Gas Exchange Characteristics in Rice Leaves Grown under the Conditions of Physiologically Low Temperature and Irradiance

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Short Communication

Gas Exchange Characteristics in Rice Leaves Grown under the Conditions of Physiologically Low Temperature and Irradiance

Keiko Ohashi, Amane Makino and Tadahiko Mae

Department of Applied Biological Chemistry, Faculty of Agriculture, Tohoku University, Tsutsumidori-Amamiyamachi, Sendai, 981- 8555 Japan

The photosynthetic rate measured at 20°C was higher in rice grown under 20/18°C day/night temperature and 350 //moI quanta m~²s~' than in rice grown under 25/

20°C and 1,000//moI quanta m~²s~', whereas there was no difference in the photosynthetic rate measured at 25°C between rice grown in these two ways. This difference was suggested to be caused by an enhanced ribulose-l,5-bis- phosphate-regeneration capacity in the low-temperature/ir- radiance-grown rice.

Key words: Gas exchange (photosynthesis) — Low irradiance — Low temperature — Oryza sativa — Ribulose- 1,5-bisphosphate (RuBP) regeneration capacity.

Leaf photosynthesis is strongly affected by temperature or light conditions. In the northern area of Japan, cool weather damage in summer is always caused not only by low temperature but also by a shortage of sunshine. Under such weather conditions, leaf photosynthesis is suppressed.

However, little is known whether plants are potentially able to acclimate to such low temperature and low irradiance conditions. Most studies on temperature acclimation of leaf photosynthesis have been done only under high or moderate light conditions (Mooney et al. 1978, Berry and Bjorkmann 1980, Badger et al. 1982, Ferrar et al. 1989, Makino et al. 1994b). Photosynthetic acclimation to low temperatures seems to be opposite that of acclimation to low irradiance. For example, although leaves develop- ing under conditions of low temperature show a relative decrease in the amount of Chi (for reviews, see Berry and Bjdrkman 1980) and a great deal of carbohydrate accumulation (Guy et al. 1992, Mercado et al. 1997), leaves develop- ing under low irradiance exhibit an increase in the amount of Chi (Evans 1987, Evans and Terashima 1988, Makino et al. 1997) and low accumulation of carbohydrate (Makino Abbreviations: Chi, chlorophyll; Cyt, cytochrome; FBPase;

fructose-1,6-bisphosphatase; pCa, ambient CO2 partial pressure;

pCi, intercellular CO2 partial pressure; PPFD, photosynthetic photon flux density; RuBP, ribulose-l,5-bisphosphate; Rubisco, ribulose-l,5-bisphosphate carboxylase/oxygenase; SPS, sucrose- phosphate synthase; Sue, sucrose.

et al. 1997). Therefore, it is important to elucidate how growth irradiance affects the photosynthetic acclimation to low temperatures. However, there is no available information on the acclimation of leaf photosynthesis in response to a combination of low temperature and low irradiance.

According to the biochemical model of leaf photosynthesis by Farquhar and von Caemmerer (1982) and Sharkey (1985), the regulation of photosynthesis in C3 plants is determined by the balance between the capacity of Rubisco and RuBP regeneration by electron transport capacity and/or Pi regeneration capacity during starch and Sue synthesis. Use of this model to analyze the capacity of photosynthetic acclimation should help to elucidate the photosynthetic limiting process under low-temperature/irradiance conditions. When plants are exposed to low temperature temporarily, photosynthetic limitation shifts to RuBP regeneration, whereas photosynthesis at normal CO2 and relatively high temperature is limited by Rubisco (Labate and Leegood 1988, Sage et al. 1990). However, little is known about how growth under low-temperature/irradiance affects the balance between the capacities of the respec- tive photosynthetic limiting processes.

In this study, we examined how growth temperature and irradiance affected the photosynthetic characteristics of leaves grown under low temperature/irradiance conditions. To deduce the in vivo balance between the capacities of Rubisco and RuBP regeneration, we first measured the rate of CO2 assimilation as a function of Ci according to the photosynthetic model of Farquhar and von Caemmerer (1982). Then, we measured the amounts of Rubisco as a determinant for CO2-limited photosynthesis, Chi as a light harvesting component, Cyt/proteins as one of rate-limiting factors for electron transport and SPS as a key enzyme during Sue synthesis (see Makino et al. 1994a). In this paper, we conducted our analysis by normalizing the changes in all parameters examined above against leaf N content, because the response of leaf photosynthesis is not only determined by a change in absolute N content in a leaf but also by changes in N partitioning among the limiting processes of photosynthesis (for a review, see Terashima and Hikosaka 1995).

