Recipient Organization
PURDUE UNIVERSITY
(N/A)
WEST LAFAYETTE,IN 47907
Performing Department
(N/A)
Non Technical Summary
Terpenoids represent one of the largest classes of plant metabolites, encompassing more than 80,000 compounds playingvarious roles in plant physiology and ecology. Terpenoids also contribute to many traits that consumers find appealing in fruits and vegetables, including color, aroma, taste, and phytonutritional content. However, these characteristics have been lost in many crops because traditional breeding programs have largely focused on agronomic traits, like yield, resistance to environmentalstresses, and post-harvest storage and handling. Therefore, the focus of this proposal is to enhance the capacity for producing terpenoid-derived flavor compounds and phytonutrients in tomato fruits. We and others previously attempted to address this problem, but those efforts led to unexpected results (e.g.reduced lycopene and carotenoid content) and raised new questions. Here, we propose to use novel approaches built on our recent work to fill gaps in basic knowledge about terpenoid metabolism in fruits. Knowledge generated in this project will provide key information aboutthe capacity of the mevalonic acid (MVA) and methylerythritol phosphate (MEP) pathwaysto provide precursors for terpenoid biosynthesis in tomato fruits. We will also test novel metabolic engineering strategiesto harness the MVA pathway for producing terpenoids in tomato, leading to more flavorful and nutritious fruits.
Animal Health Component
(N/A)
Research Effort Categories
Basic
100%
Applied
(N/A)
Developmental
(N/A)
Goals / Objectives
Our long-term goal is to create more flavorful and nutritious tomato fruits while preserving their lycopene and carotenoid content. Using a new approach, we will harness and bolster the capacity of the MVA pathway in tomato fruits through the following objectives: 1) Determine the relative contributions of the MVA and MEP pathways to extraplastidial terpenoid production over fruit ripening; 2) Enhance the capacity for the MVA pathway to supply cytoplasmic precursors for producing high-value terpenoids; and 3) Increase GPP and FPP biosynthesis in the cytoplasm to support high-value terpenoid production. Completing this project will advance basic knowledge of the roles and capacities of the MVA and MEP pathways in tomato fruits. It will also enhance the general understanding of the regulatory constraints of the MVA pathway in plants and the crosstalk with the MEP pathway. The gained information will guide producing more flavorful and nutritious fruits, leading to increased vegetable consumption, more revenue for growers, and translatable strategies to improve agronomic and consumer traits in other crops. This project is highly relevant to the FY2023-24 Program Area Priority A1103 - Foundational Knowledge of Plant Products, specifically in "Primary and/or secondary metabolic pathways regulating the biosynthesis of plant metabolites that improve the quality of food and/or feed."
Project Methods
Feeding experiments in tomato fruits will be performedto capture the entire ripening transition. Mature green fruits will be harvested just prior to ripening, cut in half longitudinally, and the volume of each half will be calculated from its diameter. To one half of each fruit, mevinolin (the MVApathway-specific inhibitor)orfosmidomycin (the MEP pathway-specific inhibitor) will be injected into the placenta to a final concentration of 100 µM, while the other half will be treated with a control solution without inhibitor. Each treatment will be replicated a minimum of five times. Fruits will then be incubated at 25? for 7 days (until Br+3 stage) before being flash frozen in liquid nitrogen and ground to a fine powder for quantification of terpenoid levels. To capture the later stages of fruit ripening, we will perform the same experiment starting from fruits collected at the Br+3 stage and analyzed 7 days later (Br+10 stage).Sesquiterpenes and monoterpenes will be extracted with methyltert-butyl ether spiked with naphthalene as internalstandard and analyzed by gas chromatography-mass spectrometry (GC-MS). Since we have observed that mono- and sesquiterpenes can accumulate as glucosides in tomato fruits, we will also analyze the abundance of potential conjugates by subjecting methanol-extracted tissues to enzymatic hydrolysis to release terpene aglycones.Sterols will be extracted and analyzed as follows: 100 mg of powdered tissue will be extracted with 4 mL chloroform:methanol (2:1) (v/v) containing 1.25 mg L-15-α-cholestan-3-α-ol as an internal standard. After incubation at 70°C for 1 h, samples will be dried and saponified with 2 mL 6% (w/v) KOH in methanol for 1 h at 90°C. Sterols will be extracted twice with 2 mL hexane:water (1:1) (v/v) and derivatization