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United States Department of Agriculture

Agricultural Research Service

2006 Annual Report

1.What major problem or issue is being resolved and how are you resolving it (summarize project aims and objectives)? How serious is the problem? Why does it matter?
The new ARS project was initiated in June 2006 following OSQR approval. The overall objective of this project is characterization of the molecular mechanisms regulating nutrient (especially carotenoid) accumulation in edible plant tissues as a first step toward genetic engineering of nutritional quality in crop plants.

Fruits and vegetables are major components of the human diet contributing a large portion of vitamins, minerals, antioxidants, and fiber. While plant-based food nutritional composition has clear and profound potential for positive human benefit, it has also proven to be a difficult set of traits to modify (via either traditional breeding or transgenic approaches) due to underlying complex biosynthetic and regulatory pathways. Synthesis and accumulation of these compounds is the result of coordinated activity of many genes that may also impact additional aspects of plant growth, development and environmental response making metabolic engineering especially challenging. Furthermore, many of these pathways appear to have endogenous regulatory feedback mechanisms designed to promote homeostasis further complicating attempts to engineer modifications in pathway outputs. Effective manipulation of crop nutrient traits for human benefit will therefore require greater knowledge of the pathways involved in their synthesis and the regulatory systems that control them. Under this project we will employ emerging genomics technologies and strategies to define the genetic basis of carotenoid accumulation and metabolic flux and we will initiate efforts toward creation of germplasm resources for evaluation of carotenoid accumulation, dietary availability and nutritive value. Carotenoids are a major focus of our effort as this class of chemicals includes compounds necessary for human health and well being (e.g. precursors to vitamin A) in addition to strong anti-oxidants associated with positive nutritive effects. Genes and germplasm resources developed under this project will be used to establish mechanisms regulating nutrient metabolism and accumulation as well as to demonstrate the availability and nutritional value of different carotenoids as constituents of the human diet.

Antioxidants including carotenoids have received considerable attention from plant and nutritional researchers in recent years due to reported links to prevention of cancer and other degenerative diseases. Cancer is the second leading cause of death in the USA with over 500,000 deaths (America Cancer Society). Diet plays an important role in the onset of cancer and it accounts for about one third of all cancer-related deaths. Thus, plant foods designed to reduce cancer risk have great potential as value-added commodities for producers and consumers. Moreover, reducing cancer risk through food-based strategies would likely result in large savings in healthcare costs and improve human health and well-being in a sustainable way.

The research activities defined under this project will help address scientifically the value of antioxidants in disease prevention and additional aspects of human nutrition. Carotenoids were selected as a focus class of compounds as they have both known nutritional value (e.g. as vitamin A precursors) and have been frequently associated with additional health benefits whose merits require further testing. To avoid an overly narrow focus we will devote some effort to additional compounds of known or suspected nutritional and/or health-promoting value in the human diet. These will include small or pilot studies on the genetic regulation of synthesis and/or accumulation of ascorbate, folic acid and selenium in edible crop tissues.

Research proposed on identifying molecular strategies for increasing nutritional content of crops has been identified as an important aspect of two components of National Program 302. These are Component 1 - Functional Utilization of Plant Genomes: Translating Plant Genomics into Crop Improvement (Problem Statement 1B: Applying Genomics to Crop Improvement) and Component II - Biological Processes that Improve Crop Productivity and Quality (Problem Statement IIC: High Value Products).

2.List by year the currently approved milestones (indicators of research progress)
FY2006: See Bridge Project 1907-21000-019-00D

FY2007: Isolation of tomato fruit tissues for transcription factor analysis.

Development of RNAi constructs for tomato COP10 functional analysis.

Create expression and repression constructs for the cauliflower OR gene.

Isolate Or complex and Or associated proteins.

Database development - creation of public database tools for analysis and dissemination of comparative transcriptome and metabolite data.

Identification of germplasm for isolation of candidate genes related to organoselenium synthesis/accumulation via comparative expression analysis.

FY2008: Gene expression profiling for transcription factor analysis.

Tomato COP10 tomato transformation.

Development of RNAi constructs for tomato COP11 functional analysis.

Generation of OR transgenic plants.

Comparative proteomics of Or and normal control plastids.

Meet with ARS human nutrition center researchers (Boston and Grand Forks) to establish ties for future analysis of nutrient quality and availability in transgenic plants likely to be developed toward the end of this project.

Database development - testing of functions developed in year 1 prior to public release with model data sets and preliminary real data as available.

Creation of gene constructs for functional analysis of organoselenium candidate genes identified in year 1.

FY2009: Informatics analysis of transcription factor gene expression data.

Functional analysis of tomato COP10 transgenics.

Tomato COP11 tomato transformation.

Phenotypic characterization of OR transgenic plants.

Development of DNA constructs for repression analysis of OR in transgenic cauliflower.

Meet with ARS human nutrition center researchers (Boston and Grand Forks) to strengthen ties for future analysis of nutrient quality and availability in transgenic plants likely to be developed toward the end of this project.

