Project description:[original title] Understanding the complexity of fruit ripening by transcriptome analysis of rin mutant fruit and in silico analysis of promoters of differentially regulated genes A tomato MADS-box transcription factor, LeMADS-RIN, controls fruit ripening and mutation in this gene results in non-ripening phenotype of fruit. This mutation down-regulates certain ripening related ethylene responses, however, other ethylene responses are normal. A complete understanding of this mutation and its effect on fruit transcriptome during ripening is not clear. In this study, microarray analysis has been used to investigate the influence of rin mutation on fruit transcriptome at different stages of ripening. A total of 2,398 genes were found to be differentially expressed in wild type fruit pericarp, which on cluster analysis indicated a major shift in their expression profiles in rin mutant fruit. A total of 1,802 genes were found to be differentially expressed between wild type and rin mutant fruits and 17% of these genes encoded regulatory elements, suggesting that mutation in LeMADS-RIN results in disturbance in the regulatory transcriptional networks during ripening. Since LeMADS-RIN has been reported to bind to the CArG box of LeACS2 promoter, in-silico analysis of 51 putative promoter sequences of the genes, that showed ripening associated up-regulation in wild type but showed impairment in up-regulation in rin mutant fruit during ripening, were searched for presence of CArG box along with ethylene and auxin responsive elements. The study revealed that only 24 putative promoter sequences harbor LeMADS-RIN specific CArG box suggesting an alternative mode of regulation by LeMADS-RIN for CArG box deficient genes.
Project description:[original title] Understanding the complexity of fruit ripening by transcriptome analysis of rin mutant fruit and in silico analysis of promoters of differentially regulated genes A tomato MADS-box transcription factor, LeMADS-RIN, controls fruit ripening and mutation in this gene results in non-ripening phenotype of fruit. This mutation down-regulates certain ripening related ethylene responses, however, other ethylene responses are normal. A complete understanding of this mutation and its effect on fruit transcriptome during ripening is not clear. In this study, microarray analysis has been used to investigate the influence of rin mutation on fruit transcriptome at different stages of ripening. A total of 2,398 genes were found to be differentially expressed in wild type fruit pericarp, which on cluster analysis indicated a major shift in their expression profiles in rin mutant fruit. A total of 1,802 genes were found to be differentially expressed between wild type and rin mutant fruits and 17% of these genes encoded regulatory elements, suggesting that mutation in LeMADS-RIN results in disturbance in the regulatory transcriptional networks during ripening. Since LeMADS-RIN has been reported to bind to the CArG box of LeACS2 promoter, in-silico analysis of 51 putative promoter sequences of the genes, that showed ripening associated up-regulation in wild type but showed impairment in up-regulation in rin mutant fruit during ripening, were searched for presence of CArG box along with ethylene and auxin responsive elements. The study revealed that only 24 putative promoter sequences harbor LeMADS-RIN specific CArG box suggesting an alternative mode of regulation by LeMADS-RIN for CArG box deficient genes. Three chronological stages of tomato (Solanum lycopersicon) fruit ripening were compared between wild type and rin mutant
Project description:The study of climacteric fruit ripening in tomato has been facilitated by the spontaneous ripening mutants Colorless non-ripening (Cnr), non-ripening (nor), and ripening inhibitor (rin). These mutants effect the genes encoding ripening transcription factors (TFs) SPL-CNR, NAC-NOR, and MADS-RIN causing pleiotropic defects to the ripening program. Here, we demonstrate that some ripening processes occur in the mutant fruit but at later stages of development compared to the wild type. The rin and nor mutant fruit exhibit similar quality traits to wildtype at later stages of ripening and senescence and delayed expression of ripening-associated genes. In addition, we propose that the Cnr mutant has a broader range of effects to fruit development than just fruit ripening. Cnr fruit show distinct differences from wild type in ripening phenotypic traits and gene expression profiles prior to the initiation of ripening. We provide new evidence that some mutants can produce more ethylene than basal levels and demonstrate ABA accumulation is also affected by the mutations. Studies have examined the relationship between the CNR, RIN, and NOR TFs based on protein-protein interactions and transcriptional regulation during fruit ripening. We describe the genetic interactions affecting specific fruit traits by using homozygous double mutants. Cnr predominantly influences the phenotype of the Cnr/nor and Cnr/rin double mutants but additional defects beyond either single mutation is evident in the transcriptome of the Cnr/nor double mutant. Our reevaluation of the Cnr, nor, and rin mutants provides new insights the utilization of the mutants in breeding and studying fruit development.
