Correspondence: Ko Noguchi. Present address: Department of Biochemistry and Plant Sciences, University of Western Australia, 35 Stirling Highway, Crawley, WA 6009, Australia. Fax: + 61 8 93801148; e-mail: email@example.com
Acclimation of respiration to the light environments is important for a plant’s carbon balance. Respiratory rates of mature leaves of Alocasia odora, a typical shade-tolerant species, were measured during the night for 14 d after reciprocal transfers between high- (330 µ mol m−2 s−1) and low-light (20 µ mol m−2 s−1) environments. Following the transfer, both the rate of CO2 efflux and that of O2 uptake of A. odora leaves adjusted to the new light environments. The O2-uptake rates changed more slowly than the CO2-efflux rates under the new environments. Leaf mass per area also changed after the transfer. We analysed whether substrate availability or ATP-consumption rates influence the respiratory acclimation. Since the addition of sucrose to leaf segments did not influence the O2-uptake rates, the change of respiratory substrate availability was not responsible for the respiratory acclimation. The addition of an uncoupler induced increases in the O2-uptake rates, and the degree of enhancement significantly decreased after the transfer from low to high irradiance. Thus, the change in ATP-consumption rates was responsible for the changes in respiratory rates in the plants transferred from low to high light. Potential rates of O2 uptake, as measured in the presence of both the substrate and the uncoupler, changed after the transfer, and strongly correlated with the O2-uptake rates, irrespective of the directions of transfer (r = 0·961). There was a strong correlation between maximal activities of NAD-isocitrate dehydrogenase and the potential rates of O2 uptake (r = 0·933), but a weaker correlation between those of cytochrome c oxidase and the potential rates (r = 0·689). These data indicate that the changes of light environments altered the respiratory rates via the change of the respiratory ATP demand, and that the altered rates of respiration will induce the changes of the respiratory capacities.
Plants often experience sudden changes in light environment. For example, gap formation causes a sudden increase in irradiance. Various leaf properties, such as respiration, photosynthesis, photoprotection, stomatal conductance and mesophyll anatomy, change responding to the changes in light environment (Bunce et al. 1977; Jurik, Chabot & Chabot 1979; Ferrar & Osmond 1986; Bauer & Thöni 1988; Eschrich, Burchardt & Essiamah 1989; Sims & Pearcy 1991, 1992; Naidu & DeLucia 1997; Mohammed & Parker 1999). These changes would contribute to optimizing the carbon balance of the plants in the new light environment. For example, when leaves are subjected to low light, a decreased respiratory rate is essential to realise a positive carbon balance. This is especially relevant to species with long-lived leaves with low photosynthetic rates, such as shade plants and ever-green trees. The changes in respiration are also important because respiration provides intermediates and energy for biosynthesis (Lambers 1985). When leaves are suddenly subjected to high irradiances they often suffer from photodamage to photosystem II (Ferrar & Osmond 1986; Mulkey & Pearcy 1992; Naidu & DeLucia 1997; Mohammed & Parker 1999). The increased respiratory rate will supply intermediates and energy for constructing chloroplasts having elevated photosynthetic capacities and sufficient protecting mechanisms.
Sims & Pearcy (1991) studied acclimation of photosynthesis and respiration in leaves of Alocasia macrorrhiza, a typical shade-tolerant species. They transferred the plants from high to low growth irradiances, and vice versa. After the transfer to the new light environment, CO2-efflux rates on a leaf area basis approached the values that were characteristic of the plants continuously grown in the ‘new’ environment within 1 week, but photosynthetic capacities did not change markedly. In the leaves of two deciduous trees, Acer saccharum and Quercus rubra, Naidu & DeLucia (1997) also observed that acclimation of respiratory rates on a leaf area basis occurred within 1 week and photosynthetic capacities changed more slowly. Although these studies did not clarify physiological mechanisms that are responsible for the respiratory acclimation, Sims & Pearcy (1991) suggested that the rapid acclimation of respiration was due to direct effects of light and/or carbohydrate availability, but not to maintenance costs of photosynthetic apparatus.
