Introduction

Tomato is an excellent model system for studying fleshy fruit development due to its importance as a food source, small genome size, diverse and extensive germplasm, well-characterized mutant stocks, high density and well-saturated genetic maps, availability of molecular tools such as expressed sequence tag (EST) resources, publicly available microarray data and efficient transient as well as stable transformation protocols (Tanksley et al. 1992; Gupta et al. 2009). Considerable progress has been made in understanding the biochemical and molecular basis of fruit ripening that has established the important role of ethylene, cell wall proteins and light during fruit ripening in tomato (Lelievre et al. 1997; Rose et al. 1997, 2000; Giovannoni 2001; Jones et al. 2002; Barry and Giovannoni 2007). Recently, several genes encoding transcription and developmental factors, such as RIN (Ripening-inhibitor), NOR (Non-ripening), CNR (Colorless non-ripening), HB-1 (HD-zip homeodomain protein1), TAGL1 (Tomato AGAMOUS-LIKE1) and AP2a (APETALA2a) have been characterized (Martin and Saftner 1995; Vrebalov et al. 2002, 2009; Manning et al. 2006; Lin et al. 2008; Itkin et al. 2009; Chung et al. 2010; Karlova et al. 2010). These proteins act upstream to the ethylene-regulated biochemical events and regulate ethylene-controlled aspects of fruit ripening in tomato. Current research on fruit ripening focuses on the identification of the new candidate genes regulating fruit ripening as well as understanding the interaction among the already characterized components. The role of various hormonal signaling pathways during fruit development and ripening in tomato and crosstalk among them are being investigated (Jones et al. 2002; Wang et al. 2005; Trainotti et al. 2007; Guillon et al. 2008; de Jong et al. 2009a, b; Martel et al. 2011). Since both RIN and ethylene have been implicated during ripening, it is imperative to identify both RIN-regulated ethylene-dependent as well as ethylene-independent aspects of ripening to improve any fruit-related agronomic trait in tomato.

In plants, MADS-box genes have been studied extensively with regard to their role in flower development (Coen and Meyerowitz 1991; Theissen et al. 2000). Several MADS-box genes have been identified to participate in fruit ripening in tomato (Vrebalov et al. 2002, 2009; Giovannoni 2004; Itkin et al. 2009; Osorio et al. 2011). Phylogenetic, molecular as well as biochemical studies for the MADS-box gene family members have been carried in tomato (Busi et al. 2003; Hileman et al. 2006; Leseberg et al. 2008). Investigations on a MADS-box gene, RIN (LeMADS-RIN), of SEPALLATA (SEP) subfamily have unraveled its indispensible role during fruit ripening in tomato (Pelaz et al. 2000; Causier et al. 2002; Vrebalov et al. 2002; Ito et al. 2008). Mutation in this gene leads to non-ripening phenotype. The mutant exhibits inhibition in the expression of the genes involved in respiration and ethylene biosynthesis. The rin mutant fruit also shows inhibition of carotenoid biosynthesis, aroma, production of flavor compounds and softening (Herner and Sink 1973; Tigchelaar et al. 1978; Knapp et al. 1989; Vrebalov et al. 2002). Though, the rin mutant fruits never ripen in response to ethylene treatment, exogenous application of ethylene was found to modulate expression of some of the ethylene-responsive genes, revealing retention of ethylene sensitivity and confirming its activity upstream to ethylene-mediated regulation of fruit development (Lincoln and Fischer 1988; Lanahan et al. 1994). Trans-activation potential of RIN has been demonstrated and it was found that this protein binds to CArG box present in the promoters of 1-aminocyclopropane-1-carboxylic acid synthases (ACS2, ACS4), β–galactosidase 4 (TBG4), endo-β-mannanase 4 (MAN4) and expansin1 (EXP1) genes, but not to the promoter of 1-aminocyclopropane-1-carboxylic acid oxidase 1(ACO1) gene (Ito et al. 2008; Fujisawa et al. 2011; Martel et al. 2011). In addition, RIN has also been shown to interact with the promoters of several ripening regulators including, NOR, CNR, HB-1, TDR4 along with its own promoter suggesting that RIN exerts its function during ripening via, at least in part, controlling the expression of other ripening regulators (Martel et al. 2011). These findings suggested that during fruit ripening, RIN gene causes direct transcriptional activation of several genes of ethylene biosynthesis, signaling and responses, cell wall metabolism and carotenoid biosynthesis pathways (Ito et al. 2008; Fujisawa et al. 2011; Martel et al. 2011).

