Identification and characterization of improved

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the seedling stage; and (2) to characterize some crucial physio- logical and ...... Hakeem KR, Ahmad A, Iqbal M, Gucel S, Ozturk M. 2011.Nitrogen-efficient.

Annals of Botany 114: 549– 559, 2014 doi:10.1093/aob/mcu135, available online at www.aob.oxfordjournals.org

Identification and characterization of improved nitrogen efficiency in interspecific hybridized new-type Brassica napus Gaili Wang1,2,†, Guangda Ding1,2,†, Ling Li1,2, Hongmei Cai2, Xiangsheng Ye2, Jun Zou1 and Fangsen Xu1,2,* 1

National Key Laboratory of Crop Genetic Improvement, and 2Microelement Research Centre, Huazhong Agricultural University, Wuhan 430070, China * For correspondence. E-mail [email protected] † These authors contributed equally to this work. Received: 16 December 2013 Returned for revision: 3 March 2014 Accepted: 19 May 2014 Published electronically: 2 July 2014

Key words: New-type, Brassica napus, interspecific hybridization, nitrogen efficiency, genetic variation, 15NH+ 4, 15 NO2 3 uptake, glutamine synthetase, nitrate reductase, nitrogen transporter, gene expression, character traits.

IN T RO DU C T IO N Nitrogen (N) is a primary constituent of nucleotides and proteins that are essential for life (Ladha and Reddy, 2003; Guo et al., 2008). The application of chemical N fertilizers resulted in a great increase in global food production in the past. In China, a 3-fold increase in fertilizer-N application in agriculture has contributed to a near 70 % increase in grain production since 1980 (Guo et al., 2010). However, it has been estimated that plants can generally consume less than half of the fertilizer-N applied in soils (Good et al., 2004). The excessive N fertilizers in soils lead to increasingly severe adverse effects to the environment, such as greenhouse gas emission, soil acidification or water eutrophication (Guo et al., 2010; Liu et al., 2013). Furthermore, N is one of the most expensive nutrients to supply, and the commercial N fertilizers represent a major cost in crop production. The cultivation of N-efficient genotypes with better N utilization efficiency (NutE) would enable a reduction in the N fertilization level without drawbacks in yield. NutE has been defined for crops as the grain yield per unit of available N in soils, and/or for other plants used for biomass production as

the fresh matter or dry matter produced per N content (Chardon et al., 2010). Comprehensive genetic variation in traits that contribute to NutE were observed among different species as well as in different varieties of the same species (Svecˇnjak and Rengel, 2006; Dawson et al., 2008; Ikram et al., 2012). It would be of great importance to identify cultivars with significant difference in NutE among plant species and/or genotypes within one species, and for a long-term goal to elucidate the physiological and molecular mechanisms on high N efficiency. The major form of inorganic N is nitrate in aerobic soils, and ammonium in flooded wetland or acidic soils. The utilization of nitrate and ammonium by plants involves several steps including uptake, assimilation, translocation, recycling and remobilization (Xu et al., 2012). The first step is the active transport across the plasma membrane of root epidermal and cortical cells (Garnett et al., 2009). Molecular data have established that multiple gene family members encoding putative transporters are present for both nitrate and ammonium transport systems (Ludewig et al., 2007; Dechorgnat et al., 2011). In arabidopsis, four nitrate transporters, i.e. NRT1.1 (CHL1), NRT1.2,

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† Background and Aims Oilseed rape (Brassica napus) is an important oil crop worldwide. The aim of this study was to identify the variation in nitrogen (N) efficiency of new-type B. napus (genome ArArCcCc) genotypes, and to characterize some critical physiological and molecular mechanisms in response to N limitation. † Methods Two genotypes with contrasting N efficiency (D4-15 and D1-1) were identified from 150 new-type B. napus lines, and hydroponic and pot experiments were conducted. Root morphology, plant biomass, N uptake parameters and seed yield of D4-15 and D1-1 were investigated. Two traditional B. napus (genome AnAnCnCn) genotypes, QY10 and NY7, were also cultivated. Introgression of exotic genomic components in D4-15 and D1-1 was evaluated with molecular markers. † Key Results Large genetic variation existed among traits contributing to the N efficiency of new-type B. napus. Under low N levels at the seedling stage, the N-efficient new-type D4-15 showed higher values than the N-inefficient D1-1 line and the traditional B. napus QY10 and NY7 genotypes with respect to several traits, including root and shoot biomass, root morphology, N accumulation, N utilization efficiency (NutE), N uptake efficiency (NupE), activities of nitrate reductase (NR) and glutamine synthetase (GS), and expression levels of N transporter genes and genes that are involved in N assimilation. Higher yield was produced by the N-efficient D4-15 line compared with the N-inefficient D1-1 at maturity. More exotic genome components were introgressed into the genome of D4-15 (64.97 %) compared with D1-1 (32.23 %). † Conclusions The N-efficient new-type B. napus identified in this research had higher N efficiency (and tolerance to low-N stress) than traditional B. napus cultivars, and thus could have important potential for use in breeding N-efficient B. napus cultivars in the field.

