soybean UCP, which reacted with a single protein of 33 kDa in all cultivars tested. Mitochondria from roots of low-H cultivars (Brookton and Amaroo) had larger amounts of UCP at the lower growth temperature (Fig. 4K, L), although these differences were not statistically significant. Similar results have been reported for potato tubers and tomato fruits (Nantes et al. 1999, Holtzapffel et al. 2002). However, in mitochondria from roots of high-H cultivars, the amounts of UCP were lower at the lower temperature (P <0.05 for Patterson, Fig. 4I, J). Respiratory efficiency and growth rates Hansen et al. (2002) suggested that a plant’s growth rate is determined by its respiratory rate and its respiratory efficiency (relative activities of phosphorylating and non-phosphorylating respiration pathways), such that both parameters respond flexibly to environmental factors, and, consequently, the plant growth rate will remain relatively stable under variable environmental conditions. According to this model, in the high-H cultivars, their high growth rate under low temperature would be supported by maintaining rapid respiratory rates and a high efficiency of ATP synthesis. Our analyses suggest that this respiratory efficiency results from lowering the amounts of AOX and UCP, and increasing the capacity of CP at the low growth temperatures. The decrease in AOX and UCP abundance at low growth temperatures in the high-H plants, may have been a consequence of maintaining rapid electron flow via the CP, because of the high energy demand for growth and nutrient uptake in these plants (Kurimoto et al. 2004). Rapid ATP turnover would be expected to avoid excessive ROS formation by maximizing electron flow from ubiquinol to COX. However, it should be noted that CP capacity also increased at low temperature in the low-H plants yet they increased their amounts of AOX and UCP. This needs to be further investigated by measuring in vivo activity of AOX and COX using the 18O-discrimination technique for roots of both high-H and low-H cultivars. Notwithstanding this, it seems that some plants are able to cope with long-term exposure to low temperatures without inducing AOX or UCP. Materials and Methods Plant materials and growth conditions Seeds of three cultivars of wheat (Triticum aestivum L. cv. Brookton, cv. Patterson, cv. Stiletto) were obtained from Dr. T. L. Setter (Department of Agriculture Western Australia, Australia) and Dr. M. Schortemeyer (the University of Western Australia, Australia). All seeds were germinated on moistened paper. The wheat cultivars were transferred into tanks containing 20 liter hydroponic nutrient solution 3 d after germination. Rice (Oryzasativa L. cv. Amaroo) seeds were grown in moistened sand for 7 d after germination and then transferred into hydroponic solutions. A total of 12–48 seedlings were placed in each tank, which were placed in a walk-in growth chamber [15 or 25°C constant day and night temperature, 14-h photoperiod, photosynthetic photon flux density 400 µmol m–2 s–1]. The nutrient solution (1/8 strength for the first 3 d after transfer and full strength thereafter) contained (full strength): 4 mM KNO3, 4 mM Ca(NO3)2, 1.5 mM MgSO4, 1.33 mM KH2PO4, 0.05 mM EDTA-Fe, 0.01 mM MnSO4, 1 µM ZnSO4, 1 µM CuSO4, 0.05 mM H3BO3, 0.5 µM Na2MoO4, 0.1 mM NaCl, 0.2 µM CoSO4. The pH of the nutrient solutions was adjusted to 6.0 with NaOH and the solutions were replaced weekly. At 13–15 d and 20–23 d after germination for T. aestivum and O. sativa, respectively, roots were harvested for measurements of root respiration and isolation of mitochondria. Measurement of root respiration Respiratory rates of intact, detached roots were measured polarographically with a Clark-type oxygen electrode (Rank Brothers, Cambridge, U.K.) at 15 and 25°C in 5 ml air-saturated nutrient solution supplemented with 10 mM 2-morpholinoethanesulfonic acid (MES) (pH 6.0). A piece of nylon mesh was used to keep the roots above the stirrer bar and electrode surface. The oxygen concentration in air-saturated buffer was assumed to be 314 and 258 µM at 15 and 25°C, respectively. After a constant rate of oxygen uptake was attained in buffer without any inhibitor, KCN (1 mM) or SHAM (5 mM) were added for estimation of CN- or SHAM-resistant respiration, respectively. After a constant rate of oxygen uptake was obtained in the presence of either inhibitor alone, the other inhibitor was added. Stock solutions were 0.4 M SHAM in ethanol and 0.1 M KCN in 0.1 M MES (pH 6.0). Isolation of mitochondria Mitochondria were isolated from wheat and rice roots as described by Day et al. (1985). For the preparation of mitochondria, approximately 20–50 g FW of roots were disrupted with a mortar and pestle in 200 ml of cold medium containing 0.3 M sucrose, 25 mM tetra-sodium pyrophosphate, 2 mM EDTA (disodium salt), 10 mM KH2PO4, 1% (w/v) PVP-40, 1% (w/v) bovine serum albumin (BSA) and 20 mM ascorbate (the pH of the medium was 7.5). The brei was filtered through four layers of miracloth and centrifuged for 5 min at 1,100×g. The supernatant was centrifuged for 20 min at 18,000×g and the pellet resuspended in approximately 240 ml of medium containing 0.3 M sucrose, 10 