Biology subjects

Chen, J.-G.

Publications and source records attributed to Chen, J.-G..

12 recordsLinked to original sources

bioRxiv2026

Neryl diphosphate-derived monoterpene biosynthesis via a biosynthetic gene cluster in the liverwort Marchantia polymorpha

Monoterpenes (C10) are a large group of specialized metabolites important for plant interactions with the environment. Their biosynthesis is well understood in seed plants, where geranyl diphosphate serves as the canonical substrate, but knowledge of monoterpene biosynthesis outside seed plants remains very limited. Here, we report neryl diphosphate (NPP)-derived monoterpene biosynthesis via a biosynthetic gene cluster in the liverwort Marchantia polymorpha. MpMTPSL2, a microbial-type terpene synthase, converts NPP into -phellandrene and D-limonene in vitro. CRISPR knockout lines showed reduced production of both monoterpenes, providing direct genetic evidence for its in planta function. MpCPT5, a cis-prenyltransferase (CPT) family member identified through co-expression with MpMTPSL2, was confirmed to encode NPP synthase, as its knockout plants abolished -phellandrene and D-limonene production. Subcellular localization analyses in protoplasts and stable transgenic plants demonstrated that both MpCPT5 and MpMTPSL2 localize to plastids, co-localizing across all cell types with markedly stronger signals in non-green plastids of oil-body cells. Consistent with this, expression of both genes under their respective promoters was nearly abolished in oil-body-deficient mutants and strongly upregulated in a gain-of-function line for oil-body formation. MpMTPSL2 and MpCPT5 are physically linked through a shared bidirectional promoter that drives their coexpression specific to oil body cells, forming a unique biosynthetic gene cluster whose coordinated expression is maintained by PRC2-mediated H3K27me3 repression. Phylogenetic analysis implies that NPP synthases in M. polymorpha and in flowering plants evolved independently from their respective long-chain CPT ancestors. These findings provide new insights into the mechanisms and evolution of monoterpene biosynthesis in non-seed plants. Significance statementMonoterpenes are a diverse group of specialized metabolites produced widely among land plants, yet our understanding of their biosynthesis outside seed plants remains limited. Here we report that in the liverwort Marchantia polymorpha, the non-canonical substrate neryl diphosphate is used for monoterpene biosynthesis. The functions of the monoterpene synthase gene MpMTPSL2 and the neryl diphosphate synthase gene MpCPT5 were demonstrated through CRISPR knockouts. These two genes are physically linked and share a bidirectional promoter. Promoter assays show both genes function in plastids within oil-body cells, revealing cell-type specificity. These findings shew new light on the mechanisms and evolution of monoterpene biosynthesis in non-seed plants.

plant 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

Diversification and conservation of DNA binding specificities of SPL family of transcription factors

O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=89 SRC="FIGDIR/small/612952v2_ufig1.gif" ALT="Figure 1"> View larger version (24K): org.highwire.dtl.DTLVardef@1154c42org.highwire.dtl.DTLVardef@1419f5borg.highwire.dtl.DTLVardef@1454a7aorg.highwire.dtl.DTLVardef@1cbd5f_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOGRAPHICAL ABSTRACTC_FLOATNO C_FIG SQUAMOSA Promoter-Binding Protein-Like (SPL) transcription factors are key regulators of plant development and stress responses. Here, we present a comprehensive DNA affinity purification sequencing (DAP-seq) analysis of 14 of the 16 SPL members in Arabidopsis thaliana, revealing two major classes based on distinct DNA-binding motifs. Gene Ontology enrichment of target genes indicated both shared and specialized functions among AtSPLs, including roles in hormone signaling, metal ion homeostasis, and developmental transitions. Comparative analysis of closely related paralogs, AtSPL9 and AtSPL15, uncovered divergence in their genomic binding locations and target gene regulation, particularly in auxin and abscisic acid pathways. These differences were supported by motif analysis and protoplast reporter assays. Extending our study across species, we analyzed DAP-seq data from maize (Zea mays) and wheat (Triticum aestivum) SPL homologs, identifying conserved target genes but species-specific DNA motifs. Notably, wheat SPLs preferentially bind to longer, palindromic motifs distinct from the GTAC core motif in Arabidopsis. Using AlphaFold3 structure modeling, we show that these motif differences may arise from enhanced protein-protein interactions that stabilize dimeric binding. Our results highlight how SPL family members diversify through DNA-binding evolution and suggest that transcription factor dimerization contributes to motif complexity and regulatory specialization across plant genomes.

plant 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

Catabolic pathway acquisition by soil pseudomonads readily enables growth with salicyl alcohol but does not affect colonization of Populus roots

Horizontal gene transfer (HGT) is a fundamental evolutionary process that plays a key role in bacterial evolution. The likelihood of a successful transfer event is expected to depend on the precise balance of costs and benefits resulting from pathway acquisition. Most experimental analyses of HGT have focused on phenotypes that have large fitness benefits under appropriate selective conditions, such as antibiotic resistance. However, many examples of HGT involve phenotypes that are predicted to provide smaller benefits, such as the ability to catabolize additional carbon sources. We have experimentally reproduced one such HGT event in the laboratory, studying the effects of transferring a pathway for catabolism of the plant-derived aromatic compound salicyl alcohol into soil isolates from the Pseudomonas genus. We find that pathway acquisition enables rapid catabolism of salicyl alcohol with only minor disruptions to existing metabolic and regulatory networks of the new host. However, this new catabolic potential does not confer a measurable fitness advantage during competitive growth in the rhizosphere. We conclude that the phenotype of salicyl alcohol catabolism is readily transferred by HGT but is selectively neutral under environmentally-relevant conditions. We propose that this condition is common and that HGT of many pathways will be self-limiting, because the selective benefits are small and negative frequency-dependent.

microbiology↗

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

Plant inositol transport influences bacterial colonization phenotypes

Plant microbiomes are assembled and modified through a complex milieu of biotic and abiotic factors. Despite dynamic and fluctuating contributing variables, specific host metabolites are consistently identified as important mediators of microbial interactions. We combine information from a large-scale metatranscriptomic dataset from natural poplar trees and experimental genetic manipulation assays in model Arabidopsis seedlings to converge on a conserved role for transport of the plant metabolite myo-inositol in mediating host-microbe interactions. While microbial catabolism of this compound is often linked to increased host colonization, we identify motility phenotypes that occur independently of catabolism, suggesting that inositol may additionally serve as a eukaryotic-derived signaling molecule to modulate microbial activities. Our data suggests host control of this compound and resulting microbial behavior are important mechanisms at play surrounding the host metabolite inositol.

microbiology↗

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↗

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↗