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Wang et al. — Nitrogen efficiency in hybridized new-type Brassica napus M AT E R I A L S A N D M E T H O D S Germplasm collection and experimental design for nitrogen efficiency analysis

A population of 150 new-type Brassica napus (ArArCcCc) inbred lines was derived from interspecific crosses of traditional B. napus (AnAnCnCn), B. rapa (ArAr) and B. carinata (BcBcCcCc), and was used for phenotypic investigation. Ten traditional B. napus lines were employed as a control. The field trial was conducted in paddy soil in Qichun county, Hubei Province in the 2009– 2010 crop season. Two N treatments were used: low N treatment with an application of N 75 kg ha – 1, and normal N treatment with an application of N 150 kg ha – 1. The amount of P, K and B fertilizers applied for each treatment was calculated according to the following nutrient rates: P2O5 90 kg ha – 1, K2O 120 kg ha – 1, borax 15 kg ha – 1. Seed yield and other yield-related traits were investigated for all the materials at the maturing stage. Then, 20 new-type B. napus inbred lines with relatively high N efficiency at the maturing stage were selected for microspore culture. Finally, a total of 46 double haploid (DH) lines were obtained from seven of the 20 new-type B. napus inbred lines, i.e. M9C102-1 (D1), M9C114-1 (D2), M9C035-1 (D3), M9C144-1 (D4), M9C135-1 (D5), M9C144-2 (D6) and M9C039-1 (D8). The 46 new-type B. napus DH lines together with two traditional B. napus genotypes Ningyou 7 (NY7) and Qingyou 10 (QY10) were grown in three independent hydroponic culture experiments to investigate phenotypic variation including biomass production, root morphology, N concentration and accumulation at the seedling stage. Plants were grown in an illuminated culture room at a cycle of 16 h/24 8C day and 8 h/22 8C night and a light intensity of 300 – 320 mmol proton m – 2 s – 1 with a relative humidity of 65– 80 %. The composition of the full-strength nutrient solution was: 0.25 mM CaCl2.2H2O, 2.0 mM KCl, 0.28 mM Na2HPO4.12H2O, 0.64 mM NaH2PO4.H2O, 2.0 mM MgSO4.7H2O, 46.0 mM H3BO3, 9.0 mM MnCl2.4H2O, 0.3 mM CuSO4.5H2O, 0.8 mM ZnSO4.7H2O, 0.1 mM (NH4)6Mo7O24 and 50.0 mM Fe-EDTA. Two N levels, high N (HN; 3.0 mM NH4NO3) and low N (LN; 0.15 mM NH4NO3) were designed with four replicates. The pH of the nutrient solution was controlled at 5.8 – 6.0 adjusted by NaOH or HCl. The solution was replaced every 5 d with quarter- and half-strength solution for the first and second time, and then with full-strength solution. Uniform seeds were germinated on moistened gauze that was fixed to a tray filled with deionized water, and were grown for 6 d at 22–24 8C in the illuminated culture room until the cotyledons were fully developed. Then the uniform seedlings were transferred carefully to HN (3.0 mM NH4NO3) and LN (0.15 mM NH4NO3) solutions, respectively. Plants were harvested after being grown for 24 d, and the phenotypes including dry weight, N concentration in the leaf, and root and root morphology were investigated. Plant materials and hydroponic experiment for characterizing nitrogen efficiency mechanisms

Two DH lines of new-type B. napus, N-efficient genotype D4-15 and N-inefficient genotype D1-1, and two traditional B. napus cultivars NY7 and QY10 were used in the hydroponic experiments. The growth conditions were the same as the above. After germinating for 6 d, uniform seedlings of the four genotypes were grown in HN solution with 3.0 mM NH4NO3

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NRT2.1 and NRT2.2, take up nitrate (Tsay et al., 2011). At least four ammonium transporters (AMT1.1, AMT1.2, AMT1.3 and AMT1.5) can take up ammonium from soil (Yuan et al., 2007). These genes play important roles in N uptake, plant growth and development under N stress conditions (Garnett et al., 2009). Moreover, the expression of nitrate and ammonium transporters is regulated by different N forms and concentrations (Sonoda et al., 2003). After being taken up by the roots, different forms of N are assimilated. The nitrate is reduced to ammonium by nitrate reductase (NR) and nitrite reductase (NiR), and ammonium, taken directly from the soil or converted from nitrate, is then incorporated into amino acids via the glutamine synthetase (GS)/glutamine-2-oxoglutarate aminotransferase (GOGAT) pathway (Bernard and Habash, 2009; Xu et al., 2012). Expression of several genes related to N metabolism is regulated by nitrate and N metabolites, as well as N deficiency (Takahashi et al., 2001; Cai et al., 2009). Brassica napus is widely used as food oil for humans and as animal feed worldwide. A disadvantage of oilseed rape is its high N fertilizer demand and the resulting N surpluses in the environment (Rathke et al., 2006). One way to reduce N surpluses is to develop N-efficient cultivars, as genotypic variation was observed for B. napus under contrasting N supplies (Svecˇnjak and Rengel, 2006; Balint et al., 2008; Kessel et al., 2012). Different traits were used in the selection of N-efficient oilseed rape cultivars, and N-efficient oilseed rape cultivars were characterized by high N uptake efficiency (NupE; Berry et al., 2010; Schulte auf’m Erley et al., 2011). Furthermore, genotypic variation in nitrogen remobilization efficiency contributes to N efficiency of oilseed rape cultivars (Ulas et al., 2013). However, it is not well documented for B. napus in the regulation of physiological processes and in the expression of key genes involved in N transport and assimilation under N starvation conditions, which are essential for developing rapeseed cultivars with enhanced NutE in low-N-availability soils. Moreover, allopolyploid B. napus (genome AACC, 2n ¼ 38) was derived from a natural cross between B. rapa (genome AA, 2n ¼ 20) and B. oleracea (genome CC, 2n ¼ 18) (Nagaharu, 1935). The short domestication history (about 400 years) and traditional breeding schedule of B. napus has led to a narrow genetic range in the population (Go´mez-Campo and Prakash, 1999). To widen the genetic diversity of B. napus (AnAnCnCn), new-type B. napus (ArArCcCc) was developed by interspecific crosses from B. rapa (ArAr), traditional B. napus (AnAnCnCn) and B. carinata (BcBcCcCc) (Xiao et al., 2010). The new-type B. napus harboured exotic genomic components from B. rapa and B. carinata and showed rich phenotypic variation with plenty of valuable traits including seed oil content, yield and yield-related traits, and flowering time (Zou et al., 2011; Fu et al., 2012). This population of new-type B. napus is of great use for an efficient breeding programme for increasing target traits such as nutrient efficiency. Our previous research showed that there were abundant genetic variability and wide genotype differences in N efficiency in the new-type B. napus (unpubl. res.). The aim of this study was: (1) to identify the variation in N efficiency within a large number of new-type B. napus genotypes at the seedling stage; and (2) to characterize some crucial physiological and molecular mechanisms that were involved in response to N limitation in B. napus.