Project description:Fruits are unique to flowering plants and play a central role in seed maturation and dispersal. Molecular dissection of fruit ripening has received considerable interest because of the biological and dietary significance of fruit. To better understand the regulatory mechanisms underlying fruit ripening, we report here the first comprehensive analysis of the nuclear proteome in tomato fruits. Nuclear proteins were isolated from tomatoes in different stages of ripening, and subjected to iTRAQ (isobaric tags for relative and absolute quantification) analysis. The proteins that changed abundance across ripening stages are involved in various cellular processes. We additionally evaluated the changes in the nuclear proteome in the ripening-deficient mutant ripening inhibitor (rin) carrying a mutation in the transcription factor RIN. A set of proteins were identified and particular attention was paid to SlUBC32 and PSMD2, the components of ubiquitin-proteasome pathway. Through chromatin immunoprecipitation and gel mobility shift assay, we provide evidence that RIN directly bound to the promoters of SlUBC32 and PSMD2. Moreover, loss of RIN function affected protein ubiquitination in nuclei. SlUBC32 encodes an E2 ubiquitin-conjugating enzyme and genome-wide survey of the E2 gene family in tomatoes identified five more E2s as the direct targets of RIN. Two E2s were demonstrated to be involved in the regulation of fruit ripening based on virus-induced gene silencing assays. Our results uncover the novel function of protein ubiquitination, identifying specific E2s as regulator in fruit ripening. These findings contribute to the unraveling of the gene regulatory networks that control fruit ripening.
Project description:In this study, we explored the metabolome and transcriptome of the ripe fruit in nine landrace accessions representing the seven genetic groups and compared them to the mature fruit of the wild progenitor S. pimpinellifolium. The goal is to shed light in understanding the factors responsible for acquiring tomato fruit quality (taste and flavour) at molecular level during the domestication process.
Project description:Ripening is an important stage of fruit development to determine its quality as a diet. A tomato (Solanum lycopersicum) MADS-box transcription factor, RIPENING INHIBITOR (RIN), has been believed to serve as a regulator of ripening lying upstream of ethylene-dependent and ethylene-independent pathways. Here, we have conducted global gene expression analysis to comprehensively identify tomato genes whose expressions are affected by the rin mutation using microarray with RNA samples from the normal and rin mutant tomato fruits at the pre-ripening (mature green) and ripening (pink coloring) stages. By analysing this microarray data, we identified 342 of positively regulated and 473 negatively regulated genes by RIN, which showed >5 and <0.2 of the fold change ratio (FC) of normal fruits at the ripening stage relative to those at the pre-ripening stage, respectively, in a RIN-dependent manner. A chromatin immunoprecipitation (ChIP) analysis of the normal ripening tomatoes with the anti-RIN antibody revealed that the positively regulated gene set contained at least 13 direct RIN targets.
Project description:The tomato MADS-box FRUITFULL (FUL) homologs, FUL1 and FUL2, interact with the main ripening regulator RIPENING INHIBITOR (RIN). To clarify their role in fruit ripening, we generated FUL1/FUL2-suppressed transgenic lines by RNAi. We found that five transgenic lines bearing fruits that did not ripen normally: lycopene accumulation and increase of ethylene production were severely inhibited. We then performed next generation RNA sequencing (RNA-Seq) analysis of the fruits of a FUL1/FUL2-suppressed line (TF18) with those of the wild type (Ailsa Craig cultivar; AC) and rin mutant. The comparison of RNA-Seq data among them indicated that FUL1/FUL2-suppression significantly affected the expression of a larger portion of ripening-induced and -repressed genes than the rin mutation did. Moreover, the effect of FUL1/FUL2-suppression was observed not only in the fruits harvested at the wild type ripening age [45 days after pollination (DAP)] but also in those at the pre-ripening age (35 DAP). This suggests that the FUL homologs play an essential role in the regulation of fruit development and ripening, the role which covers a wider range of biological processes than RIN does. Differentially expressed genes (DEGs) between the wild type and TF18 fruits included known ripening-related genes such as ACS2 and ACS4 involved in ethylene production and PSY1 in carotenoid biosynthesis, consistent with the phenotype of TF18 fruits described above. The DEGs also included many direct RIN target genes, which supports the hypothesis that the FUL homologs regulate fruit ripening in a form of MADS-box complex with RIN.