RNA-seq analysis of bread wheat grown under hydroponic Fe deficient conditions

Bread wheat (Triticum aestivum L., cv. Fielder) plants were grown under iron (Fe) deficient hydroponic conditions to analyise transcriptomic changes in leaf and root tissue. Overall design: Grain harvested from cv. Fielder plants were stratified at 4°C for two days and then surface sterilised with VitaVax 200FF (Hewitt & Whitty, Geelong, Victoria, Australia) and placed on sterile moist filter paper at 20°C for two days. Germinated seedlings were transferred to hydroponic boxes containing a nutrient replete solution: 2 mM NH4NO3, 5 mM KNO3, 2 mM Ca(NO3)2·4H2O, 2mM MgSO4·7H2O, 0.1 mM KH2PO4, 10 µM H3BO3, 5 µM MnCl2·4H2O, 5 µM ZnSO4·7H2O, 0.5 µM CuSO4·5H2O, 0.1 µM Na2MoO4·2H2O, and 50 µM NaFe3+EDTA. Following 12 days of nutrient replete growth, the solution was replaced with an Fe deficient solution (0 µM NaFe3+EDTA) and grown for a further 11 days. The nutrient solution was replaced every four days. Leaf tissues were harvested at days 0 (1st leaf), 1 (1st leaf), 7 (3rd leaf) and 11 (3rd, 4th, 5th leaves) of Fe deficiency and root tissues were harvested at day 11 of Fe deficiency. All tissues were immediately snap frozen in liquid nitrogen following sampling. Approximately 50 mg of tissue in TRI Reagent® (Merck, Darmstadt, Germany) was homogenised with stainless steel beads and a TissueLyser II (Qiagen, Hilden, Germany). Three plants were used (105_, 106_ and 112_).