Additional file 1 of Genome-wide characterization of two-component system elements in barley enables the identification of grain-specific phosphorelay genes

Additional file 1: Figure S1. Phosphorylation sites in authentic and pseudo HPs and RRs. (A) Histidyl phosphorylation site in HPs (red) and alternative amino acid in PHPs (blue). (B) Aspartate phosphorylation site in RRs (red) and alternative amino acids in PRRs (blue). Figure S2. Altered expression of barley TCS elements after treatment with hormones. Grains (5DAF) were incubated for 16 h either with abscisic acid (ABA, 50 µM), synthetic auxin 1-Naphthaleneacetic acid (NAA, 50 µM), cytokinin 6-Benzylaminopurine (BAP, 5 µM) or the ethylene precursor 1-Aminocyclopropane-1-carboxylic acid (ACC, 10 µM). Color code illustrates ΔΔCt values corresponding to log2-fold change (hormone vs. control): blue/green − 1.5 to <−0.5/ downregulated, light green/yellow/orange >−0.5 to < 0.5/ no or minor effect, red/dark red > 0.5 to 1.5/ upregulated, white- not detectable (nd). Figure S3. TCSn::GFP activity in developing barley grains after treatment with NAA and BAP. Two independent transgenic lines (E4 and E7) were examined. Grains were treated either with synthetic auxin (NAA, 50 µM) or cytokinin (BAP, 5 µM) for 16 h. Transverse sections of grains at different developmental timepoints (3-5DAF) with magnification of the ETC region at the maternal-filial boundary, bars = 100 μm. Figure S4. Heat map display of transcript levels of barley TCS genes in grains and vegetative tissues under stress treatment (Fusarium, drought, cold, heat, light/dark). Expression levels (FPKM-values) are displayed by color code. Blue- low, yellow- intermediate, red- high transcriptional levels and white boxes indicating not expressed (FPKM < 1). Abbreviations: 3ADON – Fusarium strain 3ADON; YI – Young inflorescence (see Table S4 for details). Figure S5. HP cluster genes in selected wild barley and landraces compared to cultivar ‘Morex’. (A) Phylogenetic tree of HP cluster elements of seven barley accessions: cultivar Morex, wild barley WBDC133, WBDC184 and B1K-17-07, and landraces 10TJ18, HOR21599 and HOR495. Protein sequences of AHP1 from Arabidopsis and HP1 and HP2 from rice are used for comparison. (B) Table shows reduced number of HP cluster genes in wild barley and landrace accessions. Presence of HP cluster elements in the accessions; multiple copies highlighted. Methods S1. Details of preparation of a cell type/tissue-specific cDNA library.