Biology subjects

Gehring, M.

Publications and source records attributed to Gehring, M..

14 recordsLinked to original sources

bioRxiv2026

Machine learning-assisted directed evolution of plant Rubisco

Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) is foundational to life on Earth, catalyzing carbon dioxide (CO2) fixation to generate biomass. However, Rubisco is a slow and inefficient enzyme that has proven challenging to engineer. We applied the structure-informed machine learning (ML) model ESM-IF1 to identify plausible amino acid sites in the large subunit of Nicotiana tabacum Rubisco to target for directed evolution. ML-assisted library design followed by selection in Rubisco-dependent Escherichia coli identified multiple enriched variants displaying improved catalytic efficiency. Several improved variants carried amino acid changes not found in the evolutionary lineage of plants, despite being assembly competent in plant chloroplasts, demonstrating that ML-assisted protein design can explore functional sequence space beyond what is observed from natural sequence diversity. Most prominently, the T391I substitution improved carboxylation rate by 29% and aerobic carboxylation efficiency by 43%. Our findings demonstrate the utility of ML-assisted evolution for engineering Rubisco with improved carboxylation efficiency and potential for enhancing crop productivity.

plant biology↗

bioRxiv2026

Rational design of T-DNA vectors enables predictable, single-copy integration in Arabidopsis thaliana

Agrobacterium-mediated transformation is the dominant method for plant transgenesis, yet it frequently produces multi-copy, structurally complex T-DNA insertions associated with transgene silencing, unpredictable expression, and genome instability. Here, leveraging a high-throughput phenotypic reporter, we systematically dissect how T-DNA vector architecture, plasmid biology, and regulatory element choice shape transformation outcomes in Arabidopsis thaliana. We discover a pronounced trade-off between transformation efficiency and T-DNA copy number, uncovering the virulence enhancing overdrive sequence as a major determinant of this relationship. Guided by these insights, we engineered a new T-DNA vector that balances efficient transformation with predominantly single-copy integration. Additionally, we replaced viral elements, such as the widely used CaMV 35S promoter, with Arabidopsis-derived regulatory elements to minimise undesired enhancer effects, and developed a streamlined workflow for efficient T-DNA insertion mapping in the genome. Together, these advances form the T1 vector series, an Arabidopsis-optimised T-DNA vector system that enables clean, single-copy, and readily mappable transgene integration with predictable expression in the first generation after transformation.

plant biology↗

bioRxiv2025

A transcriptional atlas of early Arabidopsis seed development suggests mechanisms for inter-tissue coordination

Successful seed development is essential for flowering plant reproduction and requires the coordination of three genetically distinct tissues: the embryo and endosperm, which are the products of fertilization, and the maternal seed coat. Our understanding of the transcriptional programs underlying tissue-specific functions and inter-tissue coordination in seeds remains incomplete. To address this, we performed single nucleus RNA-sequencing on Arabidopsis thaliana seeds at 3, 5, and 7 days after pollination. We characterize all major seed cell or nuclei types, further refine transcriptional states in the endosperm, and map signatures of selection on cell type-specific genes. Among other findings, our analyses reveal the compartmentalization of genes involved in brassinosteroid-responsive transcription factor activation, abundant endosperm expression of genes that encode short, secreted peptides (SSPs), and expression enrichment of rapidly evolving genes in endosperm and seed coat subtypes, illuminating the cell type and species specificity of seed genes.

plant biology↗

bioRxiv2024

A simple method to efficiently generate structural variation in plants

Phenotypic variation is essential for the selection of new traits of interest. Structural variants, consisting of deletions, duplications, inversions, and translocations, have greater potential for phenotypic consequences than single nucleotide variants. Pan-genome studies have highlighted the importance of structural variation in the evolution and selection of novel traits. Here, we describe a simple method to induce structural variation in plants. We demonstrate that a short period of growth on the topoisomerase II inhibitor etoposide induces heritable structural variation and altered phenotypes in Arabidopsis thaliana at high frequency. Using long-read sequencing and genetic analyses, we identified deletions and inversions underlying semi-dominant and recessive phenotypes. This method requires minimal resources, is potentially applicable to any plant species, and can replace irradiation as a source of induced large-effect structural variation.

plant biology↗

bioRxiv2024

The 5-methylcytosine DNA glycosylase ROS1 antagonizes parent-of-origin specific DNA methylation in Arabidopsis endosperm

