Differential expression of anthocyanin biosynthesis genes in suspension culture cells of Rosa hybrida cv. Charleston

Abstract

A suspension cultured cell line was established from the cultivar of Rosa hybrida 'Charleston' as a study model to understand the response of the anthocyanin biosynthesis pathway to environmental cues. The major identified anthocyanin in cell cultures was cyanidin 3-glucoside (chrysanthemin). The anthocyanin yield was enhanced by culturing cells in the EM medium with added sucrose at high concentration under additional UV-B radiation to white light. Three cDNA fragments were cloned with degenerate primers by RT-PCR and the obtained sequences shared high homology with putative key enzymes (DFR, ANS, and UF3GT) of other species. The expression levels of these three genes were promoted under optimum conditions for anthocyanin accumulation. These results suggest that expression levels of these genes were closely correlated with a temporal buildup of anthocyanins in response to environmental factors.Key words: Anthocyanin, chrysanthemin, cyanin, Rosa hybrida, suspension cell culture.Plant Biotechnology 23, 379-385 (2006) Original PaperThis article can be found at http://www.jspcmb.jp/ pelargonidin, cyanidin and peonidin type anthocyanins (Biolley et al. 1994). Recently, tissue culturing has become an important biotechnological technique to produce various secondary metabolites including anthocyanins on a mass scale for industrial and academic interest (Hirner et al. 2001;Gantet and Memelink 2002). Despite the fact that some plant species have been shown to produce anthocyanins in cell cultures, little information of the cell culture techniques that are capable of producing in vitro anthocyanins has been reported in rose.The biosynthesis of anthocyanins in plant cell culture is stimulated by not only genetic factors but also environmental factors such as light, temperature, growth regulators and nutrition (Sato et al. 1996;Zhang et al. 1997;Zhang et al. 2002). The identity of the environmental stimuli that lead to the accumulation of anthocyanins in rose is of considerable interest. To date, Sato et al. (1996) has examined the intensity of white light that induces anthocyanin production in strawberry cell culture lines; however, no information was obtained about the effect of light quality on the production of anthocyanins in cultural cells. Therefore, the originality of this research is to investigate anthocyanin biosynthesis in cultural cells exposed to UV-B (290-320 nm) radiation.In this study, we used Rosa hybrida cultivar 'Charleston', a climbing type, originated in Australia. The open flowers of 'Charleston' undergo a striking color change from yellow to red over 10-12 days under natural daylight. We established a suspension cultured cell line from this cultivar of rose. Anthocyanin content was quantified by HPLC to determine the most efficient conditions, such as light including UV-B radiation and media, for continuous production of the pigments in the rose cell suspension. The expression levels of three genes encoding putative key enzymes (DFR, ANS, and UF3GT) for anthocyanin biosynthesis were ana...

Biochemical and molecular characterization of anthocyanidin/flavonol 3-glucosylation pathways in Rosa×hybrida

“…The sequence contained a 496-bp first exon, 187-bp intron, and 908-bp second exon. The open reading frame (RhUF3GT1) showed 99.7% and 97.3% identity to the reported partial UF3GT amino acid sequence (BAF96591) from rose cell culture (Hennayake et al 2006b) and BAF35998 from 'Charleston' petal (Hennayake et al 2007), respectively. The sequence was also closely related to strawberry (Rosaceae) UF3GT (AAU12366, 87%) and apple (Rosaceae) flavonoid 3-galactosyltransferase (BAI44431, 56%).…”

“…The amino acid sequences of these glycosyltransferases are aligned using the CLUSTALW program (available at DDBJ http://clustalw.ddbj.nig.ac.jp/top-e.html) in and the tree is constructed using TREEVIEW (Page 1996). Petunia flavonol 3-galT, Petuniaϫhybrida UDP-galactose: flavonol 3-galactosyltransferase (AAD55985); RhGT4, Rosaϫhybrida RhGT4 (BAE72453); petunia UF3GT, Petuniaϫhybrida UDP-glucose: flavonoid 3-glucosyltransferase (UF3GT, BAA89008); Perilla UF3GT, Perilla frutescens UF3GT (BAA19659); Gentian UF3GT, Gentiana triflora UF3GT (BAA12737); Freesia UF3GT, Freesia hybrida UF3GT (ADK75021); Iris UF3GT, Iris hollandica UF3GT (BAD83701); Citrus UF3GT, Citrus paradise UF3GT (ACS15351); Carnation UF3GT, Dianthus caryophyllus DicGT1 (BAD52003); Apple flavonoid 3-galT, Malusϫdomestica flavonoid 3-galactosyltransferase (BAI44431); Straweberry, Fragaria ananassa UF3GT (AAU12366); Charleston partial UF3GT, rose UF3GT partial sequence from Rosaϫhybrida 'Charleston' (BAF35998, Hennayake et al 2006a); Culture cell partial UF3GT, UF3GT partial sequence from Rosaϫhybrida culture cells (BAF96591, Hennayake et al 2006b); Grape UF3GT, Vitis vinifera FIUFGT2 (BAB41026); Arabidopsis UF3GT, Arabidopsis thaliana UF3GT (AAM91139).…”

“…Mature petals of 'Charleston' and 'Ehigasa' accumulate cyanidin 3-glucoside and cyanidin 3,5-diglucoside (Hennayake et al 2006a). The gene fragments homologous to UFGT were also isolated and their expression was shown to correlate with the accumulation of cyanidin 3-glucoside both in 'Charleston' petals (Hennayake et al 2007) and culture cells (Hennayake et al 2006b). However, their full-length sequences and enzymatic function have not been revealed, and temporal regulation of anthocyanin accumulation in 'Charleston' petals is different from that in typical cultivars where anthocyanin biosynthesis is most active and the biosynthetic genes are expressed most strongly at the flower opening stage (Tanaka et al 1995).…”

“…Transgenic Rosaϫhybrida petals contain delphinidin 3-glucoside as well as delphinidin 3, 5-diglucoside (Katsumoto et al 2007). The petal color of Rosaϫhybrida 'Charleston' changes from yellow to red due to accumulation of cyanidin 3-glucoside after the flower opens (Hennayake et al 2006b). These results indicate that rose petals should have a pathway leading to anthocyanidin 3-glucoside in addition to the pathway catalyzed by A53GT.…”

“…Culture cells derived from leaves treated with UV-B radiation and media stress produced cyanidin 3-glucoside (Hennayake et al 2006b). Mature petals of 'Charleston' and 'Ehigasa' accumulate cyanidin 3-glucoside and cyanidin 3,5-diglucoside (Hennayake et al 2006a).…”