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Tsai, C.-J.

Publications and source records attributed to Tsai, C.-J..

At least 19 recordsLinked to original sources
bioRxiv2026

Spatially resolved transcriptomics of poplar reveals tissue organization across shoot-associated organs

Poplar (Populus spp.) is a model for tree biology and a platform for engineering woody biomass, biofuels, biomaterials, and bioproducts. Many relevant traits depend on tissue position, developmental stage, and cell type, yet these spatial relationships are difficult to recover from bulk or single-cell transcriptomes. Here, we generated a spatial transcriptome atlas of Populus tremula x P. alba INRA 717-1B4 across the shoot apex, axillary bud, stem, and petiole. After quality control, the atlas retained 29,687 spatial spots from 45 tissue sections and detected 58,748 genes. Histology-guided clustering and marker analysis resolved meristematic, epidermal, cortical, vascular, and organ-specific domains. Cross-organ comparisons assessed whether published markers retained tissue-associated expression across different anatomical contexts and developmental stages, while de novo analysis identified additional domain-enriched candidates. As case studies of the utility of the atlas, we examined the emergence of trichome-associated programs in the shoot apex and adaxial - abaxial expression differences in petioles. A trichome identity score based on poplar markers from the single-cell shoot atlas peaked along the inferred meristem-to-primordium trajectory, revealing spatially localized expression of trichome-associated programs during early leaf development. Petiole expression differences were concentrated in the epidermis and cortex and involved polarity-associated, auxin-responsive, and cell-wall-remodeling genes, with distinct expression profiles across leaf positions. Together, these data provide a spatial reference for investigating tissue differentiation and developmental patterning in a transformable poplar genotype.

plant biology↗
bioRxiv2026

A Comprehensive Epidermal Map from a Poplar Single-Cell Shoot Atlas Reveals New Trichome-Specific Genes

Poplar (Populus spp.) is a model system for tree biology. Specifically, P. tremula x P. alba INRA 717-1B4 (hereafter "poplar 717") has become an important platform for functional genomics and synthetic biology due to its rapid growth and ease of transgenesis. Here, we present a single-cell RNA-seq atlas of the poplar 717 shoot, including apical meristem, primary and secondary stems, and three stages of leaf development. Analysis of ca. 159,000 cells resolved 40 transcriptionally distinct clusters representing 7 major cell types, providing a high-resolution view of shoot development and tissue organization. We focused on the epidermis which constituted >15% of cells in the shoot atlas for in-depth characterization of epidermal heterogeneity. By integrating known marker genes with transcriptomic signatures consistent with established poplar leaf phytochemistry, we annotated epidermal cell subclusters corresponding to developmental stages, spatial location, and specialized cell types, including a distinct population of non-glandular trichomes. Coupling the single-cell RNA-seq atlas with bulk transcriptome data from glabrous mutants enabled the identification of novel trichome markers. Experimental validation of a representative trichome-specific promoter established a tool with potential to support cell type-targeted-metabolic engineering. We provide the poplar 717 atlas to the community through the BioPoplar Atlas Viewer (http://bio-poplar-atlas.com), providing a platform to explore the poplar transcriptome at single-cell resolution and a foundation for data-driven cell type-aware genetic engineering strategies in poplar.

plant biology↗
bioRxiv2026

Satellite DNA Editing Enables Meiosis-Independent Chromosome Engineering

Chromosome{square}scale genome engineering in plants typically relies on locus-specific recombination during meiosis to select desirable outcomes, limiting its application in species with long generation times. Here, we show that CRISPR targeting of abundant satellite DNA enables multiplexed chromosome restructuring in aspen, generating diverse large-scale structural variants in the first generation. These megabase rearrangements remain mitotically stable through clonal propagation and regeneration with no obvious growth defects.

plant biology↗
bioRxiv2025

Loss of flavonol 3-O-glucosyltransferase activity confers soybean resistance to leaf-chewing insects

