In this work, we used a newly formed allohexaploid wheat line (Allo-960, genome BBAADD), its two parents, and a natural hexaploid wheat (cv. Chinese Spring) as main experimental organisms. Allo-960 was produced by crossing a Triticum turgidum line (Black bird, genome BBAA) with a Aegilops tauschii line (30A, genome DD) . The seeds of Allo-960 and its parents were provided from George Fedak (Agriculture and Agri-Food in Canada). In this study, sixth self-pollinated generations of Allo-960 was used and labeled as neo-6×, its tetraploid parent as 4×, its diploid parent as 2×, and the natural hexaploid wheat (cv. Chinese Spring) as nat-6×. In this work, to explore the physiological mechanisms of N-deficiency tolerance alteration of the newly formed hexaploid, we utilized Allo-960 and its exact 2× (30A) and 4× (Black bird) parents to measure gene expression and physiological indices. To test generality of the results, we also used another Ae. Tauschii line, two newly formed hexaploid lines, and 4 tetraploid lines, but we only measured their chlorophyll contents to indicate the N-deficiency tolerance alteration. All wheat lines used were listed in Additional file 10: Table S1.
According to the typical concentration of available inorganic N in agricultural soils (1 to 10 mM) [24,25,26], we set 5 mM of NO3− as control N condition, and 0.1 mM as a low N stress. Seeds of all wheat lines were sown in pots containing thoroughly washed sand. Each pot contained 5 seedlings as a biological replication. All seedlings were placed in a greenhouse with a thermoperiod of 20–23/13–17 °C and a 16/8 h day/night photoperiod. The pots were watered with an altered half-strength Hoagland nutrient solution (control, 5 mM NO3−, pH 6.5) for 17 days before stress treatment. After which (i.e., at about the four-leaf stage), low N stress was applied for 7 or 31 days by supplementing nitrogen-deficient half-strength Hoagland’s solution (0.1 mM NO3−, pH 6.5). CaCl2 were used to compensate Ca2+ in low N treatment solution.
Measurement of physiological indices
After 31 days of low N stress, net photosynthetic rate (PN), stomatal conductance (gs), and transpiration rate (E), PSII efficiency (ΦPSII), efficiency of excitation capture by open PSII centres (Fv’/Fm′) and maximum quantum yield of photosystem II (Fv/Fm) were determined using a portable open flow gas exchange system LI-6400 (LI-COR, USA) according to the protocol of the instrument. The photosynthetically active radiation (PAR) was 1200 μmol m− 2 s− 1. In order to minimize the plant-to-plant variations, five seedlings were pooled as a biological replicate, and there were at least four replicates for all biochemical measurements. Chlorophyll contents were determined according to the method reported in Ni et al. 2008 . Free amino acids of dry sample were separated and measured by an automated amino acid analyzer . Total nitrogen content was measured by an elemental analyser (Vario EL Cube, Elementar, Germany).
Enzyme activity assays
The enzymes in the fresh mature leaves at the same leaf position for each wheat line were assayed using conventional methods. Ten mature leaves from five individual plants was pooled as a biological replicate, and there were four biological replicates. The activities of nitrate reductase (NR: EC 126.96.36.199), glutamine synthetase (GS: EC 188.8.131.52), and glutamate dehydrogenase (NADH-GDH: EC 184.108.40.206 and NADPH-GDH: EC 220.127.116.11) were measured according to altered methods of Debouba et al. 2006 and Surabhi et al. 2008 [29, 30]. Glycolate oxidase (GO: EC 18.104.22.168) were assayed with the method of Wu et al. 2013 . Fresh plant tissues were used to determine activities of each enzyme. NR was extracted with 1 mL of buffer solution (100 mM KH2PO4-NaOH buffer of pH 7.4, 7.5 mM cysteine, and 1 mM EDTA, 1.5% casein) at 4 °C. 0.1 mL of the supernatant was incubated in a reaction mixture containing 0.3 mL 100 mM KH2PO4-NaOH buffer (pH 7.4), 0.1 mL 3 mM NADH, and 0.1 ml 100 mM KNO3 at 30 °C for 30 mins. The reaction was stopped by adding a stopping solution including 0.25 mL sulfanilamide, 0.25 mL 0.02% N-(1-naphthy) ethylenediamine