Biology subjects

Greenham, K.

Publications and source records attributed to Greenham, K..

8 recordsLinked to original sources

bioRxiv2026

AgroGem: A Rapid and Scalable Transient Transformation System for Functional Genetics in Multiple Plant Species

Plant genetic transformation technologies are essential for functional genomics and genome engineering in plants. While transient expression systems offer a rapid alternative to stable transformation, existing platforms are often constrained by low efficiency, technical complexity, and limited scalability. Here, we developed AgroGem, an efficient Agrobacterium-mediated transient transformation system utilizing a geminiviral replicon-based T-DNA vector for Arabidopsis and Brassicaceae species. AgroGem significantly outperformed existing transient approaches, including AGROBEST and protoplast-based assays, in CRISPR-mediated editing efficiency. Moreover, AgroGem recapitulated the mutation spectra and chromatin accessibility-dependent editing patterns observed in stable transformation across both Cas9 and Cas12a systems, indicating that it captures genome editing outcomes in native chromatin contexts. Leveraging this capability, we performed high-resolution profiling of CRISPR-induced mutation outcomes across a panel of DNA repair mutants and identified distinct repair signatures, including unexpected roles for KU80 and XRCC4 in regulating non-homologous end joining (NHEJ). AgroGem also supported bimolecular fluorescence complementation assays for protein-protein interaction studies in Arabidopsis and was readily adapted to plate-based formats for high-throughput applications. Together, these results establish AgroGem as a robust, scalable, and versatile platform for genome editing, DNA repair analysis, and functional genetics in plants.

plant biology↗

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

Single-Nucleus Transcriptomics Reveals How Tissue Context Shapes the Circadian Transcriptome of the Arabidopsis Leaf

Circadian regulation enables plants to coordinate cellular processes with daily environmental cycles, yet how clocks operate across cell and tissue types remains poorly understood. To resolve circadian regulation in the mature Arabidopsis thaliana leaf, we generated a 24-hour single nucleus RNA-sequencing (snRNA-seq) circadian time course spanning seven circadian time points and ~30,000 nuclei. We recovered all major leaf cell types and identified >7,400 genes with cluster-resolved rhythmic expression. Coexpression analyses defined five major temporal expression clades, revealing cell-type-specific phase shifts, while cluster-specific gene regulatory networks identified shared and unique transcription factor targets. These analyses suggest that core clock components broadly coordinate processes such as photosynthesis and light signaling while fine-tuning hormone, redox, and stress pathways in cell-type-specific manners. To restore the spatial information lost during nuclei isolation, we designed a 100-gene Xenium spatial transcriptomics panel and profiled intact mature leaves at relative dawn and dusk. Spatial transcriptomics validated cell-type-associated expression patterns from the single-nucleus time course and further revealed spatially restricted domains that were not apparent from cluster averages alone, including vascular subdomains and guard cell-associated differences in clock transcript accumulation. Several core clock components had unequal spatial distribution across similar cell types, suggesting potential fine-tuning of the clock in discrete spatial regions. Together, these findings show that the circadian transcriptional programs in the mature leaf are not simply cell-type-specific but spatially organized within the architecture of the organ.

plant biology↗

bioRxiv2022

Natural Variation in Brachypodium distachyon Responses to Combined Abiotic Stresses

The growing world population increases demand for agricultural production, which is more challenging as climate change increases global temperature and causes more extreme weather events. High-throughput phenotyping tools can be used to measure plant responses to the environment to identify genomic regions associated with response to stress. This study examines the phenotypic variation of 149 accessions of Brachypodium distachyon under drought, heat, and the combination of both stresses. Heat alone causes the largest amounts of tissue damage and the combination of heat and drought causes the largest decrease in plant biomass compared to other treatments. Notably, Bd21-0, the reference line for B. distachyon, was identified as not having very robust growth under stress conditions, especially in the heat-drought combined treatment. Climate data from the collection locations of these accessions (climate of origin) was used to assess whether climate of origin was correlated with responses to stresses and it was found to be significantly associated with height and percent of plant tissue damage. Additionally, genome wide association mapping found a number of genetic loci associated with changes in plant height, biomass, and the amount of damaged tissue under stress. Some SNPs found to be significantly associated with a response to heat or drought are also significantly associated in the combination of stresses, while others are not, and some significantly associated SNPs were only identified in the combined stress treatment. This, combined with the phenotypic data, indicates that the effects of these abiotic stresses are not simply additive, and the responses of B. distachyon to the combined stresses differ from drought and heat alone. Significant SNPs were closely located to genes known to be involved in plant responses to abiotic stresses.

plant biology↗

bioRxiv2021

Applying cis-regulatory codes to predict conserved and variable heat and cold stress response in maize

Changes in gene expression are important for response to abiotic stress. Transcriptome profiling performed on maize inbred and hybrid genotypes subjected to heat or cold stress identifies many transcript abundance changes in response to these environmental conditions. Motifs that are enriched near differentially expressed genes were used to develop machine learning models to predict gene expression responses to heat or cold. The best performing models utilize the sequences both upstream and downstream of the transcription start site. Prediction accuracies could be improved using models developed for specific co-expression clusters compared to using all up- or down-regulated genes or by only using motifs within unmethylated regions. Comparisons of expression responses in multiple genotypes were used to identify genes with variable response and to identify cis- or trans-regulatory variation. Models trained on B73 data have lower performance when applied to Mo17 or W22, this could be improved by using models trained on data from all genotypes. However, the models have low accuracy for correctly predicting genes with variable responses to abiotic stress. This study provides insights into cis-regulatory motifs for heat- and cold-responsive gene expression and provides a framework for developing models to predict expression response to abiotic stress across multiple genotypes. One sentence summaryTranscriptome profiling of maize inbred and hybrid seedlings subjected to heat or cold stress was used to identify key cis-regulatory elements and develop models to predict gene expression responses.

