Biology subjects

Dolan, L.

Publications and source records attributed to Dolan, L..

33 records · Page 2Linked to original sources

bioRxiv2023

A versatile CRISPR-based system for lineage tracing in living plants

Individual cells give rise to diverse cell lineages during the development of multicellular organisms. Understanding the contribution of these lineages to mature organisms is a central question of developmental biology. Several techniques to document cell lineages have been used, from marking single cells with mutations that express a visible marker to generating molecular bar codes by CRISPR-induced mutations and subsequent single-cell analysis. Here, we exploit the mutagenic activity of CRISPR to allow lineage tracing within living plants. Cas9-induced mutations are directed to correct a frameshift mutation that restores expression of a nuclear fluorescent protein, labelling the initial cell and all progenitor cells with a strong signal without modifying other phenotypes of the plants. Spatial and temporal control of Cas9 activity can be achieved using tissue-specific and/or inducible promoters. We provide proof of principle for the function of lineage tracing in two model plants. The conserved features of the components and the versatile cloning system, allowing for easy exchange of promoters, are expected to make the system widely applicable. SIGNIFICANCE STATEMENTBy targeting Cas9 in a tissue- and time-specific way to correct a frameshift mutation, resulting in fluorescence labelling of nuclei, we generated a method for in vivo visual lineage tracing in two model plants. The versatile cloning system makes the system widely applicable in other plants.

cell biology↗

bioRxiv2022

Transposons repressed by H3K27me3 were co-opted as cis-regulatory elements of H3K27me3 controlled protein coding genes during evolution of plants

The mobility of transposable elements (TEs) contributes to evolution of genomes 1,2. Meanwhile, their uncontrolled activity causes genomic instability and therefore expression of TEs is silenced by host genomes 3,4. TEs are marked with DNA and H3K9 methylation that are associated with silencing in flowering plants 5, animals, and fungi 6. Yet, in distantly related eukaryotes TEs are instead marked by H3K27me3 deposited by the Polycomb Repressive Complex 2 (PRC2) 7-11, an epigenetic mark associated with gene silencing in multicellular eukaryotes 12-15. It was therefore proposed that the ancestral activity of PRC2 was the deposition of H3K27me3 to silence TEs 16. To test this hypothesis we obtained mutants deprived of PRC2 activity and used genomics to analyze the role of PRC2 in extant species along the lineage of Archaeplastida. While in the red alga Cyanidioschyzon merolae more TEs than genes were repressed by PRC2, an opposite trend was observed in bryophytes Marchantia polymorpha and Anthoceros agrestis. In the red alga, TEs silenced by H3K27me3 are in subtelomeres but in bryophytes, TEs and genes marked by H3K27me3 form coregulated transcriptional units. The latter trend was also observed in the flowering plant Arabidopsis thaliana, and we identified cis-elements recognised by transcription factors in TEs flanking genes repressed by PRC2. Together with the silencing of TEs by PRC2 in ciliates that diverged early from an ancestor common with Archaeplastida, our findings support the hypothesis that PRC2 deposited H3K27me3 to silence TEs in early lineages of eukaryotes. During evolution, TE fragments marked with H3K27me3 were selected to shape transcriptional regulation that control networks of genes regulated by PRC2. HighlightsH3K27me3 marks a decreasing proportion of TEs during evolution of plants The polycomb repressive complex 2 represses TEs in red algae and bryophytes H3K27me3-marked TEs in flowering plants contain transcription factor binding sites Transcription factors bind TEs and regulate networks of genes controlled by PRC2

