• Nitrification results in poor nitrogen (N) recovery and negative environmental impacts in most agricultural systems. Some plant species release secondary metabolites from their roots that inhibit nitrification, a phenomenon known as biological nitrification inhibition (BNI). Here, we attempt to characterize BNI in sorghum (Sorghum bicolor).
• In solution culture, the effect of N nutrition and plant age was studied on BNI activity from roots. A bioluminescence assay using recombinant Nitrosomonas europaea was employed to determine the inhibitory effect of root exudates. One major active constituent was isolated by activity-guided HPLC fractionations. The structure was analysed using NMR and mass spectrometry. Properties and the 70% inhibitory concentration (IC70) of this compound were determined by in vitro assay.
• Sorghum had significant BNI capacity, releasing 20 allylthiourea units (ATU) g−1 root DW d−1. Release of BNI compounds increased with growth stage and concentration of supply. -grown plants released several-fold higher BNI compounds than -grown plants. The active constituent was identified as methyl 3-(4-hydroxyphenyl) propionate.
• BNI compound release from roots is a physiologically active process, stimulated by the presence of . Methyl 3-(4-hydroxyphenyl) propionate is the first compound purified from the root exudates of any species; this is an important step towards better understanding BNI in sorghum.
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Nitrification is an important biological process in nitrogen (N) cycling, whereby ammonium (), either added to soils as fertilizer or formed through decomposition of organic N compounds, is oxidized to nitrite, and subsequently nitrate, by nitrifying bacteria (Nitrosomonas and Nitrobacter, respectively). Nitrogen use efficiency (NUE) for global cereal production is c. 33%. The unaccounted 67% represents a $US16.7 billion annual loss, assuming fertilizer–soil equilibrium (Raun & Johnson, 1999). Nitrification is one of the main causes of inefficient N use and N loss-associated environmental pollution as a result of leaching and denitrification. The nitrification process, resulting from subsequent denitrification, is the principal agricultural source of N2O emissions. N2O is presently the third most important gas contributing to global warming (Parker, 1972). If the nitrification process is inhibited, N recovery by crop uptake can be improved, and leaching and N2O emission can be reduced (Slangen & Kerkhoff, 1984; Subbarao et al., 2006a). Excessive nitrification can be reduced by the release of inhibitors from plant roots or by application of synthetic nitrification inhibitors (Smart & Bloom, 2001; Weiske et al., 2001). Application of synthetic nitrification inhibitors is currently the only method being used proactively to reduce soil nitrification in some agricultural production systems (Slangen & Kerkhoff, 1984; Amberger, 1989; Zerulla et al., 2001). The existence of plant-derived nitrification inhibitors has been known for several decades (Moore & Waid, 1971; Lata et al., 1999, 2004); however, previous reports have been limited in their interpretation of the effect of root exudates on soil bacteria because of the unavailability of methodology that could directly demonstrate the inhibitory capacity. Recently, a bioassay system was developed that enabled quantification of the inhibitory effect from roots. The phenomenon was termed biological nitrification inhibition (BNI) (Subbarao et al., 2006b). In situ production of nitrification inhibitors, by either plant roots or microorganisms within the rhizosphere of growing crops or pastures, is an appealing low-cost alternative to synthetic nitrification inhibitors (Fillery, 2007).
Ammonium () has a stimulatory role in the release of BNI compounds from Brachiaria humidicola and Leymus racemosus roots (Subbarao et al., 2007b,c); however, the exact roles of or the associated acidic pH that follows after its uptake (i.e. the secondary effect from ammonium uptake) in the release of BNI compounds from plant roots have not yet been clarified. Supplying N as during exudate collection will result in pH decrease over the time period of collection, because of proton release to charge balance the ammonium uptake by the roots. Conversely, supplying N as the anion nitrate results in pH increase (Raven et al., 1992). Decreased pH will affect root membrane permeability (Wollenweber, 1997), and this may cause increased release of compounds responsible for BNI. Therefore, in order to avoid fluctuations to these processes in a hydroponic study, we maintained constant pH in the nutrient solution during plant growth and root exudate collection period using a pH-stat system. Identifying the compound(s) responsible for BNI by sorghum could allow for more detailed physiological characterization of this trait in the future. Therefore, the objectives of our study were to evaluate the regulatory role of N forms (i.e. of ) in the root environment on the release of BNI compounds from roots, and to isolate, identify and characterize the compounds responsible for BNI function in terms of chemical properties, release stimulation and mode of inhibitory action.
