Functional architecture of a late pollen promoter: pollen-specific transcription is developmentally regulated by multiple stage-specific and co-dependent activator elements.

The tomato lat52 gene encodes an essential cysteine-rich protein preferentially transcribed in the vegetative cell during pollen maturation. Detailed analyses of the identity, organization and role of cis-regulatory elements in controlling the precise developmental and tissue-specific expression of lat52 during pollen development were performed. Analysis of a series of 5' promoter deletion mutants stably introduced into tobacco demonstrated differential developmental activation of deletion mutants during pollen development. All major cis-regulatory elements required for pollen-specific transcription were located within the upstream region -492 to -52. This region was shown to comprise three independent activator domains A, B and C, each sufficient to activate the minimal CaMV 35S promoter in a pollen-specific manner. 5' deletion and gain of function approaches were used to show that domain A and the previously defined motif PBII (sub-domain B1) were largely redundant in the presence of downstream sequences in mature pollen. Within domain B two novel pollen-specific sub-domains B2 and B3 were identified. Within domain C, the activity of the PBI motif (sub-domain C1) was shown to be strictly dependent upon a downstream 20 bp pollen-specific activator unit -72 to -52 (sub-domain C2), containing two novel co-dependent regulatory elements AGAAA and TCCACCATA. These results demonstrate that transcriptional activation of lat52 is controlled by a complex of pollen-specific cis-regulatory elements which cooperate to achieve maximum levels of gene expression throughout pollen maturation. Alternative models of the interaction of identified cis-regulatory elements with putative trans-acting factors within the lat52 promoter and their developmental utilization are presented.