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Presenters: Sinéad Carroll and Dylan O'Neill

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Presenters: Orin Gillespie and Suzanne Murphy

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Authors

Mi-Young Chung,

1. Boyce Thompson Institute for Plant Research, Tower Road, Cornell University Campus, Ithaca, NY 14853, USA

2. Department of Horticulture, Kyungpook National University, Daegu 702-701, Korea

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Rob Alba,

1. Boyce Thompson Institute for Plant Research, Tower Road, Cornell University Campus, Ithaca, NY 14853, USA

2. Monsanto Company, Product Safety Center, 800 N. Lindbergh Blvd, St Louis, MO 63167, USA

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James Giovannoni

Corresponding author

1. Boyce Thompson Institute for Plant Research, Tower Road, Cornell University Campus, Ithaca, NY 14853, USA

2. US Department of Agriculture Agricultural Research Service Robert W. Holy Center, Tower Road, Cornell University Campus, Ithaca, NY 14853, USA

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(fax 1 607 254 1502; e-mail jjg33@cornell.edu).

Summary

The transition of fleshy fruit maturation to ripening is regulated by exogenous and endogenous signals that coordinate the transition of the fruit to a final state of attractiveness to seed dispersing organisms. Tomato is a model for biology and genetics regulating specific ripening pathways including ethylene, carotenoids and cell wall metabolism in addition to upstream signaling and transcriptional regulators. Ripening-associated transcription factors described to date including the RIN-MADS, CLEAR NON-RIPENING, TAGL1 and LeHB-1 genes all encode positive regulators of ripening phenomena. Here we describe an APETALA2 transcription factor ( SlAP2a) identified through transcriptional profiling of fruit maturation that is induced during, and which negatively regulates, tomato fruit ripening. RNAi repression of SlAP2a results in fruits that over-produce ethylene, ripen early and modify carotenoid accumulation profiles by altering carotenoid pathway flux. These results suggest that SlAP2a functions during normal tomato fruit ripening as a modulator of ripening activity and acts to balance the activities of positive ripening regulators.

Ancillary

Supporting Information

Figure S1. Alignment of SlAP2a, SlAP2b and SlAP2c cDNA sequences.

Table S1. Carotenoid and chlorophyll content of SlAP2a RNAi lines and wild-type control tomatoes during fruit ripening. Carotenoid and chlorophyll contents of three different RNAi lines (#22, #25, and #8) and wild-type (WT) fruits were quantified by HPLC at different developmental stages (B, B3, B7 and B10 as defined in the methods). Standard error is indicated for a minimum of five fruit per sample. FW = fresh weight.

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Authors

Mi-Young Chung,

1. Boyce Thompson Institute for Plant Research, Tower Road, Cornell University Campus, Ithaca, NY 14853, USA

2. Department of Horticulture, Kyungpook National University, Daegu 702-701, Korea

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Rob Alba,

1. Boyce Thompson Institute for Plant Research, Tower Road, Cornell University Campus, Ithaca, NY 14853, USA

2. Monsanto Company, Product Safety Center, 800 N. Lindbergh Blvd, St Louis, MO 63167, USA

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James Giovannoni

Corresponding author

1. Boyce Thompson Institute for Plant Research, Tower Road, Cornell University Campus, Ithaca, NY 14853, USA

2. US Department of Agriculture Agricultural Research Service Robert W. Holy Center, Tower Road, Cornell University Campus, Ithaca, NY 14853, USA

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(fax 1 607 254 1502; e-mail jjg33@cornell.edu).

Summary

The transition of fleshy fruit maturation to ripening is regulated by exogenous and endogenous signals that coordinate the transition of the fruit to a final state of attractiveness to seed dispersing organisms. Tomato is a model for biology and genetics regulating specific ripening pathways including ethylene, carotenoids and cell wall metabolism in addition to upstream signaling and transcriptional regulators. Ripening-associated transcription factors described to date including the RIN-MADS, CLEAR NON-RIPENING, TAGL1 and LeHB-1 genes all encode positive regulators of ripening phenomena. Here we describe an APETALA2 transcription factor ( SlAP2a) identified through transcriptional profiling of fruit maturation that is induced during, and which negatively regulates, tomato fruit ripening. RNAi repression of SlAP2a results in fruits that over-produce ethylene, ripen early and modify carotenoid accumulation profiles by altering carotenoid pathway flux. These results suggest that SlAP2a functions during normal tomato fruit ripening as a modulator of ripening activity and acts to balance the activities of positive ripening regulators.

Ancillary

Supporting Information

Figure S1. Alignment of SlAP2a, SlAP2b and SlAP2c cDNA sequences.

Table S1. Carotenoid and chlorophyll content of SlAP2a RNAi lines and wild-type control tomatoes during fruit ripening. Carotenoid and chlorophyll contents of three different RNAi lines (#22, #25, and #8) and wild-type (WT) fruits were quantified by HPLC at different developmental stages (B, B3, B7 and B10 as defined in the methods). Standard error is indicated for a minimum of five fruit per sample. FW = fresh weight.

As a service to our authors and readers, this journal provides supporting information supplied by the authors. Such materials are peer-reviewed and may be re-organized for online delivery, but are not copy-edited or typeset. Technical support issues arising from supporting information (other than missing files) should be addressed to the authors.

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Authors

Mi-Young Chung,

1. Boyce Thompson Institute for Plant Research, Tower Road, Cornell University Campus, Ithaca, NY 14853, USA

2. Department of Horticulture, Kyungpook National University, Daegu 702-701, Korea

Search for more papers by this author

Rob Alba,

1. Boyce Thompson Institute for Plant Research, Tower Road, Cornell University Campus, Ithaca, NY 14853, USA

2. Monsanto Company, Product Safety Center, 800 N. Lindbergh Blvd, St Louis, MO 63167, USA

Search for more papers by this author

James Giovannoni

Corresponding author

1. Boyce Thompson Institute for Plant Research, Tower Road, Cornell University Campus, Ithaca, NY 14853, USA

2. US Department of Agriculture Agricultural Research Service Robert W. Holy Center, Tower Road, Cornell University Campus, Ithaca, NY 14853, USA

Search for more papers by this author

(fax 1 607 254 1502; e-mail jjg33@cornell.edu).

Summary

The transition of fleshy fruit maturation to ripening is regulated by exogenous and endogenous signals that coordinate the transition of the fruit to a final state of attractiveness to seed dispersing organisms. Tomato is a model for biology and genetics regulating specific ripening pathways including ethylene, carotenoids and cell wall metabolism in addition to upstream signaling and transcriptional regulators. Ripening-associated transcription factors described to date including the RIN-MADS, CLEAR NON-RIPENING, TAGL1 and LeHB-1 genes all encode positive regulators of ripening phenomena. Here we describe an APETALA2 transcription factor ( SlAP2a) identified through transcriptional profiling of fruit maturation that is induced during, and which negatively regulates, tomato fruit ripening. RNAi repression of SlAP2a results in fruits that over-produce ethylene, ripen early and modify carotenoid accumulation profiles by altering carotenoid pathway flux. These results suggest that SlAP2a functions during normal tomato fruit ripening as a modulator of ripening activity and acts to balance the activities of positive ripening regulators.

Ancillary

Supporting Information

Figure S1. Alignment of SlAP2a, SlAP2b and SlAP2c cDNA sequences.

Table S1. Carotenoid and chlorophyll content of SlAP2a RNAi lines and wild-type control tomatoes during fruit ripening. Carotenoid and chlorophyll contents of three different RNAi lines (#22, #25, and #8) and wild-type (WT) fruits were quantified by HPLC at different developmental stages (B, B3, B7 and B10 as defined in the methods). Standard error is indicated for a minimum of five fruit per sample. FW = fresh weight.

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A tomato (Solanum lycopersicum) APETALA2/ERF gene, SlAP2a, is a negative regulator of fruit ripening

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The transition of fleshy fruit maturation to ripening is regulated by exogenous and endogenous signals that coordinate the transition of the fruit to a final state of attractiveness to seed dispersing...

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Presenters: Kate McBrearty & Caoilfhionn Breathnach

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AP2 Homologs in Tomato

To gain insight into the molecular regulation of tomato fruit development, we selected potentially important transcription factor genes for suppression in transgenic tomato using an RNAi strategy. Comparison of EST frequencies in various tomato EST libraries indicated that the AP2 homolog represented by TC162117 is highly expressed in the pericarp of developing tomato fruit. Publicly available microarray expression data show that the level of mRNA represented by this contig increases steadily during tomato fruit development, to a level at day 48 that was 2.5 times the level at 7 d postanthesis ( http://ted.bti.cornell.edu/cgi-bin/array/unigene.cgi, SGN-U213383). This observation and the demonstrated important regulatory role of AP2-like transcription factors in developmental processes indicated that the AP2 homolog might play an important role in tomato fruit development as well.

We mined the Solanaceae Genomics Network (SGN) tomato Unigene database for other unigenes with homology to Arabidopsis AP2 and, where necessary, extended cDNA sequences by 5′ and 3′ rapid amplification of cDNA ends to obtain sequences covering the entire open reading frame of each gene. At completion, we had identified five distinct tomato cDNAs encoding AP2 homologs, including the one represented by the tentative consensus mentioned above. Figure 1A (see Supplemental Data Set 1 online) shows a phylogenetic tree of the encoded proteins together with AP2 homologs of Arabidopsis, Petunia, and Antirrhinum, as far as available. One of these is the previously identified AP2 from tomato represented by Unigenes SGN-U579591, 580201, and 593325, which we here call AP2a ( S. lycopersicum AP2a). For four other genes, full-length cDNA sequences were obtained by 5′ and 3′ rapid amplification of cDNA ends from available Unigene sequences. Two tomato proteins, which are homologous to the previously identified AP2A from Petunia, were designated AP2b (represented by SGN-U580135 and 589866) and AP2c (represented by SGN-U580203 and 579384). These three proteins are identical to AP2a, b, and c, respectively, published recently by Chung et al. (2010). The remaining two tomato AP2-like proteins are more distantly related to Arabidopsis AP2, are more similar to the P. hybrida AP2B ( Maes et al., 2001), and were designated AP2d (represented by SGN-U563871) and AP2e (represented by SGN-U585439 and 585539). A more elaborate phylogenetic three including these five proteins with AP2 subfamily proteins from diverse species is shown in Supplemental Figure 1 online (see Supplemental Data Set 2 online). This analysis firmly places tomato AP2a, b, and c proteins together with Arabidopsis AP2 in a subclade of the AP2 subgroup of the AP2 subfamily of transcription factors as defined by Shigyo et al. (2006), indicating that these three proteins are putative tomato orthologs of AP2. Tomato AP2d and AP2e, with P. hybrida AP2B, are more similar to Arabidopsis TOE1 and TOE2. Several other tomato AP2 subfamily proteins, or fragments thereof, were identified from the SGN Unigene database and were included in the analysis. An alignment of the tomato proteins with Arabidopsis AP2, TOE2, and AINTEGUMENTA (ANT) is shown in Supplemental Figure 2A online. The phylogenetic position of AP2d and AP2e is less clear than for the other three proteins (as shown by the low bootstrap values on the major nodes). However, all five proteins belong to the euAP2 lineage, as they all contain a putative miR172 target site and lack the characteristic ANT lineage motifs, such as the 10-amino acid and 1-amino acid insertions in AP2 domain repeats 1 and 2, respectively ( Kim and Nam, 2006).

AP2a Expression Increases during Fruit Ripening

To better understand possible functions as well as to identify possible functional redundancy through overlapping expression patterns for the tomato AP2 genes, we determined, by quantitative RT-PCR (qRT-PCR), the expression levels of the five AP2-like genes in tomato seedlings, leaves, roots, and flowers at anthesis, flowers at 2 d postanthesis (Fruit I), and fruit at different developmental stages. All expression levels as related to the expression of the β-actin gene and normalized to the first sample (seedling) are shown in Figure 1B. From this figure it is apparent that of the three genes most related to Arabidopsis AP2, AP2b, and AP2c have similar expression patterns, with highest expression in flowers and some expression in roots, leaves, and early stage fruits, but no significant expression beyond the mature green stage. By contrast, AP2a is expressed at a relatively low level in flowers and early fruit stages but is strongly upregulated between the mature green and breaker stages and is highly expressed up to the red ripe stage. AP2d and AP2e are differentially expressed, with AP2d being preferentially expressed in very early fruit development and AP2e being expressed particularly in vegetative tissues, but also in flowers. All homologs were expressed in seeds, with AP2d and AP2e being the highest expressed (see Supplemental Figure 3A online). Thus, the five identified tomato AP2 paralogs are differentially expressed in vegetative tissues, flowers, and developing fruit. The differential expression of the three closest homologs of AP2, with only AP2a being expressed in ripening fruit and AP2b and AP2c both being expressed in flowers, suggests that extensive gene duplication and subsequent subfunctionalization of these AP2 homologs through differential expression have occurred in the tomato lineage since its divergence from the Arabidopsis lineage in evolution. If this is indeed the case, the genes that, like Arabidopsis AP2, are predominantly expressed in flowers may function in floral organ identity determination, as AP2 does, while AP2a may have acquired a novel function more specific for fleshy fruit development.

Phenotype of AP2a RNAi Lines

We constructed an RNAi vector from a 468-bp cDNA fragment (underlined in Supplemental Figure 2B online) of AP2a. The resulting construct, designated AP2i-1, contains two copies of a sequence encoding most of the two AP2 domains (see Supplemental Figure 2B online, bold) in an inverted repeat orientation under the transcriptional control of the cauliflower mosaic virus 35S promoter. Transgenic tomato cv Moneymaker plants harboring the AP2i-1 construct were generated to examine the effect of the suppressed AP2a expression on development.

Out of 12 analyzed diploid transformants, three lines with AP2i-1 exhibited severe alterations both in flower development and in fruit development and ripening, and three showed a milder phenotype. Figure 2 and Supplemental Figures 3B to 3E online show representative phenotypes of flowers and fruit of AP2i-1 lines. The sepals were greatly increased in size, both in length and in width (see Supplemental Figures 3B and 3C online). Sepals were fused along the margins and curled at the tip, forming a bell shape that completely enclosed the flower, leaving the petals invisible ( Figure 2A; see Supplemental Figure 3C online). As the fruits developed, the sepals continued to increase in size (cf. the length and width of sepals in flowers and on fruit for wild-type and transgenic lines in Supplemental Figures 3B and 3C online). Because all five tomato AP2 paralogs are expressed in flowers, they might be functionally redundant in that organ; therefore, the flower phenotype may be the result of downregulation of one or more of the other AP2 homologs in addition to AP2a. To test this hypothesis, we performed qRT-PCR to compare the expression of the five AP2 homologs in AP2i-1 and wild-type sepals ( Figure 1C). We found that not only AP2a but also its closest homologs, AP2b and AP2c, were downregulated in sepals, while the expression of the other two members of the family, AP2d and AP2e, was not affected.

Additionally, the morphology of the fruit was altered in some of the AP2i-1 lines ( Figures 2B to 2D). A wild-type fruit ( Figure 2D) is round, with a smooth, consistent epidermal surface. Fruits of the AP2i-1-8 line, on the other hand, were not round but had large indentations delineating the location of each locular cavity. Another common feature of these fruits was the raised bumpy areas that were present intermittently on the epidermal surface of the fruit ( Figure 2D, arrowheads). When the seeds from these AP2i-1-8 fruits were closely examined, we found that seed morphology was also affected. In AP2i-1 lines, a variety of aberrant seed shapes was observed (see Supplemental Figures 4A to 4E online). Moreover, seed coat morphology, specifically the size and shape of the surface hairs, was strongly affected. In tomato, the seed coat hairs are the cell wall remains of the outer epidermal cells. Their size and shape varies between cultivars and cv Moneymaker has laterally banded hairs as shown in Supplemental Figures 4B and 4E online ( Chakrabarti et al., 2003).

AP2a Is Involved in Fruit Ripening

In addition to the alterations observed in the early development of the fruit, AP2i-1 lines exhibited distinct alterations in the ripening process ( Figure 2). Ripe AP2i-1 fruits are shown in Figure 2A. All fruits had ripened, yet never turned from orange to homogenous red. The fruit phenotype for wild-type and AP2i-1 lines at various stages of ripening is shown in Figure 2B (several fruits of the same truss, representing different ripening stages, are shown). In the wild type, fruit ripening ( Figure 2B, top row, left to right) begins with mature full-sized green fruit. In the next stage, the breaker (Br) stage, a visible color change just begins to occur. By the turning stage, the entire fruit has a consistent orange color. In the final stage (breaker + 7 d) of ripening, the fruit has a deep red color and starts to soften. The differences in ripening between the AP2i-1 and wild-type fruits became apparent at the turning stage. At breaker + 7 d, the fruit retained its orange color, even though other aspects of the ripening process, such as tissue softening, had begun. In Figure 2C, fruits from the wild type and two independent AP2i lines, AP2i-1-8 and AP2i-1-10, at turning (top) and breaker + 7 stage (bottom) are shown. Wild-type fruit at the turning stage was a homogenous orange. However, the color of fruit from AP2i-1 lines was patchy with sectors of different shades of yellow and orange. At the breaker + 7 d stage, wild-type fruit was homogenous red, whereas AP2i-1 lines still displayed sectors of different colors and did not turn red. The internal fruit anatomy at breaker + 7 d is shown in Supplemental Figure 4F online. The inside of wild-type fruit was red throughout. The locular cavities were filled with seeds and jelly, resulting in a juicy appearance. While AP2i-1-10 line fruits were more similar to the wild type in texture, fruits from line AP2i-1-8 appeared drier and the fruit appeared to be crumbly, with the internal structure collapsing (see Supplemental Figure 4F online). Line AP2i-1-8 showed the most severe phenotype. Additionally, the peel of AP2i-1-8 fruits was frequently cracked ( Figure 2C, arrows). We noted that the AP2i-1-8 fruits deteriorated faster than the wild-type fruits ( Figure 3A), and this could explain the observed peel cracks in the fruits shown in Figure 2C.

Because the AP2i-1 construct targets AP2a as well as AP2b and AP2c, and although the latter two show no expression in ripening fruits, we designed another RNAi fragment named AP2i-2 (highlighted in gray in Supplemental Figure 2B online), which excludes the conserved AP2 domains. In agreement with the proposed role of AP2a in fruit ripening, fruits from three out of 15 transformant lines containing this construct showed the same ripening defects ( Figure 2E), without the sepal or seed phenotypes observed in the AP2i-1 lines. This supports the proposed function of AP2a in fruit ripening, while flower- and seed-specific functions may be shared with, or are exclusively determined by, AP2b and AP2c. To confirm this, we performed qRT-PCR to compare the expression of the five AP2 homologs in AP2i-2 and wild-type sepals ( Figure 1D). We found that AP2a was downregulated in the sepals of AP2i-2, but, in contrast with AP2i-1 plants, neither of its closest homologs, AP2b or AP2c, was downregulated. To confirm downregulation of AP2a in fruits of both types of RNAi lines, we performed protein gel blot analysis using AP2a-specific peptide antiserum. In contrast with the situation in wild-type fruits, AP2a was not detected in the transgenic fruits, in both breaker stage (Br) and in 7 d after breaker stage (Br+7) fruits of AP2i-1 or AP2i-2 lines (see Supplemental Figure 3 F online).

Fruits of line AP2i-1-8 were used for more detailed characterization of the ripening-specific function of AP2a. In the progeny of this line, the described phenotypes all cosegregated with the transgene construct as determined by PCR.

AP2a Expression Is Stimulated by Ethylene, and Its Repression Resulted in Fruits Defective in Ripening but Producing Higher Levels of Ethylene

To achieve full ripening, tomato fruits require synthesis, perception, and signal transduction of the plant hormone ethylene ( Alexander and Grierson, 2002; Kevany et al., 2007). As ripening progresses, the expression of many genes is initiated or upregulated (reviewed in Alexander and Grierson, 2002). We observed that the expression of AP2a is increased during ripening. To distinguish if this regulation is ethylene dependent or ethylene independent, we incubated wild-type tomato fruits at the mature green stage with the ethylene precursor ethephon. After 6 h of induction, we observed an 8-fold upregulation of AP2a expression ( Figure 3B).

To investigate if a high level of ethylene production in the AP2i fruits may be responsible for the observed early fruit senescence ( Figure 3A), we measured the ethylene production in fruits during ripening. As shown in Figure 3C, AP2i-1-8 fruits produced 4- to 6-fold more ethylene at breaker and breaker + 7 stages than wild-type fruits at the same stage. Thus, AP2a appears to function as a negative regulator of ethylene synthesis in tomato fruits by a negative feedback loop.

AP2a Downregulation Alters Fruit Carotenoid Levels

The green-to-red color change typical of ripening tomato fruits is largely due to chlorophyll degradation and accumulation of carotenoids, including β-carotene and lycopene (reviewed in Giovannoni, 2001). Lycopene, which confers the red color to ripe tomatoes, accounts for 70 to 90% of the total carotenoids present ( Burns et al., 2003; Alba et al., 2005). To examine the underlying causes of the color differences observed between wild-type and AP2i ripe fruits in more detail, biochemical analysis of carotenoid (β-carotene, all-trans lycopene, lutein, and their precursor phytoene) levels was performed on wild-type and AP2a silenced fruits at a similar developmental stage ( Figure 4). The AP2i fruits had decreased levels of both phytoene and lycopene. By contrast, an increase in the β-carotene content was found, accounting for the orange color of the ripe AP2i fruits. The α-tocopherol content was also slightly decreased in these fruits. Elevated levels of chlorophyll were observed in the AP2i fruits compared with the wild-type fruits 7 d after the breaker stage ( Figure 4A). No difference in the lutein content was observed between the wild-type and transgenic fruits.

Gene Expression Analysis Identifies Genes Regulated by AP2a during Ripening

To identify genes that might be directly or indirectly regulated by AP2a in tomato, we compared gene expression between transgenic knockdown plants in the T1 generation, in which the transgene cosegregated correctly with the phenotype, and their wild-type siblings. For this purpose, fruit RNA from three plants derived from transformant line AP2i-1-8 and from three plants of wild-type siblings were hybridized to the Syngenta Tomato Affymetrix GeneChip. Analysis of differentially expressed genes revealed 1093 genes with significant differential expression at a P value cutoff of 0.001 (false discovery rate Q-value < 0.002), and genes with a >2-fold difference in expression were identified. After removal of apparent redundancies in the probe set, 527 genes were found to be expressed at significantly lower levels ( 2log≤−1), and 523 genes at higher levels ( 2log≥1) in the transgenic fruits. A full list of significantly differentially expressed genes can be found in Supplemental Data Set 3 online. A shortlist of differentially regulated genes, discussed below, is shown in Table 1.

Strikingly, the extreme ends of the spectrum of AP2a-regulated genes, as shown in Table 1, are made up predominantly of genes involved in heat shock responses (downregulated in RNAi fruits, 7- to 50-fold) and by genes involved in ethylene synthesis and signal transduction, as well as ethylene-(co-)regulated pathogen and wounding response genes (upregulated in RNAi plants 7- to 58-fold). Although heat shock-responsive genes have not been described in association with fruit ripening, inspection of publicly available gene expression data derived from Alba et al. (2005) and Ozaki et al. (2010) shows that many of these are upregulated from the breaker stage onwards, indicating that AP2a positively regulates these genes during ripening. Pathogenesis-related proteins that are also ethylene induced in other tissues are not known to be regulated during ripening, and the significant higher expression of these genes in fruits of RNAi plants may represent misregulation of their expression as a result of AP2a downregulation, higher ethylene production, or both. Several genes encoding pathogenesis-related proteins were identified as being enriched in the Cnr mutant fruit mRNA in a differential cDNA screen against wild-type fruit, suggesting that these genes are also negatively regulated by CNR ( Eriksson et al., 2004). Several genes that were previously characterized as being ripening induced ( Alba et al., 2005) had altered expression levels in AP2 RNAi fruits, but with opposite effects. LIPOXYGENASE C ( LOXC), PECTIN METHYLESTERASE 1.9, EXPANSIN3 ( EXP3), and LOXB were all downregulated compared with the wild-type fruits. On the other hand, MITOGEN-ACTIVATED PROTEIN KINASE1 ( MPKA1), MITOGEN-ACTIVATED PROTEIN KINASE KINASE4 ( MKK4), and MULTIPROTEIN BRIDGING FACTOR1 ( MBF1), which are all induced during ripening as well, were upregulated in the RNAi fruits compared with wild-type fruits ( Table 1). These observations suggest that AP2a may have activating and suppressing effects on genes that are normally induced during ripening.

During tomato fruit development, significant changes occur in the cell wall components and in the expression of polysaccharide-degrading enzymes (reviewed in Carrari and Fernie, 2006). We found several of the genes encoding these enzymes to be differentially regulated in the AP2 RNAi fruits. A polygalacturonase gene ( PG) is significantly upregulated compared with the wild type, as are genes encoding endo-1,3-1,4-β-d-gluconase ( CEL1), mannosidase ( MAN3), and mannan synthase ( MANS2). A PECTIN METHYLESTERASE1 ( PE1) gene and the gene encoding expansin precursor EXP3, both found to be downregulated in the Cnr mutant, were also downregulated in AP2 RNAi fruits ( Table 1). These data suggest that AP2a and CNR regulate a number of cell wall-related genes in a partially overlapping manner.

In agreement with the observed higher ethylene production in AP2 RNAi plants ( Figure 3C), we found genes involved in ethylene synthesis, specifically SAM3, ACO4, and ACS6, to be upregulated in the transgenic fruits, suggesting that, during ripening in wild-type fruits, AP2a represses ethylene production via these genes. Genes involved in ethylene signal transduction that were found to be significantly upregulated in the transgenic fruits are EIN3-LIKE2 ( EIL2), ETR5, ETR6, and EIN3 BINDING F-BOX1 ( EBF1).

In AP2i plants, several carotenoid biosynthesis genes, PHYTOENE DESATURASE ( PDS), CAROTENE-β-HYDROXYLASE ( CtR-b2), and CAROTENE ISOMERASE ( CtrISO), were significantly downregulated ( Table 1). Expression of the other carotenoid synthesis genes was detected but not significantly altered. Additionally, we found ZEP1, which encodes zeaxanthin epoxidase and is involved in abscisic acid synthesis, to be upregulated (4-fold) in the transgenic fruits. These results suggest that AP2a positively regulates carotenoid synthesis during ripening through activation of specific genes in the biosynthetic pathway.

The expression of several key transcripts related to biosynthetic genes involved in phenylpropanoid and flavonoid formation was also changed in the AP2 RNAi fruits ( Table 1; see Supplemental Data Set 3 online).

We found several genes that are possibly involved in hormone synthesis to be downregulated in the transgenic fruits. Among these are two genes that are involved in brassinosteroid synthesis: the D (dwarf) gene and a DWARF1/DIMINUTO-like gene ( DWF1). These results suggest that AP2a positively regulates brassinosteroid synthesis during ripening. Furthermore, we observed that the auxin response factor ARF4 gene was downregulated. Upregulation of a MONOPTEROS/ARF5 homolog and of two homologs of early auxin-responsive genes, gh3, and a SAUR family protein homolog and downregulation of another gh3 homolog and SAUR family homolog suggest that auxin responses are perturbed in these plants.

Finally, we found the expression of a number of genes implicated in chromoplast differentiation to be significantly downregulated. These were the STAY GREEN PROTEIN1 ( SGR1) gene, RCCR (encoding red chlorophyll catabolite reductase), a tomato homolog of the cauliflower ( Brassica oleracea) ORANGE gene ( OR), and a homolog of a target of the latter, a tomato homolog of the pepper PFTF gene.