Comparative study of wild-type and an ethylene-insensitive mutant, never-ripe (Nr), has revealed multiple points of ethylene control during fruit development (Alba et al. 2005). Fruits of Nr mutant share similarities with the fruits of the rin mutant as both exhibit greatly reduced endogenous ethylene synthesis and do not ripen even after exogenous treatment. The fact that RIN is functional in Nr mutant suggests that ethylene inhibition and non-ripening phenotypes in Nr and rin mutant fruits are independent of each other. Transcriptome analysis of rin mutant can help to investigate RIN-regulated ethylene-dependent/independent events related to fruit ripening. In tomato, rin mutation has been used to study the expression of genes involved in fruit ripening (Lincoln and Fischer 1988; Picton et al. 1993; Vrebalov et al. 2002; Meli et al. 2010). Recently, transcriptome, proteome and metabolome analyses of nor, rin and Nr mutant fruits revealed novel regulatory interactions during fruit development and further provided new insights into the molecular biology of ethylene-mediated ripening regulatory networks in tomato (Osorio et al. 2011). In the present study, an attempt was made to identify the genes which are regulated by RIN and to know the possible way by which RIN could regulate these genes. To understand these aspects a study of ripening-specific genes at genome-wide scale was carried out by performing a microarray-based analysis of five fruit ripening stages of Pusa Ruby and analyzed the genes, which followed the expression pattern of RIN gene. Architecture of promoters of ripening-related genes affected by the rin mutation was analyzed to understand the mechanism of regulation of these genes during ripening.

Materials and methods

Plant material and experimental design

Wild-type tomato plants (Solanum lycopersicum cv. Pusa Ruby; a truss tomato variety) and a homozygous line for the rin mutation (LA1795) in the unknown background as reported in Tomato Genetic Resource Center (tgrc.ucdavis.edu/) were grown in a greenhouse at 28 ± 1°C with 16-h supplemental lighting every day. To harvest fruit from mature green (MG) stage, first fruits were tagged 7 DAP and then harvested at 35 DAP stage. In our study, breaker (B) stage (when first sign of carotenoid accumulation on the external surface of fruit occurs) was observed at 40 DAP. In case of B + 3, B + 5 and B + 20 stages, fruits were harvested 3, 5 and 20 days post breaker, respectively. At B + 5 stage, fruits were fully red due to accumulation of lycopene. Since rin mutant does not show ripening phenotype, it was not possible to get breaker and red-ripe stages, hence, stages chronologically similar to MG, B, B + 3, B + 5 and B + 20 of wild-type cultivar were taken for microarray studies with the rin mutant. Three fruits per stage per plant were harvested for normal as well as rin mutant plants. Visual inspection of fruits was performed to assess developmental uniformity and fruits appearing developmentally equivalent were selected for transcriptome analyses. Fruits were cut into two halves. Locular and watery tissue was discarded and pericarp was frozen in liquid N2 and stored at −80°C till further use.

RNA extraction and microarray experiments

Total RNA was extracted from approximately 2 g of fruit tissue using hot phenol method (Reymond et al. 2000). RNA quality was checked on agarose formaldehyde gel and quantified using Nanodrop (ND-1000 spectrophotometer). Seven micrograms of RNA with 260:280 ratios of 1.9–2.0 and 260/230 ratio more than 2.0 from each sample was used for cDNA synthesis. Affymetrix GeneChip® Tomato arrays representing 10,038 probe-sets were used to study the transcriptome profiles during fruit ripening in wild-type and the rin mutant fruit. Labeling, hybridizations and microarray analysis were carried out, as described earlier (Arora et al. 2007; Kumar et al. 2010) and are briefly summarized here. Hybridization was performed in GeneChip® hybridization oven 640 for 16 h at 45°C and 60 rpm. GeneChips were washed and stained with streptavidin–phycoerythrin using the fluidics protocol EukGE_WS2V5_450 in Affymetrix fluidic station model 450. Finally, chips were scanned using the GeneChip® Scanner 3000.

Microarray data analysis

CEL files generated by GeneChip® Operating Software (GCOS) were analyzed using ArrayAssist™ 5.0. Data were normalized using GCRMA algorithm and log transformed. To obtain expression values, average values of three biological replicates were used. Cluster analysis on rows was performed using Euclidean distance metrix and Ward’s linkage rule of hierarchical clustering. Differential expression analysis was carried out using mature green fruit as reference to identify genes showing greater than twofold differences (at p value ≤ 0.05) in expression at B stage. Similarly, the preceding stage was used as reference for B + 3, B + 5 and B + 20 stages in wild-type fruit as well as in chronologically similar stages of rin mutant fruit at p values ≤ 0.05 and Benjamini Hochberg correction for FDR (False Discovery Rate) was applied. Similarly, fruit stage-specific comparison between different stages of wild-type and rin mutant fruits was carried out and analysis included Benjamini Hochberg correction for FDR at p value ≤ 0.01. Further, K-means clustering was applied to identify the co-expression profiles of differentially expressed genes during fruit ripening in wild-type fruit pericarp. All the genes, fulfilling the criteria for genes expressed differentially between wild-type and rin mutant fruits, were used for further analysis irrespective of their expression pattern during ripening in both types of plants. Annotations of genes differentially expressed during ripening were retrieved from Tomato Functional Genomic Database and Affymetrix website (http://affymetrix.com/products/arrays/specific/tomato.affx). The genes without annotations were excluded from further analysis. A total of 261 genes (p values ≤ 0.05, FC ≥ 3) that exhibited ripening-associated expression were selected to form cluster sets. Raw microarray data have been submitted to Gene Expression Ominus database at the National centre for Biotechnology Information under the series accession number (GSE20720). To compare rin microarray data with Nr mutant data, TOM1_Affy_probe mapping as well as a name search was carried out to identify the corresponding genes in the two datasets (Alba et al. 2005).

