Toshitsugu Nakano

A Large-Scale Identification of Direct Targets of the Tomato MADS Box Transcription Factor RIPENING INHIBITOR Reveals the Regulation of Fruit Ripening

This work identifies direct binding targets of the master ripening regulator RIN, a MADS box transcription factor, and examines RIN-based transcriptional regulation, finding that RIN has positive and negative regulatory roles throughout tomato fruit ripening, including in lycopene accumulation, ethylene production, chlorophyll degradation, and many other physiological processes. 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.

Identification of potential target genes for the tomato fruit-ripening regulator RIN by chromatin immunoprecipitation.

BackgroundDuring ripening, climacteric fruits increase their ethylene level and subsequently undergo various physiological changes, such as softening, pigmentation and development of aroma and flavor. These changes occur simultaneously and are caused by the highly synchronized expression of numerous genes at the onset of ripening. In tomatoes, the MADS-box transcription factor RIN has been regarded as a key regulator responsible for the onset of ripening by acting upstream of both ethylene- and non-ethylene-mediated controls. However, except for LeACS2, direct targets of RIN have not been clarified, and little is known about the transcriptional cascade for ripening.ResultsUsing immunoprecipitated (IPed) DNA fragments recovered by chromatin immunoprecipitation (ChIP) with anti-RIN antibody from ripening tomato fruit, we analyzed potential binding sites for RIN (CArG-box sites) in the promoters of representative ripening-induced genes by quantitative PCR. Results revealed nearly a 5- to 20-fold enrichment of CArG boxes in the promoters of LeACS2, LeACS4, PG, TBG4, LeEXP1, and LeMAN4 and of RIN itself, indicating direct interaction of RIN with their promoters in vivo. Moreover, sequence analysis and genome mapping of 51 cloned IPed DNAs revealed potential RIN binding sites. Quantitative PCR revealed that four of the potential binding sites were enriched 4- to 17-fold in the IPed DNA pools compared with the controls, indicating direct interaction of RIN with these sites in vivo. Near one of the four CArG boxes we found a gene encoding a protein similar to thioredoxin y1. An increase in the transcript level of this gene was observed with ripening in normal fruit but not in the rin mutant, suggesting that RIN possibly induces its expression.ConclusionsThe presented results suggest that RIN controls fruit softening and ethylene production by the direct transcriptional regulation of cell-wall-modifying genes and ethylene biosynthesis genes during ripening. Moreover, the binding of RIN to its own promoter suggests the presence of autoregulation for RIN expression. ChIP-based analyses identified a novel RIN-binding CArG-box site that harbors a gene associated with RIN expression in its flanking region. These findings clarify the crucial role of RIN in the transcriptional regulation of ripening initiation and progression.

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

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.

MACROCALYX and JOINTLESS Interact in the Transcriptional Regulation of Tomato Fruit Abscission Zone Development

Abscission in plants is a crucial process used to shed organs such as leaves, flowers, and fruits when they are senescent, damaged, or mature. Abscission occurs at predetermined positions called abscission zones (AZs). Although the regulation of fruit abscission is essential for agriculture, the developmental mechanisms remain unclear. Here, we describe a novel transcription factor regulating the development of tomato (Solanum lycopersicum) pedicel AZs. We found that the development of tomato pedicel AZs requires the gene MACROCALYX (MC), which was previously identified as a sepal size regulator and encodes a MADS-box transcription factor. MC has significant sequence similarity to Arabidopsis (Arabidopsis thaliana) FRUITFULL, which is involved in the regulation of fruit dehiscent zone development. The MC protein interacted physically with another MADS-box protein, JOINTLESS, which is known as a regulator of fruit abscission; the resulting heterodimer acquired a specific DNA-binding activity. Transcriptome analyses of pedicels at the preabscission stage revealed that the expression of the genes involved in phytohormone-related functions, cell wall modifications, fatty acid metabolism, and transcription factors is regulated by MC and JOINTLESS. The regulated genes include homologs of Arabidopsis WUSCHEL, REGULATOR OF AXILLARY MERISTEMS, CUP-SHAPED COTYLEDON, and LATERAL SUPPRESSOR. These Arabidopsis genes encode well-characterized transcription factors regulating meristem maintenance, axillary meristem development, and boundary formation in plant tissues. The tomato homologs were specifically expressed in AZs but not in other pedicel tissues, suggesting that these transcription factors may play key roles in pedicel AZ development.

