Systematic protein-DNA binding studies have shown that plant basic leucine zipper (bZIP) proteins exhibit a differential binding specificity for ACGT motifs. Here, we show that the rice transcription activator-1 (RITA-1) displays a broad binding specificity for palindromic ACGT elements, beíng able to bind A-, C-, and G-box but not T-box elements. By using gel mobility shift assays with probes differing in sequences flanking the hexameric core, we identified high-affinity A-, C-, and G-box binding sites. Quantitative and competition DNA binding studies confirmed RITA-1 specificity for these sites. Using rice protoplasts as a transient expression system, we demonstrated that RITA-1 can transactivate reporter genes possessíng high-affinity but not low-affinity RITA-1 binding sites. Our results established a direct relationship between in vivo transactivation and in vitro binding actívity. Transient expression assays that demonstrated the ability of RITA-1 to transactivate a construct containing rita-7 Sflanking sequences suggest that the factor may be autoregulated. Histochemical analysis of transgenic rice plants showed that a rifa-7-P-glucuronidase transgene is expressed in aleurone and endosperm cells of developing rice seeds. We propose that RITA-1 plays a role in the regulation of rice genes expressed in developing rice seeds.

Systematic protein-DNA binding studies have shown that plant basic leucine zipper (bZIP) proteins exhibit a differential binding specificity for ACGT motifs. Here, we show that the rice transcription activator-1 (RITA-1) displays a broad binding specificity for palindromic ACGT elements, being able to bind A-, C-, and G-box but not T-box elements. By using gel mobility shift assays with probes differing in sequences flanking the hexameric core, we identified high-affinity A-, C-, and G-box binding sites. Quantitative and competition DNA binding studies confirmed RITA-1 specificity for these sites. Using rice protoplasts as a transient expression system, we demonstrated that RITA-1 can transactivate reporter genes possessing high-affinity but not low-affinity RITA-1 binding sites. Our results established a direct relationship between in vivo transactivation and in vitro binding activity. Transient expression assays that demonstrated the ability of RITA-1 to transactivate a construct containing rita-1 5' flanking sequences suggest that the factor may be autoregulated. Histochemical analysis of transgenic rice plants showed that a rita-1-beta-glucuronidase transgene is expressed in aleurone and endosperm cells of developing rice seeds. We propose that RITA-1 plays a role in the regulation of rice genes expressed in developing rice seeds.

In order to study gene silencing in a monocot system we introduced a waxy (Wx) gene into rice. In the pollen grain of the transgenic wild-type plants, two types of Wx gene silencing were observed: Type I in which all the pollen grains showed the mutant (wx) phenotype, and Type II in which 50% of the pollen grains showed the wx phenotype. Analysis of Wx gene expression in the progeny of selfing and outcrossing indicated that Wx gene silencing was meiotically transmitted to the offspring. The number of transgene copies and transgene loci was determined by Southern blot analysis and suggested that the Wx transgene may have a paramutagenic effect on the endogenous Wx genes. In contrast to the pollen grain, the wx phenotype was not observed in the endosperm. However, the level of WAXY (WX) protein in the endosperm of Type I lines was similar to that in non-transgenic seed, while in Type II lines two classes of seeds, showing high and low levels of the protein segregated. When the same transgene was introduced into wx mutants in which no Wx transcript was detectable, the transgene behaved as a dominant Mendelian factor and no silencing was found, suggesting that the activity of the endogenous Wx gene influences the silencing phenomenon. Our study of Wx gene silencing in rice extends the well-known phenomenon of gene silencing, so far observed mainly in dicots, to a cereal.