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Keywords:

  • ammonia exchange;
  • leaf apoplast;
  • rice;
  • urea;
  • xylem sap

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. ACKNOWLEDGMENTS
  8. REFERENCES
  9. APPENDIX I

To elucidate the effects of broadcast urea on ammonia (NH3) exchange between the atmosphere and rice, we investigated the NH3 exchange flux between rice leaf blades and the atmosphere, xylem sap ammonium (inline image) concentration, leaf apoplastic inline image concentration and pH, and determined the stomatal NH3 compensation point. Paddy rice (Oryza sativa L. cv. Nipponbare) cultivation using experimental pots was conducted in the open air. Three treatments, no nitrogen (NN), standard nitrogen (SN) and high nitrogen (HN), were prepared for two supplemental fertilizations. Urea with 0, 30 and 60 kg N ha−1 for the NN, SN and HN treatments, respectively, was broadcast at panicle initiation, and urea with 0, 20 and 40 kg N ha−1 for the NN, SN and HN treatments, respectively, was broadcast at heading. The NH3 exchange fluxes between the rice leaf blades and the atmosphere (SN treatment) measured using a dynamic chamber technique showed net deposition in general; however, net emission from the old leaves occurred 1 day after the application at heading. In contrast, the xylem sap inline image concentrations increased markedly 1 day after both applications, which suggests direct transportation of inline image from the rice roots to the above-ground parts. The applications resulted in no obvious increase in the leaf apoplastic inline image concentrations. The relationship between the inline image concentration in the xylem sap and that in the leaf apoplast was uncertain, although the inline image in the xylem sap came from the roots and the inline image in the apoplast might be affected by the stomatal deposition of NH3. The stomatal NH3 compensation point of rice was estimated to be 0.1–4.1 nmol mol−1 air (20°C). The direction and intensity of the exchange flux through the stomata, interpreted on the basis of the temperature-corrected NH3 compensation point, agreed with the observed exchange flux between the rice leaf blades and the atmosphere.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. ACKNOWLEDGMENTS
  8. REFERENCES
  9. APPENDIX I

An enormous amount of ammonia (NH3) is emitted to the atmosphere through human activities. The global anthropogenic emission of NH3 (47.2 Tg N year−1 in 1993) exceeds that of nitrogen oxides (36.2 Tg N year−1) (Galloway et al. 2004). The emitted NH3 has diverse effects on the environment. In the atmosphere, some NH3 condenses with acid gases to form aerosols, such as ammonium sulfate. There is a concern about the long-range transportation of these secondary-formed aerosols as air pollutants and the impact that they may have on human health (Erisman and Schaap 2004). Although the atmospheric deposition of ammoniacal nitrogen (NHX, i.e. NH3 and ammonium ion [inline image]) is an important source of nitrogen for ecosystems, an increase in NHX deposition as a consequence of anthropogenic emission results in possible eutrophication, acidification through nitrification, and water pollution by the generated nitrate (Pierzynski et al. 1994). Increased NHX deposition also has the potential to accelerate global warming through the indirect emission of nitrous oxide (van der Gon and Bleeker 2005) and the inhibition of methane oxidation (Saari et al. 1997).

Agriculture is the main anthropogenic source of atmospheric NH3. The application of synthetic nitrogen fertilizer and livestock are two important sources of NH3. In terms of the NH3 emission induced by the application of fertilizer, crops are possible emitters in addition to the surface of arable lands. Plant emission of NH3 is explained by the stomatal compensation point for NH3s) (Farquhar et al. 1980), defined as the mole fraction of gaseous NH3 above the water film in the apoplast (Husted and Schjoerring 1995). Atmosphere–leaf NH3 fluxes through the stomata are counterbalanced when χs equals the atmospheric NH3 concentration, which results in no net exchange of NH3 through the stomata. χs varies among plant species (Loubet et al. 2002) and also depends on the different growth stages, nutrients and environmental temperatures of plants of the same species (Husted and Schjoerring 1996; Husted et al. 1996; Schjoerring et al. 1998a,b; van Hove et al. 2002). Previous studies have revealed that excessive nitrogen nutrition increases the emission of NH3 by plants (e.g. Mattsson et al. 1998; Schjoerring et al. 1998b).

The χs of rice is unknown; moreover, there has been limited research into atmosphere–rice NH3 exchange. Sekimoto and Kumazawa (1985) found that the above-ground parts of rice emitted NH3 under hydroponics with an inline image concentration of 40 mg N L−1. Furthermore, Hayashi et al. (2008) reported that urea with 30 kg N ha−1 broadcast on a paddy field at panicle initiation induced considerable NH3 emission (6.3 kg N ha−1; loss ratio of 21%); however, emissions from the surface of the paddy water accounted for only 30% of the emission, while the remaining 70% was ascribed to emissions from the rice. In contrast, broadcast urea with 10 kg N ha−1 at heading resulted in negligible emission. Rice is likely to be an NH3 emitter only with excessive nitrogen nutrition. However, atmosphere–rice NH3 exchange in the actual environment is poorly understood.

The objective of the present study was to elucidate the effects of broadcast urea on atmosphere–rice NH3 exchange. The NH3 exchange flux between rice leaf blades and the atmosphere, xylem sap inline image and leaf apoplastic properties, such as air volume, water volume, apoplastic inline image and pH, and the stomatal NH3 compensation point were investigated.

MATERIALS AND METHODS

  1. Top of page
  2. Abstract
  3. Introduction
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. ACKNOWLEDGMENTS
  8. REFERENCES
  9. APPENDIX I

Pot cultivation of paddy rice

Pot cultivation of paddy rice (Oryza sativa L. cv. Nipponbare) in the open air was conducted at the National Institute for Agro-Environmental Sciences, Japan (36°01′N, 140°07′E). Cylindrical experimental pots, each with an area of 0.05 m2, were filled with an alluvial soil (Anthrosol, Food and Agriculture Organization 1998; Aquept, Soil Survey Staff 1999). The main management practices were as follows: 11 May 2007, basal fertilization, flooding and puddling; 14 May, transplanting; 10 July, first supplemental fertilization; 13–30 July, midsummer drainage; 27 July, start of intermittent irrigation; 7 August, second supplemental fertilization.