dihydrochloride, and 0.1 mL glacial acetic acid. Finally, the absorbance was measured at 540 nm. The NR activity was expressed in μmol NO3− mg protein− 1 h− 1. GS was extracted with 1 mL of buffer solution containing 25 mM Tris-HCl (pH 7.6), 1 mM MgCl2, 1 mM EDTA, 14 mM beta-mercaptoethanol, and 1% PVP at 4 °C. 0.1 mL of supernatant was added to a reaction mixture containing 0.3 mL 250 mM imidazole-HCl buffer (pH 7.0), 0.2 mL 300 mM L-glutamic acid sodium, 0.2 mL 30 mM ATP-Na2, and 0.1 mL 500 mM MgSO4 at 25 °C for 10 mins. The reaction was stopped by adding 0.1 mL 1 M hydroxylamine hydrochloride, 0.4 mL of a solution (3.3% FeCl3, 8% TCA and 17%HCl), and rotating for 10mins. Finally, the absorbance of reactive mixture was measured at 540 nm. The GS activity was expressed as OD at 540 nm mg protein− 1 h− 1. Glutamate dehydrogenase (NADH- and NADPH-specific GDH) was extracted with 1 mL of extraction buffer (100 mM pH 8.2 Tris-HCl buffer, 14 mM beta-mercaptoethanol, and 1% PVP) at 4 °C. 0.5 mL of supernatant was added to a reaction mixture containing 0.6 mL of 0.2 M Tris-HCl buffer (pH 8.0), 0.15 mL 0.1 M α-ketoglutarate sodium, 0.15 mL 1 M NH4Cl, and 0.1 mL 3 mM NADH (NADPH). The reaction was followed by measurement of the decrease in absorbance at 340 nm. The activities were expressed as μmol NADH mg protein− 1 h− 1 or μmol NADPH mg protein− 1 h− 1. GO was extracted with 1 mL of extraction buffer containing 100 mM KH2PO4-NaOH buffer (pH 8.0) at 4 °C. 0.05 mL of supernatant was incubated in a reaction mixture containing 0.5 mL 100 mM KH2PO4-NaOH buffer (pH 8.0), 0.1 mL 1 mM FMN, and 0.1 mL 50 mM glycollic acid for 5mins. The reaction was stopped by adding 0.1 mL 2 M HCl, 0.1 mL 1.82 M NaOH, 0.33% phenylhydrazine hydrochloride, and 1 mL concentrated HCl. The absorbance was measured at 550 nm. The GO activity was expressed in μmol glyoxylate mg protein− 1 h− 1.
Measurement of H+ and NO3
The seeds were sown and germinated in 8.5-cm Petri dishes for 3 days. The dishes were placed in a growth room maintained at 25 °C day and 18 °C night temperatures under 16 h light at 300 μmol m− 2·s− 1.Then the young seedlings were transferred to a Petri dish containing the half-strength Hoagland nutrient solution with 0.1 or 5 mM NO3− (pH 6.5) for 7 days. After 7 days of low N stress, net NO3− and H+ fluxes at the surface of root maturation zone were measured using non-invasively scanning ion-selective electrode technique (SIET, SIET system BIO-003A, Younger USA Science and Technology, Falmouth, MA, USA) by Xuyue Science and Technology (Beijing, China). Six plants were randomly selected from each genotype and the roots were transferred to a Petri dish containing 10 ml the measuring solutions with 0.1 or 5 mM NO3−, and equilibrated for 2 h. Then the roots were transferred to a new Petri dish containing fresh measuring solutions with 0.1 or 5 mM NO3−, and net H+ and NO3− fluxes were monitored for 8 min. The measuring solution of H+ flux composed of 0.1 mM KCl and 0.1 mM CaCl2 in pH 6.0, and the measuring solution of NO3− flux composed of 0.1 mM CaCl2 and 0.3 mM MES in pH 6.0.
Real time qRT-PCR
When the plants were grown at low N condition for 7 days, the leaves and roots were harvested to extract total RNA using TRIzol reagent (Invitrogen). Five individual plants of neo-6× were considered as five biological replicates, and for the other three lines the three plants were pooled as a biological replicate with 3–4 biological replicates. The RNA was treated with DNaseI (Invitrogen), reverse-transcribed using SuperScriptTM RNase H-Reverse Transcriptase (Invitrogen), and then subjected to qRT-PCR analysis using gene-specific primers (Additional file 11: Table S2). Actin, RLI and GAPDH were used as normalization control genes in the assay [32,33,34]. The expression of NPF genes and NRT2 genes was assayed with the primer sequences from previous studies [34, 35], and was calculated using △△Ct method .
Statistical analysis was performed using the statistical program SPSS 13.0 (SPSS, Chicago, USA). All data were from 3 to 7 biological replicates. Statistical significance was determined by t-test.