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The relationship between rhizosphere nitrification and nitrogen-use efficiency in rice plants


Plant, Cell and Environment (2008) 31, 73–85

doi: 10.1111/j.1365-3040.2007.01737.x

The relationship between rhizosphere nitri?cation and nitrogen-use efficiency in rice

plants
YI LIN LI1,2, XIAO RONG FAN1 & QI RONG SHEN1
1 Nanjing Agricultural University, Nanjing, 210095, Jiangsu Province, People’s Republic of China and 2Institute of Soil Science, Chinese Academy of Sciences, Nanjing 210008, Jiangsu Province, People’s Republic of China

ABSTRACT
Two different rice cultivars, Yangdao 6 [Indica rice cultivar with high nitrogen-use ef?ciency (NUE)] and Nongken 57 (Japonica rice cultivar with low NUE) were used to study the relationship between NUE and nitri?cation activity in the rice seedling rhizosphere soil using a rhizobox with three compartments, and a soil-slicing method. The roots of both rice cultivars developed aerenchyma tissue [expressed as percentage porosity of root (POR)], but Yangdao 6 showed better development than Nongken 57. This root morphology change results in more radial oxygen loss (ROL) into the rhizosphere. Leaf glutamine synthetase activity (GSA) and nitrate (NO3-) reductase activity (NRA) of Yangdao 6 were signi?cantly higher than those of Nongken 57, while there was no signi?cant difference in root NRA between the cultivars. The nitri?cation activities were maximal in rhizosphere soil, followed by those in the bulk soil and the root surface for both cultivars. The rhizosphere nitri?cation activity, NO3- concentration and abundance of ammonia-oxidizing bacteria (AOB) associated with Yangdao 6 were always higher than those of Nongken 57. Therefore, we conclude that the greater N uptake by Yangdao 6 when compared to Nongken 57 can be mainly attributed to the bigger capacity for nitri?cation in Yangdao 6. Key-words: aerenchyma; radial oxygen loss. ammonia-oxidizing bacteria;

INTRODUCTION
Among the macronutrients required by plants, nitrogen (N) is consumed in the greatest quantities as it is required for the biosynthesis of amino acids and secondary metabolites. N availability, as nitrate (NO3-) or ammonium (NH4+), varies between ecosystems but it usually limits plant growth (Crawford & Glass 1998; Escobar, Geisler & Rasmusson 2006). In contrast to most agricultural soils, where NO3- is the predominant N form, the ?ooded conditions in paddy soil greatly restrain nitri?cation, the microbial formation of NO3- (Arth, Frenzel & Conrad 1998) and therefore NH4+ is the main form of N available to the young rice plants (Wang
Correspondence: Q. R. Shen. Fax: +86 25 84432420; e-mail: shenqirong@njau.edu.cn ? 2007 The Authors Journal compilation ? 2007 Blackwell Publishing Ltd

et al. 1993; Arth et al. 1998; Kronzucker et al. 1998). Consequently, rice absorbs much more NH4+ compared with NO3-, and NH4+ has received more attention as a N source for the crop (Wang et al. 1993). Many wetland plants such as rice respond to oxygen de?ciency by forming numerous adventitious roots containing aerenchyma that enhance metabolic ef?ciency and facilitate internal oxygen transport (Armstrong 1979; Armstrong et al. 1991; Jackson & Armstrong 1999). Aerenchyma refers to the tissue comprising a high proportion of gas-?lled interconnected spaces or lacunae, extending from below the ground up into the stems and leaves, making an internal aeration system (Drew, He & Morgan 2000). A welldeveloped aerenchyma system bene?ts plants for several reasons. Firstly, aerenchyma decreases the volume of respiring tissues. Secondly, aerenchyma lacunae can form an unobstructed pathway for atmospheric or photosynthetic O2 transport from shoots to root tips, supporting respiration by submerged tissues (Teal & Kanwisher 1966;Arenovski & Howes 1992). Finally, and most importantly, an aerenchyma system promotes radial oxygen loss (ROL) from the roots to the rhizosphere to restrict the accumulation of phytotoxic compounds (i.e. Fe2+, Mn2+, H2S) and maintain aerobic microbial processes, such as the conversion of NH4+ into NO3- by nitrifying bacteria (Kludze, DeLaune & Patrick 1993). In rice, the adventitious roots develop from stem nodes below ground level, and this is the main rooting system (Colmer et al. 1998). Growth in stagnant deoxygenated nutrient solution enhanced both porosity of root (POR) and development of a barrier to ROL in adventitious roots of rice (Colmer et al. 1998; Colmer 2003). The reduction in radial leakage of O2 from the basal regions of the root is of adaptative value because this would enhance the longitudinal diffusion of O2 to the root apex, which in turn, enables greater penetration of roots into anaerobic soils (Armstrong 1979). In addition to reducing ROL, one other bene?t of the physical barrier may be to decrease the in?ux of potentially toxic substances (e.g. Fe2+ and Mn2+) from the waterlogged soil into the roots. However, one possible drawback of the barrier may be an inhibition of nutrient absorption by the basal regions of the roots (Colmer & Bloom 1998). Ghosh & Kashyap (2003) studied three different rice types growing in an irrigated rice ecosystem, and found that the rice cultivars differed signi?cantly in aerenchyma tissue
73

