The objectives of this project are to 1) identify the gene regulatory and metabolic networks important for adaptation to low water availability and high-density plantings and to gain a greater understanding of the physiology underlying these adaptations and 2) develop technologies to precisely control gene insertion and replacements events for large scale engineering of pathways in model and target feedstocks. To accomplish these objectives the following experiments will be performed with primary personnel responsible listed in parenthesis and shown in project management plan:

Aim1: Identify QTL for the effect of drought and density on biomass and seed yield components of Setaria (Leakey, Cousins, Baxter, Brutnell, Mockler). This study will generate one of the most comprehensive molecular, physiological and genetic datasets to date in a field setting. This will include highly resolved transcriptional profiles, elemental profiles and isotopic discrimination generated on recombinant inbred lines of Setaria.

Aim 2: Conduct in–depth physiological profiles in roots and leaves of a subset of selected lines (Dinneny, Brutnell, Baxter, Leakey, Cousins). These detailed follow-up studies will be conducted in field, greenhouse and growth chamber settings to provide more highly resolved molecular signatures under controlled environments. These data will provide mechanistic insight into the processes regulating abiotic stress response. Additionally, the greenhouse and growth chamber studies will enable the development of more rigorous network reconstructions.

Aim 3: Integrate datasets and develop metabolic and gene networks for Setaria (Rhee, Mockler, Baxter). A key component to this proposal is to develop a comprehensive network model that integrates several tiers of –omics, QTL, eQTL and physiological data from this study with previously published dicot and monocot networks. This network model will guide the prioritization of candidate genes that when manipulated will likely have the greatest impact on biomass and seed yield under drought and high-density growth conditions.

Aim 4: Develop transformation technologies for Setaria viridis (Voytas). To rapidly accelerate the production of second generation bioenergy feedstocks it will be necessary to develop sophisticated gene and pathway manipulation systems. These tools, developed here for S. viridis, will likely be equally applicable to closely related panicoid feedstocks and thus serve as an important resource for broad bioenergy research community.

Aim 5: Functionally examine the role of candidate genes deduced by network models (Voytas, Leakey, Cousins). The function of up to 10 candidate genes will be examined through transgenics approaches. Overexpression and knockouts of candidate genes identified in Aim 3 will be performed with technologies developed in Aim 4. Detailed phenotypic characterizations of the transgenic materials will be performed in field, greenhouse and controlled growth settings.

Aim 6: Develop protocols and best practices for monitoring gene flow in transgenic Setaria (Quemada, Brutnell). As transgenics will likely be an important component to future biofuel feedstock development, it will be essential to develop a comprehensive framework for identifying and quantifying potential impacts of transgenics on ecosystems. Thus, to develop a framework for future studies we will establish metrics for quantifying transgenic risk. In summary, the proposed 6 aims will establish Setaria viridis as the model genetic system for the study of C4 feedstock grasses and provide the foundation for a systems approach to understanding and engineering bioenergy feedstock grasses.