A TRANSIENT ASSAY FOR PROMOTER ACTIVITY OF WHEAT SEED STORAGE PROTEIN GENES AND OTHER GENES EXPRESSED IN DEVELOPING ENDOSPERM

Numerous genes encoding agronomically important traits of crop plants are expressed in endosperm tissue. To facilitate the analysis of promoter regions from these genes, a transient assay was established using protoplasts from a maize (Zea mays L.) endosperm suspension culture. The activities of several different promoters in transcriptional fusions with the chloramphenicol acetyl transferase (CAT) reporter gene were compared. The maize alcohol dehydrogenase I (Adhl) promoter/intron was the most active tested, nearly 2000-times background; the cauliflower mosaic virus (CaMV) 35S promoter was about 270-times background. Use of an endpoint assay for CAT enzyme activity allowed detection of the lower activities of the promoter regions of the maize Shrunken (Sh) (8.4-times background) and Agrobacterium nopaline synthase (Nos) (3.9-times background) genes, and of a wheat alpha-gliadin gene (4.4-times background). Promoters from another family of wheat seed storage protein genes, the high molecular weight (HMW) glutenin subunits, were also active, at levels between 10- and 60-times background. This is the first report of activity from wheat seed storage protein gene promoters in a monocot transient assay. There were no statistically significant differences in activity among four different length versions of the Dy10 glutenin promoter ranging in size from 2800 to 307 bp, or between similar versions of the Dy10 and Dx5 glutenin gene promoters. These results show that maize endosperm suspension cells provide a readily available source of protoplasts for comparisons of cereal gene promoters that are active in developing endosperm tissue.