74 Y. L. Li et al. differentiation (expressed as POR), resulting in different degrees of aerobic conditions in their rhizosphere, and different radial O2 supplies from the roots. This affected the abundance and activities of ammonia-oxidizing bacteria (AOB) living in the rhizosphere, which in turn, directly in?uence the rhizosphere nitri?cation activity. NO3-–N from nitri?cation on the surface of rice roots, in the rhizosphere and in the oxidized surface soil layer is very important for the N nutrition of rice plants (Kronzucker et al. 1999, 2000). Many reports from hydroponic experiments have indicated that rice growth, yield, net N acquisition, N translocation to the shoot and nitrogen-use ef?ciency (NUE) are superior in a mixed NH4+ and NO3supply when compared with only NH4+ (Youngdahl et al. 1982; Raman, Spanswick & Walker 1995; Kronzucker et al. 1999). In our previous hydroponic study, different rice cultivars showed different NUE (expressed as the plant dry biomass relative to the plant N accumulation amount) (Koutroubas & Ntanos 2003) in a mixed NH4+ and NO3supply (with different concentration proportions) in the seedling stage (Duan et al. 2007). Unfortunately, the soil conditions are much more complicated than those found in hydroponic culture, and the contents of NH4+ and NO3- are hard to measure and maintain in soil experiments. Therefore, we designed a rhizobox system to enable the quanti?cation of nitri?cation in root surface and rhizosphere under paddy conditions. It enabled us to study the relationship between NUE and rhizosphere nitri?cation of different rice cultivars. In an earlier hydroponic culture, two rice cultivars,Yangdao 6 (an Indica rice cultivar) showed greater NUE than Nongken 57 (a Japonica rice cultivar) at both 1 and 10 mmol L-1 NO3- supply and with a mixed NH4+ and NO3- supply (1.25 mmol L-1 NH4NO3). For example, the NUE of Yangdao 6 and Nongken 57 (both 28 days old) were 130 and 90 mg mg-1 N, respectively, when they were fed with10 mmol L-1 NO3- supply (Fan et al. 2007). These two cultivars were chosen for the further research of the relationship between NUE and rhizosphere nitri?cation. Furthermore, we investigated the rice nitrate reductase activity (NRA) and glutamine synthetase activity (GSA), which are likely to be closely related to rice N nutrition and NUE. soil was air-dried and sieved (1 mm pore size), and urea (30 mg N kg-1) and KH2PO4 (93 mg kg-1) were mixed into it. Each rhizobox was ?lled with 600 g of soil, and three seedlings were planted in the middle compartment. The rhizobox was ?ooded with water to 1 cm depth after germination, and was maintained at this level throughout the growth of the rice plants. Three replicate rhizoboxes were used, and plants were harvested 44, 51 and 58 d after germination. All plant tissues were sampled 4 h after the start of the light period to assay biomass, tissue N, NRA, GSA, POR and ROL. Tissue N analysis of the rice plants was then used to determine the amounts accumulated by the plants.

Rhizobox design and soil sampling
A glass rhizobox was designed to separate the root surface soil, rhizosphere soil and bulk soil. The detail of this rhizobox was described before (see Fig. 1) (Li, Zhou & Gao 1994). Brie?y, the rhizobox was divided into three compartments with two nylon nets (30 mm mesh size), which stops roots but not water and nutrients. Rice cultivars were planted in the middle compartment, which was totally ?lled with rice roots (preliminary experiments showed root development was not limited) so that soil in this compartment was de?ned as root surface soil. The inside surfaces of the nylon nets were fully covered by roots, and thus, the soil

(a)

Rice roots Left 5 cm

Rice plant 5 cm

Right Nylon net

8 cm 12 cm (b)

8c

m

MATERIALS AND METHODS Plant materials and growth condition
Two rice cultivars (Oryza sativa L.), Yangdao 6 (Indica rice cultivar) and Nongken 57 (Japonica rice cultivar) that differ in their NUE and are very popular for cultivation in Jiangsu province, China, were used in this experiment (Fan et al. 2007). Rice plants were grown in soil in a greenhouse at 28 °C with a 16 h photoperiod. Paddy soil was collected from the farm of Jiangsu Agricultural Science Academy, and the basic properties were as follows: clay 26.4%, organic C 28.9 g kg-1, total N 1.2 g kg-1, NH4+–N 7.4 mg kg-1, NO3-–N 1.4 mg kg-1 and pH (water:soil, 10:1) 6.12. A large proportion of the soil N must be present in an organic form. The

Figure 1. Diagram of the rhizobox (a) and root surface pro?le
for Yangdao 6 and Nongken 57 growing 44 d after sowing (b).

? 2007 The Authors Journal compilation ? 2007 Blackwell Publishing Ltd, Plant, Cell and Environment, 31, 73–85

Relationship between nitri?cation and rice nitrogen-use efficiency 75 on the outer net surfaces was de?ned as rhizosphere soil (0–4 mm away from the nylon net) according to Yang, Kirk & Dobbermann (2005) and Carrasco et al. (2006). Both soils were completely separated in the rhizobox. Soil samples from the inside compartment were collected by slicing the soil into 4 mm thick vertical pro?les, and these included two types: bulk soil (>4 mm away from the nylon net) and rhizosphere soil (Shi et al. 2002). Root surface soil from the middle compartment was separated from the roots using tweezers. All soil samples were taken at the same time as the plant sampling in order to assay for NH4+, NO3-, nitri?cation activity and abundance of AOB. Soil samples were extracted with 2 mol L-1 KCl (soil:solution ratio 1:10), and the extracts were measured for NH4+ and NO3- using a continuous-?ow auto-analyser (model Autoanalyzer 3; Bran + Luebbe, Hamburg Germany). measurement was removed into 1 mL extraction buffer containing 20 mmol L-1 ethylenediaminetetraacetic acid (EDTA) and incubated at 25 °C for 15 min; another 1 mL extract for NRAact measurement, without pre-incubation. Both extracts were then added with 1 mL reaction medium (50 mmol L-1 HEPES–KOH pH 7.6, 10 mmol L-1 FAD, 1 mmol L-1 DTT, 5 mmol L-1 KNO3, 0.2 mmol L-1 NADH and either 20 mmol L-1 EDTA for RNAmax or 20 mmol L-1 MgCl2 for NRAact). The reactions were terminated after 5 min by the addition of 125 mL zinc acetate solution (0.5 mol L-1) at room temperature. The other 1 mL extract was taken in the reaction buffer without NADH in order to measure the 0 time NO2- concentration in the tissue. After a short centrifugation (4 °C, 5 min, 16 000 g), 10 mL phenazine methosulphate (PMS) was added to 950 mL of the supernatant to oxidize excess NADH. After 20 min in the dark, the NO2- formed was measured colorimetrically by adding 750 mL of 1% sulphanilamid in 3 mol L-1 HCl, and 750 mL of 0.02% N-naphthyl-ethylenediamine hydrochloride, and absorption was determined at 546 nm. For each series, blanks (without supernatant) were included. The calibration curve was prepared at the ?nal concentrations of NO2- from 0 to 20 mmol L-1. The percentage value of NRAact (NRAmax = 100%) then gives the activation state of NR. For GSA assay, the ground samples were extracted with 0.5 mmol L-1 EDTA and 50 mmol L-1 K2SO4. The homogenates were centrifuged at 20 000 g for 20 min. Then, 1.2 mL of the clear ?ltrate was added to a centrifuge tube, followed by 0.6 mL imidazole–HCl (pH 7.0; 0.25 mol L-1), 0.4 mL sodium glutamate (pH 7.0; 0.3 mol L-1), 0.4 mL ATP–Na (pH 7.0; 15 mmol L-1), 0.2 mL MgSO4 (0.5 mol L-1) and 0.2 mL hydroxylamine (1 mol L-1). After the mixture was incubated at 25 °C for 20 min, the reaction was terminated by adding 0.8 mL acidic FeCl3 [24% (w/v) trichloroacetic acid and 10% (w/v) FeCl3, in 18% HCl]. The production of g-glutamylhydroxamate was measured with a spectrophotometer at 540 nm. One unit of GSA was de?ned as the enzyme catalyzing the formation of 1 mmol g-glutamylhydroxamate per minute at 25 °C (Zhang et al. 1997).