Caterpillars and beetles are among the most economically damaging defoliating insects, and their economic damage is predicted to increase in the coming decades. Hence the use of genetically derived resistance to supplement other pest control strategies is warranted. In soybean (Glycine max (L.) Merr.), a major determinant for resistance is the quantitative trait locus, QTL-M. Glyma07g14530, the gene underlying QTL-M, encodes a feeding-inducible flavonol 3-O-glycosyltransferase (F3GlcT or UGT78D2) that glucosylates kaempferol, as well as quercetin, myricetin, and isorhamnetin. The resistant allele has a premature stop codon in it, thus preventing the glucosylation and sequestration of flavonols in the vacuole, leading to a concomitant accumulation of proanthocyanidins and manifestation of resistance. Expressing the dominant (susceptible) allele in resistant plants restores susceptibility and silencing the susceptible allele results in resistance. The discovery and characterization of GmF3GlcT helps clarify the role of flavonoids in resistance to leaf-chewing insects and facilitates the development of insect-resistant cultivars that ultimately can lower production costs and reduce insecticide applications. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=131 SRC="FIGDIR/small/679769v1_ufig1.gif" ALT="Figure 1"> View larger version (46K): org.highwire.dtl.DTLVardef@10e2e77org.highwire.dtl.DTLVardef@ca905org.highwire.dtl.DTLVardef@12cb46corg.highwire.dtl.DTLVardef@1f9328b_HPS_FORMAT_FIGEXP M_FIG C_FIG The loss of flavonol 3-O-glucosyltransferase in soybean reduces feeding damage from defoliating insects and is accompanied by a concomitant increase in proanthocyanidins (red arrowhead). SignificanceUnraveling the biochemical and genetic basis of soybean resistance to leaf-chewing insects facilitates the development of new, naturally insect resistant varieties. Such varieties contribute to on-farm profitability and reduced concerns over pesticide residues in the field.

plant biology↗
bioRxiv2025

An updated sulfate transporter phylogeny uncovers a perennial-specific subgroup associated with lignification

Sulfate-proton co-transporters (SULTRs) mediate sulfate uptake, transport, storage, and assimilation in plants. The SULTR family has historically been classified into four groups (SULTR1-SULTR4), with well-characterized roles for SULTR groups 1, 2, and 4. However, the functions of the large and diverse SULTR3 group remain poorly understood. Here, we present an updated phylogenetic analysis of SULTRs across angiosperms, including multiple early-divergent lineages. Our results suggest that the enigmatic SULTR3 group comprises four distinct subfamilies that predate the emergence of angiosperms, providing a basis for reclassifying the SULTR family into seven subfamilies. This expanded classification is supported by subfamily-specific gene structures and amino acid substitutions in the substrate-binding pocket. Structural modeling identified three serine residues uniquely lining the substrate-binding pocket of SULTR3.4, enabling three hydrogen bonds with the phosphate ion. The data support the proposed neofunctionalization of this subfamily for phosphate allocation within vascular tissues. Transcriptome analysis of Populus tremula x alba revealed divergent tissue expression preferences among SULTR subfamilies and between genome duplicates. We observed partitioned expression in vascular tissues among the four SULTR3 subfamilies, with PtaSULTR3.4a and PtaSULTR3.2a preferentially expressed in primary and secondary xylem, respectively. Gene coexpression analysis revealed coordinated expression of PtaSULTR3.4a with genes involved in phosphate starvation responses and nutrient transport, consistent with a potential role in phosphate homeostasis. In contrast, PtaSULTR3.2a was strongly coexpressed with lignification and one-carbon metabolism genes and their upstream transcription regulators. PtaSULTR3.2a belongs to a eudicot-specific branch of the SULTR3.1 subfamily found only in perennial species, suggesting a specialized role in lignifying tissues. Together, our findings provide a refined phylogenetic framework for the SULTR family and suggest that the expanded SULTR3 subfamilies have undergone neofunctionalization during the evolution of vascular and perennial plants.

plant biology↗
bioRxiv2025

Populus VariantDB v3.2 facilitates CRISPR and Functional Genomics Research

The success of CRISPR genome editing studies depends critically on the precision of guide RNA (gRNA) design. Sequence polymorphisms in outcrossing tree species pose design hazards that can render CRISPR genome editing ineffective. Despite recent advances in tree genome sequencing with haplotype resolution, sequence polymorphism information remains largely inaccessible to various functional genomics research efforts. The Populus VariantDB v3.2 addresses these challenges by providing a user-friendly search engine to query sequence polymorphisms of heterozygous genomes. The database accepts short sequences, such as gRNAs and primers, as input for searching against multiple poplar genomes, including hybrids, with customizable parameters. We provide examples to showcase the utilities of VariantDB in improving the precision of gRNA or primer design. The platform-agnostic nature of the probe search design makes Populus VariantDB v3.2 a versatile tool for the rapidly evolving CRISPR field and other sequence-sensitive functional genomics applications. The database schema is expandable and can accommodate additional tree genomes to broaden its user base.