Promoter sequence extraction and analysis

Using Affymetrix array probe IDs, target sequences were extracted from Tomato Functional Genomics Database (TFGD; http://ted.bti.cornell.edu/) (Fei et al. 2006). These sequences were used to blast search tomato BAC sequences and tomato whole genome sequence data in Sol Genomic Network (SGN) database. Sequences having ≥98% homology were used for further analysis. Gene structure of these BAC sequences was predicted using FGENESH (http://linux1.softberry.com/berry.phtml?topic=fgenesh&group=programs&subgroup=gfind) and 2 kb sequences upstream to predicted open reading frames were extracted. All the promoter sequences were analyzed using database of Plant cis-acting Regulatory DNA Elements (PLACE; Higo et al. 1999). Results were then examined for the presence of cis-regulatory elements using Microsoft Excel.

Ethylene treatment

Ethrel (2-chloroethylphosphoric acid) treatment was given to 10-day-old tomato seedlings by a 30-min dip in 5 mM aqueous solution (Katz et al. 2006). Seedlings dipped in water served as control for the experiment. Seedlings were kept at room temperature for the duration of experiment. Seedlings were frozen in liquid N2 after 30 min and 2 h of treatments. For each time point, three independent experiments were carried out.

Quantitative real-time PCR (QPCR)

Quantitative real-time PCR analysis was carried out using the same RNA samples that were used for microarray analysis described earlier. Briefly, primers were designed from the 3′ end of the selected genes using PRIMER EXPRESS version 2.0 (PE Applied Biosystems, USA) with default parameters. Each primer was checked using the BLAST program of NCBI database for its specificity, which was again confirmed by dissociation curve analysis after the PCR reaction. The information about the primers used for the expression analysis of the 14 ripening-associated SlERF genes by QPCR has been reported in our earlier study (Sharma et al. 2010). A total of 2 μg of total RNA was used for first strand cDNA synthesis using high-capacity cDNA Archive kit (Applied Biosystems, USA). The cDNA samples were used for QPCR analysis and reactions were carried out in 96-well optical reaction plates (Applied Biosystems, USA), using ABI Prism 7000 Sequence Detection System and Software (PE Applied Biosystems, USA). To normalize sample variance, GAPDH was used as endogenous control. For PCR reactions, ∆CT values were obtained by subtracting CT (cycle threshold) value of GAPDH from CT value of the gene. The relative mRNA level was then calculated by formula: relative mRNA level = 2−∆∆CT. To validate microarray data by QPCR, fold change values obtained by both microarray and QPCR for 12 genes were transformed to log2 scale. These log2 values were plotted against each other to find the correlation coefficient between the two datasets.

Results

To identify differentially expressed genes in tomato pericarp, Affymetrix GeneChip® Tomato Genome Arrays were used. Five fruit development stages namely, MG, B, B + 3, B + 5 and B + 20 were selected to identify ripening-specific transcriptional changes influenced by RIN during fruit ripening. Upon transcriptome analysis, profound changes were observed in fruit transcriptome during transition from stage MG to B, B to B + 3, B + 3 to B + 5 and B + 5 to B + 20 in the pericarp of wild-type fruits; however, in rin mutant fruit pericarp, expression variations were not significant during transition from stage MG to B and B to B + 3. The major change in transcriptome occurred during transition from B + 3 to B + 5 and B + 5 to B + 20. A total of 2,157 genes were identified that were differentially regulated during fruit ripening in wild-type, while 2,425 genes were differentially expressed in the rin mutant fruit. When unique and common sets of genes were analyzed in wild-type and the rin mutant during ripening stages, 1,184 genes were found to be differentially regulated only in wild-type while 1,452 genes were differentially regulated only in the rin mutant, and a common set of 973 genes was found to be differentially regulated in both wild-type and the rin mutant fruits during fruit ripening. Gene ontology annotations of differentially expressed genes suggested that ~16% of the differentially expressed genes encoded regulatory proteins, including transcription factors and components of various signal transduction pathways operative during ripening (Fig. 1; Supplementary Fig. Sf1; Supplementary Tables S1 and S2).

Fig. 1
figure 1

Venn diagram representing genes differentially expressed during different stages of fruit ripening in wild-type and rin mutant fruit. The differential expression is between different stages of wild-type fruits in one set and between different stages of rin mutant in another set. A total of 2,157 and 2,425 genes were differentially regulated between four transitions, i.e. MG-B, B-B + 3, B + 3-B + 5 and B + 5-B + 20 in wild-type and rin mutant fruits, respectively. A common set of 973 genes was found to be differentially regulated in both wild-type and rin mutant fruits during ripening

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Validation of microarray data by QPCR

Twelve genes, including those exhibiting high-differential expression as well as genes showing less than twofold change in their expression between wild-type and rin mutant fruits were selected for validation by QPCR analysis (Supplementary Table S3). Figure 2 shows a comparison of the microarray log2 fold change values with the QPCR log2 fold change values at the five points between rin mutant and wild-type. High correlation (R 2 = 0.96) between microarray and QPCR data was obtained which indicates that the expression analysis by both the approaches was in good agreement to each other.

