Biology subjects

Tuskan, G. A.

Publications and source records attributed to Tuskan, G. A..

18 recordsLinked to original sources

bioRxiv2026

Plant Bioengineering Atlas: A Knowledge Graph of Genes, DNA Constructs, and Plant Traits.

Plant bioengineering has generated tens of thousands of genotype-to-phenotype relationships, but this knowledge remains fragmented across narrative literature and difficult to use computationally. Inconsistent descriptions of DNA constructs, host species, and traits, including variable species names, omitted regulatory elements, and inconsistent gene symbols, impede data reuse, comparative analysis, and design-build-test-learn cycles. Here, we present the Plant Bioengineering Atlas, a literature-mined, ontology-grounded knowledge base assembled using an artificial intelligence (AI)-aided extraction pipeline. A large language model parsed open-access primary research articles to generate structured, provenance-anchored records of engineered genes, modification types, promoter-gene-terminator constructs, host species, target traits, and reported phenotypes, with every record traceable to its source. The current release contains 14,358 curated records encompassing 6,998 distinct genes across 436 plant species from 6,452 papers published between 2000 and 2026. Corpus analysis reveals that experiments are concentrated in a small group of model and crop species, disease and pathogen resistance is the most frequently engineered trait class, and constitutive regulatory parts (particularly the CaMV 35S promoter and NOS terminator) remain pervasive. Two in five records omit one or both flanking regulatory elements (i.e., promoter and terminator), while only 23.4% describe cassettes in which both elements resolve to named part classes, exposing a systematic reproducibility gap. We organize these data into a knowledge graph linking genes, constructs, species, and traits; provide access through an interactive web portal; and propose an AI-compatible documentation standard for AI-ready reporting. The Plant Bioengineering Atlas provides a foundation for data-driven hypothesis generation and AI-aided plant biodesign.

synthetic biology↗

bioRxiv2025

Telomere-to-telomere assemblies reveal complex adaptive variation of 3-ketoacyl-CoA-synthases in Populus trichocarpa likely driven by helitrons

BackgroundThe model woody plant Populus trichocarpa displays an atypical alkene-diverse wax cuticle likely driven by copy number variation (CNV) of 3-ketoacyl-CoA synthases (KCS), which has been difficult to confirm based on short-read assemblies. New long-read sequencing provides opportunities to develop telomere-to-telomere resources to detect cryptic variation, including CNVs, which are currently missed in traditional analyses. Integrating this information can improve genomic prediction for breeding and provide insights into the evolutionary basis of important traits. ResultsOur analysis of 78 telomere-to-telomere long-read haplotypes identified more than twice as many KCS genes as previously reported, along with numerous intragenic non-synonymous substitutions. Random forest predictive models highlighted the importance of Potri.010G079500 in producing very long chain alkenes; however, its absence did not predict previously reported alkene-deficient phenotypes. Instead, alkene levels are best predicted by the combinations of KCS copies. Amino acid substitutions clustered around ligand and donor binding pockets, suggesting they contribute to differing wax cuticle composition. Finally, each KCS gene and copy was linked to a helitron transposon. A phylogenetic analysis indicates they are the evolutionary mechanism for generating KCS tandem arrays. ConclusionsLong-read sequencing and telomere-to-telomere assembles revealed large-effect loci critical to genetic studies that are unattainable from short-reads. These approaches also have the potential to reveal novel insights into genome structure and function, such as the helitrons identified here. Our results highlight that, given current challenges in annotation and assembly, detailed and focused long-read sequences are key to interpreting complex genomic regions that contain tandem copy number variants.

genomics↗

bioRxiv2024

Ribozyme-based biosensor for imaging gene expression in plants

Detection of gene expression in plants is critical for understanding the molecular basis of complex plant biosystems and plant responses to environmental stresses. Here, we report the development of a split ribozyme-based biosensor that enables in vivo visualization of gene expression in plants. We demonstrated the utility of this biosensor in transient expression experiments (i.e., leaf infiltration in Nicotiana benthamiana) to detect RNAs derived from transgenes and tobacco rattle virus, respectively. Furthermore, we successfully engineered a split ribozyme-based biosensor in Arabidopsis thaliana for in vivo visualization of endogenous gene expression at the cellular level. In addition, we developed a platform for easy incorporation of different reporters into the RNA biosensor.

