Recipient Organization
Agricultural Research Service
600 E. Mermaid Lane Rm 2023
Glenside,PA 19038-8551
Performing Department
(N/A)
Non Technical Summary
Folates (vitamin B9) are a family of plant-derived vitamins essential for human growth and health. As we cannot synthesize folate ourselves, all our folate comes into our bodies through our diets.Folate deficiency leads to anemia in adults, and folate deficiency during pregnancy can lead to fetal neural tube defects. Dietary folates are found in fruits, beans, and most prominently in green leafy vegetables. Strategies to increase folate levels in plants will provide a wider range of sources to address nutritional deficiency, quality food access and product value addition. Prior efforts have focused on manipulating the metabolic pathways involved in folate biosynthesis. These approaches have had some success in increasing dietary folates, but plants internal regulatory systems keep them from over-producing folate. Genes have been identified in plants that are predicted to function in folate binding and transport. Converting folate, whichis easily degraded, to more stable storage forms may prove a more effective way of increasing folate levels in crops that we eat. These genes exist in tomato and the broad range of genetic diversity in this and many crops, suggest that natural genetic variation may well exist that could be targetedin traditional breeding or through biotechnology to yield crops with higher folate levels.This project will a) develop and test modified methods for more accurate folate analysis in plant tissues that are needed by plant breeders and the human nutrition community, b) confirm the function of folate binding proteins as a means to elevatefolate levelsin tomato and as an example of the possibilities for other fruit crops, c) test how well elevated folate is taken up by the human gut, d) determine the value of folate binding proteins as a means to enhance folate nutritional content of fruit, e) create folate-biofortified tomatoes, a crop important to small and large growers alike all via enhanced fundamental understanding of tomato folate metabolism, in particular the function of plant proteins involved in folate binding, transport, and storage.US and international consumers are the ultimate beneficiaries of this project and will achieve greater food and nutritional security through access to a more diverse and accessible nutritious food supply. Breeders and seed producers will benefit from value-added product opportunities as will those along the production, distribution, processing, wholesale and retail chain. As tomato is a high-value cash crop, there is substantial potential for impacting small and niche growers in the US and abroad. The genes targeted are highly conserved across the plant kingdom and will likely have translational potential impacting fruit producers and related industries well beyond tomato.
Animal Health Component
15%
Research Effort Categories
Basic
70%
Applied
15%
Developmental
15%
Goals / Objectives
The goal of this project is to develop folate-biofortified tomato lines by understanding and manipulating tomato genes which function in tomato stabilization and storage.Objective 1. Perform functional analysis of folate stabilization and storage proteins in tomato fruit.Objective 2. Integrate tomato folate sample preparation and analytical chemistry for highly accurate quantification of folate vitamers in tomato fruit.Objective 3. Evaluate tomato folate bioavailability using an efficient and tractable intestinal cell model where gene expression can be readily monitored to provide supporting evidence for folate uptake.
Project Methods
Methods are outline below. We note that many methods undertaken are routine in the labs of the investigators. Where methods are modified or otherwise being optimized or developed they are noted with an *.Folate sample preparation *Established protocols for folate extraction from tomato samples will be used as a starting point for optimization of folate sample preparation. Folates are subject to degradation and interconversion upon exposure to heat, UV radiation, oxidation, and acidic pH. Thus, we plan to alter folate extraction procedures by purging solvents and samples throughout the sample preparation process with an inert gas (e.g., N2 or Ar). Furthermore, we will introduce Barocycler technology which uses pressure cycling to enhance release of metabolites from their biological matrix without elevated temperatures. Value of thiese modifications will be evaluated by comparing folate vitamer profilesand amounts from original versus modified extraction procedures and presented to the research community by presentations at conferences and publications.Folate analytical chemistryFolate quantification will be carried out using multiple instruments including the Waters Acquity UPLC with QDA Mass Detector, the ABSciex ZenoTOF 7600 high-resolution tandem mass spectrometry system and a Thermo Orbitrap Fusion Tribrid instrument. The Waters system will be used for routine analyses requiring only MS analysis at unit mass resolution.The SciexZenoTOF system is equipped with an Ultimate RSLCnano liquid chromatograph and will be used for the bulk of quantitative analysis of the folate vitamers.The Orbitrap Fusion instrument is coupled with another Ultimate 3000 RSLCnano liquid chromatograph and will be utilized for the most demanding analyses requiring discovery proteomics and metabolomics. The participating labs have experience with these instruments and selection of which to use will depend on specific needs, whether routine folate assessments (Waters), quantitative