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Flower color is mainly determined by anthocyanins. Rosa hybrida lacks violet to blue flower varieties due to the absence of delphinidin-based anthocyanins, usually the major constituents of violet and blue flowers, because roses do not possess flavonoid 3',5'-hydoxylase (F3'5'H), a key enzyme for delphinidin biosynthesis. Other factors such as the presence of co-pigments and the vacuolar pH also affect flower color. We analyzed the flavonoid composition of hundreds of rose cultivars and measured the pH of their petal juice in order to select hosts of genetic transformation that would be suitable for the exclusive accumulation of delphinidin and the resulting color change toward blue. Expression of the viola F3'5'H gene in some of the selected cultivars resulted in the accumulation of a high percentage of delphinidin (up to 95%) and a novel bluish flower color. For more exclusive and dominant accumulation of delphinidin irrespective of the hosts, we down-regulated the endogenous dihydroflavonol 4-reductase (DFR) gene and overexpressed the Irisxhollandica DFR gene in addition to the viola F3'5'H gene in a rose cultivar. The resultant roses exclusively accumulated delphinidin in the petals, and the flowers had blue hues not achieved by hybridization breeding. Moreover, the ability for exclusive accumulation of delphinidin was inherited by the next generations.

Vacuolar pH plays an important role in flower coloration: an increase in the vacuolar pH causes blueing of flower color. In the Japanese morning glory (Ipomoea nil or Pharbitis nil), a shift from reddish-purple buds to blue open flowers correlates with an increase in the vacuolar pH. We describe details of the characterization of a mutant that carries a recessive mutation in the Purple (Pr) gene encoding a vacuolar Na+/H+ exchanger termed InNHX1. The genome of I. nil carries one copy of the Pr (or InNHX1) gene and its pseudogene, and it showed functional complementation to the yeast nhx1 mutation. The mutant of I. nil, called purple (pr), showed a partial increase in the vacuolar pH during flower-opening and its reddish-purple buds change into purple open flowers. The vacuolar pH in the purple open flowers of the mutant was significantly lower than that in the blue open flowers. The InNHX1 gene is most abundantly expressed in the petals at around 12 h before flower-opening, accompanying the increase in the vacuolar pH for the blue flower coloration. No such massive expression was observed in the petunia flowers. Since the NHX1 genes that promote the transport of Na+ into the vacuoles have been regarded to be involved in salt tolerance by accumulating Na+ in the vacuoles, we can add a new biological role for blue flower coloration in the Japanese morning glory by the vacuolar alkalization.

We have isolated and characterized a cDNA, PfAT208, encoding hydroxycinnamoyl-CoA: anthocyanin 3-O-glucoside-6"-O-acyltransferase (3AT) from Perilla frutescens. The identity of the cDNA was established by determination of the reaction products with recombinant enzyme overexpressed in Escherichia coli. The deduced amino acid sequence has a few regions that are conserved in a CoA-dependent acyltransferase family. The recombinant enzyme produced in yeast could utilize cyanidin 3-glucoside and cyanidin 3,5-diglucoside, putative substrates in vivo, as well as other anthocyanins. The inhibitory effects of diethyl pyrocarbonate and N-ethylmaleimide on the recombinant 3AT activities suggest that histidine and cysteine residues are important for their catalytic function. These properties are in common with anthocyanin 5-O-glucoside-6"-O-acyltransferase (5AT). In Northern analysis, a transcript of PfAT208 was detected in the young leaves of perilla red forma. The properties of other cDNAs, gentian GAT106 and petunia PhAT48, isolated during the above cloning procedure are also described.

Cytochrome P450 cDNAs, AFNS2 and TFNS5, were isolated from snapdragon and torenia petal cDNA libraries, respectively, based on the sequence homology with licorice CYP93B1 cDNA encoding (2S)-flavanone 2-hydroxylase. They were expressed in yeast and identified to encode flavone synthase II catalyzing direct conversion of flavanones to flavones probably via 2-hydroxyflavanones.

Two cDNAs, ADS1 and ADS2, were isolated from Arabidopsis. These cDNAs encoded proteins homologous to delta 9 acyl-lipid desaturases of cyanobacteria and acyl-CoA desaturases of yeast and mammals. The expression of ADS1 and ADS2 was organ-dependent. Cold temperature up-regulated the ADS2 expression, whereas it down-regulated the ADS1 expression.

Full length cDNA clones of flavonoid 3',5'-hydroxylase, dihydroflavonol 4-reductase and flavonoid 3-glucosyltransferase were cloned from petals of Gentiana triflora. Their sequences were homologous to counterparts from other plants. Flavonoid 3',5'-hydroxylase and flavonoid 3-glucosyltransferase were enzymatically characterized by expressing cDNAs in heterologous expression systems.

A full length cDNA clone encoding rose dihydroflavonol 4-reductase (DFR) was isolated from a cDNA library derived from rose petals by screening with the cDNA of Petunia hybrida DFR. Sequence comparison of the rose DFR with reported DFR genes revealed that they are homologous to each other. The amount of DFR mRNA in rose petals was developmentally regulated and paralleled anthocyanin production in petals. Sepals, thorns, styles and stamens also contained anthocyanins and DFR mRNA. No DFR mRNA was observed in mature leaves and a small amount of the transcript was detected in young leaves. A petunia cultivar, whose colour was pale pink due to a deficiency in flavonoid 3'-hydroxylase and flavonoid 3',5'-hydroxylase, was transformed with a binary vector containing a rose DFR cDNA cloned behind a constitutive promoter. Petals and anthers of the resultant transgenic petunia plants were salmon pink and contained pelargonidin, an anthocyanidin rarely found in petunia.