Characterization of the fruit specific 1-aminocyclopropane-1-carboxylic acid (ACC) synthase gene promoter in papaya (Carica papaya L.)

Papaya fruit ripening physiology is characterized by a dramatic increase in respiration accompanied by a surge in ethylene (H2C=CH 2) production. Ethylene acts as a growth regulator causing metabolic and physiological changes within the fruit. ACC synthase is believed to be the rate-limiting enzyme in the biosynthesis of ethylene.;A papaya genomic library was constructed and screened to isolate the 5'-upstream cis regulatory elements of ACC synthase. Three clones were isolated and sequenced. Sequence analysis determined that the clones represented various lengths of DNA surrounding a single ACC synthase gene. The 2.5 kb 5'-upstream region was sequenced and compared to ethylene responsive promoters from other plants. Putative fruit-specific regulatory sequence elements were identified.;Primer extension revealed three start sites, all within an 11 by region. Transcripts begin -28 bp, -22 bp, and -17 by from the translational start site. The shortest transcript occurring at -17bp from the translational start site corresponds to the longest cDNA transcript isolated during the original cloning of this gene from a cDNA library (Neupane, 1997).;Northern analysis detected a transcript of 2.1 kb and suggested very little RNA degradation. Interestingly, there is a faint second 0.3 kb RNA homologous to ACC synthase in immature green mesocarp tissue, a band of the same band appears with moderately higher intensity in leaf tissue. Because of the small band size, and its presence in tissue with restricted ACC synthase activity, it is suggested that post-transcriptional regulation of the CpACS1 gene occurs and this ∼300 by product acts as a part of a negative control system.;Electrophoretic mobility shift assays of the promoter region revealed an area in which proteins can bind in a sequence specific manner. This region includes a 16 by region upstream and adjacent to the TATA box. Utilizing the DNA region exhibiting protein binding as an affinity probe, the partial purification of a DNA binding protein was achieved. It is proposed that these cis and trans elements are at least part of the regulatory machinery that controls regulation of transcription of the ACC synthase gene in a tissue specific and developmental manner.