HPLC analyses of bronze pigments

--John Bodeau and Virginia Walbot

Although the exact enzymatic role of the Bz2 gene product is still unknown, cross-feeding studies and phenotypic similarity of bz2 mutants to bz1 plants suggest that it acts very late in the pigment biosynthetic pathway. Recent findings of acylated anthocyanins in maize following mild extraction procedures (Harborne and Self, Phytochemistry 26:2417, 1987) suggest the possibility that Bz2 encodes an acyl-transferase enzyme which acts after the Bz1 encoded enzymatic step. To test this idea we tried to identify a pigment component (a cyanidin malonylglucoside?) of wildtype plants which is reduced in bz2 plants.

We extracted soluble pigments from fresh young leaves of greenhouse-grown bz1 B Pl, bz2 B Pl, and Bz1 Bz2 B Pl plants of W23 background. Leaf slices were macerated in MAW (Methanol/Acetic Acid/Water, 8:1:1, pH~5) and allowed to equilibrate overnight at 4 C. Raw pigments were applied to a high-capacity LH-20 column run with MAW, to remove most simple sugars. Fractions with peak pigment were pooled and lyophilized. HPLC was performed with the help of Dr. Vern Singleton, and Eugene Trousdale, in the Department of Enology, University of California at Davis. Pigments were eluted on a C18 column using an acetonitrile acidic phosphate gradient. Elution peaks of cyanidin and cyanidin 3-glucoside were identified by spiking samples with these compounds.

In wildtype extracts, cyanidin 3-glucoside and trace amounts of cyanidin were present, while bz1 pigments accumulate virtually no glucoside, as expected from the previously determined identity of the Bz1 product as UDP glucose-flavonoid glucosyl-transferase. In bz2 B Pl leaf tissue no wild-type peaks appreciably decreased, including cyanidin 3-glucoside. Additionally, both bz1 and bz2 extracts accumulated high levels of an unidentified pigment which eluted at nearly the same point as purified cyanidin aglycone, and which was an extremely minor component of the wildtype extract.

While our hope of finding a “missing peak” which would provide a clue as to the function of Bz2 was frustrated, the presence of cyanidin 3-glucoside in bz2 pigment extracts provides further evidence that the Bz2 gene product acts after Bz1.

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