evolutionary biology↗

bioRxiv2022

The chromatin landscape of bryophytes

BackgroundIn animals and flowering plants specific chromatin modifications define three chromosomal domains: euchromatin comprising transcribed genes, facultative heterochromatin comprising repressed genes, and constitutive heterochromatin comprising transposons. However, recent studies have shown that the correlation between chromatin modifications and transcription vary among different eukaryotic organisms including mosses and liverworts that differ from one another. Mosses and liverworts diverged from hornworts, altogether forming the lineage of bryophytes that shared a common ancestor with all land plants. We aimed to obtain chromatin landscapes in hornworts to establish synapomorphies across bryophytes. ResultsWe mapped the chromatin landscape of the model hornwort Anthoceros agrestis. By comparing chromatin landscapes across bryophytes we defined the common chromatin landscape of the ancestor of extant bryophytes. In this group, constitutive heterochromatin was characterized by a scattered distribution across autosomes, which contrasted with the dense compartments of heterochromatin surrounding the centromeres in flowering plants. Topologically associated domains were primarily occupied by transposons with genes at their boundaries and nearly half of the hornwort transposons were associated with facultative heterochromatin and euchromatin. ConclusionsMost of the features observed in hornworts are also present in liverworts but are distinct from flowering plants. Hence, the ancestral genome of bryophytes was likely a patchwork of units of euchromatin interspersed within facultative and constitutive heterochromatin and each unit contained both transposons and genes sharing the same chromatin state. We propose this genome organization was ancestral to land plants and prevented transposons from being segregated as constitutive heterochromatin around point centromeres as in flowering plants.

genomics↗

bioRxiv2022

Meristem dormancy in a dichotomous branching system is regulated by a liverwort-specific miRNA and a clade III SPL gene in Marchantia polymorpha

The shape of modular organisms depends on branching architecture, which in plants is determined by the fates of generative centres called meristems. The branches of the liverwort Marchantia polymorpha are derived from two adjacent meristems that develop at thallus apices. These meristems may be active and develop branches or may be dormant and do not form branches. The relative number and position of active and dormant meristems defines overall shape and form of the thallus. We show that the clade III SQUAMOSA PROMOTER BINDING PROTEIN LIKE (SPL) transcription factor, MpSPL1, is required for meristem dormancy. The activity of MpSPL1 is regulated by the liverwort-specific Mpo-MR13 miRNA which in turn is regulated by PIF-mediated phytochrome signaling. An unrelated miRNA, MIR156, represses a different SPL gene (belonging to clade IV) that inhibits branching during the shade avoidance response in Arabidopsis thaliana. This suggests that a conserved mechanism of phytochrome signaling modulates branching architecture in liverworts and angiosperms and therefore likely operated in the last common ancestor. However, PIF-mediated phytochrome signaling represses the expression of different miRNA genes with different SPL targets during dichotomous, apical branching in liverworts and during lateral, subapical branching in angiosperms. We speculate that the mechanism that acts downstream of light and regulates meristem dormancy evolved independently in liverworts and angiosperms.

plant biology↗

bioRxiv2022

Reduced coenzyme Q synthesis confers non-target site resistance to the herbicide thaxtomin A