analysis of vitamers (SciexZeno TOF) or vitamer identification (Orbitrap). Precise analyses of folate forms and accumulation will enable assessment of project activities aimed toward improved folate content in tomatoes.Tomato fruit protein analysisProtein samples from tomato lines transformed with gene editing and overexpression constructs will be analyzed using targeted and untargeted (whole-proteome) methods. Targeted protein quantification in tomato fruit samples will analyze the products of the folate metabolism genes which have been silenced or overexpressed. Targeted proteomics methods include LC-MS analysis of specific peptides. Untargeted proteomics of all detectable peptides by LC-MS in tomato fruit samples will reveal the global impact of folate metabolism gene silencing or overexpression on protein expression. Folate protein analyses will be evaluated by ability to characterize protein/proteome effects of the genetic modifications undertaken in this project.Laser MicrodissectionLaser capture microdissection using the Leica LMD7 system will be used to collect specific cell types from tomato fruit tissue sections and extract and analyze proteins, nucleic acid, and metabolites useing established procedures. Outcomes will be evaluated by ability to assign accumulation of specific folate vitamers to cell and tissue types of the fruit.Caco-2 cell culture *The human Caco-2 cancer cell line is an intestinal cell model widely used to measure the absorption, metabolism, and bioavailability of food nutrients. Caco-2 cell culture protocols optimized for nutrient bioavailability studies at our research center will be applied for folate bioavailability analysis. Generation of folate bioavailability from control and genetically modified fruit tissues will be a novel data set going beyond prior efforts to quantitate folate accumulation in crop tissue and foods.Caco-2 cell in vitro digestion tomato feeding study *The response of Caco-2 cells to synthetic folate standards and to dietary folates from tomato fruit samples will be analyzed. Tomato fruit samples will be prepared for bioavailability analysis using an in vitro digestion process designed to render the fruit sample into a format simulating how the food would be converted during in vivo digestion prior to reaching the intestine. Following the 24 hour incubation of Caco-2 cells with in vitro digested tomato fruit samples, cells will be lysed and samples will be collected for protein and metabolite analysis. Targeted proteomics and metabolomics analysis of human folate metabolism proteins and folate vitamers will be performed in Caco-2 cells before and after exposure to synthetic folate standards and tomato fruit samples.Outcomes of these activities will be analyzed in terms of ability to generate new insights and hypotheses into folate metabolism and uptake in the human gut.Gene expression analysesTargeted qRT-PCR analysis of folate binding and metabolism genes in addition to whole genome analysis of transcriptome activity will be undertaken using well defined protocols and available tomato and human reference genomes for transcript mapping. All analyses will be undertaken using multiple internal gene standards for qRT-PCR and all expression analyses will use a minimum of three biological replicas. These activities will be evaluated by ability to generate reproducible gene expression data facilitating interpretation of folate vitamer and protein observations.All activities of this project noted above in this section and those prior will involve training of students and young scientists. Working with our collaborators at Tennessee State and existing internship programs at Cornell we will be able to target underrepresented groups in STEM for both training and general introduction to science in general and plant science as related to improvement of the human condition, in particular. This project is expected to result in the creation of novel tomato lines altered in expression or endogenous alleles (via gene editing) of folate binding proteins and folate metabolism genes producing fruit altered (including fortified) with bioavailable plant folates. These lines and analysis of targeted and whole genome expression profiles together with characterization of targeted and whole genome protein profiles will provide novel information regarding the potential to use folate binding and stability as means to achieve elevated fruit folate levels. Completion of activites involving use of the above Methods is expected to result in enhanced understanding of the in vivo function of tomato folate binding proteins, and the role that protein binding and polyglutamylation play in folate stability in plant cells. Improved fruit sample preparation designed to improve in vivo folate stability and mass spectrometry protocols will be applied to more accurately characterize folate vitamer diversity and abundance in tomato lines developed for overexpression or silencing of genes involved in folate stabilization and storage. Folate bioavailability protocols will be developed from existing uptake systems, including in vitro digestion of tomato fruit samples and Caco-2 intestinal cell uptake and metabolism measurements to gain insight into folate forms and protein interactions most effective in influencing nutritional availability. The ultimate measure of success will be whether development of novel alternative strategies to achieve increased folate nutritional quality in tomatoare indeed successful. If so, the highly conserved nature of foalte binding proteins means that results will have high translational potential to other fruit and non-fruit crop species.