Biology subjects

Forsythe, E. S.

Publications and source records attributed to Forsythe, E. S..

15 recordsLinked to original sources

bioRxiv2026

Gene repertoire expansion and cis-regulatory diversification shaped 26S proteasome evolution within a proteostasis-centered network

The 26S proteasome is essential for proteostasis and constitutes one of the most conserved molecular machineries in eukaryotes. Its homeostasis is maintained by a mechanistically conserved feedback loop involving kingdom-specific components. Yet, how the proteasome subunits and associated regulatory feedback loop have evolved to accommodate ever-changing cellular context is poorly understood. Here, we combine gene duplicate analysis, cis-regulatory element identification, functional validation, and protein network inference to investigate the evolution of plant 26S proteasome. Proteasome gene repertoires expanded largely independently across plant lineages, with paralogs showing shifts in post-translational modification sites and pronounced transcriptional divergence in response to environmental cues. Comparative analysis of 26S proteasome gene promoters revealed extensive diversification of proteasome-associated cis-elements across plants, with repeated enrichment of related motifs. These observations led to the identification of telomere repeat-binding proteins (TRBs) as novel transcriptional regulators of proteasome genes in A. thaliana, through association with the previously characterized PRCE motif. Finally, evolutionary rate covariation analysis identified a conserved proteasome-associated network connected through proteasome-associated cis-elements and unifying cellular proteostasis. Together, our results indicate that plant 26S proteasome is controlled by a regulatory architecture that combines conserved cis-regulatory mechanisms, lineage-specific innovation, and stress-responsive paralog specialization at the center of the proteostasis network; offering a new perspective on the evolution of one of the most essential molecular complexes.

plant biology↗

bioRxiv2025

Evolutionary rates provide genomic insights into convergent and lineage-specific evolution in carnivorous plants

Carnivorous plants (CPs) are striking examples of convergent evolution across ecological, morphological, and molecular scales, making them ideal systems for investigating genomic adaptation to extreme environments. While genomic studies have shown that gene family expansion and functional recruitment underlie key carnivorous traits, the effects of these transitions on rates of sequence evolution remain incompletely understood. Here, we present a comparative framework to examine evolutionary rate shifts across 30 carnivorous and non-carnivorous eudicots in over 12,000 gene families. Analyzing branch-lengths and evolutionary rate covariation (ERC), we detect both lineage-specific and convergent acceleration in CPs for genes associated with stress response, stimulus signaling, and light perception. Accelerated genes often belong to expanded families and show relaxation of purifying selection pressure. Despite the limited number of specific genes or gene families showing convergent acceleration across CP lineages, we find strong convergence at the functional level. Distinct sets of accelerated genes in each CP lineage are enriched for similar functional pathways, particularly light and hormone responses. ERC network analysis identifies a stress- and stimulus-responsive gene community with lineage-specific acceleration in Sarraceniaceae species and moderate acceleration in other CP lineages. Hub genes in this network are also co-expressed in a distantly related non-CP model system, suggesting repeated co-option of an ancestral regulatory module for carnivorous functions. Together, our results show that convergent genomic rate accelerations in CPs primarily involve functionally equivalent or similar but lineage-distinct genes, shaped by gene duplication, relaxed selection, and co-option of stress-response networks.

evolutionary biology↗

bioRxiv2025

Reciprocal exchange obscures phylogenetic patterns of introgression

Hybridization and introgression are major evolutionary forces in eukaryotes that have shaped the evolutionary trajectories of crops, livestock, and modern humans. Yet current theoretical and statistical frameworks provide only a partial view of the dynamics and genomic consequences of introgression. An unexplored dimension is the possibility that introgression between two lineages can occur simultaneously in both directions at a single genomic locus--a process we term reciprocal introgression. Although this type of introgression is plausible, especially in cases of bidirectional introgression across the genome, it remains largely undetectable under widely used statistical approaches, hindering evaluation of its prevalence in natural systems. Moreover, unrecognized reciprocal introgression may bias summary statistics commonly used to quantify gene flow, obscuring our ability to resolve introgression events. To address this, here, we develop theoretical expectations of the impact of reciprocal introgression on phylogenetic and site-pattern-based results. To test these expectations, we implement coalescent simulations to model genomes experiencing reciprocal introgression and apply sliding-window introgression statistics to these data. We find that reciprocally introgressed loci produce phylogenetic patterns opposite to those expected under unidirectional introgression. These mixed signals can dilute evidence of introgression or even generate false signals of introgression events that never occurred. Taken together, our results highlight the need for updated statistical approaches and pave the way toward a more complete understanding of the impacts of introgression on gene histories and organismal evolution.