Urea, fused phosphate and potassium chloride with 50 kg N ha−1, 80 kg P2O5 ha−1 and 80 kg K2O ha−1, respectively, were evenly applied and incorporated into the soil as a basal fertilization following flooding and puddling. In each pot, three seedlings at foliar age 3 were then transplanted as a hill 3 days after the basal fertilization; the primary leaf of each seedling was defined at foliar age 1.

For the two supplemental fertilizations, three treatments, no supplemental nitrogen (NN), standard supplemental nitrogen (SN) and high supplemental nitrogen (HN), were prepared. Urea with 0, 30 and 60 kg N ha−1 for the NN, SN and HN treatments, respectively, and potassium chloride with 30 kg K2O ha−1 per pot were broadcast for the first supplemental fertilization at panicle initiation. Urea with 0, 20 and 40 kg N ha−1 for the NN, SN and HN treatments, respectively, was broadcast for the second supplemental fertilization at heading. Table 1 contains a summary of the experimental conditions.

Table 1.  Fertilization and measurement conditions
TreatmentApplication rate of urea (kg N ha−1)Measurement
BasalSF1SF2§Ammonia exchangeXylem sap and apoplast
  • Basal fertilization 3 days prior to transplanting of rice seedlings.

  • First supplemental fertilization at panicle initiation.

  • §

    § Second supplemental fertilization at heading.

No supplemental nitrogen (NN; n = 3)50 0 0NoYes (n = 3)
Standard supplemental nitrogen (SN; n = 6)503020Yes (n = 3)Yes (n = 3)
High supplemental nitrogen (HN; n = 3)506040NoYes (n = 3)

Atmosphere–rice NH3 exchange

The NH3 exchange flux between the rice leaf blades and the atmosphere was measured once per day (10.30–13.30 hours in principle) from 9 to 13 July 2007, corresponding to the period of panicle initiation, including the date of the first supplemental fertilization (SF1(PI) period), and once per day from 6 to 10 August 2007, corresponding to the period of heading, including the date of the second supplemental fertilization (SF2(Hd) period). One new leaf blade and one old leaf blade per pot from the SN treatment (n = 3) were chosen for flux measurement. Leaf blades at foliar ages of 9 and 6 were used in the SF1(PI) period, and leaf blades at foliar ages of 11 and 8 were used in the SF2(Hd) period. The same leaf blades in the same period were used for the flux measurements.

A dynamic chamber technique was used for the flux measurements (Fig. 1). The chambers were made of transparent acrylic resin (inner diameter, 35 mm; length, 400 mm). For every sampling, one new or one old leaf blade was inserted into a chamber through its slit. The chambers were then fixed using clamps and stands. The opening of the slit was sealed with polyethylene tape. A two-stage filter holder (NL-I; NILU Products, Kjeller, Norway), consisting of an upstream stage with a polytetrafluoroethylene filter (T080A047A; Advantec, Tokyo, Japan) to filter out aerosols and a subsequent stage with a cellulose filter (51A; Advantec) impregnated with phosphoric acid to trap NH3, was used to collect NH3 in the chamber exhaust (Hayashi et al. 2006). Four chambers without inserted rice leaf blades were prepared to provide background values. The airflow rate was approximately 16 L min−1 (approximately 0.3 m s−1 in wind speed). Ambient air to ventilate the chambers was obtained from one open-top tank made of stainless steel to homogenize the background NH3 concentrations among the chambers. The open-top tank had a visor to keep out the rain. The air temperature and relative humidity within the tank were measured using a sensor (HMP45A; Vaisala, Helsinki, Finland) and recorded using a datalogger (CR10X; Campbell Scientific, North Logan, UT, USA). Data of global solar radiation measured at an adjacent research field were also obtained.

image

Figure 1. Schematic view of the dynamic chamber system used to measure the NH3 exchange fluxes between the rice leaf blades and the atmosphere. PTFE denotes polytetrafluoroethylene.

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The trapped NH3 on the phosphoric acid-impregnated filter was extracted by adding deionized water (Milli-Q SP TOC; Millipore, Billerica, MA, USA). The inline image concentration in the extracted solution was determined using a flow injection analyzer ([FIA] AQLA-1000; Aqualab, Tokyo, Japan). The exchange flux was expressed by:

  • image(1)

where Fex is the exchange flux (nmol m−2 leaf s−1), Cr and Cb are the NH3 concentrations (nmol m−3) in the exhaust of the leaf blade chamber and the background chamber, respectively, Q is the airflow volume (m3), t is the sampling time (s) and A is the leaf area (m2 leaf) inside the chamber.

All leaves per pot were classified as new or old, with yellowing leaves considered to be old. The approximate area of each leaf was determined as 0.75 × leaf length × maximum leaf width. The leaf areas were then summed into the new or old leaf areas per pot (m2 pot−1= m2 hill−1). The measured exchange fluxes for new or old leaves were converted into the fluxes per pot and then per hectare, assuming a density of 20 hills m−2.

