1. Volume 11, Issue 1, Pages 205-217 (January 2018)
Complete Pathway Elucidation and Heterologous Reconstitution of Rhodiola Salidroside Biosynthesis 
Michael P. Torrens-Spence, Tomáš Pluskal, Fu-Shuang Li, Valentina Carballo, Jing-Ke Weng 
Molecular Plant 
Volume 11, Issue 1, Pages (January 2018)
DOI: /j.molp
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2. Figure 1 Salidroside Biosynthesis in R. rosea.
(A) Greenhouse-grown R. rosea.
(B) Metabolic profiling of R. rosea root and crown tissues by LC-HRAM-MS revealed the enrichment of tyrosol and salidroside in the root. Extracted ion chromatogram (XIC) is shown with mass windows set to display the [M−H]− ion for tyrosol and the [M+NH4]+ ion for salidroside. The identity of the metabolites was verified in comparison with authentic standards.
(C) Putative salidroside biosynthetic pathway in Rhodiola. The previously proposed pathway is depicted in black, whereas the newly described rerouted pathway is highlighted in red. Solid black arrow represents previously characterized enzyme, whereas the dotted arrows depict proposed but previously uncharacterized enzymes.
Molecular Plant  , DOI: ( /j.molp )
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3. Figure 2 Identification and Characterization of the Rr4HPAAS.
(A) Multiple sequence alignment highlighting the sequence regions that dictate enzyme activities in select plant AAADs family members. The residue known to dictate decarboxylation versus aldehyde synthase chemistry is denoted by a red asterisk and framed in black. Columns framed in blue indicate greater than 70% conservation of residue physicochemical properties. Identical amino acids are in white font boxed in red, while similar residues are displayed in red font.
(B) A simplified maximum-likelihood (ML) phylogenetic tree of land plant AAADs. A fully annotated version of this tree is shown in Supplemental Figure 6. The three major groups of the tree have been annotated as the basal (green), TyDC (blue), and TDC (red) clades based on taxonomic distribution, clading, and conservation of the substrate-specifying active-site residue. Representative characterized enzymes are labeled at the tree branches, while the R. rosea TDC, AAS, and 4HPAAS are displayed in bold. The scale measures evolutionary distances in substitutions per amino acid.
(C) LC–UV chromatograms of the reaction product of L-tyrosine and Rr4HPAAS enzyme (with and without NaBH4 reduction) in comparison with enzyme assay conducted using PsTyDC as a control. The identity of the products was verified by comparison with authentic standards.
(D) Kinetic characterization of Rr4HPAAS against various aromatic amino acid substrates.
Molecular Plant  , DOI: ( /j.molp )
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4. Figure 3 Identification and Characterization of two R. rosea 4HPARs.
(A) A simplified ML phylogenetic tree of angiosperm ADHs. A fully annotated version of this tree is shown in Supplemental Figure 9. Major clades are annotated based on representative characterized enzymes when possible. The two R. rosea 4HPARs and the previously characterized SlPARs are labeled at the tree branches. The scale measures evolutionary distances in substitutions per amino acid.
(B) LC–MS chromatograms of the reaction product of 4-HPAA and 0.2 μg of recombinant Rr4HPAR1 after incubation for various times.
(C) LC–MS chromatograms of the reaction product of 4-HPAA and 15 μg of recombinant Rr4HPAR2 after incubation for various times. The identity of the tyrosol product was verified by comparison with an authentic standard.
Molecular Plant  , DOI: ( /j.molp )
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5. Figure 4
Identification and Characterization of R. rosea Tyrosol-Modifying UGTs.
(A) An ML phylogenetic tree of 34 R. rosea UGTs together with 88 full-length UGTs encoded by the A. thaliana genome. UGTs that show T4GT and T8GT activities are denoted by black circles and stars, respectively. Bootstrap values (based on 500 replicates) are indicated at the major nodes. The scale measures evolutionary distances in substitutions per amino acid.
(B) Relative in vivo T4GT and T8GT activities of R. rosea UGTs as examined in engineered yeast.
(C) Michaelis–Menten kinetic characterization of four R. rosea tyrosol-modifying UGTs.
Molecular Plant  , DOI: ( /j.molp )
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6. Figure 5
Heterologous Production of Tyrosine-Derived Metabolites in Transgenic N. benthamiana as Detected by LC-HRAM-MS.
(A and B) N. benthamiana transiently expressing Rr4HPAAS or Pc4HPAAS produces both salidroside and icariside D2 (A), while N. benthamiana transiently expressing PsTyDC produces tyramine (B).
(C) N. benthamiana leaves transiently co-expressing Rr4HPAAS and RrT4GT or RrT8GT produce predominantly icariside D2 or salidroside, respectively. XICs are shown with mass windows set to display the [M+NH4]+ ion for salidroside and icariside D2, and the [M+H]+ ion for tyramine. The identity of the metabolites was verified by comparison with authentic standards.
Molecular Plant  , DOI: ( /j.molp )
Copyright © 2017 The Author Terms and Conditions