Fig. 2
figure 2

Validation of microarray data by QPCR. Correlation analysis included QPCR of 12 genes and fold change values between wild-type and the rin mutant. The fold changes in gene expression were transformed to log2 scale. The microarray data log2-values (X-axis) were plotted against the QPCR log2 values (Y-axis)

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Analysis of expression of ripening-related genes during ripening

Data about biochemical changes occurring in tomato during fruit ripening is available for various tomato cultivars. We analyzed expression of several well-characterized ripening-related genes belonging to ethylene biosynthesis and signaling, carotenoid biosynthesis, cell wall metabolism, antioxidant production and genes encoding transcription factor, in Pusa Ruby. Genes such as, E8 (Les.3627.1.S1_at), ACO1 (Les.2560.1.S1_at), ACS2 (Les.3662.1.S1_at), ACS4 (Les.3661.1.S1_at), never ripe (Les.85.1.S1_at; NR), phytoene synthase 1 (Les.3171.3.S1_a_at; PSY1), plastid terminal oxidase (Les.4455.1.S1_at; GH +), lipoxygenase (Les.3980.1.S1_at; LoxC), polygalactouronase (Les.3654.1.S1_at; PG2A), endo-1,4-beta-glucanase (Les.3666.1.S1_at; Cel2), RIN (Les.4450.1.S1_at), and TDR4 (Les.4461.1.S1_at) showed ripening-associated up regulation at B stage in wild-type fruits. We also studied effect of the rin mutation on the expression of these genes. Expression of most of the genes was found to be severely inhibited at more than one stage of ripening in the rin mutant fruits. Although no such drastic decrease in the expression levels of genes E8 and TDR4 was observed, yet their expression was found to be substantially inhibited in the rin mutant fruits (Fig. 3).

Fig. 3
figure 3

Expression analysis of ripening-induced genes in wild-type and their altered expression in the rin mutant fruits. Fruit developmental stages used for expression profiling are mentioned on top of each column. On the left side gene symbols are present. On the right side of expression map color bar is present which represents log2 expression values, thereby green color representing low-level expression, black shows medium-level expression and red represents high-level expression. ACO1 1-aminocyclopropane-1-carboxylic acid oxidase 1, E8 ripening-specific E8 protein homolog, PSY1 phytoene synthase 1, GH + plastid terminal oxidase, NR never-ripe, ACS2 & ACS4 1-aminocyclopropane-1-carboxylic acid synthase 2 & 4, RIN ripening inhibitor MADS-box transcription factor, TDR4 MADS-box transcription factor TDR4, PG2A polygalacturonase-2A, Cel2 endo-1,4-beta-glucanase 2, LoxC lipoxygenase C

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Cluster analysis of ripening-associated genes

In order to find out events controlled by RIN, we did cluster analysis of the ripening-associated genes by focusing on the genes which exhibited similar expression pattern to that of RIN and analyzing the effect of rin mutation on the expression of these genes during ripening. Since RIN is known to induce expression of genes at the onset of ripening, we mainly focused on the genes that showed ≥threefold up regulation at the initiation of fruit ripening and a total of 261 ripening-induced genes were identified. Cluster analysis demonstrated high co-ordination in expression of many genes differentially expressed during ripening. These genes were categorized into four major groups using K-means clustering. These major groups were further divided into subgroups. The difference among the various subgroups present in a major group is at the initial expression levels as well as difference in their transcript accumulation levels between two stages of ripening. Genes belonging to group 1 showed high-transcript accumulation from MG to B stage, which did not deviate significantly up to B + 20 stage. This group was further divided into six subgroups, namely 1a, 1b, 1c, 1d, 1e and 1f. Several genes belonging to ethylene biosynthesis and signaling, secondary metabolite production, signal transduction and cell wall metabolism were present in the group 1. The RIN gene followed the pattern of group 1c genes. Besides RIN, important genes of ethylene biosynthesis and signaling and secondary metabolite production, including ACO1, E8, NR, protein-methionine-S-oxide reductase (Les.2899.1.S1_at), PSY1, flavonol synthase (Les.3085.1.S1_at), GH +, flavonoid glucoyltransferase (Les.2403.2.S1_at) and a 14-3-3 protein encoding genes were present in this cluster. Group 1e included genes such as ACS2, ACS4, ethylene-responsive proteinase inhibitor 1 (Les.3619.1.S1_at), histidine decarboxylase (Les.290.1.S1_at), LoxC, PG2A, and Cel2. Group 2 genes exhibited increased expression up to B + 3 stage and showed a decline from B + 3 to B + 5 followed by an increase up to B + 20 stage. Expression of group 3 genes was induced from MG to B transition that decreased up to B + 3 and showed an increase up to B + 5 followed by a decline up to B + 20 stage. Genes belonging to group 4 showed very low or negligible expression level and displayed mixed expression patterns. Expression pattern of genes, present in different groups, was also studied in the rin mutant fruit which was found to be deviated to great degree in the mutant background. For example, ripening-associated induction of genes belonging to subgroups 1e, 1f and 3a clearly exhibited inhibition in their expression during early stages of ripening in the rin mutant fruit. Along with the substantial inhibition, the co-expressed genes, in wild-type fruit, showed a clear loss of co-ordination in their co-expression in the rin mutant fruit (Fig. 4; Supplementary Table S4).