Herbicide resistance in weeds is a growing threat to global crop production. Non-target site resistance is problematic because a single resistance allele can confer tolerance to many herbicides (cross resistance), and it is often a polygenic trait so it can be difficult to identify the molecular mechanisms involved. Most characterized molecular mechanisms of non-target site resistance are caused by gain-of-function mutations in genes from a few key gene families - the mechanisms of resistance caused by loss-of-function mutations remain unclear. In this study, we first show that the mechanism of non-target site resistance to the herbicide thaxtomin A conferred by loss-of-function of the gene PAM16 is conserved in Marchantia polymorpha, validating its use as a model species with which to study non-target site resistance. To identify mechanisms of non-target site resistance caused by loss-of-function mutations, we generated 107 UV-B mutagenized M. polymorpha spores and screened for resistance to the herbicide thaxtomin A. We isolated 13 thaxtomin A-resistant mutants and found that 3 mutants carried candidate resistance-conferring SNPs in the MpRTN4IP1L gene. Mprtn4ip1l mutants are defective in coenzyme Q biosynthesis and accumulate higher levels of reactive oxygen species (ROS) than wild-type plants. Mutants are also defective in thaxtomin A metabolism, consistent with the hypothesis that loss of MpRTN4IP1L function confers non-target site resistance. We conclude that loss of MpRTN4IP1L function is a novel mechanism of non-target site herbicide resistance, and propose that other mutations which increase ROS levels or decrease thaxtomin A metabolism could confer thaxtomin A resistance in the field. AUTHOR SUMMARYModern agriculture relies on herbicides to control weed populations. However, herbicide resistance in weeds threatens the efficacy of herbicides and global crop production, similar to how antibiotic resistance poses a global health threat. Understanding the molecular mechanisms behind herbicide resistance helps to prevent resistance from evolving and to better manage herbicide resistant weeds in the field. Here, we use a forward genetic approach in the model species Marchantia polymorpha to discover novel mechanisms of herbicide resistance. We report the discovery of a novel mechanism of herbicide resistance caused by loss-of-function mutations in the MpRTN4IP1L gene. We find that Mprtn4ip1l mutants are resistant to the herbicides thaxtomin A and isoxaben, accumulate higher levels of reactive oxygen species than wild type plants, and are defective in thaxtomin A metabolism. We predict that loss-of-function mutations or treatments that increase reactive oxygen species production could contribute to thaxtomin A tolerance.

plant biology↗

bioRxiv2022

Transcriptome changes in chlorsulfuron-treated plants are caused by acetolactate synthase inhibition and not induction of a herbicide detoxification system in Marchantia polymorpha

A sensing mechanism in mammals perceives xenobiotics and induces the transcription of genes encoding proteins that detoxify these molecules. However, it is unclear if plants sense xenobiotics, and activate an analogous signalling system leading to their detoxification. Using the liverwort Marchantia polymorpha, we tested the hypothesis that there is a sensing system in plants that perceives herbicides resulting in the increased transcription of genes encoding proteins that detoxify these herbicides. Consistent with the hypothesis, we show that chlorsulfuron-treatment induces changes in the M. polymorpha transcriptome. However, these transcriptome changes do not occur in chlorsulfuron (CS)-treated target site resistant mutants, where the gene encoding the target carries a mutation that confers resistance to chlorsulfuron. Instead, we show that inactivation of the chlorsulfuron target, acetolactate synthase (ALS) (also known as acetohydroxyacid synthase (AHAS)), is required for the transcriptome response. These data are consistent with the changes in the transcriptome of chlorsulfuron-treated plants being caused by disrupted amino acid synthesis and metabolism resulting from acetolactate synthase inhibition. These conclusions suggest that chlorsulfuron is not sensed in M. polymorpha leading to induce a detoxification system. Author SummaryHerbicide use is increasing throughout the world, however we know little about how plants respond to herbicide treatment and regulate their metabolism. Some plants have evolved resistance to herbicides such as chlorsulfuron by increasing the detoxification of the herbicide compared to sensitive plants. It has been suggested that plants can directly sense the herbicide chemical which activates a detoxification response, in a similar way to the detoxification of foreign chemicals in mammalian cells. The liverwort Marchantia polymorpha is an excellent system to study plant herbicide responses due to its short generation time, ease of propagation and low genetic redundancy. We show that chlorsulfuron treatment alters the expression of many genes in M. polymorpha, however plants with a resistance-conferring mutation in the molecular target of chlorsulfuron do not show any changes in gene expression in response to chlorsulfuron treatment. This result indicates that transcriptome changes caused by chlorsulfuron depend on the inhibition of the target by chlorsulfuron. This suggests that plants do not sense chlorsulfuron and activate a detoxification system. This finding has implications for herbicide use and discovery.

plant biology↗

bioRxiv2022

Genes encoding cytochrome P450 monooxygenases and glutathione S-transferases associated with herbicide resistance evolved before the origin of land plants