Transcriptional Regulation of Fruit Ripening by Tomato FRUITFULL Homologs and Associated MADS Box Proteins

This work identifies direct targets of the MADS box transcription factors FUL1 and FUL2, key regulators of tomato fruit ripening, finding that FUL1 and FUL2 act in the transcriptional regulation of many aspects of ripening, including ripening-related ethylene production and lycopene accumulation, cooperatively with or independently of the MADS box ripening regulators RIN and TAGL1. The tomato (Solanum lycopersicum) MADS box FRUITFULL homologs FUL1 and FUL2 act as key ripening regulators and interact with the master regulator MADS box protein RIPENING INHIBITOR (RIN). Here, we report the large-scale identification of direct targets of FUL1 and FUL2 by transcriptome analysis of FUL1/FUL2 suppressed fruits and chromatin immunoprecipitation coupled with microarray analysis (ChIP-chip) targeting tomato gene promoters. The ChIP-chip and transcriptome analysis identified FUL1/FUL2 target genes that contain at least one genomic region bound by FUL1 or FUL2 (regions that occur mainly in their promoters) and exhibit FUL1/FUL2-dependent expression during ripening. These analyses identified 860 direct FUL1 targets and 878 direct FUL2 targets; this set of genes includes both direct targets of RIN and nontargets of RIN. Functional classification of the FUL1/FUL2 targets revealed that these FUL homologs function in many biological processes via the regulation of ripening-related gene expression, both in cooperation with and independent of RIN. Our in vitro assay showed that the FUL homologs, RIN, and tomato AGAMOUS-LIKE1 form DNA binding complexes, suggesting that tetramer complexes of these MADS box proteins are mainly responsible for the regulation of ripening.

Expression profiling of tomato pre-abscission pedicels provides insights into abscission zone properties including competence to respond to abscission signals

BackgroundDetachment of plant organs occurs in abscission zones (AZs). During plant growth, the AZ forms, but does not develop further until the cells perceive abscission-promoting signals and initiate detachment. Upon signal perception, abscission initiates immediately; if there is no signal, abscission is not induced and the organ remains attached to the plant. However, little attention has been paid to the genes that maintain competence to respond to the abscission signal in the pre-abscission AZ. Recently, we found that the tomato (Solanum lycopersicum) transcription factors BLIND (Bl), GOBLET (GOB), Lateral suppressor (Ls) and a tomato WUSCHEL homologue (LeWUS) are expressed specifically in pre-abscission tissue, the anthesis pedicel AZs. To advance our understanding of abscission, here we profiled genome-wide gene expression in tomato flower pedicels at the pre-abscission stage.ResultsWe examined the transcriptomes of three tomato flower pedicel regions, the AZ and flanking proximal- (Prox) and distal- (Dis) regions, and identified 89 genes that were preferentially expressed in the AZ compared to both Prox and Dis. These genes included several transcription factors that regulate apical or axillary shoot meristem activity. Also, genes associated with auxin activity were regulated in a Prox-Dis region-specific manner, suggesting that a gradient of auxin exists in the pedicel. A MADS-box gene affecting floral transition was preferentially expressed in the Prox region and other MADS-box genes for floral organ identification were preferentially expressed in Dis, implying that the morphologically similar Prox and Dis regions have distinct identities. We also analyzed the expression of known regulators; in anthesis pedicels, Bl, GOB, Ls and LeWUS were expressed in the vascular cells of the AZ region. However, after an abscission signal, Bl was up-regulated, but GOB, Ls and LeWUS were down-regulated, suggesting that Bl may be a positive regulator of abscission, but the others may be negative regulators.ConclusionsThis study reveals region-specific gene expression in tomato flower pedicels at anthesis and identifies factors that may determine the physiological properties of the pre-abscission pedicel. The region-specific transcriptional regulators and genes for auxin activity identified here may prevent flower abscission in the absence of signal or establish competence to respond to the abscission signal.

Tomato FRUITFULL homologues act in fruit ripening via forming MADS-box transcription factor complexes with RIN