mM TES (N-tris[hydroxymethyl]methyl-2-aminoethane sulfonic acid) and 0.1% (w/v) BSA (pH 7.5). The resuspended pellet was centrifuged at 1,100×g for 5 min, and the supernatant recentrifuged at 18,000×g for 20 min to yield the crude mitochondria. This pellet was resuspended in approximately 5 ml of resuspending medium (see above); 2.5 ml aliquots were then layered over 31.5 ml of solution containing 0.3 M sucrose, 10 mM TES (pH 7.5), 0.1% (w/v) BSA, 28% (v/v) Percoll and a linear gradient of 0–4.4% (w/v) PVP-40 (top to bottom) in a tube, and centrifuged for 40 min at 40,000×g. For rice root mitochondria, 32% Percoll was used. The mitochondria were found in a tight, white band near the bottom. The upper layers were aspirated and the mitochondrial fraction carefully collected. The mitochondrial fraction was diluted at least fivefold with resuspending buffer (see above), and centrifuged for 15 min at 31,000×g. The pellet was resuspended with resuspending buffer and recentrifuged for 15 min at 31,000×g. The pellet was resuspended with a small volume of resuspending buffer. The intactness of mitochondria was 84±2.0% (average ± SE) which was estimated using cytochrome c-dependent oxygen uptake (Neuburger et al. 1982). Measurement of oxygen uptake rate by isolated mitochondria Oxygen uptake rates were measured polarographically with a Clark-type oxygen electrode (Rank Brothers) at 25°C in 1 ml of air-saturated medium containing 0.3 M sucrose, 5 mM KH2PO4, 10 mM TES, 10 mM NaCl, 2 mM MgSO4 and 0.1% (w/v) BSA (pH 7.2). Maximal cytochrome c oxidase activity was measured as cyt c-dependent oxygen consumption sensitive to 0.5 mM KCN in the presence of 0.05% (w/v) Triton X-100 and 5 mM ascorbate. Maximal AOX activity was measured as oxygen uptake in the presence of 1 mM NADH, 5 mM succinate, 0.5 mM ATP, 1.5 µM myxothiazol, 5 mM pyruvate and 1.5 mM DTT. Maximal CP activity was measured as oxygen uptake in the presence of 1 mM NADH, 5 mM succinate, 0.5 mM ATP, 0.03 mM n-propyl gallate and 0.1 mM ADP. Protein was estimated by the method of Peterson (1977). Electrophoresis and immunological probing For purified mitochondria, aliquots containing 40 µg of protein were solubilized in sample buffer (2% [w/v] SDS, 62.5 mM Tris-HCl [pH 6.8], 10% [v/v] glycerol, 0.002% [w/v] bromophenol blue, and 50 mM DTT) and boiled for 5 min. Proteins were separated by SDS-PAGE as described by Kearns et al. (1992). A modified version of the method of Towbin et al. (1979) was used for immunoblotting. After electrophoresis, the separated proteins were transferred to a Hybond-C Extra nitrocellulose membrane (Amersham Pharmacia Biotech, Sydney, Australia) using a Multiphor II semi-dry blotting apparatus (Amersham Pharmacia Biotech) according to the manufacturer’s instructions. Hybridisation was detected using the BM Chemiluminescence Blotting Substrate POD system (Roche, Mannheim, Germany). The primary antibodies were used at the following dilutions: AOX, 1 : 50 (Dr. Thomas E. Elthon, University of Nebraska, Lincoln); COX subunit II, 1 : 7,500 (Daley et al. 2002) and anti-soyUCP, 1 : 10,000 (Considine et al. 2001). Proteins cross-reacting with the various antibodies were visualized using an LAS-1000 (Fuji, Tokyo, Japan) and quantified using the Image Gauge v3.0 software (Fuji). Measurement of dry weight After measurement of root respiration, the samples were dried at 70°C for at least 3 d, and then weighed. Statistical analyses Two-way ANOVA was conducted with StatView (ver. 5.0J, SAS, Cary, NC, U.S.A.), after homogeneity of variances was met. When an interaction of two-way ANOVA was smaller than 0.05, the Tukey-Kramer multiple comparison test was conducted with StatView. Bartlett’s test for homogeneity of variances was conducted according to Sokal and Rohlf (1995). Acknowledgments We would like to thank the Drs. M. Schortemeyer and T. L. Setter for providing the seeds of wheat and Dr. Christel Norman for her assistance with the Western blots. We also acknowledge Greg Cawthray and Robert Creasy for their kind help with plant culture. K. Noguchi was supported by a Grant from the Ministry of Agriculture, Forestry and Fishery, Japan (Bio-Design Program). D.A. Day, A.H. Millar and H. Lambers received support from the Australian Research Council. 4 Corresponding author: E-mail, [email protected]; Fax, +81-6-6850-5808. View largeDownload slide Fig. 1 Rate of oxygen uptake in roots of wheat and rice cultivars grown at 15°C (open) and 25°C (filled symbols). (A–D) Rates of oxygen uptake in roots in the absence of inhibitors, measured at 15 and 25°C. (E–H) Rates of oxygen uptake in roots in the presence of SHAM (SHAM-resistant respiration) measured at 15 and 25°C. (I–L) Rates of oxygen uptake in roots in the presence of KCN (CN-resistant respiration) measured at 15 and 25°C; ‘growth’ and ‘measure’ denote factors of two-way ANOVA: ‘growth condition’ and ‘measurement temperature’, respectively. *, **, ***, and **** denote significant difference at P <0.05, 0.01, 0.001, and 0.0001, respectively. ‘n.s.’ means ‘not significant’. Error bar indicates standard error of mean; n ≥ 3. View largeDownload slide Fig. 1 Rate of oxygen uptake in roots of wheat and rice cultivars grown at 