Fig. 5: Localization of SoGH1 to the cytosol and nucleus.
a, Phylogenetic analysis of GH1 enzymes from S. officinalis and other plant species belonging to the At/Os6 group of the GH1 family. The maximum-likelihood tree (Methods) was generated using an amino acid alignment of putative and characterized (bold) plant GH1 TGs. Bootstrap values less than 80% are shown beside each node. The scale bar indicates the number of amino acid substitutions per site. SFR2 (sensitive to freezing 2)-like enzymes, another subgroup of GH1 family, are used as an outgroup. The side bar to the right shows the SignalP50 score for each sequence. b, Amino acid sequence alignment (generated using ESPript 3.0)65 of the N-terminal regions of all characterized plant GH1 enzymes. Predicted signal peptides are highlighted in green. c, Confocal microscopy images of N. benthamiana leaves transiently coexpressing SoGH1 tagged with C-terminal mRFP (SoGH1:mRFP) and free GFP, both individually and merged. Images were taken 2 days after infiltration. Scale bar, 20 μm. This experiment was performed independently three times with similar results. d, Transient expression of SoGH1:mRFP in N. benthamiana. LC–MS EICs of leaf extracts coexpressing the minimal gene set for 11 with either untagged or mRFP-tagged SoGH1, along with a control leaf expressing only AstHMGR and an authentic QA-TriF(Q)RXX (12) standard, are shown. EICs displayed are m/z 1,657.7115 (calculated [M − H]− of 12). MS/MS spectra for the leaf extracts and the authentic (12) standard are shown at the bottom. The additional peak (12′) is putatively identified as a positional isomer of 12 (Supplementary Fig. 38c).