AP2a Silenced Fruits Show Increased Accumulation of Sugars and Putrescine and Reduced Free Amino Acid Levels

To identify differentially regulated metabolic pathways in AP2i-1-8 plants, we analyzed our microarray data using the Plant MetGenMAP database ( Joung et al., 2009). Among the pathways identified, we found an overrepresentation of primary metabolic processes (see Supplemental Table 1 online). Correspondingly, gas chromatography-mass spectrometry (GC-MS) profiles ( Lisec et al., 2006) revealed that the AP2a silenced fruits displayed substantial changes in the level of primary metabolites ( Figures 4C and 4D; see Supplemental Table 2 online). To visually compare alterations in sectors of metabolism and interactions between metabolite levels in the AP2i fruits and the wild type, the fold changes in metabolite levels were painted onto biochemical pathway displays (see Supplemental Figure 5 online). The majority of the amino acids (15 out of 22) were present at significantly lower levels in the AP2i fruits than in the wild type, and the most dramatic reductions were in β-Ala, Ile, Met, Phe, and Trp. The observed reduced levels of Ile and Leu (branched-chain amino acids) are in agreement with the higher expression of BRANCHED-CHAIN AMINOTRANSFERASE1 ( BCAT1; 8.6-fold), which was shown to be involved in the degradation of these two amino acids ( Maloney et al., 2010). Similarly, nine of the 15 organic acids measured displayed significant changes. However, these were predominantly increased in the AP2i line, with the exception of the tricarboxylic acid cycle intermediates citrate, fumarate and succinate, which were decreased. The increase in benzoate, dehydroascorbate, glycerate, glycolate, nicotinate, and nonanate was relatively mild. The fatty acids dodeconate and octodeconate were also present at mildly, yet significantly, higher levels in the AP2i line. Of the 15 sugars we detected, seven (Fuc, galacturonate, glucose-1,6-anhydro-glucuronate, melibiose, raffinose, and Xyl) were present at higher levels in the AP2i line, whereas maltose was present at lower levels. With the exception of maltose and raffinose, these sugars and sugar derivatives are generally associated with cell wall or ascorbate metabolism. It is interesting to note that the content of the major sugars of the fruit were consistent between genotypes. The content of sugar alcohols in the genotypes was somewhat variable, with an increase in glycerol content but a decrease in inositol in the AP2i line. By contrast, inositol 3-phosphate levels were higher in the AP2i line, whereas all other sugar phosphates were invariant. Of the six miscellaneous compounds we measured, five were present at significantly different levels between the genotypes, with gluconate-1,5-lactone, phosphorate, putrescine, 3-hydroxy-pyridine, and 3-caffeoyl quinate being elevated in the AP2 i line. Putrescine was elevated to levels that were 12-fold higher than those observed in the wild type.

Regulatory Interactions between AP2a and Other Ripening Regulators

Several transcription factors are positive regulators of tomato fruit ripening. Although AP2i fruits failed to complete ripening, they did soften in a similar way as wild-type fruits, in contrast with, for example, Cnr mutant fruits ( Thompson et al., 1999). The Cnr tomato mutant has a colorless, nonripening phenotype and mealy pericarp ( Eriksson et al., 2004; Manning et al., 2006). CNR and other ripening regulators, like RIN and TAGL1, were found to have ethylene-dependent and ethylene-independent functions ( Giovannoni, 2007; Itkin et al., 2009; Vrebalov et al., 2009). We investigated AP2a expression in nor ( Tigchelaar et al., 1973), rin, Gr, and Cnr mutant backgrounds ( Figure 5A). The expression of AP2a appeared to be reduced in all four mutants. Our results suggest that, although AP2a appears at first sight to be a negative regulator of ripening through inhibition of ethylene production, it is itself positively regulated by essential regulators of ripening, like CNR, RIN, and NOR ( Barry et al., 2005; Giovannoni, 2007; Cantu et al., 2009), and its expression is ethylene dependent, as shown by its downregulation in the Gr mutant. Next, to test whether the CNR protein could bind directly to the AP2a promoter, we expressed the full-length CNR protein tagged with glutathione S-transferase (GST) (GST-CNR) in Escherichia coli ( Figure 5B. Two AP2a promoter regions were selected, one containing the predicted (GTAC) core binding motif of the plant-specific SBP domain ( Birkenbihl et al., 2005) and the other one not containing the binding site, to be used as a negative control. To confirm that CNR binds specifically to this core element, we performed an electrophoretic mobility shift assay using the two selected promoter elements ( Figure 5B). Our data showed that CNR can bind specifically to the promoter element of AP2a in vitro and positively regulates its expression in vivo. Furthermore, CNR and AP2a were found to be colocalized in the nucleus of transfected Arabidopsis protoplast cells ( Figure 5C).

The ripening regulatory transcription factors NOR, CNR, and RIN activate the expression of the AP2a transcription factor, which in turn acts partially as a negative regulator of ripening through modulation of ethylene signaling. Our microarray analysis was performed with RNA from AP2i whole fruits and yielded only a few transcription factors that were significantly regulated ( Table 1; see Supplemental Data Set 3 online). Therefore, we decided to investigate in greater detail the expression of CNR, RIN, and TAGL1 specifically in the pericarp of the AP2i fruits by qRT-PCR. Although no significant change in the expression of RIN or TAGL1 was observed (see Supplemental Figure 6 online), CNR mRNA levels were significantly higher in the pericarp of AP2i fruits ( Figure 5D). Taking into account our previous observation that AP2a is positively regulated by CNR, these data suggest that AP2 in the pericarp of wild-type fruits suppresses the expression of CNR by a negative feedback loop.

AP2b, AP2c, or Both, Possibly with AP2a, Function in Floral Organ Development

The flower phenotype of the tomato AP2i-1 lines is reminiscent of some of the aspects of weaker ap2 phenotypes in Arabidopsis as well as of that in Antirrhinum lip1 lip2 double mutants ( Bowman et al., 1989; Keck et al., 2003). The Arabidopsis A-class genes, APETALA1 ( AP1) and AP2, confer sepal identity in the first floral whorl. While MADS-MC (Macrocalyx) is a likely ortholog of AP1 ( Vrebalov et al., 2002), a gene or genes with an A-class function similar to that of AP2 has not yet been found in tomato. Here, we found that sepals of AP2i-1 lines are elongated and show distinct leaf-like characteristics, such as a thinner blade in which venation can be easily observed. However, this sepal phenotype was not observed in the transgenic plants expressing the AP2i-2 construct for silencing, suggesting that there may be additional tomato AP2 genes that are either solely responsible for this sepal phenotype or functionally redundant with each other or with AP2a for the floral development function, similar to the redundancy observed in Antirrhinum AP2 genes. We identified five homologs of the Arabidopsis AP2 gene in tomato and showed that the observed sepal phenotype in the AP2i-1 lines is correlated with downregulation of AP2a and its closest homologs, AP2b and AP2c. Unlike in strong Arabidopsis ap2 mutants ( Bowman et al., 1989, 1991) and in Antirrhinum lip1 lip2 mutants ( Keck et al., 2003), we observed no changes in the other floral whorls. However, similar to Arabidopsis ap2 mutants, suppression of tomato AP2 homologs also affected seed shape and seed coat morphology. The AP2a suppression could cause defects in the development of the seed testa, specifically in the outer epidermal layer as in Arabidopsis ap2 mutants ( Jofuku et al., 1994; Leon-Kloosterziel et al., 1994), and may well explain the aberrant seed hair morphology that we observed in the AP2 i-1 lines.

SlAP2a Functions in Fruit Development

Our data demonstrate that AP2a plays a role in fruit development. The effect of AP2 suppression on early fruit development is a bumpy appearance of the green fruits, which is conceivably caused by a defect in the normal or coordinated expansion of the pericarp. Ripening fruits had a number of anatomical differences from wild-type fruits, such as, to a varying degree, a smaller volume of jelly, a dry and crumbly appearance (observed in one line only) of the pericarp, and cracking of the skin, indicating a defect in the expansion or elasticity of the epidermis. Differences in the ripening process were apparent from the differences in color development between wild-type and AP2i lines. Orange fruits of tomato AP2i lines had a blotchy appearance with light colored or white sectors and failed to turn completely red. Biochemical analysis of the major colored compounds of the tomato fruit, the carotenoids, indicated that AP2i fruits are affected in phytoene and lycopene production, explaining their failure to turn fully red. The reduction in color development correlates with the reduced expression of PDS, PTOX, and CRTISO in the AP2i fruits, since they encode proteins involved in the biosynthesis of the red all-trans lycopene (for review, see Bramley, 2002). PDS, PTOX, and CRTISO expression in tomato fruits increases dramatically during tomato fruit ripening ( Josse et al., 2000; Isaacson et al., 2002), as does expression of AP2a. Although reduction of the expression of CrtR-b2 was also detected, no difference in lutein accumulation was observed in the AP2i transgenic fruits (CrtR-b converts α-carotene to lutein together with CrtR-e; Bramley, 2002). Two genes involved in the biosynthesis of flavonoids, FLAVONOL SYNTHASE ( FLS) and CHALCONE SYNTHASE2 ( CHS2) ( Table 1), were also found to be downregulated in AP2i fruits, and the accumulation of tocopherols was reduced when compared with the wild-type fruits.

Carotenoids, flavonoids, and tocopherols are all bioactive compounds with potent antioxidant properties. Carotenoids can be further metabolized to abscisic acid in plants and to vitamin A in animal cells (reviewed in Cunningham and Gantt, 1998). In this study, we demonstrated that AP2a positively regulates the synthesis of several carotenoids and of tocopherol during tomato fruit ripening. One of the enzymes dramatically downregulated (24-fold) in the AP2i fruits is 3-hydroxy-3methylglutaryl-CoA (HMGR) reductase 2 ( Table 1). The HMGR reaction makes mevalonate, a necessary component in the synthesis of all isoprene-containing compounds, such as sterols, carotenoids, phytoalexins, and other growth hormones ( Rodriguez-Concepcion and Gruissem, 1999). The expression of HMG2 was shown to increase strongly during ripening and in parallel with the accumulation of lycopene ( Rodriguez-Concepcion and Gruissem, 1999).

AP2a Regulates the Expression of Genes Involved in Chromoplast Differentiation

Differentiation of chloroplasts to chromoplasts is an integral part of tomato fruit ripening, and chromoplasts are the site of lycopene production and accumulation. Chromoplast differentiation involves extensive changes in plastid ultrastructure, accompanied by similarly extensive changes in gene expression (for review, see Egea et al., 2010). A large number of genes that change expression during chromoplast differentiation or have a function in the process have been identified through transcriptome profiling and by natural mutations in genes affected in the process. Several of these are downregulated in our AP2 RNAi plants, indicating that AP2a may be a major regulator of the process. These genes are SGR1, as well as a gene encoding a predicted putative RCCR (red chlorophyll catabolite reductase), which is involved in chlorophyll degradation ( Barry et al., 2008), and a tomato homolog of the cauliflower OR gene, which encodes a plastidic DnaJ-like protein. The mutation or in cauliflower induces the production of chromoplasts in the normally white cauliflower curd, causing it to turn orange due to the accumulation of carotenoids ( Lu et al., 2006). In AP2i fruits, a tomato homolog of the pepper ( Capsicum annuum) Pftf gene, which encodes an FtsH-like protein, the homolog in cauliflower of which is positively regulated by Or ( Lu et al., 2006), is downregulated. Apart from the Or gene homolog, many other heat shock protein-encoding genes are strongly downregulated in the AP2i plants, several of which were identified as being plastid localized. DnaJ-like cochaperones interact with chaperones, such as Hsp70 (which is also downregulated in AP2i plants) and are involved in functions such as protein folding, assembly/disassembly, and translocation, which may be required in higher amounts during chromoplast differentiation. The other chloroplast-localized heat shock protein homologs may have similar functions. As downregulation of AP2a likely interferes with this process, the downregulation of the genes encoding the chloroplast-localized heat shock protein homologs in AP2 i plants would follow from this.

AP2a Is a Negative Regulator of Ethylene Production

Climacteric fruits such as tomato are distinguished from nonclimacteric fruits by their increased respiration and ethylene biosynthesis rates during ripening. Ethylene is formed from Met via S-adenosyl-l-methionine (AdoMet) and the cyclic nonprotein amino acid ACC. ACC is formed from AdoMet by the action of ACS and the conversion of ACC to ethylene is performed by ACO ( Kende, 1989). Biochemical evidence suggests that ethylene production may be influenced or regulated by interactions between its biosynthesis and other metabolic pathways (reviewed in Carrari and Fernie, 2006). For example S-adenosylmethionine is the substrate for both the polyamine pathways and ethylene. SAM, one of the enzymes in the ethylene biosynthesis pathway that is also required for the biosynthesis of polyamines, was found to be upregulated in the AP2i transgenic fruits. High levels of putrescine, which together with S-adenosylmethionine form spermidine in the polyamine biosynthetic pathway, were observed. The lower levels of Orn (a precursor of putrescine biosynthesis) found in the AP2i fruits correlate with higher levels of putrescine. The lower levels of Met measured in the AP2a silenced fruits could be attributed to the higher levels of ethylene production, since these fruits produced up to 5-fold more ethylene than the wild-type fruits. This observation suggests that the AP2a transcription factor is a negative regulator of ethylene biosynthesis in ripening fruits. While ethylene action is associated with senescence and ripening, polyamines, on the other hand, have been considered to be senescence inhibitors. Interestingly, in the Sl AP2i fruits, both ethylene and putrescine levels are elevated. In general, putrescine is the predominant polyamine during tomato ripening, and its levels are high at the immature-green stage and decline throughout the ripening process ( Rastogi and Davies, 1991), which coincides with the accumulation of AP2a transcript. Together, our data suggest that AP2a is a negative regulator of both ethylene and putrescine synthesis in wild-type fruits. Interestingly, we show that AP2a expression is induced by ethylene, suggesting that AP2a indirectly regulates ethylene biosynthesis by a negative feedback loop mechanism.

Ethylene biosynthesis is tightly regulated, and two systems of ethylene regulation have been proposed to operate in climacteric plants ( Barry et al., 2000). System 1 functions during normal vegetative growth, is ethylene autoinhibitory, and is responsible for producing basal ethylene levels that are detected in all tissues, including those of nonclimacteric fruits. System 2 operates during the ripening of climacteric fruit. It has been proposed that tomato ACS1 and ACS6 are involved in the production of system 1 ethylene in green fruits ( Barry et al., 2000). System 1 continues until the fruit ripening initiates, when ACS1 expression increases and ACS2, ACS4, ACO2, and ACO4 are induced as a result of positive feedback regulation ( Nakatsuka et al., 1998; Barry et al., 2000). We found that ACS6, but not the other ACS genes, together with ACO4 and two of the ethylene receptor genes, ETR5 and ETR6, as well as the transcription factor gene EIL2 are induced in the AP2i fruits, suggesting that AP2a is a negative regulator of their expression. A model was proposed for tomato fruit in which preclimacteric system 1 ethylene is mediated via constitutively expressed ACS1 and ACS3 and negatively regulated through a feedback loop involving ACS6 ( Nakatsuka et al., 1998). Our data suggest that AP2a might be a regulator of the switch between the two ethylene systems (1 and 2) through negative regulation of ACS6 expression. The other genes upregulated in AP2i fruits, the ETR6 receptor gene and ACO4 ( Nakatsuka et al., 1998; Kevany et al., 2007), were shown to be regulated by elevated ethylene levels, which could be the reason for their higher expression in the AP2i fruits, as opposed to regulation by AP2a directly. Since the expression of both ETR5 and EIL2 is not ethylene dependant ( Tieman et al., 2001; Kevany et al., 2007), their expression could be under direct regulation of AP2a. Finally, we also found tomato EBF1, a homolog of Arabidopsis EBF1, to be upregulated in AP2i fruits. EBF1 is involved in targeting EIN3 for degradation and thus modulates the ethylene response ( Guo and Ecker, 2003). The two tomato homologs, EBF1 and EBF2, are regulated by ethylene and auxin, and their silencing causes a constitutive ethylene response, plant senescence, and early fruit ripening ( Yang et al., 2010).

In addition to ripening, ethylene is also known to be involved in other processes, such as pathogen and wounding responses, leaf senescence, and the abiotic and biotic stress response (for review, see Alexander and Grierson, 2002). In AP2i fruits, a large group of genes encoding pathogenesis-related proteins (PR proteins and chitinases), as well as one key regulator of the biotic stress response, the MYB transcription factor gene AIM1 ( Abuqamar et al., 2009), are upregulated. Many of these genes are also likely upregulated in Cnr mutant fruits ( Eriksson et al., 2004). Our data suggest that in wild-type fruits, AP2a acts not only as repressor of ethylene biosynthesis, but also as a repressor of ethylene-induced pathogen defense and wounding responses.

In comparison with ethylene, very little is known about the role of other hormones in fruit ripening. The role of auxins has been extensively investigated in other fruits, such as strawberry ( Fragaria × ananassa) ( Harpster et al., 1998) and grape ( Vitis vinifera) ( Davies et al., 1997). Our transcriptomics data show a high level of repression of GH3, which is involved in maintaining auxin homeostasis by conjugating excess indole-3-acetic acid to amino acids ( Staswick et al., 2005). In addition to GH3, ARF4, a transcription factor gene involved in auxin signaling, was suppressed. ARF4 (also known as DEVELOPMENTALLY REGULATED12 [ DR12]; Jones et al., 2002) is developmentally regulated, and its expression increases in fruits during ripening and decreases after ethylene treatment of immature fruits. Knockdown of DR12 in transgenic tomato plants caused blotchy fruit ripening, similar to the defects observed here for AP2 RNAi plants, suggesting that at least part of the effects of AP2a downregulation is caused by deregulation of ARF4. Genes involved in the biosynthesis of brassinosteroid hormones, like DWARF ( Nomura et al., 2005) and the homolog of Arabidopsis DWF1/DIMINUTO ( Klahre et al., 1998), were downregulated in the AP2i fruits. Previously, it was shown by treatment of tomato pericarp discs with brassinosteroids that these could accelerate ripening and increase ethylene production ( Vidya Vardhini and Rao, 2002). Although brassinosteroids recently have been shown to play a role in early fruit development in cucumber ( Cucumis sativus) ( Fu et al., 2008) and in fruit ripening in grape ( Symons et al., 2006), their function in tomato fruit development or ripening remains to be determined.

Effect of AP2a Downregulation on Fruit Metabolism

From the gene ontology analysis of our transcriptomics data, it is apparent that most of the metabolic pathways that are significantly regulated are related to primary metabolism. In agreement with these data are the changes in the levels of accumulation of different primary metabolites that were measured in the AP2i fruits compared with the wild-type ripe fruits. We found that AP2a positively regulates the accumulation of Leu and Ile, probably through regulation of BCAT1. BCAT1 transcripts were shown to accumulate at the highest level in ripening fruits, and BCAT1 is likely the primary enzyme involved in the recycling of branched-chain amino acids generated by protein degradation. This statement is in agreement with data obtained in a recent study of the tomato BCAT gene family ( Maloney et al., 2010).

Taken together, our metabolic and transcriptomics data indicate that the downregulation of AP2a transcription factor has a major effect on cell wall metabolism, which is associated with fruit softening and shelf life and the biosynthesis of secondary metabolites and their primary metabolite precursors, as well as decreased flux through the tricarboxylic acid cycle.

Additionally, the downregulation of LOXC may well affect volatile production in AP2 RNAi plants during ripening. The chromoplast-located TomloxC protein has been shown to be involved in the production of flavor volatiles hexanal, hexenal, and hexenol from linoleic and linolenic acid, which may be produced through thylakoid membrane disruption at the transition from chloroplasts to chromoplasts ( Chen et al., 2004).

AP2a Participates in a Transcriptional Regulatory Network That Regulates Tomato Fruit Ripening

Unlike Arabidopsis AP2, which is mostly expressed in flowers and functions in floral organ identity, tomato AP2a has acquired a novel function in fleshy fruit development. Some of the other tomato AP2 paralogs may still be involved in floral organ identity as A-class homeotic genes, like AP2 in Arabidopsis ( Bowman et al., 1991; Coen and Meyerowitz, 1991); however, further research is needed to unravel the function of these other tomato genes.

The AP2a transcription factor appeared to have dual functions in tomato fruit ripening as a positive and negative regulator of different processes occurring during ripening. An interesting observation was that AP2a most probably functions downstream of known major positive regulators of fruit ripening, such as NOR, RIN, and CNR. At least in the case of CNR, this regulation is likely to be direct, since CNR is able to bind directly to a promoter element of AP2a. On the other hand, in the fruit pericarp, AP2a regulates the expression of CNR in a negative manner. In both the Cnr ( Eriksson et al., 2004) mutant and in AP2i transgenic fruits, several ripening-associated genes are downregulated, such as the carotene synthesis pathway genes, LOXB and LOXC, pectin methylesterase, and EXP3, indicating that AP2a has positive ripening regulatory functions besides its negative regulatory function in ethylene synthesis. Further research, such as the identification of direct and indirect targets of the key transcription factors, will likely contribute to further mapping of the transcriptional network that regulates fruit ripening, a process in which AP2a plays an essential function.

Greenhouse/Growth Conditions

Transgenic plants were transferred to soil and grown in the greenhouse at ambient temperatures (>20°C) under natural light supplemented with artificial sodium lights, according to a 16-h-light/8-h-dark cycle. Standard greenhouse culture conditions, with regular fertilizer application, were used.

Construction of RNAi Construct and Transgenic Tomato Plants

The sequences, and associated names, available on the Tomato Functional Genomics Database and The Institute for Genomic Research (TIGR) websites, are always being updated; however, old names remain linked to updated information. Therefore, the original names first notated for this research are kept for the article. The primers used (see Supplemental Table 3 online) for the RNAi construct were designed based on the sequence of EST311591 (GenBank AW442195; http://www.tigr.org/tigr-scripts/tgi/T_index.cgi?species=tomato). RT-PCR was performed with red ripe fruit RNA, using an annealing temperature of 54°C, and the products were cloned into pENTR/D-TOPO (Invitrogen) (resulting plasmid designated pARC131 and CZN098 for specific RNAi fragment) and subcloned into pHellsgate8 (designated pARC139 and CZN096, respectively) ( Helliwell et al., 2002). pARC139 and CZN096 were transformed into electrocompetent Agrobacterium tumefaciens strain EHA105, followed by transformation into Solanum lycopersicum cv Moneymaker and cv M82, respectively, as described by van Roekel et al. (1993). Tissue culture-generated nontransgenic Moneymaker and M82 plants were used for wild-type controls. Sixteen independent transgenic AP2i-1 and 14 independent transgenic AP2i-2 RNAi lines were generated and analyzed for ploidy level by flow cytometry as described elsewhere ( de Laat et al., 1987), and polyploid plants were removed from the population.

Plasmid Constructions

AP2a and CNR were amplified from the cDNA made from mixed fruit stages using the primer pairs described in Supplemental Table 4 online. The full-length open reading frames of AP2a and CNR were inserted into the pENTR-D TOPO vector following the Gateway protocol (Invitrogen). All plasmids were controlled by sequence analyses (DETT sequence kit; Amersham). Finally, expression vectors for the colocalization analysis were obtained by LR reaction following the Gateway protocol. For the C-terminal yellow fluorescent protein (YFP) and cyan fluorescent protein (CFP) fusions, CZN576 and CZN575 destination vectors, respectively, were used. For constructing CZN575, the open reading frame from sCFP3A was amplified from pSCFP3A-C1 ( Kremers et al., 2006) and was cloned into pGD120 ( Immink et al., 2002). The obtained product was digested with XbaI, which is located just before the start codon of sCFP3A, and the overhangs were blunted with Klenow. Subsequently, a Gateway conversion cassette (Invitrogen) was ligated into this vector, resulting in the destination vector CZN575, containing the expression cassette pCaMV35S:Gateway-sCFP3A:NOS terminator. For the CZN576 vector, the open reading frame from sYFP2 was amplified from pSYFP2-C1 ( Kremers et al., 2006), and the remaining cloning strategy was identical to that used for the CZN575 vector.

For the N-terminal GST fusion, the full-length CNR PCR fragment was obtained with the primers listed in Supplemental Table 3 online. The purified PCR fragment was digested with EcoRI and NotI and ligated into pGEX4T1. The resulting construct was verified by sequencing and transformed into Escherichia coli Arctic Express strain (Stratagene). The protein expression and GST purification were performed as described before ( Karlova et al., 2009).

For the electrophoretic mobility shift assay or gel retardation assay, the two AP2a promoter regions chosen were PCR amplified with the primers listed in Supplemental Table 3 online, purified, and ligated into the pGEMT vector (Promega).

Ethephon Treatment of Tomato Fruit

Ethephon (Acros Organics) treatment of intact mature green fruits involved a 1-min dip in 50 mM fresh aqueous ethephon solution for 6 h.

Gel Retardation Assays

The binding reactions for the gel retardation assays were performed as described by Winter et al. (2002). Fifty nanograms of in vitro GST-CNR purified protein were used per binding reaction. PCR fragments from the above described promoter fragment construct were biotinylated, and 40 nM of probe was used per lane. After native polyacrylamide gel electrophoresis, the DNA was blotted to positively charged nylon membranes (Hybond N⁺; Amersham), and signal was detected using the chemiluminescent nucleic acid detection module (Peirce).

Images

Micrographs of seeds were taken on a Zeiss STEMI SV8 microscope. Seed hairs were processed and viewed using a JEOL JSM-6330F field emission electron scanning microscope as described elsewhere ( Angenent et al., 1995).

Fluorescence Microscopy in Living Cells

Arabidopsis thaliana leaf protoplasts were transfected as described by Aker et al. (2006). Plasmid DNA (15 μg) was used and the protoplasts were incubated overnight at 25°C before imaging. Images were acquired using an upright confocal laser scanning microscope (Leica SPE DM5500) with a 10 × 0.30 CS ACS APO (air) lens, using the LAS AF 1.8.2 software (Leica).

Quantitative Real-Time PCR

RNA isolation from fruits or pericarp of the fruits was performed with the InviTrap Spin Plant RNA kit (Invitek) according to the manufacturer's protocol and from all other tissues by the Trizol method (Sigma-Aldrich), except from seeds, where a modified method was used. Seeds were ground in a 5:5:1 mixture of buffer (180 mM Tris pH 8.2, 90 mM LiCl, 4.5 mM EDTA, 1% SDS, and 0.1% β-mercaptoethanol), phenol, and chloroform. After phase separation by centrifugation, the watery phase was reextracted with 1 mL phenol/chloroform (1:1) and subsequently with chloroform. The extracted RNA was precipitated with one-third volume of 8 M LiCl overnight at −20°C. Precipitated RNA was centrifuged, washed with 70% ethanol, dried, and dissolved in RNase-free water.

DNaseI-treated RNA was reverse transcribed (Applied Biosystems), and 1 μL of cDNA solution was used for the real-time PCR analysis. Real-time qPCR was performed with iQ SYBR Green Supermix (Bio-Rad) using the iCycler iQ5 system (Bio-Rad) and gene-specific primers (see Supplemental Table 4 online). Two independent biological replicates were analyzed per sample (or two to three fruits from the same plant were mixed together per sample). Relative quantification of specific mRNA levels was performed using the cycle threshold (Ct) 2 -Δ(ΔCt) method (Software IQ5 2.0; Livak and Schmittgen, 2001). Expression values were normalized using the housekeeping gene actin or CAC and TIP41 in case of sepals, petals, and seeds ( Expósito-Rodriguez et al., 2008) from tomato.

Ethylene Measurements

Fruits at breaker or breaker +7 d stages were harvested and placed in open 500-mL jars for 2 h. Jars were then sealed and incubated at room temperature for 35 min, and 1.5 mL of headspace gas was injected into a Focus GS gas chromatograph (Thermo-Electron) equipped with a flame ionization detector. Samples were compared with reagent grade ethylene standards of known concentration and normalized for fruit weight.

