Location: Innovative Fruit Production, Improvement, and Protection
Project Number: 8080-21000-033-010-I
Project Type: Interagency Reimbursable Agreement
Start Date: Oct 15, 2022
End Date: Oct 14, 2026
Objective:
The objective of this project: ARS scientists previously identified a number of genes that control tree architecture. These technologies enable the manipulation of tree shape for high density planting and other novel orchard designs. Here, ARS scientists propose to work with a model tree species, poplar, to understanding how gene editing can be used to design novel tree forms by targeting multiple architectural genes simultaneously. The results will provide fundamental knowledge about the interactions of key genes and impacts on productivity, wood strength, and other characteristics. The resulting poplar trees will serve as chassis for diverse bioproduct uses where the distribution of leaves, wood, and overall tree size are limiting factors. In this project we will construct a gene regulatory map of poplar at the single cell level that will help define the expression patterns of architectural genes within the apical meristem. In Aim 2, selected architectural genes will be targeted to engineer three poplar tree forms ideally suited for bioproducts production. Last, we will assess the fitness of our chassis under variable environments in the field. As a model tree system, we will use P. tremula x P. alba INRA 717-1B4 (hereafter ‘717’), an interspecific F1 hybrid from a breeding program highly amenable to Agrobacterium transformation and CRISPR genome editing. As a haplotype-resolved assembly is near completion by the DOE JGI, 717 is an ideal study system for the proposed research from synthetic biology to bioproducts to field assessment.
Approach:
Aim 1: Utilize state-of-the-art omics to develop a cell type specific gene regulatory map of poplar development to enable high resolution biosystems design. We will leverage advances in single-cell genomics to construct a cell-type resolved atlas of transcript abundance and accessible chromatin in key stages of poplar development. Using single cell RNA-sequencing, we will identify candidate plant architecture loci and metabolic pathways that are expressed in a cell-type-specific manner.
Aim 2: Modify poplar architecture to generate three specific morphotypes. For architecture trait engineering, we will initially target known branching regulators, such as BRC, GID1, MAX (MORE AXILLARY BRANCHING), and TAC1 for knockout (KO). Mutations in different numbers and combinations of paralogs and alleles are expected to produce a spectrum of morphotypes with different branching patterns which will be characterized at multiple levels to inform the next design iteration. Characterization will include growth, biomass, branch characteristics and leaf area density. Wood composition from angled branches is known to differ from erect stems vs horizontal orientations rich in tension wood, which may affect biomass utilization.
Aim 3: Engineer bioproducts and composition in custom morphotypes. The synthesis of plant cell wall polysaccharides utilizes a significant portion of carbon fixed via photosynthesis. Most current bio-refinery processes focus on chemical or enzymatic depolymerization of the complex carbohydrates to monosaccharides. We aim to understand how processes involved in complex carbohydrate synthesis are differentially regulated in our engineered morphotypes. For our chassis with increased trichomes, we will engineer biosynthesis of the sesquiterpene, bisabolene, which when converted to bisabolane is used as a drop-in fuel and diesel substitute.
Aim 4: Employing epigenetic reprogramming to modulate morphotypes and chemotypes. The extent to which we can manipulate and modify plant metabolism and morphology is largely constrained by our ability to control transcription and chromatin state. However, we have traditionally relied on the same set of underlying tools and DNA elements that were first characterized over the last twenty years. To expand the dynamic range of these tools, we propose to identify poplar-specific effector domains in poplar TFs that modulate transcription and chromatin state.
Aim 5. Test fitness of morpho-/chemotypes under field conditions. It is well known that plants behave differently in a greenhouse versus the field. Hence, the most promising edited or transgenic genotypes will be cloned and test.