evolutionary biology↗

bioRxiv2025

Plant MutS2 proteins function in plastid ribosome quality control

Tight regulation of chloroplast translation is essential for plant growth, development and environmental response. Active translation can result in stalling and collision of ribosomes, which have negative fitness consequences. However, the ways in which chloroplasts respond to these types of translational stressors remain unknown. Here, we identify two MutS2 proteins that act as critical players in plastid ribosome-associated quality control (RQC) in Arabidopsis. We found that both MutS2A and MutS2B are required to overcome specific antibiotic-induced ribosome stalling and collisions. Further, these proteins appear to be essential for tissue greening during de-etiolation, potentially due to increased translational demand during the transition from etioplast to chloroplast. Although bacterial homologs of MutS2 have been widely recognized for their role in regulating homologous recombination, we found only weak support for this function in Arabidopsis plastids. Therefore, these proteins, which are widely conserved among photosynthetic eukaryotes, appear to be central in the resolution of ribosome collisions and may play a critical role during times of increased translational demand.

plant biology↗

bioRxiv2025

Detecting cryptic ghost lineage introgression in four-taxon genomic datasets

PremiseHybridization and introgression are pervasive evolutionary forces that have played fundamental roles in shaping the diversity of wild and domesticated plants. Four-taxon tests for introgression provide a reliable framework for detecting signatures of ancient introgression from genomic data, which have played an important role in revealing the reticulate nature of plant evolution; however, there is emerging evidence that a cryptic process known as ghost lineage introgression has the potential to dramatically skew interpretations of four-taxon introgression statistics, particularly our ability to determine the lineages involved in introgression. This ambiguity limits our ability to resolve the mechanisms and functional implications of introgression because it means we can determine neither the donor nor the recipient of introgressed alleles with confidence. MethodsHere, we develop ghostbuster, a statistical test designed to detect ghost lineage introgression in genomic data based on patterns of sequence divergence. We employ coalescent simulations to test our method and ascertain the conditions under which it accurately identifies ingroup versus ghost lineage introgression. Finally, to demonstrate the utility of ghostbuster we apply it to a previously identified introgression event in the plant family Brassicaceae. ResultOur simulations reveal that ghostbuster accurately distinguishes ghost lineage introgression from ingroup introgression across a wide range of introgression scenarios, with errors arising only when divergence events are closely spaced. Our analysis of empirical plant data reveals that the previously identified introgression likely constitutes ghost lineage introgression and, thus, was previously misinterpreted. DiscussionOur analyses of simulated and empirical data demonstrate that ghostbuster will be a helpful tool in resolving reticulate evolution in plants and other taxa. We demonstrate the biological insights that ghostbuster provides by presenting an updated model of ghost lineage introgression in Brassicaceae, impacting our understanding of the molecular evolution of crop and model species in this important plant lineage. Ghostbuster code is freely available at: https://github.com/EvanForsythe/Ghost_introgression.

evolutionary biology↗

bioRxiv2024

Correlated evolutionary rates reveal novel components and cross-compartment connectivity in plant proteostasis systems

Plant cells rely on an interconnected network of proteins interacting at many levels (e.g. physical enzyme complexes, gene regulatory modules, and biosynthetic pathways). Pairs of proteins that interact at any of these levels have been shown to exhibit phylogenetic signatures of evolutionary rate covariation (ERC), providing a basis for detecting functional interactions among proteins. Here, we apply ERCnet, a bioinformatic tool for performing genome-scale ERC analyses, to predict a plant protein-protein interactome network. We find a clustered set of proteins that exhibit strong signatures of ERC with the plastid caseinolytic protease (Clp) and other plastid proteostasis components, thus forming a functional module within the network. In addition to including proteins with known or predicted functions in protein import, transcription, translation, and degradation in plastids, the module also includes proteins with previously unknown molecular function, thus providing evidence that these proteins may contribute to plastid proteostasis in novel ways. Perhaps the most surprising members of this module are a set of proteins that are not thought to localize to the plastid at all. These proteins include a mitochondrial-localized pentatricopeptide repeat (PPR) protein with genetic evidence of interaction with the mitochondrial Clp system and two nuclear-localized actin-related proteins involved in chromatin remodeling and epigenetic regulation of nuclear genes. We speculate that these non-plastid-localized proteins act as mediators of organellar crosstalk and retrograde signaling of cellular proteostasis status in plants. In summary, our results highlight the connected nature of plant proteostasis systems and point to a promising set of novel proteostasis protein candidates.

