Plant materials and growth conditions
The process of constructing of ETW is described in two publications [16, 26]. Briefly, ETW was constructed from BBAA components of a common wheat (T. aestivum L.) cultivar (“Canthach” referred to as TAA10 in the present paper) by crossing the common wheat with durum wheat (T. turgidum, genome BBAA), followed by repeated backcrossing with TAA10 as the recurrent parent. The genome of ETW is therefore virtually identical to the BBAA subgenome of its common wheat donor [16]. The resynthesized hexaploid wheat (XX329, BBAADD genome) was synthesized by crossing and doubling of ETW with an Aegilops tauschii (TQ18, DD genome) line developed by E. Kerber [26]. We used S6 generation plants for XX329 and ETW. A typical durum wheat line ALTAR81 (T. turgidum, BBAA genome) was used as a control. All five wheat lines were sown in pots filled with washed sand each containing one seedling. All seedlings were placed in a greenhouse. The growth conditions were maintained at 21–23 °C day and 14–17 °C night under16 h light at ~ 400 μmol m− 2 s− 1. The pots were watered daily with half-strength Hoagland nutrient solution. Initial seeds of ETW, TAA10, XX329 and TQ18 were kindly provided by Prof. Moshe Feldman of the Weizmann Institute of Science, Israel. Initial seeds of ALTAR81 were bought from International Maize and Wheat Improvement Center (CIMMYT).
Sampling method and experiment design
We planted 100 pots for each genotype. At the tillering stage, we used 75 pots of plants of each genotype to conduct to proteomic analysis, biochemical measurements, metabonomic analysis, and Real-time PCR. We first collected the samples for metabonomic analysis. We took one mature leaf from each individual for each genotype, and each biological replicate was a pool of 15 leaves with five biological replicates. After sampling of metabonomic analysis, all the 75 pots of each genotype were divided into 3 sets (25 pots per set). The two sets (50 pots) were used for biochemical measurements and real time PCR, and another set (25 pots) for proteomic analysis. In addition, we used the remaining 25 pots of each genotype to measure seed quality, growth and photosynthesis. We arranged all genotypes based on randomized complete block design.
Biochemical measurements
At the tillering stage, net photosynthetic rate (PN), stomatal conductance (gs), and transpiration rate (E) were measured by a portable open-flow gas exchange system (LI-6400, LI-COR, Lincoln, NE, USA). Leaves and roots of each genotype were collected at the tillering stage, and seeds of each genotype were collected at full ripe stage. Each genotype and each tissue had four biological replicates, and each biological replicate comprised a pool of 5 plants. Amino acids in seeds were separated and measured by an automated amino acid analyser [28]. Dried samples of leaves, roots, and grains were digested three times in 65% HNO3 at 120 °C, and their contents of Ca, Fe, Mg, P, and Na were measured using an inductively coupled plasma emission spectrometer. Enzymes in freshly matured leaves from each wheat line, all the leaves represented the same leaf position, were assayed using conventional methods [29,30,31]. The activity of nitrate reductase (NR), glutamine synthetase (GS), and glutamate dehydrogenase was measured according to methods described by Debouba et al. [29] and Surabhi et al. [30], and glycolate oxidase (GO) was assayed with the method described by Wu et al. [31].
Metabonomic analysis
Mature leaves of 15 plants (one leaf per individual) at the tillering stage for each genotype were pooled as a biological replicate, and five such replicates were used in metabonomic analysis, which was conducted according to the method described by Guo et al. [32]. Briefly, leaf samples for each genotype were extracted in 0.4 mL of a mixture of methanol and chloroform (3:1, v/v). The extracted metabolites were derivatized with methoxylamine hydrochloride and N, O-bis (trimethylsilyl)-trifluoroacetamide (BSTFA) containing 1% trimethylchlorosilane (TMCS). Metabolite profiling was carried out using a GC-TOF/MS facility equipped with an Agilent 7890 gas chromatograph system and a Pegasus HT time-of-flight mass spectrometer (Chroma TOF Pegasus HT, Leco, Saint Joseph, MI, USA). Metabolic data were produced and analysed by Chroma TOF4.3X (a software package) and LECO-Fiehn Rtx5 database.
Proteomic analysis
Leaves of five plants at the tillering stage for each genotype were pooled as a biological replicate, and four such replicates were used for each genotype. Total protein was extracted from fresh leaves in TCA-acetone solution (10% TCA in acetone), and the protein samples were stored in darkness at − 20 °C for 3 h. After centrifuging (13,000×g, 4 °C, and 30 min), the pellet was suspended in buffer A (8 M urea, 4% CHAPS, 30 mM HEPES, 2 mM Na2EDTA, 10 mM DTT, and 1 mM PMSF; pH 8.0–8.3). After centrifuging again as before, 35 μL of 1 M IAM and 15.8 μL 200 mM DTT were added to 0.3 mL of the supernatant. The protein samples were quantified by the Coomassie Brilliant Blue G250 method, digested in trypsin (trypsin: protein, 1:40) at 37 °C for 16 h, and the peptides were purified using Thermo Scientific Pierce C18 pipette tips (production ID 87784). Next, label-free proteomic analysis was conducted on an LC-MS/MS system (Q-Exactive, Thermo Scientific, Germany) according to the protocol stipulated by the manufacturer. Protein searching and label-free quantification were performed using Proteome Discoverer version 2.2 (Thermo Scientific, USA) against the common wheat reference genome database of the International Wheat Genome Sequencing Consortium (IWGSC) (iwgsc_refseq ver. 1.0). Differentially expressed proteins among the wheat genotypes were identified with ANOVA (background-based) method of Proteome Discoverer version 2.2, and the generated p-values were further adjusted by the Benjamini-Hochberg method.
Real-time PCR
Total RNA from leaves at the tillering stage was isolated by TRIzol from Invitrogen, which is especially meant for plants, according to the manufacturer’s instructions. Each genotype had four biological replicates, and each biological replicate comprised a pool of five plants. The yield and quality of the RNA were checked by Nanodrop and agarose gel. 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 7:Table S5). Real-time quantitative RT-PCR was performed using SYBR Green real-time PCR Master Mix and a StepOnePlus real-time PCR system. Actin and RLI were used as normalization control genes [33, 34]. The data of gene expression were analysed using the △△Ct method [35].
Statistical analysis
The experimental design is randomized complete block design. All data were from 4 to 7 biological replicates. Statistical analysis for metabonomic and biochemical data was performed using SPSS version 13.0 (SPSS, Chicago, USA). The statistical significance was determined by the t-test at 0.05 level. Statistical analysis of proteomic data was performed using Proteome Discoverer version 2.2 based on ANOVA (background-based) method (adjusted P value < 0.05).