Evaluation of pathways to the C-glycosyl isoflavone puerarin in roots of kudzu (Pueraria montana lobata)

Abstract

Kudzu (Pueraria montana lobata) is used as a traditional medicine in China and Southeast Asia but is a noxious weed in the Southeastern United States. It produces both O- and C-glycosylated isoflavones, with puerarin (C-glucosyl daidzein) as an important bioactive compound. Currently, the stage of the isoflavone pathway at which the C-glycosyl unit is added remains unclear, with a recent report of direct C-glycosylation of daidzein contradicting earlier labeling studies supporting C-glycosylation at the level of chalcone. We have employed comparative mRNA sequencing of the roots from two Pueraria species, one of which produces puerarin (field collected P. montana lobata) and one of which does not (commercial Pueraria phaseoloides), to identify candidate uridine diphosphate glycosyltransferase (UGT) enzymes involved in puerarin biosynthesis. Expression of recombinant UGTs in Escherichia coli and candidate C-glycosyltransferases in Medicago truncatula were used to explore substrate specificities, and gene silencing of UGT and key isoflavone biosynthetic genes in kudzu hairy roots employed to test hypotheses concerning the substrate(s) for C-glycosylation. Our results confirm UGT71T5 as a C-glycosyltransferase of isoflavone biosynthesis in kudzu. Enzymatic, isotope labeling, and genetic analyses suggest that puerarin arises both from the direct action of UGT71T5 on daidzein and via a second route in which the C-glycosidic linkage is introduced to the chalcone isoliquiritigenin.

FIGURE 1

Potential pathways leading to puerarin in kudzu roots.The enzymes are: CHS, chalcone synthase; CHR, chalcone reductase; CHI, chalcone isomerase; 2‐HIS (IFS), 2‐hydroxyisoflavanone synthase; IOMT, isoflavone O‐methyltransferase; HID, trihydroxyisoflavanone dehydratase; GT, glucosyltransferase. (CHS and CHR co‐act to generate 6′‐deoxy chalcone [isoliquiritigenin], rather than acting sequentially as depicted for ease of comparison with the pathway to genistein). Hollow arrows with numbers indicate possible steps for introduction of the C‐glycosyl substituent of puerarin. Hypothetical reactions specific for puerarin biosynthesis are shown in red.

FIGURE 2

Levels of isoflavones and isoflavone pathway gene transcripts in mature root extracts from P. m. lobata and P. phaseoloides . (a‐c), HPLC chromatograms showing isoflavone profiles in P. m. lobata (a) and P. phaseoloides (b) and standards (c). mAU, milli‐absorbance units. 1, puerarin; 2, daidzin; 3, genistin; 4, ononin; 5, daidzein; 6, genistein; 7, formononetin. (d), transcript levels of CHS, CHR, CHI, 2‐HIS and 2‐HID genes in roots of P. m. lobata and P. phaseoloides , based on RNA sequencing (Adolfo et al., 2022).

FIGURE 3

Relative transcript levels of UGT candidates in root and leaf tissue of P. m. lobata.The candidate glycosyltransferase transcript levels were adjusted to the housekeeping gene elf5. The black bars represent root tissue and the light gray bars represent leaf tissue. Bars show means plus standard deviations for at least three averaged technical replicates of three biological replicates. Numbers on X axis indicate UGT candidates. Note the different scale on the insets.

FIGURE 4

Phylogenetic relationship of candidate UGTs with known plant UGTs with different regiospecificities.P. m. lobata UGTs identified here are shown with number designations. A small square identifies multi‐site UGTs. A small circle identifies UGTs that have been characterized as having activity different from the group with which they cluster. The tree was made from a protein alignment (MUSCLE) (Edgar, 2004) using ATGC PhymL 2.2.4 (Guindon et al., 2010) with 1,000 bootstrap replicates in Geneious prime 2022.0.1. The scale bar indicates the length of .5 substitutions. GenBank accession numbers are given in supplemental Table S2.

FIGURE 5

In vitro activity of recombinant UGT71T5. (a), SDS‐PAGE gel showing purification of UGT71T5. Lanes are 1, induced; 2, uninduced; 3, soluble; 4, insoluble; 5, elution 1; 6, elution 2; 7, elution 3; 8, protein MW markers. (b), extracted ion chromatogram (EIC) at m/z 415 for examination of the product of UGT71T5 with daidzein. (c), EIC at m/z 417 for examination of the product of UGT71T5 with isoliquiritigenin. (d), EIC at m/z 433 for examination of the product of UGT71T5 with genistein.

FIGURE 6

Expression of UGT71T5 in Medicago truncatula hairy roots. (a), appearance of M. truncatula hairy root cultures expressing UGT71T5. There were no observed phenotypic differences between M. truncatula hairy root cultures expressing UGT71T5 versus the GUS control gene. (b), relative transcript levels of UGT71T5 in M. truncatula hairy roots expressing UGT71T5 or GUS as compared to elf5. Bars show means and standard deviations from four technical replicates per hairy root line. (c), extracted ion chromatograms of extracts from M. truncatula hairy roots expressing UGT71T5 or GUS at m/z 415 for puerarin. (d), extracted ion chromatograms of extracts from M. truncatula hairy roots expressing UGT71T5 or GUS at m/z 431 for C‐glycosyl genistein. Data for additional independent hairy root lines are given in supplemental Figure S10.

