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Rice (Oryza sativa) plants lose significant amounts of volatile NH3 from their leaves, but it has not been shown that this is a consequence of photorespiration. Involvement of photorespiration in NH3 emission and the role of glutamine synthetase (GS) on NH3 recycling were investigated using two rice cultivars with different GS activities.
NH3 emission (AER), and gross photosynthesis (PG), transpiration (Tr) and stomatal conductance (gS) were measured on leaves of ‘Akenohoshi’, a cultivar with high GS activity, and ‘Kasalath’, a cultivar with low GS activity, under different light intensities (200, 500 and 1000 µmol m−2 s−1), leaf temperatures (27·5, 32·5 and 37·5 °C) and atmospheric O2 concentrations ([O2]: 2, 21 and 40 %, corresponding to 20, 210 and 400 mmol mol−1).
An increase in [O2] increased AER in the two cultivars, accompanied by a decrease in PG due to enhanced photorespiration, but did not greatly influence Tr and gS. There were significant positive correlations between AER and photorespiration in both cultivars. Increasing light intensity increased AER, PG, Tr and gS in both cultivars, whereas increasing leaf temperature increased AER and Tr but slightly decreased PG and gS. ‘Kasalath’ (low GS activity) showed higher AER than ‘Akenohoshi’ (high GS activity) at high light intensity, leaf temperature and [O2].
Our results demonstrate that photorespiration is strongly involved in NH3 emission by rice leaves and suggest that differences in AER between cultivars result from their different GS activities, which would result in different capacities for reassimilation of photorespiratory NH3. The results also suggest that NH3 emission in rice leaves is not directly controlled by transpiration and stomatal conductance.
The exchange of gaseous NH3 between plants and the atmosphere depends on the gradient between substomatal cavities in the leaf and the atmosphere: NH3 emission takes place when the concentration of NH3 in the atmosphere is lower than that of NH3 in the substomatal cavities of leaves, while NH3 absorption occurs in the opposite case (Schjoerring et al., 2000). The NH3 concentration at which NH3 absorption balances NH3 loss, resulting in zero net flux of NH3, is the NH3 compensation point (Farquhar et al., 1980). Generally, the concentration of NH3 is higher within the leaf than the atmosphere. Thereby, NH3 flux occurs from the leaf to the atmosphere. Net emissions range from less than 10 to more than 70 kg NH3-N ha−1 per season, depending on the plant species, nitrogen status of the plant and soil, and climatic conditions. Such emissions may lead to a significant loss (up to 5 %) of the shoot's N content (Schjoerring et al., 2000). Thus, the NH3 emission by crops may affect their productivity because N is essential for key physiological processes leading to dry-matter production.
There are several processes generating NH4+ in the leaf, including photorespiration, nitrate/nitrite reduction, lignin biosynthesis and protein turnover (Leegood et al., 1995). In the photorespiratory cycle, NH4+ is released during the decarboxylation of glycine in mitochondria (Keys et al., 1978). Nitrate is converted to NH4+ by the sequential action of the cytosolic nitrate reductase and chloroplastic nitrite reductase (Lea and Ireland, 1999). In the lignin biosynthetic pathway, a significant amount of NH4+ is generated directly in the leaf apoplast (Nakashima et al., 1997). NH4+ is released from protein degradation and amino acid deamination in the cytosol (Olea et al., 2004). Photorespiratory NH4+ production may occur at rates up to ten times that of nitrate/nitrite reduction (Lea et al., 1992; Leegood et al., 1995). Among these processes, photorespiration is probably the largest source of liberated NH4+. The NH4+ generated is in equilibrium with NH3, depending on pH in the compartment.