Biomass and total N assay of plants
The rice tissues were separated into shoots and roots, and heated at 105 °C for half an hour to kill the enzyme activities. Then, the samples were oven-dried at 70 °C for 48 h until their biomass was constant. The dried plant material was ground and digested for total N determination by the Kjeldahl method. A 5 mL aliquot from the 100 mL digested sample was then analysed using a continuous-?ow autoanalyser (model Autoanalyzer 3; Bran+Luebbe). NUE was expressed as the whole-plant dry biomass relative to N accumulation (Koutroubas & Ntanos 2003). Relative growth rate (RGR) was calculated from dry weight (DW) data obtained from 44, 51 and 58 d after germination, according to Hunt’s (1978) equation: RGR = (ln DW2 - ln DW1)/t2 - t1.

NRA and GSA of plants
Maximum NRA (NRAmax) and active NRA (NRAact) were both measured. The NRAact is measured in the presence of excess Mg2+, and is thought to be the real NRA in situ in the leaf or root tissue, the NR activation state that gives NRAact as percent of NRAmax. In addition, root nitrite (NO2-) concentrations (0 time-point) were measured to make sure that all NO2- was formed during the NR reaction and not carried over from the root tissue. Plant materials were analysed for NRA as described previously (Abd-El Baki et al. 2000) with minor modi?cations. Leaves or roots (1 g fresh weight) were ground with a mortar and pestle in liquid N. Four millilitres of extraction buffer, comprising 100 mmol L-1 N-2-hydroxyethylpiperazine-N′-2-ethanesulphonic acid (HEPES)–KOH (pH 7.6), 20 mmol L-1 MgCl2, 10 mmol L-1 ?avin adenine dinucleotide (FAD), 5 mmol L-1 dl-dithiothreitol (DTT), plus the protease inhibitors leupeptin (10 mmol L-1), Pefabloc (1 mmol L-1) and phenylmethylsulphonyl ?uoride (PMSF) (0.2 mmol L-1), and in addition 1% polyvinylpyrrolidone (PVP) and 0.05% casein were added to the frozen powder and ground continuously until thawing. The supernatant was then centrifuged for 15 min (4 °C, 16 000 g). Then, 1 mL extract for NRAmax

ROL assay of roots
The rate of O2 released through roots was estimated colorimetrically with Ti3+ citrate solution described in detail by Kludze et al. (1993). The Ti3+ citrate solution was prepared under a N2 atmosphere according to the method of Zehnder & Wuhrmann (1976).Three hundred millilitres of deoxygenated water was added to 17.7 g of sodium citrate to give a ?nal concentration of 0.2 mol L-1 sodium citrate solution. Thirty millilitres of 1.16 mol L-1 TiCl3 was then added to the sodium citrate solution, and the pH was adjusted to 5.6 by adding saturated Na2CO3. Forty millilitres of 10% Hoagland nutrient solution was poured into 50 mL test tubes, and N2 gas was bubbled through them for 30 min to remove any dissolved O2. Plant samples were washed, coated with para?lm at the bases and inserted into the tubes. Immediately

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76 Y. L. Li et al.
Means followed by different letters in the same column indicate signi?cant difference at P < 0.05 level by least signi?cant difference test. Cultivar data were pooled n = 3, except for adventitious root diameter n = 18, and was measured at the middle of every root (the root selected were at the average length of the total root).

Table 1. Maximal length, numbers and diameters of rice adventitious root; plant biomass [dry weight (DW)]; root/shoot ratio; and relative growth rate (RGR) of both Yangdao 6 and Nongken 57, 44 d, 51 d and 58 d after germination

after this, the solution surfaces were layered with 2 cm of paraf?n oil to prevent atmospheric O2 contamination. A 5 mL aliquot of Ti3+ citrate was then injected into each test tube with a syringe. Control treatments had no plants. Six hours after the introduction of roots into Ti3+ citrate solution, the test tubes were gently shaken, and the solution was sampled with a syringe through rubber tubing. Measurements of the absorbance of the partly oxidized Ti3+ citrate solution were made at 527 nm using a spectrophotometer. Released O2 was determined by extrapolation of the measured absorbance to a standard curve previously obtained from a dilution series of the Ti3+ citrate solution being used. Total ROL per plant and per root DW were then calculated with the formula (Kludze et al. 1993), respectively: ROL = c(y - z), where c is the initial volume of Ti3+ citrate added to each test tube (L), y is the concentration of Ti3+ citrate solution of control (without plants) (mmol Ti3+ L-1) and z is the concentration of Ti3+ citrate solution after 6 h with plants (mmol Ti3+ L-1).

