SEARCH

SEARCH BY CITATION

FilenameFormatSizeDescription
ppl12018-sup-0001-FigureS1.pdfPDF document21K Fig. S1. An unrooted phylogenetic tree of TKPR1 family proteins. Phylogenic analysis by MEGA 5 (Tamura et al. 2011) was performed using the neighbor-joining method and Poisson model for amino acid substitutions. Numbers at tree nodes indicate bootstrap values from 500 replications. The accession numbers for the sequences used in the alignment are listed in Table S1. Scale bar represents 0.1 substitutions per site.
ppl12018-sup-0002-FigureS2.pdfPDF document636K Fig. S2. Multiple alignments of amino acid sequences within TKPR1 family showing high sequence similarity. Asterisks indicate perfectly matched amino acids, colons indicate highly conserved amino acids and dots indicate lowly matched amino acids among the aligned proteins. The putative common NAD(P)H-binding domain is boxed. The dotted boxes indicate the putative active site motifs of the extended short-chain dehydrogenase/reductase-type protein domain. The accession numbers for the sequences used in the alignment are listed in Table S1.
ppl12018-sup-0003-FigureS3.pdfPDF document515K Fig. S3. Expression analysis of NtTKPR1 in tobacco anther section. NtTKPR1 transcripts were localized by in situ hybridization of gene-specific antisense probe and sense probe to transverse sections of wild-type anthers from stage 4 to 8. Anther stage definition is according to Koltunow et al. (1990). C, connective; E, epidermis; En, endothecium; MMC, microspore mother cell; Msp, microspores; PS, pollen sac; T, tapetum; TDS, tetrads; S, stomium. Bars = 100 µm.
ppl12018-sup-0004-FigureS4.pdfPDF document345K Fig. S4. Enzyme activity assays of recombinant NtACOS1, NtPKS1 and NtTKPR1 proteins. 16-Hydroxy palmitic acid and coenzyme A were first incubated with (His)6-tagged NtACOS1. Purified NtPKS1 and malonyl-CoA were then added for further incubation. Reaction products were identified by C18 reverse phase LC–MS/MS analysis operating in negative ionization mode. (A) Tetraketide α-pyrone (m/z 379) was detected in the NtACOS1 + NtPKS1 reactions. (B) MS/MS spectrum for the [M − H] ion at m/z 379. (C) Generation of the reduced tetraketide α-pyrone product (m/z 381) after final incubation in the presence of NtTKPR1 and NADPH. (D) MS/MS spectrum for the [M − H] ion at m/z 381. (E) Reaction steps starting from 16-hydroxy palmitic acid. In the final step, the ketone group in the alkyl chain was reduced to an alcohol function. Expected MS/MS fragmentation patterns are shown for the detected products in (B) and (D).
ppl12018-sup-0005-FigureS5.pdfPDF document357K Fig. S5. Enzyme activity assays of recombinant NtACOS1, NtPKS1 and NtTKPR1 proteins. 12-Hydroxy stearic acid and coenzyme A were first incubated with (His)6-tagged NtACOS1. Purified NtPKS1 and malonyl-CoA were then added for further incubation. Reaction products were identified by C18 reverse phase LC–MS/MS analysis operating in negative ionization mode. (A) Tetraketide α-pyrone (m/z 407) was detected in the NtACOS1 + NtPKS1 reactions. (B) MS/MS spectrum for the [M − H] ion at m/z 407. (C) Generation of the reduced tetraketide α-pyrone product (m/z 409) after final incubation in the presence of NtTKPR1 and NADPH. (D) MS/MS spectrum for the [M − H] ion at m/z 409. (E) Reaction steps starting from 12-hydroxy stearic acid. In the final step, the ketone group in the alkyl chain was reduced to an alcohol function. Expected MS/MS fragmentation patterns are shown for the detected products in (B) and (D).
