Biology subjects

Queitsch, C.

Publications and source records attributed to Queitsch, C..

At least 19 recordsLinked to original sources

bioRxiv2026

Duckweeds as Multiplexable Plant Models for Exploring Abiotic Stress Responses

In nature, plants experience complex combinations of environmental stresses. Prior studies using Arabidopsis thaliana show that transcriptional responses to combinatorial stress are unique and cannot be predicted from responses to individual treatments, making it imperative to study combinatorial stress in the monocotyledonous cereals that comprise the majority of our food supply. However, crop species are not easily amenable for such studies because of their large size, long generation time, and organismal complexity. In contrast, the monocotyledonous aquatic duckweeds are small, grow rapidly, and show reduced organismal and genomic complexity. Here, we explore the physiological responses to combinatorial stress in three duckweed species, S. polyrhiza, L. minor, and W. australiana and examine their potential to serve as multiplexable models. Focusing on S. polyrhiza, the most complex and best annotated species, we investigated transcriptional response to individual and combinatorial stress treatments. While most individual treatments elicit few transcriptional changes, combinatorial stress elicited a non-additive transcriptional response unique to each combination. Exploring the duckweed regulatory landscape, we found that regulatory regions near genes that were differentially expressed in combinatorial stress in S. polyrhiza resemble environmentally responsive regulatory elements found in terrestrial plants. Examined duckweed species show numbers and genomic distributions of regulatory elements similar to those of A. thaliana and maize, with subtle effects of genome size but none of organismal complexity. Taken together, our results establish that duckweeds respond to combinatorial stress in a manner similar to terrestrial plants and can therefore serve as high-throughput, multiplexable models for studying its molecular underpinnings.

plant biology↗

bioRxiv2026

Unpacking Chromatin Accessibility with Fiber-seq

Chromatin accessibility has long been used as a marker for regions of DNA with regulatory potential. Fiber-seq detects chromatin accessibility on individual DNA fibers, enabling analyses beyond the identification of the accessible chromatin regions (ACRs). By providing single molecule level high resolution, Fiber-seq provides unprecedented qualitative descriptions, including potential categorizations of ACRs, identification of internal transcription factor footprints and nucleosome positioning within individual DNA fibers. As with all tools, the power of this technique depends on careful experimental design and data analysis -- incorrect usage will result in incorrect conclusions. Here we offer guidelines and flag potential pitfalls when generating and analyzing Fiber-seq data, such as (1) the optimum levels of adenosine methylation per-fiber, (2) the power of per-fiber state inference, (3) the importance of controlling for read depth and methylation rates when comparing across samples, (4) the limitations of long-read sequence mapping, and (5) suggestions for identification of differentially accessible peaks across samples.

genomics↗

bioRxiv2026

Massively parallel characterization and deep learning of enhancers in plant genomes

Enhancers coordinate gene expression in response to developmental and environmental cues. Because plant enhancers lack the readily detectable molecular hallmarks of animal enhancers, their systematic functional characterization has yet to be accomplished. Here, we characterize the species- and condition-specific enhancer activity of over 350,000 sequences derived from accessible chromatin regions of the Arabidopsis, tomato, maize, and sorghum genomes. Enabled by the massive scale of the data, we developed plantGREP, a deep learning model that predicts enhancer strength and identifies the underlying functional sequence motifs. We apply plantGREP to evolve strong constitutive as well as species- and condition-specific enhancers, and to locate regions with enhancer activity upstream of developmental genes in crop genomes. These results should facilitate the targeted editing of enhancers in crop genomes and the design of cell-type-specific plant enhancers.

plant biology↗

bioRxiv2025

Dosa: A method to covalently barcode proteinsfor high throughput biochemistry

Deep mutational scanning couples a proteins activity to DNA sequencing for high throughput assessment of the effects of all single amino acid substitutions, but it largely uses indirect assays, like growth, as proxy for protein activity. Here, we covalently link variant proteins in vivo to an RNA barcode by fusing them to E. coli tRNA (m5U54) methyltransferase TrmA (E358Q), which forms a covalent bond with a tRNA stem-loop. Following cell lysis, variant proteins are separated in vitro according to their biochemical properties and identified by their barcodes. We use this method, Dosa, to analyze a large pool of FLAG epitope variants for binding to an anti-FLAG antibody, to profile the cleavage preferences of variants of enteropeptidase and human rhinovirus 3C protease, and to measure the solubility of several hundred A{beta}(1-42) variants. This method should be amenable to numerous biochemical assays with proteins produced in E. coli or mammalian cells.

