Biology subjects

Kliebenstein, D. J.

Publications and source records attributed to Kliebenstein, D. J..

At least 19 recordsLinked to original sources

bioRxiv2026

ATML1-GIR1-TPL/TPR transcriptional repression module controls glucosinolates and giant cells in Arabidopsis thaliana sepals

Glucosinolates (GSLs) are sulfur- and nitrogen-containing secondary metabolites that serve as defense compounds in Arabidopsis and other members of the Brassicales. Although the enzymatic pathway that produces GSLs is well-studied, the upstream mechanisms that control their tissue-specific synthesis are poorly understood. We identified a novel repression module that transcriptionally regulates GSL levels in sepals, the modified leaves that protect reproductive tissues within the floral bud. GLABRA2 (GL2) INTERACTING REPRESSOR1 (GIR1) interacts directly with Arabidopsis thaliana MERISTEM LAYER1 (ATML1), an HD-Zip IV transcription factor known to be required for giant cell formation in the sepal epidermis. This interaction requires a predicted Zn finger of GIR1 and the C-terminal START adjacent domain (STAD) of ATML1. The gir1 loss-of-function mutants exhibit excess giant cells, in contrast to atml1 mutants which display fewer giant cells, supporting the role of GIR1 as a negative regulator of ATML1. We confirmed that GIR1 interacts with TOPLESS (TPL) and TOPLESS-RELATED (TPR) corepressors, and coimmunoprecipitation demonstrated that GIR1 acts as an adaptor protein connecting ATML1 and TPL/TPR. RNA sequencing revealed that numerous genes involved in GSL biosynthesis, including the key transcriptional regulator MYB29, are upregulated in gir1 mutants. Consistent with the transcriptomic data, chemical analysis revealed that gir1 mutants display elevated GSL levels in sepals. Mass spectrometry imaging confirmed high GSL accumulation in gir1 sepals compared to wild type and atml1. Overall, our findings uncover a previously unrecognized link between cell expansion and GSL metabolism, suggesting strategies for engineering plants with cell-type specific GSL profiles. Significance StatementPlants belonging to the order Brassicales produce sulfur-containing glucosinolate (GSL) metabolites that serve in defense against herbivory. In cruciferous vegetables such as broccoli and kale, these compounds contribute to their unique flavors and health-promoting attributes. In agriculturally important oilseed crops, they affect the palatability of animal feeds. Here, we identified a novel transcriptional regulatory module that controls GSL biosynthesis in the epidermis of the sepal, the floral organ that protects the reproductive tissues. This regulatory module also controls cell expansion of specific cell types in the sepal, demonstrating a surprising connection between cell growth and a chemical defense pathway in plants. Our results suggest strategies for engineering crops with tissue-specific GSL profiles to fit agronomic needs.

plant biology↗

bioRxiv2026

A co-proteomic view of metabolite-specific interactions in the Botrytis cinerea-Arabidopsis pathosystem

To successfully infect their myriad hosts, generalist plant pathogens must tolerate a vast arsenal of plant specialized defense metabolites. To understand how host-specific metabolites influence plant-generalist pathogen interactions, we conducted a co-proteomic analysis of both Arabidopsis thaliana and Botrytis cinerea proteomes from the same samples during early infection. The Arabidopsis proteomic responses to Botrytis center around induction and suppression of defense metabolite pathways, particularly camalexin and glucosinolates. Several Botrytis proteins involved in key virulence pathways were induced within 32-48 hours, including potential defense metabolite detoxification proteins. Co-proteomic analysis using a panel of Arabidopsis genotypes with differing glucosinolate profiles revealed that disruptions to the glucosinolate pathway had broad changes on the Arabidopsis proteome, and that Botrytis induces specific proteins in response to presence/absence of Arabidopsis defense metabolites. Among the proteins that were induced quickly on infection and linked to the presence of glucosinolates, we validated a novel isothiocyanate hydrolase in Botrytis, BcSaxA, that catabolizes isothiocyanates in vitro. Gene expression data further indicated BcSaxA is expressed only in dicot hosts containing isothiocyanates. Our study describes a highly dynamic host proteome during infection with Botrytis and elucidates metabolite-specific infection strategies for a generalist pathogen.