ppl12018-sup-0006-FigureS6.pdfPDF document183K Fig. S6. Relative abundances of triketide and tetraketide products detected in the NtACOS1 + NtPKS1 reactions. Decanoic acid (A), 16-hydroxy palmitic acid (B) or 12-hydroxy stearic acid was first incubated with (His)6-tagged NtACOS1. Purified NtPKS1 and malonyl-CoA were then added for further incubation. Reaction products were identified by C18 reverse phase LC–MS/MS analysis operating in negative ionization mode. The tetraketide α-pyrones were reduced by NtTKPR1 (Figs 2, S4 and S5). The triketide α-pyrones were not modified by NtTKPR1.
ppl12018-sup-0007-FigureS7.pdfPDF document128K Fig. S7. Production of bisnoryangonin by NtPKS1. p-Coumaroyl CoA and malonyl CoA were incubated with purified NtPKS1. Reaction products were analyzed by C18 reverse phase LC–MS/MS analysis operating in positive ionization mode. The expected CHS product, naringenin chalcone (m/z 273), was not present but the derailment product bisnoryangonin (m/z 231), confirmed by the MS/MS fragmentation, was detected. Therefore, NtPKS1 does not function as a CHS. In vitro formation of bisnoryangonin has been observed with many recombinant PKS. It represents a non-specific condensation of two acetic units with p-coumaroyl CoA, followed by detachment of the derailment product.
ppl12018-sup-0008-FigureS8.pdfPDF document355K Fig. S8. Enzyme activity assays of recombinant NtACOS1, OsPKS1 and OsTKPR1 proteins. 16-Hydroxy palmitic acid and coenzyme A were first incubated with (His)6-tagged NtACOS1. Purified OsPKS1 and malonyl-CoA were then added for further incubation. [Correction added on 9 January 2013, after first online publication: In the preceding sentence, the protein is corrected from NtPKS1 to OsPKS1.] Reaction products were identified by C18 reverse phase LC–MS/MS analysis operating in negative ionization mode. (A) Tetraketide α-pyrone (m/z 379) was detected in the NtACOS1 + OsPKS1 reactions. (B) MS/MS spectrum for the [M − H] ion at m/z 379. (C) Generation of the reduced tetraketide α-pyrone product (m/z 381) after final incubation in the presence of OsTKPR1 and NADPH. (D) MS/MS spectrum for the [M − H] ion at m/z 381. (E) Reaction steps starting from 16-hydroxy palmitic acid. In the final step, the ketone group in the alkyl chain was reduced to an alcohol function. Expected MS/MS fragmentation patterns are shown for the detected products in (B) and (D).
ppl12018-sup-0009-FigureS9.pdfPDF document338K Fig. S9. Enzyme activity assays of recombinant NtACOS1, OsPKS1 and OsTKPR1 proteins. 12-Hydroxy stearic acid and coenzyme A were first incubated with (His)6-tagged NtACOS1. Purified OsPKS1 and malonyl-CoA were then added for further incubation. Reaction products were identified by C18 reverse phase LC–MS/MS analysis operating in negative ionization mode. (A) Tetraketide α-pyrone (m/z 407) was detected in the NtACOS1 + OsPKS1 reactions. (B) MS/MS spectrum for the [M − H] ion at m/z 407. (C) Generation of the reduced tetraketide α-pyrone product (m/z 409) after final incubation in the presence of OsTKPR1 and NADPH. (D) MS/MS spectrum for the [M − H] ion at m/z 409. (E) Reaction steps starting from 12-hydroxy stearic acid. In the final step, the ketone group in the alkyl chain was reduced to an alcohol function. Expected MS/MS fragmentation patterns are shown for the detected products in (B) and (D).
ppl12018-sup-0010-TableS1.pdfPDF document19K Table S1. Gene and expression information for the sequences used in Figs S1 and S2.
ppl12018-sup-0011-TableS2.pdfPDF document192K Table S2. List and information of primers used in this study.

Please note: Wiley Blackwell is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.