Preliminary analysis of green fluorescent compounds induced by ectopic expression of the P gene --Lin, Y, Dong, X, Grotewold, E The maize P gene, an R2R3 myb transcription factor, controls 3-deoxy flavonoid and phlobaphene biosynthesis (Grotewold et al. Cell 76: 543-553, 1994). In the pericarp, P regulates the accumulation of a subset of flavonoid biosynthetic genes (C2, Chi1 and A1). The ectopic expression of P in cultured BMS cells induces the accumulation of distinct classes of flavonoid and phenylpropanoid compounds, as well as orange-fluorescent bodies (Grotewold et al. Plant Cell 10:721-740, 1998). To gain further understanding of P function in maize cells, we used BMS cell lines transformed with ERE::P (P driven from an estradiol-inducible promoter, Bruce et al. Plant Cell 12, in press). This provides an ideal experimental system to investigate the activation of a branch of flavonoid biosynthesis, and the distribution and subcellular localization of secondary metabolites. A new type of green fluorescent vacuole-like bodies (GFVLB) was found in BMS cells transformed with ERE::P. These fluorescent vacuoles have different sizes and shapes, and they often localize in the cytoplasm close to the plasma membrane, and appear to be involved in delivering green fluorescent compounds to the cell wall. A GFVLB attached to the cell wall, which shows green fluorescence, was seen in some cells. The GFVLB may originate from SER through vesicles, because a tube-like GFVLB and a GFVLB fused with several small vacuoles were also observed in some cells. The GFVLB were found only in the BMS cells transformed with ERE::P, and were not found in either BMS cells transformed with ERE::Luc (luciferase driven from the estradiol-inducible promoter), or untransformed BMS cells. Although GFVLB exists in both induced and uninduced BMS cells with ERE::P, the number of cells containing GFVLB in the 5-days induced cells is four times higher than in uninduced cells (Fig.1).

The compounds responsible for the fluorescence of GFVLB remain to be identified, but preliminary TLC analysis has been initiated. 3-deoxy flavonoids and phenylpropanoids accumulate in ERE::P BMS cells after induction with estradiol (Fig.2 and Fig.3). Accumulation of flavan-4-ols reached a maximum at 6 days after induction, but remained at a very low level in uninduced cells. Of the five identified compounds, only ferulic acid is present at significant levels in the uninduced cells, consistent with previous findings (Grotewold et al. Plant Cell 10:721-740, 1998). After induction, ferulic acid levels stay high for several days (Fig.3D). Thus, ferulic acid and the fluorescent compounds present in GFVLB appear to share similar patterns of temporal expression. In addition, other types of fluorescent bodies were identified in both induced and uninduced BMS cells with ERE::P. They are spherical, nonphotobleaching, intensely orange fluorescent bodies very similar in properties to those 35S::P-expressing BMS cells (Grotewold et al. Plant Cell 10:721-740, 1998). The fluorescent compounds present in these bodies also show very strong red fluorescence under a 510-560 nm excision filter. The chemical nature of this second type of fluorescent compound is not known either. We believe that these fluorescent compounds will provide unique tools to investigate transport and subcellular localization of plant secondary metabolites.

Figure 1.

Figure 2.

Figure 3. HPLC Chromatograph (340nm) of ERE::P cell line before (A) and after 120 hours induction with estradiol (B). Temporal accumulation of chlorogenic acid (C) and ferulic acid (D) in the ERE::P cell-line induced with estradiol.

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