molecular biology↗

bioRxiv2024

Phylogenomic prediction of interaction networks in the presence of gene duplication

Assigning gene function from genome sequences is a rate-limiting step in molecular biology research. A proteins position within an interaction network can potentially provide insights into its molecular mechanisms. Phylogenetic analysis of evolutionary rate covariation (ERC) in protein sequence has been shown to be effective for large-scale prediction of functional relationships and interactions. However, gene duplication, gene loss, and other sources of phylogenetic incongruence are barriers for analyzing ERC on a genome-wide basis. Here, we developed ERCnet, a bioinformatic program designed to overcome these challenges, facilitating efficient all- vs-all ERC analyses for large protein sequence datasets. We simulated proteome datasets and found that ERCnet achieves combined false positive and negative error rates well below 10% and that our novel branch-by-branch length measurements outperforms root-to-tip approaches in most cases, offering a valuable new strategy for performing ERC. We also compiled a sample set of 35 angiosperm genomes to test the performance of ERCnet on empirical data, including its sensitivity to user-defined analysis parameters such as input dataset size and branch-length measurement strategy. We investigated the overlap between ERCnet runs with different species samples to understand how species number and composition affect predicted interactions and to identify the protein sets that consistently exhibit ERC across angiosperms. Our systematic exploration of the performance of ERCnet provides a roadmap for design of future ERC analyses to predict functional interactions in a wide array of genomic datasets. ERCnet code is freely available at https://github.com/EvanForsythe/ERCnet.

genomics↗

bioRxiv2022

Organellar transcripts dominate the cellular mRNA pool across plants of varying ploidy levels

Mitochondrial and plastid functions depend on coordinated expression of proteins encoded by genomic compartments that have radical differences in copy number of organellar and nuclear genomes. In polyploids, doubling of the nuclear genome may add challenges to maintaining balanced expression of proteins involved in cytonuclear interactions. Here, we use ribo-depleted RNA-seq to analyze transcript abundance for nuclear and organellar genomes in leaf tissue from four different polyploid angiosperms and their close diploid relatives. We find that, even though plastid genomes contain <1% of the number of genes in the nuclear genome, they generate the majority (69.9-82.3%) of mRNA transcripts in the cell. Mitochondrial genes are responsible for a much smaller percentage (1.3-3.7%) of the leaf mRNA pool but still produce much higher transcript abundances per gene compared to nuclear genome. Nuclear genes encoding proteins that functionally interact with mitochondrial or plastid gene products exhibit mRNA expression levels that are consistently more than ten-fold lower than their organellar counterparts, indicating an extreme cytonuclear imbalance at the RNA level despite the predominance of equimolar interactions at the protein level. Nevertheless, interacting nuclear and organellar genes show strongly correlated transcript abundances across functional categories, suggesting that the observed mRNA stoichiometric imbalance does not preclude coordination of cytonuclear expression. Finally, we show that nuclear genome doubling does not alter the cytonuclear expression ratios observed in diploid relatives in consistent or systematic ways, indicating that successful polyploid plants are able to compensate for cytonuclear perturbations associated with nuclear genome doubling.

plant biology↗

bioRxiv2022

Variation in cytonuclear expression accommodation among allopolyploid plants

Cytonuclear coevolution is a common feature among plants, which coordinates gene expression and protein products between the nucleus and organelles. Consequently, lineage-specific differences may result in incompatibilities between the nucleus and cytoplasm in hybrid taxa. Allopolyploidy is also a common phenomenon in plant evolution. The hybrid nature of allopolyploids may result in cytonuclear incompatibilities, but the massive nuclear redundancy created during polyploidy affords additional avenues for resolving cytonuclear conflict (i.e., cytonuclear accommodation). Here we evaluate expression changes in organelle-targeted nuclear genes for six allopolyploid lineages that represent four genera (i.e., Arabidopsis, Arachis, Chenopodium, and Gossypium) and encompass a range in polyploid ages. Because incompatibilities between the nucleus and cytoplasm could potentially result in biases toward the maternal homoeolog and/or maternal expression level, we evaluate patterns of homoeolog usage, expression bias, and expression level dominance in cytonuclear genes relative to the background of non-cytonuclear expression changes and to the diploid parents. Although we find subsets of cytonuclear genes in most lineages that match our expectations of maternal preference, these observations are not consistent among either allopolyploids or categories of organelle-targeted genes. Our results indicate that cytonuclear expression accommodation may be a subtle and/or variable phenomenon that does not capture the full range of mechanisms by which allopolyploid plants resolve nuclear-cytoplasmic incompatibilities.