synthetic biology↗

bioRxiv2024

Climate adaptation in P. trichocarpa: key adaptive loci identified for stomata and leaf traits

Identifying the genetic basis of traits underlying climate adaptation remains a key goal for predicting species responses to climate change, enabling the elucidation of gene targets for future climate-resilient crops. Here, we measured 14 leaf and stomatal traits under control (well-watered) and drought conditions, subsampling a diversity collection of over 1,300 Populus trichocarpa genotypes, a potential biofuel feedstock crop. Stomatal traits were correlated with the climate of origin for genotypes, such that those originating from environments subject to water deficit tended to have smaller stomata, but with higher density. Stomatal traits were also correlated with leaf morphology, with larger leaves having larger stomata and lower stomatal density mirrored in correlations to climate of origin. The direction of plastic responses - reduced stomatal size under drought - mirrors the correlations seen among genotypes with respect to the aridity of environmental origin. Genome-Wide Association Studies (GWAS) identified loci underlying trait diversity, including candidates contributing to stomatal size. We used climate of origin to predict stomatal size in genotypes with unknown trait values and found that these predicted phenotypes confirmed empirically measured allele effects. Finally, we found evidence that future climates may select for alleles contributing to decreased stomatal size, with the strength of selection depending on the availability of moisture. These findings reveal adaptive variation in stomatal and physiological traits along with underlying genetic loci, with implications for future selection and breeding - providing insights into the responses to future climate change. HighlightResearch on Populus trichocarpa reveals adaptation of physiological and stomatal traits linked to drought tolerance, with genotypes from arid regions exhibiting smaller stomata, offering insights for climate change adaptation and sustainable biofuel production.

plant biology↗

bioRxiv2024

Elemental profiling and genome-wide association mapping reveal genomic variants modulating ionomic composition in Populus trichocarpa leaves

The ionome represents elemental composition in plant tissues and can be an indicator of nutrient status as well as overall plant performance. Thus, identifying genetic determinants governing elemental uptake and storage is an important goal in plant breeding and engineering. In this study, we coupled high-throughput ionome characterization with high-resolution genome-wide association studies (GWAS) to uncover genetic loci that modulate ionomic composition in leaves of 584 black cottonwood poplar (Populus trichocarpa) genotypes. Congruence of alternate ionomic profiling platforms, i.e., inductively coupled plasma-mass spectrometry (ICP-MS), neutron activation analysis (NAA) and laser-induced breakdown spectroscopy (LIBS), was performed on leaf samples from a subset of the population. Significant agreement was observed across the three platforms with some notable exceptions for individual elements. Subsequently, we used the ICP-MS platform to profile the 584 genotypes focusing on 20 elements. GWAS performed using a set of high-density (>8.2 million) single nucleotide polymorphisms (SNP), identified multiple loci significantly associated with variations in these mineral elements. The potential causal genes for variations in the ionome were significantly enriched in genes whose homologs were previously associated to ion homeostasis in other species. Notably, a polymorphic copy of the high-affinity molybdenum transporter MOT1 was found directly associated to molybdenum content in leaf tissues. The results of the GWAS also provided evidence of physiological and genetic interactions between mineral elements in poplar. The new candidate genes predicted to play a key role in cross-homeostasis of multiple elements are new targets for engineering a variety of traits of interest in tree species.

plant biology↗

bioRxiv2024

Harnessing the power of poplar tree natural genetic variation for the development of future sustainable biofuels and bioproducts: a droughted marginal-land experiment for multi-disciplinary investigations

