Introduction to Flavonoids (1999) pp. 308-309
Bruce A. Bohm

Biosynthesis of Anthocyanins (DFR, ANS, and LDOX)

Anthocyanins are the most conspicuous products of flavonoid biosynthesis and, as such, have attracted a great deal of interest. Studies range from classical genetic analysis of flower color inheritance patterns, through establishment of their chemical structures, to efforts to understand the factors involved in their biosynthesis. Contemporary efforts to understand the metabolic pathway at the level of the gene have added immeasurably to our knowledge of this particular group of pigments, as well as to an understanding of how plant metabolic processes are controlled in general. The result of these efforts is a vast literature. What follows is only a sampling with sufficient references to the original and review literature to provide the interested reader with leads for further study. Though long out of print, no work treats the history of the subject better than Muriel Onslow's The Anthocyanin Pigments of Plants, the second edition of which was published in 1925. The work represents an outstanding synthesis of information including citations to publications from the mid 17th century. A recent work that deserves serious reading is Helen Stafford's (1990) Flavonoid Metabolism. Other timely reviews include the work of Weiring and de Vlaming (1984) on Petunia, a series of papers in a volume edited by Styles and colleagues (1989), and general reviews by Martin and Gerats (1993) and Holton and Cornish (1995). Each of the volumes of The Flavonoids series contains relevant information on chemical and biochemical aspects of anthocyanins, of course.

One of the first studies of flavonoid biosynthesis was that of Hans Grisebach (1957) who demonstrated that the carbon skeleton of cyanidin was formed from the same precursors as other flavonoid types, the A-ring from acetate and the B-ring and carbon bridge from a phenylpropanoid derivative. Over the next several years a number of workers demonstrated that chalcones, flavanones, and dihydroflavonols were all readily incorporated into anthocyanins. Genetic information was also accumulating that pointed toward involvement of these other flavonoid types (Harrison and Stickland, 1974, 1978: Stickland and Harrison, 1974, 1977). Often it was possible to induce the formation of pigment in white flowers by administering one of the pathway intermediates that lay beyond the genetic block. Other major contributors at the time were Kho and coworkers (1975, 1977) working with Petunia hybrida, Forkmann (1977, 1980) working with Matthiola incana, and McCormick (1978) working with corn. Another source of information involved the use of inhibitors that inactivated PAL, thus preventing any carbon from flowing into the phenylpropanoid and flavonoid pathways (Amrhein, 1979). Addition of a suspected intermediate on the anthocyanin pathway to the "phenylpropanoid-starved" tissue resulted in formation of pigment. With the starting and end points established it then remained to establish the detailed pathway. That part of the story comes next.

It seemed reasonable to focus attention on dihydroflavonols as likely late intermediates on the anthocyanin pathway simply because they are such prominent compounds in many plants and they served very well in precursor studies. A major step in answering the question as to what the next compound in the pathway beyond the dihydroflavonol came from studies of polyphenolic biosynthesis in cultured cells of Douglas fir (Pseudotsuga menziesii). Stafford and Lester (1982) were the first to detect dihydroflavonol reductase (DFR) activity: they also identified the product as a flavan-3,4-diol. The reaction is represented in the reduction of (2R,3R)-dihydrokaempferol [6-25] to (2R,3S,4S)-2,3-trans-5,7,4'-trihydroxyflavan-3,4-cis-diol [6-26]. The enzyme has now been purified from, or detected in, a wide spectrum of plant species including monocots, dicots, and gymnosperms; all DFR preparations require NADPH. Specificity of the enzyme varies a good deal both in what substrates it will accept and how efficiently any given dihydroflavonol will be converted to the flavandiol. For example, DFR from Callistephus and Dianthus will accept dihydrokaempferol, dihydroquercetin, and dihydromyricetin but handles dihydrokaempferol least well of the three. Matthiola, Dahlia, and Dianthus DFRs readily accept dihydromyricetin despite the absence of delphinidin glycosides in any of their species. The enzymes from Petunia, Nicotiana, and Lycopersicon (tomato) work best with dihydromyricetin, somewhat less efficiently with dihydroquercetin, and not at all with dihydrokaempferol. In these species dihydrokaempferol will accumulate if the hydroxylation system, which normally converts it to dihydroquercetin, is inactive. Incidentally, the lack of natural capacity to convert dihydrokaempferol to anthocyanins in Petunia has been "rectified" by genetic engineering using a DFR construct from corn (see Chapter Eight for details).

Flower Breeding and Genetics: Issues, Challenges and Opportunities for the 21st Century (2007) p. 322
Griesbach; Chapter 11. Petunia. Petunia x hybrida

In order to identify the DNA sequence leading to substrate specificity, chimeric DFR genes were constructed and introduced into P. x hybrida 'W80' (Johnson, et al., 1999). It was determined that the substrate binding region was between amino acids 132 and 158 with amino acid 134 critical in substrate specificity. A switch from asparagine to leucine at position 134 caused a change in substrate preference from dihydroquercetin to dihydrokaempferol.