Fig. 4
figure 4

K-means clustering of 261 ripening-induced genes during fruit ripening in wild-type and their altered expression in the rin mutant fruits. Clusters with similar trends of expression during various stages of fruit development in wild-type have been grouped together into four groups. Further, subgrouping of a cluster is done on the basis of levels of up regulation of genes from MG to B stage

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Ethylene biosynthesis and signaling during ripening

Ethylene production is inhibited in the rin mutant fruit. Moreover, even exogenous ethylene is unable to induce ripening in this mutant. Therefore, in addition to the genes belonging to ethylene biosynthesis, we also analyzed genes involved in ethylene signaling in fruits of this genotype. This analysis resulted in identification of 39 genes (including genes encoding TFs) that were differentially regulated at least at one stage of ripening in the rin mutant fruits. Some of the characterized genes of ethylene biosynthesis pathway exhibited similar expression pattern as described earlier in published data (Table 1) (Lincoln et al. 1987, 1993; Lincoln and Fischer 1988; Dellapenna et al. 1989; Lanahan et al. 1994; Zegzouti et al. 1997, 1999; Barry et al. 2000; Guo et al. 2001; Alba et al. 2005). While genes for enzymes involved in ethylene biosynthesis such as ACO1, ACS1A, ACS2, ACS4, S-adenosyl-l-methionine synthase 2 (SAM2) were upregulated at the onset of ripening in wild-type fruit, we observed down regulation of these genes in the rin mutant. Genes belonging to ethylene perception and signal transduction pathway also displayed differential expression patterns in the rin mutant fruits. While ER1, ER60, TCTR1, TCTR4, ETR3, ETR4, and E8 were downregulated, ETR5, EIN2, EIL1, and EIL2 were upregulated in the rin mutant fruits. Expression of several genes encoding ERF transcription factors, including SlERF2, 29, 30, 31, 35, 39, 60 and 79 was also found to be modulated in the rin mutant fruits. While SlERF2, 30 and 31 exhibited modulation in their expression at early stages of ripening, expression of the remaining SlERFs was modulated at the late stage (B + 20) of ripening in the rin mutant fruits (Table 1; Supplementary Table S5). In addition to ethylene, genes related to auxin, jasmonic acid, gibberellins, etc. were also found to be differentially regulated between wild-type and rin mutant fruits and the auxin-related genes were the second most represented, after ethylene-related genes in the hormone responses category during ripening (Supplementary Fig. Sf2).

Table 1 Genes belonging to ethylene biosynthesis and signaling categories differentially regulated (p ≤ 0.01) between wild-type and the rin mutant fruits during ripening
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Expression analysis of recently identified ERF genes in wild-type and rin mutant during ripening

Recently, genome-wide characterization of ERF genes resulted in identification of 85 SlERF genes in tomato (Sharma et al. 2010). Of these, 44 SlERFs were found to have corresponding probe-sets on GeneChip® and several of these genes showed differential expression during ripening in tomato. This study indicated up regulation of several ERF genes in fruit tissue at the onset of ripening. In order to establish the effects of the rin mutation on the expression of these genes during early and late ripening stages, QPCR analysis for 14 ERF genes (those exhibiting ripening-associated transcript accumulation but not presented on GeneChip®) during ripening in wild-type and rin mutant fruits was carried out (Sharma et al. 2010). Strong inhibition in the expression of SlERF11 and SlERF81 genes was observed in the rin mutant fruits. Expression of SlERF61 was found similar in both the genotypes while SlERF83 gene showed higher-expression levels in the rin mutant fruits. The remaining SlERF genes exhibited varied expression patterns between wild-type and the rin mutant fruits (Fig. 5).

Fig. 5
figure 5

QPCR analysis of genes encoding ERF transcription factors during ripening in the fruits of wild-type and the rin mutant plants. The X-axis represents the fruit developmental stages while Y-axis shows relative expression in folds between wild-type and the rin mutant fruits

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Analysis of promoter elements of 79 genes co-expressed with RIN during fruit ripening and downregulated in rin mutant fruits