15°C (open) and 25°C (filled symbols). (A–D) Rates of oxygen uptake in roots in the absence of inhibitors, measured at 15 and 25°C. (E–H) Rates of oxygen uptake in roots in the presence of SHAM (SHAM-resistant respiration) measured at 15 and 25°C. (I–L) Rates of oxygen uptake in roots in the presence of KCN (CN-resistant respiration) measured at 15 and 25°C; ‘growth’ and ‘measure’ denote factors of two-way ANOVA: ‘growth condition’ and ‘measurement temperature’, respectively. *, **, ***, and **** denote significant difference at P <0.05, 0.01, 0.001, and 0.0001, respectively. ‘n.s.’ means ‘not significant’. Error bar indicates standard error of mean; n ≥ 3. View largeDownload slide Fig. 2 Rated of oxygen uptake in isolated root mitochondria of wheat and rice cultivars grown at 15 and 25°C. (A–D) Maximal activity of cytochrome c oxidase (cytochrome c-dependent, KCN-sensitive oxygen uptake rate). (E–H) Maximal CP activity (NADH- and succinate-dependent oxygen uptake rate in the presence of n-propyl gallate and ADP). (I–L) maximal AOX activity (NADH- and succinate-dependent oxygen uptake rate in the presence of myxothiazol, DTT and pyruvate). Error bar indicates standard error of mean: n ≥ 2. View largeDownload slide Fig. 2 Rated of oxygen uptake in isolated root mitochondria of wheat and rice cultivars grown at 15 and 25°C. (A–D) Maximal activity of cytochrome c oxidase (cytochrome c-dependent, KCN-sensitive oxygen uptake rate). (E–H) Maximal CP activity (NADH- and succinate-dependent oxygen uptake rate in the presence of n-propyl gallate and ADP). (I–L) maximal AOX activity (NADH- and succinate-dependent oxygen uptake rate in the presence of myxothiazol, DTT and pyruvate). Error bar indicates standard error of mean: n ≥ 2. View largeDownload slide Fig. 3 Immunoblots of components of the respiratory chain of root mitochondria isolated from wheat and rice cultivars. Mitochondrial protein equivalent to a 40-µg BSA standard was loaded onto each lane in the presence of DTT. View largeDownload slide Fig. 3 Immunoblots of components of the respiratory chain of root mitochondria isolated from wheat and rice cultivars. Mitochondrial protein equivalent to a 40-µg BSA standard was loaded onto each lane in the presence of DTT. View largeDownload slide Fig. 4 Relative amounts of subunit II of cytochrome c oxidase (COXII: A–D), alternative oxidase (AOX: E–H), and uncoupling protein (UCP: I–L) on a mitochondrial protein basis in roots of wheat and rice cultivars. The relative amounts of these proteins were estimated by immunoblot analysis. Error bars indicate standard error of mean: n ≥ 2. Probabilities of Student’s t-test are represented. View largeDownload slide Fig. 4 Relative amounts of subunit II of cytochrome c oxidase (COXII: A–D), alternative oxidase (AOX: E–H), and uncoupling protein (UCP: I–L) on a mitochondrial protein basis in roots of wheat and rice cultivars. The relative amounts of these proteins were estimated by immunoblot analysis. Error bars indicate standard error of mean: n ≥ 2. Probabilities of Student’s t-test are represented. 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Maintenance of Growth Rate at Low Temperature in Rice and Wheat Cultivars with a High Degree of Respiratory Homeostasis is Associated with a High Efficiency of Respiratory ATP Production

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Oxford University Press
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0032-0781
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1471-9053
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10.1093/pcp/pch116
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15356327
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Abstract

Abstract Some plants have the ability to maintain similar respiratory rates (measured at the growth temperature) when grown at different temperatures. This phenomenon is referred to as respiratory homeostasis. Using wheat and rice cultivars with different degrees of respiratory homeostasis (H), we previously demonstrated that high-H cultivars maintained shoot and root growth at low temperature [Kurimoto et al. (2004)Plant Cell Environ., 27: 853]. Here, we assess the relationship between respiratory homeostasis and the efficiency of respiratory ATP production, by measuring the levels of alternative oxidase (AOX) and uncoupling protein (UCP), which have the potential to decrease respiratory ATP production per unit of oxygen consumed. We also measured SHAM- and CN-resistant respiration of intact roots, and the capacity of the cytochrome pathway (CP) and AOX in isolated mitochondria. Irrespective of H, SHAM-resistant respiration of intact roots and CP capacity of isolated root mitochondria were larger when plants were grown at low temperature, and the maximal activity and relative amounts of cytochrome c oxidase showed a similar trend. In contrast, CN-resistant respiration of intact roots and relative amounts of AOX protein in mitochondria isolated from those roots, were lower in high-H plants grown at low temperature. In the roots of low-H cultivars, relative amounts of AOX protein were higher at low growth temperature. Relative amounts of UCP protein showed similar