genomics↗

bioRxiv2025

Phenotypic tolerance for rDNA copy number variation within the natural range of C. elegans

The genes for ribosomal RNA (rRNA) are encoded by ribosomal DNA (rDNA), whose structure is notable for being present in arrays of tens to thousands of tandemly repeated copies in eukaryotic genomes. The exact number of rDNA copies per genome is highly variable within a species, with differences between individuals measuring in potentially hundreds of copies and megabases of DNA. The extent to which natural variation in rDNA copy number impacts whole-organism phenotypes such as fitness and lifespan is poorly understood, in part due to difficulties in manipulating such large and repetitive tracts of DNA even in model organisms. Here, we used the natural resource of copy number variation in C. elegans wild isolates to generate new tools and investigated the phenotypic consequences of this variation. Specifically, we generated a panel of recombinant inbred lines (RILs) using a laboratory strain derivative with [~]130 haploid rDNA copies and a wild isolate with [~]417 haploid rDNA copies, one of the highest validated C. elegans rDNA copy number arrays. We find that rDNA copy number is stable in the RILs, rejecting prior hypotheses that predicted copy number instability and copy number reversion. To isolate effects of rDNA copy number on phenotype, we produced a series of near isogenic lines (NILs) with rDNA copy numbers representing the high and low end of the rDNA copy number spectrum in C. elegans wild isolates. We find no correlation between rDNA copy number and phenotypes of rRNA abundance, competitive fitness, early life fertility, lifespan, or global transcriptome under standard laboratory conditions. These findings demonstrate a remarkable ability of C. elegans to tolerate substantial variation in a locus critical to fundamental cell function. Our study provides strain resources for future investigations into the boundaries of this tolerance.

genomics↗

bioRxiv2024

Sensitivity to photoperiod is a complex trait in Camelina sativa

Day neutrality, or insensitivity to photoperiod (day length), is an important domestication trait in many crop species. Although the oilseed crop Camelina sativa has been cultivated since the Neolithic era, day-neutral accessions have yet to be described. We sought to leverage genetic diversity in existing germplasms to identify C. sativa accessions with low photoperiod sensitivity for future engineering of this trait. We quantified variation in the photoperiod response across 161 accessions of C. sativa by measuring hypocotyl length of four-day-old seedlings grown in long-day and short-day conditions, finding wide variation in photoperiod response. Similarly, soil-grown adult plants from selected accessions showed variation in photoperiod response in several traits; however, photoperiod responses in seedling and adult traits were not correlated, suggesting complex mechanistic underpinnings. Although RNA-seq experiments of the reference accession Licalla identified several differentially regulated Arabidopsis syntelogs involved in photoperiod response, including COL2, FT, LHY and WOX4, expression of these genes in the accessions did not correlate with differences in their photoperiod sensitivity. Taken together, we show that all tested accessions show some degree of photoperiod response, and that this trait is likely complex, involving several and separable seedling and adult traits. Significance StatementDay neutrality (photoperiod insensitivity) is a common trait in domesticated crops; however, the ancient oilseed crop Camelina sativa has remained photoperiod-sensitive, which likely limits seed yields. Here, we show that photoperiod sensitivity is conserved across many C. sativa cultivars, albeit to different degrees, and we establish that photoperiod sensitivity is a complex trait, which will require genetic engineering to achieve day neutrality.

plant biology↗

bioRxiv2024

rDNA copy number variation in yeast alters response to environmental conditions

Ribosomal DNA (rDNA) in eukaryotes is maintained in hundreds of copies with rDNA copy number varying greatly among individuals within a species. In the budding yeast Saccharomyces cerevisiae, the rDNA copy number across wild isolates ranges from 90 to 300 copies. Previous studies showed that 35 rDNA copies are sufficient for ribosome biogenesis in this yeast and enable wild-type-like growth in standard laboratory growth conditions. We addressed two major questions concerning rDNA copy number variation in this yeast: (1) What are the fitness consequences of rDNA copy number variation outside and within the natural range in standard laboratory growth conditions? (2) Do these fitness effects change in different growth conditions? We used growth competitions to compare the fitness effects of rDNA copy number variation in otherwise isogenic strains whose rDNA copy number ranged from 35 to 200. In standard growth conditions, we found that fitness gradually increases from 35 rDNA copies until reaching a plateau that spans from 98 to 160 rDNA copies, well within the natural range. However, rDNA copy number-dependent fitness differed across environments. Compared to standard growth conditions, strains with higher rDNA copy numbers showed improved fitness when grown at increased temperature or in glycerol media. Moreover, the gradual fitness increase with increasing rDNA copy number in standard growth conditions gave way to a markedly lower fitness of strains with copy numbers below the natural range in these two stress conditions. These results suggest that selective pressures drive rDNA copy number in this yeast to at least ~100 copies and that a higher number of copies buffers against environmental stress. The similarity of the S. cerevisiae rDNA copy number range to the ranges reported in C. elegans, D. melanogaster, and humans points to conserved selective pressures maintaining the range of natural rDNA copy number in these highly diverse species.