Bunce et al. (1977) examined CO2-efflux rates and maximal activities of malate dehydrogenase (MDH) in leaves of Glycine max, following transfer from high to low growth irradiance, and vice versa. Both the respiratory rates and the MDH activities changed to the values that were characteristic of the plants grown continuously in the ‘new’ environments by the fourth day after the transfer. Their data indicated that the acclimation of the abundance of respiratory machinery (enzymes and transporters) occurred as well as the respiratory rates.
In general, the respiratory rate is limited by a combination of the availability of photosynthates and the rate of consumption of respiratory ATP (Lambers 1985; Amthor 1995). For example, in a constant-light environment, the ATP-consuming processes mainly limit the respiratory rate in the leaves of Alocasia odora, another typical shade-tolerant species, whereas the availability of photosynthates limits the respiratory rates in the leaves of Spinacia oleracea, a sun plant (Noguchi & Terashima 1997). Thus, to know underlying mechanisms of the respiratory acclimation, we should first examine the changes in the above two factors.
In this study, we analysed mechanisms of respiratory acclimation in the fully expanded leaves of A. odora that were transferred to a new light environment. We measured CO2-efflux rates of whole leaves and O2-uptake rates of leaf segments for 14 d after the transfer to the new light environments. We also examined the effects of sucrose, a respiratory substrate, and carbonyl cyanide p-(trifluoromethoxy)-phenylhydrazone (FCCP), an uncoupler of oxidative phosphorylation from a respiratory chain activity, on O2-uptake rates of the leaf segments to assess whether the respiratory rate was limited by the availability of substrates or by the ATP-consuming processes. To estimate changes in the abundance of respiratory machinery, we measured potential O2-uptake rates in the presence of both sucrose and FCCP. We also measured maximal activities of two respiratory enzymes, NAD-isocitrate dehydrogenase of tricarbonic acid (TCA) cycle and cytochrome c oxidase of the mitochondrial respiratory chain.
Materials and methods
Plant material and growth
Plant material and growth conditions were similar to those detailed previously (Noguchi & Terashima 1997). Alocasia odora (Lodd.) Spach. plants were grown in pots in vermiculite (diameter, 10·5 cm; depth, 17·5 cm; one plant per pot) in a controlled environment (KG-50HLA-S, Koito, Osaka, Japan). Light was supplied by cool-white fluorescence tubes (FPR96EX-N/A, National, Osaka, Japan). Black shade cloth was used to reduce irradiance levels. Photosynthetic photon flux densities were 330 and 20 µ mol m−2 s−1 in the high- and low-light environments, respectively. The day/night air temperatures were 27/20 °C, relative humidity 60% and day-length 8 h (from 0300 to 1100). The plants were used about 1 month after propagation.
Transfers and sampling schedule
When the respiratory rates of the fully expanded leaves attained constant levels, the plants were transferred from high- to low- and from low- to high-light environments. Only the fully expanded leaves were used for measurements. For the sake of simplicity, the plants transferred from low to high light are called LH plants and those transferred from high to low light are HL plants. The plants that were continuously growing in low-and high-light environments are called LL and HH plants, respectively. Ten plants were placed in the low- and high-light conditions, respectively. Half of them were transferred to the high- or low-light conditions.
Measurements of respiratory rates and sampling were made during the night on days 2 and 0 before and days 1, 3, 5, 8 and 14 after the transfer.
Measurements of CO2 efflux of intact leaves
Measurements were made according to Noguchi & Terashima (1997) with some modifications. An attached whole leaf was enclosed in a chamber and the rate of CO2 efflux was determined with an open gas-exchange system. Areas of the leaves ranged from 57·0 to 161 cm2. Air was passed through a soda lime column to remove CO2 and mixed with 5% (v/v) CO2 using mass-flow controllers (KOFLOC 3910; Kojima, Kyoto, Japan). The humidity of the air was adjusted by changing the temperature of the air that had been passed through water. The rate of air flow through the chamber was adjusted to 8·3 × 10−6 m3 s−1 (= 500 m L min−1). The concentration of CO2 and the humidity of the air were monitored with an infrared gas analyser (ZRC; Fuji, Tokyo, Japan) and a dew-point hygrometer (HYGRO M4; General Eastern, Woburn, MA, USA), respectively. A leaf temperature was measured with a copper–constantan thermocouple and adjusted to 20 ± 1 °C. The respiratory rates were measured at an ambient partial pressure of CO2 of 36 Pa. At the end of the light period (1100 h), the plants were brought into a dark room. The respiratory rates were measured for more than 3 h during the night-time from 1200 to 2200 h. Rates of CO2 efflux of A. odora leaves were almost constant during the night in all the light conditions (data not shown). Thus, we used the average values of the CO2-efflux rates during the night.