The emerging bioeconomy offers significant potential to replace fossil-fuel-based energy, manufacturing, and processing with that utilizing biomass as the raw feedstock. However, feedstock production from non-food crops such as fast-growing trees, must be delivered at scale, in a reliable and consistent manner, utilizing marginal land unsuitable for food crops and with minimum inputs. This new generation of feedstock crops has a limited history of domestication. Foundational knowledge is required to enable rapid selection and breeding for improved cultivars and varieties to enable large-scale planting of 600M ha, globally over the coming decades. Here, we describe an innovative field platform with over 1,000 unique genotypes of fast-growing poplar (Populus trichocarpa) trees, each sequenced and being subjected to a controlled drought. The 6.5 ha site provides opportunities to bring together multi-disciplinary phenotyping science linked to computational, and AI approaches, enabling the link between complex plant traits and their underlying genes to be rapidly established and translated into the development of improved climate-resilient germplasm for a future at-scale bioeconomy.

plant biology↗

bioRxiv2023

Engineered dsRNA-protein nanoparticles for effective long-distance transport, delivery and gene silencing in plants

Long-distance transport of exogenous biologically active RNA molecules in higher plants has not been reported. Here, we report that cationized bovine serum albumin (cBSA) avidly binds double-stranded beta-glucuronidase RNA (dsGUS RNA) to form nucleic acid-protein nanocomplexes. Using tobacco and poplar plants, we have shown effective uptake and long-distance transport of cBSA/dsGUS RNA nanocomplexes from basal ends of leaf petioles to leaf blades as well as from basal ends of shoots to their apexes and apical leaves. We have further demonstrated that the cBSA/dsGUS RNA nanocomplexes are highly effective in silencing both conditionally inducible DR5-GUS gene and constitutively active 35S-GUS gene in leaf, shoot and shoot meristem tissues. This cBSA/dsRNA delivery technology may provide a convenient, fast, and inexpensive tool for characterizing gene functions in plants, and potentially for in planta gene-editing.

molecular biology↗

bioRxiv2023

Functional analysis of Salix purpurea genes support roles for ARR17 and GATA15 as master regulators of sex determination

The Salicaceae family is of growing interest in the study of dioecy in plants because the sex determination region (SDR) has been shown to be highly dynamic, with differing locations and heterogametic systems between species. Without the ability to transform and regenerate Salix in tissue culture, previous studies investigating the mechanisms regulating sex in the genus Salix have been limited to genome resequencing and differential gene expression, which are mostly descriptive in nature, and functional validation of candidate sex determination genes has not yet been conducted. Here we used Arabidopsis to functionally characterize a suite of previously identified candidate genes involved in sex determination and sex dimorphism in the bioenergy shrub willow Salix purpurea. Six candidate master regulator genes for sex determination were heterologously expressed in Arabidopsis, followed by floral proteome analysis. In addition, 11 transcription factors with predicted roles in mediating sex dimorphism downstream of the SDR were tested using DAP-Seq in both male and female S. purpurea DNA. The results of this study provide further evidence to support models for the roles of ARR17 and GATA15 as master regulator genes of sex determination in S. purpurea, contributing to a regulatory system that is notably different from that of its sister genus Populus. Evidence was also obtained for the roles of two transcription factors, an AP2/ERF family gene and a homeodomain-like transcription factor, in downstream regulation of sex dimorphism.

plant biology↗

bioRxiv2022

Split selectable marker mediated gene stacking in plants

Plant synthetic biology and genetic engineering depend on the controlled expression of transgenes of interest, which relies heavily on the use of Agrobacterium-mediated transformation. Here we establish a novel split selectable marker system using protein splicing elements called "inteins" for Agrobacterium-mediated co-transformation in plants. This method enables robust co-transformation in Arabidopsis and poplar, providing a novel strategy for the simultaneous insertion of multiple genes into both herbaceous and woody plants.

plant biology↗

bioRxiv2022

PdeMIXTA04 triggers epidermal cells of placenta to differentiate into poplar catkins through forming MBW complexes with PdeMYC and PdeWD40