Xylem sap inline image concentration

Xylem sap was collected from three pots per treatment (NN, SN and HN) once per day from 10 to 12 July in the SF1(PI) period and once per day from 6 to 9 August 2007 in the SF2(Hd) period. One stem per pot was cut at a height of 5 cm above the ground. A glass vial (2 mL) was filled with a small piece of washed and dried tissue paper (Kimwipes S-200; Crecia, Tokyo, Japan) and inserted into the remaining stem. Xylem sap seeping from the cut surface was absorbed onto the tissue paper. The sampling time was 10.30–13.30 hours in principle. The cut leaves were diverted for subsequent apoplastic analysis. The amount of collected xylem sap was determined by comparing the weight of the vial before and after sampling. A known volume of deionized water was added to the vials to obtain a sufficient water volume for analysis. The inline image concentration was determined using the FIA.

Leaf apoplastic properties

New and old leaves were collected from three pots per treatment (NN, SN and HN) once per day during the SF1(PI) and SF2(Hd) periods. The analysis methods were based on early studies (Husted and Schjoerring 1995; Schjoerring and Husted 1997).

Apoplastic air volume

Leaf segments with an area of approximately 4 cm2 were weighed. The leaf segments were infiltrated with polydimethylsiloxane (a silicone oil) with a kinetic viscosity of 5 mm2 s−1 (25°C; KF-96 L-5 cs, Shin-Etsu Chemical, Tokyo, Japan) in a 50-mL polyethylene syringe with a three-way stopcock and a stopper to maintain depressurization (DIK-8390-12; Daiki Rika Kogyo, Saitama, Japan). Vacuum infiltration was carried out manually. The leaf segments were blotted with tissue paper and reweighed. The apoplastic air volume ([Vair] in cm3 g−1 leaf fresh weight [LFW]) was determined by dividing the increase in leaf weight by the density of the silicone oil (0.915 g cm−3, 25°C) and the LFW. The amount of silicone oil adsorbed to the cuticle made an insignificant contribution (Husted and Schjoerring 1995).

Apoplastic water volume

Leaf segments with an area of approximately 10 cm2 were weighed. The leaf segments were infiltrated with 50 µmol L−1 indigo-5, 5′-disulfonic acid (indigo carmine) dissolved in 50 mmol L−1phosphate buffer solution at pH 6.2 using a 50-mL syringe with the same specifications as those used for the apoplastic air volume. The infiltration was carried out manually under repeated applications of vacuum and pressure on a freezer pack. The pressures for depressurization and pressurization were approximately –0.07 and 0.15 MPa, respectively.

A piece of washed and dried tissue paper with an area of approximately 1 cm2 was inserted into the bottom of a centrifuge tube (10 mL). After infiltration, the leaf segments blotted with tissue paper were immediately placed into the centrifuge tube and the tube was centrifuged at 3,000 g for 30 min at 8°C. Assuming a solution density of 1 g cm−3, the increase in leaf weight before and after the infiltration was divided by LFW to determine the infiltration volume ([Vi] cm3 g−1 LFW), and the increase in tube weight before and after the centrifugation was used as the collected volume of the intercellular washing fluid (IWF). A volume of 150 µL of deionized water was added to the collected IWF to obtain a sufficient water volume for analysis. The absorbance of the diluted IWF at 610 nm was determined using a spectrophotometer (DU800; Beckman Coulter, Fullerton, CA, USA), and the decrease in absorbance by infiltration (Ddye) was then calculated. Ddye was determined by dividing the difference between the absorbance of the indigo carmine solution and that of the IWF by the absorbance of the indigo carmine solution (0 ≤ Ddye ≤ 1). The apoplastic water volume ([Vapo] cm3 g−1 LFW) was expressed by Husted and Schjoerring (1995) as:

  • image(2)

If the osmolarity of the indigo carmine solution is different from the symplastic osmolarity, the transportation of water across the plasmalemma affects the concentration of the dye. In order to correct this effect, Vi is used in Eq. 2 instead of Vair (Schjoerring and Husted 1997).

Leaf apoplastic inline image concentration and pH

Leaf segments with an area of approximately 10 cm2 were weighed. The cut surfaces were washed with deionized water to avoid contamination of the symplastic solution and then blotted with tissue paper. The leaf segments were then infiltrated with 350 mmol L−1 sorbitol solution. The same techniques used for the infiltration and centrifugation of the apoplastic water volume were adopted. A volume of 350 µL of deionized water (pH 6.4) was added to the collected IWF to obtain a sufficient water volume for analysis. The diluted IWF was placed into a vial (250 µL), and the pH was measured using a microelectrode (9969-10D; Horiba, Tokyo, Japan). Subsequently, the inline image concentration was determined using the FIA. The leaf apoplastic inline image concentration was calculated by multiplying the inline image concentration of IWF by the dilution factor Fdil (Husted and Schjoerring 1995):

  • image(3)
Stomatal compensation point for NH3

The stomatal compensation point for NH3 was calculated based on two equilibriums, that is, the inline image dissociation in the liquid phase and the NH3 partition between the liquid and gas phases (Husted and Schjoerring 1996):

  • image(4)

where χs is the stomatal compensation point for NH3 (nmol mol−1 air), KH is the Henry constant of NH3 (L mol−1) at the standard atmospheric pressure (1,013 hPa), Kd is the dissociation constant of inline image (mol L−1), AN is the NHX concentration in the liquid phase (mol L−1) and [H+] is the hydrogen ion concentration in the liquid phase (mol L−1). The values of KH (25°C) and Kd (25°C) are 1.7 × 10−2 and 4.8 × 10−10, respectively (Husted and Schjoerring 1996).

The effect of temperature on χs was expressed by Husted and Schjoerring (1996) as:

  • image(5)

where χ1 is the compensation point (nmol mol−1 air) at the reference temperature T1 (K), χ2 is the requested compensation point (nmol mol−1 air) at a given temperature T2 (K), inline image (52.21 kJ mol−1) and inline image (34.18 kJ mol−1) are the enthalpy of inline image dissociation and NH3 volatilization, respectively, and R is the gas constant (0.00831 kJ K−1 mol−1).