Cytochrome P450 (CYP) monooxygenases and glutathione S-transferases (GST) are enzymes that catalyse chemical modifications of a range of organic compounds. Herbicide tolerance is associated with higher levels of CYP and GST gene expression in some herbicide-resistant weed populations compared to sensitive populations of the same species. By comparing the protein sequences of 9 representative species of the Archaeplastida - the lineage which includes red algae, glaucophyte algae, chlorophyte algae, and streptophytes - and generating phylogenetic trees, we identified the CYP and GST proteins that existed in the common ancestor of the Archaeplastida. All CYP clans and all but one land plant GST classes present in land plants evolved before the divergence of streptophyte algae and land plants from their last common ancestor. We also demonstrate that there are more genes encoding CYP and GST proteins in land plants than in algae. The larger numbers of genes among land plants largely results from gene duplications in CYP clans 71, 72, and 85 and in the GST Phi and Tau classes. Enzymes that either chemically alter herbicides or confer herbicide resistance belong to CYP clans 71 and 72 and the GST Phi and Tau classes. These results demonstrate that the clan and class diversity in extant plant CYP and GST proteins evolved in the Proterozoic before the divergence of land plants and streptophyte algae from a last common ancestor. Then, early in embryophyte evolution during the Palaeozoic, gene duplication in four of the twelve CYP clans, and in two of the fourteen GST classes, led to the large numbers of CYP and GST proteins found in extant land plants. It is among the genes of CYP clans 71 and 72 and GST classes Phi and Tau that alleles conferring herbicide resistance evolved in the last fifty years.

plant biology↗

bioRxiv2022

A potent SARS-CoV-2 neutralizing antibody recognizing a conserved epitope with broad mutant variant and SARS-CoV activity.

COVID-19 is the deadliest respiratory virus pandemic since 1918 and the latest of several coronavirus epidemics and pandemics in recent years. Despite the unprecedented response by both the government and private sectors to develop vaccines and therapies, the evolution of SARS-CoV-2 variants resistant to these interventions reveals a crucial need for therapeutics that maintain their efficacy against current and future mutant variants. Here we describe a SARS-CoV-2 neutralizing antibody, ABP-310, with potent activity against all variants tested including the Omicron variant. ABP-310 also displays potent neutralizing activity against SARS-CoV, highlighting the conserved nature of the ABP-310 epitope. By targeting a conserved epitope, we believe that ABP-310 has therapeutic promise not only against the current SARS-CoV-2 variants but would be expected to maintain efficacy against future variants and possibly even novel coronaviruses.

microbiology↗

bioRxiv2021

An evidence-based 3D reconstruction of Asteroxylon mackiei the most complex plant preserved from the Rhynie chert

The 407-million-year-old Rhynie chert preserves the earliest terrestrial ecosystem and informs our understanding of early life on land. However, our knowledge of the 3D structure, and development of these plants is still rudimentary. Here we used digital 3D reconstruction techniques to produce the first complete reconstruction of the lycopsid Asteroxylon mackiei, the most complex plant in the Rhynie chert. The reconstruction reveals the organisation of the three distinct axes types - leafy shoot axes, root-bearing axes and rooting axes - in the body plan. Combining this reconstruction with developmental data from fossilised meristems, we demonstrate that the A. mackiei rooting axis - a transitional lycophyte organ between the rootless ancestral state and true roots - developed from root-bearing axes by anisotomous dichotomy. Our discovery demonstrates how this unique organ developed, and highlights the value of evidence-based reconstructions for understanding the development and evolution of the first complex plants on Earth.

paleontology↗

bioRxiv2021

Extensive N4 cytosine methylation is essential for Marchantia sperm function

4-methylcytosine (4mC) is an important DNA modification in prokaryotes, but its relevance, and even presence in eukaryotes have been mysterious. Here we show that spermatogenesis in the liverwort Marchantia polymorpha involves two waves of extensive DNA methylation reprogramming. First, 5-methylcytosine (5mC), a well-known eukaryotic DNA modification, expands from transposons to the entire genome. Notably, the second wave installs 4mC throughout genic regions, covering over 50% of CG sites in sperm. 4mC is catalyzed by two novel methyltransferases (MpDN4MT1a and MpDN4MT1b) specifically expressed during late spermiogenesis. Deletion of MpDN4MT1s eliminates 4mC, alters the sperm transcriptome, and produces sperm with swimming defects. Our results reveal extensive 4mC in a eukaryote and define a new family of eukaryotic methyltransferases, thereby expanding the repertoire of functional eukaryotic DNA modifications.