The tomato MADS-box transcription factor RIN acts as a master regulator of fruit ripening. Here, we identified MADS-box proteins that interact with RIN; we also provide evidence that these proteins act in the regulation of fruit ripening. We conducted a yeast two-hybrid screen of a cDNA library from ripening fruit, for genes encoding proteins that bind to RIN. The screen identified two MADS-box genes, FUL1 and FUL2 (previously called TDR4 and SlMBP7), both of which have high sequence similarity to Arabidopsis FRUITFULL. Expression analyses revealed that the FUL1 mRNA and FUL1 protein accumulate in a ripening-specific manner in tomato fruits and FUL2 mRNA and protein accumulate at the pre-ripening stage and throughout ripening. Biochemical analyses confirmed that FUL1 and FUL2 form heterodimers with RIN; this interaction required the FUL1 and FUL2 C-terminal domains. Also, the heterodimers bind to a typical target DNA motif for MADS-box proteins. Chromatin immunoprecipitation assays revealed that FUL1 and FUL2 bind to genomic sites that were previously identified as RIN-target sites, such as the promoter regions of ACS2, ACS4 and RIN. These findings suggest that RIN forms complexes with FUL1 and FUL2 and these complexes regulate expression of ripening-related genes. In addition to the functional redundancy between FUL1 and FUL2, we also found they have potentially divergent roles in transcriptional regulation, including a difference in genomic target sites.

The AP2/ERF transcription factor SlERF52 functions in flower pedicel abscission in tomato

In plants, abscission removes senescent, injured, infected, or dispensable organs. Induced by auxin depletion and an ethylene burst, abscission requires pronounced changes in gene expression, including genes for cell separation enzymes and regulators of signal transduction and transcription. However, the understanding of the molecular basis of this regulation remains incomplete. To examine gene regulation in abscission, this study examined an ERF family transcription factor, tomato (Solanum lycopersicum) ETHYLENE-RESPONSIVE FACTOR 52 (SlERF52). SlERF52 is specifically expressed in pedicel abscission zones (AZs) and SlERF52 expression is suppressed in plants with impaired function of MACROCALYX and JOINTLESS, which regulate pedicel AZ development. RNA interference was used to knock down SlERF52 expression to show that SlERF52 functions in flower pedicel abscission. When treated with an abscission-inducing stimulus, the SlERF52-suppressed plants showed a significant delay in flower abscission compared with wild type. They also showed reduced upregulation of the genes for the abscission-associated enzymes cellulase and polygalacturonase. SlERF52 suppression also affected gene expression before the abscission stimulus, inhibiting the expression of pedicel AZ-specific transcription factor genes, such as the tomato WUSCHEL homologue, GOBLET, and Lateral suppressor, which may regulate meristematic activities in pedicel AZs. These results suggest that SlERF52 plays a pivotal role in transcriptional regulation in pedicel AZs at both pre-abscission and abscission stages.

Tomato FRUITFULL homologs regulate fruit ripening via ethylene biosynthesis

Certain MADS-box transcription factors play central roles in regulating fruit ripening. RIPENING INHIBITOR (RIN), a tomato MADS-domain protein, acts as a global regulator of ripening, affecting the climacteric rise of ethylene, pigmentation changes, and fruit softening. Previously, we showed that two MADS-domain proteins, the FRUITFULL homologs FUL1 and FUL2, form complexes with RIN. Here, we characterized the FUL1/FUL2 loss-of-function phenotype in co-suppressed plants. The transgenic plants produced ripening-defective fruits accumulating little or no lycopene. Unlike a previous study on FUL1/FUL2 suppressed tomatoes, our transgenic fruits showed very low levels of ethylene production, and this was associated with suppression of the genes for 1-aminocyclopropane-1-carboxylic acid synthase, a rate-limiting enzyme in ethylene synthesis. FUL1/FUL2 suppression also caused the fruit to soften in a manner independent of ripening, possibly due to reduced cuticle thickness in the peel of the suppressed tomatoes. Graphical Abstract The tomato FRUITFULL homologs (FULs) interact with RIPENING INHIBITOR (RIN) and FULs knockdown inhibits fruit ripening similarly to the rin mutation.

Development and regulation of pedicel abscission in tomato

To shed unfertilized flowers or ripe fruits, many plant species develop a pedicel abscission zone (AZ), a specialized tissue that develops between the organ and the main body of the plant. Regulation of pedicel abscission is an important agricultural concern because pre-harvest abscission can reduce yields of fruit or grain crops, such as apples, rice, wheat, etc. Tomato has been studied as a model system for abscission, as tomato plants develop a distinct AZ at the midpoint of the pedicel and several tomato mutants, such as jointless, have pedicels that lack an AZ. This mini-review focuses on recent advances in research on the mechanisms regulating tomato pedicel abscission. Molecular genetic studies revealed that three MADS-box transcription factors interactively play a central role in pedicel AZ development. Transcriptome analyses identified activities involved in abscission and also found novel transcription factors that may regulate AZ activities. Another study identified transcription factors mediating abscission pathways from induction signals to activation of cell wall hydrolysis. These recent findings in tomato will enable significant advances in understanding the regulation of abscission in other key agronomic species.