RESULTS

  1. Top of page
  2. Abstract
  3. Introduction
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. ACKNOWLEDGMENTS
  8. REFERENCES
  9. APPENDIX I

Atmosphere–rice leaf blades NH3 exchange

The Fex in the SF1(PI) period was entirely net deposition (Fig. 2). However, the net deposition decreased 2 days after the application, for the old leaves in particular, despite the high background NH3 concentration 2 days after the application (13.2 nmol mol−1 air) compared with that recorded on the other days (Fig. 2). A high background concentration enhances deposition (Eqn 1). The Fex in the SF2(Hd) period was also net deposition; however, the Fex of the old leaves showed net emission 1 day after the application (Fig. 2). The Fex of the old leaves varied greatly in both the SF1(PI) and SF2(Hd) periods (Fig. 2).

image

Figure 2. Ammonia exchange fluxes between the rice leaf blades and the atmosphere before and after the urea applications by top-dressing. SN, standard supplemental nitrogen treatment; SF1(PI) period, the period at panicle initiation, including the date of the first supplemental fertilization; SF2(Hd) period, the period at heading, including the date of the second supplemental fertilization. The foliar ages of the new and old leaves were 9 and 6, respectively, in the SF1(PI) period and 11 and 8, respectively, in the SF2(Hd) period. Meteorological data show the mean values within each sampling time.

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The mean values of Fex ( standard deviation) for new and old leaves were 6.2 ± 2.4 and 12.6 ± 6.5 nmol m−2 leaf s−1 of deposition, respectively, in the SF1(PI) period and 3.9 ± 1.4 and 5.2 ± 4.3 nmol m−2 leaf s−1 of deposition, respectively, in the SF2(Hd) period. The leaf areas of new and old leaves and their sum per hill were 0.087 ± 0.005, 0.043 ± 0.004 and 0.130 ± 0.004 m2 leaf hill−1, respectively, in the SF1(PI) period and 0.148 ± 0.003, 0.076 ± 0.002 and 0.224 ± 0.002 m2 leaf hill−1, respectively, in the SF2(Hd) period. The mean values of Fex per hectare (20 hills m−2) in the SF1(PI) and SF2(Hd) periods were therefore estimated to be 0.22 ± 0.09 and 0.19 ± 0.09 mmol ha−1 s−1 of deposition, respectively.

Xylem sap inline image concentration

There is an accepted notion that rice as an ammonium-philic plant (Oji 1989) rapidly synthesizes organic nitrogen from the inline image absorbed through its roots and transports the synthesized organic nitrogen to its above-ground parts (Kiyomiya et al. 2001; Tabuchi et al. 2007). The xylem sap inline image concentrations, however, increased greatly 1 day after the applications in both the SF1(PI) and SF2(Hd) periods (Fig. 3), suggesting direct transportation of inline image to the above-ground parts of rice when the rate of inline image supply overtook the rate of organic synthesis in the roots. The inline image transportation to the above-ground parts may have induced NH3 emission from rice to the atmosphere.

image

Figure 3. Xylem sap inline image concentrations of rice before and after the urea applications by top-dressing. Xylem sap was collected from the cut surface of the stem at a height of approximately 5 cm above the ground. NN, SN and HN denote treatments with no supplemental nitrogen, standard supplemental nitrogen and high supplemental nitrogen, respectively. SF1(PI) period, the period at panicle initiation, including the date of the first supplemental fertilization; SF2(Hd) period, the period at heading, including the date of the second supplemental fertilization.

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During the SF1(PI) period, the xylem sap inline image concentrations of the SN and HN treatments 1 day after fertilizer application increased to 2.3 ± 1.3 and 2.1 ± 0.8 mmol L−1, respectively, although the concentrations of all treatments on the day of fertilization and 2 days after the application were less than 0.07 mmol L−1 (Fig. 3).

During the SF2(Hd) period, the xylem sap inline image concentrations of the SN and HN treatments 1 day after application increased to 0.78 ± 0.65 and 0.96 ± 1.35 mmol L−1, respectively, while those of all treatments 1 day before the application were less than 0.06 mmol L−1 (Fig. 3). However, the xylem sap inline image concentrations of the SN and HN treatments 2 days after the application remained relatively high (0.41–0.45 mmol L−1; Fig. 3), which seemed to be a feature specific to heading because the xylem sap inline image concentration of the NN treatment 2 days after the application was similar to the concentrations of the SN and HN treatments (Fig. 3).

The increase in xylem sap inline image concentration 1 day after the application in the SF1(PI) period was notably higher than that in the SF2(Hd) period (Fig. 3). Considering that the application rate for the SN treatment in the SF1(PI) period (30 kg N ha−1) was less than that for the HN treatment in the SF2(Hd) period (40 kg N ha−1), the larger increase in the xylem sap inline image concentration in the SF1(PI) period might be ascribed to a difference in the nitrogen demand and/or nitrogen transportation in the plant body between panicle initiation and heading.

Features of rice leaf apoplasts

Apoplastic air volume

The mean value of Vair for all treatments (NN, SN and HN; new and old leaves) was 0.13 ± 0.04 cm3 g−1 LFW (n = 144; Table 2). For another poaceous plant, van Hove et al. (2002) reported the Vair of perennial ryegrass (Lolium perenne L.) to be 0.21 cm3 g−1 LFW (Table 2), which is larger than that of the rice in the present study.

Table 2.  Apoplastic properties of poaceous plants from previous studies and in the present study
Plant species Experimental conditionApoplastic propertiesSource
Vair (cm3 g−1 LFW)Vapo (cm3 g−1 LFW)inline image (mmol L−1)pHχs (nmol mol-1 air)
  1. Vair, apoplastic air volume; Vapo, apoplastic water volume; inline image, leaf apoplast ammonium concentration; pH, leaf apoplast pH; χs, stomatal compensation point for ammonia; LFW, leaf fresh weight.

