Temperature extremes: the effects of high growth temperatures on vitamin E production and lipid remodelling in tomato

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Temperature extremes: the effects of high growth temperatures on vitamin E production and lipid remodelling in tomato

Cellular lipids have recently been shown to have a role in signalling heat stress and mitigating its impact in plants. These changes can affect the fat-soluble vitamins and an understanding of how this occurs mechanistically is the main aim of this project. Purpose: The work plan will focus on understanding whether the effect of high temperature on tocopherol and lipid metabolism is mediated by phytochromes (PHYs) in tomato. The proposed project seeks to address the following questions: 1. How does temperature affect vitamin E and lipid remodelling? 2. Are these effects mediated by PHY perception? 3. Are these effects mediated by epigenetic mechanisms? Methodology: To address these questions, we will use the following available resources: - (1) Phytochrome tomato mutants (phyA, phyB1, phyB2, phyB1B2 and triple mutants) and corresponding control (Money Maker); (2) fruit-specific PHY-silenced dwarf tomato lines (PHYAas, PHYB2as, PHYB1B2as) and corresponding control (MicroTom). These lines will enable experiments to address the role of phytochrome receptors in temperature sensing and their effect on the regulation of fruit tocopherol and lipid metabolism. Plants will be grown (four months) in quarantined glass house (GH) space available at Rothamsted in two different temperature conditions (20°C and 33°C; GH space funded by internal award to Michaelson). The following environmental, growth and fruiting parameters will be recorded: time course of temperature, humidity and radiation, time to flowering (truss 2 to 5), overall fruit set including the number of flowers and fruits, and total fresh fruit biomass. Samples will be harvested for further analysis. Source leaves and pericarp (without peel) from immature, mature green and ripe fruits (exact stage will be defined) from two trusses (3 and 4) will be dissected, weighed (approx. 3 g per sample) and frozen in liquid N2 and kept at -80°C. Aliquots of these samples for further analyses will be distributed between Rothamsted and the University of Buenos Aires (UBA). Methods are established in the laboratories of the partners for analysis of soluble metabolites, sugar/starch, tocopherol/carotenoids and lipidomics. The Rothamsted Plant Lipidomics Facility will provide a platform for the rapid profiling of structural and signaling lipids, revealing the metabolic networks that link phytochrome sensing and temperature tolerance; lipids will be profiled were using a high resolution accurate mass system (Thermo Q Exactive Plus) according to Biochim Biophys Acta 1862(8):782-785. At UBA, primary metabolism will be profiled by GC-MS as described by Bermúdez et al. (Plant J 77:676-687, 2014), and soluble sugars and starch will be quantified by HPAEC-PAD and tocopherols and carotenoids by HPLC, as described in Lira et al. (Plant Phys DOI:10.1104/pp.17.00452). Once metabolic profiles have been analysed, the most informative genotypes and samples will be selected for further expression and genetic analyses.

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