Wang et al. — Nitrogen efficiency in hybridized new-type Brassica napus for 15 d, and then were transferred to LN solution with 0.15 mM NH4NO3 for 8 d. Then the plants were harvested to analyse root morphology, dry weight, N content, GS and NR activities, and gene expression levels. C2H4N4 (Shanghai Chemical Co., www.reagent.com.cn) was added at 35.34 mg L – 1 to inhibit nitrification in the culture solution. Each treatment comprised three replicates, and the experiments were repeated twice. Root morphology investigation

After plants were harvested, shoots and roots were separated and washed with deionized water. Root morphological parameters including total root length, root volume and root surface area were quantified with root image analysis software WINRHIZO Epson Perfection V700 Photo (JZZIA, Seiko Epson Corp. Japan). Measurement of dry weight and nitrogen concentration

Determination of tissue NR and GS activities

When the fresh plants were harvested, the samples were immediately frozen in liquid N and then stored at – 80 8C. The NR activity was assayed according to the method of Silveira et al. (2001). The NR activity was expressed as mg N dioxide (NO2– ) h – 1 g – 1 fresh weight (f. wt). The GS activity was determined in a reaction mixture containing imidazole buffer (Taira et al., 2004). The frozen samples (approx. 1.0 g) were ground in an ice-cold mortar and homogenized with 3 mL of 0.05 mol L – 1 Tris – HCl buffer. After centrifugation at 15 000 rpm for 20 min at 4 8C, 0.7 mL of the supernatant was added in a reaction mixture with 1.6 mL of 0.1 mol L – 1 Tris – HCl ( pH 7.4 containing 80 mmol L – 1 HONH3Cl) and 0.7 mL of 40 mg mL – 1 ATP. The mixture was then incubated at 37 8C for 30 min. The reaction was terminated by adding acidic ferric chloride (FeCl3) which contains 2 % (w/v) trichloroacetic acid and 3.5 % (w/v) FeCl3 in 2 % HCl. After mixing and centrifuging at 5000 rpm for 10 min, the supernatant was measured at 540 nm with a spectrophotometer (722N, Shanghai Metash Instrument Co., Shanghai, China), and the GS activity was expressed as the absorbance at 540 nm (Husted et al., 2002). Quantitative RT– PCR

Gene expression levels were analysed by quantitative reverse transcription – PCR (RT – PCR). Total RNA was extracted from leaves and roots of D4-15 and D1-1 using the Trizol reagent (Invitrogen). The cDNA was synthesized from 2 mL of total RNA using M-MLV Reverse Transcriptase (Promega, Madison, WI, USA) according to the manufacturer’s protocol. The cDNA samples were used as a template to quantify target gene expression levels, and the amounts of cDNA template in

each sample were normalized against that of actin (AF111812.1). Homologous sequences of arabidopsis genes encoding nitrate transporters, NR and GS were available in GenBank. Quantitative PCR for detecting the expression of BnNR, BnNRT, BnGln and BnAMT genes was performed using the SYBR Green Real-Time PCR Master Mix Kit (TOYOBO, Japan) and the CFX96TM Real-Time PCR Detection System (Bio-Rad, Hercules, CA, USA). All PCRs were performed in three replicates. The PCR conditions were as follows: 95 8C for 1 min, followed by 45 cycles of 95 8C for 10 s, 58 8C for 15 s and 72 8C for 15 s. The primer pairs used for quantitative RT–PCR are listed in Supplementary Data Table S1. Determination of stable isotope 15N

After the two lines D4-15 and D1-1 were first grown in HN solution with 3.0 mM NH4NO3 for 15 d and then in LN solution with 0.15 mM NH4NO3 for 8 d, they were transferred to stable isotope 15N solution with 3.0 mM 15NH4NO3 or NH15 4 NO3 for 3, 11 and 24 h. Plants were sampled and divided into shoots and roots (the roots were washed in ddH2O), and then were placed in an oven at 105 8C for 30 min and dried to a constant weight at 60 8C. All the samples were ground into fine powder. 15 The 15N concentration (15NH+ NO3– ) of the samples was 4 and 15 measured using N mass spectrometry (EA-Delta VMS, Thermo Scientific, USA). Pot culture

The two genotypes D4-15 and D1-1 were grown in soil in pots supplied with two N treatments under greenhouse conditions. The basic properties of the soil were as follow: pH 6.2 (soil:water ratio of 1 : 2.5), organic matter 5.9 g kg – 1, alkaline hydrolysis N 32.3 mg kg – 1, available phosphorus 10.3 mg kg – 1, available potassium 46.3 mg kg – 1. The two N treatments were: ample N (0.2 g N kg – 1 soil) and low N (0.02 g N kg – 1 soil), with four replicates for each treatment. Each pot contained 7 kg of soil. The composition of nutrients except N was as follows (mg kg – 1 soil): P, 100; K, 166; Mg, 50; Ca, 140; B, 0.2; Mo, 0.1; and Zn, 0.1. All pots were watered with 1.5 L of distilled water and incubated for 2 weeks in a greenhouse to reach nutrient balance in the soil before planting. Plants were harvested at the maturing stage, and seed yield and yield components were investigated. Analysis of introgressed exotic genomic components in new-type B. napus

