University of Hamburg
Further analysis of the regulation of anthocyanin gene expression in maize influence of C1, R and In1 on Whp (white pollen) and C2 expression
--Kirsch, J, Techen, N, Lorbiecke, R, Brettschneider, R, Scheffler, BE, Wienand, U
The influence of C1, R and In1 (intensifier) genes on the expression of C2 and Whp has been analyzed by transient expression assays. Whp promoters were isolated either from the Line C (color converted W22 inbred line) or C1-S, and fused to a luciferase reporter gene. Two constructs of each Whp promoter (with and without the 5´ untranslated region) were used. In addition, a C2 promoter, isolated from the mutant C2-Idf, was included in the analysis. In particle bombardment assays, using the scutellum of developing embryos, the activity of the reporter gene was analyzed after co-bombardment with the regulatory genes C1 and R. In parallel, the activity of the endogenous chalcone synthase genes was monitored as red cells, due to anthocyanin accumulation in the bombarded cells. The experiments showed that the expression of the C2- and Whp-promoters was dependent on the co-bombardment of C1 and R. The activities of the Whp promoters were positively influenced by the presence of the 5´ untranslated region of the gene.

In further studies, the C2 and Whp reporter constructs were examined in the presence of a construct coding for the intensifier protein (isolated from the intensifier mutant In1-D). Bombardment of C1, R and In1-D constructs, together with the reporter constructs carrying the Whp promoters with the 5´ UTRs, led to considerably reduced promoter activities when compared to the activities without the In1-D construct. The same was true using the C2 promoter. This supports the assumption of intensifier being a repressor molecule (Burr et al., Plant Cell 8:1249-1259, 1996).

The possible interaction of C1, R and In1 was further analyzed in a yeast two hybrid system. Various constructs of C1, C1-I, R, In1 and In1-D were fused to either the activation or the binding domain of the GAL4 gene and then tested in different combinations in yeast. Whereas strong interaction could be shown for C1 and R, no comparable interaction could be observed between C1 or R with In1 and In1-D. Although, a weak reporter gene expression was detected when In1 or In-D1 constructs were used with C1 or C1-I constructs. Nevertheless strong influence of In1 and In1-D on C1 and R dependent chalcone synthase promoter activities could be shown in the transient in vivo studies. The evidence that at least a part of the intensifier repressor activity is linked with transcription is supported by nuclear import experiments, which showed that the In1-D protein is translocated into the nucleus in an onion cell system.

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