plant biology↗
bioRxiv2024

A compendium of nonredundant short Polymerase III promoters for CRISPR applications

Multiplex genome editing via CRISPR enables simultaneous gene knockout, activation, and repression, but current toolkits often rely on repetitive and lengthy Pol III promoters, limiting construct diversity and genetic stability. Here, we present a diverse collection of minimal U6 and U3 Pol III promoters that support efficient guide RNA (gRNA) expression in dicots. Using CRISPR-Cas9 editing of mEGFP in Nicotiana benthamiana and Populus tremula x alba, we demonstrate that minimal promoters can match the performance of their longer counterparts. A systematic evaluation of 14 short promoters revealed that most are functional across species, with exceptions linked to sequence variations in the upstream sequence element (USE) and TATA box. Mutagenesis confirmed the causal role of specific nucleotide changes in promoter activity. Furthermore, we show that regions outside these conserved motifs can be modified to create new-to-nature, functional promoters. This work refines the consensus for functional Pol III promoter elements and expands the toolkit for efficient, stable, and scalable CRISPR editing in dicots, with implications for synthetic promoter design.

plant biology↗
bioRxiv2024

CRISPR knockout of winter-biased SUT4 alters phenology, biomass accrual, and fertility of field-grown hybrid poplar

Climate uncertainty is intensifying the need for greater plasticity in carbohydrate reserve utilization to support winter survival and spring growth in woody perennials. In poplar, the single-copy SUT4, which encodes a tonoplast-localized sucrose transporter, and the SUT5/SUT6 genome duplicates, which encode plasma membrane-localized transporters, are expressed year-round, with SUT4 showing highest expression during cool seasons. Given its role in vacuolar sucrose efflux and winter-predominant expression, SUT4 may play a key role in modulating seasonal carbohydrate dynamics. While SUT4-knockdown and knockout effects have been studied under greenhouse conditions, their impact under field conditions remains unexplored. Here, we report a field-based study comparing CRISPR knockout mutants of winter-expressed SUT4 and SUT5/SUT6 in Populus tremula x alba. We show that sut4, but not sut56, mutants exhibited earlier autumn leaf senescence, delayed spring bud flush, reduced stem growth, and altered sugar partitioning in winter xylem and bark relative to controls. After two years in the field, all genotypes flowered before leaf flush in early spring; however, sut4 mutants produced sterile ovules despite developing normal-looking catkins. Metabolic profiling revealed disrupted sucrose and raffinose dynamics in elongating sut4 catkins, accompanied by transcriptomic signatures of elevated stress and downregulation of proanthocyanidin biosynthesis and circadian clock genes. These findings highlight the critical role of SUT4 in coordinating sugar allocation, stress responses, and seasonal development in poplar. Significance statementThis field study demonstrates that loss of SUT4, the most highly expressed sucrose transporter during cool seasons, disrupts phenology, growth, and fertility in poplar. Altered sugar and raffinose dynamics and transcriptomic signatures of stress and circadian clock gene dysregulation in the mutants underscore SUT4s role in coordinating sugar allocation and seasonal developmental transitions under natural environmental conditions.

plant biology↗
bioRxiv2024

Human Cells for Human Proteins: Isotope Labeling in Mammalian Cells for Functional NMR Studies of Disease-Relevant Proteins

In biological and biomedical research the focus progressively moves towards difficult human proteins, which often can only be expressed in higher eukaryotic cells. Nuclear magnetic resonance (NMR) could contribute significantly to the understanding of important proteins as it is one of the most information-rich methods: it allows studying structure, function and dynamics of biomolecules and, importantly, their interactions with natural ligands or drugs. However, to exploit the full potential of NMR, proteins must be isotope labeled. Although expression protocols in e.g. HEK293 cells are often established, isotope labeling is difficult and very expensive. To resolve this disparity, we have developed a comprehensive suite of protocols for isotope labeling in HEK293 cells. We demonstrate uniform 15N and 13C labeling, as well as specific labeling, with special focus on methyl bearing amino acids, including the popular ILV 13C-methyl labeling pattern. Labeling is achieved with a simple laboratory setup and affordable labeling media. These are based on either labeled amino acids, their precursors or amino extracts from microorganisms, like yeast, algae or bacteria. This enables NMR studies of important, but difficult to produce proteins, like receptors. We therefore expect that these new methods make many highly important proteins accessible to NMR studies and allow exploiting the high information content of this method for accelerating biological and pharmaceutical research.

