14-3-3 Proteins are components of the transcription complex of the ATEM1 promoter in Arabidopsis.
The AtEm1 and AtEm6 gene products accumulate exclusively in embryos during Arabidopsis seed maturation. The transcription factor ABI3 and the phytohormone abscisic acid are required for normal expression of both genes. However, the expression of these genes occurs in extremely small embryos limiting the availability of tissue to directly study DNA-protein interactions. We generated callus lines derived from embryos to determine if the regulation of Em expression was similar to wild type embryos. Expression of AtEm1 and AtEm6 was strongly induced by abscisic acid in callus derived from wild type embryos, but not in embryo callus derived from ABI3 mutant embryos (abi3-6). Epitopes to 14-3-3 proteins were found in complexes with the AtEm1 promoter in mobility shift experiments using nuclear extracts derived from both wild type and abi3-6 calli. Using phosphorylated peptides that bind to 14-3-3 proteins, we show that 14-3-3 proteins are required for the maintenance of the transcriptional complex generated in nuclear extracts. Chromatin immunoprecipitation experiments using a 14-3-3 antibody display the expected 241-bp band from the AtEm1 promoter. Hence, 14-3-3 proteins are physically present in the AtEm1 transcriptional complex in vivo and are required for the maintenance of the transcriptional complex in vitro.