Rice (Oryza sativa L. cv. Notohikari) plants were grown hydroponically in an environmentally controlled

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Photosynthesis under low temperature and irradiance 1385 growth chamber (Makino et al. 1994a). The chamber was

first operated with a 15-h photoperiod, 25/20°C day/night temperature, 60% RH, and a PPFD of l.OOO^mol quanta m~²s~' at the plant level during daytime. The basal nutrient solution was as previously described by Makino et al. (1988). From day 49 after germination, plants were grown using two growth treatments, i.e., a day/night temperature of 20/18°C under a PPFD of 350/jmol quanta m~² s~' and a day/night temperature of 25/20°C under a PPFD of 1,000 ^mol quanta m~²s~'. From day 56 after germination, N concentrations (mM) in the hydroponic solutions were 0.5 (0.25 mM NH4NO3), 2.0 (1.0 mM NH4NO3), and 8.0 (2 mM NH4NO3 plus 4.0 mM NaNO3) for each growth treatment. Gas exchange and biochemical assays were carried out on young, fully expanded leaves of 70- to 80-d-old plants.

Gas exchange was determined with an open gas-exchange system detailed by Makino et al. (1988). Measure- ments were made at two different leaf temperatures of 20°C and 25°C, a PPFD of 1,600/imol quanta m~²s~', and a leaf-to-air vapor pressure difference of 1.0 to 1.2 kPa. The first measurement was made at a pCa of 36 Pa until the steady state of the gas-exchange rate was obtained, and then/?Ca was varied to measure the rates at apCi of 20 Pa and atpCi greater than 60 Pa. Different temperature measurements were done for different leaves, respectively. Gas- exchange parameters were calculated according to the equa- tions of von Caemmerer and Farquhar (1981).

The amounts of Chi, total leaf N, and Rubisco were determined according to the methods of Makino et al.

(1994a). Cyt/content was estimated from the difference between the hydroquinone-reduced and the ferricyanide-oxi- dized spectra of the thylakoid membrane according to Evans and Terashima (1987). The difference spectrum was recorded with a Shimadzu UV-160A spectrophotometer.

The increase in the absorbance at 554 nm above the line drawn between the absorbance of 530 and 570 nm was measured and the millimolar extinction coefficient used was 20 mM"¹ cm"¹. SPS activity was measured on a subsample of each treatment group as described by Nakano et al.

(1995). The assay was carried out at 25°C under Vmax sub- strate conditions.

The relationships between the photosynthetic rate and stomatal conductance measured at 25 and 20°C and total N content of leaves grown at three N concentrations under two temperature/irradiance conditions are shown in Fig. 1.

The rate of photosynthesis at a leaf temperature of 25°C was the same for any given leaf N content in spite of different temperature/irradiance treatments. However, the rate of photosynthesis at 20°C was 20 to 25% higher in the low- temperature/irradiance-grown plants than in the high-temperature/irradiance-grown plants. However, both stomatal conductances measured at 25 and 20°C were lower in the low-temperature/irradiance-grown plants. Thus, higher

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Fig. 1 Rate of photosynthesis at pCa.=36 Pa, stomatal conductance versus total leaf N content. Measurements were made at leaf temperatures of 25°C (left) and 20°C (right), a PPFD of 1,600 /imol quanta m"² s~', and a leaf-to-air vapor pressure difference of 1.0-1.2 kPa. Plants were grown hydroponically under two temperature/irradiance conditions of 25/20°C day/night temperature, \,000fimo\ quanta m'² s~' (open circles), and 20/18°C day/

night temperature, 350^mol quanta m~²s~' (closed triangles).

rates of photosynthesis at 20°C in the low-temperature/irradiance-treatment were not caused by enhanced pCi. This means that stomatal responses play no part in the observed differences in photosynthetic rate between the two treatments. In addition, since the absolute rates of photosynthesis measured at 20°C in the low-temperature/irradiance-treatment were not higher than those measured at 25^CC, the shift of optimal temperature as suggested before (Mooney et al. 1978, Berry and Bjorkman 1980, Badger et al. 1982, Ferrar et al. 1989) did not occur in this treatment.

These results suggest that the photosynthetic capacity at 20°C in the low-temperature/irradiance-grown plants remains at higher levels relative to that in the high-temperature/irradiance-grown plants.