DNA methylation patterning is a consequence of opposing activities of DNA methyltransferases and DNA demethylases. In flowering plants, two distinct female gametes, the egg cell and the central cell, are fertilized, producing what will become the embryo and the endosperm of the seed. In Arabidopsis, a 5-methylcytosine DNA glycosylase, DME, demethylates regions in the central cell genome, leading to methylation differences between maternally- and paternally-inherited endosperm genomes after fertilization. DME is required for endosperm gene imprinting. Homologues of DME include ROS1, DML2 and DML3. It is unknown whether any of these DNA glycosylases are required for endosperm methylation patterning. We show that ROS1 prevents hypermethylation of paternally-inherited alleles in the endosperm at regions that lack maternal or paternal-allele methylation in wild-type. Thus, ROS1 promotes epigenetic symmetry between genomes in the endosperm by preventing paternal genome hypermethylation. We propose that ROS1 and DME act in a parent-of-origin-specific manner at shared endosperm targets, and consider implications for the evolution of imprinting mechanisms.

plant biology↗

bioRxiv2024

Imprinting and DNA methylation in water lily endosperm: implications for seed evolution

Endosperm is a key evolutionary innovation associated with the origin of angiosperms (flowering plants). This altruistic seed tissue supports the growth and development of the embryo by mediating the relationship of the mother plant as a nutrient source to the compatriot embryo as a nutrient sink. The endosperm is the primary site of gene imprinting in plants (where expression of an allele in offspring depends on which parent it was inherited from) and of parent-specific epigenetic modifications like DNA methylation, which are differentially patterned during male and female gamete development. Experimental results from a phylogenetically-wide array of monocot and eudicot plants suggest these parent-of-origin effects are a common feature across angiosperms. However, information about genetic imprinting and epigenetic modifications in seeds of angiosperm lineages whose origins predate the monocot-eudicot divergence (such as Nymphaeales, water lilies) is extremely limited. Additionally, Nymphaeales are an intriguing lineage in which to investigate seed genetic and epigenetic phenomena, as they are characterized by diploid endosperm and a maternal storage tissue (perisperm), both of which are unusual across angiosperm diversity. Here, we examined DNA methylation and genetic imprinting using two reproductively compatible water lily sister-species, Nymphaea thermarum and N. dimorpha. Our results suggest that maternally-expressed imprinted genes and differential DNA methylation of maternally and paternally inherited endosperm genomes are an ancestral condition for endosperm, while other seed characters like seed provisioning strategies, endosperm ploidy, and paternally-expressed imprinted genes might have evolved as coinciding, opposing strategies in the evolutionary dialogue over parental control of offspring development.

evolutionary biology↗

bioRxiv2023

CRWN nuclear lamina components maintain the H3K27me3 landscape and promote successful reproduction in Arabidopsis

The nuclear lamina, a sub-nuclear protein matrix, maintains nuclear structure and genome function. Here, we investigate the role of Arabidopsis lamin analogs CROWDED NUCLEIs during gametophyte and seed development. We observed defects in crwn mutant seeds, including seed abortion and reduced germination rate. Quadruple crwn null genotypes were rarely transmitted through gametophytes. We focused on the crwn1 crwn2 (crwn1/2) endosperm, which exhibited enlarged chalazal cysts and increased expression of stress-related genes and the MADS-box transcription factor PHERES1 and its targets. Previously, it was shown that PHERES1 is regulated by H3K27me3 and that CRWN1 interacts with the PRC2 interactor PWO1. Thus, we tested whether crwn1/2 alters H3K27me3 patterns. We observed a mild loss of H3K27me3 at several hundred loci, which differed between endosperm and leaves. These data indicate that CRWNs are necessary to maintain the H3K27me3 landscape, with tissue-specific chromatin and transcriptional consequences.

genetics↗

bioRxiv2021

Somatic DNA demethylation generates tissue-specific methylation states and impacts flowering time

Cytosine methylation is a reversible epigenetic modification to DNA. In plants, removal of cytosine methylation is accomplished by the four members of the DME family of 5-methylcytosine DNA glycosylases. Demethylation by DME is critical for seed development. Consequently, determining the function of the entire gene family in somatic tissues by mutant analysis has not been possible. Here, we bypassed the reproductive defects of dme mutants to create somatic quadruple homozygous mutants of the entire DME family. dme; ros1; dml2; dml3 (drdd) leaves exhibit hypermethylated genomes compared to both wild-type plants and rdd triple mutants, indicating functional redundancy among all four demethylases. Targets of demethylation include regions co-targeted by RNA-directed DNA methylation and, surprisingly, CG gene body methylation, indicating dynamic methylation at these little-understood sites. Additionally, many tissue-specific methylation differences are absent in drdd, suggesting a role for active demethylation in generating divergent epigenetic states across wild-type tissues. Furthermore, drdd plants display a striking early flowering phenotype, which is associated with 5 hypermethylation and transcriptional down-regulation of FLOWERING LOCUS C. Active DNA demethylation is therefore required for proper methylation patterning across somatic tissues and defines the epigenetic landscape of both intergenic and coding regions.