evolutionary biology↗

bioRxiv2021

Gene duplication and rate variation in the evolution of non-photosynthetic pathways in plastids

While the chloroplast (plastid) is known for its role in photosynthesis, it is also involved in many other metabolic pathways essential for plant survival. As such, plastids contain an extensive suite of enzymes required for non-photosynthetic processes. The evolution of the associated genes has been especially dynamic in flowering plants (angiosperms), including examples of gene duplication and extensive rate variation. We examined the role of ongoing gene duplication in two key plastid enzymes, the acetyl-CoA carboxylase (ACCase) and the caseinolytic protease (Clp), responsible for fatty acid biosynthesis and protein turnover, respectively. In plants, there are two ACCase complexes--a homomeric version present in the cytosol and a heteromeric version present in the plastid. Duplications of the nuclear-encoded homomeric ACCase gene and retargeting of one resultant protein to the plastid have been previously reported in multiple species. We find that these retargeted homomeric ACCase proteins exhibit elevated rates of sequence evolution, consistent with neofunctionalization and/or relaxation of selection. The plastid Clp complex catalytic core is composed of nine paralogous proteins that arose via ancient gene duplication in the cyanobacterial/plastid lineage. We show that further gene duplication occurred more recently in the nuclear-encoded core subunits of this complex, yielding additional paralogs in many species of angiosperms. Moreover, in six of eight cases, subunits that have undergone recent duplication display increased rates of sequence evolution relative to those that have remained single copy. We also compared substitution patterns between pairs of Clp core paralogs to gain insight into post-duplication evolutionary routes. These results show that gene duplication and rate variation continue to shape the plastid proteome.

evolutionary biology↗

bioRxiv2020

Genome-wide signatures of plastid-nuclear coevolution point to repeated perturbations of plastid proteostasis systems across angiosperms

Nuclear and plastid (chloroplast) genomes experience different mutation rates, levels of selection, and transmission modes, yet key cellular functions depend on coordinated interactions between proteins encoded in both genomes. Functionally related proteins often show correlated changes in rates of sequence evolution across a phylogeny (evolutionary rate covariation or ERC), offering a means to detect previously unidentified suites of coevolving and cofunctional genes. We performed phylogenomic analyses across angiosperm diversity, scanning the nuclear genome for genes that exhibit ERC with plastid genes. As expected, the strongest hits are highly enriched for plastid-targeted proteins, providing evidence that cytonuclear interactions affect rates of molecular evolution at genome-wide scales. Many identified nuclear genes function in post-transcriptional regulation and the maintenance of protein homeostasis (proteostasis), including protein translation (in both the plastid and cytosol), import, quality control and turnover. We also identified nuclear genes that exhibit strong signatures of coevolution with the plastid genome but lack organellar-targeting annotations, making them candidates for having previously undescribed roles in plastids. In sum, our genome-wide analyses reveal that plastid- nuclear coevolution extends beyond the intimate molecular interactions within chloroplast enzyme complexes and may be driven by frequent rewiring of the machinery responsible for maintenance of plastid proteostasis in angiosperms.

evolutionary biology↗

bioRxiv2019

Evolutionary and biochemical analyses reveal conservation of the Brassicaceae telomerase ribonucleoprotein complex

