The ectopic expression of the P and R+C1 regulators of maize flavonoid biosynthesis in cultured BMS cells induces the accumulation of distinct classes of flavonoid and phenylpropanoid compounds (Grotewold et al., Plant Cell 10:721-740, 1998). To gain insight into how many genes might be regulated by P or R and C1, proteins were radiolabeled with 35S-methionine in vivo and separated by 2-D polyacrylamide gel electrophoresis (PAGE). Only a small number of new proteins that were not found in untransformed cells were detected by this method in either the P- or R/C1-expressing cell-lines (Fig. 1). This number is of the order of magnitude expected, if the sole functions of P and R/C1 are to induce de novo some or all of the enzymes and trafficking components required for 3-deoxy and 3-hydroxy flavonoid accumulation, respectively. Significantly, there were no proteins on these gels that obviously were down-regulated by either P or R/C1. Two proteins in the 40 kD region (black arrows in Fig. 1B-C) that appear to be induced by either P or R/C1 could correspond with chalcone synthase encoded by c2 (MW 43 kD) and flavanone/flavanonol reductase encoded by a1 (MW 40 kD). We have not confirmed, however, if these enzymes co-migrate electrophoretically with the proteins indicated. The identity of two proteins that were strongly expressed only in R/C1-expressing cells (indicated by white arrows on Fig. 1B) would merit further study, because neither appears to correspond in molecular weight or pI to any of the proteins encoded by the R/C1-regulated genes f3h, a2, bz1 or bz2. No proteins specifically induced by P were confidently identified by 2D-PAGE, despite the observation that the overexpression of P has some dramatic effects on maize and Arabidopsis plants (Rabinowicz et al., MNL 71:21-22, 1997). The formation of compounds controlled by P, including C-glycosyl flavones, luteoforol and fluorescent molecules of unknown nature (Grotewold et al., Plant Cell 10:721-740, 1998) may require constitutively expressed proteins. Alternatively, P could induce proteins not detected by this method, for example proteins without methionine or of same MW as pre-existing proteins.
1. Pattern of 35S-labeled protein accumulation in BMS callus
cells transformed with 35S::R+C1 (B), 35S::P (C); or un-transformed. Arrows
in B) and C) indicate dots absent in the control (A) line. The position
of those dots in the un-transformed lines is indicated by an arrow. Empty
arrows in B) correspond to dots only seen in the R+C1 lines.
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