Yellow flowers generated by expression of the aurone biosynthetic pathway

Abstract

Flower color is most often conferred by colored flavonoid pigments. Aurone flavonoids confer a bright yellow color on flowers such as snapdragon (Antirrhinum majus) and dahlia (Dahlia variabilis). A. majus aureusidin synthase (AmAS1) was identified as the key enzyme that catalyzes aurone biosynthesis from chalcones, but transgenic flowers overexpressing AmAS1 gene failed to produce aurones. Here, we report that chalcone 4'-O-glucosyltransferase (4'CGT) is essential for aurone biosynthesis and yellow coloration in vivo. Coexpression of the Am4'CGT and AmAS1 genes was sufficient for the accumulation of aureusidin 6-O-glucoside in transgenic flowers (Torenia hybrida). Furthermore, their coexpression combined with down-regulation of anthocyanin biosynthesis by RNA interference (RNAi) resulted in yellow flowers. An Am4'CGT-GFP chimeric protein localized in the cytoplasm, whereas the AmAS1(N1-60)-RFP chimeric protein was localized to the vacuole. We therefore conclude that chalcones are 4'-O-glucosylated in the cytoplasm, their 4'-O-glucosides transported to the vacuole, and therein enzymatically converted to aurone 6-O-glucosides. This metabolic pathway is unique among the known examples of flavonoid, including anthocyanin biosynthesis because, for all other compounds, the carbon backbone is completed before transport to the vacuole. Our findings herein not only demonstrate the biochemical basis of aurone biosynthesis but also open the way to engineering yellow flowers for major ornamental species lacking this color variant.

Fig. 1.

Flavonoids and their biosynthetic pathway in A. majus. (A) Flower colors in A. majus cultivar (cv.) Snap Yellow (Left) and cv. Merryland Pink (Right). Shown are petal color on the adaxial side of cv. Snap Yellow (B) and of cv. Merryland Pink (C). Fluorescent microcopies in cv. Snap Yellow (D) and Merryland Pink (E). Cross sections show that fluorescence is restricted to the pigmented adaxial epidermis of cv. Snap Yellow (F and G). (Scale bar: 100 μm.) (H) Flavonoid biosynthetic pathway. The 4′ position of chalcones corresponds to the 6 position of aurones and the 7 position of flavanone (naringenin). Dotted arrow, aurone biosynthetic activity of AmAS1 in vitro; thick arrows, the aurone biosynthetic pathway in vivo reported in this study; thin arrows, the anthocyanin biosynthetic pathway; CHS, chalcone synthase; CHI, chalcone isomerase; F3H, flavanone 3-hydroxylase; DFR, dihydroflavonol 4-reductase; ANS, anthocyanindin synthase; 3GT, anthocyanin 3-O-glucosyltransferase; FNS, flavone synthase; AS, aureusidin synthase; 4′CGT, chalcone 4′-O-glucosyltransferase; THC, tetrahydroxychalcone; PHC, pentahydroxychalcone; Glc, glucose.

Fig. 2.

Functional characterizations of Am4′CGT. (A) A phylogenic tree for the flavonoid-related and 18 A. majus glucosyltransferases constructed by a clustal-w program (32), macvector 7.2.2 software (Accelrys, San Diego). The number on the branches indicates sequence difference (0.05 corresponds to a 5% change). UGT88A1 (AAN28841) is an Arabidopsis, function unknown, glucosyltransferase; Am3GT is an Antirrhinum anthocyanidin 3-O-GT; Db5GT (Y18871) is a Dorotheanthus betanidin 5-O-GT; and Gt3′GT (AB076697) is a Gentiana anthocyanin 3′-O-GT. (B) Expression patterns of Antirrhinum flavonoid-related structural genes during flower development. L, leaves; numbers above the lanes, a previously defined developmental flower stage (10). (C) Enzymatic activity of recombinant Am4′CGT protein against THC and PHC. Chromatograms show the absorption at 360 nm in each reaction mixture. HPLC condition is described in Materials and Methods. Standards were eluted at retention times (R.T.) 24.3 (PHC 4′-O-glucoside), 27.7 (THC 2′-O-glucoside), 30.0 (THC 4′-O-glucoside), 32.4 (PHC), and 38.2 min (THC). Arrows indicate the THC 4′-O-glucoside and PHC 4′-O-glucoside synthesized, respectively, by Am4′CGT from THC and PHC.

Fig. 3.

Phenotypic and expression analyses of transgenic Torenia Flowers. (A) Expression analysis of each transgenic line by RT-PCR/Southern blotting. Total RNAs extracted from nontransformant and transgenic flowers were reverse-transcribed then amplified by PCR with the gene-specific primers in Table 2. (B) Phenotypes of transgenic Torenia flowers. (Upper) Flower color under white light. (Lower) Cellular fluorescence from adaxial side of petal in each line. (Scale bars: 100 μm.) NT, nontransformant (T. hybrida cv. Summer Wave Blue).

Fig. 4.

Subcellular localization of Am4′CGT in Antirrhinum petal cells. (A) Control sGFP localizes in the cytoplasm and nucleus. (B) Am4′CGT-GFP fusion protein localizes in the cytoplasm. (C) AmAS1(N_1–60)-mRFP protein localizes in the vacuole lumen. (D) Merged image of B and C. (Scale bar: 20 μm.)