Ken-ichi Suzuki

Molecular and biochemical characterization of torenia flavonoid 3'-hydroxylase and flavone synthase II and modification of flower color by modulating the expression of these genes

Abstract Cytochrome P450 (P450) enzymes play important roles in the biosynthesis of flavonoids that determine flower color. Three P450s, flavonoid 3′-hydroxylase (F3′H), flavonoid 3′,5′-hydroxylase (F3′,5′H) and flavone synthase II (FNSII), are involved in torenia flavonoid biosynthesis. In this study, we isolated a full-length cDNA of F3′H from a torenia petal cDNA library. The deduced amino acid sequence of torenia F3′H has 82 and 80% identity to those of Arabidopsis and petunia F3′Hs, respectively. Phylogenetic analysis showed that F3′H and F3′,5′H genes diverted before speciation of higher plants during evolution. Expression of torenia F3′H cDNA in yeast demonstrated that torenia F3′H catalyzed hydroxylation at the 3′ position of naringenin, dihydrokaempferol, kaempferol and apigenin. Km values for these compounds were 0.83, 3.95, 2.96 and 21.5 μM, respectively. Northern analysis showed that the accumulation of anthocyanins and flavones was transcriptionally regulated and that the transcription of the FNSII gene was differently regulated from F3′H and F3′,5′H genes. The torenia, whose F3′,5′H expression had been suppressed, was further transformed with the F3′H gene driven by a constitutive promoter. Some of the transgenic torenia plants had an elevated amount of cyanidin-type anthocyanins and thus redder flower color. Co-suppression of the FNSII gene in the torenia successfully decreased the amount of flavones and increased the amount of flavanones, and yielded paler flower color.

Flower color modifications of Torenia hybrida by cosuppression of anthocyanin biosynthesis genes.

White and blue/white varieties of Torenia hybrida were successfully obtained from the blue variety cv. Summerwave (SWB) by cosuppressing expression of two of the genes involved in anthocyanin biosynthesis; chalcone synthase (CHS) and dihydroflavonol 4-reductase (DFR). Such molecular breeding is the only precise and efficient way to create flower color variation in SWB due to its male and female sterility. Flower color and the degree of suppression varied between transgenic lines, and anthocyanin biosynthesis was more consistently suppressed in the dorsal petal lobes, ventral petal lobes and corolla tube than lateral petal lobes. A pink variety was obtained by cosuppressing the flavonoid 3′,5′-hydroxylase (F3′5′H) gene. Yellow torenia was obtained from T-33, an in-house cultivar that contained both carotenoids and anthocyanins, by cosuppression of CHS or DFR genes.

Intraspecific variability of the tandem repeats in Nicotiana putrescine N-methyltransferases

The putrescine N-methyltransferase (PMT) cDNA clone previously isolated from tobacco encodes a spermidine synthase-like protein with an 11 amino acid element repeated four times in tandem at the amino terminus. Genomic Southern blot analyses indicated that this N-terminal repeat array is found in tobacco PMTs but absent in Hyoscyamus and Atropa PMTs. A truncated tobacco PMT in which this repeat array was entirely removed still retained full enzymatic activity when expressed in Escherichia coli. Three PMT genes (NsPMT1, NsPMT2, NsPMT3) isolated from Nicotiana sylvestris encode two, five, and nine tandem repeats, respectively, in the first exon, but otherwise encode highly conserved proteins. Analysis of PCR fragments amplified from the genomes of N. tabacum and its two probable progenitors shows that one of the nine repeat elements in NsPMT3 was precisely deleted in the corresponding N. tabacum gene. These results indicate that direct tandem repeats of a 33 bp sequence that encodes 11 amino acids of no obvious function were added to the ancestral Nicotiana PMT gene, and that the tandem repetition was genetically very unstable, contracting or expanding during evolution of the Nicotiana species.

Molecular breeding of flower color of Torenia hybrida

Molecular breeding is a powerful method of plant breeding because it can change a specific characteristic of a plant without changing other desirable characteristics. Flower color is predominantly influenced by two types of pigments; flavonoids and carotenoids. The anthocyanin biosynthetic pathways of many plants have been well established (Holton et al, 1995) and conserved (Fig. 1). Chalcone synthase (CHS) and dihydroflavonol 4-reductase (DFR) are the first specific enzymes in flavonoid and anthocyanin biosynthesis, respectively. The presence of flavonoid 3′5′-hydroxylase (F3′5′H), cytochrome P-450 (Holton et al, 1993), is almost critical to the production of blue to purple anthocyanins. The flower color is reddish in its absence.