Genomic DNA was extracted from young leaves of the two new-type B. napus genotypes and their parents. Simple sequence repeat (SSR) and amplified fragment length polymorphism (AFLP) markers were used to analyse the introgressed exotic genomic components. The SSR markers were selected and experiments were performed according to Xiao et al. (2010). For AFLP analysis, the genomic DNA was digested with two restriction enzymes, EcoRI and MseI. Adaptor ligation and the two successive PCRs for AFLP analysis were performed according to the method described by Vos et al. (1995). The ratio of introgressed genomic components of B. rapa and B. carinata in new-type B. napus was described by the index of sub-genomic

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Shoots and roots were dried at 60 8C to a constant mass, and then the dry weights were recorded. The dried samples were ground into fine powder, and were then digested with H2SO4 – H2O2. The concentration of N was determined using a flow injection analysis instrument (FIAstar 5000 analyzer; FOSS, Hilleroed, Denmark). Dry weight (d. wt), N concentration (NC), N accumulation (NA ¼ d. wt × NC), NupE (shoot NA/total NA) and NutE (d. wt/NA ¼ 1/NC) of each sample were calculated.

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Wang et al. — Nitrogen efficiency in hybridized new-type Brassica napus

components (ISG) for Ar, Cc or Ar + Cc, which was calculated according to the approach described by Xiao et al. (2010). Statistical analysis

The data were subjected to analysis of variance, post-hoc comparisons with Duncan’s multiple range tests at P , 0.05, principal components analysis and clustering analysis using SPSS 17.0 (SPSS Inc., Chicago, IL, USA). Ascendant hierarchical clustering was performed with Ward’s method, and the dissimilarity cut-off was chosen to define four distinct groups. RES ULT S Identification of nitrogen-efficient germplasm in new-type B. napus

45

A a

D4-15 D1-1 QY10 NY7

Shoot dry weight (mg plant –1)

300 b 250 c 200 de

d e

150 f

100 50 0

f

Dry weight, root morphology and NutE of D4-15, D1-1, QY10 and NY7 were analysed in hydroponics experiment under LN and HN conditions (Fig. 1; Table 1). For all the genotypes, shoot dry weight was decreased while root dry weight was increased under the LN level compared with the HN level (Fig. 1). The shoot and root dry weights of D4-15 under both N levels were significantly higher than those of QY10, D1-1 and NY7, as were the relative shoot and root dry weight (Fig. 1). The traditional B. napus QY10 which was N efficient showed higher shoot and root dry weight compared with D1-1 and NY7 under the LN supply (Fig. 1). Regarding N uptake, a higher concentration and accumulation were observed in both the shoot and root of the four genotypes under the HN condition than under the LN condition. The shoot and root N concentrations of D1-1 and NY7 were significantly higher than those of D4-15 and QY10 under the LN condition, but D4-15 can 2·0

B

35 b c

30 25

cd

d d

d d

20 15 10 5

Low-N

a

1·6 b

1·4 b

1·2

b

1·0 0·8 0·6

a

b ab b

0·4 0·2

0 High-N

C

1·8

a

40 Root dry weight (mg plant–1)

350

Dry weight, root morphology and NutE of the four genotypes

0 High-N

Low-N

Shoot

Root

F I G . 1. (A) Shoot dry weight, (B) root dry weight and (C) relative shoot or root dry weight of D4-15, D1-1 and NY7 under two N conditions. High-N: plants were grown in 3.0 mM NH4NO3 solution for 23 d. Low-N: plants were grown in 3.0 mM NH4NO3 solution for 15 d and then were transferred to 0.15 mM NH4NO3 solution for 8 d. Different letters indicate significant differences (P , 0.05).

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Seed yield and relative seed yield of 20 new-type B. napus inbred lines and ten traditional B. napus lines are shown in Supplementary Data Table S2. The results showed that both traditional and new-type B. napus had large variation on seed yield under the LN and HN condition. However, new-type B. napus had a higher relative seed yield, ranging from 0.41 to 0.99, than traditional B. napus (from 0.40 to 0.78), in particular for M9C104-1, M9C144-1 and M9C144-2. These three new-type B. napus lines had higher relative seed yield (.0.8) regarding of their low seed yield at HN condition, showing the characterization of B. rapa (ArAr) and B. carinata (BcBcCcCc) as having high tolerance to infertile soil and low seed yield. By microspore culture, a total of 46 new-type B. napus DH lines were successfully obtained from seven of the 20 new-type B. napus inbred lines. These DH lines and two traditional B. napus cultivars NY7 and QY10 were then grown at high- and low-N levels by hydroponic culture. The phenotypes of the 48 samples are shown in Supplementary Data Table S3. Among them, large genetic variation was observed, and the coefficient of variation ranged from 7.33 to 48.88 %. By principal component analysis, the 19 N-efficient-related traits were divided into four principal components, and the eigenvectors of shoot dry weight

(0.350), root dry weight (0.312) and shoot N accumulation (0.300) under the LN condition were higher than other indexes (Supplementary Data Table S4). Thus, shoot and root dry weight under the LN condition could be considered as the first key indicator, and shoot N accumulation under the LN condition as the secondary key indicator in identifying N-efficient genotypes. The analysis and comparisons of key indicators facilitated the clustering of 48 genotypes into four different classes (Supplementary Data Fig. S1). Class 1 and 2 include lines with high N efficiency, and Class 3 and 4 include lines with low N efficiency. Clustering results showed that D4-15 has the highest total score with 21.99, while D1-1 had the lowest total score with – 12.41, suggesting that D4-15 could be considered as a N-efficient genotype and D1-1 as a N-inefficient genotype. The traditional B. napus NY7 was N inefficient and QY10 was N efficient (Supplementary Data Fig. S1). Interestingly, D4-15 and D1-1 were derived by microspore culture from M9C144-1 and M9C102-1 (Supplementary Data Table S2), respectively.