The telomerase ribonucleoprotein complex (RNP) is essential for genome stability and performs this role through the addition of repetitive DNA to the ends of chromosomes. The telomerase enzyme is composed of a reverse transcriptase (TERT), which utilizes a template domain in an RNA subunit (TER) to reiteratively add telomeric DNA at the ends of chromosomes. Multiple TERs have been identified in the model plant Arabidopsis thaliana. Here we combine a phylogenetic and biochemical approach to understand how the telomerase RNP has evolved in Brassicaceae, the family that includes A. thaliana. Because of the complex phylogenetic pattern of template domain loss and alteration at the previously characterized A. thaliana TER loci, TER1 and TER2, across the plant family Brassicaceae, we bred double mutants from plants with a template deletion at AtTER1 and T-DNA insertion at AtTER2. These double mutants exhibited no telomere length deficiency, a definitive indication that neither of these loci encode a functional telomerase RNA. Moreover, we determined that the telomerase components TERT, Dyskerin, and the KU heterodimer are under strong purifying selection, consistent with the idea that the TER with which they interact is also conserved. To test this hypothesis further, we analyzed the substrate specificity of telomerase from species across Brassicaceae and determined that telomerase from close relatives bind and extend substrates in a similar manner, supporting the idea that TERs in different species are highly similar to one another and are likely encoded from an orthologous locus. Lastly, TERT proteins from across Brassicaceae were able to complement loss of function tert mutants in vivo, indicating TERTs from other species have the ability to recognize the native TER of A. thaliana. Finally, we immunoprecipitated the telomerase complex and identified associated RNAs via RNA-seq. Using our evolutionary data we constrained our analyses to conserved RNAs within Brassicaceae that contained a template domain. These analyses revealed a highly expressed locus whose disruption by a T-DNA resulted in a telomeric phenotype similar to the loss of other telomerase core proteins, indicating that the RNA has an important function in telomere maintenance.

genetics↗

bioRxiv2019

CyMIRA: The Cytonuclear Molecular Interactions Reference for Arabidopsis

The function and evolution of eukaryotic cells depends upon direct molecular interactions between gene products encoded in nuclear and cytoplasmic genomes. Understanding how these cytonuclear interactions drive molecular evolution and generate genetic incompatibilities between isolated populations and species is of central importance to eukaryotic biology. Plants are an outstanding system to investigate such effects because of their two different genomic compartments present in the cytoplasm (mitochondria and plastids) and the extensive resources detailing subcellular targeting of nuclear-encoded proteins. However, the field lacks a consistent classification scheme for mitochondrial- and plastid-targeted proteins based on their molecular interactions with cytoplasmic genomes and gene products, which hinders efforts to standardize and compare results across studies. Here, we take advantage of detailed knowledge about the model angiosperm Arabidopsis thaliana to provide a curated database of plant cytonuclear interactions at the molecular level. CyMIRA (Cytonuclear Molecular Interactions Reference for Arabidopsis) is available at http://cymira.colostate.edu/ and https://github.com/dbsloan/cymira and will serve as a resource to aid researchers in partitioning evolutionary genomic data into functional gene classes based on organelle targeting and direct molecular interaction with cytoplasmic genomes and gene products. It includes 11 categories (and 27 subcategories) of different cytonuclear complexes and types of molecular interactions, and it reports residue-level information for cytonuclear contact sites. We hope that this framework will make it easier to standardize, interpret and compare studies testing the functional and evolutionary consequences of cytonuclear interactions.

genomics↗

bioRxiv2019

Divergence-based introgression polarization

Introgressive hybridization results in the transfer of genetic material between species, often with fitness implications for the recipient species. The development of statistical methods for detecting the signatures of historical introgression (IG) in whole-genome data has been a major area of focus. While existing techniques are able to identify the taxa that exchanged genes during IG using a four-taxon system, most methods do not explicitly distinguish which taxon served as donor and which as recipient during IG (i.e. polarization of IG directionality). The existing methods that do polarize IG are only able to do so when there is a fifth taxon available and that taxon is sister to one of the taxa involved in IG. Here, we present Divergence-based Introgression Polarization (DIP), a method for polarizing IG using patterns of sequence divergence across whole genomes, which operates in a four-taxon context. Thus, DIP can be applied to infer the directionality of IG when additional taxa are not available. We use simulations to show that DIP can polarize IG and identify potential sources of bias in the assignment of directionality, and we apply DIP to a well-described hominin IG event.

evolutionary biology↗

bioRxiv2017

Epistatic interactions drive biased gene retention in the face of massive nuclear introgression

Phylogenomic analyses are recovering previously hidden histories of hybridization, revealing the genomic consequences of these events on the architecture of extant genomes. We exploit a suite of genomic resources to show that introgressive hybridization occurred between close relatives of Arabidopsis, impacting our understanding of species relationships in the group. The composition of introgressed and retained genes indicates that selection against incompatible cytonuclear and nuclear-nuclear interactions likely acted during introgression, while neutral processes also contributed to genome composition through the retention of ancient haplotype blocks. We also developed a divergence-based test to distinguish donor from recipient lineages without the requirement of additional taxon-sampling. Finally, to our great surprise, we find that cytonuclear discordance appears to have arisen via extensive nuclear, rather than cytoplasmic, introgression, meaning that most of the genome was displaced during introgression, while only a small proportion of native alleles were retained.

evolutionary biology↗