Biology subjects

Markel, K.

Publications and source records attributed to Markel, K..

9 recordsLinked to original sources

bioRxiv2024

Predictable modulation of plant root development using engineered cytokinin regulators

Achieving precise control over quantitative developmental phenotypes is a key objective in plant biology. Recent advances in synthetic biology have enabled tools to reprogram entire developmental pathways; however, the complexity of designing synthetic genetic programs and the inherent interactions between various signaling processes remains a critical challenge. Here, we leverage Type-B response regulators to modulate the expression of genes involved in cytokinin-dependent growth and development processes. We rationally engineered these regulators to modulate their transcriptional activity (i.e., repression or activation) and potency while reducing their sensitivity to cytokinin. By localizing the expression of these engineered transcription factors using tissue-specific promoters, we can predictably tune cytokinin-regulated traits. As a proof of principle, we deployed this synthetic system in Arabidopsis thaliana to either decrease or increase the number of lateral roots. The simplicity and modularity of our approach makes it an ideal system for controlling other developmental phenotypes of agronomic interest in plants.

plant biology↗

bioRxiv2024

Expression of a mammalian RNA demethylase increases flower number and floral stem branching in Arabidopsis thaliana

RNA methylation plays a central regulatory role in plant biology and is a relatively new target for plant improvement efforts. In nearly all cases, perturbation of the RNA methylation machinery results in deleterious phenotypes. However, a recent landmark paper reported that transcriptome-wide use of the human RNA demethylase FTO substantially increased the yield of rice and potatoes. Here, we have performed the first independent replication of those results and broader transferability of the trait, demonstrating increased flower and fruit count in the model species Arabidopsis thaliana. We also performed RNA-seq of our FTO-transgenic plants, which we analyzed in conjunction with previously-published datasets to detect several previously-recognized patterns in the functional and structural classification of the upregulated and downregulated genes. From these, we present mechanistic hypotheses to explain these surprising results with the goal of spurring more widespread interest in this promising new approach to plant engineering.

plant biology↗

bioRxiv2023

Genetically refactored Agrobacterium-mediated transformation

Members of Agrobacterium are costly plant pathogens while also essential tools for plant transformation. Though Agrobacterium-mediated transformation (AMT) has been heavily studied, its polygenic nature and its complex transcriptional regulation make identifying the genetic basis of transformational efficiency difficult through traditional genetic and bioinformatic approaches. Here we use a bottom-up synthetic approach to systematically refactor the tumor-inducing plasmid, wherein the majority of AMT machine components are encoded, into a minimal set of genes capable of plant and fungal transformation that is both controllable and orthogonal to its environment. We demonstrate that engineered vectors can be transferred to new heterologous bacteria, enabling them to transform plants. Our reductionist approach demonstrates how bottom-up engineering can be used to dissect and elucidate the genetic underpinnings of complex biological traits, and may lead to the development of strains of bacteria more capable of transforming recalcitrant plant species of societal importance.

microbiology↗

bioRxiv2023

Systematic identification of transcriptional activator domains from non-transcription factor proteins in plants and yeast

Transcription factors promote gene expression via trans-regulatory activation domains. Although whole genome scale screens in model organisms (e.g. human, yeast, fly) have helped identify activation domains from transcription factors, such screens have been less extensively used to explore the occurrence of activation domains in non-transcription factor proteins, such as transcriptional coactivators, chromatin regulators and some cytosolic proteins, leaving a blind spot on what role activation domains in these proteins could play in regulating transcription. We utilized the activation domain predictor PADDLE to mine the entire proteomes of two model eukaryotes, Arabidopsis thaliana and Saccharomyces cerevisiae (1). We characterized 18,000 fragments covering predicted activation domains from >800 non-transcription factor genes in both species, and experimentally validated that 89% of proteins contained fragments capable of activating transcription in yeast. Peptides with similar sequence composition show a broad range of activities, which is explained by the arrangement of key amino acids. We also annotated hundreds of nuclear proteins with activation domains as putative coactivators; many of which have never been ascribed any function in plants. Furthermore, our library contains >250 non-nuclear proteins containing peptides with activation domain function across both eukaryotic lineages, suggesting that there are unknown biological roles of these peptides beyond transcription. Finally, we identify and validate short, universal eukaryotic activation domains that activate transcription in both yeast and plants with comparable or stronger performance to state-of-the-art activation domains. Overall, our dual host screen provides a blueprint on how to systematically discover novel genetic parts for synthetic biology that function across a wide diversity of eukaryotes. Significance StatementActivation domains promote transcription and play a critical role in regulating gene expression. Although the mapping of activation domains from transcription factors has been carried out in previous genome-wide screens, their occurrence in non-transcription factors has been less explored. We utilize an activation domain predictor to mine the entire proteomes of Arabidopsis thaliana and Saccharomyces cerevisiae for new activation domains on non-transcription factor proteins. We validate peptides derived from >750 non-transcription factor proteins capable of activating transcription, discovering many potentially new coactivators in plants. Importantly, we identify novel genetic parts that can function across both species, representing unique synthetic biology tools.