Relative dry weight (Low-N/High-N)

552

44.28a 31.91c 37.06b 32.43c 0.35a 0.22c 0.29b 0.23c 464.08a 274.14d 387.22b 312.62c

27.07d 23.45e 26.19de 24.42de 0.23c 0.16d 0.21c 0.17d 302.19cd 203.18e 281.68d 229.42e

Surface area (cm2 per plant) Root volume (cm3 per plant) Total length (cm per plant)

Root morphology

553

accumulate more N in the shoot and root under both N levels than QY10, D1-1 and NY7 (Table 1). The NupE of the four genotypes was decreased under the LN condition, and D4-15 showed the highest value among them (Table 1). For NutE, there was no significant difference among the four genotypes under the HN condition, but it was significantly higher for D4-15 than QY10, D1-1 and NY7 under the LN condition. Compared with the HN condition, a significant increase of NutE was observed for all the genotypes under the LN condition, and D4-15 had the highest increment (Table 1). All four genotypes had a more developed root system under the LN condition than the HN condition (Table 1), suggesting that root growth was induced at the seedling stage by LN stress. Compared with QY10, D1-1 and NY7, the total root length, root volume and root surface area of D4-15 were significantly higher, suggesting an important role for a developed root system in N efficiency for D4-15.

44.87a 34.94d 40.52b 36.03c 36.34a 30.69d 34.09b 31.92c 0.85c 0.73d 0.76d 0.75d 5.92d 2.99f 4.61e 3.25f

6.77d 3.73f 5.37e 4.01f

0.87c 0.80d 0.86c 0.81d

21.80e 21.48e 21.59e 21.58e 18.02e 17.57e 17.97e 17.56e 0.94a 0.91b 0.93a 0.91b 1.09a 1.00b 1.05ab 0.98b 17.39a 9.52c 14.31b 9.95c

18.47a 10.52c 15.36b 10.95c

Root Shoot

N accumulation (mg per plant)

Total

NupE,shoot/total

Shoot

Root

NutE (mg d. wt mg N – 1)

Genotypic differences in nitrogen (15NH4+and 15NO3-) uptake

Because few growth differences were observed between the B. napus genotype NY7 and the new-type B. napus N-inefficient genotype D1-1 under both N levels (Table 1; Fig. 1), only D4-15 and D1-1 were used for the following experiments. Using stable 15 – isotope 15N (15NH+ 4 and NO3 ) labelling, we analysed genotypic differences in N uptake (Fig. 2). Significantly higher N accumulation was observed in D4-15 than in D1-1 in shoot 15N (15NH+ 4 and 15NO3– ) uptake at all the time points (P , 0.01) (Fig. 2A, C). Moreover, significantly higher uptake in root was detected for 15 D4-15 at 3 h for 15NH+ NO3– than for D1-1 4 and at 11 h for . (P , 0 05) (Fig. 2B, D). Activities of NR and GS of the two genotypes

In order to unravel the N metabolic difference in the two new-type B. napus genotypes, the activities of NR and GS in leaves and roots of D4-15 and D1-1 were determined at the seedlings stage under the LN and HN conditions (Fig. 3). The activities of GS in D4-15 leaves were significantly higher compared with D1-1 leaves under both N conditions, but no differences were detected in roots of D4-15 and D1-1 (Fig. 3C, D). As regards the activities of NR, different results were observed. The activity of NR in roots of D4-15 was significantly higher than that in D1-1 under both levels of N supply. In leaves, the activities of NR were significantly higher for D4-15 than for D1-1 under LN stress, but no differences were observed under ample N supply between the two genotypes.

4.59a 4.66a 4.62a 4.63a

2.23d 2.86b 2.47c 2.78b

5.55a 5.69a 5.56a 5.70a

2.75d 3.26b 2.93c 3.14b

High-N D4-15 D1-1 QY10 NY7 Low-N D4-15 D1-1 QY10 NY7

Genotype

Shoot

Root

N concentration (%)

Expression of nitrogen transporter genes

Subsequently, using real-time RT– PCR, we analysed the expression levels of BnAMT1;1 and BnNRT gene family members (BnNRT1;1, BnNRT2;2, BnNRT2;5, BnNRT2;6 and BnNRT2;7) in leaves and roots of D4-15 and D1-1 under the two N conditions (Fig. 4). The expression levels of these genes were higher under the LN condition than under the HN condition in both the leaves and roots of the two genotypes, except for BnAMT1;1 and BnNRT2;2. For all the genes in roots, the expression levels were significantly higher in D4-15 than in D1-1 under the N stress condition. However, ambiguous results were observed for the expression levels of all the transporter genes in leaves

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TA B L E 1. Nitrogen accumulation, nitrogen uptake efficiency (NupE), nitrogen utilization efficiency (NutE) and root morphology of D4-15, D1-1, QY10 and NY7 under high-N and low-N conditions (different superscript letters indicate significant difference at P , 0.05)