developmental biology↗

bioRxiv2019

Gene expression data support the hypothesis that Isoetes rootlets are true roots and not modified leaves

Rhizomorphic lycopsids are the land plant group that includes the first giant trees to grow on Earth and extant species in the genus Isoetes. Two mutually exclusive hypotheses account for the evolution of terminal rooting axes called rootlets among the rhizomorphic lycopsids. One hypothesis states that rootlets are true roots, like roots in other lycopsids. The other states that rootlets are modified leaves. Here we test predictions of each hypothesis by investigating gene expression in the leaves and rootlets of Isoetes echinospora. We assembled the de-novo transcriptome of axenically cultured I. echinospora. Gene expression signatures of I. echinospora rootlets and leaves were different. Furthermore, gene expression signatures of I. echinospora rootlets were similar to gene expression signatures of true roots of Selaginella moellendorffii and Arabidopsis thaliana. RSL genes which positively regulate cell differentiation in roots were either exclusively or preferentially expressed in the I. echinospora rootlets, S. moellendorffii roots and A. thaliana roots compared to the leaves of each respective species. Taken together, gene expression data from the de-novo transcriptome of I. echinospora are consistent with the hypothesis that Isoetes rootlets are true roots and not modified leaves.

plant biology↗

bioRxiv2019

Independent evolution of lateral inhibition mechanisms in different lineages of land plants: MpFEW RHIZOIDS1 miRNA-mediated lateral inhibition controls rhizoid cell patterning in Marchantia polymorpha

Lateral inhibition patterns differentiated cells during development in bacteria, metazoans and land plants. Tip-growing rhizoid cells develop among flat epidermal cells in the epidermis of the early diverging land plant Marchantia polymorpha. We show that the majority of rhizoid cells develop individually but some develop in linear, one-dimensional clusters of between two and seven rhizoid cells in wild type plants. The distribution of rhizoid cells can be accounted for within a simple model of lateral inhibition. The model also predicted that, in the absence of lateral inhibition, rhizoid cell clusters would be two-dimensional with larger clusters than those formed with lateral inhibition. Rhizoid differentiation in Marchantia polymorpha is positively regulated by the ROOT HAIR DEFECTIVE SIX-LIKE1 (MpRSL1) basic Helix Loop Helix (bHLH) transcription factor which is directly repressed by the FEW RHIZOIDS1 (MpFRH1) miRNA. To test if MpFRH1 miRNA acts during lateral inhibition we generated loss-of-function mutants that did not produce the MpFRH1 miRNA. Two-dimensional clusters of rhizoids develop in Mpfrh1loss-of-function (lof) mutants as predicted by the model for plants that lack lateral inhibition. Furthermore, clusters of up to nine rhizoid cells developed in the Mpfrh1lof mutants compared to a maximum number of seven observed in wild type. The higher steady state levels of MpRSL1 mRNA in Mpfrh1lof mutants indicate that MpFRH1-mediated lateral inhibition involves the repression of MpRSL1 activity. Together the modelling and genetic data indicate that the pattern of cell differentiation in the M. polymorpha epidermis is consistent with a lateral inhibition process in which MpFRH1 miRNA represses MpRSL1. This discovery suggests that novel mechanisms of lateral inhibition may operate in different lineages of land plants, unlike metazoans where the conserved Delta-Notch signaling system controls lateral inhibition in diverse metazoan lineages.

developmental biology↗

bioRxiv2019

An Evolutionarily Conserved Receptor-like Kinases Signaling Module Controls Cell Wall Integrity During Tip-Growth