plant biology↗

bioRxiv2021

RNA Pol IV has antagonistic parent-of-origin effects on Arabidopsis endosperm

Gene expression in endosperm - a seed tissue that mediates transfer of maternal resources to offspring - is under complex epigenetic control. We show here that plant-specific RNA Polymerase IV mediates parental control of endosperm gene expression. Pol IV is required for the production of small interfering RNAs that typically direct DNA methylation. We compared small RNAs, DNA methylation, and mRNAs in A. thaliana endosperm from reciprocal heterozygotes produced by crossing wild-type plants to Pol IV mutants. We find that maternally and paternally acting Pol IV have divergent effects on endosperm. Losses of maternal and paternal Pol IV impact sRNAs and DNA methylation at distinct genomic sites. Strikingly, maternally and paternally-acting Pol IV have antagonistic impacts on gene expression at some loci, divergently promoting or repressing endosperm gene expression. Antagonistic parent-of-origin effects have only rarely been described and are consistent with a gene regulatory system evolving under parental conflict.

plant biology↗

bioRxiv2020

Single nucleus analysis of Arabidopsis seeds reveals new cell types and imprinting dynamics

Seeds are the basis of agriculture, yet their full transcriptional complexity has remained unknown. Here, we employ single-nucleus RNA-sequencing to characterize developing Arabidopsis thaliana seeds, with a focus on endosperm. Endosperm, the site of gene imprinting in plants, mediates the relationship between the maternal parent and embryo. We identify new cell types in the chalazal endosperm region, which interfaces with maternal tissue for nutrient unloading. We further demonstrate that the extent of parental bias of maternally expressed imprinted genes varies with cell cycle phase, and that imprinting of paternally expressed imprinted genes is strongest in chalazal endosperm. These data indicate imprinting in endosperm is heterogeneous and suggest that parental conflict, which is proposed to drive the evolution of imprinting, is fiercest at the boundary between filial and maternal tissues.

plant biology↗

bioRxiv2019

Water lily (Nymphaea thermarum) draft genome reveals variable genomic signatures of ancient vascular cambium losses

For more than 225 million years, all seed plants were woody trees, shrubs, or vines (1-4). Shortly after the origin of angiosperms ~135 million years ago (MYA) (5), the Nymphaeales (water lilies) became one of the first lineages to deviate from their ancestral, woody habit by losing the vascular cambium (6), the meristematic population of cells that produces secondary xylem (wood) and phloem. Many of the genes and gene families that regulate differentiation of secondary tissues also regulate the differentiation of primary xylem and phloem (7-9), which are produced by apical meristems and retained in nearly all seed plants. Here we sequence and assemble a draft genome of the water lily Nymphaea thermarum, an emerging system for the study of early flowering plant evolution, and compare it to genomes from other cambium-bearing and cambium-less lineages (like monocots and Nelumbo). This reveals lineage-specific patterns of gene loss and divergence. Nymphaea is characterized by a significant contraction of the HD-ZIP III transcription factors, specifically loss of REVOLUTA, which influences cambial activity in other angiosperms. We also find the Nymphaea and monocot copies of cambium-associated CLE signaling peptides display unique substitutions at otherwise highly conserved amino acids. Nelumbo displays no obvious divergence in cambium-associated genes. The divergent genomic signatures of convergent vascular cambium loss reveals that even pleiotropic genes can exhibit unique divergence patterns in association with independent trait loss events. Our results shed light on the evolution of herbaceousness - one of the key biological innovations associated with the earliest phases of angiosperm evolution. Significance StatementFor ~225 million years, all seed plants were woody trees, shrubs, or vines. Shortly after the origin of flowering plants ~135 million years ago, Nymphaeales (water lilies) became one of the first seed plant lineages to become herbaceous through loss of the meristematic cell population known as the vascular cambium. We sequence and assemble the draft genome of the water lily Nymphaea thermarum, and compare it to genomes of other plants that have retained or lost the vascular cambium. By using both genome-wide and candidate-gene analysis, we find lineage-specific patterns of gene loss and divergence associated with cambium loss. Our reveal divergent genomic signatures of convergent trait loss in a system characterized by complex gene-trait relationships.

plant biology↗

bioRxiv2019

Paternally-acting canonical RNA-directed DNA methylation pathway genes sensitize Arabidopsis endosperm to paternal dosage