Materials and methods
Expt 1: assessment of BNI activity in root exudates with increasing plant age
Raising seedlings of sorghum Seeds of sorghum (Sorghum bicolor (L.) Moench var. hybrid sorgo) were germinated in trays containing sand and watered with distilled water. Plants were grown in a growth chamber with a day : night temperature regime of 30 : 28°C, a photosynthetic photon flux, averaging c. 300 µmol m−2 s−1 and a 14 : 10 h light : dark photoperiod. One-week-old seedlings were transferred to continuously aerated nutrient solution in 75 l tanks on styrofoam blocks with 10 holes and two plants per hole, supported with sponge. The composition of the nutrient solution (mg l−1) was as follows: KH2PO4, 38.31; K2SO4, 31.02; CaCl2·2H2O, 10.5; MgSO4·7H2O, 36.93; FeEDTA, 15.1; H3BO3, 0.57; CuSO4·5H2O, 0.078; MnSO4·6H2O, 2.35; Na2MoO4·2H2O, 0.126; ZnSO4·7H2O, 0.220. Nitrogen at 1 mm l−1 was added as (NH4)2SO4 to the nutrient solution. The pH of the nutrient solution was adjusted to 5.5 with 1 N NaOH twice a day. The nutrient solutions were replaced every 2 d.
Root exudates collection Root exudates were collected from intact plants at 7, 14, 21, 28, 35 and 42 dat (d after transplantation) to the nutrient solution culture. For collecting root exudates, intact plants (a sample size of two plants with three replications) were removed from the nutrient solution, the roots were rinsed with deionized water followed by distilled water, then immersed for 24 h in 2 l aerated solution of 1 mm NH4Cl (pH 6.1). After root exudate collection, roots and shoots were separated and dried at 70°C for 48 h in a forced air-circulating oven before determining dry weight. For extraction of BNI compounds, root exudates were evaporated to dryness using a rotary evaporator (Buchi, V-850, Flawil, Switzerland) under vacuum at 40°C, followed by extraction with 20 ml of methanol. The methanol extract was then further evaporated to dryness using a rotary evaporator at 35°C and the residue was extracted with 20 µl of dimethyl sulphoxide (DMSO). The DMSO extract was then used to determine the BNI activity with a bioassay as described in the next section (Subbarao et al., 2006b).
Nitrification inhibition bioassay The nitrification inhibition (NI) activity of the samples was determined using a modified bioassay that employs recombinant luminescent N. europaea (Iizumi et al., 1998; Subbarao et al., 2006b). The BNI activity of the samples is expressed in units defined in terms of the action of a standard inhibitor, allylthiourea (AT); the inhibitory effect of 0.22 µm AT in an assay containing 18.9 mm of is defined as one ATU (AT unit) of activity (Subbarao et al., 2006b).
Expt 2: effect of different forms of nitrogen ( vs ) on the release of BNI compounds
This experiment was designed to clarify the role of different N-forms (i.e. vs ) on BNI compound release from roots. The uptake of results in the acidification of the rhizosphere (Maschner et al., 1991), and the experiment was designed to separate the direct effect of from the secondary effect of acidic pH on BNI compound release. Seedlings were grown in hydroponics as described in Expt 1; plants were raised with 1 mm N as (NH4)2SO4 or KNO3 for 28 d using two 45 l tanks connected to a pH-stat system (Nissin NPH-660NDE, Tokyo, Japan). The pH-stat system used proved to be very reliable and sensitive. By addition of an acid or base to the nutrient solutions, the set pH could be maintained within 0.01 pH units. The units contained one control machine, connected to a pump for controlling acid or base flow, and two electrodes, one for pH and another for temperature, connected with the main system. The nutrient solutions were replaced every 2 d. For collection of root exudates, intact plants (a sample size of two plants with three replications) were removed from the nutrient solution after 28 d, then the roots were rinsed with deionized followed by distilled water, then immersed for 24 h in a 2 l black bottle containing an aerated solution of either 1 mm NH4Cl or distilled water; the pH of the root exudate collection solutions were maintained using the pH-stat system. BNI activity in the root exudate samples was measured as described in Expt 1.