trends to AOX. We conclude that maintenance of growth rate in high-H plants grown at low temperature is associated with both respiratory homeostasis and a high efficiency of respiratory ATP production. (Received February 28, 2004; Accepted May 7, 2004) Introduction Plant mitochondria have two ubiquinol-oxidizing pathways, the cytochrome pathway (CP) and the alternative pathway. The latter consists of one enzyme, the alternative oxidase (AOX). AOX is not coupled to H+ translocation and, therefore, ATP production, and has the potential to catalyze wasteful respiration in higher plant mitochondria. The precise role of AOX has not been defined in plants, but it is thought to prevent production of reactive oxygen species (ROS) in the respiratory chain, especially under stress conditions, by helping to prevent over-reduction of the ubiquinone pool (Purvis and Schewfelt 1993, Wagner 1995, Maxwell et al. 1999, Vanlerberghe and Ordog 2002, Millenaar and Lambers 2003). Many abiotic stresses, including sudden exposure to low temperature, induce synthesis of AOX (Wagner and Krab 1995). The increase in AOX capacity at low temperature (Elthon et al. 1986, McNulty and Cummins 1987) is often associated with de novo synthesis of AOX protein (Stewart et al. 1990, Vanlerberghe and McIntosh 1992, Gonzàlez-Meler et al. 1999). In hypocotyls and leaves of mung bean grown at low temperature (Gonzàlez-Meler et al. 1999) and chilling-sensitive maize leaves (Ribas-Carbo et al. 2000), the in vivo activity of AOX also increased. However, such increases in AOX activity and capacity were not observed in other tissues or species, such as soybean cotyledons (Gonzàlez-Meler et al. 1999) and a chilling-insensitive maize cultivar (Ribas-Carbo et al. 2000) when grown at low temperatures. Thus, there is still some confusion about the responses of AOX to low growth temperature and the role of AOX at different temperatures. Some plants have the ability to maintain their respiratory rates (measured at the growth temperature), even when grown at different temperatures, a phenomenon referred to as respiratory homeostasis (Atkin and Tjoelker 2003). We investigated root respiration and plant growth in two wheat cultivars with a high degree of homeostasis (H), and in one wheat cultivar and one rice cultivar with a low H (Kurimoto et al. 2004). The plants with high H showed a tendency to maintain their relative growth rate (RGR), irrespective of growth temperature, whereas the plants with low H grown at 15°C showed lower RGR than those grown at 25°C. We suggested that respiratory homeostasis would help to maintain RGR at lower growth temperatures. However, the contribution of AOX to respiration may influence the rate of ATP production to a major extent. We therefore asked the question: is there any difference in response of AOX to low growth temperature between high-H and low-H plants? The uncoupling protein (UCP) may also influence the efficiency of ATP synthesis, because protons leak from the intermembrane space to the mitochondrial matrix through UCP, and the proton gradient is dissipated (Sluse and Jarmuszkiewicz 2002). Low temperature induces UCP1 transcript abundance in Arabidopsis seedlings (Maia et al. 1998) and UCP protein abundance in potato tuber (Nantes et al. 1999) and tomato fruit (Holtzapffel et al. 2002). On the other hand, expressions of UCP2 in Arabidopsis seedling and UCP1 in wheat seedlings were insensitive to low temperature (Watanabe et al. 1999, Murayama and Handa 2000). More information on response of UCP to growth temperature is also needed. To investigate a possible relationship between components of the respiratory chain and the homeostasis of respiration, we selected two wheat cultivars with high H and one wheat and one rice cultivar with low H. We grew them at 15 and 25°C, and measured root oxygen uptake rates with and without respiratory inhibitors, KCN and SHAM, which inhibit the cytochrome and alternative paths, respectively. Using isolated mitochondria from the roots of these plants, NADH- and succinate-dependent rates of oxygen uptake via CP and AOX, and the maximal activity of COX, were measured. Respiratory protein abundance was examined by immunoblots for subunit II of COX, AOX and UCP. We discuss the effects of growth temperature on root electron transport system and plant growth. Results and Discussion Respiratory rates in intact roots Root respiratory rates of plants grown at 15°C were faster than those of plants grown at 25°C (Fig. 1A–D). According to Atkin et al. (2004) and Kurimoto et al. (2004), the degree of homeostasis (H) was calculated as: where Rn(m) denotes a respiratory rate of roots of n°C-grown plants, which were measured at m°C. This value occurs between 0 (no acclimation) and 1 (full acclimation). The values of H for Stiletto and Patterson were 0.65 and 0.69, respectively, which was higher than those for Brookton and Amaroo: 0.36 and 0.28, respectively. The average residual respiration (oxygen uptake in the presence of inhibitors of both CP and AOX, KCN and SHAM, respectively) was 21.2±5.7% of total control respiratory rate (data not shown). SHAM-resistant respiration of roots grown at low temperature was also