Glutamine synthetase (GS), a key enzyme in the GS–glutamate synthase cycle, plays a pivotal role in the recycling of NH4+ that is released during photorespiration by generating glutamate from NH4+ and glutamine (Leegood et al., 1995). There are two isoforms of GS in higher plants, cytosolic GS1 and chloroplastic GS2, with recycling of photorespiratory NH4+ depending on GS2 (Keys and Leegood, 2002). Despite the operation of this efficient NH4+ recycling system, part of the NH3 is lost from the leaves into the atmosphere. Previous studies of the inhibition of GS by methionine sulfoximine (MSO) and of mutants with reduced GS activity in barley (Hordeum vulgare) unambiguously demonstrated the involvement of GS in NH3 emission (Mattsson and Schjoerring, 1996; Mattsson et al., 1997). However, Husted et al. (2002) found that the antisense oilseed rape (Brassica napus) plants with reduced GS2 activity showed a similar NH3 emission rate (AER) to that of wild plants. Thus, the extent and/or the mechanism of involvement of GS in NH3 emission may differ between barely and oilseed rape plants. Further analyses with different species are required to understand the physiological mechanisms responsible for foliar NH3 emission.
Rice (Oryza sativa), one of the most important cereal crops, loses significant amounts of volatile NH3 from its leaves (da Silva and Stutte, 1980; Stutte and da Silva, 1981). We found that foliar applications of MSO and an inhibitor of photorespiration (pyrid-2-yl hydroxymethane sulfonate) to rice plants dramatically increased and decreased, respectively, AER (Kumagai et al., 2009), suggesting the involvement of photorespiration in NH3 emission and a pivotal role of GS in the recycling of NH3 in rice plants, as in barley (Mattsson et al., 1998). In another study, we found that ‘Kasalath’, a cultivar with low GS activity, had a higher AER and higher NH4+ content in its leaves than ‘Akenohoshi’, a cultivar with high GS activity (Kumagai et al., 2011). These data suggest that rice cultivars differ in the AER of their leaves and that this difference may be explained, at least in part, by different GS activities.
Kamiji and Horie (1989) reported that AER was correlated with the transpiration rate (Tr) in flag leaves of rice plants during the ripening stage and proposed that the leaves release NH3 along with water during transpiration. However, they did not consider the stomatal conductance to water vapour (gS). NH3 emission should be evaluated based on the diffusive conductance as well as the transpiration flux (Massad et al., 2008). The possible involvements of gS and Tr in AER of rice leaves remain to be investigated.
To clarify the roles of photorespiration in NH3 emission and of GS in NH3 recycling in rice plants, we investigated the response of AER in the leaves of two rice cultivars with different GS activities to light intensity, leaf temperature and O2 concentration ([O2]) with simultaneous measurements of photosynthetic gas exchanges to determine the relationships between AER and these processes. The measurement of AER under conditions with different photorespiratory rates clearly demonstrates that photorespiration is strongly involved in NH3 emission by rice leaves. In addition, it is suggested that differences in AER between the two cultivars are due to different activities of GS involved in reassimialtion of photorespiratory NH3.
‘Akenohoshi’ is a japonica–indica cross, whereas ‘Kasalath’ is a traditional indica cultivar of rice (Oryza sativa L.). Imbibed seeds of the two cultivars were sown in nursery boxes in a glasshouse on 10 May 2009. After 21 d, young seedlings were transplanted into bottomless polyvinyl chloride cylinders (8 cm in diameter and 7 cm in height) sealed with spongy tissue (one seeding per cylinder). The cylinders were floated in 400-L water baths filled with the following solution recommended by Yoshida et al. (1972), with a slight modification, in which (NH4)2SO4 was used instead of NH4NO3: 2·86 mm (NH4)2SO4, 0·51 mm K2SO4, 1·00 mm CaCl2, 1·67 mm MgSO4, 0·32 mm NaH2PO4, 0·04 mm FeCl2, 9·09 µm MnCl2, 0·08 µm (NH4)6Mo7O24, 18·2 µm H3BO3, 0·15 µm ZnSO4, 0·16 µm CuSO4 and 3·57 mm Na2SiO3. The pH of the solution was adjusted every day to between 5·0 and 5·5 using HCl and NaOH. Each solution in the water baths was renewed at 2-week intervals. The seedlings were grown in the glasshouse under natural sunlight.