Plant

0.08a 0.06a 0.09a 0.05a 0.06a 0.10a 0.56b 0.34c 0.33b 0.24c 0.23b 0.10d 47b 32de Yangdao 6 Nongken 57 51 156b 161b 1.14b 0.69d 0.69b 0.42d

Shoot

RGR (g g-1 d-1)

Root

– –

Root/Shoot ratio

Plant

0.33c 0.22d

Shoot

Root diameter (mm)

For this assay, 0.4–0.6 g of fresh adventitious roots were sampled according to published methods (Jensen et al. 1969; Kludze et al. 1993; Kim et al. 1999). POR was determined by POR = [(Pgr - Pr)/(r + P - Pr)] ? 100, where POR is the root porosity (%), Pgr is the mass of pycnometer with water and ground roots (g), Pr is the mass of pycnometer with water and roots (g), r is the mass of roots (g) and P is the mass of pycnometer with water (g).

Root

0.15c 0.05e

The structural assay of rice root aerenchyma by scanning electron microscope (SEM)
The newly formed adventitious roots, approximately 30 mm long, were collected from both cultivars 51 d after planting. Fresh samples were cut into 5 mm pieces using a razor blade for SEM preparation using a method that was described previously (David & Olga 2000). Then, the root samples were viewed and photographed on a SEM (model-3000N; Hitachi, Inc., Tokyo Japan).

Numbers of adventitious root (per plant)

35cd 26e

Nitri?cation activity assay of soil
NO2- in soil samples was determined after 24 h incubation with NaClO3 at 25 °C (Berg & Rosswall 1985). The procedure is brie?y described as follows. Each soil sample (5 g) was shaken with 2.5 mL of sodium chlorate (75 mmol L-1) at 170 rpm on an orbital shaker (model HYG-C; Taicang Laboratorial Equipment Factory, Taicang, China) for 30 min and then incubated for a further 24 h at 25 °C to prevent NO2- conversion into nitrate. After incubation, NO2- was extracted from the soil samples into a total volume of 15 mL using two solutions (?rst, 5 mL deionized H2O and then 10 mL of 2 mol L-1 KCl) by shaking in a 170 rpm speed for 30 min on an orbital shaker as described earlier. All samples were ?ltered before the NO2- assay as

Maximal root length (mm)

152b 158b

Sampling dates (d)

Yangdao 6 Nongken 57

? 2007 The Authors Journal compilation ? 2007 Blackwell Publishing Ltd, Plant, Cell and Environment, 31, 73–85

44

58

Yangdao 6 Nongken 57

Cultivar

185a 171ab

59a 41bc

1.64a 1.00bc

0.85c 0.53e

0.29a 0.17c

0.49a 0.36b

0.18c 0.17c

POR assay of plants

Biomass (g per plant)

0.77a 0.53b

0.59bc 0.47cd

0.82a 0.29e

0.03a 0.08a

– –

0.05a 0.06a

0.05a 0.06a

– –

Relationship between nitri?cation and rice nitrogen-use efficiency 77
Table 2. Mean total N accumulations, net N accumulation rates and nitrogen-use ef?ciency (NUE) of Yangdao 6 and Nongken 57 rice cultivars at different sampling dates
Total N accumulation (mg N per plant) Sampling dates (d) 44 51 58 Cultivar Yangdao 6 Nongken 57 Yangdao 6 Nongken 57 Yangdao 6 Nongken 57 Root 1.38e 0.40f 3.76c 2.05d 6.38a 4.27b Shoot 2.25e 2.49e 6.00c 5.35d 11.5a 9.74b Plant 3.63e 2.90f 9.76c 7.40d 17.9a 14.0b Net N accumulation rate (mg N g-1 DW d-1) – – 0.92b 1.26a 0.84bc 0.64c

NUE (mg mg-1 N) Root 109b 126a 61.2c 48.8d 45.0d 39.9d Shoot 81.7a 69.6b 55.7c 44.9d 42.2de 37.4e Plant 92.1a 77.3b 57.8c 46.0d 43.2de 38.1e

Means followed by different letters in the same column indicate signi?cant difference at P < 0.05 level by least signi?cant difference test. Cultivar data were pooled n = 3. DW, dry weight.

described previously for the NRA measurement. For a control measurement, soil samples were extracted as previously described after incubation with NaClO3 at -20 °C.

AOB assay of soil
The AOB abundance was counted by the most probable number (MPN) method, which is based on scoring the presence or absence of bacteria using an extinction dilution in which replicate tubes of a special culture medium are inoculated with 1 mL aliquots of a serial dilution. One prerequisite of the MPN method is that the microorganisms that are to be enumerated selectively must be able to generate some characteristic metabolite or product that can be detected easily by a special reagent.Because the NO2- produced by theAOB can be sensitively detected by the Griess–Illosvay reagent, this was used in this experiment. The culture medium contained (NH4)2SO4 (0.5 g L-1), NaCl (0.3 g L-1), FeSO4 · 7H2O (0.03 g L-1), K2HPO4 (1 g L-1), MgSO4 · 7H2O (0.3 g L-1) and CaCO3 (7.5 g L-1), and was adjusted to pH 7.8. The test cultures were incubated at a constant temperature of 25 °C for 14 d (Li, Yu & He 1996).

The numbers and diameters of adventitious roots associated with Yangdao 6 were always signi?cantly higher than those of Nongken 57, and increased with the incubation time signi?cantly (Table 1). This means the root system for Yangdao 6 developed better than that of Nongken 57. However, the maximal adventitious root length of both rice cultivars showed no signi?cant difference.

Rice total N accumulation and NUE
The total N accumulations in plants of both Yangdao 6 and Nongken 57 signi?cantly increased from 2.9 to 17.9 mg N per plant as the development of the rice plants proceeded (Table 2). Interestingly, the increases in the total N accumulated were greater than those measured for dry matter (Table 1) for both cultivars during the whole experiment. An exception was that no difference in shoot total N for the two cultivars was found at day 44. Yangdao 6 showed more N accumulation in all tissues than Nongken 57 at all three sampling stages (Table 2). In more detail, we found out that Yangdao 6 accumulated 25.2, 31.9 and 28.0% more N than Nongken 57, 44 d, 51 d and 58 d after sowing. The net N accumulation rates of Yangdao 6 were lower than those of Nongken 57 51 d after sowing, but there was no signi?cant difference for both cultivars 58 d after sowing (Table 2). By contrast, the total N and NUE in plants of both cultivars signi?cantly decreased as the plants grew; this was especially the situation for root NUE. Yangdao 6 still maintained higher NUE than Nongken 57 at the three sampling stages except for roots at day 44 (see Table 2).