Barley: Hordeum vulgare L. cv. GolfHydroponics under N limitationTillering     6.4 ± 1.1 (20°C)Husted et al. (1996)
Before anthesis     4.7 ± 0.3 (20°C)
Anthesis     3.0 ± 0.4 (20°C)
Grain filling     4.0 ± 0.7 (20°C)
Maturity     5.3 ± 0.1 (20°C)
Hydroponics; 24-day-old; 2.5 mmol L−1 nitrate0 mmol L−1 ammonium0.04 ± 0.0036.70 ± 0.18Mattsson et al. (1998)
0.5 mmol L−1 ammonium0.05 ± 0.0036.60 ± 0.11
1 mmol L−1 ammonium0.38 ± 0.116.52 ± 0.05
2.5 mmol L−1 ammonium1.04 ± 0.136.50 ± 0.22
5 mmol L−1 ammonium1.90 ± 0.226.44 ± 0.09
10 mmol L−1 ammonium2.28 ± 0.426.32 ± 0.14
Barley: Hordeum vulgare L. cv. LaevigatumHydroponics under N limitationTillering     4.2 ± 0.3 (20°C)Husted et al. (1996)
Before anthesis     2.9 ± 0.6 (20°C)
Anthesis     0.8 ± 1.7 (20°C)
Grain filling     5.3 ± 0.8 (20°C)
Maturity     3.3 ± 0.6 (20°C)
Barley: Hordeum vulgare L. cv. Maris MinkHydroponics; before anthesis; good N supplyWild type0.36 ± 0.085.32 ± 0.06   0.75 ± 0.2 (25°C)Mattsson et al. (1997)
66% glutamine synthetase activity1.08 ± 0.265.86 ± 0.14   7.72 ± 0.2 (25°C)
47% glutamine synthetase activity0.69 ± 0.125.71 ± 0.09   3.46 ± 0.2 (25°C)
Perennial ryegrass: Lolium perenne L.Field study; 1-year cultivation  0.006–0.6   5.6–7.8    0.03–14.2 (20°C)Loubet et al. (2002)
Field study; 1-year cultivation 0.210.11–0.32   0.16–0.92   6.0–6.3    0.8–6.4van Hove et al. (2002)
Hydroponics; 11-week-old3 mmol L−1 nitrate0.021 ± 0.0056.7 ± 0.1     0.6 ± 0.1 (20°C)Mattsson and Schjoerring (2002)
3 mmol L−1 ammonium0.049 ± 0.0126.2 ± 0.1     0.8 ± 0.2 (20°C)
6 mmol L−1 ammonium0.170 ± 0.0386.3 ± 0.1     2.5 ± 0.7 (20°C)
Bromegrass: Bromus erectus Huds.Hydroponics; 80–110-day-old; High N    0.14–0.24   6.0–6.3   0.32 ± 0.58 (20°C)Hanstein et al. (1999)
Hydroponics; 11-week-old3 mmol L−1 nitrate0.033 ± 0.0166.4 ± 0.1     1.0 ± 0.8 (20°C)Mattsson and Schjoerring (2002)
3 mmol L−1 ammonium0.045 ± 0.0196.6 ± 0.1     1.8 ± 1.0 (20°C)
6 mmol L−1 ammonium0.369 ± 0.0816.5 ± 0.1   10.4 ± 2.6 (20°C)
Tall oatgrass: Arrhenatherum elatius (L.) Beauv. Ex J. & C. Presl.Pot experiment; 80–110-day-old; High N –     0.19–0.30   5.7–5.9  0 .04 ± 0.59 (20°C)Hanstein et al. (1999)
Paddy rice: Oryza sativa L. cv. NipponbarePot experiment in open air; panicle initiation and heading; average on three types of supplemental fertilizations 0.13 ± 0.040.25 ± 0.220.28 ± 0.16  (6.3–6.7)      0.1–4.1 (20°C)Present study
Diluted IWFpH 5.0–6.5
Apoplastic water volume

The mean value of Vapo for all treatments was 0.25 ± 0.22 cm3 g−1 LFW (n = 101; Table 2). There was a large standard deviation, perhaps caused by errors in the method of analysis (see Appendix I). An early study reported the Vapo of perennial ryegrass to be 0.11–0.32 cm3 g−1 LFW (van Hove et al. 2002) (Table 2), similar to that of the rice in the present study.

Leaf apoplastic inline image concentration and pH

The leaf apoplastic inline image concentration of rice was estimated uniformly using the Fdil (= 1.5) derived from the mean values of Vair and Vapo. The mean inline image concentration in the leaf apoplastic solution was 0.28 ± 0.16 mmol L−1 (n = 166; Table 2), which is similar to the value of barley before anthesis (0.36 ± 0.08 mmol L−1; Mattsson et al. 1997; Table 2).

Dilution of IWF was necessary to measure the pH in the present study because the rice leaves provided only a very small amount of the IWF needed to measure the pH. The interquartile range of pH of the diluted IWF was 6.3–6.7, with a median pH of 6.5 (n = 168; Table 2). Grignon and Sentenac (1991) reported that the main range of apoplastic pH is 5.0–6.5, with the lowest and highest values just above 4 and 7, respectively.

Stomatal compensation point for NH3

The χs of rice was estimated to be 0.1–4.1 nmol mol−1 air (20°C) (Table 2). The mean value of the leaf apoplastic inline image concentration (0.28 mmol L−1) and the main range of leaf apoplastic pH (5.0–6.5; Grignon and Sentenac 1991) were used. This value was similar to the χs values of other poaceous plants, such as barley and perennial ryegrass (Hanstein et al. 1999; Husted et al. 1996; Loubet et al. 2002; Mattsson and Schjoerring 2002; Mattsson et al. 1997, 1998; van Hove et al. 2002; Table 2). However, χs varies with temperature (Eqn 5). The χs values of rice corresponding to the mean air temperature in the SF1(PI) and SF2(Hd) periods, 26.9 and 38.6°C, respectively, were estimated to be 0.3–9.2 (26.9°C) and 1.1–33.6 nmol mol−1 air (38.6°C), respectively. The same values for the leaf apoplastic inline image concentration and the range of leaf apoplastic pH were used.