At 40–60 d after transplanting, we simultaneously measured photosynthetic gas exchange and NH3 emission of the uppermost fully expanded leaves of the rice plants using the method described below. After the measurements, we sampled the leaves and measured their area. The leaves were then dried at 80 °C for 3 d in an oven before measuring their N content. At mid-day, leaves from different plants were sampled, frozen in liquid N2 and stored at –80 °C before determination of the NH4+ content and GS activity.
Photosynthetic gas exchanges and NH3 emission by the leaves were measured simultaneously using an open gas-exchange system based on an assimilation chamber (400 cm3) equipped with a water jacket and a fan. The chamber was made of transparent acryl, ensuring low water, CO2 and NH3 adsorption. For all the measurements, we maintained [CO2] and relative humidity of the entering air at 402 ± 5 µmol mol−1 and 30 ± 5 %, respectively. Air flow through the chamber was adjusted to 2·0 L min−1 throughout the measurements. Light was supplied to the chamber by a red–blue lighting system (LED-HLCN-P, Ollie Co. Ltd, Osaka, Japan). Leaf temperature was measured with T-type thermocouples and chamber temperature was maintained by the water jacket. Temperature-controlled water was circulated in the water jacket. Leaf temperature was always within ± 1 °C of the chamber temperature (data not shown). Four leaves per plant were inserted into the chamber for each measurement.
[CO2] and water vapour pressure in the reference and sample air were monitored with an infrared CO2 and H2O gas analyser (Li-6262, Li-COR, Lincoln, NB, USA). Net photosynthesis (PN), dark respiration (Rd), Tr and gS were calculated according to the method of Long and Hallgren (1985). The gross photosynthesis (PG) equalled the sum of PN and Rd. NH3 in the air entering the chamber was removed by an upstream filter consisting of a three-stage cellulose filter (51A, Advantec, Tokyo, Japan). NH3 emitted from leaves in the chamber was collected by passage through a downstream one-stage 51A filter impregnated with a mixed solution of 5 % (v/v) phosphoric acid (H3PO4) and 2 % (v/v) glycerol and then dried in NH3-free air. NH3 adsorbed by the H3PO4-impregnated filter was extracted in 10 mL of de-ionized water. The concentration of extracted NH3 was determined according to the indophenol blue method (Scheiner, 1976). AER was expressed as the amount of NH3 emitted per unit leaf area per unit time (nmol m−2 s−1). The high efficiency (>95 %) of NH3 collection in this method was confirmed in our previous study (Kumagai et al., 2011).
Responses of PG, Tr, gS and AER to light intensity (photosynthetic photon flux density, PPFD) were determined at a leaf temperature of 32·5 °C and [O2] of 21 % (i.e. ambient), with PPFD of 200, 500 and 1000 µmol m−2 s−1. Responses of PG, Tr, gS and AER to temperature were determined at a PPFD of 500 µmol m−2 s−1 and [O2] of 21 %, with leaf temperatures of 27·5, 32·5 and 37·5 °C. Responses of PG, Tr, gS and AER to [O2] were also determined at a leaf temperature of 32·5 °C and a PPFD of 500 µmol m−2 s−1, with [O2] of 2, 21 and 40 %. Each measurement was repeated at least three times.
Photorespiration (RP) was estimated by subtracting the value of PG at 21 or 40 % [O2] from that at 2 % [O2] (Yeo et al., 1994).
Dried leaves were powdered and the N content was determined using the semi-micro Kjeldahl procedure. NH4+ content was measured according to Manderscheid et al. (2005). GS activity was measured according to O'Neal and Joy (1973).
Student's t-test was applied to test the significance of differences between the data from ‘Akenohoshi’ and ‘Kasalath’. The tests were performed using version 3·1 of the Sigmastat software (Systat Software, Inc., Richmond, USA).
The N content in the leaves of ‘Akenohoshi’ was significantly higher than that of ‘Kasalath’, whereas the NH4+ content in leaves of ‘Akenohoshi’ was significantly lower than that of ‘Kasalath’ (Table 1). GS activity in the leaves of ‘Kasalath’ was significantly smaller, only 57 %, than that of ‘Akenohoshi’.
