Characterization of Two Key Flavonoid 3- O-Glycosyltransferases Involved in the Formation of Flower Color in Rhododendron Delavayi

Abstract

Flower color, largely determined by anthocyanin, is one of the most important ornamental values of Rhododendron delavayi. However, scant information of anthocyanin biosynthesis has been reported in R. delavayi. We found that anthocyanidin 3-O-glycosides were the predominant anthocyanins detected in R. delavayi flowers accounting for 93.68-96.31% of the total anthocyanins during its development, which indicated the key role of flavonoid 3-O-glycosyltransferase (3GT) on R. delavayi flower color formation. Subsequently, based on correlation analysis between anthocyanins accumulation and Rd3GTs expressions during flower development, Rd3GT1 and Rd3GT6 were preliminarily identified as the pivotal 3GT genes involved in the formation of color of R. delavayi flower. Tissue-specific expressions of Rd3GT1 and Rd3GT6 were examined, and their function as 3GT in vivo was confirmed through introducing into Arabidopsis UGT78D2 mutant and Nicotiana tabacum plants. Furthermore, biochemical characterizations showed that both Rd3GT1 and Rd3GT6 could catalyze the addition of UDP-sugar to the 3-OH of anthocyanidin, and preferred UDP-Gal as their sugar donor and cyanidin as the most efficient substrate. This study not only provides insights into the biosynthesis of anthocyanin in R. delavayi, but also makes contribution to understand the mechanisms of its flower color formation.

Keywords: Rhododendron delavayi; anthocyanin; enzyme activity; flavonoid 3-O-glycosyltransferases; flower color.

Figure 1

Anthocyanin component analyses in R. delavayi flower. (A) The phenotypes of different development stage. (B) Total anthocyanin profile at different developmental stages. (C) HPLC profiles of anthocyanin. A1, delphinidin 3-O-galacoside; A2, cyanidin 3-O-galacoside; A3, delphinidin rhamnoside; A4, cyanidin 3-O-glucoside; A5, cyanidin 3-O-arabinoside; A6/A7/A8, other cyanidin derivatives. (D) The contents of different anthocyanin at five developmental stages.

Figure 2

Expression profiles of Rd3GTs during flower development (A) and Pearson's correlation coefficient between Rd3GTs transcript levels and anthocyanin contents in R. delavayi flower (B). 1–5 represent the flowers of different developmental stages.

Figure 3

Sequence alignment and phylogenetic analyses of Rd3GT1 and Rd3GT6. (A) Sequence alignment of Rd3GT1 and Rd3GT6 with UGT78D2 (Arabidopsis thaliana, NM_121711.5) and UGT78G1 (Medicago sativa, A6XNC6). The PSPG motif that interacts with the sugar donor is underlined. (B) Phylogenetic analyses of the deduced amino acids of Rd3GT1 and Rd3GT6 and UFGTs from different plant species. GenBank accession numbers are as follows: VvFd3GT (Vitis vinifera AAB81683), Vl3GT (Vitis labrusca ABR24135), Mt3GT (Medicago truncatula XP_003610163), At3GT (Arabidopsis thaliana, NM_121711.5), Gt3GT (Gentiana triflora Q96493.1), Ph3GT (Petunia hybrida AB027454), Sm3GT (Solanum melongena Q43641), Zm3GT (Zea mays CAA31856), Hv3GT (Hordeum vulgare CAA33729), Ih3GT (Iris hollandica BAD83701), Fh3GT1 (Fressia hybrida ADK75021.1), Ih5GT (Iris hollandica BAD06874), Gt5GT (Gentiana triflora BAG32255), At5GT (Arabidopsis thaliana NP_193146), Pf5GT (Perilla frutescens BAA36421), Th5GT (Torenia hybrid BAC54093), Ph5GT (Petunia hybrida AB027455), Vh5GT (Verbena hybrida AB076698), Sb7GT (Scutellaria baicalensis BAA83484), At73B2 (Arabidopsis thaliana NM_179161.2), At73C6 (Arabidopsis thaliana NM_129234.2), Ac73G1 (Allium cepa AAP88406), GmIsoflv7GT (Glycine max ABB85236.1). Rd3GT1 and Rd3GT6 are highlighted in red stars.