RESULTS Rice plant growth
The root and shoot growths of the two cultivars were different. The root/shoot ratio of Yangdao 6 was signi?cantly higher than that of Nongken 57 at the three sampling stages (Table 1). During the whole growth periods, Yangdao 6 always grew signi?cantly better than Nongken 57 except at day 44 when there were no differences in the shoots. For example, the shoot DWs of Yangdao 6 at days 44, 51 and 58 were as 1.06, 1.39 and 1.34 times greater than Nongken 57; while the root DWs of Yangdao 6 at days 44, 51 and 58 were 3.00, 2.30 and 1.69 times greater than Nongken 57. The RGR of both rice cultivars showed no signi?cant difference (Table 1).

NRA and GSA in rice plants
In leaves, Yangdao 6 NRAmax increased more than that of Nongken 57 as the plants developed, and, except at day 44, the value of Yangdao 6 NRAmax was always higher than that of Nongken 57 (Table 3). However, in roots both cultivars showed increases from 44 to 51 d, but decreased from 51 to

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78 Y. L. Li et al.

50

Root

25.3ab 21.0bc

27.1a 19.2c

17.0c 16.7c

GSA (mmol g-1 FW h-1)

Table 3. Mean maximum NRA (NRAmax), active nitrate reductase activity (NRAact), activation state and glutamine synthetase activity (GSA) in leaves and roots of Yangdao 6 and Nongken 57 cultivars at different sampling dates

40

Yangdao 6 Nongken 57 b cd d cd

a

POR (%)

30

c

Means followed by different letters in the same column indicate signi?cant difference at P < 0.05 level by least signi?cant difference test. Cultivar data were pooled n = 3. FW, fresh weight.

Leaf

192ab 148c

225a 154bc

211a 165bc

20

10

0.008a 0.005b

Root

0.006b 0.006b

0.004c 0.002c

0 44 51 58

NO2(mmol g-1 FW)

Days after sowing

0.008bc 0.005c

0.012a 0.008bc

Leaf

0.008bc 0.008b

Figure 2. Porosity of root (POR) measured for Yangdao 6 and
Nongken 57 rice cultivars at different sampling dates. Data represent means of three replicates. Bars indicate SD. For each datum, bars with different letters indicate signi?cant differences (P < 0.05).

65.9ab 68.7ab

Root

67.5ab 75.1a

55.0bc 41.6c

NRAmax (mmol g-1 FW h-1)

58 d and no signi?cant difference was shown between the two cultivars. But for NRAact, both cultivars showed an increase in leaves from 44 to 51 d, but a decrease from 51 to 58 d (Table 3). While in root, the NRAact of both cultivars increased slightly at three sampling times. The NRAact in Yangdao 6 leaves was always larger than that in Nongken 57, but not signi?cantly different in roots of both cultivars. Another important result was that in both cultivars, the values of NRA in leaves could be 5- to 20-fold higher than in roots. The NR activation state of Yangdao 6 leaves was about 50% at days 44 and 51, but decreased to 28.3% at day 58. For Nongken 57 leaves, the activation state was around 26% at days 41 and 58, but 45.8% at day 51 (Table 3). There was no signi?cant difference in NR activation between the roots of both cultivars. However, there were no changes of GSA in leaves and roots of both cultivars in three sampling stages; even GSA in Yangdao 6 leaves showed some decrease during the stages, but not signi?cant (Table 3). While for the comparison of GSA in two cultivars, the same as NRA, it showed greater in Yangdao 6 leaves than in Nongken 57 leaves at each sampling stage, and there was no difference in roots.

Activation state (%)

50.2a 26.0b

Leaf

Root

0.08a 0.06ab 0.77a 0.41c 1.54b 0.88c 0.14a 0.14a

0.04b 0.04b

NRAact (mmol g-1 FW h-1)

0.49bc 0.21d

Root

Leaf

0.97c 0.81c

Leaf

2.10a 1.27b

0.11b 0.10b

0.07c 0.06c

0.60b 0.32cd

0.07a 0.07a

28.3b 26.4b

51.7a 45.8a

ROL, rice POR and aerenchyma development
As the plants grow, rice POR (Fig. 2) and total ROL per plant (Table 4) increased and were always signi?cantly higher for Yangdao 6 than for Nongken 57. These differences between two cultivars became greater at later sampling times (Fig. 2 and Table 4). However, the ROL per DW showed that Yangdao 6 increased but Nongken 57 decreased as growing. At days 44 and 51, the ROL per DW of Nongken 57 was signi?cantly higher than that of Yangdao 6, but the reverse happened at day 58. This suggested that at days 44 and 51, the root size decided the amount of total ROL and at day 58, both root size and ROL

Yangdao 6 Nongken 57

Yangdao 6 Nongken 57 51

Sampling dates (d)

44

58

Yangdao 6 Nongken 57

Cultivar

? 2007 The Authors Journal compilation ? 2007 Blackwell Publishing Ltd, Plant, Cell and Environment, 31, 73–85

Relationship between nitri?cation and rice nitrogen-use efficiency 79
Table 4. Mean radial oxygen loss (ROL) measured for Yangdao 6 and Nongken 57 rice cultivars at different sampling dates

ROL (mmol h-1) Sampling dates (d) 44 51 58 Total release (per plant) 0.59de 0.41e 1.24b 0.77d 2.10a 0.97c Root dry weight (DW) basis (g-1) 3.97c 8.20a 5.45b 7.66a 7.38a 5.75b

Cultivar Yangdao 6 Nongken 57 Yangdao 6 Nongken 57 Yangdao 6 Nongken 57

Means followed by different letters in the same column indicate signi?cant difference at P < 0.05 level by least signi?cant difference test. Cultivar data were pooled n = 3.