Carotenoid Analysis

Pooled fruits from independent plants were harvested and homogenized. Aliquots from these samples were taken separately for extraction and analysis of primary and secondary metabolite content by HPLC coupled with photodiode array detection and MS. The extraction, HPLC separation, photodiode array detection, and quantification of carotenes, carotenoids, and xanthophylls were previously described ( Fraser et al., 2000). In this study, 0.5 to 1 g of frozen fruit tissue was extracted with chloroform and methanol (2.5:1 by volume), mixed, and incubated for 20 min on ice. Then, 1 volume of deionized water was added. A partition was formed after mixing by centrifugation. The organic hypophase was removed, and the aqueous phase was reextracted with chloroform (2.5 times by volume). HPLC separations were performed using a C30 reverse-phase column (250 × 4.6 mm) purchased from YMC. The mobile phases used were methanol (A), water/methanol (20/80 by volume) containing 0.2% ammonium acetate (B), and tert-methyl butyl ether (C). The gradient used was 95% A:5% B, isocratically for 12 min and then stepped to 80% A:5% B:15%C, from which a linear gradient to 30% A:5% B:65% C over 30 min was performed. A Waters Alliance model 2695 injection and solvent delivery system was used. Detection was performed continuously from 220 to 700 nm with an online photodiode array detector (Waters 966). Identification was performed by cochromatography and comparison of spectral properties with authentic standards and reference spectra. Annotated chromatograms of this HPLC system are provided elsewhere ( Fraser et al., 2007). Quantitative determination of carotenoids was performed by comparison with dose-response curves (0.2 to 1.0 mg) constructed from authentic standards. Purchasing, preparation, and characterization of authentic standards are as described previously ( Fraser et al., 2000).

Peptide Antibody Generation

For polyclonal AP2a antibodies, the peptide CRTQNNGFHHYFMRP was generated and used for rabbit immunization by Genosphere Biotechnologies.

Protein Gel Blot Analysis

Plant material (tomato fruits) was ground in liquid nitrogen, and 0.2 g was dissolved in sample buffer (100 mM Tris-HCl, pH 6.8, 4% SDS, 0.2% bromphenol blue, 20% glycerol, and 100 mM DTT) and boiled for 5 min. Protein gel blotting was performed as described before ( Karlova et al., 2006). Proteins were detected with anti-AP2 antibodies (2000× diluted).

Phylogenetic Analysis

Protein homology searches in the National Center for Biotechnology Information (NCBI) GenBank protein database were done using the BLASTP algorithm ( Altschul et al., 1997). Homology searches in the TIGR Tomato Gene Index were done with the TBLASTN algorithm. Multiple protein alignments of the full-length proteins were produced with the ClustalW program of the DNAstar Megalign software package (DNASTAR). Phylogenetic trees were produced with the ClustalX package ( Thompson et al., 1997). Phylogenetic reconstruction was obtained by the neighbor-joining method ( Saitou and Nei, 1987) together with bootstrap analysis using 1000 replicates. Kimura correction for multiple substitutions was applied ( Kimura and Takahata, 1983). The tree was visualized using the TreeView package ( Page, 1996).

Microarray Analysis

Transcript profiling was performed on fruits of wild-type tomato and AP2-1 RNAi mutant lines, at the breaker +7 d stage (U.S. standards for grades of fresh tomatoes, 1991). RNA was isolated with the Trizol method (Sigma-Aldrich) and hybridized to the Syngenta Tomato Affymetrix GeneChip. Three biological replicates and two technical replicates were analyzed from either unmodified tomato variety Moneymaker or from an AP2-1 RNAi line. Twelve experiments (slides) were analyzed. Data were analyzed using the GeneData Expressionist suite of tools. All 12 experiments passed quality controls and were used for analysis. The Bioconductor RMA algorithm was used for background subtraction according to a Bayesian model using the PM Features Only option ('bgversion = 2' in the BioConductor routine) for quantile normalization of the arrays ( Bolstad et al., 2003) and for condensing probes into probe regions (hereafter referred to as genes) ( Irizarry et al., 2003).

A Welch t test was performed to identify genes with significant differences in expression level between the two data sets. P values were adjusted for false discovery rate 0.05, as described by Benjamini and Hochberg (1995).

Accession Numbers

Sequence data from this article can be found in Swiss-Prot or Protein Bank/NCBI databases under the following accession numbers: Arabidopsis ANT protein, Q38914; At RAP27/TOE1, Q9SK03; At TOE2, Q9LVG2; At SMZ, Q6PV68; At SNZ, Q6PV67; At TOE3, Q9FH95; At AP2, AAC13770; At WRI1, NP_191000; At AIL1, Q1PFE1; At AIL5, Q6PQQ3; At AIL6, Q52QU2; At AIL7, Q6J9N8; At BBM, Q6PQQ4; At PUCHI, NP_197357; At PLT1, Q5YGP8; At PLT2, Q5YGP7; Tomato AP2a, ACD62792, Sl AP2c, HQ586951, Sl AP2b, HQ586952, Sl AP2d, HQ586953; Sl AP2e, HQ586954; Petunia PHAP2A, AAD39439, and PHAP2B, AAD39440; Antirrhinum LIP1, AAO52746 and LIP2, AAO52747; Malus domestica AHAP2, AAL57045; Zea mays GLOSSY15, AAC49567; IDS1, NP_001104904. The accession numbers of genes used in the microarray analysis are provided in Table 1 and Supplemental Data Set 3 online.

Supplemental Data

The following materials are available in the online version of this article.

View Abstract

# data[].attributes.attachment_link.title
Transcriptome and Metabolite Profiling Show That APETALA2a Is a Major Regulator of Tomato Fruit Ripening

# data[].attributes.attachment_link.description
Fruit ripening in tomato ( Solanum lycopersicum ) requires the coordination of both developmental cues as well as the plant hormone ethylene. Although the role of ethylene in mediating climacteric ripening has been established, knowledge regarding the developmental regulators that modulate the involvement of ethylene in tomato fruit ripening is still lacking.

# data[].attributes.body
Presenters: Orla Daly & Hussah Aljanahi

# data[].attributes.attachment_link.readable_text
Journal name:

Nature

Volume:

485,

Pages:

635-641

Date published:

DOI:

doi:10.1038/nature11119

The genome of the inbred tomato cultivar 'Heinz 1706' was sequenced and assembled using a combination of Sanger and 'next generation' technologies ( Supplementary Information section 1). The predicted genome size is approximately 900 megabases (Mb), consistent with previous estimates3, of which 760  Mb were assembled in 91 scaffolds aligned to the 12 tomato chromosomes, with most gaps restricted to pericentromeric regions ( Fig. 1A and Supplementary Fig. 1). Base accuracy is approximately one substitution error per 29.4  kilobases (kb) and one indel error per 6.4  kb. The scaffolds were linked with two bacterial artificial chromosome (BAC)-based physical maps and anchored/oriented using a high-density genetic map, introgression line mapping and BAC fluorescence in situ hybridization (FISH).

Figure 1: Tomato genome topography and synteny.

A, Multi-dimensional topography of tomato chromosome 1 (chromosomes 2-12 are shown in Supplementary Fig. 1). a, Left: contrast-reversed, 4′,6-diamidino-2-phenylindole (DAPI)-stained pachytene chromosome; centre and right: FISH signals for repeat sequences on diagrammatic pachytene chromosomes (purple, TGR1; blue, TGR4; red, telomere repeat; green, Cot 100 DNA (including most repeats)). b, Frequency distribution of recombination nodules (RNs) representing crossovers on 249 chromosomes. Red stars mark 5  cM intervals starting from the end of the short arm (top). Scale is in micrometres. c, FISH-based locations of selected BACs (horizontal blue lines on left). d, Kazusa F2-2000 linkage map. Blue lines to the left connect linkage map markers on the BAC-FISH map ( c), and to the right to heat maps ( e) and the DNA pseudomolecule ( f). e, From left to right: linkage map distance (cM/Mb, turquoise), repeated sequences (% nucleotides per 500  kb, purple), genes (% nucleotides per 500  kb, blue), chloroplast insertions; RNA-Seq reads from leaves and breaker fruits of S. lycopersicum and S. pimpinellifolium (number of reads per 500  kb, green and red, respectively), microRNA genes (transcripts per million per 500  kb, black), small RNAs (thin horizontal black and red lines, sum of hits-normalized abundances). Horizontal grey lines represent gaps in the pseudomolecule ( f). f, DNA pseudomolecule consisting of nine scaffolds. Unsequenced gaps (approximately 9.8  Mb, Supplementary Table 13) are indicated by white horizontal lines. Tomato genes identified by map-based cloning ( Supplementary Table 14) are indicated on the right. For more details, see legend to Supplementary Fig. 1. B, Syntenic relationships in the Solanaceae. COSII-based comparative maps of potato, aubergine (eggplant), pepper and Nicotiana with respect to the tomato genome ( Supplementary Information section 4.5 and Supplementary Fig. 14). Each tomato chromosome is assigned a different colour and orthologous chromosome segment(s) in other species are shown in the same colour. White dots indicate approximate centromere locations. Each black arrow indicates an inversion relative to tomato and '+1' indicates a minimum of one inversion. Each black bar beside a chromosome indicates translocation breakpoints relative to tomato. Chromosome lengths are not to scale, but segments within chromosomes are. C, Tomato-potato syntenic relationships dot plot of tomato (T) and potato (P) genomic sequences based on collinear blocks ( Supplementary Information section 4.1). Red and blue dots represent gene pairs with statistically significant high and low ω ( K a/K s) in collinear blocks, which average K s  ≤  0.5, respectively. Green and magenta dots represent genes in collinear blocks which average 0.5  <  K s  ≤  1.5 and K s  >  1.5, respectively. Yellow dots represent all other gene pairs. Blocks circled in red are examples of pan-eudicot triplication. Inserts represent schematic drawings of BAC-FISH patterns of cytologically demonstrated chromosome inversions (also in Supplementary Fig. 15).

The genome of S. pimpinellifolium LA1589 was sequenced and assembled de novo using Illumina short reads, yielding a 739  Mb draft genome ( Supplementary Information section 3). Estimated divergence between the wild and domesticated genomes is 0.6% (5.4 million single nucleotide polymorphisms (SNPs) distributed along the chromosomes ( Fig. 1A and Supplementary Fig. 1)).

Tomato chromosomes consist of pericentric heterochromatin and distal euchromatin, with repeats concentrated within and around centromeres, in chromomeres and at telomeres ( Fig. 1A and Supplementary Fig. 1). Substantially higher densities of recombination, genes and transcripts are observed in euchromatin, whereas chloroplast insertions ( Supplementary Information sections 1.22 and 1.23) and conserved microRNA (miRNA) genes ( Supplementary Information section 2.9) are more evenly distributed throughout the genome. The genome is highly syntenic with those of other economically important Solanaceae ( Fig. 1B). Compared to the genomes of Arabidopsis 4 and Sorghum 5, tomato has fewer high-copy, full-length long terminal repeat (LTR) retrotransposons with older average insertion ages (2.8 versus 0.8 million years (Myr) ago) and fewer high-frequency k-mers ( Supplementary Information section 2.10). This supports previous findings that the tomato genome is unusual among angiosperms by being largely comprised of low-copy DNA6, 7.

The pipeline used to annotate the tomato and potato8 genomes is described in Supplementary Information section 2. It predicted 34,727 and 35,004 protein-coding genes, respectively. Of these, 30,855 and 32,988, respectively, are supported by RNA sequencing (RNA-Seq) data, and 31,741 and 32,056, respectively, show high similarity to Arabidopsis genes ( Supplementary Information section 2.1). Chromosomal organization of genes, transcripts, repeats and small RNAs (sRNAs) is very similar in the two species ( Supplementary Figs 2-4). The protein-coding genes of tomato, potato, Arabidopsis, rice and grape were clustered into 23,208 gene groups (≥2 members), of which 8,615 are common to all five genomes, 1,727 are confined to eudicots (tomato, potato, grape and Arabidopsis), and 727 are confined to plants with fleshy fruits (tomato, potato and grape) ( Supplementary Information section 5.1 and Supplementary Fig. 5). Relative expression of all tomato genes was determined by replicated strand-specific Illumina RNA-Seq of root, leaf, flower (two stages) and fruit (six stages) in addition to leaf and fruit (three stages) of S. pimpinellifolium ( Supplementary Table 1).

sRNA sequencing data supported the prediction of 96 conserved miRNA genes in tomato and 120 in potato, a number consistent with other plant species ( Fig. 1A, Supplementary Figs 1 and 3 and Supplementary Information section 2.9). Among the 34 miRNA families identified, 10 are highly conserved in plants and similarly represented in the two species, whereas other, less conserved families are more abundant in potato. Several miRNAs, predicted to target Toll interleukin receptor, nucleotide-binding site and leucine-rich repeat (TIR-NBS-LRR) genes, seemed to be preferentially or exclusively expressed in potato ( Supplementary Information section 2.9).

Comparative genomic studies are reported in Supplementary Information section 4. Sequence alignment of 71  Mb of euchromatic tomato genomic DNA to their potato8 counterparts revealed 8.7% nucleotide divergence ( Supplementary Information section 4.1). Intergenic and repeat-rich heterochromatic sequences showed more than 30% nucleotide divergence, consistent with the high sequence diversity in these regions among potato genotypes8. Alignment of tomato-potato orthologous regions confirmed nine large inversions known from cytological or genetic studies and several smaller ones ( Fig. 1C). The exact number of small inversions is difficult to determine due to the lack of orientation of most potato scaffolds.

A total of 18,320 clearly orthologous tomato-potato gene pairs were identified. Of these, 138 (0.75%) had significantly higher than average non-synonymous ( K a) versus synonymous ( K s) nucleotide substitution rate ratios ( ω ), indicating diversifying selection, whereas 147 (0.80%) had significantly lower than average ω , indicating purifying selection ( Supplementary Table 2). The proportions of high and low ω between sorghum and maize ( Zea mays) are 0.70% and 1.19%, respectively, after 11.9  Myr of divergence9, indicating that diversifying selection may have been stronger in tomato-potato. The highest densities of low- ω genes are found in collinear blocks with average K s  >  1.5, tracing to a genome triplication shared with grape (see below) ( Fig. 1C, Supplementary Fig. 6 and Supplementary Table 3). These genes, which have been preserved in paleo-duplicated locations for more than 100  Myr10, 11, are more constrained than 'average' genes and are enriched for transcription factors and genes otherwise related to gene regulation ( Supplementary Tables 3 and 4).

Sequence comparison of 31,760 Heinz 1706 genes with >5× S. pimpinellifolium read coverage in over 90% of their coding regions revealed 7,378 identical genes and 11,753 with only synonymous changes. The remaining 12,629 genes had non-synonymous changes, including gains and losses of stop codons with potential consequences for gene function ( Supplementary Tables 5-7). Several pericentric regions, predicted to contain genes, are absent or polymorphic in the broader S. pimpinellifolium germplasm ( Supplementary Table 8 and Supplementary Fig. 7). Within cultivated germplasm, particularly among the small-fruited cherry tomatoes, several chromosomal segments are more closely related to S. pimpinellifolium than to Heinz 1706 ( Supplementary Figs 8 and 9), supporting previous observations on recent admixture of these gene pools due to breeding12. Heinz 1706 itself has been reported to carry introgressions from S. pimpinellifolium 13, traces of which are detectable on chromosomes 4, 9, 11 and 12 ( Supplementary Table 9).

Comparison of the tomato and grape genomes supports the hypothesis that a whole-genome triplication affecting the rosid lineage occurred in a common eudicot ancestor11 ( Fig. 2a). The distribution of K s between corresponding gene pairs in duplicated blocks suggests that one polyploidization in the solanaceous lineage preceded the rosid-asterid (tomato-grape) divergence ( Supplementary Fig. 10).

Figure 2: The Solanum whole genome triplication.

a, Speciation and polyploidization in eudicot lineages. Confirmed whole-genome duplications and triplications are shown with annotated circles, including ' T' (this paper) and previously discovered events α , β, γ 10, 11, 14. Dashed circles represent one or more suspected polyploidies reported in previous publications that need further support from genome assemblies27, 28. Grey branches indicate unpublished genomes. Black and red error bars bracket indicate the likely timings of divergence of major asterid lineages and of ' T', respectively. The post-' T' subgenomes, designated T1, T2, and T3, are further detailed in Supplementary Fig. 10. b, On the basis of alignments of multiple tomato genome segments to single grape genome segments, the tomato genome is partitioned into three non-overlapping 'subgenomes' (T1, T2, T3), each represented by one axis in the three-dimensional plot. The ancestral gene order of each subgenome is inferred according to orthologous grape regions, with tomato chromosomal affinities shown by red (inner) bars. Segments tracing to pan-eudicot triplication ( γ ) are shown by green (outer) bars with colours representing the seven putative pre- γ eudicot ancestral chromosomes10, also coded a- g.

Comparison with the grape genome also reveals a more recent triplication in tomato and potato. Whereas few individual tomato/potato genes remain triplicated ( Supplementary Tables 10 and 11), 73% of tomato gene models are in blocks that are orthologous to one grape region, collectively covering 84% of the grape gene space. Among these grape genomic regions, 22.5% have one orthologous region in tomato, 39.9% have two, and 21.6% have three, indicating that a whole-genome triplication occurred in the Solanum lineage, followed by widespread gene loss. This triplication, also evident in potato ( Supplementary Fig. 11), is estimated at 71 (±19.4)  Myr on the basis of the K s of paralogous genes ( Supplementary Fig. 10), and therefore predates the ~7.3  Myr tomato-potato divergence. On the basis of alignments to single grape genome segments, the tomato genome can be partitioned into three non-overlapping 'subgenomes' ( Fig. 2b). The number of euasterid lineages that have experienced the recent triplication remains unclear and awaits complete euasterid I and II genome sequences. K s distributions show that euasterids I and II, and indeed the rosid-asterid lineages, all diverged from common ancestry at or near the pan-eudicot triplication ( Fig. 2a), suggesting that this event may have contributed to the formation of major eudicot lineages in a short period of several million years14, partially explaining the explosive radiation of angiosperm plants on Earth15.

Fleshy fruits ( Supplementary Fig. 12) are an important means of attracting vertebrate frugivores for seed dispersal16. Combined orthology and synteny analyses indicate that both genome triplications added new gene family members that mediate important fruit-specific functions ( Fig. 3). These include transcription factors and enzymes necessary for ethylene biosynthesis ( RIN, CNR, ACS) and perception ( ETR3/NR, ETR4)17, red light photoreceptors influencing fruit quality ( PHYB1/PHYB2) and ethylene- and light-regulated genes mediating lycopene biosynthesis ( PSY1/PSY2). Several cytochrome P450 subfamilies associated with toxic alkaloid biosynthesis show contraction or complete loss in tomato and the extant genes show negligible expression in ripe fruits ( Supplementary Information section 5.4).

Figure 3: Whole-genome triplications set the stage for fruit-specific gene neofunctionalization.

The genes shown represent a fruit ripening control network regulated by transcription factors ( MADS-RIN, CNR) necessary for production of the ripening hormone ethylene, the production of which is regulated by ACC synthase ( ACS). Ethylene interacts with ethylene receptors ( ETRs) to drive expression changes in output genes, including phytoene synthase ( PSY), the rate-limiting step in carotenoid biosynthesis. Light, acting through phytochromes, controls fruit pigmentation through an ethylene-independent pathway. Paralogous gene pairs with different physiological roles ( MADS1/RIN, PHYB1/PHYB2, ACS2/ACS6, ETR3/ETR4, PSY1/PSY2), were generated during the eudicot ( γ , black circle) or the more recent Solanum ( T, red circle) triplications. Complete dendrograms of the respective protein families are shown in Supplementary Figs 16 and 17.

Fruit texture has profound agronomic and sensory importance and is controlled in part by cell wall structure and composition18. More than 50 genes showing differential expression during fruit development and ripening encode proteins involved in modification of cell wall architecture ( Fig. 4a and Supplementary Information section 5.7). For example, a family of xyloglucan endotransglucosylase/hydrolases (XTHs) has expanded both in the recent whole-genome triplication and through tandem duplication. One of the triplicated members, XTH10, shows differential loss between tomato and potato ( Fig. 4a and Supplementary Table 12), suggesting genetically driven specialization in the remodelling of fruit cell walls.

Figure 4: The tomato genome allows systems approaches to fruit biology.

a, Xyloglucan transglucosylase/hydrolases (XTHs) differentially expressed between mature green and ripe fruits ( Supplementary Information section 5.7). These XTH genes and many others are expressed in ripening fruits and are linked with the Solanum triplication, marked with a red circle on the phylogenetic tree. Red lines on the tree denote paralogues derived from the Solanum triplication, and blue lines are tandem duplications. b, Developmentally regulated accumulation of sRNAs mapping to the promoter region of a fruit-regulated cell wall gene (pectin acetylesterase, Solyc08g005800). Variation of abundance of sRNAs (left) and messenger RNA expression levels from the corresponding gene (right) over a tomato fruit developmental series (T1, bud; T2, flower; T3, fruit 1-3  mm; T4, fruit 5-7  mm; T5, fruit 11-13  mm; T6, fruit mature green; T7, breaker; T8, breaker  +  3  days; T9, breaker  +  7  days). The promoter regions are grouped in 100-nucleotide windows. For each window the size class distribution of sRNAs is shown (red, 21; green, 22; orange, 23; blue, 24). The height of the box corresponding to the first time point shows the cumulative sRNA abundance in log scale. The height of the following boxes is proportional to the log offset fold change (offset = 20) relative to the first time point. The expression profile of the mRNA is shown in log ₂ scale. The horizontal black line represents 1  kb of the promoter region. 0 to 12 represent arbitrary units of gene expression.

Similar to soybean and potato and in contrast to Arabidopsis, tomato sRNAs map preferentially to euchromatin ( Supplementary Fig. 2). sRNAs from tomato flowers and fruits19 map to 8,416 gene promoters. Differential expression of sRNAs during fruit development is apparent for 2,687 promoters, including those of cell-wall-related genes ( Fig. 4b) and occurs preferentially at key developmental transitions (for example, flower to fruit, fruit growth to fruit ripening, Supplementary Information section 2.8).

The genome sequences of tomato, S. pimpinellifolium and potato provide a starting point for comparing gene family evolution and sub-functionalization in the Solanaceae. A striking example is the SELF PRUNING ( SP) gene family, which includes the homologue of Arabidopsis FT, encoding the mobile flowering signal florigen20 and its antagonist SP, encoding the orthologue of TFL1. Nearly a century ago, a spontaneous mutation in SP spawned the 'determinate' varieties that now dominate the tomato mechanical harvesting industry21. The genome sequence has revealed that the SP family has expanded in the Solanum lineage compared to Arabidopsis, driven by the Solanum triplication and tandem duplication ( Supplementary Fig. 13). In potato, SP3D and SP6A control flowering and tuberization, respectively22, whereas SP3D in tomato, known as SINGLE FLOWER TRUSS, similarly controls flowering, but also drives heterosis for fruit yield in an epistatic relationship with SP 23, 24, 25. Interestingly, SP6A in S. lycopersicum is inactivated by a premature stop codon, but remains functionally intact in S. pimpinellifolium. Thus, allelic variation in a subset of SP family genes has played a major role in the generation of both shared and species-specific variation in solanaceous agricultural traits.

The genome sequences of tomato and S. pimpinellifolium also provide a basis for understanding the bottlenecks that have narrowed tomato genetic diversity: the domestication of S. pimpinellifolium in the Americas, the export of a small number of genotypes to Europe in the 16th century, and the intensive breeding that followed. Charles Rick pioneered the use of trait introgression from wild tomato relatives to increase genetic diversity of cultivated tomatoes26. Introgression lines exist for seven wild tomato species, including S. pimpinellifolium, in the background of cultivated tomato. The genome sequences presented here and the availability of millions of SNPs will allow breeders to revisit this rich trait reservoir and identify domestication genes, providing biological knowledge and empowering biodiversity-based breeding.

A total of 21 gigabases (Gb) of Roche/454 Titanium shotgun and mate pair reads and 3.3  Gb of Sanger paired-end reads, including ~200,000 BAC and fosmid end sequence pairs, were generated from the 'Heinz 1706' inbred line ( Supplementary Information sections 1.1-1.7), assembled using both Newbler and CABOG and integrated into a single assembly ( Supplementary Information sections 1.17 and 1.18). The scaffolds were anchored using two BAC-based physical maps, one high density genetic map, overgo hybridization and genome-wide BAC FISH ( Supplementary Information sections 1.8-1.16 and 1.19). Over 99.9% of BAC/fosmid end pairs mapped consistently on the assembly and over 98% of EST sequences could be aligned to the assembly ( Supplementary Information section 1.20). Chloroplast genome insertions in the nuclear genome were validated using a mate pair method and the flanking regions were identified ( Supplementary Information sections 1.22-1.24). Annotation was carried out using a pipeline based on EuGene that integrates de novo gene prediction, RNA-Seq alignment and rich function annotation ( Supplementary Information section 2). To facilitate interspecies comparison, the potato genome was re-annotated using the same pipeline. LTR retrotransposons were detected de novo with the LTR-STRUC program and dated by the sequence divergence between left and right solo LTR ( Supplementary Information section 2.10). The genome of S. pimpinellifolium was sequenced to ×40 depth using Illumina paired end reads and assembled using ABySS ( Supplementary Information section 3). The tomato and potato genomes were aligned using LASTZ ( Supplementary Information section 4.1). Identification of triplicated regions was done using BLASTP, in-house-generated scripts and three-way comparisons between tomato, potato and S. pimpinellifolium using MCSCAN ( Supplementary Information sections 4.2-4.4). Specific gene families/groups (genes for ascorbate, carotenoid and jasmonate biosynthesis, cytochrome P450s, genes controlling cell wall architecture, hormonal and transcriptional regulators, resistance genes) were subjected to expert curation/analysis ( Supplementary Information section 5). PHYML and MEGA were used to reconstruct phylogenetic trees and MCSCAN was used to infer gene collinearity ( Supplementary Information section 5.2).

Primary accessions

GenBank/EMBL/DDBJ

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This work was supported by: Argentina: INTA and CONICET. Belgium: Flemish Institute for Biotechnology and Ghent University. China: The State Key Laboratory of Plant Genomics, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences; Ministry of Science and Technology (2006AA10A116, 2004CB720405, 2006CB101907, 2007DFB30080) Ministry of Agriculture ('948' Program: 2007-Z5); National Natural Science Foundation (36171319); Postdoctoral Science Foundation (20070420446). European Union: FP6 Integrated Project EU-SOL PL 016214. France: Institute National de la Recherche Agronomique and Agence Nationale de la Recherche. Germany: the Max Planck Society. India: Department of Biotechnology, Government of India; Indian Council of Agricultural Research. Italy: Ministry of Research (FIRB-SOL, FIRB-Parallelomics, ItaLyco and GenoPOM projects); Ministry of Agriculture (Agronanotech and Biomassval projects); FILAS foundation; ENEA; CNR-ENEA project L. 191/2009. Japan: Kazusa DNA Research Institute Foundation and National Institute of Vegetable and Tea Science. Korea: KRIBB Basic Research Fund and Crop Functional Genomics Research Center (CFGC), MEST. Netherlands: Centre for BioSystems Genomics, Netherlands Organization for Scientific Research. Spain: Fundación Genoma España; Cajamar; FEPEX; Fundación Séneca; ICIA; IFAPA; Fundación Manrique de Lara; Instituto Nacional de Bioinformatica. UK: BBSRC grant BB/C509731/1; DEFRA; SEERAD. USA: NSF (DBI-0116076; DBI-0421634; DBI-0606595; IOS-0923312; DBI-0820612; DBI-0605659; DEB-0316614; DBI 0849896 and MCB 1021718); USDA (2007-02773 and 2007-35300-19739); USDA-ARS. We acknowledge the Potato Genome Sequencing Consortium for sharing data before publication; potato RNA-Seq data was provided by C. R. Buell from the NSF-funded Potato Genome Sequence and Annotation project; tomato RNA-Seq data by the USDA-funded SolCAP project, N. Sinha and J. Maloof; the Amplicon Express team for BAC pooling services; construction of the Whole Genome Profiling (WGP) physical map was supported by EnzaZaden, RijkZwaan, Vilmorin & Cie, and Takii & Co. Keygene N.V. owns patents and patent applications covering its AFLP and Whole Genome Profiling technologies; AFLP and Keygene are registered trademarks of Keygene N.V. The following individuals are also acknowledged for their contribution to the work described: J. Park, B. Wang, C. Niu, D. Liu, F. Cojutti, S. Pescarolo, A. Zambon, G. Xiao, J. Chen, J. Shi, L. Zhang, L. Zeng, M. Caccamo, D. Bolser, D. Martin, M. Gonzalez, P. A. Bedinger, P. A. Covey, P. Pachori, R. R. Pousada, S. Hakim, S. Sims, V. Cahais, W. Long, X. Zhou, Y. Lu, W. Haso, C. Lai, S. Lepp, C. Peluso, H. Teramu, H. De Jong, R. Lizarralde, E. R. May and Z. Li. M. Zabeau is thanked for his support and encouragement and S. van den Brink for her secretarial support. We dedicate this work to the late C. Rick who pioneered tomato genetics, collection of wild germplasm and the distribution of seed and knowledge.