faster than that in plants grown at high temperature, except for cv. Patterson (Fig. 1E–H), which showed similar rates measured at 15 and 25°C. SHAM-resistant respiration of intact tissues can be considered as the potential flux via the CP, but it should be noted that this rate may underestimate the actual capacity of the electron transport pathway (Day et al. 1996). A similar response of SHAM-resistant respiration has been observed in other species. SHAM-resistant respiration was higher in leaves of an Arctic plant grown at lower temperature (McNulty and Cummins 1987). SHAM-resistant respiration also increased in maize leaves grown at 5°C for 5 d (Ribas-Carbo et al. 2000), and cucumber leaves exposed to 8°C for 8 h (Ordentlich et al. 1991). In vivo activity of CP in soybean cotyledons grown at 14°C was faster than that at 28°C (Gonzàlez-Meler et al. 1999). In contrast to the CP, CN-resistant respiration showed different responses to growth temperature between high-H and low-H cultivars (Fig. 1I–L). Again, while CN-resistant respiration is an estimate of the potential flux via AOX, it may not accurately reflect AOX capacity of intact tissues. In roots of cv. Amaroo with low H, the CN-resistant respiration of roots grown at the lower temperature was significantly greater that in those grown at the higher temperature (Fig. 1L). This increase in CN-resistant respiration at low temperature was also reported in other studies (Elthon et al. 1986, McNulty and Cummins 1987, McCaig and Hill 1977). However, the CN-resistant respiration of roots in high-H cultivars showed a different response to growth temperature, being higher in roots grown at 25°C, except at 15°C in the roots of Patterson (Fig. 1I, J). This raised the question of whether these different responses of high-H and low-H plants were due to a difference in mitochondrial electron transport capacities. Respiratory rates in isolated mitochondria The maximal COX activity (cytochrome c-dependent and KCN-sensitive oxygen consumption in detergent solubilised mitochondria) was consistently higher in mitochondria isolated from roots grown at the lower temperature than those at the higher temperature (Fig. 2A–D). Analysis of the results by two-way ANOVA showed that the results were significant, with the probabilities of two factors (growth condition and cultivar) being 0.037 and less than 0.0001, respectively, whereas that of interaction was 0.114. A similar trend was observed with state 3 respiratory rates via the CP (oxygen uptake in the presence of ADP and n-propylgallate) with NADH plus succinate as substrates (Fig. 2 E–H). Two-way ANOVA analysis showed that these results were significant, with a probability of 0.018 for growth condition and 0.015 for cultivar. The probability of interaction was more than 0.05. In cv. Stiletto grown at 15°C, this rate, which represents the maximal CP activity of isolated mitochondria, was about three times that of mitochondria from plants grown at 25°C (Fig. 2E). Lower growth temperature also increased the maximal CP activity of mitochondria isolated from tobacco suspension cells (Vanlerberghe and McIntosh 1992). In contrasts, maximal AOX activity in the high-H and low-H cultivars seemed to respond differently to growth temperature. In the low-H cultivars (Brookton and Amaroo), maximal AOX activity was higher in mitochondria from roots grown at the lower temperature (Fig. 2K, L), whereas, in mitochondria from the roots of high-H cultivars, Stiletto and Patterson, maximal AOX activity was lower in mitochondria from plants grown at the lower temperature compared with those from plants grown at the higher temperature (Fig. 2I, J). However, two-way ANOVA of maximal AOX activity (Fig. 2I–L) yielded probabilities greater than 0.05 for the two factors and their interaction, and comparisons between conditions were not significant with the Tukey-Kramer multiple comparison test. Further analyses are needed to clarify these different responses of AOX between high- and low-H. Nonetheless, these results appear to agree with the CN-resistant oxygen uptake rates observed with intact roots in the high-H cultivars (Fig. 1 I–L), which are also able to maintain their RGR at the lower growth temperature (Kurimoto et al. 2004). These high-H cultivars might sustain more efficient ATP production via their respiratory system at low growth temperatures (i.e., they show lower maximal AOX activity and enhanced CP activity). It is probable, therefore, that the higher RGR was associated with a higher efficiency of ATP production as well as a faster overall respiratory rates in high-H plants. Respiratory chain protein abundance To extend the activity measurements described above, we estimated relative amounts of some key electron transport chain proteins using specific antibodies and purified mitochondria: subunit II of cytochrome c oxidase (COXII), AOX and UCP (Fig. 3, 4). COXII abundance was evaluated using a polyclonal antibody raised against soybean COXII (Daley et al. 2002), which reacted with a single protein at 30 kDa in all of the cultivars (Fig. 3). The relative amount of COXII on a mitochondrial protein basis showed a similar trend between cultivars as did