will be performed on the dried residues using 100 μL of BSTFA and analyzed by GC-MS. Ubiquinone will be extracted from 200 mg of ground tissue with 3 mL of 95% (v/v) ethanol spiked with 4 nmol ubiquinone-4 internal standard and incubated overnight at 4°C. After centrifugation to pellet debris, 1.5 mL of water will be added to the supernatant and partitioned twice with 4.5 mL hexane. The hexane layers will be combined, dried under nitrogen gas, resuspended to a final volume of 1 ml in 90:10 methanol:dichloromethane, and analyzed by high performance liquid chromatography-diode array spectrophotometry (HPLC-DAD).We will also analyze carotenoids, including lycopene. To minimize photo-oxidative reactions, experiments will be performed under yellow lights using 2.5 g of ground tissue, which will be combined with 0.5 g sodium bicarbonate and 1 g Celite. Carotenoids will be extracted using a 1:1 solution of acetone/petroleum ether (0.1% BHT). The suspension will be filtered through filter paper, and the remaining tissue homogenate will be re-extracted three more times. All acetone/petroleum ether fractions will be combined and saponified using 40% (w/v) KOH in methanol. The ether layer will be collected and combined with 10 mL of a saturated NaCl solution to remove hydrophilic compounds. The collectedpetroleum ether phase will be filtered through a column of sodium sulfate to remove residual water and brought to a final volume of 100 ml. Petroleum ether fractions (4 mL) will be dried under nitrogen gas, re-solubilized in 1:1 (v/v) methanol/ethyl acetate, and quantified by HPLC-DAD.We will co-expressAtPMKwithAtHMGR1to increase MVA pathway flux in tomato fruits.To achieve near stoichiometric expression ofthe two genesin tomato fruits, we will generate a 2A-linker-based polyprotein construct, which undergoes self-cleaving by "ribosome skipping" to produce two separated proteins. The synthesized recombinant dual gene,AtHMGR1-2A-AtPMK, will be inserted into pTwist-ENTR and cloned using GatewayTMtechnology into the plant binary destination vector pH2GW7 in which the 35S Cauliflower Mosaic Virus (CaMV) promoter has been replaced with the fruit ripening-specific tomatoPGpromoter. The binary vector will be introduced intoAgrobacteriastrain c58C1/pMP90 and used for transient expression in tomato fruits and for stable transformation.In transient experiments,AtHMGR1-AtPMKwill be co-expressed withthe gene encodingAmNES/LIS-1, a cytoplasmic bifunctional enzyme that produces nerolidol and linalool from FPP and GPP, respectively. Productionof nerolidol will serve as a marker to assess whether the cytoplasmic pool of FPP increased and is metabolically available inAtHMGR1-AtPMKfruits relative to controls. Similarly, linalool will serve as a marker should there be any GPP produced in the cytoplasm. Tomato fruits will be injectedwithAgrobacteriacarrying an empty vector, ourAmNES/LIS-1reporter construct, or pH2GW7:AtHMGR1-2A-AtPMKalone, and in combination. For individually testing constructs,Agrobacteriumsuspensions will be used at OD600= 1. When constructs will be used in combination, theAgrobacteriumsuspensions for each strain will be diluted to OD600= 0.5 and mixed in equal concentration.Agrobacteriawill be injectedvia the pedicel of MP1fruits on the vine at the Br+3 stage. MVA and MEP pathway-derived terpenoids will be analyzed at the Br+10 stage.To generate stable lines, thepH2GW7:AtHMGR1-2A-AtPMKconstruct will be transformed into wild-type MP1tomato cotyledons. An empty-vector control will be generated in parallel. Hygromycin-resistant transformants will be screened for the presence of transgenes by PCR using gene-specific primers on genomic DNA and for transgene expression in fruit by qRT-PCR. Multiple independent lines showing the highest levels ofAtHMGR1andAtPMKexpression will be self-pollinated and the T1generation will be produced, checked for 3:1 segregation to verify that only one copy of the T-DNA was incorporated, and metabolically profiled for levels of terpenoids. Subsequently, transformants with the highest levels ofAtHMGR1andAtPMKexpression be carried out to generate homozygous T3lines.To increase GPP and FPP levels in the cytoplasm, we will expressRcG/FPPSin tomato fruits. TheRcG/FPPSgene will be synthesized,cloned into the plant binary destination vector pK2GW7, in which the CaMV promoter has been replaced with the fruit ripening-specific tomatoPGpromoter.The pK2GW7:RcG/FPPSconstruct will alsobe used to generate stable transgenic plants.Kanamycin-resistant transformants will be screened based onRcG/FPPSexpression in fruits, and multiple independent lines will be generated and metabolically profiled for terpenoid compounds as described above. Subsequently, transformants with the highest levels ofRcG/FPPSexpression be carried out to generate homozygous T3lines.