Tomato FRUITFULL homologues act in fruit ripening via forming MADS-box transcription factor complexes with RIN

Abstract

The tomato MADS-box transcription factor RIN acts as a master regulator of fruit ripening. Here, we identified MADS-box proteins that interact with RIN; we also provide evidence that these proteins act in the regulation of fruit ripening. We conducted a yeast two-hybrid screen of a cDNA library from ripening fruit, for genes encoding proteins that bind to RIN. The screen identified two MADS-box genes, FUL1 and FUL2 (previously called TDR4 and SlMBP7), both of which have high sequence similarity to Arabidopsis FRUITFULL. Expression analyses revealed that the FUL1 mRNA and FUL1 protein accumulate in a ripening-specific manner in tomato fruits and FUL2 mRNA and protein accumulate at the pre-ripening stage and throughout ripening. Biochemical analyses confirmed that FUL1 and FUL2 form heterodimers with RIN; this interaction required the FUL1 and FUL2 C-terminal domains. Also, the heterodimers bind to a typical target DNA motif for MADS-box proteins. Chromatin immunoprecipitation assays revealed that FUL1 and FUL2 bind to genomic sites that were previously identified as RIN-target sites, such as the promoter regions of ACS2, ACS4 and RIN. These findings suggest that RIN forms complexes with FUL1 and FUL2 and these complexes regulate expression of ripening-related genes. In addition to the functional redundancy between FUL1 and FUL2, we also found they have potentially divergent roles in transcriptional regulation, including a difference in genomic target sites.

TomatoFRUITFULLhomologs regulate fruit ripening via ethylene biosynthesis

“…Our previous study found that FUL1 and FUL2 are plausible candidates for components of the ripening regulator complex. 20) In this study, we demonstrated that the suppression of FUL1 and FUL2 inhibited ripening, at least partially through ethylene-dependent pathways. These results are consistent with the hypothesis that the FUL homologs and RIN compose a complex that regulates ripening.…”

“…1) and the results of our previous study, 20) we hypothesized that FUL1 and FUL2 act redundantly in the regulation of fruit ripening. Therefore, we decided to suppress FUL1 and FUL2 simultaneously, and constructed an RNAi vector using a FUL2 fragment that shows 78% identity to the corresponding sequence of FUL1.…”

“…20) We found that RIN forms heterodimers with FUL1 and FUL2 and that the heterodimers can bind to a typical target DNA motif for MADS-box proteins in vitro. Moreover, in vivo analysis revealed that the FUL homologs bind to the promoters of the two genes that encode a rate-limiting enzyme for ripening-associated ethylene production, 1-aminocyclopropane-1-carboxylic acid (ACC) synthase (ACS2 and ACS4).…”

“…Based on the following evidence, we have hypothesized that the FUL homologs regulate ethyleneinducible ripening processes: (i) RIN is a dominant regulator of ripening-associated ethylene production, 5) and FUL1 and FUL2 form complexes with RIN, 10,20) (ii) ChIP assays have indicated that FUL1 and FUL2 bind to the ACS gene promoters that are target sites of RIN.…”

“…16,18) We observed lower expression of the genes involved in cuticle biosynthesis in the pre-ripening stage fruit of the suppressed line. FUL2 is expressed strongly at the pre-ripening stage, but FUL1 is not, 20) suggesting that FUL2 is responsible for cuticle biosynthesis before ripening.…”