Measurement of O2 uptake of leaf segments
Measurements were made according to Noguchi & Terashima (1997). The rates of O2 uptake of leaves were measured polarographically with a Clark type liquid-phase O2 electrode (Rank Brothers, Cambridge, UK) at 20 °C in 4 mL of the air-saturated solution containing 50 mm 2-[4-(2-hydroxyethyl)-1-piperazinyl] ethanesulfonic acid (HEPES), 10 mm 2-morpholinoethanesulfonic acid (MES) (pH 6·6) and 0·2 mm CaCl2. The leaf segments (≈ 1·5 cm2 each) were cut paradermally (in parallel with epidermis) into two pieces with a new razor blade. The slicing was essential for the experiments with exogenous reagents. As the respiratory rate of these slices was almost fully inhibited by a combination of 20 mm salicylhydroxamic acid and 1 mm KCN, the exogenous application of inhibitors was effective. Because the slicing transiently enhanced the respiratory rate, the slices were soaked for more than 20 min in the same buffer before the measurements. After the soaking, the O2-uptake rates of the slices were the same as those in the segments (1 cm × 1·5 cm) of leaves that were incubated for more than 20 min in the buffer before the measurements. A piece of nylon net was used to keep the slices above a stirrer bar and an electrode. The rates of O2 uptake were calculated assuming that the concentration of oxygen in the air-saturated buffer at 20 °C was 276 µ mol L−1. After a constant rate of O2 uptake was attained in the buffer without any reagents, sucrose (110 mm, final concentration) or FCCP (1 µm, final concentration) was added and the changes in the rate were analysed. Stock solutions were 1 m sucrose in the above buffer and 1 mm FCCP in ethanol.
Measurement of dry weight
Segments were cut from a leaf with a fresh razor blade at night. After measurement of areas of the segments, the segments were dried at 70 °C for more than three days and then weighed.
Determination of maximal enzymatic activities
We measured the maximal activities of two respiratory enzymes, NAD-isocitrate dehydrogenase, a TCA-cycle enzyme, and cytochrome c oxidase of the mitochondrial respiratory chain. The maximal activities of NAD-isocitrate dehydrogenase were determined according to Noguchi, Sonoike & Terashima (1996). Frozen leaf segments were ground to powder in liquid nitrogen and extracted in a buffer containing 100 mm HEPES-KOH (pH 7·5), 10 mm KH2PO4, 0·5 mm EDTA, 10% (v/v) glycerol and 10 mm dithiothreitol with a chilled mortar and pestle. The ratio of leaf to buffer was 1 : 7 (mg fresh weight µ L−1 volume). The homogenate was filtered through 20 µm mesh to remove tissue debris. For the assay of NAD-isocitrate dehydrogenase, 20 µL of the filtrate was added to 680 µL of a reaction mixture containing 50 mm HEPES-KOH (pH 7·6), 1 mm MnSO4, 1·33 mm NAD+, 17·1 mm isocitrate and 0·05% (v/v) Triton X-100. The increase in absorbance at 340 nm due to the reduction of NAD+ was monitored for 20 min at 20 °C (UV-200S; Shimadzu, Kyoto, Japan). A molar extinction coefficient of 6·22 m m−1 cm−1 was used (Cox 1969).
The maximal activities of cytochrome c oxidase were measured according to Hendry (1993). Frozen leaf segments were ground to a powder in liquid nitrogen and extracted in a buffer that contained 100 mm KPi (pH 7·5), 250 mm sorbitol, 0·2 mm NaEDTA with a chilled mortar and pestle. The ratio of leaf to buffer was 1 : 7 (mg fresh weight µL−1 volume). Each homogenate was filtered through 20 µm mesh to remove tissue debris. The filtered homogenate was centrifuged at 15 000 g for 3 min at 4 °C. The pellet was resuspended in 50 mm KPi buffer. For the assay of cytochrome c oxidase, 10 µL of the resuspended crude extract was transferred to a cuvette that contained 570 µL of 50 mm KPi (pH 7·5) and 100 µL of reduced cytochrome c. The decrease in absorbance at 550 nm was monitored for 20 min at 20 °C. A molar extinction coefficient of 20 m m−1 cm−1 was used (Hendry 1993). The reduced cytochrome c was prepared by desalting 0·5 mL of 50 mm KPi buffer that contained 5 mg horse heart cytochrome c and 11 mg ascorbic acid with a Sephadex G 50 column (1·3 × 5 cm).
