Ammonia emission from rice leaves in relation to photorespiration and genotypic differences in glutamine synthetase activity

doi:10.1093/aob/mcr245

Ann Bot. 2011 November; 108(7): 1381–1386.

Published online 2011 September 20. doi: 10.1093/aob/mcr245

PMCID: PMC3197464

Ammonia emission from rice leaves in relation to photorespiration and genotypic differences in glutamine synthetase activity

Etsushi Kumagai,¹^* Takuya Araki,² Norimitsu Hamaoka,¹ and Osamu Ueno³

¹Graduate School of Bioresource and Bioenvironmental Science, Kyushu University, 6-10-1 Hakozaki, Higashi-ku, Fukuoka 812-8581, Japan

²Faculty of Agriculture, Ehime University, 3-5-7 Tarumi, Matsuyama 790-8566, Japan

³Faculty of Agriculture, Kyushu University, 6-10-1 Hakozaki, Higashi-ku, Fukuoka 812-8581, Japan

^*For correspondence. Present address: NARO Tohoku Agricultural Research Center, 4 Akahira, Shimokuriyagawa, Morioka 020-0198, Japan. E-mail ekumagai/at/affrc.go.jp

Received May 27, 2011; Revisions requested July 1, 2011; Accepted August 2, 2011.

Abstract

Background and Aims

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.

Methods

NH₃ emission (AER), and gross photosynthesis (P_G), transpiration (Tr) and stomatal conductance (g_S) 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).

Key Results

An increase in [O₂] increased AER in the two cultivars, accompanied by a decrease in P_G due to enhanced photorespiration, but did not greatly influence Tr and g_S. There were significant positive correlations between AER and photorespiration in both cultivars. Increasing light intensity increased AER, P_G, Tr and g_S in both cultivars, whereas increasing leaf temperature increased AER and Tr but slightly decreased P_G and g_S. ‘Kasalath’ (low GS activity) showed higher AER than ‘Akenohoshi’ (high GS activity) at high light intensity, leaf temperature and [O₂].

Conclusions

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.

Keywords: Ammonia assimilation, ammonia emission, glutamine synthetase, nitrogen, Oryza sativa, photorespiration, rice cultivars

INTRODUCTION

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 (g_S). NH₃ emission should be evaluated based on the diffusive conductance as well as the transpiration flux (Massad et al., 2008). The possible involvements of g_S 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.

MATERIALS AND METHODS

Plant materials and cultivation

‘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.

Simultaneous measurements of photosynthesis, transpiration and NH₃ emission

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 (P_N), dark respiration (R_d), Tr and g_S were calculated according to the method of Long and Hallgren (1985). The gross photosynthesis (P_G) equalled the sum of P_N and R_d. 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 P_G, Tr, g_S 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 P_G, Tr, g_S 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 P_G, Tr, g_S 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.

Estimation of photorespiration

Photorespiration (R_P) was estimated by subtracting the value of P_G at 21 or 40 % [O₂] from that at 2 % [O₂] (Yeo et al., 1994).

Determination of N and NH₄⁺ contents and GS activity in leaves

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).

Statistical analysis

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).

RESULTS

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’.

Table 1.

Nitrogen (N) content, NH₄⁺ content and glutamine synthetase (GS) activity in the two rice cultivars.

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. P_G, Tr and g_S 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.

Fig. 1.

Effects of increasing (A–D) light intensity (PPFD), (E–H) leaf temperature and (I–L) [O₂] on the NH₃ emission (AER), gross photosynthesis (P_G), transpiration (Tr) and stomatal conductance to water vapour (g_S) in two rice cultivars. (more ...)

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 P_G and g_S of both cultivars (Fig. 1F, H). No significant differences in P_G, Tr and g_S 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 P_G in both cultivars (Fig. 1J). Tr showed little response to the increase in [O₂] (Fig. 1K). The value of g_S decreased slightly as [O₂] increased (Fig. 1L). There were no significant differences in P_G, Tr and g_S between the cultivars.

There were high and significant positive correlations between R_P 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’.

Fig. 2.

Relationships between the photorespiration (R_P) and NH₃ emission (AER) for the two rice cultivars. ***Significant correlation at P < 0·001.

DISCUSSION

Our results clearly indicated that an increase in [O₂] enhanced AER by both rice cultivars (Fig. 1I). Simultaneously, P_G decreased because of enhanced R_P (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 R_P 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 P_G both increased (Fig. 1A, B). Under these circumstances, R_P would also increase, keeping the ratio of R_P to P_G constant (Leegood et al., 1995). When leaf temperature increased, AER also increased, whereas P_G decreased slightly (Fig. 1E, F). This decrease in P_G would be due partly to the enhanced R_P 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 P_G. 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 g_S as light intensity increased. We also found that AER, Tr and g_S 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 g_S slightly (Fig. 1H). Furthermore, the increase in [O₂] increased AER (Fig. 1I), but did not greatly influence Tr (Fig. 1K) and decreased g_S slightly (Fig. 1L). These data suggest that AER in rice leaves is not directly controlled by the transpiration flux and g_S. 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 g_S 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.

ACKNOWLEDGEMENTS

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.

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