Expt 3: influence of concentration in the root exudate collection solution on the release of BNI compounds from roots
Seedlings of sorghum were grown in hydroponics as described in Expt 1, with 1 mm N as (NH4)2SO4 or KNO3 for 28 d. Root exudates were collected from intact plants after 28 dat. For collecting root exudates, intact plants (a sample size of two plants with three replications) were removed from the nutrient solution, the roots were rinsed with deionized followed by distilled water, then immersed for 24 h in aerated 2 l NH4Cl treatment solutions (0.0, 0.1, 1.0 and 5.0 mm) with and without the use of the pH-stat system; after collection of root exudates, roots were separated from the shoot, dried at 70°C for 48 h in a forced air-circulating oven before determining dry weight. The pH changes were also calculated in the solution by taking the initial and final pH measurements for the exudate collections not using the pH-stat systems. Root exudate samples were processed for the determination of BNI activity in a similar way to Expt 1.
Expt 4: isolation and characterization of BNI compound (s) from the root exudates
Chemicals All the solvents and other chemicals used were purchased from Wako (Osaka, Japan) and Sigma (St Louis, MO, USA) Pure Chemical Industries Ltd unless otherwise stated.
Plant materials Seeds of sorghum were germinated and grown in hydroponics as described in Expt 1. Nitrogen at 1 mm was added as (NH4)2SO4 to the nutrient solution. The pH of the nutrient solution was adjusted to 5.5 with 1 N NaOH or 1N HCl twice a day. The nutrient solutions were replaced every 2 d.
Collection of nitrification inhibition activity (BNI) from roots Root exudates were collected from intact plants after 28 d following transplantation to nutrient solution culture, in a similar way to Expt 1 using aerated solutions of 1 mm NH4Cl. Nearly 70 l of root exudate were collected for the concentration and extraction of BNI activity. The BNI activity from the root exudate was extracted as described earlier in Expt 1; this amounted to c. 1000 ATU.
Isolation of BNI compounds The crude methanol extracts (containing BNI activity, c. 1000 ATU) were diluted with water to reach a volume of 5% methanol before loading into a reverse-phase column (25 cm × 2.8 cm, Wakosil 40C18, Wako). The column was eluted with 210 ml each of 5, 10, 20, 30, 40, 50, 75 and 100% MeOH, and 30 fractions of 7 ml each were collected using a fraction collector (CHF121SA, Advantec, Tokyo, Japan). All the above fractions were dried in vacuo at 35°C using a centrifugal evaporator and the residues were collected in MeOH (1 ml); aliquots of these fractions (50 µl) were dried again in vacuo at 35°C in a centrifugal evaporator using 0.5 ml Eppendorf and dissolved in 10 µl DMSO; 2 µl was used to determine BNI activity. The active fractions were further purified by HPLC on a Jasco Gulliver HPLC system consisting of a PU-1580 receprocal pump, UV-1570/1575 UV detector, and 807-IT integrator equipped with TSKgel Super-ODS (4.6 mm × 100 mm or 10 mm × 100 mm) C18 column (Tosoh, Tokyo, Japan), monitored at 280 nm. The column was eluted with a linear gradient mobile-phase system of aqueous acetonitrile (10% to 30%) and all the peaks and troughs were checked for BNI activity in the bioassay. Finally the major active peak from 30% methanol fractions was isolated using an isocratic HPLC (15% acetonitrile) programme.
Instrumental analysis The mass (MS) spectra were recorded on a GCMS-QP2010 spectrometer (Shimadzu, Kyoto, Japan) by direct electron ionization (EI) at an ionization energy of 70 eV. The 1H NMR, 13C NMR and heteronuclear multiple bond correlation (HMBC) spectra were recorded at 298 K on an Avance 800 spectrometer (Bruker Biospin, Karlsruhe, Germany) using the pulse sequences and software provided by the manufacturer.