All the data were analysed with StatView (ver. 5·0 J, SAS Institute Inc., Cary, NC, USA).
On a leaf area basis, both the CO2-efflux and O2-uptake rates of high-light leaves (HH) were higher than those of low-light leaves (LL) throughout the experimental period (Fig. 1). When A. odora plants were transferred to the high- or low-light environment, the respiratory rates of their leaves changed. The CO2-efflux rates of the whole leaves showed quicker responses to the new environments than the O2-uptake rates of the leaf segments, both in HL and in LH plants. The O2-uptake rates of HL and LH plants changed little at day 1 after the transfer. However, the CO2-efflux rates of HL and LH plants changed significantly at day 1 after the transfer.
We also expressed the respiratory rates on a leaf dry mass basis (Fig. 2), which reduced the differences in both CO2-efflux and O2-uptake rates between HH and LL plants, but the difference was still significant. After the transfer to the low- or high-light environment, both the rates changed. Expressed on a dry mass basis, the O2-uptake rates also showed slower responses to the new environments than the CO2-efflux rates, both in HL and in LH plants. In order to express the respiratory rates on a dry mass basis, we used leaf mass per area (LMA). The LMA of HH plants was almost twice that of LL plants throughout the experimental period (Fig. 3). After the transfer to the low- or high-light conditions, LMA also changed. Although the LMA of HL plants decreased, the difference in LMA between HH and HL was small. The LMA of LH plants increased at day 3 after the transfer, and was about 1·5 times greater than the LMA of LL plants at day 14 after the transfer. In order to assess what factors influence the changes of respiration, values on a fixed (i.e. area) basis would be better than those on a variable (i.e. dry mass) basis. Thus, we expressed all the values on a leaf area basis for further analyses.
In order to assess whether either the rates of ATP consumption or the substrate availability limited the respiratory rate after the transfer, we examined the effect of an uncoupler, FCCP, or a substrate, sucrose, on the O2-uptake rates of leaves. FCCP increased the O2-uptake rates in HH and LL plants to a large extent (Fig. 4a & b), which suggests that the low ATP-consumption rate limited the respiratory rates in both HH and LL plants. In the presence of FCCP, the percentage increase in O2 uptake of the leaves of LL plants was higher than that of HH plants throughout the experimental period. If the limitation by the rates of ATP consumption was responsible for the changes of the respiratory rate, then the degree of the increase in O2 uptake by the addition of FCCP is expected to be considerably different before and after the transfer. However, in HL plants, the effects of FCCP were not significantly greater than those in HH plants. On the other hand, in LH plants, the effect of FCCP was significantly smaller than that in LL plants. The differences of the effects of FCCP between LL and LH plants were, however, much smaller than expected. Assuming that the changes in the respiratory rates of LH plants were explained only by the changes in ATP consumption, the O2-uptake rates in the presence of FCCP of LH plants should be the same as those of LL plants throughout the experimental period. It is then possible to calculate the expected values of the percentage increase in O2 uptake of LH plants after the addition of FCCP from the O2-uptake rates in LL plants in the presence of FCCP and the O2-uptake rates of LH plants in the absence of FCCP. The expected percentage increases in O2 uptake after the addition of FCCP were as low as 11% at day 5 and 0% at day 14 in LH plants, whereas the actual values were much higher (Fig. 4b). Sucrose hardly influenced the respiration in any of the plants, and thus the substrate level did not limit the respiratory rates in these plants (Fig. 4c & d).