per unit of root contributed to the total ROL. The plant ROL rates were signi?cantly and positively correlated with POR (r = 0.895; P < 0.01; Table 5). The total ROL and POR results indicated that Yangdao 6 could transport more O2 into the root, and therefore, more NO3- could be transformed from NH4+ around the root surface than Nongken 57. To investigate this idea more closely, we looked at the detailed root anatomy. The SEM pictures of root aerenchyma showed that in the range of 0–5 mm from the newly formed adventitious root tip, there were few air spaces, and the cortical cells were arranged in columns or ?les of cells that extended from the endodermis to the epidermal layers in each cultivar (Fig. 3a,d). By contrast, in the range 5–10 mm from the root tip, signi?cant gas spaces were observed in Yangdao 6, but not in Nongken 57 (Fig. 3b,e). At 15 mm from the root tip, well-developed gas spaces were found in both rice cultivars (Fig. 3c,f). These SEM results also showed that the gas spaces (Yangdao 6) in the cortex were separated by bridges of cells spanning the space between the stele and epidermis, giving the appearance of wheel spokes (Fig. 3c). There were two distinct patterns for the origins of the lacunae in each rice cultivar: radial lysigeny in Yangdao 6 and tangential lysigeny in Nongken 57 according to the de?nitions of Seago et al. (2005). The availability of a route for gas diffusion through roots, and therefore, the amount of aerenchyma can be measured by the percentage POR (Maricle & Lee 2002). Therefore, we could clearly conclude that the aerenchyma tissue of Yangdao 6 was better developed, and therefore, more ef?cient for gas diffusion than that of Nongken 57 (Figs 2 & 3). This result establishes that Yangdao 6 has the capacity to conduct more O2 by diffusion to the root tips than Nongken 57 under paddy soil conditions.

increased with distance from the rice roots 44, 51 and 58 d after sowing. A very distinct depleted zone of NH4+ was clearly found. For example, among the three sampling dates, the average NH4+–N concentrations of the soils in the root surface of Yangdao 6 and Nongken 57 were 3.9 and 2.4 mg kg-1 soil, respectively; in the rhizosphere soil, they were 4.7 and 4.5 mg kg-1 soil, while in the bulk soil they were 12.6 and 11.1 mg kg-1 soil for the two cultivars, respectively. During the different sampling dates, NO3- concentration signi?cantly decreased with growth period for both rice cultivars (Fig. 4b). Almost no NO3- was detected in the last sample, perhaps showing that more NO3- was being taken up by the rice plants at the later growth stage. The NO3-–N concentrations of the soils at different distances from the root surface showed almost no concentration gradient. The NO3- concentrations of the root surface, rhizosphere and bulk soils growing with Yangdao 6 were higher than those of Nongken 57 at day 44, but almost no difference in the later sampling times.

Nitri?cation activity and AOB abundance in root surface, rhizosphere and bulk soil
The nitri?cation activity in the root surface and rhizosphere soil samples associated with both rice cultivars increased with the age of the plants, but there was no signi?cant change in the bulk soil (Fig. 5a). Generally, the nitri?cation activity was highest in the rhizosphere, lower in the bulk soil and lowest in the root surface except at day 58. This difference could be understood as a result from a higher number of AOB in the rhizosphere than in the root surface (see Fig. 5b), which might be attributed to a lower pH caused by acidic root exudates and to a lower NH4+ concentration in the root surface (Fig. 4a) (Arth & Frenzel 2000). And the nitri?cation activity and AOB in Yangdao 6 soil samples were always signi?cantly higher than those for Nongken 57 especially in the root surface. Both the nitri?cation activity and AOB numbers were signi?cantly and positively correlated with rice biomass, total N accumulation, leaf NR and POR (P < 0.01; Table 5).

NH4+–N and NO3-–N concentrations in root surface, rhizosphere and bulk soil
The concentrations of NH4+ associated with both cultivars showed no signi?cant differences over the whole experimental period, except the bulk soil for Nongken 57 which decreased with time (Fig. 4a). The concentrations of NH4+

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80 Y. L. Li et al.
Leaf GSA

Table 5. Correlation matrix pooled for both cultivars between plant biomass, total N accumulation, nitrogen-use ef?ciency (NUE), leaf nitrate reductase activity (NRA), leaf glutamine synthetase activity (GSA), porosity of root (POR), rhizosphere nitri?cation activity and ammonia-oxidizing bacteria (AOB) abundance

0.126NS -0.096NS 0.430NS 0.176NS 0.182NS 0.366NS 0.144NS 0.028NS

DISCUSSION The anatomical differences between the two cultivars contribute to root acclimatization to paddy soil
According to Armstrong (1979), the effectiveness of gas transport is dependent on: (1) the physical resistance to diffusion (which is directly proportional to root length and is inversely proportional to fractional POR); and (2) O2 demand along the diffusion path (which is a function of respiratory uptake and radial O2 leakage from the root to the soil). Oxygen ef?ux from root to soil account for 30–40% of O2 transportation from shoot to root (Armstrong 1979;Armstrong & Beckett 1987). ROL from rice roots estimated by Kludze et al. (1993) showed a signi?cant and positive correlation with POR, which was consistent with the results obtained in our study (Table 5). Furthermore, four genotypes of rice were found to differ in their POR (lowest was 16%, highest was 30%) when grown in stagnant conditions (Colmer et al. 1998). Similar results were found in wheat, and Huang et al. (1994) showed that there were genotypic differences in wheat root aerenchyma development under waterlogged conditions. These authors showed that the genotype with greater waterlogging resistance had better developed aerenchyma. According to Gibberd, Colmer & Cocks (1999), the adventitious roots (mainly newly formed) are the main functional root system in waterlogged soils. As shown in Table 1 and Fig. 2, the root DWs and POR values of Yangdao 6 were signi?cantly higher than those of Nongken 57. In addition, the adventitious root numbers and diameters of Yangdao 6 were signi?cantly higher than those of Nongken 57 (see Table 1), although the maximal root length of both rice cultivars showed no signi?cant differences at all three sampling times (Table 1). A larger root system and greater POR (better-developed aerenchyma) give the rice plants more ef?ciency in O2 transport and more ROL from root to soil via aerenchyma (Table 4). Furthermore, the POR and root systems of both cultivars increased with the development of the rice plants, which gave more ROL into the root surface and rhizosphere soil (see Table 4). These differences in POR and aerenchyma of rice plants resulted in their local niche environment differences in root surface and rhizosphere.

