In order to study the tissue-specific regulation of the P1-rr allele, maize plants were transformed with various portions of the P1-rr upstream regulatory region linked to the GUS reporter gene (P::GUS constructs). Unexpectedly, plants with strong transgene expression in floral organs also had transgene expression in vegetative organs, including the coleoptile, leaves, and roots of seedlings; and the sheath, auricle, and blade of adult leaves. The intensity of GUS staining ranged from light to very dark in seedling organs and from light to moderate in adult leaves. In the coleoptile, GUS activity was primarily localized to the two lateral vascular bundles. For seedling roots, the most intense staining was at the site of lateral branch formation and in the central cylinder (pith and vascular cells) of primary, lateral, and seminal adventitious roots. Leaves of primary regenerants grown in culture boxes stained uniformly blue, while leaves from greenhouse-grown seedlings, which have a thicker cuticle, stained unevenly due to poor substrate penetration. No blue staining was observed in comparable non-transformed plant material. These patterns of vegetative transgene expression were observed in 14 out of 27 independent transformation events, suggesting that such expression is not an artifact of transgene "position effect".

Similarly, when maize plants were transformed with constructs containing the P1-rr promoter driving either P1-rr or P1-wr cDNA sequences (P::P constructs), the transgenes promoted pigmentation in the expected floral organs­pericarp, cob, husk, silk and tassel glumes, as well as in the vegetative organs of plants that had strong transgene expression in floral organs. Non-floral pigmentation was observed in the leaf midrib, sheath, ligule and auricle; in the pith of the culm; and in the lateral veins of the coleoptile. No pigment was discernible in the leaf blade, except for in the midrib. Interestingly, this pattern of transgene expression is comparable to that caused by Ufo1 (unstable factor for orange), a dominant allele that induces phlobaphene production in vegetative organs when combined with a P1-wr allele (Styles et al., MNL 61:100, 1987). Similar to Ufo1, strong transgene expression that results in dark vegetative pigmentation is associated with retarded plant growth.

The expression of P::GUS and P::P transgenes in vegetative organs prompted examination of non-transgenic plants for endogenous p1-conferred vegetative pigmentation. Indeed, P1-rr plants have a light orange tint to the leaf midrib, auricle and ligule that is absent in P-ww plants of the same genetic background. Also, a light orange uniform sheen can be observed on the adaxial surface of the sheath of P1-rr plants, especially after the plants have dried. No P1-rr-regulated pigmentation was visually discernible in the leaf blade, lateral veins of the coleoptile, or roots. Scrutiny of P1-wr plants for pigmentation revealed red sheath and auricle margins; this phenotype is analogous to the pigmented margins of P1-wr husk and tassel glumes. Thus, vegetative pigmentation patterns conferred by P1-rr and P1-wr alleles (uniform vs. marginal, respectively) are consistent with the allele-specific patterns of the floral organs.

Our observation of p1 expression in vegetative organs is further supported by biochemical and molecular analyses. D. Styles and O. Ceska (Can J Genet Cytol 23: 691-704, 1981) reported detection of p1-regulated 3-deoxy flavonoids in coleoptiles, and we reproducibly observe very low levels of p1 transcript in coleoptiles and auricles by RNA blot analysis. Through the use of a more sensitive rt-PCR method, we were able to detect transcripts in other vegetative organs. The primer set used for the PCR reactions is complementary to p1, as well as p2, a recently identified p1 gene homolog (P. Zhang, personal communication). Amplification products of the p1 and p2 transcripts can be distinguished by size due to an 80 bp deletion in the 5� untranslated region of p2. Amplified products corresponding to p1 mRNA were detected in coleoptile, seedling leaf, seedling root, and sheath from P1-rr and P1-wr plants, while no p1-specific amplification products were detected in P1-ww samples. Taken together, these results indicate that the endogenous p1 gene is expressed at low levels in vegetative organs, suggesting that p1 gene expression is floral-preferred rather than floral-specific.
 
 

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