Direct targets of the tomato-ripening regulator RIN identified by transcriptome and chromatin immunoprecipitation analyses

Abstract

The physiological and biochemical changes in fruit ripening produce key attributes of fruit quality including color, taste, aroma and texture. These changes are driven by the highly regulated and synchronized activation of a huge number of ripening-associated genes. In tomato (Solanum lycopersicum), a typical climacteric fruit, the MADS-box transcription factor RIN is one of the earliest-acting ripening regulators, required for both ethylene-dependent and ethylene-independent pathways. Although we previously identified several direct RIN targets, many additional targets remain unidentified, likely including key ripening-associated genes. Here, we report the identification of novel RIN targets by transcriptome and chromatin immunoprecipitation (ChIP) analyses. Transcriptome comparisons by microarray of wild-type and rin mutant tomatoes identified 342 positively regulated genes and 473 negatively regulated genes by RIN during ripening. Most of the positively regulated genes contained possible RIN-binding (CArG-box) sequences in their promoters. Subsequently, we selected six genes from the positively regulated genes and a ripening regulator gene, CNR, and assayed their promoters by quantitative ChIP-PCR to examine RIN binding. All of the seven genes, which are involved in cell wall modification, aroma and flavor development, pathogen defense and transcriptional regulation during ripening, are targets of RIN, suggesting that RIN may control multiple diverse ripening processes. In particular, RIN directly regulates the expression of the ripening-associated transcription factors, CNR, TDR4 and a GRAS family gene, providing an important clue to elucidate the complicated transcriptional cascade for fruit ripening.

The Tomato FRUITFULL Homologs TDR4/FUL1 and MBP7/FUL2 Regulate Ethylene-Independent Aspects of Fruit Ripening

“…A full list of significantly differentially expressed genes can be found in Supplemental Data Set 1 online. Our results confirm downregulation of both FUL1 (log 2 = 24.5) and FUL2 (log 2 = 22.7) and also yielded a number of genes that were previously reported to be involved in tomato fruit ripening, including RIN and CNR targets (Thompson et al, 1999;Fujisawa et al, 2012;Qin et al, 2012). A short list, containing the up-and downregulated genes discussed in the text, can be found in Table 1.…”

“…Expression of several known targets of RIN, such as Carbonic Anhydrase3, Gibberellin 20-oxidase-2, Polygalacturonase Ae (PG2A), 1-deoxy-D-xylulose 5-phosphate synthase1 (DXS1), expansin B2, and Phosphoglycerate dehydrogenase, was also affected in the FUL1/2 knockdown plants, suggesting that there is overlap between the targets. Yet, other known targets of RIN, such as the ethylene biosynthesis genes ACO1, ACS2, and ACS4, and the ripening-related genes E4 and E8, lipoxygenase C, or the carotenoid biosynthesis gene Phytoene synthase1 (PSY1) were not significantly affected (Fujisawa et al, 2012). In addition to the earlier identified regulators of tomato fruit ripening, a large number of up-or downregulated genes was not implicated in ripening before but likely play a role in ripening-associated processes as well.…”

The regulatory mechanism of fruit ripening revealed by analyses of direct targets of the tomato MADS-box transcription factor RIPENING INHIBITOR

“…10 In the report, we analyzed RIN binding to DNA from ripening tomato fruits by ChIP-chip using anti-RIN antibodies and a microarray for tomato gene promoters. This ChIP-chip analysis detected 1,046 RIN binding sites and, in combination with transcriptome data for wild type and rin mutant tomato fruits, 6 we identified 241 genes as direct RIN targets. These targets are positively or negatively regulated by RIN, generally contain a RIN binding site in their promoters, and include 11 known ripeningrelated genes.…”

“…6 Shown are the subcategories in which the ratio of classified genes was significantly (p < 0.001 by Fisher's exact test) higher in the sets of direct RIN target genes than that in the tomato genome. Gene symbols and products: ACO6, …”

“…This suggests that RIN acts as a master regulator of ripening and recent studies revealed that RIN may directly regulate the expression of two dozen ripening-associated genes. [5][6][7][8][9] However, our knowledge of RIN target genes has been limited, considering the central role of RIN in regulation of ripening. In our recent work, published in The Plant Cell, we report a large-scale identification of direct RIN targets by chromatin immunoprecipitation coupled with DNA microarray (ChIP-chip) and transcriptome analysis.…”