Database development - loading of data and assessment of functions/tools. Release to the public.

Plant transformation of gene constructs for functional analysis of organoselenium candidate genes for which constructs were developed in year 2.

FY2010: Create DNA constructs for functional characterization of ripening/carotenoid regulatory genes.

Functional analysis of tomato COP11 transgenics.

Plant transformation of OR repression constructs.

Identify candidate transgenic or breeding materials in consultation with ARS Nutrition center collaborators for small pilot nutritional analysis.

Database development - user surveys and assessment/modification for optimal utility.

Identification and analysis of T-DNA knock outs of putative organoselenium genes identified in year 2.

Characterization of transgenic plants altered in expression of candidate organoselinium genes.

FY2011: Creation and analysis of transgenic plants for testing of putative ripening/carotenoid transcription factors.

Characterization of and analysis of OR repression and over-expression transformants.

Generate experimental and control tissues in replicates for analysis with ARS human nutrition center collaborators (pilot studies).

Continued characterization of transgenic plants altered in expression of candidate organoselinium genes.

4a.List the single most significant research accomplishment during FY 2006.
This is a new project in its third month. The following is listed under the bridge project report that covered the majority of FY06 under item 4a. Under this project we developed carotenoid and transcriptional profiling data for a collection of tomato carotenoid mutants. Correlative analysis of this data revealed that transcription of most genes in the carotenoid synthesis pathway is under feedback regulation. These results have been reported at a number of international conferences and have led to research in several labs (and our own) focused on defining the underlying molecular mechanisms of this regulatory system. These results have significant implications relevant to attempts to modify carotenoid accumulation in crop plants as they suggest on the one hand that endogenous monitoring and regulatory systems may counter some strategies for engineering crop carotenoid accumulation. Alternatively, they also imply that manipulation of carotenoid levels may be achieved more simply and effectively if appropriate regulators are identified and targeted for manipulation.

4b.List other significant research accomplishment(s), if any.
This is a new project in its third month so there are limited additional accomplishments to report to date.

4c.List significant activities that support special target populations.

4d.Progress report.
This report serves to document research conducted under a Specific Cooperative Agreement between ARS and the Boyce Thompson Institute for Plant Research (BTI) entitled "Identification and functional genomics of genes impacting phytonutrient levels in fruit tissues" 21000-025-03S. This is a new project started in FY06 and emphasizing functional characterization of putative phytonutrient regulatory genes in collaboration with Greg Martin at the Boyce Thompson Institute. In the last year we have used previously generated microarray expression data from developing tomato fruit combined with analysis of gene activity in the Never-ripe (Nr) ripening mutant to identify candidate regulators of carotenoid synthesis/accumulation. Four candidate genes were identified and verified for accurate expression via RNA gel-blot or quantitative RT-PCR analysis and sequences from each were employed in construction of RNAi gene repression constructs. All four constructs have been used to generate transgenic tomato plants that are currently growing to maturity in the BTI greenhouses and will be subject to phenotypic analysis at maturity.

This report serves to document research conducted under a Non Funded Cooperative Agreement between ARS and Boyce Thompson Institute for Plant Research (BTI) entitled "Functional genomics analysis of fruit flavor and nutrition pathways" 21000-025-04N. Activity under this project was initiated in FY06 and is centered on assessment of a collection of diverse tomato germplasm for levels of carotenoids and ascorbate (vitamin C). The primary objective is to identify genes whose expression correlates with variation in the population in an effort to identify novel regulatory genes of carotenoid and vitamin C synthesis and/or accumulation. Candidate genes will be mapped to the tomato genetic map to determine linkage to previously localized nutrient QTLs and a subset of promising genes selected for functional analysis in transgenic tomato plants. In this first year of the project activities have centered on production and collection of plant tissues and initial assessment of carotenoid and ascorbate levels in the population. Preliminary results indicate that the population does indeed represent variation in carotenoid levels that span a range of at east an order of magnitude.

5.Describe the major accomplishments to date and their predicted or actual impact.
Again, we are only 3 months into this new project but the major accomplishments during the last year under this and the prior bridge project are as stated in the bridge project report and as follows.

The major accomplishments include the insights into feedback regulation of the carotenoid synthesis pathway described in 4a. These results impact scientists focused on metabolic engineering of carotenoid pathways in edible plant tissues and are fully reside under National Program 302, component 1 (Functional Utilization of Plant Genomes: Translating Plant Genomics into Crop Improvement).

6.What science and/or technologies have been transferred and to whom? When is the science and/or technology likely to become available to the end-user (industry, farmer, other scientists)? What are the constraints, if known, to the adoption and durability of the technology products?
Data has been transferred under this and the preceding bridge project to other scientists and researchers via publications and the tomato expression and metabolite databases at

7.List your most important publications in the popular press and presentations to organizations and articles written about your work. (NOTE: List your peer reviewed publications below).

Last Modified: 9/1/2015
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