Project description:Fruits of transgenic tomato (Solanum lycopersicum) plants engineered with ripening-induced, yeast S-adenosylmethionine decarboxylase (ySAMdc) gene, accumulate the higher polyamines spermidine (Spd) and spermine (Spm) and demonstrate ameliorated phytonutrient content, juice quality, and prolonged vine life. Enhanced nitrogen-carbon interactions were revealed by comprehensive Nuclear Magnetic Resonance (NMR) spectroscopy-based metabolite profiling of the transgenics, suggesting that Spd and Spm are perceived as nitrogenous metabolites by the fruit cells (Mattoo et al., 2006). The recent work by our colleagues identified the effects of Spd/Spm accumulation on various functional classes of tomato genes affected during ripening by probing 1522 ESTs on a custom-made array (Srivastava et al., 2007). In this study we monitored alterations of genome-wide transcriptional patterns in pericarp of Spd/Spm-accumulating tomatoes by means of direct comparison with azygous controls using DNA-microarray technology. Consistent with the ySAMdc expression pattern, very minor transcriptional alterations were detected in mature green developmental stage. For both breaker and red stages, large mutual and unique gene sets displayed altered levels of transcript. Ontological term analysis of up- and down-regulated transcript groups revealed processes in cell metabolism that are regulated by increased levels of Spd/Spm in ripening tomato fruits. These processes mainly involve carbohydrate and amino acids metabolism and protein synthesis. Additionally, transcript levels of representative genes encoding structural enzymes for related biosynthetic pathways show strong relationship to specific metabolites that were identified as regulated in Spd/Spm-accumulating transgenics.
Project description:DNA methylation is a conserved epigenetic mark that influences diverse biological processes in many eukaryotes. Recently, DNA methylation was proposed to regulate fleshy fruit ripening. Fleshy fruits can be distinguished by their ripening process as climacteric fruits, such as tomatoes, or non-climacteric fruits, such as strawberries. Tomatoes undergo a global decrease in DNA methylation during ripening, due to increased expression of a DNA demethylase gene. The dynamics and biological relevance of DNA methylation during ripening of non-climacteric fruits, or other climacteric fruits, are unknown. Here, we generated and characterized single-base resolution maps of the DNA methylome in strawberry fruit, from immature to ripe stages. We observed an overall loss of DNA methylation during strawberry fruit ripening. Thus, ripening-induced DNA hypomethylation occurs not only in climacteric fruit, but also in non-climacteric fruit. However, we discovered that the mechanisms underlying DNA hypomethylation during ripening of tomato and strawberry are distinct. Unlike in tomatoes, DNA demethylase genes were not up-regulated during ripening of strawberries. Instead, genes involved in RNA-directed DNA methylation were down-regulated during strawberry ripening. Further, ripening-induced DNA hypomethylation was associated with decreased siRNA levels, consistent with reduced RdDM activity. Therefore, we propose that DNA hypomethylation during strawberry ripening is caused by diminished RdDM activity. Finally, hundreds of ripening-related genes displayed altered expression that was associated with, and thus potentially regulated by, DNA hypomethylation during ripening. Our findings provide new insight into the DNA methylation dynamics during the ripening of non-climateric fruit and reveal a novel function of RdDM in regulating an important process in plant development.
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. We monitored global gene expression in normal (PK331 cultivar) and rin mutant (PK353 cultivar) tomatoes at the pre-ripening (mature green, G) and ripening (pink coloring, P) stages using microarray with three biological replicates for each sample.
Project description:Fruits of transgenic tomato (Solanum lycopersicum) plants engineered with ripening-induced, yeast S-adenosylmethionine decarboxylase (ySAMdc) gene, accumulate the higher polyamines spermidine (Spd) and spermine (Spm) and demonstrate ameliorated phytonutrient content, juice quality, and prolonged vine life. Enhanced nitrogen-carbon interactions were revealed by comprehensive Nuclear Magnetic Resonance (NMR) spectroscopy-based metabolite profiling of the transgenics, suggesting that Spd and Spm are perceived as nitrogenous metabolites by the fruit cells (Mattoo et al., 2006). The recent work by our colleagues identified the effects of Spd/Spm accumulation on various functional classes of tomato genes affected during ripening by probing 1522 ESTs on a custom-made array (Srivastava et al., 2007). In this study we monitored alterations of genome-wide transcriptional patterns in pericarp of Spd/Spm-accumulating tomatoes by means of direct comparison with azygous controls using DNA-microarray technology. Consistent with the ySAMdc expression pattern, very minor transcriptional alterations were detected in mature green developmental stage. For both breaker and red stages, large mutual and unique gene sets displayed altered levels of transcript. Ontological term analysis of up- and down-regulated transcript groups revealed processes in cell metabolism that are regulated by increased levels of Spd/Spm in ripening tomato fruits. These processes mainly involve carbohydrate and amino acids metabolism and protein synthesis. Additionally, transcript levels of representative genes encoding structural enzymes for related biosynthetic pathways show strong relationship to specific metabolites that were identified as regulated in Spd/Spm-accumulating transgenics. Plants of both genotypes were grown in randomized blocks (3 blockes 8-10 plants each). At each of 3 developmental stages (mature green, breaker and pink-red) a single fruit was harvested from a randomly chosen plant of each genotype within each block. In each harvest different plants in each block were taken to represent each genotype. Following harvest, equal pericarp samples were cut from the equator region of each fruit. These samples were pooled according to genotype prior to RNA extraction, allowing equal representation of each block in the final sample. A total of 3 harvests taken from each genotype were eventually analyzed– each harvest representing a biological repeat.
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:Bacteria utilize sophisticated mechanisms to detect and adapt to challenging environmental conditions by activating specific genes that help counteract stressors. Over 150 small proteins (≤ 50 amino acids long) are documented in Escherichia coli, but only a fraction of them are well-studied. Many of these proteins do not have an associated phenotype and their biological roles remain elusive. Here, we investigate small proteins induced in response to magnesium limitation using ribosome profiling, RNA sequencing, and transcriptional reporter assays. We uncover 17 small proteins with increased translation initiation, a majority of which are transcriptionally upregulated by the PhoQ-PhoP two-component signaling system, central for magnesium homeostasis. Next, we describe small protein-specific deletion and overexpression phenotypes, which underscore the significance of their expression to cellular physiology in low magnesium stress. Most remarkably, our study reveals the small protein YoaI as a connector of the major signaling networks – PhoR-PhoB and EnvZ-OmpR in E. coli.
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:Ethylene receptor protein quantification is essential to study their functions, but is impaired by low resolutive tools such as antibodies that are mostly nonspecific. Here we report a proteomic method that enables the quantification of all tomato ethylene receptors, which can be applied to other organisms. Testing this method, we found that “Never-Ripe” tomatoes stay orange while a mutated receptor accumulated at ripening, further blocking the ethylene signal .