Affiliations

1. Kazusa DNA Research Institute, 2-6-7 Kazusa-kamatari, Kisarazu, Chiba 292-0818, Japan.

Shusei Sato,

Satoshi Tabata,

Hideki Hirakawa,

Erika Asamizu,

Kenta Shirasawa,

Sachiko Isobe,

Takakazu Kaneko,

Yasukazu Nakamura,

Daisuke Shibata &

Koh Aoki

2. Institute of Research in Biomedicine, c/  Josep Samiter 1-5, 08028 Barcelona, Spain

3. Plant Research International, Business Unit Bioscience, Droevendaalsesteeg 1, 6708 PB Wageningen, The Netherlands.

Erwin Datema,

Sandra Smit,

Elio G. W. M. Schijlen,

Jose van de Belt,

Jan C. van Haarst,

Sander A. Peters,

Marjo J. van Staveren,

Marleen H. C. Henkens,

Paul J. W. Mooyman,

Thamara Hesselink &

Roeland C. H. J. van Ham

Consortia

1. The Tomato Genome Consortium

2. Kazusa DNA Research Institute

Shusei Sato,

Satoshi Tabata,

Hideki Hirakawa,

Erika Asamizu,

Kenta Shirasawa,

Sachiko Isobe,

Takakazu Kaneko,

Yasukazu Nakamura,

Daisuke Shibata &

Koh Aoki

3. Keygene N.V.

Marco G. M. van Schriek,

Richard M.C. Feron,

Jan van Oeveren,

Peter de Heer,

Lorena daPonte,

Saskia Jacobs-Oomen,

Mike Cariaso,

Marcel Prins,

Michiel J. T. van Eijk,

Antoine Janssen &

Mark J. J. van Haaren

4. Plant Research International

Erwin Datema,

Sandra Smit,

Elio G. W. M. Schijlen,

Jose van de Belt,

Jan C. van Haarst,

Sander A. Peters,

Marjo J. van Staveren,

Marleen H. C. Henkens,

Paul J. W. Mooyman,

Thamara Hesselink &

Roeland C. H. J. van Ham

Competing financial interests

The authors declare no competing financial interests.

Corresponding authors

Correspondence to:

The genomic data generated by the whole project are available in GenBank as accession number AEKE00000000, and the individual chromosome sequences as numbersCM001064-CM001075. TheRNA-Seq data are available in the Sequence Read Archive under accession number SRA049915, GSE33507, SRA050797 and SRA048144. Further information on data access can be found in Supplementary Information section 2.2.

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# data[].attributes.attachment_link.title
The tomato genome sequence provides insights into fleshy fruit evolution

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Tomato (Solanum lycopersicum) is a major crop plant and a model system for fruit development. Solanum is one of the largest angiosperm genera and includes annual and perennial plants from diverse habitats. Here we present a high-quality genome sequence of domesticated tomato, a draft sequence of its closest wild relative, Solanum pimpinellifolium, and compare them to each other and to the potato genome (Solanum tuberosum).

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Presenters: Mark Mc Dermott & Eoin O Cinneide

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Looking for lost flavor in tomatoes

Commercially available tomatoes are renowned these days for sturdiness, but perhaps not for flavor. Heirloom varieties, on the other hand, maintain the richer flavors and sweeter tomatoes of years past. Tieman et al. combined tasting panels with chemical and genomic analyses of nearly 400 varieties of tomatoes. They identified some of the flavorful components that have been lost over time. Identification of the genes that have also gone missing provides a path forward for reinstating flavor to commercially grown tomatoes.

Science, this issue p. 391

Abstract

Modern commercial tomato varieties are substantially less flavorful than heirloom varieties. To understand and ultimately correct this deficiency, we quantified flavor-associated chemicals in 398 modern, heirloom, and wild accessions. A subset of these accessions was evaluated in consumer panels, identifying the chemicals that made the most important contributions to flavor and consumer liking. We found that modern commercial varieties contain significantly lower amounts of many of these important flavor chemicals than older varieties. Whole-genome sequencing and a genome-wide association study permitted identification of genetic loci that affect most of the target flavor chemicals, including sugars, acids, and volatiles. Together, these results provide an understanding of the flavor deficiencies in modern commercial varieties and the information necessary for the recovery of good flavor through molecular breeding.

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A chemical genetic roadmap to improved tomato flavor

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Commercially available tomatoes are renowned these days for sturdiness, but perhaps not for flavor. Heirloom varieties, on the other hand, maintain the richer flavors and sweeter tomatoes of years past. Tieman et al. combined tasting panels with chemical and genomic analyses of nearly 400 varieties of tomatoes.

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Presenters: Sahar and Taylor

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A MADS-Box Gene Necessary for Fruit Ripening at the Tomato Ripening-Inhibitor (Rin) Locus

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Tomato plants harboring the ripening - inhibitor ( rin ) mutation yield fruits that fail to ripen. Additionally, rin plants display enlarged sepals and loss of inflorescence determinacy. Positional cloning of the rin locus revealed two tandem MADS-box genes ( LeMADS - RIN and LeMADS - MC ), whose expression patterns suggested roles in fruit ripening and sepal development, respectively.

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Presenters:

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Proceedings of the National Academy of Sciences

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iUnited States Department of Agriculture, Robert W. Holley Center and Boyce Thompson Institute for Plant Research, Cornell University, Ithaca, NY 14853

Edited by Robert L. Fischer, University of California, Berkeley, CA, and approved July 8, 2015 (received for review March 3, 2015)

Significance

This work shows that active DNA demethylation governs ripening, an important plant developmental process. Our work defines a molecular mechanism, which has until now been missing, to explain the correlation between genomic DNA demethylation and fruit ripening. It demonstrates a direct cause-and-effect relationship between active DNA demethylation and induction of gene expression in fruits. The importance of these findings goes far beyond understanding the developmental biology of ripening and provides an innovative strategy for its fine control through fine modulation of epimarks in the promoters of ripening related genes. Our results have significant application for plant breeding especially in species with limited available genetic variation.

Abstract

In plants, genomic DNA methylation which contributes to development and stress responses can be actively removed by DEMETER-like DNA demethylases (DMLs). Indeed, in Arabidopsis DMLs are important for maternal imprinting and endosperm demethylation, but only a few studies demonstrate the developmental roles of active DNA demethylation conclusively in this plant. Here, we show a direct cause and effect relationship between active DNA demethylation mainly mediated by the tomato DML, SlDML2, and fruit ripening- an important developmental process unique to plants. RNAi SlDML2 knockdown results in ripening inhibition via hypermethylation and repression of the expression of genes encoding ripening transcription factors and rate-limiting enzymes of key biochemical processes such as carotenoid synthesis. Our data demonstrate that active DNA demethylation is central to the control of ripening in tomato.

Genomic DNA methylation is a major epigenetic mark that is instrumental to many aspects of chromatin function, including gene expression, transposon silencing, or DNA recombination ( 1- 4). In plants, DNA methylation can occur at cytosine both in symmetrical (CG or CHG) and nonsymmetrical (CHH) contexts and is controlled by three classes of DNA methyltransferases, namely, the DNA Methyltransferase 1, Chromomethylases, and the Domain Rearranged Methyltransferases ( 5- 7). Indeed, in all organisms, cytosine methylation can be passively lost after DNA replication in the absence of methyltransferase activity ( 1). However, plants can also actively demethylate DNA via the action of DNA Glycosylase-Lyases, the so-called DEMETER-Like DNA demethylases (DMLs), that remove methylated cytosine, which is then replaced by a nonmethylated cytosine ( 8- 11). Initially identified as enzymes necessary for maternal imprinting in Arabidopsis thaliana ( 12), the role of DMLs has since been established in various processes such as limiting extensive DNA methylation at gene promoters ( 13), determining the global demethylation of seed endosperm ( 8, 14) and promoting plant responses to pathogens ( 15). Of note, Arabidopsis ros1, dml2, and dml3 single, double, or triple mutants showed little or no developmental alterations ( 9, 16, 17), suggesting that active DNA demethylation is not critical for development in this species. However, as mentioned above, genomic DNA methylation is an important mechanism that influences gene expression, and methylation at promoters is known to inhibit gene transcription ( 5, 18). Hence, it is likely that the active removal of methylation marks is an important mechanism during plant development and plant cell fate reprogramming, leading to the hypomethylation of sites important for DNA-protein interaction and gene expression, as already observed in human cells ( 19).

Indeed, accumulating evidence suggests that active DNA demethylation might play a greater role in controlling gene expression in tomato. In support of this idea, recent work describing the methylome dynamics in tomato fruit pericarp revealed substantial changes in the distribution of DNA methylation over the tomato genome during fruit development, and demethylation during ripening at specific promoters such as the NON RIPENING ( NOR) and COLORLESS NON RIPENING ( CNR) promoters ( 20, 21). This observation is consistent with previous studies indicating that genome cytosine methylation levels decrease by 30% in pericarp of fruits during ripening, although DNA replication is very limited at this stage ( 22).

Here, we investigated active DNA demethylation as a possible mechanism governing the reprogramming of gene expression in fruit pericarp cells at the onset of fruit ripening.

Results

The Tomato Genome Contains Four DNA Glycosylase Genes with Specific Expression Patterns.

The tomato genome contains four putative DML genes encoding proteins with characteristic domains of functional DNA glycosylase-lyases ( 23) (SI Appendix, Fig. S1 A and C and Table S1). SlDML1 and -2 are orthologous to the Arabidopsis AtROS1 (Repressor of Silencing 1) gene and SlDML3 to AtDME (DEMETER), whereas SlDML4 has no closely related Arabidopsis ortholog (). All four SlDML genes are ubiquitously expressed in tomato plants, although SlDML4 is expressed at a very low level in all organs analyzed. In leaves, flowers, and young developing fruits, the four genes present coordinated expression patterns characterized by high expression levels in young organs that decrease when organs develop. However, unlike SlDML1, SlDML3, and SlDML4, which are barely expressed during fruit ripening, SlDML2 mRNA abundance increases dramatically in ripening fruits, suggesting an important function at this developmental phase ( Fig. 1).

Transgenic Plants with Reduced DML Gene Expression Present Various Fruit and Plant Phenotypes.

The physiological significance of tomato DMLs was addressed through RNAi-mediated gene repression using the highly conserved Helix-hairpin-Helix-Gly/Pro rich domain (HhH-GPD) specific to DML proteins as a target sequence (). Our goal was to repress simultaneously all tomato SlDML genes, anticipating potential functional redundancy among these four genes; 23 independent T0 transgenic lines were generated and 22 showed alterations of fruit development, including delayed ripening, modified fruit shape, altered color, shiny appearance, parthenocarpy, or combinations of these phenotypes ().

Lines 2 and 8, which showed delayed and inhibited ripening phenotypes, were chosen to investigate the possible link between ripening and DNA demethylation. In both cases, 10-25 T1 and T2 plants were grown that showed maintenance and strengthening of the nonripening phenotypes in subsequent generations coincident with the presence of the transgene. The loss of the RNAi transgene in segregating lines led to reversion to a wild-type (WT) phenotype, indicating a lack of memory effect across generations when fruit ripening is considered ( and ). In plants of both RNAi lines, analysis of SlDML gene residual expression in 20 days postanthesis (dpa) fruits indicates that only SlDML1 and SlDML2 are repressed to 40-60% of the WT level, whereas SlDML3 and SlDML4 are either unaffected or induced compared with WT (). This is most likely attributable to the lower homology level of these two genes, with SlDML1 in the part of the gene used for the RNAi construct (). During ripening, SlDML2 expression is reduced to 10% of WT at the Breaker (Br) stage and remains low at 55 dpa (Br + 16) but increases slightly at 70 dpa (Br + 31) ( and ), coincident with the partial ripening observed in transgenic RNAi fruits ( and ). Whether the increase in SlDML2 expression at late ripening stages is attributable to a weaker effect of the RNAi remains unclear. None of the three remaining genes, SlDML1, SlDML3, and SlDML4, which are weakly expressed during ripening, displayed significantly reduced expression compared with WT fruits of the same age, indicating that observed ripening phenotypes are likely attributable to SlDML2 gene repression. This hypothesis was further confirmed using virus induced gene silencing (VIGS) to specifically repress the SlDML2 gene; 17.5% of the fruits injected with a PVX/SlDML2 vector presented non ripening sectors contrary to those injected with a control PVX virus that all ripened normally ( and ). Indeed, SlDML2 was down-regulated in nonripening sectors of fruits injected with the PVX/SlDML2 vector, whereas none of the three other SlDML genes was repressed (), demonstrating that the specific knock down of SlDML2 is sufficient to inhibit ripening.

It was noteworthy that some plants from line 2 developed additional phenotypes affecting plant growth, leaf shape, flower development, and fruit carpel number that were not observed in T0 and T1 generations ( and ). The screening of additional lines revealed other independent transgenic lines that presented flower, fruit, and plant phenotypes similar to line 2 (). These observations indicate that the severity of the phenotypes increases over generations and suggest that DMLs may also be involved in other aspects of tomato plant development beyond fruit ripening.

All Aspects of Fruit Ripening Are Delayed and Limited in RNAi Transgenic Lines.

Fruits of transgenic lines 2 and 8 were further analyzed to investigate the consequences of DNA demethylation on the ripening process. Indeed, in fruits of both transgenic lines, the onset of fruit ripening was delayed from 10 to 20 d compared with WT or Azygous revertant fruits, and ripening of transgenic fruits was never completed even after 45 d or longer maturation times ( and ). The ripening defect is further demonstrated by the late and extremely reduced total carotenoids and lycopene accumulation and the delayed chlorophyll degradation (). Primary metabolite composition was also modified, as visualized by principal component analysis (PCA) using the absolute concentration of 31 primary metabolites issued from ¹H-NMR analysis ( and ). The first two principal components (PCs), explain more than 54% of total variability. During early development (20, 35, and 39 dpa), WT and transgenic samples follow parallel trajectories as highlighted by the PCA in which the second PC (PC2) explains 21% of the total variability. However, at 55-dpa and later ripening stages, PC1, which accounts for 33.67% of the global variability, separates WT fruits from all other samples. Hence, WT fruit samples harvested at 55-dpa and older stages are clearly distinct from transgenic fruit samples of the same age. Metabolic differences between ripening WT and transgenic fruits are mainly attributable to overaccumulation of malate and reduction or delayed accumulation of compounds typical of ripening fruits, including glucose, fructose, glutamate, rhamnose, and galactose (). Climacteric rise of ethylene production was also dramatically reduced in fruits of both DML RNAi lines, although low ethylene accumulation occurred to a degree and timing consistent with the late and limited ripening process of RNAi fruits (SI Appendix, Fig. S6).

Fruit-Ripening Defects Are Correlated with the Repression and Hypermethylation of Genes Necessary for This Developmental Process.

To demonstrate a causal relationship between fruit ripening defects of transgenic lines and the impairment of active DNA demethylation, the expression of CNR ( 21), RIPENING INHIBITOR (RIN) ( 24), NOR ( 25), and PHYTOENE SYNTHASE 1 ( PSY1) ( 26, 27) genes was assessed in RNAi transgenic plants. These genes were selected among others because they are necessary for the overall ripening process ( CNR, RIN, NOR), or specifically govern carotenoid accumulation ( PSY1), an important quality trait of mature tomato fruit. Moreover, the promoter regions of these genes showed reduced methylation levels during fruit ripening in WT tomato ( 20, 21). It is noteworthy that CNR gene induction was delayed 15 d in transgenic fruits, and all three other genes showed a dramatic reduction in expression level consistent with the ripening defect of the transgenic lines ( andSI Appendix, Fig. S7). To assess whether repression of CNR, RIN, NOR, and PSY1 gene expression in ripening fruits results from the maintenance of a high cytosine methylation status of their promoter upon down-regulation of SlDML2, methylsensitive-PCR (McrBC-PCR) analysis of the corresponding promoters was performed. This approach revealed a ripening-associated demethylation of the RIN, NOR, and PSY1 promoters in WT and Azygous revertant fruits but not in SlDML RNAi fruits (). No detectable variations of methylation in the CNR promoter during ripening of WT fruits were revealed with this method. The putative differentially methylated regions (DMRs) in the NOR and PSY1 promoter regions were subsequently analyzed by gene specific bisulfite pyrosequencing ( 28). Methylation analysis of the CNR promoter was targeted to a region known to be methylated at all stages (CNR1) (SI Appendix, Fig. S9C), used here as a control for methylation and to a previously identified DMR (CNR2) () ( 20, 21). For all three promoters, cytosines that became demethylated in ripening WT fruits but not in transgenic fruits of the same age were identified ( andSI Appendix, Fig. S9). Two distinct situations were observed: ( i) sequences corresponding to putative RIN binding sites (RIN BS) in the CNR and NOR promoters ( 20), where methylation is high at 20 and 35 dpa in all plants analyzed and drops to very low levels during ripening of WT fruits but is maintained to high levels in RNAi fruits of the same age; and ( ii) sequences that are hypermethylated in transgenic fruits at all stages analyzed compared with WT fruits. These latter sequences include a newly identified DMR in the PSY1 promoter and cytosines upstream and downstream to the RIN BS in the NOR and CNR promoters. These data demonstrate the absolute requirement of promoter demethylation in critical genes for ripening to occur. The data also suggest multiple patterns of cytosine demethylation occurring either specifically during ripening or at earlier stages.

Discussion

Previously reported analysis of DNA cytosine methylation and RIN binding during fruit development in WT and in the rin and Cnr tomato-ripening mutants suggested a significant role for DNA methylation during ripening and a feedback loop between methylation and ripening transcription factors ( 20, 21, 29). Here, we demonstrate for the first time to our knowledge that active DNA demethylation is an absolute requirement for fruit ripening to occur and show a direct cause and effect relationship between hypermethylation at specific promoters and repression of gene expression. In this context, SlDML2 appears to be the main regulator of the ripening associated DNA demethylation process. ( i) SlDML2 is the only SlDML gene induced concomitantly to the demethylation and induction of genes that control fruit ripening; ( ii) the specific knockdown of SlDML2 in VIGS-treated fruits leads to inhibition of fruit ripening similar to DML-RNAi fruits; and ( iii) the hypermethylated phenotype described in the Cnr and rin mutants ( 20) is associated with the specific repression of SlDML2, with none of the other SlDML genes being down-regulated ( and Dataset S1).

Indeed, we cannot formally rule out that SlDML1, which is repressed in the transgenic RNAi lines, also participates in the genomic DNA demethylation in fruits. However, SlDML1 is mainly expressed at early stages of fruit development and only at very low levels during fruit ripening. Hence, this protein may also be involved in demethylation events but mainly those occurring at the early stages of fruit development.

In addition to genes encoding major fruit ripening regulators, those encoding enzymes involved in various aspects of fruit ripening are also likely to be demethylated, as suggested by the observation that PSY1 gene expression also requires demethylation. Combined transcriptomic, methylomic, and metabolomic analysis of the transgenic lines described here will now be required to determine the network of genes and metabolic processes primarily targeted by demethylation in tomato fruit.

SlDML2 is the likely focal point of a feedback regulation on ripening-associated DNA demethylation, because this gene is clearly down-regulated in fruits of the rin, nor, and Cnr mutants, contrary to the other SlDML genes that are normally expressed ( and Dataset S1). It is plausible that timing and extent of demethylation may represent an important source of variation in the diversity of kinetics and intensity of ripening found among tomato varieties, thus presenting a frontier for further investigation. Controlling the timing and kinetics of active DNA demethylation in fruits may therefore provide new strategies to enhance fruit shelf life. In addition, engineering DNA demethylation in tomato fruits would be an innovative and novel strategy for the improvement of traits of agronomical relevance in a species with little genetic diversity ( 30). Finally, the recent demonstration that hypermethylation of a Myb promoter blocks anthocyanin accumulation during pear and apple ripening ( 31, 32) supports the notion of a more general role for demethylation in fruits. However, whether this mechanism occurs similarly during the ripening of all fleshy fruit species now requires further investigation.

Materials Methods

Plant Material and Experimental Plan.

All experiments were performed using a cherry tomato variety ( Solanum lycopsersicum, cv WVA106) that was grown in greenhouse conditions, except for VIGS experiments, which were performed on Solanum lycopsersicum, cv Ailsa Craig grown in growth chambers as described ( 21). For the array experiments, fruit pericarp of Ailsa Craig and near-isogenic mutants rin, nor, and Cnr were collected at 13 stages of fruit development and ripening with three independent biological replicates per line and immediately frozen in liquid nitrogen for RNA extraction and array analysis. Details of tomato transformation, selection of line 2 and 8 used in this study, and of VIGS experiments are provided inSI Appendix, Materials and Methods.

For all analysis, two independent transgenic T2 plants (DML2A and -B and DML8A and -B for lines 2 and 8, respectively) and an azygous plant obtained from line 8 were used. Additional T2 plants were eventually used as controls for the phenotypes of these four plants. T2 plants from line 2 presented dramatic alterations of flower development, not visible in previous generations, and were backcrossed to allow fruit development. This resulted in a limited number of fruits (see below). For this reason, not all developmental stages could be analyzed for this line.

The experimental plan was designed to span tomato fruit development and ripening in cv West Virginia 106 (WVA106) and transgenic DML RNAi plants over a period of 85 d from fruit set to account for the strongly delayed ripening phenotype of the transgenic fruits. At stages following mature green, the DML RNAi fruits diverge from the WT, because they are significantly delayed in ripening induction and almost completely ripening inhibited. Because it was not possible to select stages equivalent to the Br (39 dpa) or red ripe stages in the transgenic lines, we have chosen to analyze fruits identically staged, which allows comparing changes in the context of a developmental parameter (days postanthesis) that can be precisely measured. Two independent cultures were performed. ( i) Plants from line 2 and the relevant WT control (WT1), fruits were harvested at 20, 35, 55 (Br + 16), 70 (Br + 31), and 85 (Br + 46) dpa. Because the fruit yield was reduced in line 2, a sufficient number of fruits at the Br stage could not be harvested and older fruits were preferentially selected to allow the analysis of late effects of demethylation inhibition. ( ii) Line 8 was grown together with its own WT control (WT2) and an azygous plant. Because there were more fruits available for this line, the Br stage (39 dpa) was harvested in addition of the stages used for line 2.

For all fruit samples, two individual T2 plants were used, and for each sample, a minimum of six fruits separated in three biological replicates were processed and stored at −80 °C until used.

Acknowledgments

We thank Marie Mirouze for critical reading of the manuscript and Antoine Daunay and Nicolas Mazaleyrat for technical support in bisulfite pyrosequencing analysis. We acknowledge Syngenta and specifically Dr. Charles Baxter for help with the tomato GeneChip studies and Alex Marshall for help with the array analysis, as well as Cécile Cabasson and Jim Craigon for help in statistical analysis. Metabolomic profiling was performed on the Metabolome Facility of Bordeaux Functional Genomics Center and supported by the French National Infrastructure for Metabolomics and Fluxomics (MetaboHUB) funded by the Agence Nationale de Recherche (ANR, Project ANR-11-INBS-0010). R.L. was the recipient of a grant from the Chinese Scholarship Council and P.G. of a Fulbright grant. G.B.S., T.C.H., and N.H.C. acknowledge financial support from the Biotechnology and Biological Sciences Research Council, UK (Grants BB/F005458/1 and BB/J015598/1), and G.B.S. acknowledges support from the European Cooperation in Science and Technology (COST) Action FA1106. Y.H., J.K., and C.W. were supported by Hangzhou Normal University and the National Natural Science Foundation of China (NFSC, Grant 31370180). M.B. and M.L. received support from the Laboratoire d'Excellence (LABEX) entitled "Towards a Unified theory of biotic Interactions; the roLe of environmental Perturbations" (TULIP, ANR-10-LABX-41) and from the networking activities within the European COST Action FA1106. J.J.G. was supported by National Science Foundation Grant IOS-1322714.

Footnotes

Author contributions: T.C.H., Y.H., and P.G. designed research; R.L., A.H.-K., L.S., E.T., D.R., A.M.-B., S.H., M.L., J.K., C.W., C.D.-G., N.H.C., M.M., T.C.H., and P.G. performed research; A.H.-K. targeted bisulfite pyrosequencing; R.L., A.H.-K., E.T., D.R., A.M.-B., S.H., T.C.H., J.T., M.B., Y.H., G.B.S., J.J.G., and P.G. analyzed data; and E.T., G.B.S., J.J.G., and P.G. wrote the paper.

The authors declare no conflict of interest.

This article is a PNAS Direct Submission.

Data deposition: Full array datasets of WT (Ailsa craig) and of the rin, nor, and Cnr mutants have been deposited with the Sol Genomics Network and are accessible under the following link: ftp://ftp.solgenomics.net/microarray/, file name rinnorcnr microarrays Liuetal PNAS2015. These array datasets have been used to analyze the expression of the four DML genes in the tomato rin, nor, and Cnr mutants and in WT fruits. The results are shown in and Dataset S1.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1503362112/-/DCSupplemental.

Freely available online through the PNAS open access option.

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A DEMETER-like DNA demethylase governs tomato fruit ripening

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iUnited States Department of Agriculture, Robert W. Holley Center and Boyce Thompson Institute for Plant Research, Cornell University, Ithaca, NY 14853 Edited by Robert L. Fischer, University of California, Berkeley, CA, and approved July 8, 2015 (received for review March 3, 2015) This work shows that active DNA demethylation governs ripening, an important plant developmental process.

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Presenters: James Burke and Johnny Farrell

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Presenters: Emma Whelan & Rebecca Sheridan

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CRISPR/Cas9-mediated mutagenesis of the RIN locus that regulates tomato fruit ripening - ScienceDirect

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Site-directed mutagenesis using genetic approaches can provide a wealth of resources for crop breeding as well as for biological research. The clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated 9 endonuclease (CRISPR/Cas9) system is a novel strategy used to induce mutations in a specific genome region; the system functions in a variety of organisms, including plants.

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Presenters: Danielle Preston and cathal phelan

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Production of RIN-Specific Antibodies and Validation of the rin Mutation Predicted Chimeric Protein

To study the endogenous function and regulation of the RIN gene, we developed polyclonal antibodies that can specifically detect the RIN protein. RIN shares a highly conserved N-terminal DNA-binding domain with other members of the MADS-box family, whereas the C-terminal portion, whose functions include protein-protein interactions and transcriptional activation, is more variable ( Kaufmann et al., 2005). To obtain antibodies that would specifically recognize RIN and not other of the over 100 members of the tomato MADS family ( www.solgenomics.net), we raised the antibodies against the C-terminal portion of RIN. A His-tagged recombinant protein encoding the RIN C-terminal portion was produced in Escherichia coli and purified on a His-binding column. The purified protein was used to raise polyclonal antibodies from rabbit. To test the specificity of the antibodies, protein extracts from tomato fruits of cultivar Ailsa Craig (Ac) and lines nearly isogenic for rin and rin plus a 35S:: RIN transgene ( Vrebalov et al., 2002) were isolated and used in a western-blot analysis. As shown in Figure 1A, RIN antibodies recognize a 28-kD band in the wild-type (Ac) fruit extract but not in the rin mutant extract. This size corresponds to the expected M r of the RIN protein. The fact that this band is not detected in rin mutant protein extract but is present in the isogenic rin 35S:: RIN line confirms that it corresponds to the RIN protein. A faint higher M r band (45 kD) is detected in the rin mutant protein extract ( Fig. 1A, 5-min exposure). As described previously ( Vrebalov et al., 2002), the rin mutation is caused by a genomic deletion removing the 3′-untranslated region encoding exon of the RIN gene thus creating a translational fusion with the adjacent MACROCALYX ( MC) MADS-box gene. Because the chimera still possess the majority of the RIN C-terminal region used to raise the RIN antibodies, said antibodies should detect the chimeric RIN-MC protein of the rin mutant. The 45-kD band observed in the rin pericarp protein extract ( Fig. 1A, 5-min exposure) corresponds to the predicted size of the chimeric RIN-MC protein ( Vrebalov et al., 2002).