We tested whether the abundance of the respiratory components changed after the transfer. Figure 5a shows the O2-uptake rates in the presence of sufficient substrate and uncoupler. These rates represent the respiratory potential of the leaves. The respiratory potential of HH leaves was significantly greater than that of LL leaves throughout the period of measurements. The difference in the respiratory potential between HL and HH, and LH and LL, became significant by day 3 after the transfer. The correlation between the O2-uptake rates and the respiratory poten- tial was very strong [Pearson’s correlation coefficient (r) = 0·961, P < 0·0001](Fig. 6a). The maximal activities of NAD-isocitrate dehydrogenase in the HL and LH plants showed changes after the transfer to the new light environments (Fig. 5b). The changing patterns of NAD-isocitrate dehydrogenase activity in these plants were similar to those of the respiratory potential; there was a good correlation between the respiratory potential and the maximal activity of NAD-isocitrate dehydrogenase (r = 0·933, P < 0·0001) (Fig. 6b). On the other hand, the maximal activity of cytochrome c oxidase in the HL and LH plants did not show the very clear changes (Fig. 5c); Pearson’s correlation coefficient was lower (r = 0·689, P < 0·001, Fig. 6b).
The present data clearly indicate that, in Alocasia leaves, the changes in light environments altered the O2-uptake rates via the changes of ATP-consumption rates. The altered rates of respiration will modify the abundance of respiratory machinery.
The respiratory rates of the HL and LH plants acclimated to the new light environments (Fig. 1). Comparing the CO2-efflux and O2-uptake rates, there was a clear difference in the period that was required to acclimate to the new light environments. In LH plants, biosynthesis of photosynthetic and photoprotective machineries would be induced (Ferrar & Osmond 1986) and nitrogen assimilation for biosynthesis would be enhanced. Therefore, in LH plants, NADH from glycolysis and the TCA cycle would be consumed by biosynthesis and nitrogen assimilation, rather than by the respiratory electron chain for ATP production. Nitrate assimilation and biosynthesis of the reduced compounds typical of plant biomass often increase the respiratory quotient (RQ; CO2 efflux/O2 uptake) (Lambers, Chapin & Pons 1998). This may account for the observation that the CO2-efflux rates showed a faster response to high irradiances than the O2-uptake rates in LH plants. In HL plants, components that were involved in photosynthesis and photoprotection would become surplus to the demand at the low irradiances, and some of these components would be broken down gradually. In HL plants therefore a part of the respiratory substrates would be degradation products of cellular constitutions, such as photosynthetic enzymes and transporters. These reduced substrates, compared with sucrose, would lower RQ (Lambers et al. 1998). Estimated RQ with the rates of CO2 efflux and O2 uptake was quite different between HH and LL plants. The difference of RQ may be attributed to the difference of rates of nitrate assimilation or biosynthesis. As the attached whole leaf was used for the measurements of the CO2-efflux rates and the leaf segments for those of the O2-uptake rates, we could not calculate an exact value for RQ. Accurate and simultaneous measurements of the rates of CO2 efflux and O2 uptake will be needed to assess the changes of RQ in LH and HL plants and the difference between HH and LL plants. However, our data clearly suggest the changes in RQ after the transfer to the new irradiances.
After the transfer to the low- or high-light condition, LMA changed (Fig. 3). The leaves of two deciduous tree species showed similar changes of LMA after the transfer from low- to high-light environment (Naidu & DeLucia 1997). In the leaves of HL plants, decreases in the amounts of photosynthates and degradations of cellular components may account for the decrease in LMA, whereas the opposite is expected to occur in the leaves of LH plants.
In the constant-light environments, the respiratory rates of A. odora leaves were not influenced by the abundance of carbohydrates (Fig. 3 in Noguchi & Terashima 1997; Fig. 4c & d in this study). Exogenous carbohydrates also did not influence the O2-uptake rates in HL and LH plants. It is concluded that the respiration of A. odora leaves was limited by the rates of ATP-consuming processes under the constant-light environments (Fig. 4 in Noguchi & Terashima 1997; Fig. 4a & b in this study). Therefore, we expected that the effects of FCCP would largely change after the transfer in HL and LH plants. However, the differences of the percentage increase of O2 uptake were small (Fig. 4a & b). If the respiratory capacities change in parallel with the respiratory rates in HL and LH plants, the large change of ATP-consumption rates will not result in the large change of FCCP effects. Thus, these small changes of FCCP effects will be ascribed to the changes of the amounts of the respiratory components (Figs 5 & 6). It is, however, worth noting that the present data with FCCP may not represent the true degree of adenylate control in vivo, because FCCP also affects pH in cellular components.
