Consequences of the ectopic expression of the Myb-domain protein P
--Rabinowicz, P; Ma, H; Grotewold, E

The P gene encodes a Myb-domain protein required for the transcriptional regulation of a subset of the maize anthocyanin biosynthetic genes regulated by C1/Pl + R/B. Whereas the accumulation of the P-regulated phlobaphene pigments seems to be restricted to the floral organs of some grasses including maize and sorghum, anthocyanin pigments can be expressed in all plant tissues and are widely found in the plant kingdom. The regulators of anthocyanin biosynthesis are functionally conserved, and the ectopic expression of R and C1 induces the accumulation of anthocyanins in a number of plant species.

As a way to determine whether phlobaphene accumulation could be induced in plant species normally lacking these pigments, we investigated the effect of expressing P in the dicot Arabidopsis thaliana. A functional P cDNA was ectopically expressed in Arabidopsis from the constitutive 35S promoter following an in planta Agrobacterium-mediated transformation.

Kanamycin-resistant seedlings were selected, and to our surprise, we found a large number of very small plants present only in the material derived from 35S::P-transformed plants, but absent in plants transformed with unrelated plasmids (Fig. 1). Northern analysis of mRNA derived from total plant tissue demonstrated that the severity of the phenotype is dependent on high levels of P expression. P expression levels were 20-100 fold higher than in maize P-rr pericarps. Every organ we looked for was present in the 35S::P plants, but was reduced in size. Microscopic investigation of these plants indicates that the reduced size is the product of the combination of smaller and fewer cells (Fig. 2A, B). As far as we can tell from comparing leaves of equal size from wild type and mutant plants, the density and shape of trichomes were not affected (Fig. 2C, D). This is in contrast to what has been determined for Arabidopsis plants ectopically expressing R, where an increased number of trichomes was observed (Larkin et al., The Plant Cell 5:1065-1076, 1994). 35S::P expressing plants often accumulate red pigments in the stem and the leaves. We have not yet carried out extensive biochemical characterization of these pigments, yet the color is very similar to the anthocyanin pigments that accumulate in R+C1-expressing Arabidopsis plants (Lloyd et al., Science 258: 1773-1775, 1992). Provided that these pigments are anthocyanins, we believe that they could be induced by stress, from which the 35S::P plants clearly suffer. In maize, P does not activate anthocyanin biosynthesis, because it cannot bind to the promoters of anthocyanin-specific genes such as bz1 (Grotewold et al., Cell 76:543-553, 1994).

What precedents are available for the small plant phenotype induced by P in Arabidopsis? A dominant genetic factor, called Ufo (for Unstable Factor Orange), has been identified by Dr. Charles Burnham that induces phlobaphene accumulation in maize vegetative tissues (Styles et al., MNL 61:100, 1987). Northern and RNase protection experiments indicate that Ufo induces the accumulation of P-homologous sequences in leaf sheath tissue at levels comparable to pericarp tissues (Grotewold, unpublished). Plants carrying the Ufo allele show a significant retardation in tissue growth, proportional to the amount of pigment present (Styles et al., 1987).

Ectopic expression of P from the strong 35S promoter in Black Mexican Sweet suspension cells does not appear to affect cell size, although the cultures grow more slowly. Yet, some unknown selection mechanisms allowed us to recover only transgenic lines with levels of expression of P comparable to the levels of P mRNA in P-rr pericarps (Grotewold et al., manuscript in preparation).

These observations are somehow consistent with what happens in the 35S::P transgenic Arabidopsis plants: P expression somehow interferes with cellular processes. Why does P cause such dramatic effects when expressed in plant tissues where it is not normally found?

1. P ectopic expression could interfere with the normal expression of gene(s) with important cellular functions. This could be mediated by the conserved Myb domain present in a large number of other plant proteins, or by other regions of the P protein. It is remarkable, however, that ectopic expression of C1 (with an 80% identical Myb domain with P) does not show similar effects.

2. P-regulated compounds, such as phenolics or isovitexin (a C-glycosyl flavone), could have an effect on cell growth/division. Although it is known that these compounds are regulated by P in maize, the effect seen in Arabidopsis suggests that their accumulation in this dicot would also be induced by P. This remains to be determined.

3. P could have regulatory functions, in addition to controlling phlobaphene accumulation.

Figure 1. Effect of the ectopic expression of P in Arabidopsis. About four week old plants containing the 35S::P transgene (two left pots) compared to wild type plants (right pot).

Figure 2. Leaf morphology of P-expressing Arabidopsis plants. A and B correspond to optical micrographs of sections of wild type (A) and 35S::P (B) leaves. Trichomes are not preserved in the fixation conditions used. The arrow in B indicates the central vasculature. C and D correspond to scanning electron micrographs of leaf trichomes of wild type (C) and 35S::P transgenic plants (D).

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