Differentiation of plant epidermal cells is a keen research topic in plant biology. Our study on Populus deltoides revealed that epidermal cells of the female flower placenta protruded to form catkin fibers immediately after pollination. We discovered that PdeMIXTA04 was explicitly expressed in placenta of female poplar flowers. Heterologous expression of PdeMIXTA04 in Arabidopsis demonstrated that it significantly promoted the leaf epidermal cells to differentiate into trichomes. Compared with the wild type, significant increases in trichome density and trichome branches were observed on leaves of all 35S:PdeMIXTA04 transgenic lines. Furthermore, transformation of PdeMIXTA04 in the trichomeless Arabidopsis mutant (gl1) restored trichome development to that of the wild type. GUS expression in poplar, driven by the promoter of PdeMIXTA04, also confirmed trichome-specific expression. We then screened a yeast library with PdeMIXTA04 and captured two interacting genes, PdeMYC and PdeWD40. Interactions between these two proteins were verified by yeast two-hybrid (Y2H), biomolecular fluorescence complementation (BiFC), dual-luciferase (dual-LUC), and pull-down assays, indicating that PdeMIXTA04 functions through the MYB-BHLH-WD40 (MBW) ternary complex. Our work presents evidence of PdeMIXTA04 as a candidate gene for editing to resolve catkins associated pollution and provides distinctive understanding of the molecular mechanism triggering differentiation of plant epidermal cells.

molecular biology↗

bioRxiv2022

High resolution mapping reveals hotspots and sex-biased recombination in Populus trichocarpa

Fine-scale meiotic recombination is fundamental to the outcome of natural and artificial selection. Here, dense genetic mapping and haplotype reconstruction were used to estimate recombination for a full factorial Populus trichocarpa cross of seven males and seven females. Genomes of the resulting 49 full-sib families (N=829 offspring) were re-sequenced, and high-fidelity biallelic SNP/INDELs and pedigree information were used to ascertain allelic phase and impute progeny genotypes to recover gametic haplotypes. The fourteen parental genetic maps contained 1820 SNP/INDELs on average that covered 376.7 Mb of physical length across 19 chromosomes. Comparison of parental and progeny haplotypes allowed fine-scale demarcation of cross-over (CO) regions, where 38,846 CO events in 1,658 gametes were observed. CO events were positively associated with gene density and negatively associated with GC content and long terminal repeats. One of the most striking findings was higher rates of COs in males in 8 out of 19 chromosomes. Regions with elevated male CO rates had lower gene density and GC content than windows showing no sex bias. High-resolution analysis identified 67 candidate CO hotspots spread throughout the genome. DNA sequence motifs enriched in these regions showed striking similarity to those of maize, Arabidopsis and wheat. These findings, and recombination estimates, will be useful for ongoing efforts to accelerate domestication of this and other biomass feedstocks, as well as future studies investigating broader questions related to evolutionary history, perennial development, phenology, wood formation, vegetative propagation, and dioecy that cannot be studied using annual plant model systems.

genetics↗

bioRxiv2021

An intein-mediated split-nCas9 system for base editing in plants

Virus-assisted delivery of the clustered regularly interspaced short palindromic (CRISPR)/CRISPR-associated (Cas) system represents a promising approach for editing plant genomes. However, the relatively large size of the CRISPR/Cas9 system is challenging to package into viral vectors with confined packaging capacity. To address this technical challenge, we developed a strategy that splits the required CRISPR-Cas9 components across a dual-vector system in which CRISPR-Cas reassembles into an active form following co-infection to achieve targeted genome editing in plant cells. An intein-mediated split system was adapted and optimized in plant cells by successfully demonstrating split-eYGFPuv expression. Using a plant-based biosensor, we demonstrated for the first time that the split-SpnCas9 is capable of inducing efficient base editing in plant cells and identified several valid split sites for future biodesign strategies. Overall, this strategy provides new opportunities to bridge different CRISPR/Cas9 tools including base editor, prime editor, and CRISPR activation with virus-mediated gene editing.

plant biology↗

bioRxiv2021

Biomass formation and sugar release efficiency of Populus modified by altered expression of a NAC transcription factor

