Biology subjects

Cuperus, J.

Publications and source records attributed to Cuperus, J..

12 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↗

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

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

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

Impact of random 50-base sequences inserted into an intron on splicing in Saccharomyces cerevisiae

Intron splicing is a key regulatory step in gene expression in eukaryotes. Three sequence elements required for splicing - 5 and 3 splice sites and a branch point - are especially well- characterized in Saccharomyces cerevisiae, but our understanding of additional intron features that impact splicing in this organism is incomplete, due largely to its small number of introns. To overcome this limitation, we constructed a library in S. cerevisiae of random 50-nucleotide elements (N50) individually inserted into the intron of a reporter gene and quantified canonical splicing and the use of cryptic splice sites by sequencing analysis. More than 70% of approximately 140,000 N50 elements reduced splicing by at least 20% compared to the intron control. N50 features, including higher GC content, presence of GU repeats and stronger predicted secondary structure of its pre-mRNA, correlated with reduced splicing efficiency. A likely basis for the reduced splicing of such a large proportion of variants is the formation of RNA structures that pair N50 bases - such as the GU repeats - with other bases specifically within the reporter pre-mRNA analyzed. However, neither convolutional neural network nor linear models were able to explain more than a small fraction of the variance in splicing efficiency across the library, suggesting that complex non-linear interactions in RNA structures are not accurately captured by RNA structure prediction methods given the limited number of variants. Our results imply that the specific context of a pre-mRNA may determine the bases allowable in an intron to prevent secondary structures that reduce splicing.

synthetic 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↗

bioRxiv2021

Synthetic Promoter Designs Enabled by a Comprehensive Analysis of Plant Core Promoters

Targeted engineering of plant gene expression holds great promise for ensuring food security and for producing biopharmaceuticals in plants. However, this engineering requires thorough knowledge of cis-regulatory elements in order to precisely control either endogenous or introduced genes. To generate this knowledge, we used a massively parallel reporter assay to measure the activity of nearly complete sets of promoters from Arabidopsis, maize and sorghum. We demonstrate that core promoter elements - notably the TATA-box - as well as promoter GC content and promoter-proximal transcription factor binding sites influence promoter strength. By performing the experiments in two assay systems, leaves of the dicot tobacco and protoplasts of the monocot maize, we detect species-specific differences in the contributions of GC content and transcription factors to promoter strength. Using these observations, we built computational models to predict promoter strength in both assay systems, allowing us to design highly active promoters comparable in activity to the viral 35S minimal promoter. Our results establish a promising experimental approach to optimize native promoter elements and generate synthetic ones with desirable features.

plant biology↗

bioRxiv2020

A single cell view of the transcriptome during lateral root initiation in Arabidopsis thaliana

Root architecture is a major determinant of fitness, and is under constant modification in response to favorable and unfavorable environmental stimuli. Beyond impacts on the primary root, the environment can alter the position, spacing, density and length of secondary or lateral roots. Lateral root development is among the best-studied examples of plant organogenesis, yet there are still many unanswered questions about its earliest steps. Among the challenges faced in capturing these first molecular events is the fact that this process occurs in a small number of cells with unpredictable timing. Single-cell sequencing methods afford the opportunity to isolate the specific transcriptional changes occurring in cells undergoing this fate transition. Using this approach, we successfully captured the transcriptomes of initiating lateral root primordia, and discovered many previously unreported upregulated genes associated with this process. We developed a method to selectively repress target gene transcription in the xylem pole pericycle cells where lateral roots originate, and demonstrated that expression of several of these targets was required for normal root development. We also discovered novel subpopulations of cells in the pericycle and endodermal cell files that respond to lateral root initiation, highlighting the coordination across cell files required for this fate transition. One sentence summarySingle cell RNA sequencing reveals new molecular details about lateral root initiation, including the transcriptional impacts of the primordia on bordering cells.

plant biology↗

bioRxiv2020

The regulatory landscape of Arabidopsis thaliana roots at single-cell resolution

In plants, chromatin accessibility - the primary mark of regulatory DNA - is relatively static across tissues and conditions. This scarcity of accessible sites that are dynamic or tissue-specific may be due in part to tissue heterogeneity in previous bulk studies. To assess the effects of tissue heterogeneity, we apply single-cell ATAC-seq to A. thaliana roots and identify thousands of differentially accessible sites, sufficient to resolve all major cell types of the root. However, even this vast increase relative to bulk studies in the number of dynamic sites does not resolve the poor correlation at individual loci between accessibility and expression. Instead, we find that the entirety of a cells regulatory landscape and its transcriptome each capture cell type identity independently. We leverage this shared information on cell identity to integrate accessibility and transcriptome data in order to characterize developmental progression, endoreduplication and cell division in the root. We further use the combined data to characterize cell type-specific motif enrichments of large transcription factor families and to link the expression of individual family members to changing accessibility at specific loci, taking the first steps toward resolving the direct and indirect effects that shape gene expression. Our approach provides an analytical framework to infer the gene regulatory networks that execute plant development.

genomics↗

bioRxiv2020

Identification of plant enhancers and their constituent elements by STARR-seq in tobacco leaves

Genetic engineering of cis-regulatory elements in crop plants is a promising strategy to ensure food security. However, such engineering is currently hindered by our limited knowledge of plant cis-regulatory elements. Here, we adapted STARR-seq -- a technology for the high-throughput identification of enhancers -- for its use in transiently transformed tobacco leaves. We demonstrate that the optimal placement in the reporter construct of enhancer sequences from a plant virus, pea and wheat was just upstream of a minimal promoter, and that none of these four known enhancers was active in the 3'-UTR of the reporter gene. The optimized assay sensitively identified small DNA regions containing each of the four enhancers, including two whose activity was stimulated by light. Furthermore, we coupled the assay to saturation mutagenesis to pinpoint functional regions within an enhancer, which we recombined to create synthetic enhancers. Our results describe an approach to define enhancer properties that can be performed in potentially any plant species or tissue transformable by Agrobacterium and that can use regulatory DNA derived from any plant genome. One-sentence summaryWe developed a high-throughput assay in transiently transformed tobacco leaves that can identify enhancers, characterize their functional elements and detect condition-specific enhancer activity.

plant biology↗