It has been proposed by some researchers that at high irradiance, atmospheric CO2 partial pressures and optimal temperature, photosynthesis is well balanced by Rubisco capacity and RuBP regeneration capacity (for a review, see Evans 1989). However, short-term low temperature disrup- ts this balance between Rubisco and RuBP regeneration capacities, and the limitation of photosynthesis shifts to RuBP regeneration capacity (Sage et al. 1990). Therefore, if plants have a positive acclimation to temperature change, the stimulation of RuBP regeneration capacity rather than that of Rubisco capacity should be reasonable for growth at low temperature. In fact, some gas exchange studies have suggested that change in RuBP regeneration

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1386 Photosynthesis under low temperature and irradiance with growth temperature is relatively great (Mooney et al.

1978, Badger et al. 1982, Ferrar et al. 1989). Therefore, we first investigated whether the rate of photosynthesis limited by RuBP regeneration capacity was affected in the low-temperature/irradiance-grown plants. According to the photosynthetic model by Farquhar and von Caemmerer (1982), RuBP regeneration capacity can be deduced by measuring the photosynthetic rate under saturating CO2 conditions.

Figure 2 shows the relationships between CO2-saturated photosynthesis measured at two leaf temperatures of 25 and 20°C and total N content of leaves grown at three N concentrations under two growth environments. Interes- tingly, both rates of CO2-saturated photosynthesis at 25 and 20° C in the low-temperature/irradiance-grown plants were higher than in the high-temperature/irradiance-grown plants, whereas this high rates of CO2-saturated photosynthesis was not observed in both rice plants grown under low temperature and high irradiance (Makino et al. 1994b) and rice plants grown under high temperature and low irradiance (Makino et al. 1997). These results suggest that growth under the low-irradiance/temperature conditions promoted RuBP regeneration capacity, which resulted in relatively high photosynthesis at a normal level of CO2

(Fig. 1).

To elucidate whether this enhanced RuBP regeneration capacity in the low-temperature/irradiance-treatment was associated with changes in the amounts of the photosynthetic key components, we next examined the difference in the relationships between several key photosynthetic enzymes and components and total leaf N content. Since photosynthesis at low temperature and low irradiance is limited by RuBP regeneration but not by Rubisco, it was possible that N from Rubisco, the most abundant leaf protein, is reallocated to electron-transport components and/or key enzymes of Sue synthesis during long-term low temperature. Contrary to this expectation, however, our results showed that the amounts of Rubisco, Chi, Cyt/, and the

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Fig. 2 Relationship between the rate of photosynthesis at 60 Pa and total leaf N content. Symbols are the same as in Fig. 1.

Measurements were made at leaf temperatures of 25 °C (left panel) and 20°C (right panel), a PPFD of 1,600//mol quanta m "²s " ' , and a leaf-to-air vapor pressure difference of 1.0-1.2 kPa.

activity of SPS' were all the same at any given leaf N content regardless of treatment (Fig. 3). Although the Chi a/b ratio was slightly lower in the low-temperature/irradiance- grown plants than in the high-temperature/irradiance- grown plants (data not shown), this difference does not ex- plain the difference between them in the photosynthetic rate at 20°C. In addition, the activation state of Rubisco at 20°C tended to be slightly higher in the low-temperature/irradiance-grown plants (data not shown), but this difference was too small to account for the difference in the observed photosynthetic rates. Thus, the stimulation of RuBP regeneration in the low-temperature/irradiance-grown plants was not associated with any increase in the key enzymes and components related to RuBP regeneration examined here. This means that the stimulation of the RuBP regeneration capacity by low temperature and irradiance might be caused by the modification or change in the activation state of enzymes or components related to RuBP regeneration,

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Fig. 3 Rubisco, Chi, Cyt/content and SPS activity versus total leaf N. Symbols are the same as in Fig. 1.

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Photosynthesis under low temperature and irradiance 1387

and/or the change in the levels of the photosynthetic metabolites. Therefore, we will next examine those factors by using a gas exchange system. For example, in vivo activation of SPS and FBPase and the levels of the metabolites will be analyzed under conditions when photosynthesis is strongly limited by RuBP regeneration.

In this study, we were able to obtain new information showing that rice plants grown under physiologically low temperature/irradiance conditions promote RuBP regeneration capacity to maintain the photosynthetic rates at relatively high levels.

We wish to thank Dr. Hiromi Nakano for his advice and valuable comments. This work was supported by Grants-in-Aid for Bio Design Program (BDP-98-I-1-1) from the Ministry of Agriculture, Forestry and Fisheries, Japan, and for Scientific Research (No. 09660061) from the Ministry of Education, Science and Culture, Japan, and for Research for the Future from the Japan Society for the Promotion of Science (JSPS-RFTF 96L00604).

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(Received July 14, 1998; Accepted September 25, 1998)