RIN Expression and Protein Accumulation Are Tightly Correlated through Fruit Development

We next examined the behavior of RIN protein and mRNA accumulation during pericarp development and ripening using western- and northern-blot analyses. Figure 1B shows RIN protein and RNA accumulation at different stages of pericarp development and ripening. RIN protein and mRNA are absent during the initial preripening phase of pericarp development (0-35 DPA), but accumulate early during ripening. RIN protein is detected slightly before the breaker (BK) stage and its expression is maintained throughout ripening (up to 15 d after BK). Comparison of protein and RNA expression profiles indicates a tight correlation between the two. The simplest explanation for this observation is that the RIN protein is short lived in vivo and thus accumulation of the RIN protein and its corresponding effects are largely transcriptionally regulated, though we recognize that more intricate explanations could also account for this result.

Identification of Promoters That Associate with RIN

We suspect that RIN may act as master regulator of ripening by influencing the expression of numerous genes. Although its role in influencing ripening-associated traits, such as cell wall degradation, carotenoid accumulation, and ethylene production, is clearly supported by the strong phenotype of the rin mutant ( Vrebalov et al., 2002), the mechanism and specific target genes by which it exerts its effect have only begun to be addressed. To better understand the mechanism by which RIN influences ripening, a ChIP strategy was employed ( Wang et al., 2002). The above-described anti-RIN antibodies were used to isolate RIN-bound chromatin fragments from BK-stage tomato fruits. Quantitative PCR (qPCR) was used to measure an enrichment ratio at different loci. Association of RIN to a particular locus should result in an increase in the enrichment level following RIN immunoprecipitation compared to the level prior to immunoprecipitation (input) or following a nonspecific immunoprecipitation (using preimmune serum). The specificity of the immunoprecipitation was further assessed by performing the ChIP on rin fruits that we expect to have a nonfunctional RIN protein due to the rin mutation. To explore the depth of RIN activity, the ability of RIN to associate with important classes of ripening genes was examined. Candidate genes were selected among: (1) ripening-related transcription factors, (2) ethylene synthesis enzymes, (3) ethylene signal transduction components, (4) ethylene-responsive genes, (5) cell wall metabolism enzymes, and (6) carotenoid biosynthesis pathway enzymes.

DNA-Binding Dynamics of RIN during Fruit Development and Ripening

In light of previous results, two models can be proposed regarding the temporal action of RIN in controlling gene expression during fruit ripening. RIN could bind to and influence the expression of its target genes in early ripening (around BK stage and a few days before or after) but this binding might not be required to complete the ripening process. Alternatively, RIN might be required in a continuous fashion to both initiate and maintain the expression of ripening genes throughout the ripening process. Fujisawa et al. (2011) report active RIN binding in pink early ripening fruit to a number of ethylene synthesis and cell wall associated genes that could be consistent with either hypothesis. They also observed RIN binding to its own promoter at the ripe fruit stage, an observation that is consistent with the latter hypothesis. To better distinguish between these two hypotheses and to further validate the accuracy of our ChIP analyses, we performed the ChIP assay on a developmental series of both ripening wild type and identically aged rin fruits. Specifically, ChIP enrichment level of RIN, ACS2, PG2a, and PSY1 promoters was assayed in 17 DPA, 27 DPA, mature green, BK, 5-d post BK (BK + 5), and 10-d post BK (BK + 10) fruits. The nonbound target ACO1 promoter was also examined as a negative control. Western-blot analysis ( Fig. 3A) shows the amount of RIN protein present in each of the stages used for the ChIP assay. As anticipated, the amount of RIN protein increases at the onset of ripening (mature green to BK) and remains high in the following stages. Figure 3B shows that a strong enrichment of all RIN targets is detected following RIN-ChIP on the BK, BK + 5 (red), and BK + 10 (red) stages when RIN protein is most prevalent. This result indicates that RIN binding occurs throughout ripening and better supports the hypothesis in which continuous RIN binding is required for RIN-mediated gene expression. This mechanism of action is also consistent with the observed RNA and protein expression profile of RIN ( Fig. 1B). The tight correlation between RIN protein accumulation ( Fig. 3A) and RIN ChIP promoter enrichment ( Fig. 3B) also serves to validate the accuracy and specificity of the RIN ChIP analysis.

CNR Is Required for RIN-Binding Activity

Like the rin mutation, the Cnr mutation has a broad inhibitory impact on ethylene- and nonethylene-mediated ripening activities ( Fraser et al., 2001; Eriksson et al., 2004). The Cnr mutation is caused by changes in the methylation status of the CNR promoter ( Manning et al., 2006). We showed that RIN protein associates with the CNR promoter and examination of the CNR promoter region reveals the presence of a CArG cis-element located 2-bp upstream of a cytosine whose methylation status is changed in the Cnr mutant. We were interested to see if the change in methylation level of the CNR promoter in the Cnr mutant impacts this binding. The ChIP assay was performed on Cnr fruits at the BK + 2 stage and enrichment levels of the CNR and other promoters were measured. Interestingly, no enrichment of the CNR promoter was observed in Cnr fruit following RIN ChIP ( Fig. 4B) even though RIN protein is present ( Fig. 4A). Other target loci should still be bound by RIN since the Cnr mutation affects only its own methylation status. Nevertheless, we found that RIN association with all tested target genes was lost in the Cnr mutant ( Fig. 4B). This result suggests that RIN interaction with its target loci depends on CNR or a CNR-regulated gene product. Importantly, the lack of ChIP enrichment was not due to a lack of RIN protein accumulation in the Cnr mutant as western-blot analysis of Cnr mutant tissue clearly demonstrated the presence of RIN protein in the samples used for ChIP ( Fig. 4A). The level of RIN protein accumulation in Cnr, although lower than in wild-type fruit, is likely sufficient to confer ripening activity in that a comparable level of RIN protein accumulation is observed in the 35S::RIN complementation lines that are able to ripen ( Fig. 1A; Vrebalov et al., 2002). Eriksson et al. (2004) reported that PG2a, Exp, ACO1, E8, E4, and PSY1 expression are all down-regulated in the Cnr mutant consistent with the lack of RIN binding observed in Figure 4B.

Two-Hybrid Screen Analysis Fails to Recover CNR But Does Capture Other Known Fruit Development and Ripening Regulators

To identify proteins that interact with RIN and thus indirectly ask whether the RIN and CNR proteins could directly interact with each other we performed a two-hybrid screen using reagents and procedures from Hybrigenics Inc. In summary, when the RIN protein was deployed as bait against a wild-type tomato fruit prey library, the primary proteins that interacted were almost all MADS-box proteins and included MPB7, TAGL1, TAG1, and TDR4 ( Table I). These interacting proteins are known to be associated with fruit development or ripening ( Pnueli et al., 1994; Lozano et al., 2009; Vrebalov et al., 2009). Importantly, CNR was not recovered as a significant RIN interacting protein ( Table I), suggesting that CNR's influence on RIN-binding activity may not be mediated by a direct interaction between the two proteins. We did not perform a two-hybrid assay with RIN and CNR as prey and bait to directly test this question.

RIN Regulation of Other Transcription Factors

Recent efforts have identified a number of transcription factors from multiple gene families (MADS-box, NAC, HB-zip, APETALA2, SBP) that are necessary for broad fruit ripening effects. We show here that RIN interacts with the promoters of several of these transcription factors influencing the major ripening processes and phenotypes of tomato, demonstrating the central role played by RIN in this regulatory process. The observed binding of RIN to the NOR promoter is interesting given the similar and strong ripening inhibition conferred by both mutations, suggesting interaction between the RIN and NOR genes. Regulation of a NAC domain transcription factor by a MADS-box protein was previously reported by Sablowski and Meyerowitz (1998) who showed that the floral identity dimer AP3/PI directly regulates the NAC domain protein, NAP. Our result suggests that the binding of RIN to the NOR could be required to promote NOR expression. Our result also suggests that a positive feedback loop is involved in RIN regulation, since an enrichment of the RIN promoter was observed. Several other examples of MADS regulatory feedback loops have been described ( Schwarz-Sommer et al., 1992; Tröbner et al., 1992; Hill et al., 1998). Arabidopsis AGL15 was shown to have a direct inhibitory effect on its own expression in embryonic tissue ( Heck et al., 1995). Tilly et al. (1998) showed that the expression of AP3, a B-class floral identity MADS-box gene, is directly influenced by the binding of the AP3-PI dimer to one or more CArG boxes present in its promoter. PI gene expression is similarly controlled by the AP3-PI dimer but does not possess a CArG box motif in its promoter ( Chen et al., 2000). This latter observation could indicate that the AP3-PI dimer is recruited to the promoter of PI through another DNA-binding protein, or could result from indirect regulation. Our results suggests that RIN may also operate in such a regulatory loop and further interaction with ethylene synthesis gene and regulatory promoters (see below) may explain the autocatalytic production of ethylene occurring during climacteric ripening ( Barry et al., 2000). Another interesting observation is the binding of RIN to the CNR promoter. The SBP family is known to be involved in the regulation of the SQUAMOSA family of MADS-box proteins ( Klein et al., 1996); however, little is known regarding their own regulation at the transcriptional level. Our results suggest that RIN, a MADS-box protein, may be involved in CNR regulation.

RIN and Ethylene Regulation

A hallmark of climacteric fruit ripening is the production of elevated ethylene at the onset of ripening. rin fruits are unable to produce climacteric ethylene, hence our interest in testing the binding of RIN to ethylene-producing genes. Interestingly, both the ACS2 and ACS4 gene promoters show enrichment following RIN ChIP, indicating that RIN associates with their respective promoters. ACS2 and ACS4 have previously been reported to be bound by RIN by Ito et al. (2008) and Fujisawa et al. (2011), respectively. The ACO1 promoter was not significantly bound by RIN in our assay. A regulatory link might however be postulated between RIN and ACO1 expression since RIN binds to the promoter of HB1. HB1 has been shown to interact with the promoter of ACO1 and be necessary for its expression during fruit ripening ( Lin et al., 2008). Our results suggest that RIN binding to the promoter of HB1 could indirectly influence ACO1 expression.

Other loci of components of the ethylene-signaling cascade also associate with RIN, including the ethylene receptor NR. Ito et al. (2008) and Fujisawa et al. (2011) also tested interaction between RIN and the NR promoter but failed to detect a significant enrichment following ChIP. This discrepancy might be due to a difference in the promoter region tested. The ChIP primers used by Ito et al. (2008) and Fujisawa et al. (2011) fall inside a large intron (2.35 kb) located between the 5′-untranslated region coding exon and the ATG coding exon of the NR gene. We selected our ChIP primers in the 3-kb region located upstream of the first exon of the gene. Together these results suggest that RIN association with the NR gene is mediated by its interaction with the genomic region located upstream of the first exon of the gene and not by interaction with putative CArG boxes in the intron located upstream of the ATG. Interestingly, the promoter of the NR gene possesses two putative CArG box elements. Further studies are needed to determine if these motifs are involved in RIN recruitment to the promoter.

CNR-Dependent RIN-Binding Activity

We showed that RIN-binding activity was greatly diminished in the Cnr mutant background ( Fig. 4). This observation suggests that RIN may not engage in direct DNA binding with its target promoter but might be recruited to them through the DNA-binding ability of other members of a complex. No direct interaction between RIN and CNR proteins could be detected by two-hybrid assay, however we cannot exclude the possibility that CNR and RIN are part of the same protein complex in vivo and that the absence of CNR from this complex prevents its recruitment to target loci. Alternatively, CNR could be required for the expression of a cofactor needed for proper DNA-binding activity of the RIN-containing complex. CNR is a member of the SBP family of transcription factors ( Klein et al., 1996) known to regulate the transcription of certain MADS-box genes. One possible candidate for bridging CNR and RIN activity is the MADS-box gene TDR4. As reported here, TDR4 protein is able to interact directly with RIN in a two-hybrid assay and TDR4 gene expression is highly diminished in the Cnr mutant ( Eriksson et al., 2004). Although a direct interaction between CNR and the TDR4 promoter has yet to be reported, it is interesting to note that TDR4 belongs to the SQUAMOSA class of MADS-box gene, a class of gene known to be regulated by SBP-box proteins, of which CNR is a member. Other potential partners of RIN were uncovered by a two-hybrid screen for all available interacting partners in maturing fruit ( Table I), including the recently described MADS-box gene TAGL1 ( Itkin et al., 2009; Vrebalov et al., 2009). The expression of this gene is independent from RIN and its suppression by RNAi produces a ripening phenotype similar to that of RIN-silenced lines ( Vrebalov et al., 2002, 2009). Furthermore, Itkin et al. (2009) showed that TAGL1 interacts directly with the ACS2 promoter, a gene also bound by RIN ( Ito et al., 2008).

RIN-Binding Motif

Previous studies have reported the ability of RIN to bind to CArG motif in vitro and that a number of promoters that are bound by RIN appear to be enriched in CArG sites ( Ito et al., 2004; Fujisawa et al., 2011). We analyzed the promoter region (3-kb upstream of the transcription start site) of the genes used in this study for the presence of putative CArG boxes defined as the consensus sequence C(C/T)(A/T) ₆(A/G)G. All but two promoters possesses one or more CArG boxes (data not shown). The two promoters for which no CArG box could be detected are the CNR and the EXP1 promoters, both of which associate with RIN in our ChIP assay. Relaxing the parameter of the search to allow one mismatch in the CArG box consensus sequence allows the identification of two additional motifs in EXP1 but not CNR promoters. The promoters of ACO1 and PDS also possess CArG boxes sequences but fail to associate with RIN in vivo. Based on these results, it is not clear whether RIN association to its target promoter is solely mediated by CArG boxes or if other cis-elements are required for specificity of recruitment. A search for other common motifs using the MEME software (Multiple Em for Motif Elicitation, http://meme.nbcr.net/meme/intro.html) did not reveal any motif significantly overrepresented in the ChIPed promoters. More than one motif could be involved in the recruitment of the RIN complex, a mechanism that has previously been described for other transcription factors with a large number of target genes ( Chakravarthy et al., 2003; Kaufmann et al., 2009). Although this work has focused on the ability of RIN to bind to the 5′ promoter region of ripening genes, it would be of interest in future studies to determine if RIN could also be recruited to other regions of the gene (coding, 3′ region) especially in the cases of genes such as PDS where RIN upstream binding has not been detected.

Significance of RIN Binding

Our ChIP results suggest that RIN binds to a wide variety and large number of ripening-associated genes spanning all major classes of ripening pathways. Recent work by others have provided data concerning the number of binding sites of transcription factors on a genomic scale using ChIP-sequencing technology, and have shown that transcription factors generally bind to a very large number of target genes. For example, the Arabidopsis SEP3 protein (a close homolog of the RIN protein) was shown to bind to about 4,000 sites throughout the genome ( Kaufmann et al., 2009). Interestingly, only a fraction of the bound regions contained a typical CArG box consensus motif, while many other motifs associated with other known transcription factors were also enriched. Similarly, Zheng et al. (2009) showed that the MADS-box protein AGL15 was bound to 2,000 sites, only 64% of which had a clearly defined CArG cis-element. The HY5 protein involved in regulating light signal transduction was also shown to bind to more than 3,000 sites ( Lee et al., 2007). Considering these studies, the ability of RIN to bind to numerous ripening-associated genes is not unexpected, though the meaning of such global binding remains unclear. An interesting question raised by such a high number of bound target genes is its relation with gene expression. It is becoming clear that binding of a transcription factor to a specific promoter does not immediately activate transcription ( Wyrick and Young, 2002). Instead, it is thought that some transcription factors may bind to their targets yet will not affect transcription until other conditions are met (e.g. interaction with other transcription factors). In line with this model, the expression of only a small subset of AGL15-bound targets are influenced by the presence of AGL15 ( Zheng et al., 2009). Similarly, the expression of only 6% of HY5-bound targets are affected in a hy5 mutant ( Lee et al., 2007). Previous studies and qRT-PCR analyses presented here ( Fig. 2) clearly demonstrate that RIN is required for the regulation of most of the genes that we have identified by ChIP as target of RIN. However, it is unclear whether RIN binding alone is sufficient to affect expression. Indeed a few genes do not show a clear correlation between RIN binding and changes in gene expression. The HB1 promoter is bound by RIN in our ChIP assay but its expression decreases during normal ripening and is elevated in post-BK rin mutant fruits. This observation may indicate that alternative regulators operate in the absence of RIN. We also note that ACO1 doesn't seem to be bound by RIN even if its expression increases during ripening. These discrepancies highlight the fact that RIN binding does not necessarily result in a change in gene expression and that other cofactors might be required for efficient expression of RIN-bound targets (for example, RIN itself presumably responds to additional regulators beyond the product of its own transcription/translation as evidenced in Fig. 4 where RIN binding is absent in the Cnr mutant but RIN protein still accumulates to a reduced level). RIN is a member of the SEP3 clade of MADS-box proteins that are known to be involved in the bridging of other MADS-box proteins to form higher-order complexes involved in regulating flower development (ABCE model; Robles and Pelaz, 2005; Immink et al., 2009). RIN activity could similarly be influenced by the formation of transcriptional complexes through protein-protein interactions with other transcriptional regulators such as those recovered in the two-hybrid results presented here ( Table I).

Our analysis expands the work of Ito et al. (2008) and Fujisawa et al. (2011) to demonstrate that RIN interacts not just with cell wall metabolism and ethylene synthesis promoters but also ethylene signaling, ethylene response, and carotenoid synthesis genes in addition to promoters of other ripening-related transcription factors. In the specific cases of ethylene and carotenoid biosynthesis RIN interacts with the promoters of the rate-limiting steps in each pathway (ACS and PSY, respectively). Furthermore, demonstration of CNR necessity for RIN promoter binding yet absent evidence for direct RIN-CNR interaction provides an initial foundational insight on which to expand our understanding of transcriptional ripening control.

Plants

Wild type and rin ( rin/ rin) mutant tomato ( Solanum lycopersicum 'Ac') were grown under normal greenhouse condition until maturity. Fruits were staged based on the number of days from anthesis to BK stage as defined by the detection of orange color at the base of wild-type fruits. Age of the nonripening cultivars rin and Cnr were calculated based on DPA using wild type as a reference (hence BK stage in rin correspond to fruits having the same age as wild-type BK fruits).

Constructs

The construct pET-RIN-KC was obtained by PCR amplification of pET-RIN using primers RIN KC-F (5′-TATAGGTACCGGTGAGGATTTGGGACAATTG-3′) and pET28a R (5′-TATAGGTACCCATTTGCTGTCCACCAGTC-3′), digesting with KpnI and DpnI, and ligating. pET-RIN was obtain by PCR, amplifying full-length RIN cDNA using primers RIN F (5′-TTTTGGATCCGAATTCATGGGTAGAGGGA-AAGTAG-3′) and RIN R (5′-TTTTCTCGAGTCAAAGCATCCATCCAGGTA-CAAC-3′), digesting with EcoRI and Xho I restriction enzymes, and cloning into pET28a vector (Novagen) previously digested with the same enzymes.

Recombinant Protein Purification and Antibodies Production

Escherichia coli BL21 star (DE3; 2TInvitrogen2T) cells containing pETRIN-KC were grown in Luria-Bertani broth at 30°C overnight. The next day a fresh Luria-Bertani broth culture was inoculated with the overnight culture diluted to 0.1 ODR ₆₀₀R and grown for 3 h at 30°C. Isopropylthio-β-galactoside was then added to a final concentration of 1 mm and the culture incubated for another 3 h at 30°C. The cells were pelleted by centrifugation and resuspended in TALON equilibration buffer 1× pH 8 (Clontech) containing 2 mg/mL of lysozyme. Cells were lysed by sonication on ice using a Branson 450 sonicator (settings: power 4.5, duty 50%). The lysate was centrifuged at 14,000 g for 10 min at 4°C and the supernatant discarded. The pellet was solubilized in TALON equilibration buffer 1× pH 8 containing 6 m guanidine. Purification of the HIS-tagged RIN-KC protein was performed according to the TALON His batch/gravity-flow column purification protocol (Clontech). One milligram of purified His-RIN-KC protein, quantified using the bicinchoninic acid assay (Pierce), was sent to Covance Research Products for injection into rabbits to raise antibodies.

Protein Extraction from Fruit

Tomato (cv Ac) wild type and rin fruits at different stages of development were frozen and ground in liquid nitrogen. Proteins were isolated from ground tissue using the protocol described by Wang et al. (2006). Protein was quantified using the bicinchoninic acid assay (Pierce).

Western Blot

Twenty-five-microgram aliquots of protein extracts were separated in SDS-PAGE gels and transferred to a nitrocellulose membrane using standard procedures. Immunoblotting analyses were performed using the rabbit polyclonal RIN antibodies at a 1:1,000 dilution and 1:100,000 anti-rabbit HRP secondary antibodies (Sigma).

ChIP

ChIP was adapted from Fiil et al. (2008, CSH protocol), Manzara et al. (1991), and Nelson et al. (2006). For chromatin cross-linking, tomato fruit pericarp tissue was diced and place in a 50-mL Falcon tube filled with MC buffer (10 mm KHPOR ₄R pH 7, 50 mm NaCl, 0.1 m Suc, 1% formaldehyde). Vacuum was applied for 10 min at a time for four times (5-min rest between vacuum applications) and one additional time in presence of 0.125 m Gly to stop the cross-linking. Tissue was then rinsed with water, frozen in liquid nitrogen, and ground with mortar and pestle. For each ChIP, 3 g of the cross-linked tissue was resuspended for 30 min at 4°C in 45 mL of buffer 1 (0.4 m Suc, 10 mm Tris pH 8, 5 mm β-mercapto-ethanol [BME], 1× plant protease inhibitors [Sigma]), filtered through two layers of Miracloth (Calbiochem), and centrifuged for 20 min at 3,000 g. Pellets were resuspended in 1 mL of buffer 2 (0.25 m Suc, 10 mm Tris pH 8, 10 mm MgClR ₂R, 1% Triton X-100, 5 mm BME, 1× plant protease inhibitor [Sigma]), transferred to a 1-mL eppendorf, and centrifuged for 10 min at 12,000 g. Pellets were resuspended in 300 μL of buffer 3 (1.7 m Suc, 10 mm Tris pH 8, 2 mm MgClR ₂R, 0.15% Triton X-100, 5 mm BME, 1× plant protease inhibitors), carefully layered on top of 1.5-mL of buffer 3 (in a 2-mL tube), and centrifuged for 60 min at 16,000 g. The resulting pellet was resuspended in 1 mL of freezing buffer (100 mm NaCl, 50 mm HEPES pH 7.6, 25% glycerol, 1 mm EDTA, 5 mm BME, protease inhibitor), frozen in liquid nitrogen, and thawed on ice. A total of 250 μL of lysing buffer (2.5 m NaCl, 50 mm HEPES pH 7.6, 1 mm EDTA, 5 mm BME) was added to the resupsended pellet and incubated for 30 min at 4°C. The solution was then sonicated on ice (Branson Sonifier 450) for 10 s at a time (duty 15%, power 3) for a total of 40 s (30 s on ice between sonication), centrifuged 10 min at 16,000 g, and the chromatin-containing supernatant was transferred to a 2-mL tube. Supernatant was diluted 2-fold with 50 mm Tris-Cl pH 8, 1 mm EDTA, 0.1% Triton X-100, and incubated for 1 h at 4°C on a rotating wheel with 25 μL of preimmune serum and 40 μL of blocked protein A sepharose beads (GE Healthcare), blocked with 10 μg/mL of bovine serum albumin and 10 μg/mL of salmon sperm DNA. The mixture was centrifuged for 10 min at 16,000 g and the supernatant separate in three tubes for INPUT, preimmune, and RIN ChIP treatments. ChIP was performed on the preimmune and RIN ChIP samples by incubation with 25 μL of blocked proteinA sepharose and 3 μL of rabbit preimmune serum or RIN antibodies serum, respectively, for 16 h at 4°C on a rotating wheel. Samples were centrifuged 2 min at 2,000 g and the supernatant discarded. The sepharose beads were then washed five times with washing buffer (150 mm NaCl, 50 mm Tris pH 8, 1 mm EDTA, 1% Triton X-100) by rotation for 10 min at room temperature followed by 2-min centrifugation at 2,000 g. After the last wash was removed, 100 μL of 10% Chelex resin solution (BioRad) was added to the beads and the INPUT sample and boiled for 10 min. After cooling at room temperature, samples were incubated for 45 min at 55°C with 20 μg/μL of proteinase K. Samples were then boiled for 10 min and centrifuged for 10 min at 16,000 g (4°C) and the supernatant recovered. An additional 100 μL of water was added to each tube, vortexed, centrifuged, and the supernatant was then pooled with the previous supernatant solution. Samples were further purified using a Qiagen PCR purification column (Qiagen) following the manufacturer's instructions, except that the final elution was 100 μL.

qPCR

qPCR was performed using SYBR green on an AB 7900 real-time PCR platform following the manufacturer's instructions. Briefly, 2 μL of gDNA sample obtained as described above was mixed with 5 μL of SYBR Green and 300 nm of promoter-specific primer. Relative-fold enrichments are calculated by dividing the amount of gene-specific amplification by 18S amplification. Primers used in the qPCR reaction are listed in Supplemental Table S1. At least three biological replicates of each data point were used to calculate statistical significance of the enrichment. Error bars represent the se calculated from those samples.

qRT-PCR

RNA was extracted from fruit tissue at different stages of development (days after anthesis) using the plant RNA kit (Invitrogen) and following the manufacturer's instructions. RNA was then digested with RQ1 DNase (Promega), and further purified on Qiagen RNA purification columns. qRT-PCR was performed using SYBR Green on the AB7900 using 18S as the internal control. Gene-specific primers used in the qRT-PCR assay are listed in Supplemental Table S1. ses were calculated based on a minimum of three biological replicates.

Two-Hybrid

The yeast-two-hybrid assay was performed by Hybrigenics Inc ( www.hybrigenics.com) using the RIN protein (amino acid 1 to amino acid 185) as bait to screen a cDNA prey library derived from wild-type (cv Ac) fruits of stages from immature through red ripe (BK + 10). Calculation of predicted biological score (PBS) has been described previously ( Rain et al., 2001; Formstecher et al., 2005).

View Abstract

# data[].attributes.attachment_link.title
The Tomato MADS-Box Transcription Factor RIPENING INHIBITOR Interacts with Promoters Involved in Numerous Ripening Processes in a COLORLESS NONRIPENING-Dependent Manner

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Fruit ripening is a complex developmental process responsible for the transformation of the seed-containing organ into a tissue attractive to seed dispersers and agricultural consumers. The coordinated regulation of the different biochemical pathways necessary to achieve this change receives considerable research attention.

