Bottlenecks for metabolic engineering of isoflavone glycoconjugates in Arabidopsis

Abstract

In view of their perceived chemopreventive activities against hormone-dependent cancers, cardiovascular disease, and postmenopausal ailments, there is considerable interest in engineering plants to contain isoflavone phytoestrogens. However, attempts to date have only resulted in low levels of isoflavone accumulation in non-legumes. Introducing soybean isoflavone synthase (IFS) into Arabidopsis thaliana leads to accumulation of low levels of genistein glycosides. Leaves of wild-type A. thaliana contain high levels of similar conjugates of the flavonols quercetin and kaempferol, which could be increased by threefold on introduction of an alfalfa chalcone isomerase transgene. Levels of genistein were not increased by expressing both IFS and alfalfa chalcone isomerase, but levels of flavonol conjugates were reduced to a greater extent than could be accounted for by flux into isoflavone. Introduction of IFS into the tt6/tt3 double mutant blocked in flavonol, and anthocyanin synthesis resulted in high levels of genistein. The bottleneck for constitutive isoflavone production in Arabidopsis is, therefore, competition for flavanone between IFS and endogenous flavonol synthesis, and the flavonol pathway is reciprocally but disproportionately affected by IFS.

Fig 1.

Biosynthesis of flavonoids and isoflavonoids, indicating competing pathways for naringenin in transgenic Arabidopsis expressing IFS. Formation of liquiritigenin requires the activity of a chalcone reductase (CHR) that co-acts with chalcone synthase (CHS) to form isoliquritigenin. CHR is present in legumes, but not in Arabidopsis. Daidzein cannot be made in Arabidopsis without introduction of CHR and a legume CHI. Steps blocked in the tt3, tt5, and tt6 mutations are shown. F3H, flavonoid 3-hydroxylase; DFR, dihydroflavonol reductase.

Fig 2.

Expression of IFS and accumulation of genistein conjugates in transgenic Arabidopsis. (A) Binary soybean IFS construct. ³⁵S, cauliflower mosaic virus ³⁵S promoter; B = BamHI; P = PstI; T, pea rubisco 3′ terminator. (B) RNA gel blot analysis of soybean IFS transcripts in Arabidopsis transformants and empty vector controls. (C and D) HPLC profiles of nonhydrolyzed extracts from leaves of an empty vector control plant (C) and line 6E expressing soybean IFS (D). (E and F) HPLC profiles of β-glucosidase-treated extracts from leaves of an empty vector plant (E) and line 6E (F). Most peaks correspond to flavonol glycosides. Genistein conjugates are labeled 1, 2, and 3; free genistein is labeled 4. (G–I) Mass spectrometry total ion chromatographs (TIC) and corresponding mass spectra (Insets) of partially purified genistein conjugate peaks 2 and 3 (G), partially purified genistein conjugate peak 4 (H), and authentic genistein (I).

Fig 3.

Relations between IFS activity and genistein and flavonol levels in transgenic Arabidopsis. (A) Genistein and total flavonols (kaempferol plus quercetin) in T₂ plants from 18 independent IFS primary transformants. Regression analysis, R² = 0.264. (B) IFS activity and genistein levels in T₃ progeny of line 15b. R² = 0.843. (C) Genistein and total flavonols in the same lines as analyzed in B, R² = 0.584. (D) Time course of genistein (•) and kaempferol (▴) levels in a homozygous 15b line.

Fig 4.

Expression of alfalfa CHI in Arabidopsis. (A) Binary alfalfa CHI construct. B, BamHI; S, SalI; T, rubisco 3′ terminator. (B) Complementation of the tt5 mutation by transformation with alfalfa CHI. (C) RNA gel blot analysis of alfalfa CHI transcripts in independent transformants in WT (Col-0) and pap1-D backgrounds. (D) Transgene CHI activities in independent transformants in Col-0 or Col-0/pap1-D backgrounds. (E and F) HPLC profiles of nonhydrolyzed extracts from leaves of an empty vector plant (E) and from lines 4–11 expressing alfalfa CHI (F). (G and H) HPLC profiles of β-glucosidase-treated extracts from leaves of an empty vector plant (G) and from lines 4–11 (H). Compounds are: a, Rha-Gluc-Rha-quercetin; b, Rha-Gluc-Rha-kaempferol; c, Rha-Rha-Q; d, Rha-Rha-K; e, Sinapic acid; f, Gluc-Rha-Q; g, rutin; l, kaempferol, p, quercetin; k and n, unidentified kaempferol conjugates; o, unidentified quercetin conjugate; h, i, j, and m, other unknown flavonol conjugates.

Fig 5.

Isoflavone levels in Arabidopsis impaired in flavonol production. (A–D) HPLC profiles of hydrolyzed extracts from leaves of the tt6/tt3 mutant and WT Ler with and without expression of soybean IFS (T₃ generation). (A) tt6/tt3. (B) tt6/tt3 expressing IFS. (C) Ler WT. (D) Ler expressing IFS. Compounds are: G, genistein; K, kaempferol; Q, quercetin. (E and F) Levels of flavonols (E) and genistein (F) in individual T₃ plants of Ler (eight designations) and tt6/tt3 (nine designations) expressing IFS. White bars, quercetin; black bars, kaempferol.