AOB abundance Nitri?cation activity POR Total ROL Leaf NRAmax

0.926** 0.810** -0.523*

0.927** 0.883** -0.577* 0.923**

0.867** 0.733** -0.362NS 0.883** 0.895**

0.932** 0.907** -0.703** 0.854** 0.143NS 0.763**

0.869** 0.859** -0.778** 0.824** 0.027NS 0.790** 0.901**

* and **, signi?cant at P < 0.05 and 0.01, respectively. Cultivar data were pooled n = 6. Data are correlation coef?cients (r values). NS, not signi?cant.

Total N accumulation

NUE

-0.625** -0.814**

0.924**

The importance of rice NO3- nutrition in paddy soil
Although the amounts of NO3- are small in lowland paddy soil, this form of N is of exceptional importance for the growth of rice plants (Kronzucker et al. 1999, 2000). In radiotracer 13N experiments using hydroponically grown rice seedlings, it was found that lowland rice was ef?cient in absorbing and assimilating NO3- when compared with NH4+, and compared with other plant species (Kronzucker et al. 1999, 2000). Using model calculations and experiments, Kirk & Kronzucker (2005) estimated that NO3- uptake accounted for 34% of total N uptake. With an increase in O2 concentration around the rice root, the AOB abundance and

Plant biomass Total N accumulation NUE Leaf NRAmax Total ROL POR Nitri?cation activity AOB abundance

Parameter

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Relationship between nitri?cation and rice nitrogen-use efficiency 81

(a)

(d)

(b)

(e)

(c)

(f)

Figure 3. Scanning electron microscope (SEM) micrographs of transverse sections of roots (51-day-old, newly formed adventitious
roots, approximately 30 mm long) from Yangdao 6 and Nongken 57. Transverse sections of Yangdao 6 at (a) 5 mm, (b) 10 mm and (c) 15 mm behind the root tip; transverse sections of Nongken 57 at (d) 5 mm, (e) 10 mm and (f) 15 mm behind the root tip. Bars = 300 mm (a, b, d, f), 500 mm (c) and 200 mm (e).

activities were increased and the nitri?cation activity was enhanced. Thus, it suggested that more NO3- were transferred from NH4+ around the surface of rice roots, even though the result of NO3- in soils (Fig. 4b) did not agree with

this suggestion. The leaf NRAmax values of both Yangdao 6 and Nongken 57 increased signi?cantly with the plant growth, and NRAact also increased from 44 to 51 d (Table 3). It also suggests that the available NO3- at the root surface is

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82 Y. L. Li et al.
18 16

(a)

NH4+-N (mg kg–1 soil)

14 12 10 8 6 4 2 0 44 51 58

Yangdao 6 Nongken 57

Bulk soil a b ab b ab c

Root surface Rhizosphere soil d d d d de d de f de ef fg g

Yangdao 6 rhizosphere soil were always higher than those in Nongken 57 (Fig. 4b), this provides more available NO3for uptake by the rice plant. With the more available NO3-, the NH4+ assimilation in Yangdao 6 was enhanced more than in Nongken 57 (see GSA results in Table 3). Both POR and the aerenchyma results, plus the NRA and GSA data, suggest that Yangdao 6 could uptake and use more NO3giving greater N accumulation than Nongken 57 in the same type of soil.

44 51 58 Days after sowing

44 51 58

The growth difference of two cultivars was contributed by nitri?cation and ammoni?cation difference in paddy soil
Chemolithotrophic nitri?cation is the main form occurring in terrestrial ecosystems. This is a two-step process, consisting of the conversion of ammonia to NO2-, which is in turn

2.5 2.0 1.5 1.0

(b)

NO3–-N (mg kg–1 soil)

a

Root surface Rhizosphere soil Bulk soil a a b b b

Nitrification activity (mmol kg–1 h–1)

20

Rhizosphere soil a Yangdao 6 b Nongken 57 b Root surface cd g gf h h i c e g ef h de fg

(a)

15

c .5 0.0 44 51 58 44 51 58 44 51 58 Days after sowing e ef fg g c d f g cd fg g

Bulk soil e gh

10

5

Figure 4. Mean (a) NH4+ and (b) NO3- concentrations measured at different distances from the rice root in the ?ooded paddy soil growing Yangdao 6 and Nongken 57 rice cultivars at different sampling dates. Data represent means of three replicates. Bars indicate SD. For each datum, bars with different letters indicate signi?cant differences (P < 0.05).

0 44 51 58 44 51 58 44 51 58

Days after sowing 4 AOB abundance (106 g–1 dry soil) Rhizosphere soil 3 Root surface 2 d de 1 g 0 44 51 58 44 51 58 Days after sowing
Figure 5. Mean nitri?cation activity (a, short-term estimation)
and ammonia-oxidizing bacteria (AOB) abundance [b, most probable number (MPN) method] measured at different distances from the rice root in ?ooded paddy soil growing either Yangdao 6 and Nongken 57 rice cultivars at three sampling dates. Data represent means of three replicates. Bars indicate SD. For each datum, bars with different letters indicate signi?cant differences (P < 0.05).