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Presenters :Kirstin Patterson
& Lisa O'Dowd

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Large-Scale Identification of RIN Binding Sites and Potential Direct RIN Target Genes by ChIP-chip

To identify, at the large scale, direct RIN target genes whose promoters are bound by RIN, we performed a ChIP-chip analysis using anti-RIN antibodies and a Roche NimbleGen custom microarray carrying probes designed for the putative promoters (2-kb upstream regions from the translation initiation sites) of the 35,802 predicted genes provided by the International Tomato Annotation Group (ITAG2). The chromatin for ChIP was prepared from wild-type tomato fruits at the ripening stage and immunoprecipitated with anti-RIN antibodies. The ChIPed and input (no ChIP) DNAs from three biologically independent preparations were amplified by random priming to prepare enough DNA for the microarray analysis. The quality of the ChIPed DNAs was confirmed by quantitative ChIP-PCR ( qChIP-PCR) testing for enrichment of a known RIN binding sequence, the promoter region of the gene for 1-aminocyclopropane-1-carboxylic acid synthase ( ACS2; Ito et al., 2008; Fujisawa et al., 2011) (see Supplemental Figure 1 online). We then hybridized the ChIPed and input DNAs to the NimbleGen microarrays. In each experiment, we calculated the log ₂-scale fold change ( FC) values of the ChIPed DNA relative to the input DNA for each probe and detected ChIP-chip peak regions, which were defined by consecutively arranged probes with high FC at statistically significant levels (false discovery rate [ FDR] ≤ 0.05) (for more details, see Methods). We regarded a ChIP-chip peak region that was detected in at least two of the three independent experiments as a RIN binding site. Using these criteria, we identified 1046 RIN binding sites, each of which was assigned the average FC value, ranging from 0.9 to 3.1 (corresponding to 1.9- to 8.6-fold enrichment), as a peak score (see Supplemental Table 1 online). The average width of these sites was 469 bp. Of the sites, 1002 were mapped in silico onto the 12 tomato chromosomes ( Figure 1), whereas the remaining sites were mapped onto chromosome 0 (the Solanaceae Genomics Network [ http://solgenomics.net] identifier Solyc00), which is an artificial pseudomolecule composed of scaffolds that could not be placed on either of the 12 tomato chromosomes ( Tomato Genome Consortium, 2012). Using the information about genomic positions of the RIN binding sites, we identified 1200 genes as potential direct RIN targets that carried one or more RIN binding sites in the transcriptional regulatory region (2-kb upstream putative promoter) or in other gene regions, such as exons or introns, or downstream from the translation termination site (1 kb), in the region of overlap with the promoter region of a neighboring gene ( Table 1; see Supplemental Data Set 1 online).

Detection of Conserved DNA Sequences in the RIN Binding Sites

Previously, our in vitro analysis revealed that RIN binds to DNA sequences known as the C-A/T-rich-G (CArG) box, which is the general target of MADS box proteins ( Ito et al., 2008). To detect conserved DNA sequences in the RIN binding sites in vivo, we first searched the RIN binding sites for CArG-box sequences. As summarized in Table 1, we found that a total of 620 (59%) RIN binding sites contained a DNA sequence that met the patterns for any of the possible three RIN biding CArG-box motifs, C(C/T)(A/T) ₆(A/G)G, C(A/T) ₈G, or C(C/T)(A/T)G(A/T) ₄(A/G)G ( Ito et al., 2008; Fujisawa et al., 2011). The 620 sites were located in the 2-kb promoters or other gene regions of 718 potential target genes (see Supplemental Data Set 1 online). The frequency of occurrence of the motif, C(C/T)(A/T) ₆(A/G)G, which seems to be preferentially bound by RIN ( Ito et al., 2008), was significantly higher in the RIN binding sites than in the whole set of 2-kb promoters (P < 0.001 by χ ² test), whereas the frequency of the other two motifs was not (see Supplemental Table 2 online). In the 620 RIN binding sites, the CArG boxes tended to be located closer to the centers ( Figure 2A). To predict the in vivo RIN binding CArG-box motif, we cataloged and analyzed CArG-box sequences that lie in the 2-kb promoters along with the strands encoding the genes. The analysis of the collected 1063 strand-oriented CArG-box sequences yielded a 10-bp motif consensus sequence, C(T/A/C)(A/T) ₆(A/T/G)G ( Figure 2B). Unlike a previous in vitro assay for RIN binding ( Ito et al., 2008), no conservation was observed in the flanking regions upstream and downstream of the 10-bp core.

Furthermore, we searched the RIN binding sites for known plant TF binding sequences, based on the motifs deposited in the Arabidopsis Gene Regulatory Information Server database ( Davuluri et al., 2003). Sixteen out of the 87 examined motifs exhibited a significantly (P < 0.001) higher frequency of occurrence in the RIN binding sites than that expected from the 2-kb promoters of the set of all predicted genes (see Supplemental Table 2 online). In addition to the CArG-box-type motifs, a remarkably high frequency of occurrence was observed for the GCC-box motif (GCCGCC), although the numbers of these motifs in the RIN binding sites were much fewer than the CArG boxes. The GCC-box motif is a target of the AP2/ERF TF family proteins ( Stockinger et al., 1997; Shinozaki and Yamaguchi-Shinozaki, 2000; Hao et al., 2002). Unfortunately, we could not detect any significantly conserved DNA motifs, even the CArG-box, in the RIN binding sites using authentic programs for de novo motif discovery.

Combined ChIP-chip and Transcriptome Analysis for Identification of RIN Direct Target Genes Whose Expression Was Positively or Negatively Regulated by RIN during Ripening

In our earlier study, to select genes with RIN-dependent expression, we calculated the gene expression FC ratio of the ripening (pink coloring [ P]) stage relative to the preripening (mature green [ G]) stage (FC WT for the wild type and FC rin for the rin mutant; for more details, see Methods) from a microarray data set for tomato fruit ripening ( Fujisawa et al., 2012). We also calculated the expression change score ( ECS), defined as the ratio of FC WT relative to FC rin, and used the ECS to evaluate the dependency of gene expression on the wild-type RIN gene.

In this study, we reevaluated our microarray expression data, using lower FC and ECS stringency thresholds, to identify RIN-dependent targets more broadly. Using a threshold of FC WT > 2 (q-value < 0.05) and ECS > 2, we selected 1000 of the 17,307 genes with valid expression data as genes positively regulated by RIN. We next examined whether the set of genes positively regulated by RIN overlapped with the identified RIN direct binding targets. Of the 1200 potential targets, 851 were covered in the microarray expression data, but the remaining 349 were not because there were no probes specific to these loci in the microarray. Of the potential targets, 137 were included in the set of positively regulated genes ( Figure 3A; see Supplemental Data Set 2 online). Similarly, we selected 1948 genes negatively regulated by RIN using a threshold of FC WT < 0.5 (q-value < 0.05) and ECS < 0.5. Of the potential targets, 104 were included in the negatively regulated gene set and then identified as negatively regulated direct targets of RIN ( Figures 3A and 3B; see Supplemental Data Set 2 online). The remaining 863 positively regulated and 1844 negatively regulated genes were not selected as direct RIN targets and may be regulated by an indirect effect of RIN. Furthermore, 610 of the 851 potential targets were also not selected despite RIN binding to a site close to them because their expression changes did not meet the criteria for selection and thus are likely not transcriptionally regulated by RIN.

Detection of Known and Novel Ripening-Related Direct RIN Targets

To test whether our combined ChIP-chip and expression analysis was capable of detecting RIN targets, we first determined whether known targets of RIN were identified in the analysis. The positively and negatively regulated subsets of direct RIN targets included 11 out of 24 known RIN target genes ( Ito et al., 2008; Fujisawa et al., 2011, 2012; Martel et al., 2011; Qin et al., 2012) (see Supplemental Data Set 2 online). Five more known direct RIN targets, genes for β-d-xylosidase ( XYL1), alcohol dehydrogenase ( ADH2), phytoene synthase ( PSY1), TDR4, and HB-1, were also included in the set of identified genes when ChIP-chip peak detection was conducted at a lower stringency ( FDR ≤ 0.2) (see Supplemental Figure 2 online). Of the 11 known targets, nine were analyzed by qChIP-PCR in our earlier studies ( Fujisawa et al., 2011, 2012). We found overlaps between the RIN binding sites and the already confirmed RIN binding CArG boxes in five (genes for 1-aminocyclopropane-1-carboxylic acid synthase [ ACS4], an endo-β-1,4-glucanase [ Cel2], an osmotin-like protein [ NP24], CNR, and RIN itself) of the nine targets ( Figure 4A). In the remaining targets detected, the RIN binding sites did not overlap with but were located near (within 400 bp) the enriched CArG boxes already confirmed ( Figure 4A). The results indicated that our ChIP-chip would identify successfully a large portion of the direct RIN targets in the tomato genome.

The set of genes identified as direct RIN targets in this study did not include more than half of known direct RIN targets due to the following reasons. A known target, a gene for hydroperoxide lyase ( HPL), was identified as a potential direct RIN target by our ChIP-chip, but its expression levels were not altered enough in either the wild type or rin mutant fruits to meet the criteria for selection. In addition, another known RIN target, a gene for Never-ripe ( NR; also called ETR3), was also not detected in this study because our ChIP-chip microarray did not cover the RIN binding site likely located beyond 2 kb upstream of the first exon of NR ( Martel et al., 2011). Our failure to detect the other known targets might be caused by a difference in sensitivity between quantitative PCR-based assays and microarray-based ChIP assays (as mentioned in the next section), or by the ChIP-chip microarray, if it does not cover the entire functional promoter regions.

Based on gene annotations, on the other hand, we found that the positively regulated RIN targets included two important ripening-related genes for senescence-inducible chloroplast stay-green protein 1 ( SGR1) and AP2a ( Figure 4B). By contrast, many ripening-associated genes (e.g., genes for 1-aminocyclopropane-1-carboxylic acid oxidase [ ACO1], 1-d-deoxyxylulose 5-phosphate synthase [ DXS], and geranylgeranyl pyrophosphate synthase [ GGPS2]; Fujisawa et al., 2012) were not identified as direct RIN targets, although their expression patterns are RIN dependent. It is likely that these genes are not directly regulated by RIN during ripening but are regulated by other factors, such as ethylene signaling or RIN-regulated TFs.

Verification of RIN Binding to the Sites Detected by ChIP-chip

We used diagnostic qChIP-PCR to verify RIN binding to the sites detected by ChIP-chip. As representatives, we monitored the enrichment of RIN binding sites that showed the 10 highest ChIP-chip peak scores among the sites mapped onto the 12 tomato chromosomes (excluding the sites of chromosome 0 [Solyc00]). The highest peak score among those of the sites mapped onto the 12 chromosomes was 2.56 (see Supplemental Data Set 3 online). The qChIP-PCR amplified the DNA fragments of nine of the 10 sites but not that of the remaining one, likely due to the difficulty of PCR amplification of the site. All of the amplified sites showed enrichment of close to or more than twofold by qChIP-PCR (see Supplemental Figure 3 online). A positive correlation between ChIP-chip peak scores and qChIP-PCR enrichments was confirmed (Pearson's r = 0.78), indicating that our ChIP-chip results are reliable. The scores determined by ChIP-chip were lower than the enrichment levels measured by qChIP-PCR; this may be attributable to the differences in dynamic range between the two analyses. This lower sensitivity of ChIP-chip compared with qChIP-PCR might also cause the failure to identify some known targets in the set of direct RIN targets by ChIP-chip, as described above.

Furthermore, we verified RIN binding to the sites in the promoters of the negatively regulated subset. As described above, the combined ChIP-chip and transcriptome analysis detected RIN binding sites in the promoters or other gene regions of both positively and negatively regulated RIN targets, suggesting both activator and repressor roles of RIN. Whereas many positive targets have been reported previously ( Ito et al., 2008; Fujisawa et al., 2011, 2012; Martel et al., 2011; Qin et al., 2012), HB-1 is the only reported negatively regulated target ( Martel et al., 2011). To verify RIN binding to the sites of the negatively regulated subset, we used diagnostic qChIP-PCR to monitor the enrichment level of RIN binding sites in the promoters of three randomly selected negatively regulated direct RIN targets. All sites examined were indeed enriched in the ChIPed DNA relative to the input at similar levels to those detected by ChIP-chip (see Supplemental Figures 4A and 4B online). Taken together, our ChIP-chip results were generally reproducible, regardless of positive or negative regulation by RIN.

Predicted Functions of the Direct RIN Target Genes

To provide an overview of the functional roles of RIN, we next estimated the functions of the direct RIN targets by searching for similarity to Arabidopsis thaliana proteins and then classified these proteins based on their predicted biological pathways. We found that the products of the direct RIN targets were involved in a total of 67 biological pathways, especially rich in "metabolic pathways," including "biosynthesis of secondary metabolites" (see Supplemental Data Set 4 online). We also classified the direct RIN targets using the Munich Information Center for Protein Sequences ( MIPS) functional catalog database based on the similarity to the Arabidopsis genes (see Supplemental Data Set 5 online). As found for the pathway search described above, the MIPS classification also showed that RIN targets were implicated in a wide range of fruit ripening processes. In particular, the classification of the positively regulated subset showed a significantly (P < 0.001 by Fisher's exact test) higher frequency of genes involved in two functional categories, "metabolism" and "systemic interaction with the environment," than the frequency in the whole genome ( Figure 5). In the "metabolism" category, genes involved in the subcategories related to Met metabolism were significantly enriched in the positively regulated subset (see Supplemental Data Set 6 online). Because Met is a precursor of ethylene, this enrichment is likely associated with ethylene production during ripening. In addition to known direct RIN targets involved in ethylene synthesis ( ACS2, ACS4, and E8), the positively regulated subset included two genes for homocysteine S-methyltransferase ( HMTs), which belong to the S-adenosyl-Met-homocysteine cycle upstream of an ethylene synthesis pathway, and a gene for 1-aminocyclopropane-1-carboxylic acid oxidase ( ACO6) catalyzing the ethylene synthesis reaction (annotated as the categories related to the Met metabolism; see Supplemental Data Sets 5 and 6 online). Furthermore, in the category of "systemic interaction with the environment," genes belonging to such subcategories as "plant defense response" and "response to wounding" were also significantly enriched in the positively regulated subset (see Supplemental Data Set 6 online). These subcategories, which were associated with various stress responses, are frequently observed in tomato fruit ripening ( Fujisawa et al., 2012). By contrast, the negatively regulated subset showed a significantly higher frequency of genes classified into the categories "interaction with the environment" and "systemic interaction with the environment" than the frequency in the whole genome ( Figure 5; see Supplemental Data Set 6 online). Both categories shared the negatively regulated genes involved in response to plant hormones such as auxin and gibberellin (see Supplemental Data Sets 5 and 6 online).

Pathway Analysis for Carotenoid Synthesis Based on the Expression Profiles of RIN Targets

Our functional estimation found that a number of the direct RIN target genes were implicated in lycopene accumulation, a process characteristic of tomato fruit ripening. To analyze how RIN participates in lycopene accumulation during ripening, we mapped the direct RIN targets and their isozyme genes with their expression profiles on the carotenoid pathway and the 2- C-methyl-d-erythritol 4-phosphate/1-deoxy-d-xylulose 5-phosphate ( MEP/DOXP) pathway (also called the nonmevalonate pathway). The MEP/DOXP pathway is one of the terpenoid backbone biosynthesis pathways and is considered to be the main route for plastidic carotenoid production during tomato fruit ripening ( Rodriguez-Concepcion and Gruissem, 1999). We found that in the MEP/DOXP pathway, a gene for 4-diphosphocytidyl-2- C-methyl-d-erythritol kinase ( ISPE; also called CDPMEK) was positively regulated by RIN in a direct manner ( Figure 6). By contrast, DXS and GGPS2 were not included in the targets of RIN but were positively regulated in a RIN-dependent manner, suggesting that DXS and GGPS2 expressions are regulated by an indirect effect of RIN ( Figure 6). Similarly, another DXS and a gene for isopentenyl pyrophosphate isomerase ( IPI) were negatively regulated by an indirect effect of RIN ( Figure 6). In the carotenoid pathway from phytoene to α- and β-carotenes, RIN positively regulates a known target, PSY1 ( Martel et al., 2011), which encodes a rate-limiting enzyme of carotenoid synthesis ( Fraser et al., 2002), although our ChIP-chip analysis surveying the 2-kb promoter regions could not detect any significantly enriched peaks ( FDR ≤ 0.05) in the PSY1 promoter. Also, RIN positively regulated genes for ζ-carotene isomerase ( Z-ISO) and carotenoid isomerase ( CRTISO) in a direct manner. In addition, RIN positively regulated genes for another phytoene synthase ( PSY2) and ζ-carotene desaturase ( ZDS) and negatively regulated genes for lycopene β-cyclase ( CrtL-b) and lycopene ϵ-cyclase ( CrtL-e) by an indirect effect ( Figure 6).

Expression Analysis of the TF Genes Directly Regulated by RIN

The positively and negatively regulated subsets of the direct RIN targets contained 14 and 17 TF genes, respectively, which were assigned a MIPS subcategory of "transcription" (see Supplemental Data Set 7 online). MADS box, NAC, SBP, and bZIP family genes were found in the positively regulated subset but not in the negatively regulated subset. By contrast, IAA and WRKY family genes were found in the negatively regulated subset but not in the positively regulated subset. Genes belonging to the AP2/ERF, bHLH, GRAS, HB, and zinc-finger families were found in both subsets, but the numbers of AP2/ERF family genes in the negatively regulated subset were relatively larger than those in the positively regulated subset. Apart from the known ripening regulator genes, such as RIN, NOR, CNR, and AP2a, there were no TF genes whose roles in fruit ripening have been characterized. In this study, we focused on the 14 positively regulated TF genes ( Table 2) because they were expected to be associated with phenomena activated during ripening. Of the 14 TF genes, only four ( CNR, NOR, a SCL32-like GRAS gene [Solyc07g052960], and RIN itself) were identified previously as direct RIN targets; the remaining 10 genes were newly identified as direct RIN targets ( Table 2). Expression analysis by quantitative RT-PCR ( qRT-PCR) revealed that nine targets (the RIN binding sites by ChIP-chip for the AP2a promoter are shown in Figure 4; the binding sites for the other eight targets are shown in Supplemental Figure 5 online) showed increased expression levels in the wild-type fruits for at least one of the ripening ( P and red ripe [R]) stages relative to the levels at the preripening (G) stage. The increases were statistically significant (P value < 0.05) compared with the changes in the rin mutant fruits at the same age as observed in RIN and NOR ( Figure 7). By contrast, the expression level of Solyc02g085630 during ripening was not increased in the wild-type fruits but decreased gradually in the rin mutant ( Figure 7).

Ethylene Responsiveness of Positively Regulated Direct RIN Target TFs

To examine ethylene responsiveness of the TF genes that were identified as positively regulated direct targets of RIN in the previous and current studies, we monitored their expression levels in tomato fruits treated with an ethylene receptor inhibitor, 1-methylcyclopropene ( 1-MCP). Tomato fruits harvested at the breaker stage were treated with 1-MCP for 4 d and subjected to a qRT-PCR analysis of the nine novel and five known target TF genes. The treatment severely inhibited color development of tomato fruits during ripening ( Figure 8A). The qRT-PCR confirmed that expression of an ethylene-inducible gene, ACS2, was suppressed in the 1-MCP-treated fruits compared with the control ( Figure 8B), indicating the successful inhibition of ethylene signaling by the treatment. The qRT-PCR also showed that the treatment significantly decreased the expression of six novel target TFs and NOR, TDR4, Solyc07g052960, and RIN itself in the 1-MCP-treated tomato fruits ( Figure 8B). In particular, the treatment substantially decreased the expression levels of nine TFs to 50% or less of the control. As for RIN, its expression level was decreased to 20% of the control ( Figure 8B). By contrast, the treatment did not significantly change the expression levels of five target TFs, including CNR, whereas the level of a gene for calcium/calmodulin-regulated SR/CAMTA TF, SR3L, was increased ( Figure 8B).

Structural Features of the RIN Binding Sites Detected by ChIP-chip

Our ChIP-chip analysis showed that 59% of the RIN binding sites contained CArG-box motifs. These CArG boxes were frequently observed close to the center of the CArG box-containing sites ( Figure 2A), suggesting that these CArG boxes are possibly recognized by RIN. However, RIN binding to sites without a CArG box was also confirmed. Similar cases have been described in previous reports on ChIP-chip analyses for several types of Arabidopsis TFs ( Lee et al., 2007; Kaufmann et al., 2009; Oh et al., 2009; Zheng et al., 2009; Tao et al., 2012), but the reason for detecting such sites remains unclear. We assume that RIN may bind to non-CArG-box motif sites via an additional DNA binding factor that forms a complex with RIN. Indeed, Arabidopsis MADS box proteins interact with various types of TFs and chromatin remodeling proteins in vivo ( Smaczniak et al., 2012). Our results also showed that the RIN binding sites overrepresented a number of TF binding motifs, especially the GCC-box motif. The GCC box is recognized by ERFs ( Fujimoto et al., 2000). The overrepresentation indicates the possibility that DNA binding factors, such as ERF family proteins, may form a complex with RIN and bring RIN to the genome sites with the DNA binding factor-specific motifs.

RIN Affects a Wide Range of Fruit Ripening Processes through Transcriptional Regulation of Key Genes

Our results demonstrated that direct targets of RIN are involved in various pathways for biosynthesis, metabolism, and signaling, such as carotenoid accumulation, ethylene synthesis, and chlorophyll degradation, suggesting the contribution of RIN to a wider range of fruit ripening processes than previously known. In particular, RIN appears to regulate carotenoid biosynthesis and its upstream MEP/DOXP pathway through the direct regulation of many target genes. The role of several carotenogenesis genes targeted by RIN has been shown in the reports on their mutants ( Fray and Grierson, 1993; Isaacson et al., 2002). Lycopene accumulation in the ripening tomato fruit seems to be explained by our result ( Figure 6), demonstrating both the upregulation of the genes involved in the upstream pathway of lycopene and downregulation of the genes in the downstream lycopene cyclization to generate α- or β-carotenes, as suggested previously ( Alba et al., 2005). We demonstrate that RIN regulates these up- and downregulations directly and indirectly and thus consider that RIN acts to enhance the levels of carotenoid (lycopene in common cultivars) in the ripening tomato fruit.

Among the identified direct RIN targets, SGR1 is responsible for the tomato green-flesh mutation, which causes the production of brown fruits as the result of carotenoid accumulation and lack of chlorophyll degradation occurring simultaneously ( Barry et al., 2008). SGR1 is a tomato homolog of SGR family proteins that destabilize the light-harvesting chlorophyll binding protein complexes of the thylakoid membranes ( Park et al., 2007). Our finding that SGR1 is a direct RIN target agrees with the ripening-related expression of SGR1, as described previously ( Barry et al., 2008), suggesting that RIN regulates chlorophyll degradation during ripening via transcription of SGR1.

TF Genes Directly Targeted and Positively Regulated by RIN during Fruit Ripening

At the onset of ripening, the increase and decrease in expression of numerous ripening-associated genes occurs after the rise in RIN expression. This implies that, in addition to the direct regulation of RIN, other TFs under the control of RIN participate in transcriptional regulation. Here, we identified at least nine additional TF genes that are positively regulated during ripening as direct targets of RIN. The target TFs are expected to regulate the expression of their own targets downstream of RIN. We observed slight increases in expression levels of several target TF genes also in the rin mutant fruits, suggesting that these targets are affected by other developmental factors in addition to RIN. Several of the RIN targets have been reported to be associated with fruit ripening. For example, AP2a, a member of the AP2/ERF family, is a negative regulator of ethylene synthesis during ripening ( Chung et al., 2010; Karlova et al., 2011). Our finding that AP2a is a direct RIN target suggests that RIN plays a role in its regulation, but ethylene and other factors are also involved ( Karlova et al., 2011). Interestingly, the subsets of direct RIN targets share several genes, such as CRTISO, SGR1, and a lipoxygenase gene ( TomLoxC), with the set of AP2a-affected genes ( Chung et al., 2010; Karlova et al., 2011). This suggests that AP2a activity is required to express a subset of the direct RIN targets. Another RIN target, SR3L, which is a homolog of calcium-signaling TF genes, has been recently reported to be affected in expression by RIN and ethylene during ripening ( Yang et al., 2012). Calcium is associated with various ripening processes, such as ripening rate, respiration rate, ethylene production, and fruit softening ( Ferguson, 1984). Although the exact role of SR3L in fruit ripening remains uncertain, RIN may contribute to calcium-mediated ripening processes by regulating SR3L expression. The roles of the remaining novel RIN target TF genes, and also most of the negatively regulated TF genes, in tomato fruit ripening remain unclear. Functional analysis of the target TFs will lead to a more comprehensive understanding of the ripening regulatory mechanism.

The Interplay between RIN and Ethylene May Have a Crucial Role in the Regulation of Fruit Ripening

The analysis of ethylene responsiveness using the ethylene receptor inhibitor 1-MCP revealed that ethylene is implicated in the upregulation during ripening of at least 10 TF genes that are direct RIN targets and is especially critical for the upregulation of nine TFs, including RIN; more than half of their expression levels during ripening depend on ethylene action. This observation indicates that ethylene is required for activating RIN-mediated ripening regulation. The ethylene effect on the expression of TDR4 and AP2a is consistent with results reported previously ( Karlova et al., 2011; Osorio et al., 2011). By contrast, it is surprising that ethylene is involved in the upregulation of main ripening regulators, such as RIN and NOR, because they are believed to lie upstream of both the ethylene-dependent and -independent pathways in the fruit ripening regulatory mechanism ( Giovannoni, 2007; Lozano et al., 2009). The induction of RIN at the onset of ripening is not influenced by exogenous ethylene ( Vrebalov et al., 2002), indicating that ethylene likely affects the enhancement but not the initial induction of RIN expression. Concerning ethylene production during ripening, Yokotani et al. (2009) demonstrated that ethylene production for tomato fruit ripening is initially induced by a developmental factor and then massively increased by ethylene autocatalytic regulation. They also suggest that RIN is a possible inducer of the initial ethylene production. Other ripening regulators, such as NOR, CNR, TAGL1, and HB-1, are also involved in the regulation of ethylene levels during ripening ( Thompson et al., 1999; Giovannoni, 2004; Lin et al., 2008; Itkin et al., 2009; Vrebalov et al., 2009). In addition to this study, recent molecular and biochemical studies suggest that RIN participates in ethylene production by inducing many of the ethylene synthesis/signaling genes (e.g., ACS2 and ACS4), by upregulating NOR and CNR and by downregulating HB-1 ( Ito et al., 2008; Fujisawa et al., 2011, 2012; Martel et al., 2011; Qin et al., 2012; Zhou et al., 2012). Furthermore, TAGL1 and HB-1 may directly regulate the expression of ACS2 ( Itkin et al., 2009) and ACO1 ( Lin et al., 2008), respectively. Furthermore, ethylene production is negatively regulated by AP2a and ERF6 ( Chung et al., 2010; Karlova et al., 2011; Lee et al., 2012). Taking the observations and the existing ripening regulatory model ( Giovannoni, 2007; Lozano et al., 2009) into account, we propose a revised model for tomato fruit ripening ( Figure 9). In this model, RIN induced at the onset of ripening initiates both the production of climacteric ethylene and the ethylene-independent transcriptional regulatory pathway. The RIN-induced and subsequent autocatalytic ethylene further induces the ethylene responsive genes via the ethylene signal transduction pathway. The signaling pathway also enhances RIN expression in a positive feedback loop. We previously proposed that the expression of RIN is autoregulated ( Fujisawa et al., 2011). The two enhancement systems, the autoregulation and the ethylene-mediated positive feedback loop, may bring about a rapid and remarkable upregulation (up to a several hundred-fold increase) of RIN, leading to the subsequent rise in ethylene level and a rapid up- and downregulation of the vast number of genes associated with fruit ripening. The proper ethylene level for each ripening stage may be fine-tuned by both the positive loop regulated by RIN and the negative regulation of ethylene production by AP2a and ERF6. Thus, a robust linkage between RIN and ethylene signaling is central to the regulation of fruit ripening. The positive feedback loop requires an additional factor that is implicated in the ethylene signal transduction pathway and directly regulates RIN expression during ripening. Identifying the factor implicated directly or indirectly in the ethylene signal transduction pathway will substantially unveil the regulatory mechanism acting between RIN and ethylene during fruit ripening.

