Computational protocol: Acyl substrate preferences of an IAA-amido synthetase account for variations in grape (Vitis vinifera L.) berry ripening caused by different auxinic compounds indicating the importance of auxin conjugation in plant development

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Protocol publication

[…] GH3-related plant sequences were identified by BLASTP searches of the non-redundant protein database (P value ≤10−20) using the Vitis vinifera L. GH3-1 protein sequence as the query. Non-redundant sequences that, based on comparison with the grapevine GH3-1 protein, were considered full-length were included in the analysis. NCBI or GenBank accession numbers for all sequences used in the phylogenetic analysis are listed in Supplementary Table S1 at JXB online. The sequences were aligned using the ClustalX (version 2.0.10) program () in the multiple alignment mode and the Neighbor–Joining unrooted tree was generated with PHYLIP 3.6 (). The Invitrogen Vector NTI AlignX software (version Advanced 10) was used to determine sequence similarities and identities. [...] To identify suitable protein amounts and incubation times to be used for the determination of steady-state kinetic parameters for GH3-1 and GH3-2 the following standard assay conditions were used: 50 mM TRIS/HCl (pH 8.6), 3 mM MgCl2, 3 mM ATP, 1 mM DTT, 1 mM aspartic acid (Asp) or tryptophan (Trp), 1 mM IAA, 25 °C, 50 μl volume. The reactions were initiated by adding 0–1 μg of purified GH3-1 or GH3-2 from glycerol stocks [10% (v/v) glycerol, 1 mM DTT, 5–10 mg ml−1 protein, stored at –80 °C for up to one month] and stopped after 0–20 min by adding 10 μl 50% (v/v) HCl. After the addition of 250–750 pmol of labelled IAA–-Asp or IAA–Trp as internal standards the samples were extracted twice with 150 μl ethyl acetate. The extract was dried and the residue resuspended in 30 μl 60% (v/v) MeOH, 1% (v/v) acetic acid to be analysed by LC-MS. Steady-state parameters for both enzymes were determined using the standard assay but varying the concentration of one substrate at a time: (0–20 mM) Asp, (0–20 mM) Trp, (0–5 mM) ATP, (0–2 mM) auxins (IAA, NAA or BTOA). Reactions were stopped after 0, 2, 5, and 10 min, appropriate labelled standards were added and the samples were acidified to pH 1–2. Samples were extracted as described above and subjected to LC-MS analysis (below). The reactions products were quantified, initial velocities were calculated from the linear range and fit to the Michaelis–Menten equation using non-linear regression (SigmaPlot 11.0). […]

Pipeline specifications

Software tools BLASTP, Clustal W, PHYLIP, Vector NTI Advance, SigmaPlot
Applications Miscellaneous, Phylogenetics, Protein sequence analysis
Organisms Vitis vinifera