BNI activity of free acid and synthesized methyl 3-(4-hydroxyphenyl) propionate 3-(4-hydroxyphenyl) propionic acid was purchased from Sigma in free acid form. Methyl esters of 3-(4-hydroxyphenyl) propionic acid were synthesized by acid esterification (Gopalakrishnan et al., 2007). Briefly, 3-(4-hydroxyphenyl) propionic acid (0.5 g) was dissolved in 10 ml of acidified (0.1 N HCl) methanol. The solution was incubated at 37°C for 24 h. The volatiles were removed with a centrifugal evaporator. The reaction product was then purified using a HPLC, and the molecular mass was confirmed by EIMS. The BNI activity of 3-(4-hydroxyphenyl) propionic acid and its methyl esters were assayed using the bioassay system, and 70% inhibitory concentration (IC70; mean of three replications) was calculated using a dose–response curve.
Expt 5: influence of nitrogen forms ( vs ) on the release of methyl 3-(4-hydroxyphenyl) propionate from sorghum roots
Seedlings were grown in hydroponics and exudates collected with three replications as described in Expt 2. For the extraction of methyl 3-(4-hydroxyphenyl) propionate, the root exudate was evaporated to dryness using a rotary evaporator under vacuum at 40°C, followed by extraction with 20 ml of methanol. The methanol-soluble fraction was then evaporated using a centrifugal evaporator at 35ºC; the residue was then extracted again with 2 ml acetonitrile, and passed through a millipore filter (Millex-R-LG, Bedford, MA, USA) before injecting the sample into HPLC; the HPLC column was eluted with an isocratic phase system (15% acetonitrile) and monitoring was performed at a wavelength of 280 nm. The concentration of methyl 3-(4-hydroxyphenyl) propionate in the root exudate sample was determined from the HPLC peak area and calculated against a standard of pure methyl 3-(4-hydroxyphenyl) propionate (Sigma).
Expt 6: mode of inhibitory action of BNI compounds from the root exudates
The mode of inhibitory action of root exudates and methyl 3-(4-hydroxyphenyl) propionate on N. europaea was evaluated by incubating the pure cultures of N. europaea in the presence or absence of hydroxylamine in the assay medium using a previously reported protocol (Subbarao et al., 2006b). Hydroxylamine is the AMO (ammonia monooxygenase) enzymatic product resulting from ammonia oxidation, which is a substrate for the HAO (hydroxylamine oxidoreductase) enzyme; therefore hydroxylamine addition allows for determination of whether the HAO enzymatic pathway has also been blocked. The modes of inhibitory action of the known synthetic nitrification inhibitor dicyandiamide (DCD) were also tested. Root exudates and methyl 3-(4-hydroxyphenyl) propionate were dissolved in DMSO and 2 µl of each were used after adding 198 µl of distilled water in the 250 µl of bacterial culture. The sample was then incubated for 15 min before 20 µl of 10 mm hydroxylamine or distilled water was added. DCD, 200 µl (2200 µm), of the water-soluble inhibitor solution was added to the 250 µl of bacterial culture and it was incubated for 15 min before 20 µl of 10 mm hydroxylamine or distilled water (as a control) was added. The mean of the 3-bioluminescence measurements made during the 10 min incubation period was taken as the activity level. Every incubation was replicated three times. The effect of addition of the hydroxylamine to the reaction mixture was evaluated. The inhibition percentage of AMO and HAO by root exudates and methyl 3-(4-(hydroxyphenyl) propionate were evaluated using DMSO as a control; for DCD, water was used as a control. Data were subjected to analysis of variance and least significant difference at P < 0.001 was determined (Fisher LSD).
Expt 1: release of BNI compounds from sorghum roots
The inhibitory effect of root exudates was found to increase with plant age when expressed on a per unit root DW basis (Fig. 1). The results showed that the release of BNI compounds increased fivefold, from 6.22 ATUg−1 root DW d−1 at 7 dat (dat to hydroponics) to 30.1 ATUg−1 root DW d−1 at 35 dat. The pH of the growth solution decreased by 0.3 to 0.5 units at 14 dat and 0.7 to 1 unit at later growth stages for every 12 h.
