Cold Spring Harbor Laboratory

JOHNSTON, IOWA

Pioneer Hi-Bred International, Inc.

Differential regulation of flavonoid biosynthetic genes by the P gene

--Erich Grotewold, Thomas Peterson, Bruce Drummond, Brad Roth and Ben Bowen

The P gene regulates 3-deoxy flavonoid biosynthesis in maize. The 3-deoxy flavonoid flavan-4-ol undergoes a non-enzymatic polymerization to form the red phlobaphene pigment found in pericarps and cobs carrying a P-rr allele (red pericarp and red cob) (Styles, ED and Ceska, Can. J. Genet. Cytol. 19:289, 1977). P regulates the accumulation in the pericarp of transcripts of the C2, CHI and A1 genes (Grotewold, E et al., PNAS 88:4587, 1991). C2, CHI, and A1 encode three enzymes required for biosynthesis of both flavan-4-ol (a 3-deoxy flavonoid) and anthocyanins (which are derived from 3-hydroxy flavonoids). On the other hand, A2, Bz1 and Bz2 encode enzymes specifically required for anthocyanin biosynthesis; the P gene has little or no effect on the expression of these genes in the pericarp (Grotewold, unpublished).

Two alternatively spliced P transcripts (P-cDNA1 and P-cDNA2) encode proteins with amino terminal regions homologous to the myb family of transcriptional activators (Grotewold et al., PNAS 88:4587, 1991). To determine which of the cloned P transcripts encodes the product responsible for the regulatory function of P, transient expression experiments using the particle gun were performed.

When P-cDNA1 (1802bp) under the control of the 35S promoter was co-bombarded into embryonic callus cells with a luciferase gene under the control of the A1 promoter (P dependent expression), a dramatic increase in luciferase activity was detected (over 200 fold). However, a very low level induction (3-6 fold) was obtained when the A1 promoter was replaced by the Bz1 promoter (P independent expression). When P-cDNA2 was used instead of P-cDNA1, there was no detectable activation of either the A1 or Bz2 promoters.

These experiments not only point to P-cDNA1 as the P encoded regulator, but also indicate important differences in the ways in which P and C1, two maize myb homologous genes, regulate expression of the structural genes for flavonoid biosynthesis. Whereas C1 (a myb homologous regulator of anthocyanin biosynthesis) requires a member of the R or B gene families for its regulatory activity, the regulation by P is clearly R/B independent. Furthermore, P does not efficiently activate the Bz1 promoter, even in the presence of R. However, competition experiments suggest that the product encoded by P-cDNA1 may interact with Bz1 promoter sequences.

We tried a complementation test by shooting P-cDNA1 into pericarps carrying a P gene deletion. No red cells were detected, even though many red cells are found when the same pericarps were bombarded with R+C1. Possibly the cells bombarded with P-cDNA1 produce the colorless flavan-4-ol, but it does not polymerize into the red phlobaphene pigment because we are not able to reproduce the conditions present in a naturally maturing ear; pigment formation is thought to require specific physiological conditions of the tissue such as maturation or aging (Styles, ED and Ceska, Maydica 34:227, 1989). The alternative possibility is that the product encoded by P-cDNA1 does not activate the C2 and/or CHI genes. Experiments are in progress to test whether P-cDNA1 can activate the promoters of these genes.

We thank Dr. Heinz Saedler, H and Dr. Alfons Gierl, A for the A2 probe and Dr. Virginia Walbot, V for the Bz2 probe.

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