The cost of PSII repair during 5 d and its contribution to increments of respiratory ATP production for these periods were 1·7 m mol ATP m−2 and 1%, respectively. Thus, the recalculated repair cost of PSII would still be too low to account for the increase in ATP consumption and to influence the respiratory acclimation in LH plants. Therefore, ATP in LH plants was probably consumed for the biosynthesis of new cellular components. The transfer of pea plants from low- to high-light environment increased the amount of components of photosynthesis in the leaves, such as Rubisco, cytochrome f, chloroplast ATPase (Chow & Anderson 1987a, 1987b).
The respiratory potential in HL and LH plants changed after the transfer to the new light environments (Fig. 5a). The very strong correlation between the O2-uptake rates and the respiratory potential indicates that the abundance of the respiratory components (enzymes and transporters) changed after the transfer. In fact, the maximal activity of NAD-isocitrate dehydrogenase changed after the transfer in HL and LH plants (Fig. 5b) and the correlation between the respiratory potential and the maximal activity of NAD-isocitrate dehydrogenase was very strong (Fig. 6b). Thus, we concluded that the changes of ATP-consumption rates caused the changes of the O2-uptake rates, and that the changed rates of respiration will alter the abundance of respiratory components. These results agree with the data of Bunce et al. (1977), which showed the good correlation between the respiratory rates and the MDH activities of soybean leaves.
The maximal activity of cytochrome c oxidase did not show distinct changes after the transfer to the new light environments (Fig. 5c). In the respiratory electron transport chain, there are two pathways from ubiquinol to oxygen, the alternative cyanide-resistant pathway and the cytochrome pathway. The distinctly different changes of the maximal activities of cytochrome c oxidase and those of the respiratory potential may be caused by the alternative pathway, although A. odora plants show little in vivo activity of the alternative pathway under constant-light environments (Noguchi et al. 2001a). Millenaar et al. (2000) showed that O2-uptake rates and maximal activity of cytochrome c oxidase decreased in parallel after transfer from high-to low-light environments in roots of Poa annua. They also showed that the alternative pathway activity did not change after the transfer, whereas the cytochrome pathway activity decreased. Our results need to be clarified by analysing the in vivo activities of cytochrome and alternative pathways after the transfer of A. odora plants.
The respiratory potential of LL plant was higher than the O2-uptake rate of LH plant even 14 d after the transfer (Figs 1b & 5a). Why did the abundance of respiratory components increase in LH plants? This may be because the costs of the respiratory components would be much lower than those of photosynthetic components (Makino & Osmond 1991) and because the sufficient respiratory components would allow respiratory rates to increase rapidly under a stress environment. For example, the leaves of A. odora show a rapid and transient increase of the CO2-efflux rates upon injury (Noguchi, unpublished results). Although physiological mechanisms of this phenomenon are still unknown, this response may be important to protect a leaf from herbivores.
We examined the respiratory acclimation of A. odora leaves to the changes of light environments. The changes in the light environments caused the changes in the respiratory demands and rates. The altered rates of respiration will induce the changes in respiratory capacities. These excess capacities will permit transient demands for rapid respiration.
We thank Dr Sachiko Funayama-Noguchi and Dr Shin-Ichi Miyazawa for the helpful advice and the members of the Terrestrial Ecological Laboratory, Institute of Biological Sciences, University of Tsukuba and the members of the Ecophysiological Group, Department of Biology, Graduate School of Science, Osaka University for kind advice and help. This study was supported by a Fellowship from the Japan Society for the Promotion of Science for Japanese Junior Scientists, the Sasakawa Scientific Research Grant from the Japan Science Society, Grant-in-aid for Basic Studies (No. 10440235) and Grant-in-aid under Creative Basic Research Program (09NP1501), both from the Ministry of Education, Science, Sports and Culture, Japan, and a Grant from the Ministry of Agriculture, Forestory and Fishery, Japan (Bio-Design Program).
Received 30 January 2001;received inrevised form 20 April 2001;accepted for publication 20 April 2001