Gametophytic expression of GUS activity controlled by the potato Lhca3.St.1 promoter in tobacco pollen

A beta-glucuronidase (GUS) gene fusion with the promoter from the potato Lhca3.St.1 gene, encoding a light-harvesting complex protein, shows expression in pollen of transgenic tobacco plants. This was established by histochemical and fluorometric assays, and confirmed by RT-PCR, Western analysis and GUS activity following gel electrophoresis, The activity is minimal in early pollen development and high in mature pollen at anthesis, It shows gametophytic segregation in pollen grains. Pollen from tobacco plants hemizygous for a single Lhca3.St.1-GUS locus exhibited 1:1 segregation ratios, whereas dihybrids segregated in the expected 3:1 ratio. A sequential combination of histochemical staining for GUS activity and Alexander's stain for pollen viability, offers a convenient system to eliminate artefacts in the histochemical staining, identify inviable pollen and accurately score pollen segregation. The dual staining approach also confirmed gametophytic segregation in tobacco plants hemizygous for the GUS gene under transcriptional control of a doubled CaMV 35S promoter. Gametophytic segregation establishes that the GUS activity is determined by the genetic status of the pollen rather than of the parent plant, and indicates de novo GUS gene expression and translation during pollen maturation. The unanticipated activity of the Lhca3.St.1 promoter in pollen has implications for the biosafety assessment of transgenic plants intended for agricultural use. Even when a promoter is not generally considered to be active in pollen, its expression in pollen should be evaluated if effects on pollinating insects or food safety concerns for products such as honey can be anticipated.