Expt 2: influence of or supply on the release of BNI compounds
Sorghum grew well with both N forms (i.e. or ) when pH of the nutrient solutions was maintained using the pH-stat system, and total dry matter production was similar for both the N treatments (data not shown). Release of BNI compounds from roots was stimulated by the presence of in the root exudate collection solution for both - and -raised plants; however, the BNI activity in the root exudate was threefold higher from -grown plants compared with -grown plants (18.75 vs 6.78 ATU g−1 root DW d−1) (Fig. 2). In the absence of (i.e. root exudates collected using distilled water) BNI activity was detected in the root exudates of -grown plants (5 ATU g−1 root DW d−1), and was only just above detection limits for -grown plants (1.65 ATU g−1 root DW d−1).
Expt 3: influence of concentrations on the release of BNI compounds
Release of BNI compounds increased with increasing concentration of in the collection medium, and this increase was greater when the plants were raised with N supplied as (Fig. 3). When BNI activity was collected using 1 or 5 mm NH4Cl, plants raised with N supplied as showed higher BNI activity (c. 3.5-fold) than -supplied plants. When the pH was controlled using the pH-stat system, release of BNI activity was lower than when pH was not controlled for both - and -raised plants (Fig 3). For -grown plants, without pH-stat, the root exudate solution pH was c. 1.5 units lower at 1.0 mm and 2.2 units lower at 5 mm NH4Cl treatment after the 24 h collection period; for -grown plants, the figures were 0.9 and 1.3 units lower, respectively.
Expt 4: isolation and characterization of BNI compounds from root exudates
The plants used for the isolation of BNI compounds were found to release, on average, 20 ± 1.2 ATU g−1 root DW d−1. The crude extracts with the activity of BNI compounds were further fractionated by reverse-phase column chromatography. Activity-guided fractionations indicated that 10, 30 and 50% methanol effluents contained BNI activity (Fig. 4). Other fractions (20, 40, 75 and 100%) did not show BNI activity. The active constituent of the 30% fraction was successfully purified and results are presented here. Others active fractions, 10 and 50%, have not been purified yet.
The physicochemical properties of the compound are as follows – umber gum: 1H NMR (800.3 MHz, CD3OD) δ 2.56 (2H, t, J= 7.6 Hz, H-2), 2.81 (2H, t, J= 7.6 Hz, H-3), 3.63 (3H, s, OCH3), 6.68 (2H, m, H-3′ and H-5′), 7.00 (2H, m, H-2′ and 6′); 13C NMR (201.3 MHz, CD3OD) δ 31.2 (C-3), 37.1 (C-2), 52.0 (OCH3), 116.2 (C-3′ and C-5′), 130.3 (C-2′ and C-6′), 132.7 (C-1′), 156.9 (C-4′), 175.4 (C-1); EIMS m/z (%) 180 [M]+ (25), 149 (9), 120 (46), 107 (100).
Assignments of the chemical shifts of 1H and 13C NMR spectra were confirmed by HMBC. The NMR spectral data of the isolated compound were identical to those reported for methyl 3-(4-hydroxyphenyl) propionate (Pouchert & Behnke, 1992) and of the authentic preparation. The attachment position of the methyl group was confirmed by HMBC correlation from carbonyl to methyl protons. Thus, the isolated compound was identified as methyl 3-(4-hydroxyphenyl) propionate (Fig. 5).The molecular weight was confirmed by EIMS data. To study the inhibitory properties of the purified compound on Nitrosomonas further, we synthesized methyl ester of 3-(4-hydroxyphenyl) propionic acid. These preparations showed inhibitory effect on Nitrosomonas. Seventy per cent inhibition (IC70) was used for evaluating the effectiveness of the inhibitory effect of this compound. The IC70 value for methyl 3-(4-hydroxyphenyl) propionate (i.e. the BNI compound isolated from root exudate during this study) is 9.0 µm, and the free-acid form did not have inhibitory activity even at high concentration (> 10 000 µm; data not shown).