Woody biomass is an important feedstock for biofuel production. Manipulation of wood properties that enable efficient conversion of biomass to biofuel reduces cost of biofuel production. Wood cell wall composition is regulated at several levels that involve expression of transcription factors such as wood-/secondary cell wall-associated NAC domains (WND or SND). In Arabidopsis thaliana, SND1 regulates cell wall composition through activation of its down-stream targets such as MYBs. The functional aspects of SND1 homologs in the woody Populus have been studied through transgenic manipulation. In this study, we investigated the role of PdWND1B, Populus SND1 sequence ortholog, in wood formation using transgenic manipulation through over-expression or silencing under the control of a vascular-specific 4-coumarate-CoA ligase (4CL) promoter. As compared to control plants, PdWND1B-RNAi plants were shorter in height, with significantly reduced stem diameter and dry biomass, whereas there were no significant differences in growth and productivity of PdWND1B over-expression plants. Conversely, PdWND1B over-expression lines showed a significant reduction in cellulose and increase in lignin content, whereas there was no significant impact on lignin content of down-regulated lines. Stem carbohydrate composition analysis revealed a decrease in glucose, mannose, arabinose, and galactose, but an increase in xylose in the over-expression lines. Transcriptome analysis revealed upregulation of several downstream transcription factors and secondary cell wall related structural genes in the PdWND1B over-expression lines that corresponded to significant phenotypic changes in cell wall chemistry observed in PdWND1B overexpression lines. Relative to the control, glucose release and ethanol production from stem biomass was significantly reduced in over-expression lines but appeared enhanced in the RNAi lines. Our results show that PdWND1B is an important factor determining biomass productivity, cell wall chemistry and its conversion to biofuels in Populus.

plant biology↗

bioRxiv2021

Plant-based biosensors for detecting CRISPR-mediated genome engineering

CRISPR/Cas has recently emerged as the most reliable system for genome engineering in various species. However, concerns about risks associated with CRISPR/Cas9 technology are increasing on potential unintended DNA changes that might accidentally arise from CRISPR gene editing. Developing a system that can detect and report the presence of active CRIPSR/Cas tools in biological systems is therefore very necessary. Here, we developed the real-time detection systems that can spontaneously indicate CRISPR-Cas tools for genome editing and gene regulation including CRISPR/Cas9 nuclease, base editing, prime editing and CRISPRa in plants. Using the fluorescence-based molecular biosensors, we demonstrated that the activities of CRISPR/Cas9 nuclease, base editing, prime editing and CRIPSRa can be effectively detected in transient expression via protoplast transformation and leaf infiltration (in Arabidopsis, poplar, and tobacco) and stable transformation in Arabidopsis.

plant biology↗

bioRxiv2020

Host auxin machinery is important in mediating root architecture changes induced by Pseudomonas fluorescens GM30 and Serendipita indica

Auxin is a key phytohormone that is integral to plant developmental processes including those underlying root initiation, elongation, and branching. Beneficial microbes have been shown to have an impact on root development, potentially mediated through auxin. In this study, we explore the role of host auxin signaling and transport components in mediating the root growth promoting effects of beneficial microbes. Towards this end, we undertook co-culture studies of Arabidopsis thaliana plants with microbes previously reported to promote lateral root proliferation and produce auxin. Two types of beneficial microbes were included in the present study; a plant growth promoting bacterial species of interest, Pseudomonas fluorescens GM30, and a well-studied plant growth promoting fungal species, Serendipita indica (Piriformospora indica). Following co-culture, lateral root production was found restored in auxin transport inhibitor-treated plants, suggesting involvement of microbe and/or microbially-produced auxin in altering plant auxin levels. In order to clarify the role of host auxin signaling and transport pathways in mediating interactions with bacterial and fungal species, we employed a suite of auxin genetic mutants as hosts in co-culture screens. Our result show that the transport proteins PIN2, PIN3, and PIN7 and the signaling protein ARF19, are required for mediating root architecture effects by the bacterial and/or fungal species. Mutants corresponding to these proteins did not significantly respond to co-culture treatment and did not show increases in lateral root production and lateral root density. These results implicate the importance of host auxin signaling in both bacterial and fungal induced changes in root architecture and serve as a driver for future research on understanding the role of auxin-dependent and auxin-independent pathways in mediating plant-microbe interactions in economically important crop species.