FIGURE 7

Down‐regulation of UGT71T5 in P. m. lobata hairy roots. (a), relative transcript levels of UGT71T5 and other isoflavonoid pathway genes in UGT71T5‐RNAi and GUS control lines. CHI 1, chalcone isomerase 1; CHI 2, chalcone isomerase 2; 2‐HIS, 2‐hydroxyisoflavanone synthase; 2‐HID, 2‐hydroxyisoflavanone dehydratase. Bars show means and standard deviations from at least three technical replicates. (b), (Iso)flavonoid levels in UGT71T5 knockdown lines as compared to the GUS control. Data are means from three biological replicates. * = significant at p < .05 and ** = significant at p < .01, unpaired t‐test.

FIGURE 8

Consequences of down‐regulation of 2‐HIS in hairy roots of P. m. lobata. (a‐b), phenotypic comparison of kudzu hairy roots growing on solid medium. (a), GUS control roots and (b), 2‐HIS RNAi roots showing yellow coloration. (c), relative transcript levels of 2‐HIS and other genes in the flavonoid pathway in 2‐HIS RNAi and GUS control lines. CHI 1, chalcone isomerase 1; CHI 2, chalcone isomerase 2; 2‐HIS, 2‐hydroxyisoflavanone synthase; 2‐HID, 2‐hydroxyisoflavanone dehydratase. Data show means and standard deviations for at least three technical replicates. (d), levels of flavonoid compounds in 2‐HIS RNAi lines as compared to the GUS control. Data are means from 3 biological replicates. ** = significant at p < .01 and *** = significant at p ≤ .0005, unpaired t‐test. (e), EICs for (iso)liquiritigenin glycosides at m/z 417 in extracts from GUS control and 2‐HIS RNAi hairy roots. (f), EICs at m/z 417 after treatment of flavonoid extracts from 2‐HIS RNAi hairy roots with β‐glucosidase. (g), UV spectra of potential chalcone/flavanone glycosides at m/z 417 in extracts from 2‐HIS RNAi hairy roots. Spectra correspond to compounds in panel F at the following retention times: 1, 9.4 min; 2, 11 min; 3, 14.7 min; 5, 15 min; 6, 15.2 min. 4 and 7 are liquiritigenin and isoliquiritigenin standards, respectively. Data for additional independent hairy root lines are given in supplemental Figure S12.

FIGURE 9

UGT71T5 is involved in C‐glycosylation of early puerarin precursors in P. m. lobata hairy roots. (a‐c), identification of chalcone/flavanone C‐glycosides using a triple‐TOF high resolution mass spectrometer. Extracted ion chromatograms at m/z 417 of flavonoid extracts from (a), 2‐HIS RNAi; (b), UGT71T5 RNAi; (c), GUS control kudzu hairy roots. (d), extracted ion chromatogram of extract from 2‐HIS RNAi hairy roots before and after incubation with recombinant 2‐HIS at m/z 271 for 2‐hydroxyisoflavanone. (e), extracted ion chromatogram of extract from 2‐HIS RNAi hairy roots before and after incubation with recombinant 2‐HIS at m/z 433 for 2‐hydroxyisoflavanone C‐glycoside.

FIGURE 10

Down‐regulation of 2‐HID reduces daidzein levels in kudzu hairy roots. (a), relative transcript levels of 2‐HID and other enzymes of isoflavone biosynthesis in 2‐HID RNAi lines. CHI 1, chalcone isomerase 1; CHI 2, chalcone isomerase 2; 2‐HIS, 2‐hydroxyisoflavanone synthase; 2‐HID, 2‐hydroxyisoflavanone dehydratase. Data show means and standard deviations for at least three technical replicates. (b), levels of flavonoid compounds in 2‐HID RNAi lines as compared to a GUS control. Data are means from three biological replicates. * = significant at p < .05, unpaired t‐test. (c), EIC of hairy root extracts from 2‐HID RNAi (top) and GUS control (bottom) at m/z 271 (for 2‐hydroxyisoflavanone). (d), EIC of hairy root extracts from 2‐HID RNAi (top) and GUS control (bottom) at m/z 433 (for a C‐glycoside of 2‐hydroxy‐isoflavanone).

FIGURE 11

A model for the biosynthesis of puerarin involving UGT71T5 in both free and channel‐associated forms. (a), The enzymatic synthesis of puerarin through channel‐associated UGT71T5 proceeding via C‐glycosylation of isoliquiritigenin. (b), The enzymatic synthesis of puerarin following the formation of daidzein with free UGT71T5. Enzyme names as in legend to Figure 1. NE, non‐enzymatic.