Figure 4

Expression profiles of Rd3GT1 and Rd3GT6 in different tissues of R. delavayi. Pe, petals; To, toruses; Sc, scapes; Pi, pistils; St, stamens; Ro, roots; Le, leaves.

Figure 5

Complementation of the pigmentation of UGT78D2 mutant seedlings with Rd3GT1 and Rd3GT6. (A) Phenotypes of wild-type, mutant, and transgenic Arabidopsis seedlings. (B) Contents of anthocyanins in Arabidopsis seedlings. (C) Expressional analysis of Rd3GT1 and Rd3GT6 by reverse transcription polymerase chain reaction. (D) HPLC analyses of anthocyanins in Arabidopsis seedlings. Data correspond to means of three biological replicates. Asterisks indicate significant differences between means of mutant and wild-type as well as transgenic plants calculated by Student's t-test (^***p < 0.001).

Figure 6

Effect of Rd3GT1 and Rd3GT6 on anthocyanin accumulation in transgenic tobacco flowers. (A) Tobacco flowers of wild-type and transgenic lines. (B) Contents of anthocyanin accumulation in transgenic tobacco flowers with HPLC. (C) Expression confirmation of Rd3GT1 and Rd3GT6 in flowers of transgenic tobacco. (D) HPLC analyses of anthocyanins in transgenic tobacco flowers. 1, Cyanidin 3-O-rutinoside 5-O-glucoside; 2, Cyanidin 3-O-arabinoside; 3, Cyanidin 3-O-rutinoside; 4, Cyanidin 3-O-xyloside; 5, Cyanidin 3-O-(6-O-malonyl-beta-D-glucoside). Results correspond to means from three biological replicates. Asterisks indicate significant differences between means of wild-type and transgenic plants calculated by Student's t-test (^**p < 0.01); ^***p < 0.001.

Figure 7

HPLC profiles of Rd3GT1 and Rd3GT6 reaction products with UDP-Glu and different anthocyanidin. (A) Cyanidin. P1/P5/S1, cyanidin 3-O-glucoside; P2, cyanidin 4'-O-glucoside, (B) Delphinidin. P3/P6/S2, delphinidin 3-O-glucoside; P4, delphinidin 4'-O-glucoside, (C) Pelargonidin. P7/S3, pelargonidin 3-O-glucoside, (D) Peonidin. P8/S4, peonidin 3-O-glucoside, (E) Petunidin. P9/S5, petunidin 3-O-glucoside, and (F) Malvinidin. P10/S6, malvinidin 3-O-glucoside. Cya-3-O-G, cyanidin 3-O-glucoside; Del-3-O-G, delphinidin 3-O-glucoside; Pel-3-O-G, pelargonidin 3-O-glucoside; Peo-3-O-G, peonidin 3-O-glucoside; Pet-3-O-G, petunidin 3-O-glucoside; Mal-3-O-G, malvinidin 3-O-glucoside.

Figure 8

Relative activity of Rd3GT1 and Rd3GT6 toward several substrates and sugar donor. (A) Relative activity of Rd3GT1 and Rd3GT6 toward UDP- Gal and six anthocyanidins. The relative activity was calculated by Rd3GT1 activity toward delphinidin as 100%. Cya, cyanidin; Del, delphinidin; Pel, pelargonidin; Peo, peonidin; Pet, petunidin; Mal, malvinidin. (B) HPLC profiles of Rd3GT6 reaction products with UDP-Rha and cyanidin/delphinidin. P1, cyanidin 3-O-rhamnoside; P2, cyanidin 4'-O-rhamnoside; P3, delphinidin 3-O-rhamnoside; P4, delphinidin 4'-O-rhamnoside.

Figure 9

Proposed pathway leading to anthocyanin biosynthesis in the flowers of R. delavayi.