Wang et al. — Nitrogen efficiency in hybridized new-type Brassica napus

554

Wang et al. — Nitrogen efficiency in hybridized new-type Brassica napus

D4-15 D1-1

**

2·0

**

1·5

**

1·0

15NH + 4

0·5

0·4

*

0·3 0·2 0·1

**

3·5 3·0 2·5

** 2·0 1·5

** 1·0

accumulation in roots (mg plant–1)

0·7

C

15NO – 3

accumulation in shoots (mg plant–1)

0·5

0

0 4·0

0·5 0

B

3

11 Time (h)

24

D

0·6 0·5

*

0·4 0·3 0·2 0·1 0

3

11 Time (h)

24

15 . F I G . 2. 15N (15NH+ NO2 4 and 3 ) accumulation in the two genotypes. Plants were grown in HN solution (3 0 mM NH4NO3) for 15 d, followed by growth in LN solution (0.15 mM NH4NO3) for 8 d, and then transferred to stable isotope 15N solution with 3.0 mM 15NH4NO3 or 3.0 mM NH15 4 NO3. Shoots (A, C) and roots (B, D) were harvested at 3, 11 and 24 h after being grown in the 15N solution. *P , 0.05, **P , 0.01.

of the two genotypes under low N supply. Interestingly, the expression levels of both BnNRT2;5 and BnNRT2;7 were greatly induced by LN stress and were significantly higher than those of any other N transporter genes under the LN environment (Fig. 4G, H, K, L), indicating their key roles in N uptake under the LN condition. Expression of GS and NR family genes

Using real-time RT – PCR, the expression levels of GS gene family members (BnGln1;1, BnGln1;2, BnGln1;3, BnGln1;4, BnGln1;5 and BnGln2) and NR gene family members (BnNR1 and BnNR2) in leaves and roots of the two genotypes were also detected under both levels of N supply (Figs 5 and 6). The expression levels of most of the GS gene family members in leaves and roots of the two genotypes were downregulated by LN stress, but different expression patterns were observed between D4-15 and D1-1. The expression levels of BnGln1;1, BnGln1;2 and BnGln1;5 were significantly higher in both leaves and roots of D1-1 than in those of D4-15 under the LN condition, but no significant differences were observed for the expression of BnGln1;3 and BnGln2 in either leaves or roots of the two

genotypes under the same N condition (Fig. 5). Interestingly, the expression of Gln1;1 and Gln1;4 was induced by LN stress in leaves of D4-15, suggesting their important roles in N assimilation in the N-efficient line D4-15 under the LN condition. The expression levels of BnNR1 in both leaves and roots of D4-15 and D1-1 were downregulated by LN stress, but BnNR2 was downregulated in leaves but upregulated in roots by LN stress (Fig. 6). Furthermore, the expression levels of BnNR1 in both leaves and roots of D4-15 were significantly lower than those in D1-1 under the two N conditions. However, the expression levels of BnNR2 in both leaves and roots of D4-15 were significantly higher than those in D1-1 under the LN condition. In particular, the expression of BnNR2 in roots under the LN condition showed nearly a 4-fold increase in D4-15 compared with D1-1 (Fig. 6D). The responses of D4-15 and D1-1 to N starvation at the maturing stage

The results in hydroponics are often not reproducible in soil. Thus, in order to test further the responses of D4-15 and D1-1 to N starvation at the maturing stage, a pot experiment was conducted. Seed yield and yield components of the two genotypes

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Large phenotypic variation exist in new-type B. napus

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F I G . 3. Activities of nitrate reductase (NR; A, B) and glutamine synthetase (GS; C, D) of leaf (A, C) and root (B, D) of D4-15 and D1-1 under both levels of N supply. High-N: plants were grown in 3.0 mM NH4NO3 solution for 23 d. Low-N: plants were grown in 3.0 mM NH4NO3 solution for 15 d and then were transferred to 0.15 mM NH4NO3 solution for 8 d. *P , 0.05, **P , 0.01.

were investigated (Table 2). The results showed that the N-efficient line D4-15 produced significantly more yield per plant than the N-inefficient line D1-1 under HN and LN conditions, and the relative seed yield, which was defined as the ratio of seed yield under the LN level to that under the HN level, was significantly higher for D4-15 (0.52) than for D1-1 (0.29). A significant decrease was observed for pod number per plant of the two lines under the LN condition compared with the HN condition. However, D4-15 could produce nearly twice as many pods per plant as D1-1 under the LN level. The seed numbers per pod and 1000-seed weight of D4-15 were significantly higher than those of D1-1 under both N levels.

Evaluation of introgression of exotic genomic components in new-type B. napus

The ratio of introgressed exotic genomic components in new-type B. napus was evaluated using SSR and AFLP molecular markers (Table 3). About 1140 polymorphic bands were produced from the two new-type lines and their parents. The ISG (Ar) in D4-15 and D1-1 were 32.74 and 17.65 %, respectively, and tthe ISG (Cc) in D4-15 and D1-1 were 32.23 and 14.58 %, respectively. In total, the ISG (Ar + Cc) in D4-15 was 64.97 %, which was .2-fold higher than in D1-1 (32.23 %). This result revealed that the genome of the new-type B. napus changed considerably after the introgression of exotic genome components from B. rapa and/or B. carinata, and more exotic genome components were introgressed into the genome of D4-15 than D1-1.