Chromatin immunoprecipitated (ChIPed) DNA was prepared using anti-RIN antibodies from input chromatin extracted from ripening tomato ( Solanum lycopersicum) fruits basically as previously described ( Ito et al., 2008; Fujisawa et al., 2011). For microarray hybridization, the ChIPed and input DNAs were amplified with the GenomePlex Complete Whole Genome Amplification kit 2 (Sigma-Aldrich) according to the manufacturer's instructions. Each of the amplified ChIPed DNA samples was labeled with Cy3 and competitively hybridized with the amplified input DNA labeled with Cy5 onto a NimbleGen custom-designed tomato gene promoter microarray (Roche NimbleGen). The 720K array had 694,802 probes (52- to 70-mers tiled throughout nonrepetitive sequences at a median spacing of 80 bp), designed from the 2-kb upstream regions (putative promoters; a total of ∼71.6 Mb) of all 35,802 tomato predicted genes provided by ITAG version 2 (ITAG2; http://solgenomics.net/genomes/Solanum_lycopersicum/index.pl) ( Tomato Genome Consortium, 2012). Sequence source for probes was a draft genome sequence (∼781 Mb) of the tomato chromosomes released by the International Tomato Genome Sequencing Consortium (version 2.31; http://solgenomics.net/about/tomato_sequencing.pl) ( Tomato Genome Consortium, 2012). Labeling of the ChIPed and input DNAs, two-color competitive hybridization, and peak detection were performed by Roche NimbleGen. Extraction of signal intensity data from scanned images of the ChIP-chip array and subsequent peak detection were performed with NimbleScan software (Roche NimbleGen). Three independent biological samples were used in separate hybridizations (including one dye swap).

Data Analysis

Normalization and peak detection were performed by Roche NimbleGen as per their protocols ( http://www.nimblegen.com/products/chip/data_guide.html). In each ChIP-chip experiment, the FC ratio of the ChIP signal value to the input signal value for each probe was converted to a log ₂ scale. The log ₂ ratio was scaled to center the data around zero by subtracting the bi-weight mean for the log ₂ ratio values for all features on the array from each log ₂ ratio value. The details of the procedure for peak detection were described by Johnson et al. (2008).

Peaks were determined using a 500-bp sliding window with a probe height cutoff. A peak was recognized if the number of probes above a specific probe height cutoff value in the window was ≥4 (or two if there were fewer than four probes per window). As the window slid along the data track, the recognized peak could extend. The probe height cutoff values were defined as a percentage of a hypothetical maximum, which is the mean of the log ₂ ratio values of the probes + 6 sd. The default value of the probe height cutoff was 90%, and the peak detection scan was repeated. For each repetition, the probe height cutoff values were decreased by 1% per detection scan, down to 15% of the theoretical maximum. The probe height cutoff value at which a peak was first recognized was used as the score of the peak. Peaks were categorized based on their scores; peaks recognized during the first detection scan were in the category of cutoff = 90%, peaks recognized during the second scan but not during the first scan were in the category of cutoff = 89%, and so on, down to cutoff = 15%. The number of peaks in each category was used in the FDR calculation.

The FDR, which represents the probability of finding a peak of similar significance by chance ( Johnson et al., 2008), was estimated for each peak using a permutation-based algorithm to find statistically significant peaks. For the FDR calculation, simulated noise data were made by the following method: The original data were scrambled (randomly permuted) and peaks were counted using the same peak detection procedure. We then computed the ratio of the average number of peaks recognized across 20 scrambled data tracks in a detection scan with a probe height cutoff to the number of peaks recognized in the original data track in the scan with the same cutoff and used the ratio as FDR for peaks that fall in the range of the cutoff.

Next, to identify reproducible RIN binding sites, the genomic start and end positions of the peaks ( FDR ≤ 0.05) were compared among three experiments, and a region that was covered by the peak in at least two experiments was searched. The region was finally noted as a RIN binding site, and the width of the region was defined as the peak width.

The enrichment of DNA fragments bound by RIN was measured by qChIP-PCR as described previously ( Fujisawa et al., 2011, 2012). To confirm the ChIPed DNA quality, a pair of oligonucleotide primers for a known RIN binding site in the ACS2 promoter ( Fujisawa et al., 2011) was used. The oligonucleotide sequences of primer pairs used for validation of the enrichment of RIN binding sites identified by ChIP-chip are listed in Supplemental Data Set 3 online. The measurements (cycle threshold [C T]) for the CArG-box sites were normalized with those for the Actin gene, which is free from RIN binding and was used for qChIP-PCR ( Ito et al., 2008; Fujisawa et al., 2011). The quantitative PCR was conducted as described below in the gene expression analyses section.

Functional Gene Annotation

A similarity search of the tomato ITAG2 predicted gene products was conducted as described previously ( Fujisawa et al., 2011) using the BLASTP program ( Altschul et al., 1997) with an e-value cutoff of <0.01 against the Arabidopsis protein database provided by The Arabidopsis Information Resource (TAIR10; http://Arabidopsis.org/). Based on the similarity, the ITAG2 genes were functionally annotated using a functional catalog database provided by MIPS (Helmholtz Zentrum München) ( http://www.helmholtz-muenchen.de/en/mips/projects/funcat/index.html) ( Ruepp et al., 2004). For each MIPS functional category, significance of enrichment of the genes in the positively or negatively regulated subsets of direct RIN targets relative to those in the tomato genome (the whole set of ITAG2 genes) was calculated by Fisher's exact test. A pathway search for direct RIN targets was conducted using the Kyoto Encyclopedia of Genes and Genomes pathway database for Arabidopsis ( http://www.genome.jp/kegg/pathway.html).

Gene Expression Analyses

To select differentially expressed genes during ripening, we analyzed a microarray data set (accession number GSE28564 in the Gene Expression Omnibus database at the National Center for Biotechnology Information) ( http://www.ncbi.nlm.nih.gov/geo/) for gene expression of the wild-type and rin mutant tomato fruits basically as reported earlier ( Fujisawa et al., 2012). In brief, the FC of the expression level of each tomato gene was calculated as the relative signal intensity of the probes in the wild type at the ripening ( P) stage to that at the preripening ( G) stage (FC WT) or in the rin mutant fruits at the same age as the wild-type P stage relative to those at the G stage (FC rin). Note that the rin mutant tomatoes harvested at the same age as the wild-type P fruit expressed the mutated RIN gene and did not normally reach the pink stage. The significance of FC WT was tested using the P value from three independent experiments estimated by the t test with the FDR control (q-value) as previously described ( Fujisawa et al., 2012). Furthermore, to select genes positively and negatively regulated by RIN, we analyzed the ECS (the ratio of FC WT relative to FC rin) ( Fujisawa et al., 2012). We finally selected genes that met the criteria of FC WT > 2 (q-value < 0.05) and ECS > 2 as positively regulated genes by RIN and genes that met the criteria of FC WT < 0.5 (q-value < 0.05) and ECS < 0.5 as negatively regulated genes by RIN.

Gene expression levels in tomato fruits were confirmed by qRT-PCR as previously described ( Fujisawa et al., 2011, 2012). Briefly, 1 μL of cDNA synthesis reaction mixture was used as template for analysis using Thunderbird SYBR qPCR mix (Toyobo). Three biological replicates were analyzed for each stage of the respective lines. A pair of primers specific to each gene was designed using the PRIMER3 program ( http://frodo.wi.mit.edu/primer3/) with the following settings: 130 to 150 bp for product size range, 22-mer for optimal primer size, and 62°C for optimal annealing temperature. The oligonucleotide sequences of gene-specific primer pairs used in this study for RIN, CNR, TDR4, and a SCL32-like GRAS gene (Solyc07g052960) were described previously ( Fujisawa et al., 2011, 2012), and for the other genes, the primer sequences are listed in Supplemental Data Set 3 online. The amplification efficiency of the primer pairs was checked using the Sequence Detection Software (version 1.3.1) included in the 7300 system (Applied Biosystems). The data were represented as FC relative to the G stage in each line calculated by the 2 −ΔΔCT method ( Livak and Schmittgen, 2001) using a primer pair for a gene encoding clathrin adaptor complexes medium subunit ( CAC) as a reference ( Expósito-Rodríguez et al., 2008). The significance of difference in FC (log ₂ scale) between the wild-type and rin mutant fruits at each P and G stage was evaluated by Student's t test.

Tomato fruits at the breaker stage were placed into an airtight 1-liter plastic container with 5 ppm of 1-MCP that was generated by dissolving 24 mg of 1-MCP-releasing powder (SmartFresh, 0.14% of active ingredient by weight; Rohm and Haas) in 25 μL of water. The treatment was conducted for 4 d in an incubator under 16-h light at 26°C and 8-h dark at 20°C conditions. The 1-MCP solution was changed every other day. After the treatment, the fruits were sliced, frozen in liquid nitrogen, and subjected to RNA isolation followed by qRT-PCR as described above. The effect of 1-MCP was confirmed by qRT-PCR using a pair of primers for the protein-coding sequence of ACS2, whose expression is suppressed by 1-MCP treatment ( Yokotani et al., 2009).

Accession Numbers

Sequence data from this article can be found in the Solanaceae Genomics Network or GenBank/EMBL databases under the following accession numbers: RIN, AF448522; CNR, DQ672601; TDR4, AY098732; Actin, U60482; and CAC, SGN-U314153. The accession numbers of the other data are shown in Supplemental Data Set 3 online. Sequence data of the ITAG2 identifiers for tomato genes from this article are shown in Supplemental Data Sets 1 to 3 online. The ChIP-chip data are MIAME compliant and have been deposited in a MIAME-compliant database (Gene Expression Omnibus accession number GSE40257) at the National Center for Biotechnology Information ( http://www.ncbi.nlm.nih.gov/geo/).

Supplemental Data

The following materials are available in the online version of this article.

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

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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.

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Abstract

Fleshy fruits have evolved to be attractive to frugivores in order to enhance seed dispersal, and have become an indispensable part of the human diet. Here we review the recent advances in the understanding of transcriptional regulation of fleshy fruit development and ripening with a focus on tomato. While aspects of fruit development are probably conserved throughout the angiosperms, including the model plant Arabidopsis thaliana, it is shown that the likely orthologues of Arabidopsis genes have distinct functions in fleshy fruits. The model for the study of fleshy fruit development is tomato, because of the availability of single gene mutants and transgenic knock-down lines. In other species, our knowledge is often incomplete or absent. Tomato fruit size and shape are co-determined by transcription factors acting during formation of the ovary. Other transcription factors play a role in fruit chloroplast formation, and upon ripening impact quality aspects such as secondary metabolite content. In tomato, the transcription factors NON-RIPENING (NOR), COLORLESS NON-RIPENING (CNR), and RIPENING INHIBITOR (MADS-RIN) in concert with ethylene signalling regulate ripening, possibly in response to a developmental switch. Additional components include TOMATO AGAMOUS-LIKE1 (TAGL1), APETALA2a (AP2a), and FRUITFULL (FUL1 and FUL2). The links between this highly connected regulatory network and downstream effectors modulating colour, texture, and flavour are still relatively poorly understood. Intertwined with this network is post-transcriptional regulation by fruit-expressed microRNAs targeting several of these transcription factors. This important developmental process is also governed by changes in DNA methylation levels and possibly chromatin remodelling.

Fruit types and equivalence of structures and tissues

Fruits are plant organs specialized for seed dispersal. Most fruits can be placed within a few broad categories based on a range of features including their morphology, method of dehiscence, and number of carpels that constitute the organ ( Fig. 1), and this is important because we can use this information to associate morphological and anatomical features with underlying conserved genetic mechanisms. The mature ovary wall or pericarp can be divided into an exocarp, mesocarp, and endocarp ( Fig. 1A), and fruits can be dry or fleshy, dehiscent or indehiscent, and with free (apocarpous) or fused (syncarpous) carpels. In some cases, the edible part of the fruit develops from extra-carpellary tissues ( Esau, 1977; Ireland et al., 2013) as is the case in apple or strawberry ( Fig. 1B). In dry fruits, such as cereal grains, the pericarp may remain intact and fused to the seed coat to form a caryopsis. Alternatively, the ovary wall and seed coat are separate and the pericarp may develop wings or other modifications for dispersal, for example in maple. Dry fruits may also be dehiscent, where the pericarp or outer tissue of the fruit splits to release the seeds. In legumes, such as pea, the pod splits and the carpels open to release their seeds. Other forms of dehiscence include the formation of pores in the apical region of the fruit, for example poppy capsules. Fleshy fruits are generally indehiscent. There are several well-known classes which include berries where the seeds are enclosed in a dense fleshy pulp, for example tomato, grapes, and bananas, drupes where the endocarp is stony and protects a single seed, for example peach and nectarine, and pomes such as apple and pear where the edible organ results from expansion of accessory tissues. There are also aggregate fruits such as raspberry, which is a collection of drupes ( Fig. 1B). Dry and fleshy fruits appear very different, but they are composed of similar tissues with varying degrees of lignification, cell numbers, and sizes. In tomato and many fleshy fruits, the pericarp cells undergo substantial expansion during fruit development, whereas in dry fruits fewer cell layers are apparent with less expanded and more lignified cells, while drupes, have a lignified endocarp. This morphological and anatomical continuum is reflected in common genetic mechanisms regulating development and ripening ( Fig. 1C).

Ripening involves major metabolic changes regulated by hormones

The timing and strategy for seed dispersal are critical for ensuring the survival of the next generation, and fruits have evolved complex mechanisms to maximize the efficacy of this process. Ripening frequently involves profound changes in metabolism of the tissue surrounding the seeds to aid their dispersal, including drastic alterations in colour, texture, and sugar content, that have been exploited by humans for crop domestication ( Klee and Giovannoni, 2011; Seymour et al., 2013).

Fruits can be classified into two groups, climacteric and non-climacteric fruits, by whether or not they show a rapid rise in respiration and a burst of ethylene production at the onset of the ripening process. Those that show enhanced respiration or a respiratory climacteric are known as climacteric fruits and include tomatoes, bananas, apples, pears, mangoes, and papaya. These fruits also show a steep rise in the production of the plant hormone ethylene at the onset of ripening. In contrast, in non-climacteric fruit such as strawberry, grape, and citrus, the respiratory burst and rise in ethylene production are absent. The pathway of ethylene biosynthesis is now well understood and the major steps involve the conversion of S-adenosylmethionine (SAM) to 1-aminocyclopropane-1-carboxylic acid (ACC) by ACC synthase (ACS) and then by ACC oxidase (ACO) to ethylene ( Alexander and Grierson, 2002). In climacteric fruit tissues, ethylene biosynthesis proceeds at a low level during development (System 1), but at the onset of ripening it becomes autocatalytic (System 2). For many years, ethylene has been shown to be necessary for the initiation of ripening in climacteric fruits, and other plant hormones, including auxin, have been implicated in the control of ripening in non-climacteric forms ( Seymour et al., 1993). Therefore, it has been assumed that two different types of ripening control mechanism are operating in fleshy fruits. However, more recent information from studies in melon, pepper, and other fruits suggests that the differences between climacteric and non-climacteric fruits are less distinct than once was presumed (reviewed by Paul et al., 2012; McAtee et al., 2013).

Cantaloupe melons have been classed as climacteric fruit because they ripen rapidly and have a short shelf life. In contrast, honeydew types ripen slowly. Genetic studies indicate, that in a cross between non-climacteric and climacteric melon types, two loci were found to be responsible for the different ripening phenotypes, with the non-climacteric behaviour being due to a recessive allele linked to ethylene insensitivity ( Périn et al., 2002). In other studies, crosses between two non-climacteric melon types have yielded progeny showing climacteric ripening behaviour, again suggesting that a number of distinct loci are responsible for these effects ( Pech et al., 2008). Ethylene signal transduction is also linked to processes controlled by other plant hormones, and this is covered elsewhere in this special issue ( Kumar et al., 2014).

Our knowledge of the role of hormones, other than ethylene, during ripening is rather limited. However, a few studies point to a role for auxin, abscisic acid (ABA), and brassinosteroids in the ripening of both climacteric and non-climacteric fruits (reviewed by Gillaspy et al., 1993; Srivastava and Handa, 2005; McAtee et al., 2013). Early studies in strawberries showed that when achenes are removed from immature fruit, precocious ripening of the receptacle occurs, which can be stopped by the application of exogenous auxin ( Given et al., 1988). In both grapes and tomato, low auxin levels also seem to be required at the onset of ripening ( Gillaspy et al., 1993; Böttcher et al., 2010). Concentrations decline to low levels at the onset of ripening, which is associated with an increase in the conjugated form of indole acetic acid (IAA; IAA-Asp) ( Mapelli et al., 1978; Buta and Spaulding, 1994; Böttcher et al., 2010). Consistent with this, in tomato, a reduction of free IAA by overexpression of a Capsicum chinense auxin-conjugating (IAA-amido synthetase) enzyme encoded by a Gretchen Hagen 3-like gene, CcGH3, leads to an increased sensitivity to ethylene at an earlier stage of development ( Liu et al., 2005). Böttcher et al. (2010) hypothesized that, in grapes and tomato, the ratio between IAA and its conjugated forms, rather than the level of free IAA, might be important for the regulation of ripening. Interestingly, a cross-talk between auxin and ethylene also occurs later during ripening in climacteric fruit such as peaches and tomato ( Jones et al., 2002; Trainotti et al., 2007). A role for ABA during ripening has also been described, and it appears that in fruit having a lower requirement for ethylene to ripen, ABA might have a stronger role ( McAtee et al., 2013). In both climacteric and non-climacteric fruit, there is an increase in ABA levels at the onset and/or during the ripening process which, in climacteric fruit, precede the production of ethylene ( Kondo and Inoue, 1997; Jiang et al., 2000; Zhang et al., 2009). In tomato, silencing of a gene which encodes a key enzyme in ABA biosynthesis affected several metabolic pathways of fruit ripening ( Sun et al., 2012). Similarly, in strawberry, down-regulation of an ABA biosynthetic gene correlates with retardation of ripening ( Jia et al., 2011). Finally recent studies in grape ( Symons et al., 2006) and tomato ( Vidya Vardhini and Rao, 2002) showed that brassinosteroids might be another player during ripening as their levels increase at the onset of ripening in grape, and exogenous application of this hormone can promote ripening in both species, as well as ethylene production in tomato. Brassinosteroids are actively produced during tomato fruit development ( Montoya et al., 2005), and transgenic up-regulation of the signal transduction pathway resulted in higher carotenoids and soluble solids in ripe fruit ( Liu et al., 2014).

Colour changes and alterations in metabolites

The most obvious ripening-related changes are alterations in fruit colour brought about by the accumulation of pigments such as betalains (occuring only in the Caryophyllales), carotenoids, and anthocyanins. Carotenoids are terpenoid derivatives that are part of the normal photosynthetic apparatus, which is functional in fruit tissues (see Rambla et al., 2014), and are thought to have a photo-protective role in the cell. In fruits such as tomato, there is a substantial accumulation of certain carotenoid pigments during the ripening process, and this occurs as the thylakoid membranes in the chloroplast break down and the plastids become chromoplasts. These plastid changes are initiated by signals which have yet to be identified, but evidence indicates that the chloroplast to chromoplast transition is synchronous for all plastids in a tomato cell ( Egea et al., 2011). The onset of ripening is followed by the increased transcription of several nuclear genes that encode enzymes involved in the biosynthesis of carotenoids (see Bramley and Fraser, 2013). The best studied of these gene products is phytoene synthase (PSY1) that catalyses the first committed step in the carotenoid biosynthetic pathway. and down-regulation of PSY1 abolishes normal carotenoid accumulation ( Bartley et al., 1992; Fray and Grierson, 1993). Phytoene is used as the precursor for the formation of the red pigment lycopene. The enzymes that metabolize lycopene are normally turned off at ripening. The developmental control of this pathway now appears to involve a number of factors including ripening regulation of gene expression by ethylene signalling, developmental regulators, and carotenoid metabolites ( Klee and Giovannoni, 2011; Kachanovsky et al., 2012; Seymour et al., 2013). Besides carotenoids, flavonoids, predominantly accumulating in the peel, also play a role in determining the colour of tomato fruits ( Schijlen et al., 2006; Ballester et al., 2010). As with the synthesis of carotenoids in tomato, the ripening-related accumulation of the red, blue, and purple anthocyanin pigments in fruits such as grape is also under strong genetic control. The pathway of anthocyanin biosynthesis is well known and involves a range of enzymes in the flavonoid pathway including chalcone synthase ( Jaakola, 2013). In cultivated tomato, this pathway ends at naringenin, giving the peel its yellowish colour, but introduction of the dominant ANTHOCYANIN FRUIT ( AFT) gene from Solanum chilense increases the anthocyanin levels ( Jones et al., 2003). Up-regulation of anthocyanins in the flesh of transgenic tomato fruits impacts not only colour, but also shelf-life ( Zhang et al., 2013).

Fruit softening

Cell wall remodelling plays a major role in the texture changes in fleshy fruits and involves the coordinated expression of a large number of genes. In tomato, >50 cell wall structure-related genes are expressed during fruit development ( Tomato Genome Consortium, 2012). Changes occur in the structure of all the major cell wall polysaccharides, with differences in the degree and nature of wall modifications depending on the tissue ( Hyodo et al., 2013). Evidence from transgenic tomato experiments indicates that the extent of fruit softening can be reduced by silencing genes encoding polygalacturonase (PG), β-galactosidase, and expansin (; Smith et al., 2002; Powell et al., 2003). In apple, reduced PG expression leads to firmer fruits ( Atkinson et al., 2012). In other fruits such as strawberry, PG and pectate lyase were shown to be involved in the softening process ( Jiménez-Bermúdez et al., 2002; Posé et al., 2013). However, in all cases, down-regulating a single gene, or in some cases two of these genes ( Powell et al., 2003), had only a very limited effect on texture changes. This indicates that either multiple enzyme activities are involved, or we have yet to identify the main protagonists, or both. Whether texture genes are predominantly under the control of a specific class of transcription factors in a way analogous to the situation with flavonoid biosynthesis and MYELOBLASTOSIS (MYB) transcription factors (see below) is still unknown.

Tomato as a model species and the genomics revolution

Tomato is a good model system to investigate the mechanistic basis of fruit ripening because it has diploid genetics, a range of well-characterized single gene mutants [available from the Tomato Genomic Resource Center (TGRC)], recombinant inbred lines (RILs; Eshed and Zamir, 1994), and mapping populations and an excellent and well-annotated genome sequence ( Tomato Genome Consortium, 2012). Additionally it is easily transformed, and mechanistic hypotheses can be tested using stable transgenic lines or by virus-induced gene silencing (VIGS) ( Liu et al., 2002). Several databases are available for exploring genome and expressed sequence tag (EST) sequences (Sol Genomics Network; Bombarely et al., 2011) and for gene expression analysis (Tomato Expression Database; Fei et al., 2006). Furthermore, decades of work have been undertaken on the biochemical changes underlying processes such as softening, colour changes, and the regulation of ripening ( Seymour et al., 2013).

Along with tomato, the sequencing of numerous fleshy fruit genomes including papaya ( Ming et al., 2008), strawberry ( Shulaev et al., 2011), grape ( Jaillon et al., 2007), apple ( Velasco et al., 2010), cucumber ( Huang et al., 2009), cacao ( Argout et al., 2011), banana ( D'Hont et al., 2012), melon ( Garcia-Mas et al., 2012), kiwifruit (S. Huang et al., 2013), pear ( Wu et al., 2013), sweet orange (Q. Xu et al., 2013), watermelon (Y. Xu et al., 2013), and pepper ( Kim et al., 2014) has now provided the tools to reveal the underlying mechanisms governing fruit development and ripening.

Transcription factors involved in fruit patterning and early fruit development

Many ripe fruits are composed of matured ovaries, and therefore it should be no surprise that many aspects of fruit size, shape, and further developmental changes dependent on organ identity are determined at an early stage. During flower development, the apical meristem typically produces the primordia of the four floral whorls, of which the inner fourth whorl, the carpel primordia, fuses to form the ovary, with ovules originating at the carpel margins. Ovary identity and size, and, as a consequence-at least partially-final fruit size and proper later development or ripening, are thus determined at a very early stage and have been found to be controlled by transcription factors.

One major determinant in the selection of larger fruit sizes in domesticated tomato was the increase in the number of locules, from two to four in wild tomato species to eight or more in some cultivated lines ( Cong et al., 2008). Two genes underlying quantitative trait loci (QTLs) encode transcription factors and determine this increase in locule number by causing an increase in the number of carpels forming a single ovary ( Lippman and Tanksley, 2001). These transcription factor genes, and those discussed in subsequent sections, are listed in Table 1. The gene with the strongest effect, FASCIATED ( FAS), encodes a YABBY transcription factor and is expressed, although not exclusively, in carpel primordia ( Cong et al., 2008). The best known example of YABBY function in the Arabidopsis flower is CRABS CLAW ( CRC), which is involved in carpel and nectary development ( Bowman and Smyth, 1999). However, FAS is an orthologue of Arabidopsis YABBY2 (Z. Huang et al., 2013), which is involved in organ polarity. The fas mutation causes lower expression of the gene and higher locule number without apparently changing the protein ( Cong et al., 2008). The second QTL, for locule number ( lc), is jointly controlled by two single-nucleotide polymorphisms near the tomato orthologue of Arabidopsis WUSCHEL ( WUS), a homeodomain transcription factor gene ( Mayer et al., 1998). However, the identification of WUS as the causative gene underlying the QTL has not yet been established ( Muños et al., 2011).

Carpel identity and number, fruit patterning

Overall ripening regulation

Ethylene response and signalling

Chloroplast development, flavonoid/anthocyanin biosynthesis

Floral organ identity is governed by MADS-box transcription factors according to the extended ABC model, or variations thereof ( Smaczniak et al., 2012). Following this model, organ identity in the four whorls is determined by the combined expression of particular MADS-box genes, and the functional interactions of their products, in each of the four whorls. Carpel identity is determined by the C-type genes, AGAMOUS ( AG) and SHATTERPROOF1/2 ( SHP1 and SHP2), in Arabidopsis ( Favaro et al., 2003; Pinyopich et al., 2003). Arabidopsis ag mutant flowers lack an ovary (and thus fruit) and have lost determinacy (i.e. the floral meristem continues to develop from the centre of the flower) ( Bowman et al., 1989). Knock-down of TOMATO AGAMOUS 1 ( TAG1) by RNA interference (RNAi) results in stamen defects and loss of determinacy, leading to nested flowers-in-flowers ( Pnueli et al., 1994) or a fruit-in-fruit phenotype ( Pan et al., 2010). Knock-down of the tomato SHP orthologue TOMATO AGAMOUS-LIKE 1 ( TAGL1) appears to affect carpel identity by leading to loss of style trichomes and a thinner fruit pericarp ( Vrebalov et al., 2009). Moreover, ectopic expression of either TAGL1 or TAG1 results in fleshy sepals accumulating lycopene, further supporting their role as typical C-type genes ( Pan et al., 2010; Pineda et al., 2010). It has to be noted here that RNAi leads to variable degrees of, and rarely complete, knock-down of gene expression, and thus the relative contributions of the two genes to carpel identity may be difficult to establish.

Tomato fruit patterning, determinacy, and early development appear to be regulated by one or more miR156-targeted SQUAMOSA promoter binding protein-like (SPL/SBP) transcription factors, since knocking down their expression by ectopic expression of Arabidopsis MIR156b led to extra carpels and new fruit-like structures growing at the stylar end of the fruit. Two genes associated with meristem maintenance, encoding the class I KNOTTED1-like homeobox (KNOX)-like LeT6/TKN2 and the No Apical Meristem/Cup-shaped Cotyledon (NAC) transcription factor GOBLET, were up-regulated in ovaries of these transgenic plants ( Silva et al., 2014). Mouse ear ( Me), a dominant mutation mapping at the location of TKn2, leading to misexpression of an aberrant TKn2 mRNA, also leads to extra carpels, suggesting that tomato SPLs regulate carpel number and determinacy through down-regulation of TKn2 ( Parnis et al., 1997). In Arabidopsis, the down-regulation of miR156-targeted SPL genes has no clear effect on gynoecium determinacy or carpel number, suggesting the existence of a distinct regulatory mechanism in tomato compared with Arabidopsis ( Xing et al., 2013).

