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

Vijaya Shukla,Mark L Tucker,Sairam V Rudrabhatla,Autar K Mattoo,James J Giovannoni,Rakesh K Upadhyay

doi:10.1038/s41598-017-06622-0

Abstract

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.

Highlights

Small heat shock proteins are ubiquitous ancient proteins with conserved structural features, which evolved before the divergence of Archaea, Bacteria, and Eukarya[1,2,3,4,5,6,7]
Never-ripe 2 (Nr-2) mutation is a semi-dominant mutation in a tomato ethylene receptor orthologous to Arabidopsis AtETR1 ethylene receptor[29], while the non-ripening (Nor) mutation is linked to a change in the expression of a NAC transcription factor, both affect ethylene and ripening-dependent gene expression[30]
Transcripts of all the three SlHSP genes are highly up-regulated during ripening, abundance of each increasing from mature green (MG) to BR stage [7.28fold for SlHSP17.6, 5.35-fold for SlHSP20.0 and 17.40-fold for SlHSP20.1], with SlHSP20.1 being more abundant at the BR + 8 stage (Fig. 1a)

Summary

Introduction

Small heat shock proteins (sHSPs) are ubiquitous ancient proteins with conserved structural features, which evolved before the divergence of Archaea, Bacteria, and Eukarya[1,2,3,4,5,6,7]. Diverse sHSPs are found with distinct subfamilies, and their up-regulation by heat has presented an avenue to study their role in thermotolerance[7] They are induced by other stresses[12]. To gain an insight into possible ethylene-mediated regulation of three clustered tomato sHSP genes, we studied their gene expression in wild-type Ailsa Craig variety and its isogenic ripening mutants – rin/rin, nor/nor and Nr/Nr, and in the wild-type Ohio8245 processing tomato line and its transgenic line harboring an anti-sense ACC synthase 2 gene (2AS-AS). We demonstrate that all the three tomato sHSP genes (17.6, 20.0 and 20.1) harbor functional and interactive RIN-binding, CArG motifs in their promoters. The relevance of these findings to the control of ripening is discussed

Methods

Results

Conclusion