genetics↗

bioRxiv2024

Small DNA elements that act as both insulators and silencers in plants

Insulators are cis-regulatory elements that separate transcriptional units, whereas silencers are elements that repress transcription regardless of their position. In plants, these elements remain largely uncharacterized. Here, we use the massively parallel reporter assay Plant STARR-seq with short fragments of eight large insulators to identify more than 100 fragments that block enhancer activity. The short fragments can be combined to generate more powerful insulators that abolish the capacity of the strong viral 35S enhancer to activate the 35S minimal promoter. Unexpectedly, when tested upstream of weak enhancers, these fragments act as silencers and repress transcription. Thus, these elements are capable of both insulating or repressing transcription dependent upon regulatory context. We validate our findings in stable transgenic Arabidopsis, maize, and rice plants. The short elements identified here should be useful building blocks for plant biotechnology efforts.

plant biology↗

bioRxiv2024

A florigen-expressing subpopulation of companion cells expresses other small proteins and reveals a nitrogen-sensitive FT repressor

The precise onset of flowering is crucial to ensure successful plant reproduction. The gene FLOWERING LOCUS T (FT) encodes florigen, a mobile signal produced in leaves that initiates flowering at the shoot apical meristem. In response to seasonal changes, FT is induced in phloem companion cells located in distal leaf regions. Thus far, a detailed molecular characterization of the FT-expressing cells has been lacking. Here, we used bulk nuclei RNA-seq and single nuclei RNA (snRNA)-seq to investigate gene expression in FT-expressing cells and other phloem companion cells. Our bulk nuclei RNA-seq demonstrated that FT-expressing cells in cotyledons and true leaves showed differences especially in FT repressor genes. Within the true leaves, our snRNA-seq analysis revealed that companion cells with high FT expression form a unique cluster in which many genes involved in ATP biosynthesis are highly upregulated. The cluster also expresses other genes encoding small proteins, including the flowering and stem growth inducer FPF1-LIKE PROTEIN 1 (FLP1) and the anti-florigen BROTHER OF FT AND TFL1 (BFT). In addition, we found that the promoters of FT and the genes co-expressed with FT in the cluster were enriched for the consensus binding motifs of NITRATE-INDUCIBLE GARP-TYPE TRANSCRIPTIONAL REPRESSOR 1 (NIGT1). Overexpression of the paralogous NIGT1.2 and NIGT1.4 repressed FT expression and significantly delayed flowering under nitrogen-rich conditions, consistent with NIGT1s acting as nitrogen-dependent FT repressors. Taken together, our results demonstrate that major FT-expressing cells show a distinct expression profile that suggests that these cells may produce multiple systemic signals to regulate plant growth and development.

plant biology↗

bioRxiv2024

The regulatory potential of transposable elements in maize

The genomes of flowering plants consist largely of transposable elements (TEs), some of which modulate gene regulation and function. However, the repetitive nature of TEs and difficulty of mapping individual TEs by short-read-sequencing have hindered our understanding of their regulatory potential. We demonstrate that long-read chromatin fiber sequencing (Fiber-seq) comprehensively identifies accessible chromatin regions (ACRs) and CpG methylation across the maize genome. We uncover stereotypical ACR patterns at young TEs that degenerate with evolutionary age, resulting in TE-enhancers preferentially marked by a novel plant-specific epigenetic feature: simultaneous hyper-CpG methylation and chromatin accessibility. We show that TE ACRs are co-opted as gene promoters and that ACR-containing TEs can facilitate gene amplification. Lastly, we uncover a pervasive epigenetic signature - hypo-5mCpG methylation and diffuse chromatin accessibility - directing TEs to specific loci, including the loci that sparked McClintocks discovery of TEs.

genomics↗

bioRxiv2024

Florigen-producing cells express FPF1-LIKE PROTEIN 1 that accelerates flowering and stem growth in long days with sunlight red/far-red ratio in Arabidopsis

