Engineering topics

Schmitz, Robert J.

Publications and source records attributed to Schmitz, Robert J..

6 records

DOE OSTI2023

Engineering custom morpho- and chemotypes of Populus for sustainable production of biofuels, bioproducts, and biomaterials

Humans have been modifying plant traits for thousands of years, first through selection (i.e., domestication) then modern breeding, and in the last 30 years, through biotechnology. These modifications have resulted in increased yield, more efficient agronomic practices, and enhanced quality traits. Precision knowledge of gene regulation and function through high-resolution single-cell omics technologies, coupled with the ability to engineer plant genomes at the DNA sequence, chromatin accessibility, and gene expression levels, can enable engineering of complex and complementary traits at the biosystem level. Populus spp., the primary genetic model system for woody perennials, are among the fastest growing trees in temperate zones and are important for both carbon sequestration and global carbon cycling. Ample genomic and transcriptomic resources for poplar are available including emerging single-cell omics datasets. To expand use of poplar outside of valorization of woody biomass, chassis with novel morphotypes in which stem branching and tree height are modified can be fabricated thereby leading to trees with altered leaf to wood ratios. These morphotypes can then be engineered into customized chemotypes that produce high value biofuels, bioproducts, and biomaterials not only in specific organs but also in a cell-type-specific manner. For example, the recent discovery of triterpene production in poplar leaf trichomes can be exploited using cell-type specific regulatory sequences to synthesize high value terpenes such as the jet fuel precursor bisabolene specifically in the trichomes. By spatially and temporally controlling expression, not only can pools of abundant precursors be exploited but engineered molecules can be sequestered in discrete cell structures in the leaf. The structural diversity of the hemicellulose xylan is a barrier to fully utilizing lignocellulose in biomaterial production and by leveraging cell-type-specific omics data, cell wall composition can be modified in a tailored and targeted specific manner to generate poplar wood with novel chemical features that are amenable for processing or advanced manufacturing. Precision engineering poplar as a multi-purpose sustainable feedstock highlights how genome engineering can be used to re-imagine a crop species.

09 BIOMASS FUELS↗

DOE OSTI2023

Haplotype‐resolved genome assembly of Populus tremula × P. alba reveals aspen‐specific megabase satellite DNA

SUMMARY Populus species play a foundational role in diverse ecosystems and are important renewable feedstocks for bioenergy and bioproducts. Hybrid aspen Populus tremula × P. alba INRA 717‐1B4 is a widely used transformation model in tree functional genomics and biotechnology research. As an outcrossing interspecific hybrid, its genome is riddled with sequence polymorphisms which present a challenge for sequence‐sensitive analyses. Here we report a telomere‐to‐telomere genome for this hybrid aspen with two chromosome‐scale, haplotype‐resolved assemblies. We performed a comprehensive analysis of the repetitive landscape and identified both tandem repeat array‐based and array‐less centromeres. Unexpectedly, the most abundant satellite repeats in both haplotypes lie outside of the centromeres, consist of a 147 bp monomer PtaM147, frequently span >1 megabases, and form heterochromatic knobs. PtaM147 repeats are detected exclusively in aspens (section Populus ) but PtaM147‐like sequences occur in LTR‐retrotransposons of closely related species, suggesting their origin from the retrotransposons. The genomic resource generated for this transformation model genotype has greatly improved the design and analysis of genome editing experiments that are highly sensitive to sequence polymorphisms. The work should motivate future hypothesis‐driven research to probe into the function of the abundant and aspen‐specific PtaM147 satellite DNA.

Zhou, Ran↗

DOE OSTI2023

Exploring Plant Cis –Regulatory Elements at Single–Cell Resolution: Overcoming Biological and Computational Challenges to Advance Plant Research

Cis-regulatory elements (CREs) are important sequences for gene expression and for plant biological processes such as development, evolution, domestication, and stress response. However, studying CREs in plant genomes has been challenging. The totipotent nature of plant cells, coupled with inability to maintain plant cell types in culture and the inherent technical challenges posed by the cell wall have limited our understanding of how plant cell types acquire and maintain their identities and respond to the environment via CRE usage. Furthermore, advances in single cell epigenomics have revolutionized the field identifying cell-type-specific CREs. These new technologies have the potential to significantly advance our understanding of plant CRE biology, and shed light on how the regulatory genome gives rise to diverse plant phenomena. However, there are significant biological and computational challenges associated with analyzing single cell epigenomic datasets. In this review, we discuss the historical and foundational underpinnings of plant single-cell research, challenges and common pitfalls in analysis of plant single-cell epigenomic data, and highlight biological challenges unique to plants. Additionally, we discuss how the application of single-cell epigenomic data in various contexts stands to transform our understanding of the importance of CREs in plant genomes.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2022

Multiplex knockout of trichome-regulating MYB duplicates in hybrid poplar using a single gRNA

As the focus for CRISPR/Cas-edited plants moves from proof-of-concept to real-world applications, precise gene manipulation will increasingly require concurrent multiplex editing for polygenic traits. A common approach for editing across multiple sites is to design one guide RNA (gRNA) per target; however, this complicates construct assembly and increases the possibility of off-target mutations. In this study, we utilized one gRNA to target MYB186, a known positive trichome regulator, as well as its paralogs MYB138 and MYB38 at a consensus site for mutagenesis in hybrid poplar (Populus tremula × P. alba INRA 717-1B4). Unexpected duplications of MYB186 and MYB138 resulted in eight alleles for the three targeted genes in the hybrid poplar. Deep sequencing and polymerase chain reaction analyses confirmed editing across all eight targets in nearly all of the resultant glabrous mutants, ranging from small indels to large genomic dropouts, with no off-target activity detected at four potential sites. This highlights the effectiveness of a single gRNA targeting conserved exonic regions for multiplex editing. Additionally, cuticular wax and whole-leaf analyses showed a complete absence of triterpenes in the trichomeless mutants, hinting at a previously undescribed role for the nonglandular trichomes of poplar.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2021

IMITATION SWITCH is required for normal chromatin structure and gene repression in PRC2 target domains

Significance Polycomb Repressive Complex 2 (PRC2) methylates histones to regulate multicellular development, maintenance of stem cell identity, X-chromosome inactivation, and other important processes. Given these essential roles, there is significant interest in identifying components that function with PRC2 to establish and maintain transcriptionally repressive heterochromatin. Here we document an unexpected new role for a well-studied and conserved chromatin remodeling factor, ISWI. We found that the Neurospora ISWI homolog is required for normal facultative heterochromatin structure and gene repression at PRC2 target regions, and we defined requirements for ATP-dependent catalytic activity and accessory regulatory proteins. These findings provide mechanistic insights into the formation and function of facultative heterochromatin in a model eukaryote.

Kamei, Masayuki↗

DOE OSTI2020

AlphaBeta: computational inference of epimutation rates and spectra from high-throughput DNA methylation data in plants

Stochastic changes in DNA methylation (i.e., spontaneous epimutations) contribute to methylome diversity in plants. Here, we describe AlphaBeta, a computational method for estimating the precise rate of such stochastic events using pedigree-based DNA methylation data as input. We demonstrate how AlphaBeta can be employed to study transgenerationally heritable epimutations in clonal or sexually derived mutation accumulation lines, as well as somatic epimutations in long-lived perennials. Application of our method to published and new data reveals that spontaneous epimutations accumulate neutrally at the genome-wide scale, originate mainly during somatic development and that they can be used as a molecular clock for age-dating trees.

59 BASIC BIOLOGICAL SCIENCES↗