Gene identification: The OsBG1 coding sequence from NCBI (Q10R09.1) also known as Os03g0175800 was used as a query for Big Grain genes in Ensemble plants. (http://plants.ensembl.org/index.html). Protein domain identification was found using the biomart section of Ensembl plants v42 to search both the rice and wheat genomes for genes using the Interpro ID IPR039621. All DNA and Proteins sequences are available at Ensembl plants. Expression profiles of the Big Grain like genes was downloaded from the Wheat Expression Browser, (http://www.wheat-expression.com) . Phylogenetic tree was constructed using the amino acid sequences of the Big Grain genes in MEGA X . Sequences were aligned using the MUSCLE algorithm and the tree was constructed using 500 iterations using the Maximum Likelihood Tree option. Heat map was constructed using heatmapper (http://www.heatmapper.ca/) .
Wheat variety Fielder (USDA, available from CIMMYT, under the name BW 35215) plants were grown in controlled environment chambers (Conviron) at 20°C day/15°C night with a 16 hr day photoperiod (approximately 400 µE m− 2 s− 1). Immature seeds were harvested for transformation experiments at 14–20 days post-anthesis (dpa). Isolated immature wheat embryos were co-cultivated with Agrobacterium tumefaciens for 2 days in the dark . Subsequent removal of the embryonic axis and tissue culture was performed as described by . Individual plantlets were hardened off following transfer to Jiffy-7 pellets (LBS Horticulture), potted up into 9 cm plant pots containing M2 and grown on to maturity and seed harvest in controlled environment chambers, as above.
Plasmid construction for genetic modification
TaBG1-A was synthesized from the public sequence available at the time for the wheat cultivar Chinese Spring with attL1 and attL2 sites for direct recombination into binary gateway vectors. TaBG1-A was then recombined into the binary vector pSc4ActR1R2 using a Gateway LR Clonase II Kit (Thermofisher) to create pMM24. TaBG1-A was expressed in wheat cv Fielder from the rice Actin promoter and transcripts terminated by the Agrobacterium tumefaciens nopaline synthase terminator (tNOS).
pMM24 was verified by restriction digest and sequencing before being electro-transformed into A. tumefaciens. Plasmids were re-isolated from Agrobacterium cultures and verified by restriction digest prior to use in wheat experiments .
DNA analysis of transformed wheat plants
Plantlets which regenerated under G418 selection in tissue culture were transferred to Jiffy-7 pellets and validated using an nptII copy number assay relative to a single copy wheat gene amplicon, GaMyb, normalized to a known single copy wheat line. Primers and Taqman probes were used at a concentration of 10µM in a 10µl multiplex reaction using ABsolute Blue qPCR ROX mix (Thermofisher) with the standard run conditions for the ABI 7900 HT. The relative quantification, ΔΔCT, values were calculated to determine nptII copy number in the T0 and subsequent generations . Homozygous transgenic lines were identified on the basis of nptII copy number and segregation analysis. WT Fielder plants were null segregates.
Expression of TaBG1 in wheat: Total RNA was isolated from wheat shoots using a RNeasy Kit (Qiagen). Following treatment with DNaseI (Thermofisher), cDNA synthesis was conducted on 500 ng of total RNA using Omniscript RT Kit (Qiagen). The cDNA was diluted 1:2 with water and 0.5 µL was used as template in each RT-PCR reaction. Transcript levels were quantified using SYBR Green JumpStartTaq ReadyMix (SIGMA) with the standard run conditions for the ABI 7900 HT. Three technical replicates were performed on each of the three biological replicates. Two reference genes TaUbiquitin and TaEF1α were used for the normalization using the ΔΔCT. The sequence of primers used in Q-PCR assays are TaUbiquitin F (5’-CCTTCACTTGGTTCTCCGTCT-3’), TaUbiquitin R (5’-AACGACCAGGACGACAGACACA-3’), TaEF1α F (5’-TGGTGTCATCAAGCCTGGTATGGT-3’), TaEF1α R (5’-ACTCATGGTGCATCTCAACGGACT-3’), TaBG1 F (5’-GCTGCTGGACGCGATATAC-3’) and TaBG1 R (5’-CTGCTTCTTGGCCTTCTTCT-3’).
Transgenic lines and corresponding null segregants were grown on TS5 low fertility soil to control total nitrogen with a starting nitrogen level of 0.1 mg/l (Bourne Amenity, Kent, UK). Ammonium nitrate was then added to each pot to reach a final concentration in the pots equivalent to field fertilizer application of 70, 140 or 210 kg N /ha. Plants were grown in a climate controlled glasshouse with 10000 lux sodium supplemental light for a 16hr day and 20°C/15°C day night temperatures. At least 21 plants per line were grown per treatment in a randomized design.