maximal COX activity (Fig. 2A–D). Roots of plants grown at 15°C possessed a larger amount of COXII protein than those grown at 25°C, irrespective of H (Fig. 4 A–D), although these difference were not significant except for Amaroo (P <0.05). In contrast, the relative amount of AOX showed a different response between high-H and low-H cultivars (Fig. 3, 4E–H). We used the monoclonal AOA antibody raised against voodoo lily AOX (Elthon et al. 1989) and detected two bands (32 and 33 kDa) in the roots of the three wheat cultivars, and a single band of 33 kDa in the roots of rice cv. Amaroo (Fig. 3). In the roots of low-H cultivars (Brookton and Amaroo), the relative amount of AOX was higher in plants grown at the lower growth temperature (P <0.1), whereas, in the roots of high-H cultivars grown at 15°C, the reverse was seen (P <0.05 for Stiletto, Fig. 3E–H). There are several reports showing increases in the amount of AOX protein with a decrease in temperature in higher plants. These include mitochondria isolated from maize mesocotyles (Stewart et al. 1990), tobacco suspension-cultured cells (Vanlerberghe and McIntosh 1992), mung bean hypocotyls and leaves (Gonzàlez-Meler et al. 1999), and tomato fruit (Holtzapffel et al. 2002). In rice, mRNA levels of AOX1a and AOX1b increased at low temperature (Ito et al. 1997). Thus, AOX was thought to prevent the generation of ROS under the condition with a highly reduced ubiquinone pool (Purvis and Schewfelt 1993, Wagner and Krab 1995). However, many of these studies involved the sudden imposition of cold on cells or plants grown at a higher temperature, while here, we have grown plants for several weeks at the specified temperature. Under these conditions, the amount of AOX decreased in the roots of high-H cultivars grown at 15°C. UCP abundance showed a similar trend to that of AOX protein (Fig. 3, 4 I–L). The amount of UCP was estimated using a polyclonal antibody raised against soybean UCP, which reacted with a single protein of 33 kDa in all cultivars tested. Mitochondria from roots of low-H cultivars (Brookton and Amaroo) had larger amounts of UCP at the lower growth temperature (Fig. 4K, L), although these differences were not statistically significant. Similar results have been reported for potato tubers and tomato fruits (Nantes et al. 1999, Holtzapffel et al. 2002). However, in mitochondria from roots of high-H cultivars, the amounts of UCP were lower at the lower temperature (P <0.05 for Patterson, Fig. 4I, J). Respiratory efficiency and growth rates Hansen et al. (2002) suggested that a plant’s growth rate is determined by its respiratory rate and its respiratory efficiency (relative activities of phosphorylating and non-phosphorylating respiration pathways), such that both parameters respond flexibly to environmental factors, and, consequently, the plant growth rate will remain relatively stable under variable environmental conditions. According to this model, in the high-H cultivars, their high growth rate under low temperature would be supported by maintaining rapid respiratory rates and a high efficiency of ATP synthesis. Our analyses suggest that this respiratory efficiency results from lowering the amounts of AOX and UCP, and increasing the capacity of CP at the low growth temperatures. The decrease in AOX and UCP abundance at low growth temperatures in the high-H plants, may have been a consequence of maintaining rapid electron flow via the CP, because of the high energy demand for growth and nutrient uptake in these plants (Kurimoto et al. 2004). Rapid ATP turnover would be expected to avoid excessive ROS formation by maximizing electron flow from ubiquinol to COX. However, it should be noted that CP capacity also increased at low temperature in the low-H plants yet they increased their amounts of AOX and UCP. This needs to be further investigated by measuring in vivo activity of AOX and COX using the 18O-discrimination technique for roots of both high-H and low-H cultivars. Notwithstanding this, it seems that some plants are able to cope with long-term exposure to low temperatures without inducing AOX or UCP. Materials and Methods Plant materials and growth conditions Seeds of three cultivars of wheat (Triticum aestivum L. cv. Brookton, cv. Patterson, cv. Stiletto) were obtained from Dr. T. L. Setter (Department of Agriculture Western Australia, Australia) and Dr. M. Schortemeyer (the University of Western Australia, Australia). All seeds were germinated on moistened paper. The wheat cultivars were transferred into tanks containing 20 liter hydroponic nutrient solution 3 d after germination. Rice (Oryzasativa L. cv. Amaroo) seeds were grown in moistened sand for 7 d after germination and then transferred into hydroponic solutions. A total of 12–48 seedlings were placed in each tank, which were placed in a walk-in growth chamber [15 or 25°C constant day and night temperature, 14-h photoperiod, photosynthetic photon flux density 400 µmol m–2 s–1]. The nutrient solution (1/8 strength for the first 3 d after transfer and full strength thereafter) contained (full strength): 4 mM KNO3, 4 mM Ca(NO3)2, 1.5 mM MgSO4, 1.33 mM KH2PO4, 0.05 mM EDTA-Fe, 0.01 mM MnSO4, 1 µM ZnSO4, 1 