Seasonal changes in spring induce flowering by expressing the florigen, FLOWERING LOCUS T (FT), in Arabidopsis. FT is expressed in unique phloem companion cells with unknown characteristics. The question of which genes are co-expressed with FT and whether they have roles in flowering remains elusive. Through tissue-specific translatome analysis, we discovered that under long-day conditions with the natural sunlight red/far-red ratio, the FT-producing cells express a gene encoding FPF1-LIKE PROTEIN 1 (FLP1). The master FT regulator, CONSTANS (CO), controls FLP1 expression, suggesting FLP1s involvement in the photoperiod pathway. FLP1 promotes early flowering independently of FT, is active in the shoot apical meristem, and induces the expression of SEPALLATA 3 (SEP3), a key E-class homeotic gene. Unlike FT, FLP1 facilitates inflorescence stem elongation. Our cumulative evidence indicates that FLP1 may act as a mobile signal. Thus, FLP1 orchestrates floral initiation together with FT and promotes inflorescence stem elongation during reproductive transitions.

plant biology↗

bioRxiv2023

Cooperativity and additivity in plant enhancers

Enhancers are cis-regulatory elements that shape gene expression in response to numerous developmental and environmental cues. In animals, several models have been proposed to explain how enhancers integrate the activity of multiple transcription factors. However, it remains largely unknown how plant enhancers integrate transcription factor activity. Here, we use Plant STARR-seq to characterize three light-responsive plant enhancers--AB80, Cab-1, and rbcS-E9--derived from genes active in photosynthesis. Saturation mutagenesis reveals mutations, many of which cluster in short regions, that strongly reduce enhancer activity in the light, in the dark or in both conditions. When tested in the light, these mutation-sensitive regions do not function on their own; rather, cooperative interactions with other such regions are required for full activity. Epistatic interactions occur between mutations in adjacent mutation-sensitive regions, and the spacing and order of mutation-sensitive regions in synthetic enhancers affects enhancer activity. In contrast, when tested in the dark, mutation-sensitive regions act independently and additively in conferring enhancer activity. Taken together, this work demonstrates that plant enhancers show evidence for both cooperative and additive interactions among their functional elements. This knowledge can be harnessed to design strong, condition-specific synthetic enhancers.

plant biology↗

bioRxiv2023

De novo designed Hsp70 activator dissolves intracellular condensates

Protein quality control (PQC) is carried out in part by the chaperone Hsp70, in concert with adapters of the J-domain protein (JDP) family. The JDPs, also called Hsp40s, are thought to recruit Hsp70 into complexes with specific client proteins. However, the molecular principles regulating this process are not well understood. We describe the de novo design of a set of Hsp70 binding proteins that either inhibited or stimulated Hsp70s ATPase activity; a stimulating design promoted the refolding of denatured luciferase in vitro, similar to native JDPs. Targeting of this design to intracellular condensates resulted in their nearly complete dissolution. The designs inform our understanding of chaperone structure-function relationships and provide a general and modular way to target PQC systems to condensates and other cellular targets.

biochemistry↗

bioRxiv2023

Features that Govern Terminator Strength in Plants

The 3 end of a gene, often called a terminator, modulates mRNA stability, localization, translation, and polyadenylation. Here, we adapted Plant STARR-seq, a massively parallel reporter assay, to measure the activity of over 50,000 terminators from the plants Arabidopsis thaliana and Zea mays. We characterize thousands of plant terminators, including many that outperform bacterial terminators commonly used in plants. Terminator activity is species-specific, differing in tobacco leaf and maize protoplast assays. While recapitulating known biology, our results reveal the relative contributions of polyadenylation motifs to terminator strength. We built a computational model to predict terminator strength and used it to conduct in silico evolution that generated optimized synthetic terminators. Additionally, we discover alternative polyadenylation sites across tens of thousands of terminators; however, the strongest terminators tend to have a dominant cleavage site. Our results establish features of plant terminator function and identify strong naturally occurring and synthetic terminators.

plant biology↗

bioRxiv2023

Decreasing LTP2 expression increases phenotypic variation in early seedling traits of Arabidopsis thaliana

Isogenic individuals can display seemingly stochastic phenotypic differences, limiting the accuracy of genotype-to-phenotype predictions. The extent of this phenotypic variation depends in part on genetic background, raising questions about the genes involved in controlling stochastic phenotypic variation. Focusing on early seedling traits in Arabidopsis thaliana, we found that hypomorphs of the cuticle-related gene LTP2 greatly increased variation in seedling phenotypes, including hypocotyl length, gravitropism and cuticle permeability. Many ltp2 hypocotyls were significantly shorter than wild-type hypocotyls while others resembled the wild type. Differences in epidermal properties and gene expression between ltp2 seedlings with long and short hypocotyls suggest a loss of cuticle integrity as the primary determinant of the observed phenotypic variation. We identified environmental conditions that reveal or mask the increased variation in ltp2 hypomorphs, and found that increased expression of its closest paralog LTP1 is necessary for ltp2 phenotypes. Our results illustrate how decreased expression of a single gene can generate starkly increased phenotypic variation in isogenic individuals in response to an environmental challenge.

