Additional file 1: of Biochemical properties and subcellular localization of six members of the HXK family in maize and its metabolic contribution to embryo germination

Information Figure S1 and Table S1. Figure S1. Molecular phylogenetic analysis for plant HXKs. Table S1. ID numbers of the HXK gene families used for the phylogenetic analysis. Table S2. Comparison of conserved amino acids at catalytic and substrate binding domains between ZmHXKs with AtHXK1 [33]. Figure S2. SDS-PAGE of full versions of recombinant ZmHXKs. Purification process of recombinant: (A) ZmHXK4, (B) ZmHXK5, (C) ZmHXK6, (D) ZmHXK7, and (E) ZmHXK8. St: Molecular weight standard, P: Pellet clarified with urea, S: Soluble supernatant, U: Unbound, W: Wash, EX: Elution. Figure S3. Purification of truncated versions of recombinant ZmHXKs. Purification process of recombinant: (A) ZmHXK4Δ30, (B) ZmHXK5Δ30, (C) ZmHXK6Δ30 and (D) ZmHXK9Δ30. All fractions were separated by SDS-PAGE and stained with Coomasie blue. (E) Western Blot of full and truncated versions of ZmHXK4-6. The proteins were detected using the anti-V5-HRP. St: Molecular weight standard, T: Total cell extract S: Soluble supernatant, U: Unbound, W: Wash, EX: Elution. Figure S4. Inhibitory effects of ADP, NAG and G6P on ZmHXKs. (A, B, C) ZmHXKΔ4, (D, E, F) ZmHXKΔ5, (G, H, I) ZmHXKΔ6, (J, K, L) ZmHXK7, and (M, N, O) ZmHXK8. Figure S5. Expression profile of full and truncated versions of ZmHXKs in the JT 20088 yeast mutant. Table S3. Subcellular prediction of ZmHXKs.Figure S6. Evaluation of cytosolic and mitochondrial purity using specific antibodies. The purity of the cytosolic (Cyt), mitochondrial washed (wMit) and mitochondrial Percoll purified (pMit) fractions (10 µg) was evaluated by Western blot using Agrisera (Vännäs, Sweden) antibodies. These are representative membranes of at least three replicates. Figure S7. Changes in the amino acids of ZmHXK9 that could explain its low activity. The sequences were aligned using SeaView 4 [33]. Table S4. List of primers used for qPCR analysis, subcloning each maize HXK and PCR analysis in the yeast mutant. Uniprot1 ( https://www.uniprot.org/uniprot The UniProt Consortium. UniProt: the universal protein knowledgebase. Nucleic Acids Res. 2017;45:D158–9), PLAZA2 ( https://bioinformatics.psb.ugent.be/plaza/ ), EnsemblPlants3 ( http://plants.ensembl.org/index.html ) and NCBI4 ( https://www.ncbi.nlm.nih.gov/ ). Table S2 Comparison of conserved amino acids at catalytic and substrate binding domains between maize HXKs with AtHXK1 [33]. Figure S2: (A) ZmHXK4, (B) ZmHXK5, (C) (PDF 1360 kb)