biophysics↗
bioRxiv2024

Active state structures of a bistable visual opsin bound to G proteins

Opsins are G protein-coupled receptors (GPCRs) that have evolved to detect light stimuli and initiate intracellular signaling cascades. Their role as signal transducers is critical to light perception across the animal kingdom. Opsins covalently bind to the chromophore 11-cis retinal, which isomerizes to the all-trans isomer upon photon absorption, causing conformational changes that result in receptor activation. Monostable opsins, responsible for vision in vertebrates, release the chromophore after activation and must bind another retinal molecule to remain functional. In contrast, bistable opsins, responsible for non-visual light perception in vertebrates and for vision in invertebrates, absorb a second photon in the active state to return the chromophore and protein to the inactive state. Structures of bistable opsins in the activated state have proven elusive, limiting our understanding of how they function as bidirectional photoswitches. Here we present active state structures of a bistable opsin, jumping spider rhodopsin isoform-1 (JSR1), in complex with its downstream signaling partners, the Gi and Gq heterotrimers. These structures elucidate key differences in the activation mechanisms between monostable and bistable opsins, offering essential insights for the rational engineering of bistable opsins into diverse optogenetic tools to control G protein signaling pathways.

biochemistry↗
bioRxiv2024

Activating an invertebrate bistable opsin with the all-trans 6.11 retinal analogue

Animal vision depends on opsins, a category of G protein-coupled receptor (GPCR) that achieves light sensitivity by covalent attachment to retinal. Typically binding as an inverse agonist in the 11-cis form, retinal photoisomerizes to the all-trans isomer and activates the receptor, initiating downstream signaling cascades. Retinal bound to bistable opsins isomerizes back to the 11-cis state after absorption of a second photon, inactivating the receptor. Bistable opsins are essential for invertebrate vision and non-visual light perception across the animal kingdom. While crystal structures are available for bistable opsins in the inactive state, it has proven difficult to form homogeneous populations of activated bistable opsins either via illumination or reconstitution with all-trans retinal. Here we show that a non-natural retinal analogue, all-trans retinal 6.11 (ATR6.11), can be reconstituted with the invertebrate bistable opsin, Jumping Spider Rhodopsin-1 (JSR1). Biochemical activity assays demonstrate that ATR6.11 functions as an agonist of JSR1. ATR6.11 binding also enables complex formation between JSR1 and downstream signaling partners. Our findings demonstrate the utility of retinal analogues for biophysical characterization of bistable opsins, which will deepen our understanding of light perception in animals.

biochemistry↗
bioRxiv2023

Sucrose or starch? The influence of tonoplast sucrose transporter perturbation on carbon partitioning for growth, defense, and winter protection in coppiced poplar

Non-structural carbohydrate reserves of stems and roots underpin overall tree fitness. These reserves also contribute to productivity under short-rotation management practices such as coppicing for bioenergy. While sucrose and starch comprise the predominant carbohydrate reserves of Populus, utilization is understood primarily in terms of starch turnover. The tonoplast sucrose transport protein SUT4 modulates sucrose export from source leaves to distant sinks during photoautotrophic growth, but the possibility of its involvement in remobilizing carbohydrates from sinks during heterotrophic growth has not been explored. Here, we used PtaSUT4-knockout mutants of Populus tremula x P. alba (INRA 717-1B4) in winter (cool) and summer (warm) glasshouse coppicing experiments to strain carbon demand and test for SUT4 involvement in reserve utilization. We show that epicormic bud emergence, subsequent growth, and xylem hexose increases were delayed or reduced in sut4 mutants following lower temperature winter coppicing. Depletion of stem reserves during post-coppice regrowth was not impaired in sut4, but sut4 winter maintenance costs may have been higher in metabolic terms. Bark accrual of abundant defense metabolites, including salicinoids, chlorogenic acids, and flavonoid products, was prioritized in the summer, but attenuated in sut4 mutants. Summer sprout growth was not reduced in sut4 compared to controls. Together, our results point to shifting priorities for SUT4 modulation of seasonal trade-offs between growth and other priorities in the stem and emerging buds during reserve utilization in Populus.

plant biology↗
bioRxiv2023

Neurodevelopmental disorders and cancer networks share pathways; but differ in mechanisms, signaling strength, and outcome