Seed development is sensitive to parental dosage, with excess maternal or paternal genomes creating reciprocal phenotypes. Paternal genomic excess frequently results in extensive endosperm proliferation without cellularization and seed abortion. We previously showed that loss of the RNA Pol IV gene nrpd1 in tetraploid fathers represses seed abortion in paternal excess crosses. Here we show genetically that RNA-directed DNA methylation (RdDM) pathway activity in the paternal parent is sufficient to determine the viability of paternal excess seeds. We compared transcriptomes, DNA methylation, and small RNAs from endosperm of balanced crosses (diploid x diploid) and lethal (diploid x tetraploid) and viable paternal excess (diploid x tetraploid nrpd1). Endosperm from both lethal and viable paternal excess seeds share widespread transcriptional and DNA methylation changes at genes and TEs. Interploidy seed abortion is thus unlikely to be caused by either transposable element or imprinted gene mis-regulation, and its repression by loss of paternal RdDM is associated with only modest gene expression changes. Finally, using allele-specific transcription data, we present evidence for a transcriptional buffering system that increases expression of maternal alleles and represses paternal alleles in response to excess paternal genomic dosage. These findings prompt reconsideration of models for dosage sensitivity in endosperm.

plant biology↗

bioRxiv2018

A variably imprinted epiallele impacts seed development

The contribution of epigenetic variation to phenotypic variation is unclear. Imprinted genes, because of their strong association with epigenetic modifications, represent an opportunity for the discovery of such phenomena. In mammals and flowering plants, a subset of genes are expressed from only one parental allele in a process called gene imprinting. Imprinting is associated with differential DNA methylation and chromatin modifications between parental alleles. In flowering plants imprinting occurs in a seed tissue - endosperm. Proper endosperm development is essential for the production of viable seeds. We previously showed that in Arabidopsis thaliana intraspecific imprinting variation is correlated with naturally occurring DNA methylation polymorphisms. Here, we investigated the mechanisms and function of allele-specific imprinting of the class IV homeodomain-Leucine zipper (HD-ZIP) transcription factor HDG3. In imprinted strains, HDG3 is expressed primarily from the methylated paternally inherited allele. We manipulated the methylation state of endogenous HDG3 in a non-imprinted strain and demonstrated that methylation of a proximal transposable element is sufficient to promote HDG3 expression and imprinting. Gain of HDG3 imprinting was associated with earlier endosperm cellularization and changes in seed weight. These results indicate that epigenetic variation alone is sufficient to explain imprinting variation and demonstrate that epialleles can underlie variation in seed development phenotypes.\n\nAuthor SummaryThe contribution of genetic variation to phenotypic variation is well-established. By contrast, it is unknown how frequently epigenetic variation causes differences in organismal phenotypes. Epigenetic information is closely associated with but not encoded in the DNA sequence. In practice, it is challenging to disentangle genetic variation from epigenetic variation, as what appears to be epigenetic variation might have an underlying genetic basis. DNA methylation is one form of epigenetic information. HDG3 encodes an endosperm specific transcription factor that exists in two states in A. thaliana natural populations: methylated and expressed and hypomethylated and repressed. We show that pure epigenetic variation is sufficient to explain expression variation of HDG3 - a naturally lowly expressed allele can be switched to a higher expressed state by adding DNA methylation. We also show that expression of HDG3 in strains where it is normally hypomethylated and relatively repressed causes a seed development phenotype. These data indicate that naturally circulating epialleles have consequences for seed phenotypic variation.

plant biology↗

bioRxiv2017

A Small RNA Pathway Mediates Global Allelic Dosage In Endosperm

Balance between maternal and paternal genomes within the triploid endosperm is necessary for normal seed development. The majority of genes in Arabidopsis endosperm are expressed in a 2:1 maternal:paternal ratio, reflecting endosperm genomic DNA content. Here we find that the 2:1 transcriptional ratio is not, unexpectedly, a passive default but is actively regulated. We describe an inverse relationship between the parent-of-origin of small RNAs and mRNAs in endosperm on a genome-wide scale. Disruption of the Pol IV small RNA pathway causes the entire transcriptome to become more maternally biased. Furthermore, paternal inheritance of a RNA Pol IV mutation is sufficient to rescue seed abortion caused by excess paternal genome dosage. These results indicate that maintenance of the maternal:paternal transcriptome ratio in endosperm is an active process and reveal a function for RNA Pol IV in mediating the global transcriptional balance between maternally and paternally inherited genomes in endosperm.

plant biology↗