Project description:Fruit taste is determined by sugars, acids and in some species, bitter chemicals. Attraction of seed-dispersing organisms in nature and breeding for consumer preferences requires reduced fruit bitterness. A key metabolic shift during ripening prevents tomato fruit bitterness by eliminating α-tomatine, a renowned defence-associated Solanum alkaloid. Here, we combined fine mapping with information from 150 re-sequenced genomes and genotyping a 650 tomato core collection to identify nine bitter-tasting accessions including the ‘high α-tomatine’ Peruvian landraces reported by Rick and colleagues (1994). These ‘bitter’ accessions contain a deletion in GORKY, a nitrate/peptide family (NPF) transporter mediating α-tomatine subcellular localization during fruit ripening. GORKY exports α-tomatine and its derivatives from the vacuole to the cytosol and this facilitates the conversion of the entire α-tomatine pool to non-bitter forms, rendering the fruit palatable. Hence, GORKY activity was a significant innovation in the process of tomato fruit domestication and breeding. The experiment was carried out to further prove that GORKY is localized to tonoplast in ripe fruit.
Project description:Lycopersicon esculentum cv. Moneymaker tomato plants were grown for 4 weeks in greenhouse conditions (16h/8h light/darkness, 27 °C). L. esculentum was randomly sorted into two groups of 16 plants each and a third reference group of 6 plants. All plants were contained individually in mesh bags. One group of 16 plants was infested with 25 female adult tomato psyllids that were 2-3 days old per plant. The other two groups of 16 and 6 plants were not infested (healthy control, and reference pool). After 3 days, the adults were removed from the infested plants to allow for egg eclosion and eventually nymphal development. In this study, leaves infested and healthy plants were harvested for RNA extraction at 0, 3, 11, and 17 days after infestation with tomato psyllids. The reference pool was harvested on day 0. Tissue (all-leaflets) from at least three plants per group was collected, flash frozen in liquid nitrogen and pooled for each time point (0, 3, 11 and 17 days after infestation) to represent treatments for before feeding (0 d), adult feeding and egg deposition (3 d), 1st and 2nd instar feeding (11 d) and 3rd to 5th instar feeding (17 d ), respectively. The whole experiment was repeated three times (three biological replicates). Plant tissues were ground using a cold mortar and pestle. Total tomato leaf RNA was extracted using the hot-phenol protocol. The RNA was precipitated, pooled, cleaned with a kit and stored at –80C. Two populations of single-stranded cDNAs will be generated from the aliquots and labeled with the cyanine Cy3 and Cy5 fluorophores. RNA isolated from the reference pool (0 d) will be pooled from each replicate and will be labeled with Cy5; this is the reference sample for each hybridization. RNA isolated from leaves infested with tomato psyllids and healthy uninfested plants from each time point will be query samples labeled with Cy3. The two samples of labeled cDNA will be simultaneously hybridized to the same microarray. Keywords: Reference design