Clustered class-I small heat-shock protein (sHSP) chaperone genes, SlHSP17.6, SlHSP20.0 and SlHSP20.1, in tomato are demonstrated to be transcriptionally regulated by ethylene during mature green (MG) fruit transition into ripening. These genes are constitutively expressed at MG fruit stage in two different tomato genotypes as well as in their ripening mutants, including rin, nor and Nr, and an ethylene-deficient transgenic line, ACS2-antisense. Notably, ethylene treatment of the MG fruit led to significant sHSP gene suppression in both wild-types, ACS2-antisense, nor/nor and Nr/Nr, but not the rin/rin mutant. Inability of ethylene to suppress sHSP genes in rin/rin mutant, which harbors MADS-RIN gene mutation, suggests that MADS-RIN transcription factor regulates the expression of these genes. Treatment of the wild type and ACS2-antisense fruit with the ethylene-signaling inhibitor, 1-methylcyclopropane (1-MCP), reversed the sHSP gene suppression. Transcripts of representative ethylene-responsive and ripening-modulated genes confirmed and validated sHSP transcript profile patterns. In silico analysis in conjunction with chromatin immunoprecipitation demonstrated MADS-RIN protein binding to specific CArG motifs present in the promoters of these chaperone genes. The results establish MADS-RIN protein as a transcriptional regulator of these chaperone genes in an ethylene-dependent manner, and that MADS-RIN protein-regulation of sHSPs is integral to tomato fruit ripening.
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