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. 2013 Feb;25(2):371-86.
doi: 10.1105/tpc.112.108118. Epub 2013 Feb 5.

A large-scale identification of direct targets of the tomato MADS box transcription factor RIPENING INHIBITOR reveals the regulation of fruit ripening

Masaki Fujisawa  1 , Toshitsugu Nakano, Yoko Shima, Yasuhiro Ito
Affiliations

A large-scale identification of direct targets of the tomato MADS box transcription factor RIPENING INHIBITOR reveals the regulation of fruit ripening

Masaki Fujisawa et al. Plant Cell. 2013 Feb.

Abstract

The fruit ripening developmental program is specific to plants bearing fleshy fruits and dramatically changes fruit characteristics, including color, aroma, and texture. The tomato (Solanum lycopersicum) MADS box transcription factor RIPENING INHIBITOR (RIN), one of the earliest acting ripening regulators, is required for both ethylene-dependent and -independent ripening regulatory pathways. Recent studies have identified two dozen direct RIN targets, but many more RIN targets remain to be identified. Here, we report the large-scale identification of direct RIN targets by chromatin immunoprecipitation coupled with DNA microarray analysis (ChIP-chip) targeting the predicted promoters of tomato genes. Our combined ChIP-chip and transcriptome analysis identified 241 direct RIN target genes that contain a RIN binding site and exhibit RIN-dependent positive or negative regulation during fruit ripening, suggesting that RIN has both activator and repressor roles. Examination of the predicted functions of RIN targets revealed that RIN participates in the regulation of lycopene accumulation, ethylene production, chlorophyll degradation, and many other physiological processes. Analysis of the effect of ethylene using 1-methylcyclopropene revealed that the positively regulated subset of RIN targets includes ethylene-sensitive and -insensitive transcription factors. Intriguingly, ethylene is involved in the upregulation of RIN expression during ripening. These results suggest that tomato fruit ripening is regulated by the interaction between RIN and ethylene signaling.

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Figures

Figure 1.
Figure 1.
Distribution of the RIN Binding Sites Detected by ChIP-chip on the Tomato Chromosomes. Genomic positions of the RIN binding sites on the 12 tomato chromosomes are indicated by red bars with the log2 scale peak score. Positions of the promoters (top, forward strand; bottom, complementary strand) where ChIP-chip probes were designed are indicated by blue bars.
Figure 2.
Figure 2.
Position and Conservation of the CArG Boxes in the RIN Binding Sites. (A) Histogram of the distance between the center of the RIN binding sites and CArG boxes found in the sites. (B) The consensus sequence of the CArG boxes in the RIN binding sites of the gene promoters.
Figure 3.
Figure 3.
Identification of Direct RIN Targets Whose Expression Was Regulated by RIN. (A) Venn diagram of potential direct RIN targets and genes positively and negatively regulated by RIN selected by microarray analysis. (B) Distribution of RIN binding sites in the gene regulatory and transcribed regions of the positively and negatively regulated subsets.
Figure 4.
Figure 4.
RIN Binding Sites in the Promoters of Ripening-Associated Genes. RIN binding sites detected by ChIP-chip in the promoters of known direct RIN targets (A) and previously unidentified direct RIN targets (B) involved in fruit ripening. Genomic position and log2 scale peak score of each RIN binding site is indicated above the 2-kb gene promoters (horizontal lines). Boxed arrows with gene identifier indicate the orientation of genes. Thin vertical lines indicate the positions of three types of CArG boxes in the promoter. Asterisks below the thin vertical lines in (A) indicate the position of CArG boxes that were confirmed to be enriched by the previous qChIP-PCR.
Figure 5.
Figure 5.
Functional Classification of the Direct RIN Targets. For the analysis, we used genes that were assigned MIPS information based on similarity to Arabidopsis genes (135 positively regulated targets, 101 negatively regulated targets, and 28,440 genes as whole genome). Bars represent the ratio of the genes included in the categories. Asterisks indicate a significant enrichment (P < 0.001 by Fisher's exact test) compared with the genome.
Figure 6.
Figure 6.
A Diagram of the Terpenoid Backbone Synthesis and Carotenoid Biosynthesis Pathways with Expression Profiles of Direct and Indirect RIN Target Genes during Ripening. Arrows indicate reaction flows in the pathways. The names of enzymes that catalyze the reaction are indicated on the left or below the arrows when the enzymes are encoded by direct or indirect RIN target genes. Boxes represent the predicted gene identifiers encoding the enzymes. FC value of the genes in the wild-type (FCWT) and rin mutant (FCrin) fruits during ripening are shown in parentheses. A known direct RIN target, PSY1 (Solyc03g031860; indicated by an asterisk), is also shown in red and was not detected by our ChIP-chip analysis. CRTISO, carotenoid isomerase; CrtL-b, lycopene β-cyclase; CrtL-e, lycopene ϵ-cyclase; DXS, 1-deoxy-d-xylulose 5-phosphate synthase; IPI, isopentenyl pyrophosphate isomerase; ISPE, 4-diphosphocytidyl-2-C-methyl-d-erythritol kinase; P and PP, phosphate and pyrophosphate, respectively; PSY, phytoene synthase; ZDS, ζ-carotene desaturase; Z-ISO, ζ-carotene isomerase.
Figure 7.
Figure 7.
Expression Targets during Ripening of Wild-Type and rin Mutant Fruits of TF Genes That Are Direct RIN Targets. The change in expression level of the genes in the fruits at the ripening (P and R) stages is shown as FC relative to that in the G stage in the wild type and rin mutant. Bars represent the mean of three biological replicates. Error bars represent sd of the mean. Asterisks indicate a statistically significant (P < 0.05) difference in FC between the wild-type and rin mutant fruits at each P or R and G stage.
Figure 8.
Figure 8.
Expression of the TF Genes That Are Direct RIN Targets in Tomato Fruits Treated with 1-MCP. (A) Tomato fruits at the breaker (Br) stage and after 4 d of treatment (Br + 4 d) with 1-MCP. Fruits harvested at the Br stage and treated with water for 4 d were used as a control. (B) Expression analysis of direct RIN target TF genes in the control and 1-MCP–treated tomato fruits. The change in expression levels of the genes in the fruits treated with 1-MCP is shown as FC relative to the control. Bars represent the mean of three biological replicates. Error bars represent sd of the mean. Asterisks indicate a statistically significant (P < 0.05) difference in FC between the 1-MCP–treated fruits and control.
Figure 9.
Figure 9.
A Schematic Representation of the Proposed Model for a Regulatory Mechanism of Tomato Fruit Ripening, Including a Mechanism That Maintains Ethylene Levels via RIN and Other Factors. Bold line arrows indicate an ethylene-mediated positive feedback loop that enhances RIN expression. It is unclear whether the loop regulates the expression of the other ripening regulators (such as NOR and TDR4) affected by ethylene during ripening directly or indirectly (via RIN). Arrows indicate the direction of the transcriptional regulatory pathways. Blunt-ended lines indicate repression. Circle arrows on RIN and TAGL1 indicate autoregulation and on ethylene indicate autocatalytic ethylene production.
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