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Title: Transcriptional Profiling of high pigment-2dg Tomato Mutant Fruit      
Transcriptome or Gene expression
Phenotypes of the tomato (Solanum lycopersicum L.) high pigment-2dg (hp-2dg) mutant are caused by a mutation in the gene encoding DEETIOLATED1, a negative regulator of light signaling. Homozygous hp-2dg plants display a plethora of distinctive developmental and metabolic phenotypes in comparison to their normal isogenic counterparts. This mutant is however best known for the increased levels of lycopene and other plastid-accumulating functional metabolites. In this study we analyzed the transcriptional alterations in mature-green, breaker and early-red fruits of hp-2dg/hp-2dg plants in relation to their normal counterparts using microarray technology. Results show that a large portion of the genes that are affected by hp-2dg mutation, display a tendency for up- rather than down-regulation. Ontology assignment of these differentially regulated transcripts, revealed a consistent upregulation of those related to chloroplast biogenesis and photosynthesis in hp-2dg mutants throughout fruit ripening. A tendency of upregulation was also observed in structural genes involved in phytonutrient biosynthesis. However, this upregulation was not as consistent, positioning plastid biogenesis as an important determinant of phytonutrient overproduction in hp-2dg mutant fruits. Microscopic observations revealed a highly significant increase in chloroplasts size and number in pericarp cells of mature-green hp-2dg/hp-2dg fruits in comparison to their normal counterparts. This increase could be observed from early stages of fruit development. Therefore, the molecular trigger that drives phytonutrient overproduction in hp-2dg mutant fruits should be initially traced at early stages of fruit development. Keywords: Comparative Transcriptional Profiling of tomato fruit pericarp tissue Overall design: Representative plants of hp-2dg tomato mutants and their normal (WT) counterparts were planted in a randomized-block design (3 blocks, 5 plants in each block) in an environmentally controlled greenhouse at the Volcani Center, Bet Dagan, Israel, during the winter seasons of 2004 and 2005 (minimal temperature kept at 15°C with no supplemental light). 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. Transcriptional profiling of tomato pericarp tissue of hp-2dg/hp-2dg plants was obtained using direct comparisons with their normal counterparts in each developmental stage separately. Two-color hybridizations were carried out to the TOM1 microarray slide (CGEP website at Cornell). Three microgram of amplified mRNA products were subjected to reverse transcription and then labeled with Cy3 and Cy5 (Amersham Biosciences, Uppsala, Sweden) by the indirect amino-allyl method (Invitrogen, Carlsbad, CA, USA). Hybridization of labeled targets to the tomato microarray slide TOM1 was carried out at 42°C for 14 to 16 h using a covered hybridization oven (Amersham Pharmacia, Buckinghamshire, England). Microarray slides were washed seven times for 10 min each: at room temperature in 2XSSC/0.1% SDS- twice, room temperature in 0.2XSSC/0.1% SDS- twice, room temperature with 0.2XSSC- twice, and once in 0.2XSSC at 42°C. Excess fluids were drained from the arrays by centrifugation (5 min at 450g). For each developmental stage (mature green, breaker and early-red), 4 hybridizations were carried out, representing 3 independent biological repeats and one technical repeat of swapped dyes. Separate images for each fluorescence dye were acquired using ScanArray 4000 genetic analyzer [GSI Lumonics (Packard / Perkin Elmer), Ramsey MN, USA]. The analyzer scan was carried out at 10 µm resolution per pixel, adjusting the photomultiplier and laser power to achieve an optimal distribution of signals with minimal saturation. Quantification was carried out using QuantArray version 3 software, applying histogram method (Billerica, Packard BioScience, MA, USA). Data analysis was performed using Genespring® 7.2 software (Silicon Genetics, Redwood City, CA, USA), applying per-spot and per-chip normalizations: For each hybridization Lowess curve was fit to the log-intensity versus log-ratio plot. 40% of the data was used to calculate the Lowess fit at each point. This curve was used to adjust the value of the normal genotype for each measurement (control channel). If the control channel was lower than 10 then 10 was used instead. Low-quality data were identified and discarded when their signal to noise ratio was <2.
Solanum lycopersicum
National Center for Biotechnology Information
NCBI BioProject