Neurodevelopmental disorders (NDDs) and cancer are connected, with immunity as their common factor. Their clinical presentations differ; however, individuals with NDDs are more likely to acquire cancer. Schizophrenia patients have [~]50% increased risk; autistic individuals also face an increased cancer likelihood. NDDs are associated with specific brain cell types at specific locations, emerging at certain developmental time windows during brain evolution. Their related mutations are germline; cancer mutations are sporadic, emerging during life. At the same time, NDDs and cancer share proteins, pathways, and mutations. Here we ask exactly which features they share, and how despite their commonality, they differ in outcomes. Our pioneering bioinformatics exploration of the mutations, reconstructed disease-specific networks, pathways, and transcriptome profiles of autism spectrum disorder (ASD) and cancers, points to elevated signal strength in pathways related to proliferation in cancer, and differentiation in ASD. Signaling strength, not the activating mutation, is the key factor in deciding cancer versus NDDs.

systems biology↗
bioRxiv2022

In vitro floral development in poplar: Insights into seed trichome and trimonoecy regulation

Woody perennials including Populus spp. (poplars) have a juvenile phase that ranges from several years to decades in length. This and the year-long floral development process are major impediments to breeding and to fundamental research of reproductive traits. Here we report a CRISPR-empowered in vitro flowering system and demonstrate its application using three reproductive traits: sex, seed trichomes, and a previously undescribed potential for trimonoecy in poplar.

plant biology↗
bioRxiv2022

ULTRAFAST STRUCTURAL CHANGES DIRECT THE FIRST MOLECULAR EVENTS OF VISION

Vision is initiated by the rhodopsin family of light-sensitive G protein-coupled receptors (GPCRs). A photon is absorbed by the 11-cis retinal chromophore of rhodopsin which isomerises within 200 femtoseconds to the all-trans conformation, thereby initiating the cellular signal transduction processes that ultimately lead to vision. However, the intramolecular mechanism by which the photoactivated retinal induces the activation events inside rhodopsin remains elusive. In this work, we use ultrafast time-resolved crystallography at room temperature to determine how an isomerised twisted all-trans retinal stores the photon energy required to initiate protein conformational changes associated with the formation of the G protein-binding signalling state. The distorted retinal at 1 ps time-delay of photoactivation has pulled away from half of its numerous interactions with its binding pocket, and the excess of the photon energy is released through an anisotropic protein breathing motion in the direction of the extracellular space. Strikingly, the very early structural motions in the protein side chains of rhodopsin appear in regions involved in later stages of the conserved Class A GPCR activation mechanism. Our work sheds light on the earliest stages of vision in vertebrates and points to fundamental aspects of the molecular mechanisms of agonist-mediated GPCR activation.

biochemistry↗
bioRxiv2022

Multiplex editing of the Nucleoredoxin1 tandem array in poplar: Decrypting outcomes due to CRISPR/Cas9-mediated translocations and complex inversions

The CRISPR-Cas9 system has been deployed for precision mutagenesis in an ever-growing number of species, including agricultural crops and forest trees. Its application to closely linked genes with extremely high sequence similarities has been less explored. Here, we used CRISPR-Cas9 to mutagenize a tandem array of seven Nucleoredoxin1 (NRX1) genes spanning ~100 kb in Populus tremula x alba. We demonstrated efficient multiplex editing with one single gRNA in 42 transgenic lines. The mutation profiles ranged from small indels and local deletions in individual genes to large genomic dropouts and rearrangements spanning tandem genes. We also detected complex rearrangements including translocations and inversions resulting from multiple cleavage and repair events. Target capture sequencing was instrumental for unbiased assessments of repair outcomes to reconstruct unusual mutant alleles. The work highlights the power of CRISPR-Cas9 for multiplex editing of tandemly duplicated genes to generate diverse mutants with structural and copy number variations to aid functional characterization.

plant biology↗
bioRxiv2021

Multiplex knockout of trichome-regulating MYBs in poplar affects light sensitivity and triterpene accumulation

As the focus for CRISPR 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 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 Populus tremula x P. alba INRA 717-1B4. Unexpected duplications of MYB186 and MYB138 resulted in a total of eight alleles for the three targeted genes in the hybrid poplar. Deep sequencing and PCR 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 non-glandular trichomes of poplar. ONE SENTENCE SUMMARYTargeting conserved sequences with a single gRNA allowed efficient mutagenesis of a multigene family and the recovery of trichomeless and triterpene-free poplar mutants.

plant biology↗