Expt 5: influence of or supply on the release of methyl 3-(4-hydroxyphenyl) propionate from roots
The release of methyl 3-(4-hydroxyphenyl) propionate was 3.5-fold higher in -grown plants (56.6 µm g−1 root DW d−1) than in -grown plants (17 µµ g−1 root DW d−1) when root exudates were collected in 1 mm NH4Cl solution for 24 h (Fig. 6). When the root exudates were collected in distilled water, the amount of the compound was low (7.6 µm g−1 root DW d−1) in comparison to NH4Cl collection from -grown plants, and the compound was not detected in the distilled water-collected root exudates of -grown plants (Fig. 6).
Expt 6: mode of inhibitory action of methyl 3-(4-hydroxyphenyl) propionate
Nitrosomonas europaea recovered completely from the DCD inhibitory effect when the AMO product hydroxylamine was added to the assay medium, and the same effect occurred for the BNI compound isolated from the sorghum root exudate, methyl 3-(4-hydroxyphenyl) propionate (Table 1). By contrast, the inhibitory effect of crude root exudates on N. europaea did not recover with the addition of hydroxylamine to the assay medium indicating that both the enzymatic pathways are likely to be inhibited (Table 1).
Table 1. Effect of hydroxylamine on the inhibitory action of biological nitrification inhibition (BNI) compounds in the sorghum (Sorghum bicolor) root exudates, methyl 3-(4-hydroxyphenyl) propionate and dicyandiamide (a synthetic nitrification inhibitor) in a culture of Nitrosomonas europaea
Bioluminescence (RLU ml−1)
Values are means ± SE; **P < 0.001; ns, not significantly different.
Sorghum root exudates had previously been found to inhibit nitrification in soils (Alsaadawi, 1988) and, using the bioassay system, the direct inhibitory effect on Nitrosomonas bacteria was confirmed (Subbarao et al., 2007a); however, this response had not been characterized in detail. Our results demonstrate for the first time that the production and release of inhibitory compounds from sorghum roots is enhanced by low pH, stimulated by the presence of , and influenced by plant growth stage. Using an activity-guided fractionation and purification approach, one of the active compounds responsible for inhibitory activity was determined, and release from roots has been shown to be specifically stimulated by the presence of in the root zone.
stimulates the release of BNI compound from sorghum roots
Different forms of N supplied to sorghum roots ( or ) influence the release of BNI compounds, and is a stimulus for BNI compound release (Fig. 2). Previously, the presence of has been reported to stimulate BNI compound release from B. humidicola and L. racemosus roots (Subbarao et al., 2007b,c). In both studies, plants were cultivated with N supplied as or and then root exudates were collected in 1 mm NH4Cl solution or distilled water and pH was not maintained at a steady state. Because uptake leads to alkalization of the growth media (because of H+ influx or OH− efflux from the plants) and uptake results in acidification (H+ efflux; Raven et al., 1992), the final pH after 24 h would be very different between exudate collections using the two forms. Differences in pH in the exudate collection medium, in particular low pH, could result in differences in plasma membrane permeability (Yan et al., 1998). Thus, enhanced plasma membrane permeability resulting from low rhizosphere pH (because of uptake of ) could be partly responsible for higher BNI release in root exudate solutions containing . Therefore, earlier studies (Subbarao et al., 2007b,c) indicating the stimulatory role of in BNI compound release from roots of B. humidicola and L. racemosus were not conclusive.
In the present study, a pH-stat system was used to keep the pH in the growth and exudate collection medium constant, removing the secondary effects of rhizosphere pH associated with the uptake from N forms on BNI compound release from roots. Our results show that is the major contributing factor in the release of BNI compounds and that rhizosphere pH is also important but influences BNI compound release from roots to a lesser extent (Fig. 2). The effect of different concentrations of in the exudate collection medium on the release of BNI compounds from - and -grown plants was also studied (Fig. 3). Continuous exposure of during plant growth was also found to be an important factor resulting in greater release of BNI compounds. The results also show that high concentrations of can stimulate greater release of BNI compounds under pH-stat conditions. This demonstrates conclusively that is the key contributing factor to the production and release of BNI compounds by sorghum. Presently, it is unknown why this link could be ecologically significant, or what benefit the exudation of these compounds could have to whole-plant nutrition.
