Mol Gen Genet (1994) 244:391400 © Springer-Verlag1994 P. Gallusci • F. Salamini • R. D. Thompson Differences in cell type-specific expression of the gene Opaque2 in maize and transgenic tobacco Received: 16 November 1993 / Accepted: 15 February 1994 Abstract The Opaque 2 (02) gene encodes a transcrip- tional activator of the basic region/leucine zipper fami- ly, which controls the synthesis of a major storage protein class in maize endosperm, the 22 kDa ~-zeins, and of several other non-zein polypeptides including b32. We demonstrate, by analysing 02 mRNAs in differ- ent organs of maize plants, that the 02 gene is only active in the endosperm. Its transcription is precisely controlled during seed development: 02 mRNAs are first detected 10 days after pollination and accumulate in the endosperm over a period of 20 days. When intro- duced into tobacco plants, the 02 promoter directs the expression of the 13-glucuronidase (GUS) reporter gene in endosperm, but also in the embryo, cotyledons and pollen. The first 185 bp of the 02 promoter is sufficient for developmentally regulated expression in tobacco seeds. A distinct cis-acting element, located between po- sitions -185 and -520, directs expression in the cotyledons of tobacco seedlings. The possible origins of this breakdown in promoter specificity in the het- erologous host are discussed. Key words Opaque2 • Maize endosperm • Transgenic tobacco • Promoter Introduction Zeins, the major seed storage proteins of maize, are al- cohol-soluble proteins that accumulate in the en- dosperm during seed maturation. The most abundant zeins have molecular weights of 20-22 kDa (~-zeins), and are encoded by a large multigene family comprising about 100 genes (Hagen and Rubenstein 1981; Wilson and Larkins 1984). Zein gene expression is tissue specif- Communicated by J. Schell P. Gallusci • F. Salamini - R.D. Thompson ([~) Max-Planck-Institut ffir Ziichtungsforschung, Carl-von-Linn~-Weg 10, D-50829 K61n, Germany ically and developmentally controlled, and occurs in the endosperm between 10 and 45 days after pollination (Murphy and Dalby 1971). Several mutants affect speci- fically either the accumulation of one group of a-zeins, such as opaque 7 and opaque 2, or of all ~-zeins, such as opaque 6 andfloury 2 (for review see Motto et al. 1989). The opaque 2 (o2) mutation reduces the accumula- tion of the 22 kDa zeins (Jones et al. 1977a, b), by lower- ing the rate of transcription of the corresponding genes (Kodrzycki et al. 1989). The mutation also affects the accumulation of a few other non-zein polypeptides, in- cluding the b32 protein (Soave and Tardani 1981), which is an endosperm-specific ribosome-inactivating protein (Bass et al. 1992). The 02 gene has been cloned (Schmidt et al. 1987; Motto et al. 1988) and shown to encode a transcriptional activator of the basic domain/ leucine zipper family (Hartings etal. 1989; Schmidt et al. 1990). In transient expression assays, the 02 protein activates transcription from the b32 gene pro- moter (Lohmer et al. 1991) and from a 22 kDa zein pro- moter (Ueda et al. 1992), most probably by interacting with specific binding sites (Lohmer et al. 1991 ; Schmidt et al. 1992). However, additional proteins may be re- quired for the activation in vivo of the zein and b32 promoters (Pysh et al. 1993). The 02 gene product does not influence cell-type determination but enhances the expression of a set of genes in a very specific type of cells. Therefore, the transcription of the 02 gene must be pre- ceded by a series of developmental decisions leading to formation of distinct cell types of the triploid endosperm lineage, such as the aleurone, subaleurone and central endosperm cells. To identify factors involved in early steps of endosperm development, a study of mecha- nisms controlling the transcription of the 02 gene was initiated. The functional analysis of maize gene promoters is difficult due to the lack of a routine transformation sys- tem for this species. To circumvent this problem, tran- sient expression systems based on endosperm cell cul- tures have been proposed and adopted (Ueda and Mess- ing 1991; Manzocchi et al. 1989). In these cell cultures,