plant biology↗

bioRxiv2026

Natural Variations of Spring Wheat Nitrogen and Carbon Assimilation under Different Inorganic Nitrogen Forms and CO2 levels

Wheat (Triticum aestivum L.) production depends upon nitrogen fertilization and may be threatened by the climate conditions anticipated in the next few decades. The natural variation in the ability of wheat to assimilate carbon and different inorganic nitrogen forms, nitrate (NO3-) and ammonium (NH4+), into vegetative growth remains unexplored. Here, we evaluated growth under either NO3- or NH4+ as a sole nitrogen source for 875 spring hexaploid wheat accessions that represent the genetic diversity within the global germplasm. These accessions varied over 8-fold in vegetative biomass but grew similarly under moderate levels of either nitrogen form. At high, potentially toxic concentrations of NH4+, however, they lost approximately 20% of their biomass. We characterized the influence of changing CO2 levels in bi-parental Nested Association Mapping populations. Genetic backgrounds determined wheat biomass responses to CO2 enrichment and nitrogen form. Genome-Wide Association and linkage mapping identified certain loci as consistently associated with biomass accumulation under different nitrogen forms and CO2 levels. These results will assist breeding efforts to develop food crops that are resilient to the worlds climate changes.

plant biology↗

bioRxiv2026

A multi-plant transcriptomic atlas reveals conserved and lineage specific defense architectures in response to Botrytis cinerea

Generalist pathogens pose a challenge to plant immunity by infecting diverse hosts while harboring extensive intraspecific genetic variation. Whether evolutionary distant plant lineages rely on a shared immune strategy or deploy distinct, lineage-specific defenses when confronted by these genetically variable members of the same pathogen species remains unresolved. Here, we employed a large-scale co-transcriptomic approach to map the immune landscape of ten diverse eudicot species infected with 72 genetically distinct Botrytis cinerea isolates. We identified a limited core of evolutionarily conserved defense orthologs, along with a vast landscape of lineage-specific transcriptional rewiring. While the broad physiological outcome such as metabolic reprogramming, cell wall modification, and suppression of growth-associated processes was shared across hosts, the regulatory pathways governing this were largely lineage-specific. Crucially, this immune landscape is dynamically shaped by pathogen diversity. Nearly three-quarters of host transcriptional responses were isolate-dependent, with the magnitude of defense activation defined by specific host-isolate combinations rather than a universal species-level response. Even host responses to shared virulence factors, including broadly expressed pathogens phytotoxins, were lineage specific. These findings show that plant immunity to generalist pathogens is built on conserved physiological outcomes executed through rapidly evolving, lineage-specific regulatory programs. This distinct regulatory architecture creates an immune landscape heavily modulated by specific host-isolate combinations, highlighting the necessity of integrating pathogen diversity into models of plant defense evolution and resistance breeding. Significance StatementAchieving durable, broad-spectrum crop protection remains difficult because plant immunity models often rely on limited species and overlook natural genetic diversity. Effective crop protection requires understanding how defense networks operate across diverse lineages. We tested this by measuring immune responses of ten phylogenetically diverse eudicots infected with 72 genetically distinct Botrytis cinerea isolates. We found that plants share conserved physiological defense outcomes achieved through highly divergent, lineage-specific regulatory networks. Host responses were strongly shaped by pathogen genetic diversity, with identical isolates eliciting different transcriptional responses in different hosts. This demonstrates that plant immunity quantitatively senses pathogen variation and disease outcomes emerge from specific host-isolate combinations. These findings explain the limits of resistance transfer and inform strategies for durable disease control.