plant biology↗

bioRxiv2022

Proteostasis governs differential temperature sensitivity across embryonic cell types

The genetic program of embryonic development is remarkably robust, but temperature stress can degrade its ability to generate animals with invariant anatomy. While the stereotyped, consistent phenotypes associated with environmental stress during vertebrate development suggest that some cell types are more sensitive to stress than others, the basis of this sensitivity is unknown. Here, we characterize hundreds of individual zebrafish embryos under temperature stress using whole-animal single cell RNA-seq to identify cell types and molecular programs within them that drive phenotypic variability. We find that temperature perturbs the normal proportions and gene expression programs of numerous cell types and also introduces asynchrony in their development. The notochord is particularly sensitive to temperature stress, which we show is due to a specialized cell type, sheath cells. Further analyses show that sheath cells accumulate misfolded protein at elevated temperature, leading to a cascading structural failure of the notochord and irreversible anatomic defects in the embryo. Our study demonstrates that whole-animal single cell RNA-seq can characterize mechanisms important for developmental robustness and pinpoint molecular programs within specific cell types that constitute key failure points.

genomics↗

bioRxiv2022

AGO1 and HSP90 buffer different genetic variants in Arabidopsis thaliana

Argonaute 1 (AGO1), the principal protein component of microRNA-mediated regulation, plays a key role in plant growth and development. AGO1 physically interacts with the chaperone HSP90, which buffers cryptic genetic variation in plants and animals. We sought to determine whether genetic perturbation of AGO1 in Arabidopsis thaliana would also reveal cryptic genetic variation, and if so, whether AGO1-dependent loci overlap with those dependent on HSP90. To address these questions, we introgressed a hypomorphic mutant allele of AGO1 into a set of mapping lines derived from the commonly used Arabidopsis strains Col-0 and Ler. Although we identified several cases in which AGO1 buffered genetic variation, none of the AGO1-dependent loci overlapped with those buffered by HSP90 for the traits assayed. We focused on one buffered locus where AGO1 perturbation uncoupled the traits days to flowering and rosette leaf number, which are otherwise closely correlated. Using a bulk segregant approach, we identified a non-functional Ler hua2 mutant allele as the causal AGO1-buffered polymorphism. Introduction of a non-functional hua2 allele into a Col-0 ago1 mutant background recapitulated the Ler-dependent ago1 phenotype, implying that coupling of these traits involves different molecular players in these closely related strains. Taken together, our findings demonstrate that even though AGO1 and HSP90 buffer genetic variation in the same traits, these robustness regulators interact epistatically with different genetic loci, suggesting that higher-order epistasis is uncommon. Article SummaryArgonaute 1 (AGO1), a key player in plant development, interacts with the chaperone HSP90 which buffers environmental and genetic variation. We found that AGO1 buffers environmental and genetic variation in the same traits; however, AGO1-dependent and HSP90-dependent loci do not overlap. Detailed analysis of a buffered locus found that a non-functional HUA2 allele decouples days to flowering and rosette leaf number in an AGO1-dependent manner, suggesting the AGO1-dependent buffering acts at the network level.

plant biology↗

bioRxiv2021

A minimal rDNA array replicates early, delays genome replication, and uncouples anaphase entry from S phase completion

Eukaryotes maintain hundreds of copies of ribosomal DNA (rDNA), many more than required for ribosome biogenesis, suggesting a yet undefined role for large rDNA arrays outside of ribosomal RNA synthesis. We demonstrate that reducing the Saccharomyces cerevisiae rDNA array to 35 copies, which is sufficient for ribosome function, shifts rDNA from being the latest replicating region in the genome to one of the earliest. This change in replication timing results in delayed genome-wide replication and classic replication defects. We present evidence that the requirement for rDNA to replicate late, which is conserved among eukaryotes, also coordinates the completion of genome replication with anaphase entry through the proper sequestration of the mitotic exit regulator Cdc14p in the rDNA-containing nucleolus. Our findings suggest that, instead of being a passive repetitive element, the large late-replicating rDNA array plays an active role in genome replication and cell cycle control.

molecular biology↗