Transcriptional and translational control of gene expression during pollen development
thesisposted on 15.12.2014, 10:33 by Neil. Bate
The tomato lat52 gene encodes an essential cysteine-rich protein required for normal pollen tube growth which is preferentially transcribed in the vegetative cell during pollen maturation. The identity, organisation and role of cis-regulatory elements which control the development and cell-specific expression of lat52 were investigated in detail. A series of 5' promoter deletion mutants fused to the E. coli -glucoronidase gene uidA (gus) were stably introduced into tobacco. Detailed analysis of the accumulation of GUS activity during anther development demonstrated that lat52 promoter activity during early to mid-pollen maturation was regulated specifically by the region -492 to -101.;Transgenic studies and transient expression analysis demonstrated that the major cis-regulatory elements responsible for high-level pollen-specific transcriptional activity were located between -492 and -42. Fusion of the region -492 to -52 to a minimal CaMV35S promoter enhanced reporter gene expression in a pollen-specific manner. This gain of function approach was used to show that the previously defined sequence motifs PBII and PBIII did not contribute to pollen-specific transcription in a germinating pollen. Analysis of the region -100 to -52 led to the identification of two novel co-dependent cis-regulatory elements within the minimal pollen-specific activator unit -71 to -52.;In addition to multiple upstream cis-regulatory elements which control the level and specificity of lat52 transcription, the 5' untranslated region (5'-UTR) conferred a dramatically increased translational yield to heterologous transcripts in a pollen-specific and highly developmentally regulated manner during the final stages of pollen maturation in transgenic tobacco. Taken together, these data suggest that lat52 expression is developmentally regulated in a tri-phasic manner during tobacco pollen development.