The supply of nitrogen as or is known to have many other influences on root systems. The morphological effect of increased root branching with supply of compared with has been thoroughly characterized in many species (Martins-Lucao et al., 2000; Mahmood et al., 2002). Supply of has also previously been associated with increased root respiration and sugar exudation (Martins-Lucao et al., 2000). The genetic response of roots to differing forms of N supply has also been extensively studied. Supplying has been associated with various aspects of plant growth and metabolism, for example, carbon and nitrogen partitioning (Cramer & Lewis, 1993), solute composition and energy status of roots (Lang & Kaiser, 1994), and leaf morphogenesis (Walch-Liu et al., 2000). Given the many different morphological, chemical and genetic responses that have previously been associated with differing forms of N supply, a link between acquisition and the production and/or release of compounds for the purpose of nitrification inhibition, thereby retaining more N as in soil, is not altogether surprising, but at this stage it is poorly understood. Isolation and identification of BNI compounds from root exudates may help to further our understanding of the stimulatory role of on the production and/or release of BNI compounds from sorghum roots. Whether the stimulatory role of in the root zone for the release of BNI compounds is the result of its uptake or assimilation remains unclear from this study, and will be the subject of further investigations.
The properties of methyl 3-(4-hydroxyphenyl) propionate with respect to BNI
The active BNI compound in the sorghum root exudate, methyl 3-(4-hydroxyphenyl) propionate, showed a strong inhibitory effect on Nitrosomonas activity in the assay, as indicated by the IC70 value of 9.0 µm. This is the first time that a nitrification inhibitor has been isolated from the root exudate of any plant species. There have been previous reports of nitrification inhibitors extracted from different plant parts. Gopalakrishnan et al. (2007) isolated ferulic acid and p-coumaric acid from the root tissue of B. humidicola; however, these compounds were not present in root exudates. Other examples of plant tissue-extracted substances that can inhibit nitrification include neem oil from the neem tree (Azadirachta indica) (Kumar et al., 2007), and the extracts of karanj (Pongamia glabra) seed, bark and leaves of neem (Sahrawat et al., 1974). Crude extracts from the roots of Astragalus mongholicus have also been reported to inhibit soil nitrification (Mao et al., 2006).
The inhibitory properties of 3-(4-hydroxyphenyl) propionic acid and chemically synthesized methyl 3-(4-hydroxyphenyl) propionate were further confirmed by in vitro assay. The nitrification enzymes and related electron carrier system of N. europaea are located in the periplasm or inner membrane (Iizumi & Nakamura, 1997). The results suggest that methyl 3-(4-hydroxyphenyl) propionate may have such polarity that can reach the active site of a bacterial cell. By contrast, the presence of a polar –COOH group of the free acids might make their permeability too low to approach the action point. This implies that ester forms of the compound are preferable to prevent nitrification by the bacterial cell. Similarly, it has also been reported that the esters of phenolic acids are more toxic to fungi than their corresponding free acids (Daayf et al., 2000). Methyl 3-(4-hydroxyphenyl) propionate can be formed from the free-acid form in the presence of methanol under acidic conditions. To rule out the possibility that the methyl ester is an artefact formed during the sample preparation process, after rotary evaporation the root exudate residue was extracted with chloroform instead of methanol. Subsequent HPLC analysis determined a concentration of 60 µm g−1 root DW d−1 of methyl 3-(4-hydroxyphenyl) propionate for -supplied plants when root exudates were collected in 1 mm NH4Cl solution for 24 h. This result confirmed that methyl 3-(4-hydroxyphenyl) propionate is not an artefact. Our results thus indicate that methyl ester (i.e. methyl 3-(4-hydroxyphenyl) propionate) is naturally synthesized and released from sorghum roots. However, the isolated compound, methyl 3-(4-hydroxyphenyl) propionate, has not previously been reported to be released from plants or for its inhibitory effects on nitrification.