DISCUSSION

  1. Top of page
  2. Abstract
  3. Introduction
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. ACKNOWLEDGMENTS
  8. REFERENCES
  9. APPENDIX I

NH3 exchange and meteorological conditions

The large net deposition in the SF1(PI) period compared to that in the SF2(Hd) period (Fig. 2) could be ascribed to the effects of differences in relative humidity, air temperature and solar radiation. An increase in relative humidity enhances NH3 deposition to the cuticle (Erisman 1994). The cuticular resistance at 50% relative humidity corresponds to approximately 800 s m−1, while resistance at 80% and 90% relative humidity exponentially decrease to approximately 100 and 10 s m−1, respectively (Erisman 1994). The term resistance is a parameter used for the resistance model to estimate deposition velocity, expressed as the inverse of the sum of aerodynamic, sub-laminar and surface resistances (Hicks et al. 1987). The cuticular resistance is a component of the surface resistance. The relative humidity in the SF1(PI) period except on the first day was high (approximately 80%) because of the cloudy and occasionally drizzly weather, while the relative humidity in the SF2(Hd) period was low (approximately 45%) because of the fine and very hot weather (Fig. 2). The high relative humidity in the SF1(PI) period seemed to enhance the cuticular deposition of NH3.

Furthermore, an increase in air temperature enhances the NH3 compensation point (Eqn 5), with the potential to reduce deposition through the stomata. The mean air temperatures over the sampling time (10.30–13.30 hours) in the SF1(PI) and SF2(Hd) periods were 26.9 and 38.6°C, respectively. The temperature effect lowered the compensation point in the SF1(PI) period to one-quarter of that in the SF2(Hd) period (Eqn 5). Hence, the lower air temperature in the SF1(PI) period is likely to have enhanced deposition through the stomata.

In contrast, the intensity of global solar radiation in the SF1(PI) period was low (Fig. 2). The contribution of the stomatal exchange to the atmosphere–leaf exchange appeared to be weaker in the SF1(PI) period because the stomatal aperture is reduced under weak solar radiation (Ishihara et al. 1971), which could counterbalance the enhancement effect of the lower air temperature on the stomatal deposition in the SF1(PI) period. However, the NH3 exchange as deposition in the SF1(PI) period was greater than that in the SF2(Hd) period (Fig. 2). It is, therefore, considered that the high relative humidity that enhanced the cuticular deposition had the strongest effect among the measured meteorological conditions on the atmosphere–leaf NH3 exchange in the SF1(PI) period.

Differences in the NH3 exchange between new and old leaves

Old leaves generally showed larger deposition than new leaves (Fig. 2), although old leaves need much less nitrogen. However, both the stomatal and cuticular depositions are passive phenomena for plant leaves. The greater deposition of old leaves is, therefore, not necessarily related to the nitrogen demand.

Enhancement in stomatal and/or cuticular deposition is a possible cause of the large deposition recorded in the old leaves. It is, however, known that the stomatal aperture of old rice leaves is clearly lower than that of new rice leaves (Ishihara et al. 1971). Hence, stomatal deposition must be reduced for old leaves. The results suggested that the enhancement in the cuticular deposition, perhaps resulting from age-related degradation of the cuticle, outweighed the reduction in the stomatal deposition.

inline image in rice plants

Considering the direction of water movement from the roots to the leaves, xylem sap inline image is governed by the inline image absorbed through the roots, and leaf apoplastic inline image can be affected by NH3 exchange through the stomata. The xylem sap inline image concentrations increased markedly 1 day after fertilizer application (Fig. 3), while the leaf apoplastic inline image concentrations showed no clear changes following the application (Fig. 4). In terms of inline image concentration, there was no direct correlation between the xylem sap and the leaf apoplastic solution.

image

Figure 4. Leaf apoplastic inline image concentrations before and after the urea applications by top-dressing. NN, SN, and HN denote treatments with no supplemental nitrogen, standard supplemental nitrogen and high supplemental nitrogen, respectively. SF1(PI) period, the period at panicle initiation, including the date of the first supplemental fertilization; SF2(Hd) period, the period at heading, including the date of the second supplemental fertilization. The foliar ages of the new and old leaves were 9 and 6, respectively, in the SF1(PI) period and 11 and 8, respectively, in the SF2(Hd) period.

Download figure to PowerPoint

In addition, the xylem sap inline image concentration of the NN treatment increased slightly 1 day after the application (0.25 ± 0.02 mmol L−1; Fig. 3). Considering the direction of the flow of xylem sap, from the roots to the leaves, and the strong nitrogen demand of the NN treatment, a feasible cause was the root uptake of the deposited NH3 onto the flooded water of the NN pots, which could have originated from the volatilized NH3 from the adjacent SN and HN pots.

As a reference, the bulk tissue inline image concentration of the new leaves at heading collected from each pot in the HN treatment (n = 3) was estimated to be 12.3 ± 1.2 mmol L−1; the leaf water content was assumed to be 75% in this calculation according to the measurements of Ishihara et al. (1974). The bulk tissue inline image concentration was more than 40-fold higher than the mean apoplastic inline image concentration (0.28 mmol L−1).