plant biology↗

bioRxiv2020

Genetic and Genomic Resources to Study Natural Variation in Brassica rapa

ABSTRACTThe globally important crop Brassica rapa, a close relative of Arabidopsis, is an excellent system for modeling our current knowledge of plant growth on a morphologically diverse crop. The long history of B. rapa domestication across Asia and Europe provides a unique collection of locally adapted varieties that span large climatic regions with various abiotic and biotic stress tolerance traits. This diverse gene pool provides a rich source of targets with the potential for manipulation towards the enhancement of productivity of crops both within and outside the Brassicaceae. To expand the genetic resources available to study natural variation in B. rapa, we constructed an Advanced Intercross Recombinant Inbred (AI-RIL) population using B. rapa subsp. trilocularis (Yellow Sarson) R500 and the B. rapa subsp. parachinensis (Cai Xin) variety L58. Our current understanding of genomic structure variation across crops suggests that a single reference genome is insufficient for capturing the genetic diversity within a species. To complement this AI-RIL population and current and future B. rapa genomic resources, we generated a de novo genome assembly of the B. rapa subsp. trilocularis (Yellow Sarson) variety R500, the maternal parent of the AI-RIL population. The genetic map for the R500 x L58 population generated using this de novo genome was used to map QTL for seed coat color and revealed the improved mapping resolution afforded by this new assembly.Competing Interest StatementThe authors have declared no competing interest.View Full Text

plant biology↗

bioRxiv2020

Expansion of the circadian transcriptome in Brassica rapa and genome-wide diversification of paralog expression patterns

An important challenge of crop improvement strategies is assigning function to paralogs in polyploid crops. Gene expression is one method for determining the activity of paralogs; however, the majority of transcript abundance data represents a static point that does not consider the spatial and temporal dynamics of the transcriptome. Studies in Arabidopsis have estimated up to 90% of the transcriptome to be under diel or circadian control depending on the condition. As a result, time of day effects on the transcriptome have major implications on how we characterize gene activity. In this study, we aimed to resolve the circadian transcriptome in the polyploid crop Brassica rapa and explore the fate of multicopy orthologs of Arabidopsis circadian regulated genes. We performed a high-resolution time course study with 2 h sampling density to capture the genes under circadian control. Strikingly, more than two-thirds of expressed genes exhibited rhythmicity indicative of circadian regulation. To compare the expression patterns of paralogous genes, we developed a program in R called DiPALM (Differential Pattern Analysis by Linear Models) that analyzes time course data to identify transcripts with significant pattern differences. Using DiPALM, we identified genome-wide divergence of expression patterns among retained paralogs. Cross-comparison with a previously generated diel drought experiment in B. rapa revealed evidence for differential drought response for these diverging paralog pairs. Using gene regulatory network models we compared transcription factor targets between B. rapa and Arabidopsis circadian networks to reveal additional evidence for divergence in expression between B. rapa paralogs that may be driven in part by variation in conserved non coding sequences. These findings provide new insight into the rapid expansion and divergence of the transcriptional network in a polyploid crop and offer a new method for assessing paralog activity at the transcript level. SignificanceThe circadian regulation of the transcriptome leads to time of day changes in gene expression that coordinates environmental conditions with physiological responses. Brassica rapa, a morphologically diverse crop species, has undergone whole genome triplication since diverging from Arabidopsis resulting in an expansion of gene copy number. To examine how this expansion has influenced the circadian transcriptome we developed a new method for comparing gene expression patterns. This method facilitated the discovery of genome-wide expansion of expression patterns for genes present in multiple copies and divergence in temporal abiotic stress response. We find support for conserved sequences outside the gene body contributing to these expression pattern differences and ultimately generating new connections in the gene regulatory network.

genomics↗

bioRxiv2019

Members of the Arabidopsis auxin receptor gene family are essential early in embryogenesis and have broadly overlapping functions

The TIR1/AFB auxin co-receptors mediate diverse responses to the plant hormone auxin. The Arabidopsis genome encodes six TIR1/AFB proteins representing three of the four clades that were established prior to angiosperm radiation. To determine the role of these proteins in plant development we performed an extensive genetic analysis involving the generation and characterization of all possible multiply mutant lines. We find that loss of all six TIR1/AFB proteins results in defects in embryogenesis as early as the 8-cell stage, and possibly earlier. Mutant embryos progress but exhibit frequent cell division errors followed by proliferation of the suspensor, and eventually seed abortion. Despite this dramatic phenotype, a single wild-type allele of TIR1 or AFB2 is sufficient to support growth throughout plant development. Further, gametophytic expression of the TIR1/AFB genes is not essential for development of the male or female gametophyte. Our analysis reveals extensive functional overlap between even the most distantly related TIR1/AFB genes except for AFB1. Surprisingly, the AFB1 protein has a specialized function in rapid auxin-dependent inhibition of root growth and early phase of root gravitropism. This activity may be related to a difference in subcellular localization compared to the other members of the family.

plant biology↗