genomics↗

bioRxiv2025

Structure and sequence evolution in the pennycress (Thlaspi arvense) pangenome

O_LIEukaryotic genomes harbor many forms of variation, including nucleotide diversity and structural polymorphisms, which experience natural selection and contribute to genome evolution and biodiversity. However, harnessing this variation for agriculture hinges on our ability to detect, quantify, catalog, and utilize genetic diversity. C_LIO_LIHere, we explore seven complete genomes of the emerging biofuel crop pennycress (Thlaspi arvense) drawn from across the speciess current genetic diversity to catalogue variation in genome structure and content. C_LIO_LIAcross this new pangenome resource, we find contrasting evolutionary modes in different genomic regions. Gene-poor, repeat-rich pericentromeric regions experience frequent rearrangements, including repeated centromere repositioning. In contrast, conserved gene-dense chromosome arms maintain large-scale synteny across accessions, even in fast-evolving immune genes where microsynteny breaks down across species but the macrosynteny of gene cluster positioning is maintained. C_LIO_LIOur findings highlight that multiple elements of the genome experience dynamic evolution that conserves functional content on the chromosome scale but allows rearrangement and presence-absence variation on a local scale. This diversity is invisible to classical reference-based approaches and highlights the strength and utility of pangenomic resources. These results provide a valuable case study of rapid genomic structural evolution within a species and powerful resources for crop development in an emerging biofuel crop. C_LI

genomics↗

bioRxiv2025

Combined generalist and host-specific transcriptional strategies enable host generalism in the fungal pathogen Botrytis cinerea

How generalist pathogens infect phylogenetically diverse hosts remains a central question in plant-pathogen biology. In particular, the extent to which broad host range is enabled by genetic variation versus transcriptional plasticity is unclear. To investigate how variation and plasticity contribute to generalism, we studied the generalist necrotrophic fungus Botrytis cinerea that infects more than 1,500 plant species. Using a cross-infection matrix of 72 B. cinerea isolates infected on 57 plant genotypes distributed across 15 eudicot species, we identified general and host-dependent fungal components of lesion formation. Transcriptome profiling at 48 hours post-inoculation revealed two distinct pathogen gene modules: (1) a set of general lesion-associated genes enriched in primary metabolism, showing similar expression across hosts but varied among isolates; and (2) a set of high-entropy, host specific-inducible genes, organized into distinct co-regulated modules that respond dynamically to specific host cues. Both gene sets were genomically dispersed, lacking structural clustering, and were under different levels of selective constraints. Our results demonstrate that B. cinerea employs a modular transcriptional strategy that integrates a core metabolic program along with a plastic, host-responsive regulatory network to achieve broad host colonization. This study presents the most comprehensive cross-species co-transcriptomic dataset to date for any fungal phytopathogen, highlighting transcriptional plasticity as a key mechanism underlying generalism in plant-fungal interactions. Moreover, the identification of conserved fungal gene targets across diverse hosts offers a foundation for developing broad-spectrum resistance strategies in multiple crops.

genomics↗

bioRxiv2025

Convergence and constraint in glucosinolate evolution across the Brassicaceae

Diversity in plant specialized metabolites plays critical roles in plant-environment interactions. In longer evolutionary scales, e.g. between families or orders, this diversity arises from whole-genome and tandem duplication events. Less is known about the evolutionary patterns shaping chemical diversity at shorter scales, e.g. within a family. Utilizing the aliphatic glucosinolate pathway we explored how the terminal structural modification enzyme GSL-OH evolved across the Brassicaceae and the genomic processes that control presence-absence variation of its products (R)-2-hydroxy-but-3-enyl and (S)-2-hydroxy-but-3-enyl. We implemented a phylo-functional approach where we functionally validated GSL-OH orthologs across the Brassicaceae and used that information to map the genomic origin and trajectory of the loci. This uncovered a complex mechanism involving at least three ancestral loci with extensive gene loss across all species creating unequal retention across the phylogenetic relationships. Convergent evolution in enantiomeric specificity was observed where several independent species had tandem duplicates diverged towards producing the R or S enantiomers. To explore potential biological differences between the enantiomers, we performed Trichoplusia ni insect assays and Botrytis cinerea detached leaf assays. We found that plants with the S enantiomer were more susceptible to B. cinerea infection while plants with the R enantiomer seemed more susceptible to T. ni herbivory. This variation in activity between the precursor and different enantiomeric products may shape the recurrent evolution of their production.

plant biology↗

bioRxiv2024

Trans-regulatory loci shape natural variation of gene expression plasticity in Arabidopsis