Cluster analysis resulted in identification of 112 genes (including subgroups 1a–f and 3a), which exhibited expression profile similar to that of RIN in wild-type fruits and their expression was altered in the rin mutant fruits. Following these two criteria, BLASTN search was performed using coding sequence of these 112 genes as query sequences but putative promoter sequences for only 79 genes could be extracted from Solanaceae Genome Network (SGN) tomato BAC and tomato whole genome sequences databases. Analysis of putative promoter sequences for the presence of various cis-regulatory elements resulted in the identification of binding sites for MADS-box TFs (CArGCW8GAT) and ERFs (EREGCC, GCCCORE, and ERELEE4). When compared with the cis-element data for genes with no substantially altered expression during ripening in wild-type, these promoter sequences clearly showed a marked increase in frequency of CArG box (2.91 in comparison to 1.91) and ERE elements (1.43 in comparison to 1.06) in genes exhibiting RIN-like expression pattern (Table 2). Of these 79 genes, promoters of 52 genes had both CArG box and ERE elements. To get more insights into RIN-regulated ethylene-dependent as well as ethylene-independent events underlying ripening and to validate our cis-element data, effect of exogenously applied ethylene on the expression of these genes was studied. Ethylene treatment was given to 10-day-old tomato seedlings. For this, 25 genes involved in the ethylene biosynthesis and signaling, auxin signaling, carotenoid production, cell wall metabolism, and genes encoding regulatory proteins along with a few genes with unknown function were selected. Out of these, nine genes did not respond to externally applied ethylene at both the stages while six genes showed up regulation at only one of the stages of treatment. The remaining genes were upregulated at both the stages of ethylene treatment (Table 3; Supplementary Table S6). Moreover, we compared the microarray data of both rin and Nr mutants mainly focusing on the genes that displayed altered expression in rin mutant fruit. Some of the genes important for hormone responses, carotenoids biosynthesis, cell wall metabolism and genes encoding regulatory components of signaling pathways, ACO1, GH +, PG-2A, Pti5, Pti6, etc. were affected by both rin and Nr mutations, suggesting that these genes might be regulated indirectly by RIN via ethylene (Supplementary Table S5).

Table 2 Frequency of cis-regulatory elements in the 2-kb putative promoter region of genes differentially expressed during fruit ripening stages between rin mutant and wild-type fruit pericarp
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Table 3 Study of ethylene responsiveness of ripening-related genes by QPCR analysis along with the status of CArG and ERE elements in their promoters
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Discussion

The transcriptome analysis of a cultivated Indian cultivar Pusa Ruby and the rin mutant fruits was carried out to get insight into RIN-regulated ethylene-dependent as well as ethylene-independent events underlying the ripening process. Five stages, namely MG, B, B + 3, B + 5 and B + 20 of fruit ripening in wild-type and chronologically similar stages of the rin mutant fruits have been selected in this study. Analysis of the expression of some of the well-characterized genes related to ethylene biosynthesis and signaling (E8, ACS2, ACS4, ACO1, and NR), carotenoid biosynthesis and volatile production (PSY1, GH +, and LoxC), cell wall metabolism (PG2A and Cel2) and genes encoding transcription factors (RIN and TDR4) indicated that similar kind of molecular changes occur at the onset of ripening in Pusa Ruby as already described in the literature for commonly studied cultivar, Ailsa Craig (Dellapenna et al. 1987, 1989, 1990; Lincoln and Fischer 1988; Giuliano et al. 1993; Fraser et al. 1994; Lashbrook et al. 1994; Griffiths et al. 1999; Barry et al. 2000; Lois et al. 2000; Seymour et al. 2002; Vrebalov et al. 2002; Alba et al. 2005; Katz et al. 2006; Osorio et al. 2011). Inhibited expression of these genes in the rin mutant is in accordance with the earlier report suggesting the necessity of functional RIN protein along with the ethylene for their up regulation during ripening (Osorio et al. 2011). Further, we have also compared the expression pattern of some of the already well-characterized genes during ripening between Pusa Ruby and Micro-tom varieties and similar type of expression variance during ripening for the most of the genes indicated that the various aspects of ripening are highly conserved in different cultivars of tomato. Whereas difference in the expression pattern in case of a few genes also suggests that altered expression of some of the genes in the rin mutant (LA1795) fruits could also be due to allelic variation between the two genotypes used for fruit transcriptomes comparison in the present study (Supplementary Table S7).

Transcriptome dynamics of tomato fruit pericarp in wild-type and rin mutant

In the recent past, several reports on the transcriptional changes occurring during ripening process in fleshy and non-fleshy fruits have appeared (Alba et al. 2005; Anjanasree et al. 2005; Lemaire-Chamley et al. 2005; Kok et al. 2007; Kolotilin et al. 2007; Pilati et al. 2007; Trainotti et al. 2007; Janssen et al. 2008; Mounet et al. 2009; Srivastava et al. 2010; Ozaki et al. 2010; Osorio et al. 2011; Rohrmann et al. 2011). In this study, analysis of differentially expressed genes during fruit development in wild-type and the rin mutant resulted in identification of a set of 1,184 genes differentially regulated during ripening only in wild-type fruit and indicated the importance of these genes during ripening. A common set of 973 genes, exhibiting differential regulation in both wild-type and the rin mutant fruits during ripening, indicated that these genes might not be controlled by RIN. A separate set of 1,452 genes differentially regulated only in the rin mutant indicated that loss of function of RIN altered the expression pattern of these genes. The presence of a large number of differentially expressed genes at the onset of fruit ripening in wild-type fruit while very few at chronologically similar stage in the rin mutant revealed the impact of the loss of function of RIN gene on the transcriptome of rin mutant fruit. This study also confirmed the earlier report which implies the importance of trans-activation property of RIN protein at the onset of ripening in wild-type fruit (Ito et al. 2008; Fujisawa et al. 2011; Martel et al. 2011).