µM CuSO4, 0.05 mM H3BO3, 0.5 µM Na2MoO4, 0.1 mM NaCl, 0.2 µM CoSO4. The pH of the nutrient solutions was adjusted to 6.0 with NaOH and the solutions were replaced weekly. At 13–15 d and 20–23 d after germination for T. aestivum and O. sativa, respectively, roots were harvested for measurements of root respiration and isolation of mitochondria. Measurement of root respiration Respiratory rates of intact, detached roots were measured polarographically with a Clark-type oxygen electrode (Rank Brothers, Cambridge, U.K.) at 15 and 25°C in 5 ml air-saturated nutrient solution supplemented with 10 mM 2-morpholinoethanesulfonic acid (MES) (pH 6.0). A piece of nylon mesh was used to keep the roots above the stirrer bar and electrode surface. The oxygen concentration in air-saturated buffer was assumed to be 314 and 258 µM at 15 and 25°C, respectively. After a constant rate of oxygen uptake was attained in buffer without any inhibitor, KCN (1 mM) or SHAM (5 mM) were added for estimation of CN- or SHAM-resistant respiration, respectively. After a constant rate of oxygen uptake was obtained in the presence of either inhibitor alone, the other inhibitor was added. Stock solutions were 0.4 M SHAM in ethanol and 0.1 M KCN in 0.1 M MES (pH 6.0). Isolation of mitochondria Mitochondria were isolated from wheat and rice roots as described by Day et al. (1985). For the preparation of mitochondria, approximately 20–50 g FW of roots were disrupted with a mortar and pestle in 200 ml of cold medium containing 0.3 M sucrose, 25 mM tetra-sodium pyrophosphate, 2 mM EDTA (disodium salt), 10 mM KH2PO4, 1% (w/v) PVP-40, 1% (w/v) bovine serum albumin (BSA) and 20 mM ascorbate (the pH of the medium was 7.5). The brei was filtered through four layers of miracloth and centrifuged for 5 min at 1,100×g. The supernatant was centrifuged for 20 min at 18,000×g and the pellet resuspended in approximately 240 ml of medium containing 0.3 M sucrose, 10 mM TES (N-tris[hydroxymethyl]methyl-2-aminoethane sulfonic acid) and 0.1% (w/v) BSA (pH 7.5). The resuspended pellet was centrifuged at 1,100×g for 5 min, and the supernatant recentrifuged at 18,000×g for 20 min to yield the crude mitochondria. This pellet was resuspended in approximately 5 ml of resuspending medium (see above); 2.5 ml aliquots were then layered over 31.5 ml of solution containing 0.3 M sucrose, 10 mM TES (pH 7.5), 0.1% (w/v) BSA, 28% (v/v) Percoll and a linear gradient of 0–4.4% (w/v) PVP-40 (top to bottom) in a tube, and centrifuged for 40 min at 40,000×g. For rice root mitochondria, 32% Percoll was used. The mitochondria were found in a tight, white band near the bottom. The upper layers were aspirated and the mitochondrial fraction carefully collected. The mitochondrial fraction was diluted at least fivefold with resuspending buffer (see above), and centrifuged for 15 min at 31,000×g. The pellet was resuspended with resuspending buffer and recentrifuged for 15 min at 31,000×g. The pellet was resuspended with a small volume of resuspending buffer. The intactness of mitochondria was 84±2.0% (average ± SE) which was estimated using cytochrome c-dependent oxygen uptake (Neuburger et al. 1982). Measurement of oxygen uptake rate by isolated mitochondria Oxygen uptake rates were measured polarographically with a Clark-type oxygen electrode (Rank Brothers) at 25°C in 1 ml of air-saturated medium containing 0.3 M sucrose, 5 mM KH2PO4, 10 mM TES, 10 mM NaCl, 2 mM MgSO4 and 0.1% (w/v) BSA (pH 7.2). Maximal cytochrome c oxidase activity was measured as cyt c-dependent oxygen consumption sensitive to 0.5 mM KCN in the presence of 0.05% (w/v) Triton X-100 and 5 mM ascorbate. Maximal AOX activity was measured as oxygen uptake in the presence of 1 mM NADH, 5 mM succinate, 0.5 mM ATP, 1.5 µM myxothiazol, 5 mM pyruvate and 1.5 mM DTT. Maximal CP activity was measured as oxygen uptake in the presence of 1 mM NADH, 5 mM succinate, 0.5 mM ATP, 0.03 mM n-propyl gallate and 0.1 mM ADP. Protein was estimated by the method of Peterson (1977). Electrophoresis and immunological probing For purified mitochondria, aliquots containing 40 µg of protein were solubilized in sample buffer (2% [w/v] SDS, 62.5 mM Tris-HCl [pH 6.8], 10% [v/v] glycerol, 0.002% [w/v] bromophenol blue, and 50 mM DTT) and boiled for 5 min. Proteins were separated by SDS-PAGE as described by Kearns et al. (1992). A modified version of the method of Towbin et al. (1979) was used for immunoblotting. After electrophoresis, the separated proteins were transferred to a Hybond-C Extra nitrocellulose membrane (Amersham Pharmacia Biotech, Sydney, Australia) using a Multiphor II semi-dry blotting apparatus (Amersham Pharmacia Biotech) according to the manufacturer’s instructions. Hybridisation was detected using the BM Chemiluminescence Blotting Substrate POD system (Roche, Mannheim, Germany). The primary antibodies were used at the following dilutions: AOX, 1 : 50 (Dr. Thomas E. Elthon, University of Nebraska, Lincoln); COX subunit II, 1 : 7,500 (Daley et al. 2002) and anti-soyUCP, 1 : 10,000 (Considine et al. 2001). Proteins cross-reacting with the various antibodies were visualized using an LAS-1000 (Fuji, Tokyo, Japan) and quantified using the Image Gauge