Rooting cells and pollen tubes - key adaptative innovations that evolved during the colonization and subsequent radiation of plants on land - expand by tip-growth. Tip-growth relies on a tight coordination between the protoplast growth and the synthesis/remodeling of the external cell wall. In root hairs and pollen tubes of the seed plant Arabidopsis thaliana, cell wall integrity (CWI) mechanisms monitor this coordination through the Malectin-like receptor kinases (MLRs) such as AtANXUR1 and AtFERONIA that act upstream of the AtMARIS PTI1-like kinase. Here, we show that rhizoid growth in the early diverging plant, Marchantia polymorpha, is also controlled by an MLR and PTI1-like signaling module. Rhizoids, root hairs and pollen tubes respond similarly to disruption of MLR and PTI1-like encoding genes. Thus, the MLR/PTI1-like signaling module that controls CWI during tip-growth is conserved between M. polymorpha and A. thaliana suggesting it was active in the common ancestor of land plants.

plant biology↗

bioRxiv2019

Lateral organ diversification in plants mediated by the ALOG protein family of transcription factors

Land plant shoot structures evolved a diversity of lateral organs as morphological adaptations to the terrestrial environment, in which lateral organs independently evolved in each lineage in the sporophyte or gametophyte generation. The gametophyte meristem of the basally-diverging plant Marchantia polymorpha produces axes with non-photosynthetic scale-like lateral organs instead of leaves. Here we report that an ALOG (Arabidopsis LSH1 and Oryza G1) family protein in Marchantia, MpTAWAWA1 (MpTAW1), regulates meristem maintenance and lateral organ development. A mutation in MpTAW1, preferentially expressed in lateral organs, induces lateral organs with mis-specified identity and increased cell number, and furthermore, causes defects in apical meristem maintenance. Remarkably, MpTAW1 expression rescued the elongated-spikelet phenotype of a rice mutant of MpTAW1 homologue. This suggests that ALOG genes are co-opted to specify lateral organ identities in both gametophyte and sporophyte shoots by repressing lateral organ growth. We propose that the recruitment of ALOG-mediated lateral organ modification was in part responsible for the convergent evolution of independently-evolved lateral organs among highly divergent plant lineages and contributed to the morphological diversification of land plants.

plant biology↗

bioRxiv2019

Neofunctionalisation of basic helix loop helix proteins occurred when plants colonised the land

ROOT HAIR DEFECTIVE SIX-LIKE (RSL) genes control the development of structures - rhizoids, root hairs, gemmae, mucilage papillae - that develop from single cells at the surface of diverse groups of land plants. RSL proteins constitute a subclass (VIIIc) of the basic helix loop helix (bHLH) class VIII transcription factor family. We set out to determine if the function of RSL genes in the control of cell differentiation in land plants was inherited from streptophyte algal ancestor. The Charophyceae are a monophyletic class of streptophyte algae with tissue-like structures and rhizoids. We identified the single class VIII bHLH gene from the charophyceaen alga Chara braunii (CbbHLHVIII). Phylogenetic analysis suggests that this protein is sister to the RSL (bHLH subclass VIIIc) proteins and together they constitute a monophyletic group. Expression of CbbHLHVIII does not compensate for loss of the RSL function in either Marchantia polymorpha or Arabidopsis thaliana. Furthermore, CbbHLHVIII is expressed at sites of morphogenesis in C. braunii - the apices, nodes and gametangia - but not in rhizoids. This indicates that C. braunii class VIII protein is functionally different from land plant RSL proteins; they control rhizoid development in land plants but not in the charophycean algae. These data are consistent with the hypothesis that RSL proteins and their function in the differentiation of cells at the plant surface evolved in the lineage leading to land plants after the divergence of the land plants and C. braunii from their last common ancestor. This may have occurred by neofunctionalisation at or before the colonisation of the land by streptophytes.

evolutionary biology↗