Transient regulation of three clustered tomato class-I small heat-shock chaperone genes by ethylene is mediated by SIMADS-RIN transcription factor

Authors: SHUKLA, VIJAYA - Pennsylvania State University; UPADHYAY, RAKESH - Pennsylvania State University; Tucker, Mark; Giovannoni, James; RUDHRABHATLA, SAIRAM - Pennsylvania State University; Mattoo, Autar

Submitted to: Scientific Reports
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 6/27/2017
Publication Date: 7/25/2017
Citation: Shukla, V., Upadhyay, R.K., Tucker, M.L., Giovannoni, J.J., Rudhrabhatla, S.V., Mattoo, A.K. 2017. Transient regulation of three clustered tomato class-I small heat-shock chaperone genes by ethylene is mediated by SIMADS-RIN transcription factor. Scientific Reports. 7:6474. https://doi.org/10.1038/s41598-017-06622-0.
DOI: https://doi.org/10.1038/s41598-017-06622-0

Interpretive Summary: Ripening of fruits is a genetically programmed event in the life of a horticultural crop. The shelf life of ripe fruit is usually short while over-ripe fruit is unacceptable to consumers, loses nutritional value and becomes susceptible to disease. Such postharvest decay results in close to a 30% loss of fruit. Ripening is initiated by the gaseous hormone ethylene. Features of the ethylene signaling pathway are being studied worldwide in order to develop the means to control the fruit ripening process. Studies with fruit ripening mutants in tomato have brought to light the function of a protein called RIN that regulates the initiation of tomato fruit ripening by affecting transcription of important genes. A plethora of genes are known to regulate fruit ripening. Among these is a class of small heat shock protein chaperone genes. There is limited information on how these genes are regulated. In this manuscript, we present data showing that ethylene causes transient and negative regulation of a clustered group of small heat shock protein genes prior to the initiation of fruit ripening. Using wild-type control lines, tomato fruit ripening mutants, and an ethylene-deficient transgenic tomato line, we demonstrated that the RIN protein binds to specific regions of the clustered small heat shock proteins and transiently blocks their transcription. Identification of such targets that regulate the fruit ripening-initiation is critical for development of future strategies to regulate the ripening process of fruits. These findings will be of interest to a diverse and broad readership including scientists involved in developing new strategies to enrich horticultural crops with a genetic potential for prolonged postharvest shelf life.

Technical Abstract: An intronless cluster of three class I small heat shock protein (sHSP) chaperone genes, Sl17.6, Sl20.0 and Sl20.1, resident on the short arm of chromosome 6 in tomato, was previously characterized (Goyal et al., 2012). This shsp chaperone gene cluster was found decorated with cis sequences known to be responsive to hormones (ABA, ethylene, GA, methyl jasmonate), developmental regulation (MYB), abiotic (CBF, DRE), and biotic (WB/W box, WRKY super family) stressors. Since ethylene is a fruit ripening hormone, we investigated if it regulates transcription of shsp genes. Mature green tomatoes treated with ethylene or an inhibitor of ethylene signaling, 1-methylcyclopropane (1-MCP), confirmed that ethylene suppressed the expression of the shsp chaperone gene cluster while treatment with 1-MCP overcame this suppression. Next, shsp transcript levels in three near isogenic tomato ripening mutants, rin, nor and Nr, were found constitutively expressed to different degrees at mature green stage while ethylene treatment led to their inhibition in nor/nor mutant, less so in Nr/Nr mutant, but the rin/rin mutant was totally refractile. These results suggested that RIN protein may be involved in regulating these genes. We quantified transcript levels of a few representative ethylene responsive and ripening-modulated genes, ACS6, PG, TAG1 and TAGL11, RIN, NR and SR3L, to confirm and validate our transcript profile patterns. In silico analysis indicated the presence of RIN binding sites, namely CArG motifs, in the promoter of these class I shsps genes. Chromatin immunoprecipitation assay confirmed RIN protein binding to specific CArG motifs found in the promoters of the three shsp chaperone genes. Taken together these results demonstrate that RIN protein regulates transcription of class I shsp genes in an ethylene-dependent manner.