An increase in PPFD from 200 to 1000 µmol m−2 s−1 increased AER in both cultivars (Fig. 1A). AER in ‘Kasalath’ was significantly higher than that in ‘Akenohoshi’ at 500 and 1000 µmol m−2 s−1 PPFD. PG, Tr and gS of the two cultivars also increased with increasing PPFD (Fig. 1B–D), and the three parameters were significantly higher in ‘Akenohoshi’ than in ‘Kasalath’ at 1000 µmol m−2 s−1 PPFD.
An increase in leaf temperature from 27·5 to 37·5 °C also increased AER in both cultivars (Fig. 1E). AER in ‘Kasalath’ was significantly higher than that in ‘Akenohoshi’ at 32·5 and 37·5 °C. Tr of both cultivars also increased as leaf temperature increased (Fig. 1G). In contrast, the increase in leaf temperature caused slight decreases in PG and gS of both cultivars (Fig. 1F, H). No significant differences in PG, Tr and gS were observed between the cultivars at any temperature.
An increase in [O2] increased AER in both cultivars, but the increase was greater in ‘Kasalath’ than in ‘Akenohoshi’ (Fig. 1I), significantly so at 21 and 40 % [O2]. The increase in [O2] caused a decrease in PG in both cultivars (Fig. 1J). Tr showed little response to the increase in [O2] (Fig. 1K). The value of gS decreased slightly as [O2] increased (Fig. 1L). There were no significant differences in PG, Tr and gS between the cultivars.
There were high and significant positive correlations between RP and AER for both ‘Kasalath’ and ‘Akenohoshi’ (Fig. 2; P < 0·001). However, the slope of the regression line was higher in ‘Kasalath’ than in ‘Akenohoshi’.
Our results clearly indicated that an increase in [O2] enhanced AER by both rice cultivars (Fig. 1I). Simultaneously, PG decreased because of enhanced RP (Fig. 1J), which would be caused by accelerated oxygenation activity relative to the carboxylation activity of ribulose-1,5-bisphoshate carboxylase/oxygenase (Leegood et al., 1995). In both cultivars, high positive correlations were found between RP and AER (Fig. 2). These results show that photorespiration is strongly involved in NH3 emission from rice leaves. Similar responses of AER to [O2] were observed in soybean (Glycine max; Weiland and Stutte, 1985) and spring wheat (Triticum aestivum; Morgan and Parton, 1989). In oilseed rape, however, AER did not change despite a 300 % increase in the ratio of O2 to CO2 (Husted et al., 2002). These data suggest that the extent of involvement of the photorespiratory process in foliar NH3 emission differs among species. Recently, it was reported that GS2 was localized in mitochondria of Arabidopsis thaliana leaves (Taira et al., 2004). It may be possible that mitochondrial GS2 efficiently assimilates NH4+ generated during photorespiration (Linka and Weber, 2005). Oilseed rape belongs to the family Brassicaceae together with Arabidopsis. Although GS2 has not been reported in mitochondria of species other than Arabidopsis so far, these contradictory results may be due to the specific difference in the intracellular localization of GS.
When PPFD increased, AER and PG both increased (Fig. 1A, B). Under these circumstances, RP would also increase, keeping the ratio of RP to PG constant (Leegood et al., 1995). When leaf temperature increased, AER also increased, whereas PG decreased slightly (Fig. 1E, F). This decrease in PG would be due partly to the enhanced RP caused by a decline in CO2 solubility in water compared with that of O2 at higher temperatures (Jordan and Ogren, 1984; Brooks and Farquhar, 1985). However, other factors may also be involved in NH3 emission, because AER increased more rapidly in response to increasing leaf temperature than the corresponding decrease in PG. In plants grown at high temperature, protein degradation is accelerated and accompanied by the release of NH4+ (Lawlor, 1979). Temperature influences foliar NH3 emission by affecting the thermodynamic equilibrium between the aqueous NH3 in the apoplast and the gaseous NH3 in the substomatal cavity (Massad et al., 2008). Thus, such non-photorespiratory factors may also affect the temperature-dependent increase in AER.