The study of transcriptional regulation of early fleshy fruit development in species other than tomato is hampered by the lack of or difficulty of transformation protocols for functional studies and/or the lack of available mutants. Thus the information on gene function from these species is often incomplete and derived from expression studies or from heterologous expression in other species. For example, a peach ( Prunus persica) SHP orthologue, PpPLE, induces carpel-like sepals in transgenic tomato ( Tadiello et al., 2009). Analogies and differences in the regulatory network of FRUITFULL ( FUL) and SHP or their orthologues, and their roles in development in dry versus fleshy fruits have been reviewed elsewhere ( Ferrándiz and Fourquin, 2014). In developing peach, a fleshy fruit with a strongly lignified endocarp (stone fruit), PpPLE is expressed at a higher level in cultivars showing the split-pit phenotype, which is correlated with increased lignification ( Tani et al., 2007). Heterologous expression of a grape ( Vitis vinifera) AG orthologue, VviAG1, in tomato caused fleshy sepals, as do the tomato orthologues ( Mellway and Lund, 2013). In apple ( Malus domestica), one of two FUL orthologues, MdMADS2.1, is associated with fruit firmness at the ripe stage ( Cevik et al., 2010). Suppression of a SEPALLATA1/2-like ( SEP-like) gene either in apple ( MdMADS8/9) or in strawberry ( Fragaria×ananassa) ( FaMADS9) leads to a greatly reduced fruit flesh, indicating a role for these MADS-domain genes during early fruit development. Interestingly those genes also have a role during fruit ripening (see next section), showing the plasticity of function of MADS-box genes ( Seymour et al., 2011; Ireland et al., 2013;).

Spontaneous mutations affecting fruit ripening are frequently in genes encoding transcription factors

In tomato, ripening is regulated by a number of transcription factors in conjunction with the plant hormone ethylene. The presence of an intricate regulatory network underlying the process is evident from a large number of mutations affecting ethylene signalling or transcription factor activity that lead to defective ripening. However, the topology and internal interactions of this network are far from understood. Ripening-associated transcription factors have been found to regulate the biosynthesis of ethylene. For example, three transcription factors, the MADS-domain protein RIPENING-INHIBITOR (RIN) ( Vrebalov et al., 2002), COLORLESS NON-RIPENING (CNR), an SBP transcription factor ( Manning et al., 2006), and the product of the gene underlying the non-ripening ( nor) mutation ( Tigchelaar et al., 1973), which was identified as a NAC domain family transcription factor ( Martel et al., 2011), were proposed to function early in the transcriptional activation cascade regulating ripening-related processes. The rin and Cnr mutations effectively block the ripening process and result in fruits that fail both to produce elevated ethylene and to respond to exogenous application of the gas ( Manning et al., 2006). These data suggested that both genes lie upstream of ethylene production and have functions that are ethylene dependent and independent. Ripening traits such as autocatalytic ethylene production, softening, and carotenoid accumulation are inhibited in rin and Cnr mutant fruit ( Fraser et al., 2001; Vrebalov et al., 2002; Manning et al., 2006). Recently a systems biology approach was used to study the role of the nor and rin loci in tomato fruit ripening. This study confirms that nor has a more global effect on ethylene/ripening-related gene expression than rin and might even act upstream of RIN in the transcriptional network controlling tomato fruit ripening ( Osorio et al., 2011). Ethylene biosynthesis was altered in both mutants. Expression of genes for autocatalytic ethylene biosynthesis, SlACS2 and SlACS4, is suppressed in the rin mutant ( Barry et al., 2000). RIN was also found to modulate the aroma formation in tomato fruit by direct regulation of LIPOXYGENASE ( LOX) genes ( Qin et al., 2012). Interestingly, SEPALLATA ( SEP)-type MADS-domain ( RIN-like) genes appear to be global regulators of ripening with conserved functions in both climacteric and non-climacteric fruits. Homologues of RIN accumulate during the ripening of non-climacteric fruit such as strawberry ( Vrebalov et al., 2002) and pepper ( Lee et al., 2010). Seymour et al (2011) showed that suppression of a SEP-like gene, FaMADS9, in strawberry resulted in delayed ripening, similar to the rin mutant in tomato. In grape, another fruit considered as non-climacteric, protein-protein interactions, expression pattern, and partial complementation of the tomato rin mutation suggest that VviSEP4 may have a function similar to that of RIN in ripening ( Mellway and Lund, 2013). The apple MADS8/9 genes were found to control fruit ripening characters such as starch degradation and ethylene-modulated ripening traits. Moreover the apple MADS9 gene was shown to act as a transcriptional activator of ACS1, but unlike RIN ( Martel et al., 2011) it can also transactivate the ACO1 promoter ( Ireland et al., 2013). To date, very little is known about the involvement of SEP-like genes in monocot fruit ripening. However, expression data in banana and oil palm fruit indicate that SEP homologues from the SEP3 subgroup play a role during their ripening ( Elitzur et al., 2010; Tranbarger et al., 2011).

Similar to the SEP-like regulatory genes, the tomato CNR gene was also implicated in the positive regulation of several ripening-related genes, including PSY1, LOX, and ACO1 ( Eriksson et al., 2004). The absence of phytoene and other carotenoid precursors explains the abolishment of carotenoid biosynthesis in the Cnr mutant ( Fraser et al., 2001). The Cnr mutation is an epigenetic change that increases cytosine methylation in an upstream region of the promoter of a SQUAMOSA promoter-binding protein-encoding gene. This epimutation severely decreases gene expression and blocks normal fruit ripening ( Manning et al., 2006).

Ethylene-regulated transcription factors (EILs and ERFs) involved in fruit ripening

Ripening of climacteric fruits is characterized by an autocatalytic increase in respiration and ethylene biosynthesis just prior to the initiation of ripening. Ethylene signalling can be regulated at several levels, which include ethylene biosynthesis and its perception through ethylene receptors encoded by ETHYLENE RESPONSE ( ETR) genes, which activate a signal transduction cascade through release of the block exerted by CONSTITUTIVE TRIPLE RESPONSE 1 (CTR1) on ETHYLENE INSENSITIVE 2 (EIN2). This release then activates EIN3/EIN3-like ( EIL) primary transcription factor genes ( Tieman et al., 2001), resulting in the expression of secondary transcription factors, the ethylene response factors or ERFs (reviewed in Adams-Phillips et al., 2004; Bapat et al., 2010). The final result of the signalling is regulation of target gene expression by EILs or ERFs. Several genes that regulate tomato ripening through ethylene signal transduction have been identified. These are, among others, genes encoding the ethylene receptor genes, NEVER-RIPE ( Wilkinson et al., 1995; Yen et al., 1995), ETR6 ( Kevany et al., 2007), and GREEN-RIPE ( Gr), a gene encoding a protein of unknown function ( Barry et al., 2005; Barry and Giovannoni, 2006)

The ERF genes belong to the large AP2/ERF multigene family and mediate ethylene-dependent gene expression by binding to the GCC motif in the promoter region of target genes ( Pirrello et al., 2012). ERFs have been shown to play a role in plant development, including in tomato ripening. Li et al. (2007) demonstrated that LeERF1 positively mediated the ethylene signalling in tomato seedlings and fruits. ERF1 RNAi fruits showed longer shelf-life compared with the wild-type fruits. Another member of the ERF family, Sl-ERF.B3, has been shown to act as a transcriptional activator on GCC box-containing promoters, and plants expressing a dominant chimeric repressor ERF.B3-SRDX displayed constitutive ethylene responses in the absence of ethylene. The multiple symptoms related to enhanced ethylene sensitivity correlated with the altered expression of ethylene biosynthesis and signalling genes and suggested the involvement of Sl-ERF.B3 in a feedback mechanism that regulates components of ethylene production and responses ( Liu et al., 2013). Reduced expression of another ERF gene, SlERF6, by RNAi enhanced both carotenoid and ethylene levels during ripening, demonstrating an important role for SlERF6 in fruit ripening, integrating the ethylene and carotenoid synthesis pathways ( Lee et al., 2012). Although the function of many of the ERF transcription factor genes in tomato has been studied in detail, not much is known about their direct ethylene-responsive target genes. With the development of in vivo chromatin immunoprecipitation (ChIP), followed by high-throughput sequencing, the identification of these ERFs targets will no doubt soon be revealed.

The transcriptional regulatory network controlling tomato fruit ripening

Several transcription factors that are highly and often specifically expressed during tomato fruit development have been shown also to regulate the ripening process; for example, the MADS-domain proteins TOMATO AGAMOUS-LIKE1 (TAGL1) ( Itkin et al., 2009; Vrebalov et al., 2009), MADS1 ( Dong et al., 2013), FUL1/TDR4 and FUL2/MBP7 ( Bemer et al., 2012), HD-Zip homeobox protein LeHB-1 ( Lin et al., 2008), and AP2/ERF protein APETALA2a (AP2a) ( Chung et al., 2010; Karlova et al., 2011). SlAP2a and SlMADS1 were shown to act as negative regulators of fruit ripening ( Chung et al., 2010; Karlova et al., 2011; Dong et al., 2013). SlAP2a negatively regulates ethylene biosynthesis and signalling. RNAi-mediated repression of SlAP2 resulted in alterations in fruit shape, orange-coloured ripe fruits, and altered carotenoid content, as well as faster fruit senescence and higher levels of ethylene production ( Chung et al., 2010; Karlova et al., 2011). Transcriptomic and metabolic analysis of the AP2i silenced fruits indicates that AP2a plays an important role during tomato fruit development and in ripening, controlling aspects of primary and secondary metabolism, ethylene biosynthesis, and signalling pathways, and also in the differentiation of chromoplasts. Furthermore, ripening regulators such as RIN and CNR were shown to function upstream of SlAP2 and to regulate its expression positively. CNR directly binds to the promoter of SlAP2a in vitro. Interestingly, in the pericarp of SlAP2 RNAi fruits, mRNA levels of CNR were elevated, indicating that SlAP2a and CNR are part of a negative regulatory feedback loop, which remains to be characterized further. In the AP2i transgenic fruits, several ripening-associated genes encoding proteins such as those in the carotene biosynthesis pathway, LOXB and LOXC, pectin methylesterase, and EXP3 are down-regulated, indicating that AP2a has positive ripening regulatory functions besides its negative regulatory function in ethylene biosynthesis ( Chung et al., 2010; Karlova et al., 2011). Recently it was shown that AP2 is involved in the development of the dry fruits of Arabidopsis, regulating dehiscence zone development in the silique, where it acts as a negative regulator of replum growth ( Ripoll et al., 2011).

The MADS-box gene SlMADS1, another SEP-clade member, is a negative regulator of tomato fruit ripening and has been shown to be highly expressed in mature green fruits, with decreasing expression during fruit ripening ( Dong et al., 2013). In SlMADS1 RNAi-silenced plants, fruits started ripening earlier and, like AP2a RNAi fruits, they also showed increased production of ethylene compared with the wild-type fruits. Elevated ethylene was consistent with the observed up-regulation of ethylene-related and ripening-related genes in these SlMADS1-silenced fruits. Interestingly an interaction between RIN and SlMADS1 proteins was observed by a yeast two-hybrid assay. These results suggest that SlMADS1 plays a role in fruit ripening as a repressor of ethylene biosynthesis and signalling by directly or indirectly interacting with RIN ( Dong et al., 2013).

TAGL1 interacts with RIN ( Leseberg et al., 2008), is highly expressed during fruit ripening ( Itkin et al., 2009; Vrebalov et al., 2009), and is thus a candidate for controlling ripening processes in concert with RIN. Indeed TAGL1 has been reported to function as a positive regulator of fruit development and ripening ( Itkin et al., 2009; Vrebalov et al., 2009). TAGL1 knock-down plants produced yellow-orange fruits with reduced carotenoids and a thin pericarp. They had low ethylene levels due to decreased expression of the RIN target ACS2, and TAGL1 therefore appears to work together with RIN to regulate ripening by inducing System 2 autocatalytic ethylene production. Itkin et al. (2009) studied TAGL1 overexpression in the rin mutant background and found evidence for RIN-dependent and RIN-independent functions of TAGL1. Down-regulation of a TAGL1/SHP orthologue from strawberry, FaSHP, by agroinfiltration of an RNAi construct in fruit resulted in delayed ripening and repression of several ripening-related genes, suggesting a similar function in this species ( Daminato et al., 2013).

Two other tomato MADS-domain proteins interacting with RIN are FRUITFULL 1 and 2 (FUL1/TDR4 and FUL2/MBP7). Silencing the encoding FUL genes separately resulted in very mild alterations in tomato fruit pigmentation. FUL1 and FUL2 appear to have redundant functions in ripening, since simultaneous silencing of these genes resulted in an orange ripe fruit with highly reduced lycopene. Expression of genes involved in cell wall modification, cuticle production, volatile production, and glutamate accumulation were altered. In contrast to previously identified ripening regulators, FUL1 and FUL2 do not regulate ethylene biosynthesis but influence ripening in an ethylene-independent manner ( Bemer et al., 2012). These data suggest that FUL1/2 and TAGL1 may regulate different subsets of the known RIN targets, probably in a protein complex with the latter, although recent data suggest that tomato FUL genes may have broader functions ( Fujisawa et al., 2014). RIN and TAGL1 were found to be up-regulated in the pericarp of FUL1/2 RNAi fruits, pointing to a negative feedback loop from FUL1/2 to these genes ( Bemer et al., 2012). Notably, Arabidopsis FUL, which has a function in the development of the silique, also represses the TAGL1 orthologues SHP1/2 ( Ferrándiz et al., 2000), suggesting some conservation of the regulatory network between Arabidopsis and tomato. Interestingly a FUL homologue of bilberry ( Vaccinium myrtillus) was found to regulate colour development and anthocyanin-related gene expression during berry ripening ( Jaakola et al., 2010). These data indicate that FUL genes play important roles in both dry and fleshy fruit development (see Ferrándiz and Fourquin, 2014).

Another transcription factor gene highly expressed in tomato fruits, LeHB-1, encodes a homeobox protein that binds in vitro to the promoter of the ethylene biosynthesis gene LeACO1. The silencing of LeHB-1 in tomato fruit using VIGS greatly reduced LeACO1 mRNA levels, and inhibited ripening. Ectopically expressing the gene, using a virus vector, induced alterations in floral organs, including the formation of fleshy sepals that showed several features of fruit ripening ( Lin et al., 2008). Recently a new tomato NAC domain protein gene, SlNAC4, was shown to be highly expressed in sepals and at the onset of fruit ripening. Reduced expression of SlNAC4 by RNAi resulted in delayed fruit ripening, decreased ethylene synthesis, suppressed chlorophyll degradation, and reduced carotenoids ( Zhu et al., 2014). These transgenic tomato fruits also displayed significant down-regulation of ripening-associated genes, indicating that SlNAC4 functions as a positive regulator of fruit ripening. Positive and negative interactions of known major ripening-related transcription factors in tomato and other fleshy fruits are summarized in Fig. 2.

Transcription factors regulating particular aspects of fruit quality

Biochemical and molecular data demonstrated that the MYB transcription factor MYB12 plays an important role in regulating the flavonoid pathway in tomato fruit, and reduced expression leads to reduction of flavonoids in the peel and to pink fruit colour ( Adato et al., 2009; Ballester et al., 2010). Unlike cultivated tomato, where the flavonoid/anthocyanin pathway downstream of naringenin is apparently inactive, many other fleshy fruits accumulate anthocyanins as well as flavonoids in the skin and/or flesh. In several species (grape, apple, pear, and strawberry), different parts of the flavonoid/anthocyanin pathway have been shown to be regulated by various homologous R2R3 MYB transcription factors. These function in complexes with basic helix-loop-helix (bHLH) transcription factors and WD40 domain proteins. Both for grape and for apple, several MYB transcription factors involved in fruit skin anthocyanin biosynthesis, and in some cases their (putative) bHLH and WD40 partners, have been identified (reviewed in Jaakola, 2013). Using a combination of co-expression analysis, yeast two-hybrid interactions, and Arabidopsis mutant complementation, Schaart et al. (2013) identified FaMYB9/FaMYB11, FabHLH3, and FaTTG1 as encoding the likely representatives of these three protein classes regulating proanthocyanidin biosynthesis in strawberry. A related transcription factor, FaMYB10, was shown to be involved in both early and late anthocyanin biosynthesis in the receptacle, in a ripening-regulated manner ( Medina-Puche et al., 2014). The S. chilense Aft mutation, leading to anthocyanin accumulation in the peel, co-segregates with two MYB genes, ANTHOCYANIN 2 ( AN2) and ANTHOCYANIN 1 ( ANT1) ( Mes et al., 2008). The latter gene had previously been shown to be capable of anthocyanin biosynthesis up-regulation when it was activation tagged in Micro-Tom tomato ( Mathews et al., 2003). Both genes are up-regulated in S. lycopersicum Aft fruits, compared with negligible expression levels in wild-type fruits, suggesting that the activity of either one or both induces anthocyanin biosynthetic pathway genes ( Povero et al., 2011). A homologue of AN2 located at a similar genomic position, A, regulates anthocyanin biosynthesis in purple immature pepper fruits ( Borovsky et al., 2004).

Links between the higher level ripening regulatory genes, intermediary regulators, and downstream effectors are still largely unexplored. The first evidence that FUL controlled flavonoid accumulation during fruit ripening was reported in bilberry by Jaakola et al. (2010) where silencing of VmTDR4 ( VmFUL) inhibited anthocyanin accumulation in the flesh of the berry. In tomato, lines with reduced FUL expression had much reduced flavonoid accumulation in the form of naringenin chalcone in the peel, indicating conservation of function for FUL between tomato and bilberry ( Fujisawa et al., 2014).

During tomato fruit ripening, chloroplasts differentiate to chromoplasts, which are the site of lycopene production and accumulation. Powell et al. (2012) identified the gene underlying the tomato Uniform ripening ( U) locus as encoding a Golden2-like (SlGLK2) transcription factor, which regulates tomato fruit chloroplast development. The SlGLK2 transcript was found to accumulate in green fruits and is more abundant in the shoulder than in the blossom (stylar) end of the fruit, consistent with the chlorophyll distribution. Ectopic expression of SlGLK2 in both U/U and u/u genotypes resulted in homogeneously dark green unripe fruits. A related transcription factor, APRR2-like, is also involved in regulating fruit chloroplast number and aspects of ripening in tomato ( Pan et al., 2013).

A role for auxin in chloroplast development and fruit quality became evident from the observation that down-regulation of AUXIN RESPONSE FACTOR 4 ( ARF4) leads to dark green fruits with increased starch levels and blotchy ripening ( Jones et al., 2002). As starch accumulated during development is the main source of soluble sugars in ripe fruit, the increased starch levels resulted in significantly increased glucose and fructose contents in ripe fruits ( Sagar et al., 2013).

Role of microRNAs targeting transcription factors in tomato fruit development and ripening

Many transcription factor mRNAs are the targets of microRNAs (miRNAs). High-throughput degradome library sequencing, or parallel analysis of RNA ends (PARE), was used to discover many new miRNA-mRNA target pairs in tomato fruits ( Karlova et al., 2013). Many of the newly identified miRNA targets (~30%) encoded transcription factors, which hints at the importance of miRNAs in regulating tomato fruit development. Among those identified as targets were several ARF genes. These data suggest that miRNAs in tomato are involved in the initiation of fruit set and growth by controlling the expression of ARF genes ( Karlova et al., 2013). Fruit formation and fruit yield were affected in tomato by overexpression of miR156 ( Zhang et al., 2011). miR156 was found to target CNR, while AP2a was identified as a target of miR172. AP2a and CNR are important regulators of fruit ripening (see above) and their expression increases in the breaker stage; however, miRNA-dependent cleavage of AP2a and CNR mRNAs also increases at the same stage ( Karlova et al., 2013). Apparently both miR156 and miR172 modulate the intact mRNA levels of their targets during ripening, without completely abolishing them. These data suggest that miR156 and miR172 in tomato fruit ripening may fine-tune the expression of CNR and AP2a.

Mode of action of the transcription factors regulating fleshy fruit development

Transcription factor genes govern the development and ripening of both dry and fleshy fruits. In fleshy fruits, the links between these high-level regulators, such as RIN, FUL, TAGL1 ( SHP), CNR, and NOR, and downstream effectors are still poorly understood.

In Arabidopsis, SHP activates a bHLH transcription factor, INDEHISCENT (IND), that controls the production of the lignified layer, and also another bHLH factor, ALCATRAZ (ALC), that is responsible for the generation of the separation layer ( Liljegren et al., 2004; Ferrándiz and Fourquin, 2014). ALC may be involved in the up-regulation of cell wall remodelling genes ( Ferrándiz, 2002). In tomato there do not appear to be direct IND and ALC orthologues, which have been found only in the Brassicaceae, although these genes probably diverged from more conserved HECATE-like or SPATULA-like ancestors ( Ferrándiz and Fourquin, 2014).

Transcriptomics analyses in mutant or knock-down backgrounds give an impression of the downstream genes, but do not provide information about the direct targets of the TFs. ChIP experiments have demonstrated that direct targets of RIN include RIN itself, FUL1/ TDR4, FUL2/MBP7, NOR, CNR, and HB1. In addition, RIN was unable to bind to its targets in the absence of a functioning CNR allele ( Martel et al., 2011), possibly because it is missing other MADS-domain dimerization partners, which are targets of CNR. MADS-box genes are known to operate in a combinatorial manner to specify cell fates ( Smaczniak et al., 2012), and it seems likely that they act as dimers or tetramers to control fruit ripening. Yeast two-hybrid screens demonstrate that RIN interacts with FUL1/TDR4, TAGL1, TAG1, and FUL2/MPB7 ( Leseberg et al., 2008; Martel et al., 2011). More recent experiments indicate that possible tetramers include RIN-TAGL1 with RIN-FUL1 or RIN-FUL2. Interestingly RIN directly targets CNR ( Martel et al., 2011), which in turn is likely to interact with FUL, since the Cnr mutant shows very low FUL1 expression ( Eriksson et al., 2004). Therefore, reduced expression of FUL1 in the Cnr mutant may affect tetramer formation and explain why CNR is needed for RIN to bind many of its target genes. The need for a functional tetramer may also explain why there are similar defects in FUL1/FUL2, RIN, and TAGL1 mutants ( Vrebalov et al., 2002, 2009; Itkin et al., 2009; Bemer et al., 2012; Fujisawa et al., 2014). Thus FUL and TAGL1 may have distinct or (partially) overlapping ripening functions that are both dependent on RIN function.

RIN binds to at least 241 direct targets showing both positive and negative regulation ( Martel et al., 2011; Fujisawa et al., 2012; Qin et al., 2012). In floral development many genes involved in hormone biosynthesis and signalling are under direct control of MADS-domain proteins ( Dornelas et al., 2011). This appears also to be the case for fruit ripening. In tomato, RIN interacts directly with the promoters of genes involved in ethylene biosynthesis, ACS2 and ACS4, and in ethylene perception, NR ( Martel et al., 2011; Fujisawa et al., 2012, 2013). This provides direct evidence for the link between RIN and ethylene control of ripening. This is further supported by data that show that ethylene is involved in the up-regulation of RIN expression during ripening, and inhibiting ethylene signalling inhibits RIN transcription ( Fujisawa et al., 2013). RIN also directly targets genes involved in cell wall remodelling and carotenoid biosynthesis such as PG2a and PSY1 ( Martel et al., 2011).

FUL1 and FUL2 working in concert with RIN or alone appear to be even more promiscuous than RIN itself, although it needs to be established to what extent this conclusion is based on specific down-regulation of FUL1 and FUL2 alone as opposed to more general MADS down-regulation ( Fujisawa et al., 2014). The FUL genes encode transcription factors that have been shown to target overlapping and non-overlapping sets of 860 and 878 direct targets, respectively. The interactions between FUL and RIN occur in a number of pathways including the biosynthesis of carotenoids, where ChIP experiments indicate that FUL regulates the entire carotenoid pathway, but RIN is specialized to regulate genes involved in lycopene accumulation ( Fujisawa et al., 2014). A majority of FUL1 and FUL2 targets appear not to be RIN targets, and this is illustrated by the influence of FUL on the flavonoid pathway in tomato and other fruits.

Our understanding of the interactions between ripening regulators and downstream effectors is still fragmentary. The observation that MADS-box genes are direct targets of MADS complexes provides a system where positive autoregulation will give stable high expression and negative regulation leads to sharp signal pulses ( Kaufmann et al., 2010; Dornelas et al., 2011).

Tomato epigenetics and transcriptional regulation

Epigenetic modifications of DNA (cytosine methylation and histone modification, among others) play important roles in regulating gene expression by affecting transcription factor binding and activity or, conversely, being affected by transcription factors that recruit chromatin remodelling proteins ( Kaufmann et al., 2010). Differential epigenetic modifications or 'epigenetic reprogramming' play roles in many plant developmental processes such as vernalization, flowering, gametogenesis, and seed development ( Feng et al., 2010; Wollmann and Berger, 2012). Notably, in tomato, the Cnr mutation is epigenetic, resulting in hypermethylation of an upstream region of the CNR gene ( Manning et al., 2006). Interestingly, genome-wide DNA cytosine methylation, one of the hallmarks of chromatin modification, appears to decrease during tomato pericarp fruit ripening ( Teyssier et al., 2008). More specifically, in the regulatory region of the CNR gene in the tomato cultivar Liberto, changes in methylation were associated with fruit development and ripening, with specific sites in the promoter showing lower levels of cytosine DNA methylation in ripening fruits ( Manning et al., 2006). More recently it has been shown that inducing genome-wide cytosine demethylation leads to premature ripening, and differential methylation sites occur near RIN-binding sites ( Zhong et al., 2013). Whether and how this differential methylation directly affects the binding of transcription factors is still unknown.

Conclusions and outlook

As discussed in this review, our knowledge about the regulatory genes controlling fruit development and ripening is becoming substantial, although in fruit we have yet to explore in any detail the types of regulatory interactions that are found in organisms such as yeast ( MacQuarrie et al., 2011). With an increased understanding of the fruit ripening network we see numerous feed-forward and feed-back loops that are needed for a delicate modulation of the process ( Fig. 2). For instance the vast and fast up-regulation of genes during the breaker stage is modulated, and probably requires this modulation, by negative regulators such as AP2a, MADS1, and possibly miRNAs. Recent genome-wide in vivo transcription factor binding profiles by ChIP-seq will shed light on the molecular interactions and the topology of the gene regulatory network. These studies should be combined with transcriptome data, because it is known from several studies that although there is good evidence for a direct connection between transcription factor binding and direct target gene regulation, transcription factor binding may greatly exceed the expected number of target binding sites ( Kaufmann et al., 2009; MacQuarrie et al., 2011). The reason for these observations is not clear, but may involve titration of transcription factors in the nucleus to limit their availability, or the requirement for the simultaneous, combinatorial binding of multiple transcription factors. The developmental switch that is apparent in tomato fruit development and that appears to be essential to prime the fruit for initiation of ripening processes by ethylene is still largely an enigma. Interestingly, genome-wide DNA cytosine methylation, one of the hallmarks of chromatin modification, appears to decrease during tomato fruit ripening. This leads to the suggestion that initiation of ripening requires an increase of binding site accessibility for the top-level transcriptional regulators that then, by gene activation or repression, set in motion the regulatory network that controls fleshy fruit ripening.

Acknowledgements

This work was supported by the Netherlands Organisation for Scientific Research (NWO), VENI-ALW grant no. 863.11.016 to RK, and a BBSRC TomNet grant to GBS. We thank Michiel Lammers (Wageningen) for producing Fig. 1C. We also acknowledge the support of the COST Action FA1106 for making it possible for the authors to attend the symposium and generate this article.

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Transcriptional control of fleshy fruit development and ripening | Journal of Experimental Botany | Oxford Academic

# data[].attributes.attachment_link.description
Fleshy fruits have evolved to be attractive to frugivores in order to enhance seed dispersal, and have become an indispensable part of the human diet. Here we review the recent advances in the understanding of transcriptional regulation of fleshy fruit development and ripening with a focus on tomato.