Cluster analysis of ripening-associated genes

For elucidation of functional relationships among genes, co-expression analysis has turned out to be a powerful approach. In tomato, several studies have demonstrated the presence of tightly regulated co-expression modules in general as well as in tissue-specific manner during fruit development (Alba et al. 2005; Lemaire-Chamley et al. 2005; Carrari and Fernie 2006; Mintz-Oron et al. 2008; Srivastava et al. 2010; Ozaki et al. 2010). Ozaki et al. (2010) identified 199 tightly regulated co-expression modules associated with various biological processes in tomato. Alba et al. (2005) showed that the Nr mutation results in loss of tight regulation of co-expressed genes, which is the reason for non-ripening phenotype of its fruits. In the present study, the presence of RIN along with several functionally related genes belonging to various categories, such as ethylene biosynthesis and response (NR, E8, ACO1, ACS2, and ACS4), carotenoid biosynthesis (PSY1 and GH +) and cell wall metabolism (PG2A and Cel2) in subgroups 1c and 1e indicates the high level of co-ordination among their expression during fruit ripening. Since RIN protein has been shown to bind to the CArG box present in the promoters of several of these genes, it is possible that other genes of this group might also serve as putative targets for RIN protein (Fujisawa et al. 2011; Martel et al. 2011). Cluster analysis demonstrated that the tight regulation among functionally related genes was lost in the rin mutant background, suggesting that ripening-related changes in transcriptome of tomato fruit pericarp depend a great deal on the availability of functional RIN protein during ripening (Ito et al. 2008).

Analysis of putative RIN-regulated promoters

In order to find out whether the 79 genes which had expression pattern similar to that of RIN and showed down regulation in the rin mutant are part of ethylene-dependent or ethylene-independent aspect of ripening, we followed a similar approach as described by Lenka et al. (2009). Higher frequency of occurrence of CArG box element within the 79 promoters indicated that there is higher probability of direct interaction of RIN with promoters of these ripening-related genes. These results are in accordance with the earlier report where 64% of a total of 2,000 sites of MADS-box protein AGL15 had clearly defined CArG box element (Zheng et al. 2009). High frequency of ERE elements demonstrated that ethylene would also be involved in their regulation during ripening. Effect of exogenously applied ethylene on the selected genes provided confidence to the in silico data on cis-elements as five out of seven genes (Les.3666.1.S1_at, Les.3627.1.S1_at, Les.4461.1.S1_s_at, Les.5579.1.S1_at, LesAffx.187.1.S1_at, LesAffx.15004.1.S1_at, and LesAffx.57437.1.S1_at) which did not show any up regulation in their expression either at early or late stage of treatment lacked ERE elements. This finding suggests that these genes might serve as direct targets for RIN protein. Three genes (Les.3171.3.S1_a_at, Les.3388.2.S1_at, LesAffx.58502.1.S1_at) lacked ERE element but their ethylene inducibility indicated that these genes would be regulated by both ethylene as well as RIN, as has also been reported in case of ACS2 and PG genes (Lincoln et al. 1987; Barry et al. 2000; Ito et al. 2008; Karlova et al. 2010; Fujisawa et al. 2011; Li et al. 2011a). Ethylene inducibility of the remaining genes, having ERE elements, suggests that these genes would be involved in ethylene-dependent aspect of ripening. Recent studies where several fold enrichment of CArG boxes in the promoters of several ripening-associated, including ACS2, ACS4, E4, E8, NR, PG, TBG4, LeEXP1, and LeMAN4, was observed, also supports our finding that along with these genes other co-expressed genes reported in the present study may also serve as sites for the direct interaction of RIN with their promoters in vivo (Fujisawa et al. 2011; Martel et al. 2011). In addition to interaction of RIN with the ripening-related genes, other genes not showing such expression, such as HB-1, can also serve as targets for RIN as reported by Martel et al. (2011). It was proposed that since RIN is SEP3 class protein, known to act as bridging protein in the formation of higher complex with other MADS-box proteins, it is possible that RIN as a part of such complex could bind to the promoters of target genes without necessarily activating their expression. Further, requirement of additional regulators, such as CNR, for the interaction of RIN to the target genes justifies that RIN, along with other regulators, might be involved in indirect regulation of ripening-related genes during fruit ripening (Martel et al. 2011). Similarly, altered expression of a large number of genes only in rin mutant fruits, in the present study indicates that this set of genes can further be utilized to identify additional putative targets for RIN, in future. Similar to the earlier published studies, genes identified in this study broaden the scope for research into the molecular mechanisms of RIN-regulated ethylene-dependent as well as ethylene-independent aspects of fruit ripening (Lincoln et al. 1987; Zegzouti et al. 1997, 1999; Fujisawa et al. 2011; Martel et al. 2011).