Grain cross sections
The wheat grains were soaked in distilled water and subjected to a vacuum system for 10 minutes. The seeds were then fixed overnight with 3% EM grade glutaraldehyde in 0.1M Cacodylate buffer and oscillated overnight at room temperature. Seeds were washed twice in 0.1M cacodylate buffer for 10 minutes for each wash and then stored in 0.1M Cacodylate buffer prior to post tissue-fixation with 1% Osmium Tetroxide in 0.1M Cacodylate Buffer for two days at 4°C.
Seeds were then washed twice in distilled water for 10 minutes each time and dehydrated with 50%, 70%, 90% (2 x 15 minute for each) and 100% ethanol (3 x 30 minutes) respectively. To further remove water from the samples, propylene oxide (100%) was added (2 changes for 30 minutes at room temperature) followed by the addition of propylene oxide: resin (48g TLV Resin, 16g VH1 Hardener, 36g TLV Hardener VH2) solution at 3:1 ratio (1 hour, room temperature) and 1:1 ratio (overnight with lids off, room temperature) respectively. A pure resin preparation (TAAB Low Viscosity Resin – medium recipe) was then added (2 changes, 2 hours per change) prior to embedding in the oven at 60°C for 48 hours. Samples were sectioned with a Histo diamond knife at 1000nm (1micron) thick using Leica EM UC6 (Leica Biosystems, Wetzlar, Germany), placed on APES coated slide and stained using 1% Toluidine Blue in 1% Sodium Borate.Sections were imaged using ZEISS Axiophot microscope and captured using Micromanager Software. Cell area was measured by tracing individual cells taken from images taken at 200X magnification using ImageJ .
Elemental analysis: Sample digestates were diluted 1-in-10 using Milli-Q water prior to elemental analysis. The concentrations of 28 elements were obtained using inductively coupled plasma-mass spectrometry (ICP-MS; Thermo Fisher Scientific iCAPQ, Thermo Fisher Scientific, Bremen, Germany); Ag, Al, As, B, Ba, Ca, Cd, Cr, Co, Cs, Cu, Fe, K, Mg, Mn, Mo, Na, Ni, P, Pb, Rb, S, Se, Sr, Ti, U, V, Zn. Operational modes included: (i) a helium collision-cell (He-cell) with kinetic energy discrimination to remove polyatomic interferences, (ii) standard mode (STD) in which the collision cell was evacuated, and (iii) a hydrogen collision-cell (H2-cell). Samples were introduced from an autosampler incorporating an ASXpress™ rapid uptake module (Cetac ASX-520, Teledyne Technologies Inc., Omaha, NE, USA) through a PEEK nebulizer (Burgener Mira Mist, Mississauga, Burgener Research Inc., Canada). Internal standards were introduced to the sample stream on a separate line via the ASXpress unit and included Sc (20 µg L-1), Rh (10 µg L-1), Ge (10 µg L-1) and Ir (5 µg L-1) in 2% trace analysis grade HNO3 (Fisher Scientific UK Ltd). External multi-element calibration standards (Claritas-PPT grade CLMS-2; SPEX Certiprep Inc., Metuchen, NJ, USA) included Ag, Al, As, B, Ba, Cd, Ca, Co, Cr, Cs, Cu, Fe, K, Mg, Mn, Mo, Na, Ni, P, Pb, Rb, S, Se, Sr, Ti (semi-quant), U, V and Zn, in the range 0–100 µg L-1 (0, 20, 40, 100 µg L-1). A bespoke external multi-element calibration solution (PlasmaCAL, SCP Science, Courtaboeuf, France) was used to create Ca, K, Mg and Na standards in the range 0–30 mg L-1. Boron, P and S calibration utilized in-house standard solutions (KH2PO4, K2SO4 and H3BO3). In-sample switching was used to measure B and P in STD mode, Se in H2-cell mode and all other elements in He-cell mode. Sample processing was undertaken using Qtegra™ software (Thermo Fisher Scientific) with external cross-calibration between pulse-counting and analogue detector modes when required.
Samples were measured using the dumas method. The samples were dried for 17 hours at 100°C and then milled on a 1mm hammer mill. Prior to testing the sample were dried at 104°C for 3 hours and 1g of sample was loaded on the instrument (Leco TruMacN Dumas gas analyser), following the manufacturer’s instructions. Samples were converted to gases by heating in a combustion tube at 1150°C. Interfering components are removed from the resulting gas mixture. The nitrogen compounds in the gas mixture or a representative part of the mixture, are converted to molecular nitrogen which is quantitatively determined by a thermal conductivity detector. The nitrogen content is then calculated by a microprocessor.
Analysis of variance (ANOVAs) or Wilcox Tests were run using the aov and TukeyHSD functions in the R environment with the null hypothesis of no difference between lines . Tukey’s post hoc test was added to identify each significant interaction between the lines tested. Data was plotted using R ggplot2 .