As it is thought that photorespiration does not substantially occur at 2 % [O2], AER derived from photorespiration at ambient [O2] (21 %) was estimated by subtracting AER measured at 2 % [O2]. The values were 3·71 ± 0·14 and 5·96 ± 0·21 nmol m−2 s−1 in ‘Akenohoshi’ and ‘Kasalath’, respectively. These values accounted for 57 and 67 % of the total AER derived from all the processes, including NH4+-generating processes other than photorespiration. Morgan and Parton (1989) reported corresponding values of 15–50 % in spring wheat. Based on data by Weiland and Stutte (1985), the value is approx. 35 % for soybean. Thus, the extent of involvement of photorespiration in NH3 emission from rice leaves might be greater than that in other species.
Husted and Schjoerring (1996) reported that in oilseed rape plants NH3 emission increased linearly with gS as light intensity increased. We also found that AER, Tr and gS increased with increases in PPFD. However, the patterns of increase differed somewhat among the three parameters (Fig. 1A, C, D). The increase in temperature increased AER and Tr (Fig. 1E, G) but decreased gS slightly (Fig. 1H). Furthermore, the increase in [O2] increased AER (Fig. 1I), but did not greatly influence Tr (Fig. 1K) and decreased gS slightly (Fig. 1L). These data suggest that AER in rice leaves is not directly controlled by the transpiration flux and gS. Stutte and Weiland (1978) showed that the rates of NH3 emission in several species were more closely correlated with temperature than with Tr. Husted and Schjoerring (1996) reported that gS was not the only factor responsible for the increase in AER caused by increasing temperature.
Our study demonstrated that ‘Kasalath’ emits NH3 at a higher rate than ‘Akenohoshi’ from their leaves under conditions of enhanced photorespiration. The amount of NH4+ in leaf tissues was higher in ‘Kasalath’ than in ‘Akenohoshi’, whereas GS activity showed the opposite trend (Kumagai et al., 2011; Table 1). Thus, the observed cultivar differences in AER may be due to differences in GS activity; ‘Akenohoshi’, with high GS activity, was able to reassimilate more of the NH3 released by photorespiration than ‘Kasalath’, with low GS activity. Obara et al. (2000) reported that ‘Kasalath’ (an indica cultivar) has lower GS2 activity in its leaves than japonica and javanica cultivars of rice. The GS activity in leaves may therefore be one of the factors that determine differences in AER among rice cultivars.
Our study showed that ‘Akenohoshi’, with a higher leaf N content than ‘Kasalath’, loses less NH3 from its leaves than ‘Kasalath’, as we found in our previous study (Kumagai et al., 2011). Based on the data in Fig. 1A, we estimated the amounts of N loss through foliar NH3 emission during the life span of the two cultivars on the assumption of a 40-d leaf life span, an 8-h daylength with 1000 µmol m−2 s−1 mean daily PPFD (sufficiently high to support photorespiration), and a mean daily temperature of 32·5 °C. The amounts of N loss per unit leaf area would be 0·165 and 0·195 g m−2 in ‘Akenohoshi’ and ‘Kasalath’, respectively. These N losses account for 12 and 21 % of the leaf N contents in ‘Akenohoshi’ and ‘Kasalath’, respectively. According to the concept of the NH3 compensation point (Farquhar et al., 1980), our measurements of AER, which were carried out by use of NH3-free air, would lead to an overestimation relative to naturally occurring conditions in which the atmosphere inevitably contains some NH3. Nevertheless, it seems likely that NH3 emission will have a considerable influence on the N economy of rice plants, and suggests that reduced NH3 emission should become a target trait in future rice breeding programmes.
This study was supported by a grant from the Japanese Society for the Promotion of Science Research Fellowship for Young Scientists to E.K. (No. 08J03273). We thank Mr Masayuki Tsuji, Department of Technology Education, Fukuoka University of Education, for his help in the manufacture of the assimilation chamber. We are greatly indebted to Dr David Lawlor and anonymous reviewers for invaluable comments and suggestions.