Relationship between NH3 exchange and stomatal compensation point

The range of the NH3 background concentration in the SF1(PI) period was 3.6–13.2 nmol mol−1 air (Fig. 2); the respective upper and lower limits were larger than the upper and lower limits of the estimated χs in the SF1(PI) period (0.3–9.2 nmol mol−1 air). Hence, the NH3 background concentration is likely to have exceeded the χs, resulting in NH3 deposition through the stomata. Furthermore, the high relative humidity during the SF1(PI) period enhanced the cuticular deposition of NH3. As a result of the enhanced stomatal and cuticular deposition, the rice leaves acted as absorbers of atmospheric NH3. This interpretation agrees with the observation that the Fex showed net deposition throughout the SF1(PI) period (Fig. 2).

In contrast, the range of the NH3 background concentration in the SF2(Hd) period was 5.2–11.3 nmol mol−1 air (Fig. 2), within the range of the estimated χs in the SF2(Hd) period (1.1–33.6 nmol mol−1 air). Therefore, the rice leaves probably acted as absorbers and emitters of atmospheric NH3 in response to changes in the magnitude of the relationship between the background NH3 concentration and the χs. This interpretation is in agreement with the observation that the Fex showed net deposition in the SF2(Hd) period; however, the fluxes were smaller than those in the SF1(PI) period, and the Fex of old leaves showed a temporal net emission (Fig. 2).

Conclusion

In the present study, the NH3 exchange between rice leaf blades and the atmosphere at panicle initiation and at heading showed net deposition in general, even with supplemental fertilizations of urea by top-dressing; however, net emission from old leaves occurred 1 day after the second supplemental fertilization at heading (Fig. 2). As shown for methane emissions from the base of the leaf sheath and the surface of the stem (Nouchi et al. 1990), parts other than the rice leaf blade were involved in the atmosphere–rice gas exchange. These and/or other, as yet unascertained, parts are also likely to contribute to the atmosphere–rice NH3 exchange.

By contrast, the xylem sap inline image concentrations increased markedly 1 day after the applications at both panicle initiation and at heading (Fig. 3). This result suggests that part of the inline image absorbed by the rice roots was directly transported to the above-ground parts of the plant under excessive nitrogen nutrition. However, the applications resulted in no obvious increases in leaf apoplastic inline image concentrations (Fig. 4). The relationship between the inline image concentrations in the xylem sap and the leaf apoplasts remains to be determined.

The stomatal NH3 compensation point of rice was estimated to be 0.1–4.1 nmol mol−1 air (20°C). The temperature-corrected NH3 compensation point at panicle initiation was generally below the background NH3 concentrations at which NH3 deposition through the stomata took place. In contrast, the temperature-corrected NH3 compensation point at heading was occasionally above the background NH3 concentrations at which both stomatal emission and deposition of NH3 could occur. These interpretations agreed with the observed NH3 exchange fluxes between rice leaf blades and the atmosphere.

ACKNOWLEDGMENTS

  1. Top of page
  2. Abstract
  3. Introduction
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. ACKNOWLEDGMENTS
  8. REFERENCES
  9. APPENDIX I

We would like to express our appreciation to Dr Toshihiro Hasegawa, National Institute for Agro-Environmental Sciences (NIAES), for his helpful comments on the physiological properties of rice leaf blades and to Dr Hiroko Akiyama, NIAES, for providing the data on global solar radiation.

REFERENCES

  1. Top of page
  2. Abstract
  3. Introduction
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. ACKNOWLEDGMENTS
  8. REFERENCES
  9. APPENDIX I
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  • Food and Agriculture Organization 1998: World Reference Base for Soil Resources. World Soil Resources Reports 84, Food and Agriculture Organization, Rome.
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  • Hanstein S, Mattsson M, Jaeger H-J, Schjoerring JK 1999: Uptake and utilization of atmospheric ammonia in three native Poaceae species: leaf conductances, composition of apoplastic solution and interactions with root nitrogen supply. New Phytol., 141, 7183.
  • Hayashi K, Nishimura S, Yagi K 2006: Ammonia volatilization from the surface of a Japanese paddy field during rice cultivation. Soil Sci. Plant Nutr., 52, 545555.
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  • Hicks BB, Baldocchi DD, Meyers TP, Hosker Jr RP, Matt DR 1987: A preliminary multiple resistance routine for deriving dry deposition velocities from measured quantities. Water Air Soil Pollut., 36, 311330.
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  • Husted S, Schjoerring JK 1996: Ammonia flux between oilseed rape plants and the atmosphere in response to changes in leaf temperature, light intensity, and air humidity: interactions with leaf conductance and apoplastic inline image and H+ concentrations. Plant Physiol., 112, 6774.
  • Ishihara K, Ishida Y, Ogura T 1971: The relationship between environmental factors and behavior of stomata in the rice plant. 3. On the aperture of the stomata and their diurnal movement in the leaf at different position on the stem. Proc. Crop Sci. Soc. Jpn., 40, 505512 (in Japanese with English summary).
  • Ishihara K, Ishida Y, Ogura T 1974: On the diurnal variation of leaf water content on an areal basis in the rice plant. Proc. Crop Sci. Soc. Jpn., 43, 7782 (in Japanese with English summary).
  • Kiyomiya S, Nakanishi H, Uchida H et al . 2001: Real time visualization of 13N-translocation in rice under different environmental conditions using positron emitting tracer imaging system. Plant Physiol., 125, 17431754.
  • Loubet B, Milford C, Hill PW, Tang YS, Cellier P, Sutton MA 2002: Seasonal variability of apoplastic inline image and pH in an intensively managed grassland. Plant Soil, 238, 97110.
  • Mattsson M, Schjoerring JK 2002: Dynamic and steady-state responses of inorganic nitrogen pools and NH3 exchange in leaves of Lolium perenne and Bromus erectus to changes in root nitrogen supply. Plant Physiol., 128, 742750.
  • Mattsson M, Häusler RE, Leegood RC, Lea PJ, Schjoerring JK 1997: Leaf-atmosphere NH3 exchange in barley mutants with reduced activities of glutamine synthetase. Plant Physiol., 114, 13071312.
  • Mattsson M, Husted S, Schjoerring JK 1998: Influence of nitrogen nutrition on ammonia emission from plant leaves. Nutr. Cycl. Agroecosyst., 51, 3540.
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  • Oji Y 1989: Differential preference of plants for ammonium or nitrate. Nippon Nogeikagaku Kaishi, 63, 13821385 (in Japanese).
  • Pierzynski GM, Sims JT, Vance GF, eds 1994: Soil nitrogen and environmental quality. In Soils and Environmental Quality, pp. 55102. CRC Press, Florida.
  • Saari A, Martikainen PJ, Ferm A et al . 1997: Methane oxidation in soil profiles of Dutch and Finnish coniferous forests with different soil texture and atmospheric nitrogen deposition. Soil Biol. Biochem., 29, 16251632.
  • Schjoerring JK, Husted S 1997: Measurement of ammonia gas emission from plants. In Modern Methods in Plant Analysis, Vol.19, Plant Volatile Analysis. Eds HFLinskens and JFJackson, pp. 7395, Springer-Verlag, New York.
  • Schjoerring JK, Husted S, Mattsson M 1998b: Physiological parameters controlling plant–atmosphere ammonia exchange. Atmos. Environ., 32, 491498.
  • Schjoerring JK, Husted S, Poulsen MM 1998a: Soil–plant–atmosphere ammonia exchange associated with Calluna vulgaris and Deschampsia flexuosa. Atmos. Environ., 32, 507512.
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  • Tabuchi M, Abiko T, Yamaya T 2007: Assimilation of ammonium ions and reutilization of nitrogen in rice (Oryza sativa L.). J. Exp. Bot., 58, 23192327.
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APPENDIX I

