Auronidins are a previously unreported class of flavonoid pigments that challenges when anthocyanin biosynthesis evolved in plants

Abstract

Anthocyanins are key pigments of plants, providing color to flowers, fruit, and foliage and helping to counter the harmful effects of environmental stresses. It is generally assumed that anthocyanin biosynthesis arose during the evolutionary transition of plants from aquatic to land environments. Liverworts, which may be the closest living relatives to the first land plants, have been reported to produce red cell wall-bound riccionidin pigments in response to stresses such as UV-B light, drought, and nutrient deprivation, and these have been proposed to correspond to the first anthocyanidins present in early land plant ancestors. Taking advantage of the liverwort model species Marchantia polymorpha, we show that the red pigments of Marchantia are formed by a phenylpropanoid biosynthetic branch distinct from that leading to anthocyanins. They constitute a previously unreported flavonoid class, for which we propose the name "auronidin," with similar colors as anthocyanin but different chemistry, including strong fluorescence. Auronidins might contribute to the remarkable ability of liverworts to survive in extreme environments on land, and their discovery calls into question the possible pigment status of the first land plants.

Keywords: CRISPR; Marchantia; anthocyanin; flavonoid; liverwort.

Fig. 1.

Selected NMR information from various 1D and 2D NMR spectra used for structural elucidation of auronidin 4-neohesperidoside (1). The main annotated spectrum (¹H-¹³C heteronuclear single quantum coherence [HSQC]) shows ¹J_CH cross-peaks representing the carbon atoms, which are directly connected to hydrogen atoms. The gray arrows in the included structure show selected long-range ¹H-to-¹³C bonding correlations observed as cross-peaks in the heteronuclear multiple bond correlation spectrum, while the 2-sided pink arrows represent ¹H-to-¹H through-space neighborships observed as cross-peaks in the rotating frame Overhauser spectroscopy spectrum. The ¹H NMR spectrum is shown on top and the ¹³C projection of the HSQC spectrum on the left. Pigment 1 is dissolved in CD₃OD–CF₃CO₂D (95:5, vol/vol), and the NMR spectra are recorded at 25 °C. SI Appendix, Table S1 provides the chemical shift values for the proton and carbon atoms.

Fig. 2.

Proposed biosynthetic relationships among aurones, auronidins, and anthocyanidins and comparison of the UV/visible-light spectra of corresponding glycosides. (A) Marchantia plants with a knockout mutation of the single chi gene showed no detectable flavones but were unaltered in auronidin production compared with WT plants. Plants with a knockout mutation of the candidate ppo aurone biosynthetic gene showed reduced auronidin content but were unaltered in flavone production. (B) Structures and corresponding UV/visible-light spectra recorded in acidified methanol of an aurone, aureusidin 4-glucoside (3) (λ_max = 402 nm); an auronidin, auronidin 4-neohesperidoside (1) (λ_max = 495 nm); and an anthocyanin, cyanidin 3-glucoside (4) (λ_max = 529 nm).

Fig. 3.

¹H NMR spectra, colors, and fluorescence of auronidin 4-neohesperidoside from Marchantia in solvents with different acidities, demonstrating lack of some typical anthocyanin properties. (A and B) Auronidin 4-neohesperidoside (1) dissolved in methanol containing various amounts of TFA. The aromatic regions of the corresponding ¹H NMR spectra (A) show that the color changes from orange to pink-purple (B) are in accordance with increased amounts of the anionic form of 1 in an acid-base equilibrium when the acid content is reduced. The lack of additional peaks in the NMR spectra prove the absence of various anthocyanin equilibrium forms expressing different colors. (C) Auronidin 4-neohesperidoside dissolved in deuterated dimethyl sulfoxide containing 5% TFA in 365-nm UV light. (D) Comparison of auronidin 4-neohesperidoside and the anthocyanin cyanidin 3-glucoside dissolved in methanol containing 0.5% TFA (acidic) or 0.5% 0.2 M NaOH (alkaline). The samples were illuminated by a 365-nm UV lamp (Top) or ambient light (Bottom). The anthocyanin in alkaline is not shown, as it degraded in that solvent.

Fig. 4.

Identification of transcripts associated with MpMYB14-regulated auronidin production. (A) Differential transcript abundance for WT vs. myb14 mutant Marchantia lines during nutrient deprivation stress, as determined using RNA-seq DESeq2, with an adjusted P value <0.001 and log2 fold increase >1.0 cutoff. The number of transcripts up- or down-regulated are shown on the left, and details of the 20 up-regulated transcripts having the lowest adjusted P value are shown in the table on the right. (B) Comparison of the 121 up-regulated transcripts from the WT-stress (WTS) vs. myb14-stress (myb14S) analysis to those up-regulated between a 35S:MYB14 transgenic and WT, with an adjusted P value <0.001 and log2 fold increase >1.0 cutoff. Details of the 22 transcripts up-regulated in both comparisons are shown on the right. In both A and B, candidate transcripts related to phenylpropanoid biosynthesis are highlighted in green.

Fig. 5.

Marchantia genetic mutants showing that auronidins are produced by a different branch of the flavonoid pathway than that producing anthocyanins. WT and mutant Marchantia plants were induced to synthesize red auronidin pigments by nutrient deprivation. Lines contain mutations in flavonoid biosynthetic (chi, chil, and ppo) or regulatory (myb14) genes. (A) Representative pigmentation phenotypes. (B and C) Flavone and auronidin contents. n = 3 biological replicates ± 95% confidence interval. ND, not detected. Means that are significantly different are indicated by different letters (a, b, c, and d).