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Abstract

Two cultivars of Perilla frutescens, red and green formas are known to differ in anthocyanin accumulation in leaves and stems. cDNA clones encoding the enzymes involved in anthocyanin biosynthesis, chalcone synthase (CHS), flavanone 3-hydroxylase (F3H), dihydroflavonol 4-reductase (DFR), and UDP glucose: flavonoid 3-O-glucosyltransferase (3GT), were isolated from cDNA libraries derived from the leaves of a red forma of P. frutescens by screening with partial fragments amplified by means of polymerase chain reaction (PCR) and heterologous cDNAs as probes. The deduced amino acid sequences of these four genes exhibited 40-90% identity with those reported for the corresponding gene from other unrelated species. Southern blot analysis for these genes and two other structural genes, the leucoanthocyanidin dioxygenase (LDOX, anthocyanidin synthase) and anthocyanin acyltransferase (AAT) genes, indicated that each gene comprises a small multi-gene family. More than three copies of the CHS gene are present, two copies of the other genes being present. The expression of five genes, the exception being the CHS gene, was detected only in red leaves of the red forma of P. frutescens, i.e. not in green leaves of the green forma plant. The CHS gene was expressed in both red and green leaves, but 10-fold more in red leaves than in green leaves. These results suggest that the expression of all structural genes examined is coordinately regulated in a forma-specific manner. Under weak-light conditions, the accumulation of both anthocyanin and mRNAs of biosynthetic enzymes was lower in leaves of the red forma. High-intensity white light coordinately induced the accumulation of transcripts of all six genes examined in the mature leaves of red P. frutescens.

Molecular Cloning and Biochemical Characterization of a Novel Anthocyanin 5-O-Glucosyltransferase by mRNA Differential Display for Plant Forms Regarding Anthocyanin

“…Underlining indicates the signature motif generally found in a family of glycosyltransferases. 3r4 -5gt, 5-GT of p3R4 from P. frutescens (this study); 3r6 -5gt, 5-GT homologue of p3R6 from P. frutescens (this study); hgt8 -5gt, 5-GT of pSHGT8 from V. hybrida (this study); per-3gt, 3-GT from P. frutescens (10); gen-3gt, 3-GT from Gentiana triflora (19); mai-3gt, 3-GT from maize (28). gradient elution of 10 -50% acetonitrile with 0.5% trifluoroacetic acid for 20 min, followed by an isocratic elution of 50% acetonitrile with 0.5% trifluoroacetic acid at a flow rate of 0.6 ml/min at 40°C with a monitoring absorbance of 520 nm.…”

“…Miscellaneous Techniques-DNA sequencing and nucleic acid hybridization analysis were carried out as described previously (10). Protein concentration was determined by the method of Bradford (21) using bovine serum albumin as a standard.…”

“…Recently, we have isolated and characterized a series of genes encoding enzymes involved in anthocyanin biosynthesis from P. frutescens (10). These results indicated that the expression of these structural genes was coordinately regulated in an anthocyanin form-specific manner: the expression was observed only in a reddish-purple form but not in a green form (10).…”

“…These results indicated that the expression of these structural genes was coordinately regulated in an anthocyanin form-specific manner: the expression was observed only in a reddish-purple form but not in a green form (10). These results prompted us to examine the possibility of isolating new genes, which are expressed in a form-specific manner, by applying a differential screening system.…”

“…1 in P. frutescens var. crispa (10), in which malonyshisonin is the major anthocyanin (8,11). Cyanidin is glucosylated at the 3-position by UDP-glucose: anthocyanidin 3-O-glucosyltransferase (3-GT).…”