v3.0 software (Fuji). Measurement of dry weight After measurement of root respiration, the samples were dried at 70°C for at least 3 d, and then weighed. Statistical analyses Two-way ANOVA was conducted with StatView (ver. 5.0J, SAS, Cary, NC, U.S.A.), after homogeneity of variances was met. When an interaction of two-way ANOVA was smaller than 0.05, the Tukey-Kramer multiple comparison test was conducted with StatView. Bartlett’s test for homogeneity of variances was conducted according to Sokal and Rohlf (1995). Acknowledgments We would like to thank the Drs. M. Schortemeyer and T. L. Setter for providing the seeds of wheat and Dr. Christel Norman for her assistance with the Western blots. We also acknowledge Greg Cawthray and Robert Creasy for their kind help with plant culture. K. Noguchi was supported by a Grant from the Ministry of Agriculture, Forestry and Fishery, Japan (Bio-Design Program). D.A. Day, A.H. Millar and H. Lambers received support from the Australian Research Council. 4 Corresponding author: E-mail, [email protected]; Fax, +81-6-6850-5808. View largeDownload slide Fig. 1 Rate of oxygen uptake in roots of wheat and rice cultivars grown at 15°C (open) and 25°C (filled symbols). (A–D) Rates of oxygen uptake in roots in the absence of inhibitors, measured at 15 and 25°C. (E–H) Rates of oxygen uptake in roots in the presence of SHAM (SHAM-resistant respiration) measured at 15 and 25°C. (I–L) Rates of oxygen uptake in roots in the presence of KCN (CN-resistant respiration) measured at 15 and 25°C; ‘growth’ and ‘measure’ denote factors of two-way ANOVA: ‘growth condition’ and ‘measurement temperature’, respectively. *, **, ***, and **** denote significant difference at P <0.05, 0.01, 0.001, and 0.0001, respectively. ‘n.s.’ means ‘not significant’. Error bar indicates standard error of mean; n ≥ 3. View largeDownload slide Fig. 1 Rate of oxygen uptake in roots of wheat and rice cultivars grown at 15°C (open) and 25°C (filled symbols). (A–D) Rates of oxygen uptake in roots in the absence of inhibitors, measured at 15 and 25°C. (E–H) Rates of oxygen uptake in roots in the presence of SHAM (SHAM-resistant respiration) measured at 15 and 25°C. (I–L) Rates of oxygen uptake in roots in the presence of KCN (CN-resistant respiration) measured at 15 and 25°C; ‘growth’ and ‘measure’ denote factors of two-way ANOVA: ‘growth condition’ and ‘measurement temperature’, respectively. *, **, ***, and **** denote significant difference at P <0.05, 0.01, 0.001, and 0.0001, respectively. ‘n.s.’ means ‘not significant’. Error bar indicates standard error of mean; n ≥ 3. View largeDownload slide Fig. 2 Rated of oxygen uptake in isolated root mitochondria of wheat and rice cultivars grown at 15 and 25°C. (A–D) Maximal activity of cytochrome c oxidase (cytochrome c-dependent, KCN-sensitive oxygen uptake rate). (E–H) Maximal CP activity (NADH- and succinate-dependent oxygen uptake rate in the presence of n-propyl gallate and ADP). (I–L) maximal AOX activity (NADH- and succinate-dependent oxygen uptake rate in the presence of myxothiazol, DTT and pyruvate). Error bar indicates standard error of mean: n ≥ 2. View largeDownload slide Fig. 2 Rated of oxygen uptake in isolated root mitochondria of wheat and rice cultivars grown at 15 and 25°C. (A–D) Maximal activity of cytochrome c oxidase (cytochrome c-dependent, KCN-sensitive oxygen uptake rate). (E–H) Maximal CP activity (NADH- and succinate-dependent oxygen uptake rate in the presence of n-propyl gallate and ADP). (I–L) maximal AOX activity (NADH- and succinate-dependent oxygen uptake rate in the presence of myxothiazol, DTT and pyruvate). Error bar indicates standard error of mean: n ≥ 2. View largeDownload slide Fig. 3 Immunoblots of components of the respiratory chain of root mitochondria isolated from wheat and rice cultivars. Mitochondrial protein equivalent to a 40-µg BSA standard was loaded onto each lane in the presence of DTT. View largeDownload slide Fig. 3 Immunoblots of components of the respiratory chain of root mitochondria isolated from wheat and rice cultivars. Mitochondrial protein equivalent to a 40-µg BSA standard was loaded onto each lane in the presence of DTT. View largeDownload slide Fig. 4 Relative amounts of subunit II of cytochrome c oxidase (COXII: A–D), alternative oxidase (AOX: E–H), and uncoupling protein (UCP: I–L) on a mitochondrial protein basis in roots of wheat and rice cultivars. The relative amounts of these proteins were estimated by immunoblot analysis. Error bars indicate standard error of mean: n ≥ 2. Probabilities of Student’s t-test are represented. View largeDownload slide Fig. 4 Relative amounts of subunit II of cytochrome c oxidase (COXII: A–D), alternative oxidase (AOX: E–H), and uncoupling protein (UCP: I–L) on a mitochondrial protein basis in roots of wheat and rice cultivars. The relative amounts of these proteins were estimated by immunoblot analysis. Error bars indicate standard error of mean: n ≥ 2. Probabilities of Student’s t-test are represented. 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Plant and Cell Physiology – Oxford University Press

Published: Aug 15, 2004

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