Open Access Research article

Evolution of specifier proteins in glucosinolate-containing plants

Jennifer C Kuchernig1, Meike Burow2 and Ute Wittstock1*

Author Affiliations

1 Institute of Pharmaceutical Biology, Technische Universität Braunschweig, Mendelssohnstr. 1, D-38106 Braunschweig, Germany

2 DynaMo Centre of Excellence and VKR Research Centre for Pro-Active Plants, Department of Plant and Environmental Sciences, Faculty of Science, University of Copenhagen, Thorvaldsensvej 40, 1871, Frederiksberg C, Denmark

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BMC Evolutionary Biology 2012, 12:127  doi:10.1186/1471-2148-12-127

Published: 28 July 2012

Additional files

Additional file 1:

Table S1. Glucosinolate classes and hydrolysis product types in species of the Brassicales. The plant organs indicated were analyzed for glucosinolates by HPLC after derivatization to desulfoglucosinolates. For the analysis of glucosinolate hydrolysis products, fresh plant material was homogenized in aqueous solution, and dichloromethane extracts of the homogenates were analyzed by GC-MS. Glucosinolates were assigned to structural classes according to the structure of their side chains as follows: A1, aliphatic: methylthio-/methylsulfinyl-; A2, aliphatic: terminal double bond; A3, aliphatic: terminal double bond and 2-hydroxy-group; A4, aliphatic: other than A1-A3; B1, benzyl-; B2, 4-hydroxybenzyl-; B3, 2-hydroxy-2-phenylethyl-; B4, Phe- and Tyr-derived other than B1-B3; C, indolic. Glucosinolate hydrolysis product types are abbreviated as follows: I, isothiocyanate; N, simple nitrile; EN, epithionitrile; OX, oxazolidine-2-thione; T, organic thiocyanate. An X indicates the identification of at least one representative of a glucosinolate class or a hydrolysis product type in the given plant material (n.i., not investigated). Most species were of the Brassicaceae. For species of other families, the family is given below the species name.

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Additional file 2:

Figure S1. Glucosinolate hydrolysis products in Tropaeolum minus . Fresh leaves (A) or flowers (B) were homogenized in aqueous buffer, and dichloromethane extracts of the homogenates were analyzed by GC-MS. Representative chromatograms (total ion current) are shown. 1, benzylisothiocyanate; 2, phenylacetonitrile; IS, internal standard (phenylcyanide). Chromatograms in A and B were recorded with three years difference explaining the changed retention times.

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Additional file 3:

Figure S2. Alignment of previously known specifier proteins for primer design. Protein names are as given in Table 1. Amino acid sequences were aligned with the ClustalW option implemented in MEGA 5.05 [39]. White letters on black background indicate amino acid residues that are identical in at least 80% of the sequences, white letters on gray background indicate amino acid residues that are similar in at least 80% of the sequences. In case of AtNSP1 and AtNSP2, partial amino acid sequences starting with amino acid 120 and 121, respectively, are shown. For all other proteins, full-length amino acid sequences are included. Green boxes indicate regions of high amino acid identity that were used to design degenerate primers P1, P2, P5, and P6 (Table S2).

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Additional file 4:

Table S2. Oligonucleotide primers for cDNA isolation. Sequences are given starting with the 5' end. Mixed nucleotide codes are: D, A + G + T; K, G + T; M, A + C; R, A + G; S, C + G; W, A + T; Y, C + T. Protein names are as given Table 1.

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Additional file 5:

Figure S3. Assignment of newly identified specifier proteins to the ESP, TFP or NSP type. Activity was tested using crude extracts of bacteria expressing specifier protein cDNAs, 1 mM allylglucosinolate (A-K) or 2 mM benzylglucosinolate (L-V) and 0.005 units myrosinase in 50 mM MES buffer, pH 6.0. Shown are total ion current traces of GC-MS chromatograms recorded of dichloromethane extracts of the assay mixtures after 30 min incubation. Products of allylglucosinolate breakdown are simple nitrile (1), organic thiocyanate (2), isothiocyanate (3), and epithionitrile (4). Products of benzylglucosinolate are simple nitrile (5), and isothiocyanate (6). (X) indicates the peak of the internal standard. The following cDNAs were used: A, L, no cDNA (empty vector control); B, M, ApTFP; C, N, ChESP; D, O, CiESP; E, P, DaESP; F, Q, DlESP; G, R, ItESP; H, S, SpESP; I, T, ChNSP; J, U, ItNSP; K, V, SpNSP.

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Additional file 6:

Table S3. Amino acid and nucleotide sequence identities of specifier proteins/ specifier protein cDNAs. Amino acid sequence identities are shown on the right, nucleotide sequence identities on the left as determined in separate comparisons of each sequence pair by ClustalW implemented in MEGA Vers. 5.05.

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Additional file 7:

Figure S4. Phylogenetic tree of specifier proteins from Brassicaceae. Full-length amino acid sequences of 19 biochemically characterized NSPs, ESPs and TFPs (Table 1) as well as three putative specifier proteins (Table 3) and one homolog of unknown function (At3g07720) were subjected to phylogenetic analysis using the Maximum Likelihood algorithm with 1000 bootstrap repetitions.

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Additional file 8:

Figure S5. Neighbor joining tree of specifier protein cDNAs from Brassicaceae. Full-length nucleotide sequences of 19 biochemically characterized NSPs, ESPs and TFPs (Table 1) as well as three putative specifier proteins (Table 3) and one homolog of unknown function (At3g07720) were subjected to phylogenetic analysis using the Neighbor joining algorithm with 1000 bootstrap repetitions. Bootstrap values are given at the nodes. A homolog from Vitis vinifera (Vitaceae) which does not contain glucosinolates was used as an outgroup. Alignment gaps (e.g. JAL domains that are present only in NSPs) are regarded as non-informative posititions in this analysis. Branch lengths refer to the number of substitutions per site. A scale bar is given below the tree.

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Additional file 9:

Figure S6. Maximum Parsimony tree of specifier protein cDNAs from Brassicaceae. Full-length nucleotide sequences of 19 biochemically characterized NSPs, ESPs and TFPs (Table 1) as well as three putative specifier proteins (Table 3) and one homolog of unknown function (At3g07720) were subjected to phylogenetic analysis using the Maximum Parsimony algorithm with 1000 bootstrap repetitions. Bootstrap values are given at the nodes. A homolog from Vitis vinifera (Vitaceae) which does not contain glucosinolates was used as an outgroup. Alignment gaps (e.g. JAL domains that are present only in NSPs) are regarded as non-informative posititions in this analysis.

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Additional file 10:

Table S4. Amino acid and nucleotide distances of specifier proteins/ specifier protein cDNAs. Amino acid distances are shown on the right, nucleotide distances on the left as determined by MEGA Vers. 5.05 using the Equal-input + G model (daa) and the Kimura-Two-Parameter model + G + I (dna). Alignment gaps (e.g. JAL domains that are present only in NSPs) are regarded as non-informative posititions in this analysis.

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Additional file 11:

Table S5. Non-synonymous vs. synonymous substitution rate ratios of specifier protein cDNAs.dN and dS were calculated by MEGA 5.05 from pairwise comparisons based on the Kimura-2-parameter model and used to determine ω as the dN / dS ratio.

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Additional file 12:

Table S6. Sources of seeds for growing plants for phytochemical analysis and cDNA isolation.

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