(b)

increasing, following to the relationship between the NR active state and external NO3- supply that has been shown in hydroponic conditions (Kaiser & Huber 2001). Many researches have indicated that rice growth, yield and N acquisition are enhanced signi?cantly when both NH4+ and NO3- sources are provided simultaneously, when compared with growth on NH4+ alone in solution culture (Cox & Reiseenauer 1973; Youngdahl et al. 1982; Raman et al. 1995). However, this synergistic model of uptake is still poorly understood. When NO3- and NH4+ were provided together at the same total N concentration as in single N species experiments, the uptake and assimilation of NO3were repressed, but those of NH4+ were stimulated to the extent that net N in?ux was doubled compared with plants fed solely with NH4+. As very little free NH4+ is translocated to the shoot in rice (Kronzucker et al. 1998), this indicates that NO3- enhances NH4+ assimilation in some way, possible through the NO3--speci?c induction of additional pathways for NH4+ assimilation (Kronzucker et al. 1999; Britto & Kronzucker 2004). Because the NO3- concentrations in

b c

a c

Bulk soil d d de de de ef

f g g

f g

44 51 58

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Relationship between nitri?cation and rice nitrogen-use efficiency 83 converted to nitrate. These steps are carried out by two different groups of organisms, the AOB and the nitriteoxidizing bacteria (NOB), respectively (Warrington 1878). It is thought that ammonia oxidation is thought to be the rate-limiting step for nitri?cation (De Boer, Klein Gunnewiek & Troelstra 1990). There are no known autotrophic bacteria that can catalyse the production of nitrate from ammonia (Kowalchuk & Stephen 2001). Therefore, rice plants and AOB are expected to compete for the same substrate, that is to say the NH4+ formed from mineralization of organic N or the N fertilizer. The main factors affecting nitri?cation activities in paddy ?elds are NH4+ and O2 concentration in the paddy soil. Some studies have shown that nitri?cation activity is directly related to the NH4+ concentration in ?ooded water (Katyal, Xarter & Vlek 1988; Jensen, Revsbech & Nielsen 1993). When the NH4+ concentration in the ?ooded soil is not the factor to limit the growth of nitri?ers, then an increase of O2 concentration in ?ooded water will signi?cantly enhance nitri?cation activities (Jensen et al. 1993). In our experiments, nitri?cation activities measured in the root surface were much lower than those measured in the rhizosphere and bulk soil (Fig. 5a), mainly because of a sharp decrease in pH at the root surface associated with NH4+ uptake by rice roots (Colmer & Bloom 1998) and the depletion of NH4+ in the root surface (Fig. 4a). In our experiments, the ROL could be 0.41–2.10 mmol h-1 per plant at the root surface. Radial O2 ?uxes decreased with distance from the root tip, with the lowest oxygen release at a distance of about 30–60 mm from the root tip (Gilbert & Frenzel 1998). Revsbech et al. (1999) reported the O2 pro?le in the rhizosphere and root of a 6-week-old rice transplant. The O2 concentration was 75 mmol L-1 at the root surface and nearly zero at 0.4– 0.75 mm from the root surface (Revsbech et al. 1999). The in situ measurements suggested the O2 consumption was rather huge than our expectation. In addition, the POR and root systems of both cultivars increased with the development of the rice plants (Table 1; Fig. 2), which led to much more radial O2 diffusion into the root surface and rhizosphere soils (Table 4), and then the nitri?cation activities increased correspondingly (Fig. 5a). As Yangdao 6 had a larger root system and greater POR than Nongken 57 (Table 1; Fig. 2), this supports the idea that the former has a greater ef?ciency in O2 transport from root to soil via aerenchyma than the latter. Furthermore, there was no signi?cant difference in the NH4+ concentrations of the rhizosphere soil between both the rice cultivars (Fig. 4a). Thus, the main factor limiting nitri?cation activities in paddy ?elds is likely to be soil O2 concentration, which means more AOB surviving around the root surface and rhizophere soil of Yangdao 6 than Nongken 57 (see Fig. 5b), and so the nitri?cation activities of Yangdao 6 soil were higher than those of Nongken 57 throughout the experimental period. The NH4+ in the soil was generated by the conversion of urea and organic N. A large proportion of organic N was present in the soil at the start of the experiment (see Materials and methods). The process of ammoni?cation of the organic N pool is important for the generation of inorganic N forms in the soil. This process is O2 dependent and so the conversion of organic N into NH4+ should be higher in the whole root surface and rhizosphere of Yangdao 6 which effused more O2 than those of Nongken 57 (Table 4).Therefore, more NH4+ was available to Yangdao 6 roots than to Nongken 57 at day 44 (Fig. 4a). The increased O2 availability around the roots of Yangdao 6 would enhance microbial ammoni?cation and nitri?cation. The reason for the NH4+ decrease in the bulk soil of Nongken 57 at day 58 was not clear (Fig. 4a). The rice plant, soil and microorganism are a macrocosm, and every part performs its own task to maintain the whole system. Rice plant roots with differing physiological and morphological characteristics (such as POR, aerenchyma and length density) can determine the radial O2 diffusion abilities. Differing radial O2 supplies from the roots then impact on the abundance and activities of AOB living in the rhizosphere, which in turn directly in?uence the nitri?cation activity in the rhizosphere (Ghosh & Kashyap 2003). Ureafertilized rice plants actually receive a mixture of NH4+ and NO3- with ratios depending on the root-associated nitri?cation activity (Aurelio et al. 2003). Differing nitri?cation activities will lead to varying NO3- concentrations in the rhizosphere available for root absorption and assimilation (Fig. 4b). Finally, rice with higher rhizosphere nitri?cation activities (e.g. Yangdao 6) showed better dry matter accumulation, N accumulation and NUE when compared with plants with relatively lower nitri?cation activities (e.g. Nongken 57; see Tables 1 & 2). We can therefore conclude that nitri?cation and AOB in the rhizosphere are major contributors to rice N nutrition, and are important for growth and NUE in the crop.

ACKNOWLEDGMENTS
This research work was ?nancially supported by the National Nature Science Foundation of China (project nos. 30671234, 40471074). We thank Dr Miller at Rothamsted Research, UK for his suggestions and careful English revision.

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COTTON LECTIN AND THE RHIZOSPHERE COMPETENCE OF TRICHODERMA SPP.
Nitrification and denitr... 暂无评价 9页 1...used in this study were isolated from rhizosphere...The relationship between cotton lectin agglutination ...
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nitrification | rhizosphere | rhizosphere影响因子 | rhizosphere期刊 | rhizosphere 是sci吗 | efficience | coefficience | efficien |