There is much genetic variation in traits that contribute to NutE (Svecˇnjak and Rengel, 2006; Balint et al., 2008; Dawson et al., 2008; Kessel et al., 2012). Identification of N-efficient genotypes from a natural population is an important first step to improve N efficiency of crops and reduce environmental pollution (Hakeem et al., 2011). Previous studies have extensively characterized various crop performances under high- and low-N environments, such as arabidopsis, rice, maize and B. napus (Ikram et al., 2012; Kessel et al., 2012; Wei et al., 2012; Abdel-Ghani et al., 2013). In the current research, the performances of new-type B. napus and traditional B. napus were analysed under high- and low-N conditions at both the seedling stage and maturing stage. The results showed that N availability greatly influenced the variation of all the measured traits and most of the computed traits, and large phenotypic variation for N efficiency was observed in new-type B. napus (Supplementary Data Tables S2 and S3). NutE is defined differently, depending on whether vegetative biomass productivity or grain production are important traits to be considered (Dawson et al., 2008). In this study, principal components analysis and clustering analysis using SPSS 17.0 were performed to determine key indicators under high- and low-N conditions (Supplementary Table S4, Fig. S1). Finally, two extreme genotypes (D4-15 and D1-1) were noted and were used for further study. Developed root systems and efficient transport systems contribute greatly to plant growth under low nitrogen environments

Nitrogen uptake depends on the extent and effectiveness of the root system. Differences in N uptake may have been caused by differences in root growth. For example, Kamh et al. (2005) showed that the variety with a high yield under an LN environment had greater root growth following stem extension in a comparison of two B. napus varieties. Similarly, Ye et al. (2010) analysed two B. napus genotypes differing in N use in nutrient solution and reported a greater root volume and root active absorbing area in the N-efficient genotype. The same results were observed in the present study. The N-efficient line D4-15 had a more developed root system compared with NY7 and D1-1 under both N levels (Table 1). In terms of N uptake among the four genotypes, the results showed that D4-15 could accumulate more N in both shoots and roots than NY7 and D1-1 under the LN condition (Table 1). The result was confirmed in the 15N experiment by adding two different forms 15 of 15N (15NH+ NO3– ) to the nutrient solution (Fig. 2). A 4 and larger root system and higher NupE of D4-15 facilitated the accumulation of more N in the shoot than in D1-1, and a larger shoot of D4-15 might result in a change in sink strength which then would drive N uptake. It is reported that nitrate and ammonium transporters play important roles in N acquisition by regulating root growth and development under N starvation (Remans et al., 2006; Engineer and Kranz, 2007; Shi et al., 2010). For example, Remans et al. (2006) found that NRT2.1 had a key dual function in coordinating root development with external NO3– availability. In this study, we examined the expression of six nitrate and ammonium transporters under N stress conditions, and found that the

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F I G . 4. Expression of BnAMT1;1 and NRT family member genes (BnNRT1;1, BnNRT2;2, BnNRT2;5, BnNRT2;6 and BnNRT2;7) in leaves and roots of D4-15 and D1-1 under both levels of N supply. High-N: plants were grown in 3.0 mM NH4NO3 solution for 23 d. Low-N: plants were grown in 3.0 mM NH4NO3 solution for 15 d and then were transferred to 0.15 mM NH4NO3 solution for 8 d. *P , 0.05, **P , 0.01.

expression levels of all of these genes were significantly higher in roots of the N-efficient genotype D4-15 than in roots of the N-inefficient genotype D1-1 under the LN condition (Fig. 4), and some genes were greatly induced by LN stress, such as BnNRT2;5 and BnNRT2;7 (Fig. 4G, H, K, L). Our results suggest that efficient transport systems and developed root systems play important roles in B. napus N acquisition under N starvation. However, the mechanism by which gene expression regulates shoot and root growth in B. napus needs further study, and more evidence of its molecular biology needs to be uncovered. The importance of NR and GS in nitrogen assimilation and the regulation mechanisms of gene expression at the seedling stage of new-type B. napus

Different forms of N are assimilated after being taken up by plant roots (Xu et al., 2012). Reports show that NR and GS play important roles in N assimilation for plants grown under LN conditions (Shi et al., 2010; Hakeem et al., 2011). Using

two B. napus genotypes differing in N efficiency in nutrient solution, Ye et al. (2010) found that higher NR and GS activity were detected in the more N-efficient genotype. Similar results were observed in the present study. The activities of NR were higher in roots and leaves of the N-efficient line D4-15 compared with the N-inefficient line D1-1 under the LN condition (Fig. 3). It is reported that NR could play an indirect role in the absorption of NO3– , regulating the levels of NO3– and amino acids in root cells (Hakeem et al., 2011). In our experiment, the pattern of NO3– uptake by the root and the NO3– concentration and accumulation in roots and shoots were coincident with the pattern of NR activities (Fig. 2; Table 1). The activities of GS were significantly higher in D4-15 leaves than in D1-1 leaves, but no significant differences were observed in GS activities between D4-15 and D1-1 roots (Fig. 3). The reasons might be that a more efficient transport system of D4-15 resulted in more N accumulated in leaves (Fig. 4G), which led to higher GS activities for assilimation in leaves of D4-15. Our results show that the N-efficient genotype D4-15 had a higher nitrate reduction in leaves and roots and

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F I G . 5. Expression of glutamine synthetase (GS) family member genes (BnGln1;1, BnGln1;2, BnGln1;3, BnGln1;4, BnGln1;5 and BnGln2) in leaves and roots of D4-15 and D1-1 under both levels of N supply. High-N: plants were grown in 3.0 mM NH4NO3 solution for 23 d. Low-N: plants were grown in 3.0 mM NH4NO3 solution for 15 d and then were transferred to 0.15 mM NH4NO3 solution for 8 d. *P , 0.05, **P , 0.01.