Identification of new regulators of ethylene responses and effect of RIN on their expression during ripening

Induction of ripening-associated genes involved in ethylene biosynthesis, such as ACS1A, ACS2, ACS4, and ACO1, in wild-type fruit is in accordance with the earlier findings while inhibition in their expression in the rin mutant fruits explained as to why no climacteric production of ethylene is observed in fruits of the rin mutant (Lincoln et al. 1993; Barry et al. 1996, 2000; Nakatsuka et al. 1998; Vrebalov et al. 2002; Yokotani et al. 2004; Li et al. 2011a, b). Modulated expression of genes encoding three ethylene receptors proteins (ETR3-5) as well as downstream components of ethylene signal pathways, such as TCTR1, 4, EIN2, EIL1, 2, 4 and several ERFs suggests that ethylene signal transduction also gets affected during fruit development in the rin mutant. Modulated expression of several ethylene-responsive genes, including ER1, ER5, ER24, ER49 and ER60 indicated that due to lesser ethylene production, expression of these genes is altered in the rin mutant fruits (Zegzouti et al. 1997; Klee 2002). Recently, RIN has been shown to interact with the promoters of genes involved in ethylene signaling and responses and this interaction was found to be lost in the rin mutant background suggesting that beside low-ethylene levels, unavailability of functional RIN protein could also be one of the reasons for their down regulation in rin mutant fruits (Martel et al. 2011). In addition, several ERF genes have been identified and characterized in tomato (Tournier et al. 2003; Pirrello et al. 2006; Li et al. 2007; Chen et al. 2008; Chung et al. 2010; Karlova et al. 2010). In this study, expression analysis of ripening-associated ERF genes, which are not present on GeneChip®, in the rin mutant fruits, indicates that more than one ERF gene would be involved in regulation of ripening in tomato and RIN might play significant role in their regulation. Significant alteration in the expression of SlERFs (SlERF29, 35, 39, 60, 61, 79, and 83) at late ripening stage (B + 20) or no substantial inhibition in the expression of SlERF61 and SlERF83 or up regulation of SlERF31 during ripening in rin mutant fruits suggests that these genes would be involved in regulation of fruit ripening in the RIN-independent manner. Modulated expression of already characterized SlERFs, SlAP2a (as represented SlERF42) and SlERF2, in rin mutant fruit suggests that RIN might play some role in the regulation of these genes during ripening (Zhang et al. 2009; Chung et al. 2010; Karlova et al. 2010). Strong inhibition in the expression of SlERF11 and SlERF81 genes in the rin mutant fruit further indicates that these genes might be directly regulated by RIN during ripening. Beside ethylene, differential expression of several genes related to auxin, jasmonic acid, gibberellin, etc. in the present study is in accordance with an earlier finding that emphasized on the involvement of other hormones and existence of complex interactive hormonal network during ripening (Osorio et al. 2011). Further, altered expression of genes, such as ACO1, GH +, PG-2A, Pti5, and Pti6, etc. in both rin and Nr mutants indicated that these genes would be regulated by RIN directly or via ethylene during ripening, thus confirming findings reported earlier as well as in this report (Lin et al. 2008; Martel et al. 2011). The absence of ACS2 and ACS4 genes among genes downregulated in Nr mutant and the presence of ACO1 in microarray data of genes downregulated in both the mutants confirms the earlier findings that RIN does not directly regulate ACO1 gene but it regulates ACS2 and ACS4 genes directly, hence, suggesting that microarray data of rin and Nr might be useful to identify the putative targets for RIN protein (Ito et al. 2008; Fujisawa et al. 2011; Li et al. 2011a, b).

In conclusion, we showed that similar to other spontaneous mutations, such as for Nr, high-pigment −dg (hp −2dg) and colorless non-ripening (cnr), the rin mutation also results in profound change in fruit transcriptome during ripening (Eriksson et al. 2004; Alba et al. 2005; Kolotilin et al. 2007; Osorio et al. 2011; Rohrmann et al. 2011). Functional classification of genes modulated in the rin mutant fruits suggested that this mutation significantly alters the regulatory networks during ripening. Cluster analysis identified genes exhibiting expression pattern similar to that of RIN gene and also unraveled the loss of tight regulation among ripening-associated co-expressed genes in the rin mutant fruits. Analysis of expression of genes belonging to ethylene biosynthesis and responses in depth suggested that besides ethylene biosynthesis, altered ethylene signal transduction responses could be the reason for non-ripening phenotype of fruits of the rin mutant, even upon exposure to external ethylene. In silico analysis of cis-elements in the promoters of ripening-induced genes identified putative targets of the RIN protein. The correlation between ethylene inducibility of these genes with the presence or the absence of cis-elements provided insights into RIN-regulated ethylene-dependent as well as ethylene-independent events of ripening. Further, functional validation of the ripening-associated genes identified in this study will help in dissecting the complexity of RIN-regulated ripening events at molecular level and the information generated here will augment the genetic manipulation of fruit ripening process targeting increased shelf-life and better fruit qualities.