Fruit Ripening Regulation of α-Mannosidase Expression by the MADS Box Transcription Factor RIPENING INHIBITOR and Ethylene

alpha-Mannosidase (alpha-Man), a fruit ripening-specific N-glycan processing enzyme, is involved in ripening-associated fruit softening process. However, the regulation of fruit ripening specific expression of alpha-Man is not well understood. We have identified and functionally characterized the promoter of tomato (Solanum lycopersicum) alpha-Man to provide molecular insights into its transcriptional regulation during fruit ripening. Fruit ripening-specific activation of the alpha-Man promoter was revealed by analysing promoter driven expression of beta-glucuronidase (GUS) reporter in transgenic tomato. We found that RIPENING INHIBITOR (RIN), a MADS box family transcription factor acts as positive transcriptional regulator of alpha-Man during fruit ripening. RIN directly bound to the alpha-Man promoter sequence and promoter activation/alpha-Man expression was compromised in rin mutant fruit. Deletion analysis revealed that a promoter fragment (567 bp upstream of translational start site) that contained three CArG boxes (binding sites for RIN) was sufficient to drive GUS expression in fruits. In addition, alpha-Man expression was downregulated in fruits of Nr mutant which is impaired in ethylene perception and promoter activation/alpha-Man expression was induced in wild type following treatment with a precurs or of ethylene biosynthesis, 1-aminocyclopropane-1-carboxylic acid (ACC). Although, alpha-Man expression was induced in rin mutant after ACC treatment, the transcript level was less as compared to ACC-treated wild type. Taken together, these results suggest RIN-mediated direct transcriptional regulation of alpha-Man during fruit ripening and ethylene may acts in RIN-dependent and -independent ways to regulate alpha-Man expression.