higher ammonium assimilation in leaves than the N-inefficient genotype D1-1 under LN supply, suggesting that NR and GS may play vital roles in the N efficiency of new-type B. napus under LN conditions. As NO3– is assimilated via conversion to NO2– , then of NH+ 4 into amino acids, the internal pools of amino acids within plants may indicate the N status by providing a signal that is somehow sensed and can feed back to regulate N uptake and assimilation by the plant (Miller et al., 2008). The feedback regulation can occur by changing the expression of transporters, and may also involve the post-translational modification of protein levels (Bernard and Habash, 2009). For example, Finnemann and Schjoerring (2000) found that GS1 was regulated posttranslationally by reversible phosphatases. In this research, although the activities of GS and NR were significantly higher in

leaves of D4-15 than in D1-1 under LN supply (Fig. 3), the gene expression levels were different (Figs 5 and 6). The reason might be that D4-15 had higher N accumulation and higher activities of NR and GS in vivo which resulted in a larger internal pool of downstream N metabolites. Thus, certain N-sensing systems were started, and the feedback regulation may occur by post-translationally modifying NR and GS protein levels. Application potential of interspecific hybridized new-type Brassica napus in breeding cultivars with enhanced NutE and seed yield

The role of cultivated B. napus as a commercial oil crop in Asia, Europe, North America and Australia has progressively

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F I G . 6. Expression of nitrate reductase (NR) family member genes (BnNR1 and BnNR2) in leaves and roots of D4-15 and D1-1 under both levels of N supply. High-N: plants were grown in 3.0 mM NH4NO3 solution for 23 d. Low-N: plants were grown in 3.0 mM NH4NO3 solution for 15 d and then were transferred to 0.15 mM NH4NO3 solution for 8 d. *P , 0.05, **P , 0.01.

TA B L E 2. Seed yield and yield components of the two genotypes under high- and low-N supply Genotype High-N D4-15 D1-1 Low-N D4-15 D1-1

Seed yield (g per plant)

No. of pods (per plant)

No. of seeds (per pod)

1000-seed weight (g)

18.94a 13.53b

358a 349a

12.56a 10.69bc

4.44a 3.86b

9.79c 3.63d

218b 121c

11.40ab 9.63c

4.27a 3.59b

Different superscript letters indicate significant difference at P , 0.05.

TA B L E 3. Replacement of sub-genomes in new-type Brassica napus estimated with AFLP and SSR markers New-type B.napus D4-15 D1-1

Index of sub-genome Ar

Index of sub-genome Cc

Index of subgenome Ar + Cc

32.74 17.65

32.23 14.58

64.97 32.23

(Go´mez-Campo and Prakash, 1999), which was not beneficial in breeding B. napus cultivars with higher seed yield and oil content, and even better tolerance to biotic and abiotic stresses. It is reported that the introgression of Ar and/or Cc genomic components from B. rapa and B. carinata into new-type B. napus (ArArCcCc) can lead to considerable differences in the gene expression profiles (Chen et al., 2008). In this research, large genetic variation was observed among agronomic traits contributing to N efficiency of new-type B. napus (Supplementary Tables S2 and S3). Further research on D4-15, QY10, NY7 and D1-1 demonstrated that the N-efficient new-type B. napus line showed higher values than the N-inefficient line and the traditional B. napus genotypes on several phenotypic traits under both LN and HN conditions by hydroponic culture, suggesting that the N-efficient new-type B. napus line had a better overall growth potential under both N levels at the seedling stage (Tables 1 and 2). However, at the maturing stage, there was no significant difference in seed yield between D4-15 derived from M9C144-1 by microspore culture and QY10 under low N conditions, but D4-15 had a higher relative seed yield (0.87) than QY10 (0.71), indicating that D4-15 had higher N efficiency (tolerance to LN stress) than QY10 which would be of importance in breeding N-efficient B. napus cultivars in the field (Supplementary Data Table S2). By evaluation of introgression of exotic genomic components in D4-15 and D1-1 with SSR and AFLP molecular markers, we found that more exotic genome components were introgressed into the genome of D4-15 (64.97 %) than D1-1 (32.23 %) (Table 3). This may be one of the key factors that contribute to the better performance of D4-15 than D1-1 at both the seedling stage and maturing stage under an LN environment. However, more research is needed to confirm whether the differentially expressed N-related genes were genetically localized in the genomic region containing the exotic genome components that were introgressed. Moreover, depending on the morphological and physiological features, it would be very interesting to use D4-15 together with D1-1 or NY7 as parent lines of populations to perform quantitative trait locus mapping of traits related to NutE in B. napus in the future.

S U P P L E M E N TARY D ATA Supplementary data are available online at www.aob.oxford journals.org and consist of the following. Figure S1: systemic classification of 46 new-type Brassica napus and two traditional B. napus cultivars. Table S1: primer sequences used for real-time RT – PCR. Table S2: seed yield and relative seed yield of 20 newtype Brasscia napus inbred lines and ten traditional Brasscia napus lines. Table S3: phenotypic analysis of 46 new-type Brassica napus genotypes and two traditional B. napus genotypes in the hydroponic experiment. Table S4: principal components analysis of 46 new-type Brassica napus genotypes with 19 nitrogen-efficient indicators in the hydroponic experiment.

AC KN OW L E DG MEN T S increased due to better production potential and seed quality improvement. However, the short domestication history and intensive breeding has resulted in a narrow genetic base of B. napus

This work was supported by grants from the National Natural Science Foundation (30830073) and the National Basic Research and Development Program (2011CB100301), China.

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Wang et al. — Nitrogen efficiency in hybridized new-type Brassica napus LIT E RAT URE CITED

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