Genome-wide Analysis of Small Signaling Peptides in Medicago truncatula with an Emphasis on Macro-nutrient Regulation of Root and Nodule Development

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PI: Wolf Scheible (The Samuel Roberts Noble Foundation) Co-PIs: Michael Udvardi and Patrick Xuechun Zhao (The Samuel Roberts Noble Foundation) and Hideki Takahashi (Michigan State University) Key Collaborators: Michael Sussman (University of Wisconsin-Madison), Hiroo Fukuda (University of Tokyo, Japan), and Fiona McAlister (Southern Oklahoma Technology Center, Ardmore, OK) Small signaling peptides (SSPs) emerge as an important class of regulatory molecules in plants, especially in the control of plant growth and development in response to environmental cues. Only a few of the many SSPs encoded in plant genomes have been characterized functionally in plants. In this respect, SSP-encoding genes represent some of the potentially most important "dark matter" of plant genomes. Medicago truncatula is a premier model legume species, which is closely related to the most important forage species in the USA, Medicago sativa (alfalfa), and to food legumes such as soybean and common bean. Legumes are key components of sustainable agricultural systems because they form symbioses with bacteria called rhizobia that reduce molecular nitrogen to ammonium in specialized root organs called nodules. Symbiotic nitrogen fixation provides legumes with a source of nitrogen, obviating the need for synthetic nitrogen fertilizers for these important crop species. Recently, it has become clear that SSPs can control both nodule and root development in legumes and the hunt is on for more SSPs that control important developmental traits in legumes of importance to agriculture. A priority objective of this project therefore is to identify novel SSPs that affect root and nodule development in M. truncatula, with a long-term view of using SSPs in non-transgenic approaches to improve plants for agriculture. Datasets and biological materials generated in this project will be made available through appropriate biological databases and stock centers. A database (MtSSPdb) will be developed and maintained for both project and public access to store, link and present the information on the comparative genomics of peptide-coding genes and to integrate and manage all experimental data emerging from this project. With regard to outreach and training, the project will leverage existing programs to provide research training for postdoctoral associates and graduate, undergraduate and high school students. A multidisciplinary strategy will be implemented, including bioinformatics, chemical genomics, genetics, biochemistry, molecular and developmental biology. Activities of this project comprise a genome-wide survey of SSP-encoding genes in Medicago truncatula and the identification of macronutrient (N, P, S, and K) -responsive SSP-encoding genes from transcriptome data. A library of genome-encoded, synthetic peptides will be established as a community resource for biochemical genetics and used to screen for developmental and molecular phenotypes in M. truncatula. Constitutive overexpression lines will be produced in Arabidopsis thaliana and M. truncatula for selected SSP-encoding genes that are prioritized because they have been shown to be either strongly responsive to macronutrients (N, P, S, K), produce strong visual/molecular phenotypes when chemically synthesized peptides are exogenously applied to M. truncatula, and/or have their mature peptides predicted to be produced through posttranslational modifications that make chemical synthesis and/or exogenous application impracticable. The synthetic peptides will be further tested for their effects on root nodule development and nitrogen fixation in M. truncatula. The Medicago HAPMAP genotypes will be employed to identify genetic loci associated with natural diversity in response to bioactive peptides, and specific SSPs will also be tested for their efficacy in improving alfalfa performance in the field. Identification of a peptide receptor through genetic or chemo-proteomic approaches is another activity included in this project.