Organisms regulate gene expression in response to environmental cues, a process known as plasticity, to adjust to changing environments. Research into natural variation and the evolution of plasticity frequently studies cis-regulatory elements with theory suggesting they are more important evolutionarily than trans-regulatory elements. Genome-wide association studies have supported this idea, observing a predominance of cis-loci affecting plasticity. However, studies in structured populations provide a contrasting image, raising questions about the genetic architecture of natural variation in plasticity. To circumvent potential statistical difficulties present in genome-wide association studies, we mapped loci underlying transcriptomic plasticity in response to salicylic acid using recombinant inbred lines generated from two random Arabidopsis thaliana accessions. We detected extensive transgressive segregation in the salicylic acid response, suggesting that plasticity to salicylate in Arabidopsis is polygenic. Most loci (>75%) underlying this variation act in trans, especially for loci influencing plasticity. Trans-acting loci were enriched in genome hotspots, with predominantly small effect sizes distributed across many genes. This could potentially explain their under-discovery in genome-wide association studies. This work reveals a potentially important role for trans-acting loci in plastic expression responses, with implications for understanding plant adaptation to different environments.

genetics↗

bioRxiv2024

An efflux pump family distributed across plant commensal bacteria conditions host- and organ-specific detoxfication of a host-specific glucosinolate

In nature, plants recruit a diverse microbial community, the plant microbiome, that is distinct from the surrounding soil community 1-4. To understand the forces that shape the plant microbiome we need to characterize the microbial traits that contribute to plant colonization. We used barcoded mutant libraries to identify bacterial genes that contribute to the colonization of a monocot and a eudicot host 5-7. We show that plant colonization is influenced by dozens of genes. While some of these colonization genes were shared between the two host plant species, most were highly specific, benefiting the colonization of a single host and organ. We characterized an efflux pump that specifically contributes to Arabidopsis shoot colonization. This efflux pump is prevalent across Pseudomonadota genomes, yet benefits the bacterial association with only a small subset of Arabidopsis thaliana accessions. Leveraging genomic diversity within Arabidopsis thaliana, we confirmed that specific glucosinolate breakdown products are detoxified by this family of efflux pumps. The broad prevalence of this efflux pump family suggests that its members contribute to protection of commensal bacteria from collateral damage of plant glucosinolate-based defense responses to herbivores and necrotrophic pathogens.

microbiology↗

bioRxiv2024

The polygenic strategies of Botrytis cinerea for virulence across eight eudicot host species

Diverse qualitative and quantitative genetic architectures can successfully influence fungal virulence and host range. To model the quantitative genetic architecture of a generalist pathogen with an extensive host range, we conducted a genome-wide association study (GWAS) of the virulence of Botrytis cinerea across eight hosts. This approach revealed 4772 candidate genes, about one-third of the Botrytis genome, contributing to virulence with small effect sizes. The candidate genes are evenly spread across the core chromosomes with no indication of bipartite genomic architecture. The GWAS-identified polymorphisms and genes show that B. cinerea relies on genetic variants across hundreds of genes for infecting diverse hosts, with most genes influencing relatively few hosts. When pathogen genes associated to multiple hosts, they typically influenced more unrelated than related host species. Comparative genomics further suggested that the GWAS-identified genes are largely syntenic with other specialist Botrytis species and not novel to B. cinerea. Overall, B. cinereas generalist behavior is derived from the sum of the genome-wide genetic variation acting within gene networks that differentially coordinate the interaction with diverse hosts.

genetics↗

bioRxiv2024

Allopolyploidy expanded gene content but not pangenomic variation in the hexaploid oilseed Camelina sativa

Ancient whole-genome duplications (WGDs) are believed to facilitate novelty and adaptation by providing the raw fuel for new genes. However, it is unclear how recent WGDs may contribute to evolvability within recent polyploids. Hybridization accompanying some WGDs may combine divergent gene content among diploid species. Some theory and evidence suggest that polyploids have a greater accumulation and tolerance of gene presence-absence and genomic structural variation, but it is unclear to what extent either is true. To test how recent polyploidy may influence pangenomic variation, we sequenced, assembled, and annotated twelve complete, chromosome-scale genomes of Camelina sativa, an allohexaploid biofuel crop with three distinct subgenomes. Using pangenomic comparative analyses, we characterized gene presence-absence and genomic structural variation both within and between the subgenomes. We found over 75% of ortholog gene clusters are core in Camelina sativa and <10% of sequence space was affected by genomic structural rearrangements. In contrast, 19% of gene clusters were unique to one subgenome, and the majority of these were Camelina-specific (no ortholog in Arabidopsis). We identified an inversion that may contribute to vernalization requirements in winter-type Camelina, and an enrichment of Camelina-specific genes with enzymatic processes related to seed oil quality and Camelinas unique glucosinolate profile. Genes related to these traits exhibited little presence-absence variation. Our results reveal minimal pangenomic variation in this species, and instead show how hybridization accompanied by WGD may benefit polyploids by merging diverged gene content of different species.

