Functional Analysis of Genes Encoding Ubiquitin Proteasome System Components Affecting Poplar Wood Traits
Daniela Rodriguez-Zaccaro1,2 * (email@example.com), Andrew Groover1,2, Justin Walley3, and Nitzan Shabek1
1University of California–Davis; 2U.S. Forest Service, Pacific Southwest Research Station, Davis, CA; and 3Iowa State University
Wood vessel trait candidate genes coding for E3 ubiquitin ligase enzymes will be functionally characterized and examined through CRISPR-cas9 genome editing, TurboID proximity labeling, and drought and ABA treatments. Specifically, the data generated in this project will be further used to study the transcriptome, proteome, interactome, and ubiquitinome in poplar wood forming tissues.
Wood is the water-conducting tissue of tree stems. Like most angiosperm trees, poplar wood contains water-conducting vessel elements whose anatomical properties affect water transport and growth rates as well as susceptibility to cavitation and hydraulic failure during drought. Despite their key role in determining the hydraulic physiology of trees, the genetic regulation of vessel element morphological traits is poorly understood. In a preliminary study, a dosage- based genome-wide screen found significant associations (or dosage QTL regions) between wood vessel traits and specific regions of the genome. Poplar wood forming tissues were then sampled to conduct a gene coexpression network analysis. Height-corrected vessel frequency was significantly correlated to a group of co-expressed genes that code for E3 ubiquitin ligase components of the ubiquitin proteasome system. The team found that some of these genes are located within a chromosome 9 dosage QTL region identified in its previous screen, suggesting that these could affect vessel trait variation in a dosage-dependent manner. From these genes, the team selected vessel trait-related candidates for further characterization, including E3 ubiquitin ligases makorin (MKRN), SKP1-interacting protein 2 (SKIP2), and Big Brother (BB). Based on these preliminary findings, future aims involve the novel functional characterization of key components of ubiquitin-proteasome regulation in poplar wood forming tissue. To meet this goal, researchers will generate CRISPR-cas9 mutants for poplar ubiquitin E3 ligase candidate genes to determine changes in wood phenotype, gene expression, protein abundance and ubiquitinomes. The team will also use a TurboID proximity labeling strategy in poplar to identify the interacting partners for candidate proteins and characterize their specific structure and function. Similarly, the transcriptome, proteome, interactome, and ubiquitinome of trees that were grown under drought or treated with ABA will be determined. Ultimately, these strategies will shed light on the role of the ubiquitin proteasome system in wood formation, vessel trait variation, and tree responses to the environment.
This project is supported by the U.S. Department of Energy, Office of Science, Biological and Environmental Research, Genomic Science Program grant no. DE-SC0023158. F.D.R.Z. was supported in part by an appointment to the Science Education Programs at National Institutes of Health (NIH), administered by ORAU through the U.S. Department of Energy Oak Ridge Institute for Science and Education.