plant biology↗

bioRxiv2019

The somatic genetic and epigenetic mutation rate in a wild long-lived perennial Populus trichocarpa

BackgroundPlants can transmit somatic mutations and epimutations to offspring, which in turn can affect fitness. Knowledge of the rate at which these variations arise is necessary to understand how plant development contributes to local adaption in an eco-evolutionary context, particularly in long-lived perennials. ResultsHere, we generated a new high-quality reference genome from the oldest branch of a wild Populus trichocarpa tree with two dominant stems which have been evolving independently for 330 years. By sampling multiple, age-estimated branches of this tree, we used a multi-omics approach to quantify age-related somatic changes at the genetic, epigenetic and transcriptional level. We show that the per-year somatic mutation and epimutation rates are lower than in annuals and that transcriptional variation is mainly independent of age divergence and cytosine methylation. Furthermore, a detailed analysis of the somatic epimutation spectrum indicates that transgenerationally heritable epimutations originate mainly from DNA methylation maintenance errors during mitotic rather than during meiotic cell divisions. ConclusionTaken together, our study provides unprecedented insights into the origin of nucleotide and functional variation in a long-lived perennial plant.

genomics↗

bioRxiv2019

Inference of Gene Regulatory Network Uncovers the Linkage Between Circadian Clock and Crassulacean Acid Metabolism in Kalanchoë fedtschenkoi

The circadian clock drives time-specific gene expression, allowing for associated biological processes to be active during certain times of the 24 h day. Crassulacean acid metabolism (CAM) photosynthetic plants represent an interesting case of circadian regulation of gene expression as CO2 fixation and stomatal movement in CAM plants display strong circadian dynamics. The molecular mechanisms behind how the circadian clock enabled these physiological differences is not well understood. Therefore, we set out to investigate whether core circadian elements in CAM plants were re-phased during evolution, or whether networks of phase-specific genes were simply connected to different core elements. We utilized a new metric for identifying candidate core genes of a periodic gene network and then applied the Local Edge Machine (LEM) algorithm to infer regulatory relationships between the candidate core clock genes and orthologs of known core clock genes in K. fedtschenkoi. We also used LEM to identify stomata-related gene targets for K. fedtschenkoi core clock genes and constructed a subsequent gene regulatory network. Our results provide new insights into the mechanism of circadian control of CAM-related genes in K. fedtschenkoi, facilitating the engineering of CAM machinery into non-CAM plants for sustainable crop production in water-limited environments.

plant biology↗

bioRxiv2019

A Willow Sex Chromosome Reveals Convergent Evolution of Complex Palindromic Repeats

BackgroundSex chromosomes in a wide variety of species share common characteristics, including the presence of suppressed recombination surrounding sex determination loci. They have arisen independently in numerous lineages, providing a conclusive example of convergent evolution. Mammalian sex chromosomes contain multiple palindromic repeats across the non-recombining region that facilitate sequence conservation through gene conversion, and contain genes that are crucial for sexual reproduction. Plant sex chromosomes are less well understood, and in particular it is not clear how coding sequence conservation is maintained in the absence of homologous recombination.\n\nResultsHere we present the first evidence of large palindromic structures in a plant sex chromosome, based on a highly contiguous assembly of the W chromosome of the dioecious shrub Salix purpurea. Two consecutive palindromes span over a region of 200 kb, with conspicuous 20 kb stretches of highly conserved sequences among the four arms. The closely-related species S. suchowensis also has two copies of a portion of the palindrome arm and provides strong evidence for gene conversion. Four genes in the palindrome are homologous to genes in the SDR of the closely-related genus Populus, which is located on a different chromosome. These genes show distinct, floral-biased expression patterns compared to paralogous copies on autosomes.\n\nConclusionThe presence of palindromic structures in sex chromosomes of mammals and plants highlights the intrinsic importance of these features in adaptive evolution in the absence of recombination. Convergent evolution is driving both the independent establishment of sex chromosomes as well as their fine-scale sequence structure.

genomics↗