genomics↗

bioRxiv2023

Altered defense patterns upon retrotransposition highlights the potential for rapid adaptation by transposable elements

Transposable elements can be activated in response to environmental changes and lead to changes in DNA sequence. Their target sites of insertions have previously been thought to be random, but this theory has lately been contradicted. For instance, mobilization is favored towards genes involved in regulatory processes. This makes them interesting as potential players in rapid responses required under stressful environmental conditions. In this paper, we report the in-depth characterization of an Arabidopsis thaliana Col-0-based line whose altered DNA methylation pattern made it vulnerable for transposable element movement. We identified a transposable element retrotransposition into a transporter of glucosinolate defense compounds. As a consequence of this transposable element movement, the plants showed tissue-specific changes in glucosinolate profiles and levels accompanied by rewiring of glucosinolate- and defense-related transcriptional changes. As this single transposable element had strong impact on the plants resistance to insect herbivory, our findings highlight the potential for transposable elements to play a role in plant adaptation.

plant biology↗

bioRxiv2023

Polygenic pathogen networks control of host and pathogen transcriptional plasticity in the Arabidopsis-Botrytis pathosystem

Bidirectional flow of information shapes the outcome of the host-pathogen interactions and depends on the genetics of each organism. Recent work has begun to use co-transcriptomic studies to shed light on this bidirectional flow, but it is unclear how plastic the co-transcriptome is in response to genetic variation in both the host and pathogen. To study co-transcriptome plasticity, we conducted transcriptomics using natural genetic variation in the pathogen, Botrytis cinerea, and large effect genetic variation abolishing defense signaling pathways within the host, Arabidopsis thaliana. We show that genetic variation in the pathogen has a greater influence on the co-transcriptome than mutations that abolish defense signaling pathways in the host. Genome wide association mapping using the pathogens genetic variation and both organisms transcriptomes allowed an assessment of how the pathogen modulates plasticity in response to the host. This showed that the differences in both organisms responses were linked to trans-eQTL hotspots within the pathogens genome. These hotspots control gene sets in either the host or pathogen and show differential allele sensitivity to the hosts genetic variation rather than qualitative host specificity. Interestingly, nearly all the trans-eQTL hotspots were unique to the host or pathogen transcriptomes. In this system of differential plasticity, the pathogen mediates the shift in the co-transcriptome more than the host.

plant biology↗

bioRxiv2023

Epigenomic divergence underlies sequence polymorphism and the evolutionary fate of duplicate paralogs in A. thaliana.

Processes affecting rates of sequence polymorphism are fundamental to molecular evolution and the evolutionary fate of gene duplicates. The relationship between gene activity and sequence polymorphism can influence the likelihood that functionally redundant gene copies are co-maintained in stable evolutionary equilibria versus other outcomes such as neo-functionalization. Here we investigate genic variation in epigenome-associated polymorphism rates in Arabidopsis thaliana and consider whether these affect the evolution of gene duplicates. We compared the frequency of sequence polymorphism and patterns of genetic differentiation between genes classified by exon methylation patterns: unmethylated (unM), gene-body methylated (gbM), and transposon-like methylated (teM) states, which reflect divergence in gene expression. We found that the frequency of polymorphism was higher in teM (transcriptionally repressed, tissue-specific) genes and lower in gbM (active, constitutively expressed) genes. Comparisons of gene duplicates were largely consistent with genome-wide patterns - gene copies that exhibit teM tend to accumulate higher sequence polymorphism, evolve faster, and are in chromatin states associated with reduced DNA repair. This relationship between expression, the epigenome, and polymorphism may lead to the breakdown of equilibrium states that would otherwise maintain genetic redundancies. Epigenome-mediated polymorphism rate variation may therefore aid the pseudogenization of duplicate paralogs or increase the evolution of novel gene functions in duplicate paralogs maintained over evolutionary time.

