1a.Objectives (from AD-416):
The objective of this cooperative research project is to identify and functionally test genes that are important in the regulation of plant compounds with known or potential human health benefits. In doing so, we will identify candidates for manipulation through traditional breeding or transgenic strategies for enhancement of phytonutrients in plant-based foods and we will additionally develop basic knowledge regarding the genetic mechanisms underlying the synthesis and accumulation of target compounds. Specific compounds of interest under this project will be carotenoids, flavonoids, ascorbate, and folic acid.
1b.Approach (from AD-416):
The general approach of this cooperative research project will entail continued utilization of emerging genomics technologies to identify genes related to phytonutrient accumulation in tomato with a secondary effort on melon. We will continue to screen and expand set of tomato and melon germplasm developed as part of the prior project which varied in target nutrient and phytonutrient levels for genes whose expression is coordinated with changes in metabolite targets of interest. Considerable germplasm appropriate for this study is available and already in hand in the ARS lab and from collaborators in ongoing projects. Genes will continue to be screened initially via transient viral induced gene silencing (VIGS) which is an expertise of our collaborator. Genes coming through the VIGS screens with an impact on phytonutrient levels will be used in development of a smaller number of stable transformants for comprehensive analysis. The ARS lab is proficient in tomato gene expression profiling using microarrays and more recently RNAseq technologies and in the creation and analysis of stable transformed tomato lines. Candidate melon genes (or their homologs) will be initially analyzed in tomato. As stated, the BTI cooperator’s lab is proficient in creation and high throughput transient expression of DNA constructs for testing function of candidate genes in tomato. The complementary skills and mutual interests of the ARS and BTI groups make this an ideal collaboration.
This project is focused on characterization of fruit and vegetable nutritional metabolites and the genes that control their synthesis and accumulation. Identification of biosynthesis and regulatory genes that impact fruit and vegetable nutrients and the biological process that are associated with them (e.g. fruit ripening), will provide new targets for selection and breeding toward a more nutrient diverse and nutrient dense food supply. The seed companies that generate them, the farmers who grow them, the processors who use them, and the consumers who eat them, will all benefit from nutrient enhanced crops. In the last year, project activities have involved continued generation of plant material for analysis including tomato fruit from multiple genotypes and maturity stages. Replica tissues were collected in the field and greenhouse to distinguish genetic from environmental variation. Harvested and frozen tissues were extracted for RNA and assayed by RNA-seq transcriptome profiling (to measure gene expression) and target nutritional metabolites have been measured (pro-vitamin A and additional carotneoids, flavonoids, ascorbic acid /vitamin C and folic acid/vitamin B9). Several candidate genes potentially impacting fruit nutritional quality have been identified and DNA constructs to be used in functional analysis have been developed.