Modes of action of the compound for nitrification inhibition
Some progress has been made towards understanding the modes of inhibition of Nitrosomonas spp. by synthetic chemical inhibitors. Two enzymatic pathways are involved in N. europaea for nitrite formation. Ammonia is first converted to hydroxylamine by AMO, and hydroxylamine is then converted into nitrite by hydroxylamine oxidoreductase (HAO). Ammonia monooxygenase has a broad substrate range for catalytic oxidation, and the inhibitory effect of synthetic nitrification inhibitors is the result of competition for the active site of AMO, but they do not inhibit HAO (Murakami et al., 1995; Arp & Stein, 2003). In our study, a known synthetic nitrification inhibitor, DCD, was found to inhibit the AMO pathway by more than 80%, and it does not have any inhibition function on HAO (Table 1). This confirms that it is specific to the AMO enzymatic pathway, as previously reported by McCarty (1999). In contrast to synthetic inhibitors such as DCD, root exudates inhibited the function of both the enzymes (Table 1). Root exudates of B. humidicola and L. racemosus have also been reported to inhibit the function of both the enzymes (Subbarao et al., 2007b,c); however, the chemical identity of these BNI compounds has not yet been resolved. Methyl 3-(4-hydroxyphenyl) propionate, however, did not inhibit the function of both enzymes. The isolated compound, methyl 3-(4-hydroxyphenyl) propionate, has a similar mode of inhibitory action to the synthetic inhibitor DCD, suggesting that other compounds that have not yet been purified must be responsible for inhibition of the HAO enzyme.
How might be a stimulus for BNI compound synthesis and/or release?
The presence of has a direct influence on the release of the methyl 3-(4-hydroxyphenyl) propionate from roots (Fig. 6), and on the release of other unidentified compounds (Fig. 2). The isolated BNI compound is phenolic in nature and might be synthesized through a modified phenyl propanoid metabolism pathway. This can be speculated because the structure of the compound has similarity with methyl p-coumarate, only it does not have double bond in the side-chain of position 2. These types of compound are derived mainly from L-phenylalanine in the initial step of phenyl propanoid synthesis mediated by phenylalanine ammonia-lyase (PAL, EC 184.108.40.206) (Jozef et al., 2007). Ammonium is eliminated along with the pro-S hydrogen atom from L-phenylalanine to form secondary phenylpropanoid products in the reaction catalysed by PAL. Ammonium ion released by the PAL enzyme can be assimilated via the GS/GOGAT system (Razal et al., 1996). It has been reported that low PAL activity accumulates high amounts of endogenous ammonia, and that PAL activity and endogenous concentrations of ammonia are known to affect each other in a plant cell (Maldonado et al., 2002). There is a possibility that endogenous may be a regulator in adjusting PAL activity or other related enzymes, which could lead on to other metabolic changes, including the production of BNI compounds, as well as giving a signal to the membrane for BNI compound release. We now know that assimilation of and associated metabolic processes have a definite contribution to the release of BNI compounds from roots; however, little is known about the in-depth induction mechanisms of on the release of BNI compounds from sorghum roots. Future studies will be required to understand this process.
The release of nitrification inhibitors from the roots of crop and pasture species may be a useful tool for decreasing the emission of nitrogenous greenhouse gasses from soil and reducing off-farm impacts associated with nitrate leaching. The isolation of a compound from the root exudates of sorghum that is responsible for nitrification inhibition has many implications for understanding the BNI phenomenon. In particular, it will allow for more detailed physiological characterization of BNI compound release mechanisms with regard to the effects of assimilation. Our ongoing research is aimed at further characterization of the BNI phenomenon in sorghum.
We thank Dr Matthias Wissuwa, Takeshi Watanabe and Dr Juan Pariasca Tanaka for their valuable suggestions during this research. We also thank Ms Ikuko Maeda for technical assistance in NMR analysis. This work was supported by a Grant-in-Aid for Scientific Research from the Ministry of Agriculture, Forestry and Fisheries of Japan.