genomics↗

bioRxiv2022

Differential genetic variation underlying Ammonium and Nitrate responses in Arabidopsis thaliana

Nitrogen is an essential element required for plant growth and productivity. Understanding the mechanisms and natural genetic variation underlying nitrogen use in plants will facilitate engineering plant nitrogen use to maximize crop productivity while minimizing environmental costs. To understand the scope of natural variation that may influence nitrogen use, we grew 1135 Arabidopsis thaliana natural genotypes on two nitrogen sources, nitrate and ammonium, and measured both developmental and defense metabolite traits. By using different environments and focused on multiple traits, we identified a wide array of different nitrogen responses. These responses are associated with a large number of genes, most of them not previously associated with nitrogen responses. Only a small portion of these genes appear to be shared between environments or traits while most of the detected genes are predominantly specific to a developmental or defense trait under a specific nitrogen source. Finally, by using a large population we were able to identify unique nitrogen responses, like preferring ammonium or nitrate, that appear to be generated by combinations of loci rather than a few large effect loci. This suggests that it may be possible to obtain novel phenotypes in complex nitrogen responses by manipulating sets of genes with small effects rather than solely focusing on large effect single gene manipulations. One Sentence SummaryUsing a large collection of natural genotypes, and studying both developmental and metabolic responses, we found a large number of genes that are involved in the plants nitrogen response.

plant biology↗

bioRxiv2020

Genetic variation, environment and demography intersect to shape Arabidopsis defense metabolite variation across Europe

Plants face a variety of challenges within their ever-changing environment. Diverse metabolites are central to the plants ability to overcome these challenges. Understanding the environmental and genetic factors influencing the variation in specialized metabolites is the key to understand how plants survive and develop under changing environments. Here we measure the variation in specialized metabolites across a population of 797 natural Arabidopsis thaliana accessions. We show a combination of geography, environmental parameters, demography, and different genetic processes that creates a specific pattern in their accumulation and distribution. By identifying and tracking causal polymorphisms at multiple loci controlling metabolites variation we show that each locus displays extensive allelic heterogeneity with signatures of both parallel and convergent evolutionary processes. These loci combine epistatically and show differing relationships to environmental parameters leading to different distributions. This provides a detailed perspective about the complexity of the forces and mechanisms that shape the accumulation and distribution of a family of specialized metabolites critical for plant fitness.

plant biology↗

bioRxiv2020

Genetic diversity increases food-web persistence in the face of climate warming

Genetic diversity provides the raw material for species to adapt and persist in the face of climate change. Yet, the extent to which these genetic effects scale at the level of ecological communities remains unclear. Here we experimentally test the effect of plant genetic diversity on the persistence of an insect food web under a current and future warming scenario. We found that plant genetic diversity increased food-web persistence by increasing the intrinsic growth rates of species across multiple trophic levels. This positive effect was robust to a 3{degrees}C warming scenario and resulted from allelic variation at two genes that control the biosynthesis of chemical defenses. Our results suggest that the ongoing loss of genetic diversity may undermine the persistence and functioning of ecosystems in a changing world. One Sentence SummaryThe loss of genetic diversity accelerates the extinction of inter-connected species from an experimental food web.

ecology↗

bioRxiv2020

Innovation, conservation and repurposing of gene function in plant root cell type development

Plant species have evolved myriads of solutions to adapt to dynamic environments, including complex cell type development and regulation. To understand this diversity, we profiled tomato root cell type translatomes and chromatin accessibility. Using xylem differentiation in tomato, relative to Arabidopsis, examples of functional innovation, repurposing and conservation of transcription factors are described. Repurposing and innovation of genes are further observed within an exodermis regulatory network and illustrate its function. Translatome analyses of rice, tomato and Arabidopsis tissues suggest that root meristems are more conserved, and that the functions of constitutively expressed genes are more conserved than those of cell type/tissue-enriched genes. These observations suggest that higher-order properties of cell type and pan-cell type regulation are conserved between plants and animals. One Sentence SummaryPan-species cell type translatome and chromatin accessibility data reveal novelty, conservation and repurposing of gene function.

genomics↗