  1. Top of page
  2. Abstract
  3. Introduction
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. ACKNOWLEDGMENTS
  8. REFERENCES
  9. APPENDIX I

Problems in the analysis of rice leaf apoplasts

Infiltration

A criterion by which to judge the completeness of infiltration is that the leaf color turns blackish when apoplastic air is replaced with solution. A test using the leaves of spinach (Spinacia oleracea L.) and kidney bean (Phaseolus vulgaris L.) showed that complete infiltration was achieved by several repeated applications of vacuum and pressure within several minutes. However, the rice leaves required 20–30 applications of vacuum and pressure for complete infiltration, which took from 40 min to 3 h. The rice leaf cuticle was strongly hydrophobic, which prevented infiltration of the solution. A long infiltration time is undesirable because changes in the IWF quality might be induced by factors other than dilution by infiltration.

The time required for infiltration was cut in half by using a solution with 1% surfactant. Therefore, the hydrophobia of the cuticle was likely to be related to the resistance to deep infiltration. However, the addition of a surfactant resulted in further difficulties in the collection of IWF. In addition to yellowing the IWF, the surfactant appeared to damage the plasmalemma. The surfactant, we conclude, was useless for apoplastic analysis.

Erroneous Ddye and/or Vi resulted in erroneous Vapo (Eqn 2). For Ddye, a decrease in the concentration of indigo carmine owing to factors other than dilution, for example, decomposition and adsorption, results in possible overestimation of Ddye and then Vapo. Rice leaves are very thin and their absolute water content is therefore small. The very small amount of IWF obtained from the rice leaves required further dilution to obtain a sufficient volume of IWF for the absorbance measurement, which was a possible cause of the large errors in the Ddye and Vapo values.

In terms of the infiltration technique for rice leaves, a method to quickly complete the infiltration needs to be developed. For example, infiltration under exposure to strong light to prevent stomatal closure, although not applied in the present study, may be effective because rapid infiltration through the unclosed stomata can be achieved. Automation of the infiltration method is also desirable in response to repeated applications of vacuum and pressure.

Centrifugation

Early studies used centrifugation at 2,000 g or less (e.g. Burkey et al. 2006; Husted and Schjoerring 1995). In the present study, however, centrifugation at 3,000 g was used because centrifugation at 2,000 g provided little IWF for the rice. A higher centrifugal acceleration is advantageous in the collection of IWF, although contamination of the symplastic solution is likely to occur because of damage to the plasmalemma. A test of centrifugation at accelerations ranging from 3,000 to 8,000 g with an interval of 1,000 g was conducted using the potassium ion (K+) concentration in IWF as an indicator of contamination of the symplastic solution. The K+ concentration was determined using capillary electrophoresis (G1600A; Agilent Technologies, Santa Clara, CA, USA). As a result, an increase in K+ concentration was avoided until centrifugation at 4,000 g (data not shown). In addition, the rice leaves showed no changes in their shape after centrifugation, even at 8,000 g.

The amount of IWF obtained by centrifugation at 3,000 g was only half of that of the infiltration volume for both the indigo carmine and sorbitol solutions; a test of centrifugation at 4,000 g showed a similar result. Furthermore, the IWF seeping from the rice leaves stuck to the cuticle, which prevented efficient collection of the IWF. In contrast, a test using the leaves of spinach and kidney bean showed that IWF formed droplets and was easily collected. The cuticle of the rice leaf seemed to change from hydrophobic to hydrophilic with infiltration of the solution.

In the method used in the present study, a piece of tissue paper was used effectively to absorb and collect the IWF of rice; however, it should be noted that tissue paper itself has a background inline image level. Furthermore, overstuffing of leaf segments in a centrifuge tube prevented collection of the IWF by centrifugation because the IWF, like the capillary water, abided in the interspace of the leaves.

Rice leaves have a low water content and low recovery of IWF; however, apoplastic analysis requires a certain amount of IWF. Hence, a method to efficiently centrifuge IWF using a relatively large number of leaf segments should be developed.