Analysis of the P-rr promoter in transgenic maize
--Li, X; Sidorenko, L; Tagliani, L; Chopra, S; Bowen, B and Peterson, T

The maize P-rr gene encodes a myb-like transcription activator which activates the C2, CHI1, and A1 genes to produce a red phlobaphene pigment in floral tissues, such as mature cob glumes, pericarps, and husks. To characterize the tissue-specific activity of the P-rr promoter, we produced transgenic maize plants carrying different segments of the P-rr promoter fused to the GUS reporter gene. Plasmid construct P1.0b::GUS, which contains the P-rr region from -1252 to +352 relative to the transcription start, produced 96 stable callus events and 714 transgenic plants. The majority of these plants expressed GUS specifically in pericarps, husks, and silks; GUS activity was not detectable in endosperm, embryo, and pedicel, or vegetative tissues of mature plants. Interestingly, GUS was expressed specifically in transgenic anther wall. To our knowledge there is no previous report of P-rr expression in these cells. With the exception of the anther wall expression, the majority of transgenic plants demonstrated floral-specific GUS expression pattern similar to the pattern of P-rr. In conclusion, the 1.5 kb fragment contains the elements required for floral-specific expression of the P gene.

We also generated 12 stable callus lines and 76 plants for construct Pb::GUS (-233 to +326), and 15 stable callus lines and 94 transgenic plants for construct P1.2b::GUS (5' 1.2- kb SalI fragment fused to the basal plasmid Pb::GUS). Interestingly, 77% and 54% of transgenic T0 stable transgenic maize transformed with the P1.0b::GUS or P1.2b::GUS, respectively, expressed GUS in their floral tissues. In contrast, only 18% of transgenic Pb::GUS plants expressed GUS in floral tissues. In conclusion, the P1.0 and P1.2 fragments not only enhanced Pb strength in driving expression of the reporter gene in transiently assayed suspension cells and pericarps (X. Li et al., MNL 69:9, 1995), but also boosted tissue-specific Pb activity in stable transgenic maize plants. The pattern of tissue specific expression in plants transformed with P1.2b::GUS was more variable compared to those transformed with P1.0b::GUS. Occasionally, only parts of glumes, husks, and silks of P1.2b::GUS showed GUS blue staining, unlike the uniform GUS expression in transgenic plants containing the P1.0b::GUS constructs. Perhaps, the irregular expression of P1.2::GUS may be due to the presence of the 5' 1.2 kb SalI fragment which is a site of epigenetic modification in the P-pr allele as demonstrated by Das and Messing (Genetics 136:1121-1141, 1994) and Lund et al. (Plant J. 7:797-807, 1995).

The Pb, P1.0b, and P1.2b constructs above all contain the maize Adh1 intron in front of the GUS gene to boost expression. Three constructs which were similar, but without the Adh1 intron, produced only a few GUS positive plants. These constructs without the Adh1 intron also had very low activity in transient assays. These results suggest that transient assays should be used as a preliminary test of promoter activity before generating stable transgenic maize. 

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