genetics↗

bioRxiv2023

Cynipid wasps systematically reprogram host metabolism and restructure cell walls in developing galls

Many insects have evolved the ability to manipulate plant growth to generate extraordinary structures called galls in which insect larva can develop while being sheltered within and feeding on the plant. In particular, Cynipid (Hymenoptera: Cynipidae) wasps have evolved to form some of the most morphologically complex galls known and generate an astonishing array of gall shapes, colors, and sizes. However, the biochemical basis underlying these remarkable cellular and developmental transformations remains poorly understood. A key determinant in plant cellular development is the deposition of the cell wall to dictate the physical form and physiological function of newly developing cells, tissues, and organs. However, it is unclear to what degree cell walls are restructured to initiate and support the formation of new gall tissue. Here, we characterize the molecular alterations underlying gall development using a combination of metabolomic, histological, and biochemical techniques to elucidate how leaf cells are reprogrammed to form galls. Strikingly, gall development involves an exceptionally coordinated spatial deposition of lignin and xylan to form de novo gall vasculature. Our results highlight how Cynipid wasps can radically change the metabolite profile and restructure the cell wall to enable the formation of galls, providing new insights into the mechanism of gall induction and the extent to which plants can be entirely reprogrammed to form novel structures and organs.

plant biology↗

bioRxiv2022

The trans-regulatory landscape of gene networks in plants

The effector domains of transcription factors play a key role in controlling gene expression; however, their functional nature is poorly understood, hampering our ability to explore this fundamental dimension of gene regulatory networks. To map the trans-regulatory landscape in a complex eukaryote, we systematically characterized the putative effector domains of over 400 Arabidopsis thaliana transcription factors for their capacity to modulate transcription. We demonstrate that transcriptional effector activity can be integrated into gene regulatory networks capable of elucidating the functional dynamics underlying gene expression patterns. We further show how newly characterized domains can enhance genome engineering efforts and reveal how plant transcriptional activators share regulatory features conserved across distantly related eukaryotes. Our results provide a framework to systematically characterize the regulatory role of transcription factors at a genome-scale in order to understand the transcriptional wiring of biological systems.

plant biology↗

bioRxiv2020

Comparative analysis of early divergent land plants and construction of DNA tools for hyper-expression in Marchantia chloroplasts

Chloroplast genes are present at high ploidy in plants, and capable of driving very high levels of gene expression if mRNA production and stability are properly regulated. Marchantia polymorpha is a simple model plant that allows rapid transformation studies, however post-transcriptional regulation in plastids is poorly characterized in this liverwort. We have mapped patterns of transcription in Marchantia chloroplasts. Furthermore, we have obtained and compared sequences from 51 early-divergent plant species, and identified putative sites for pentatricopeptide repeat protein binding that are thought to play important roles in mRNA stabilisation. Candidate binding sites were tested for their ability to confer high levels of reporter gene expression in Marchantia chloroplasts, and levels of protein production and effects on growth were measured in homoplasmic transformed plants. We have produced novel DNA tools for protein hyper-expression in a facile plant system that is a test-bed for chloroplast engineering.

plant biology↗

bioRxiv2020

Pavlov's pea plants? Not so fast. An attempted replication of Gagliano et al. (2016)

Gagliano et al. (Learning by association in plants, 2016) reported associative learning in pea plants. Associative learning has long been considered a behavior performed only by animals, making this claim particularly newsworthy and interesting. In the experiment, plants were trained in Y-shaped mazes for three days with fans and lights attached at the top of the maze. Training consisted of wind consistently preceding light from either the same or the opposite arm of the maze. When plant growth forced a decision between the two arms of the maze, fans alone were able to influence growth direction, whereas the growth direction of untrained plants was not affected by fans. Importantly, some plants were trained to grow towards the fan and others to grow away, demonstrating the flexibility of associative learning. However, a replication of their protocol failed to demonstrate the same result, calling for further verification and study before mainstream acceptance of this paradigm-shifting phenomenon. This replication attempt used a larger sample size and fully blinded analysis.

plant biology↗

bioRxiv2020

Systematic tools for reprogramming plant gene expression in a simple model, Marchantia polymorpha.

We present the OpenPlant toolkit, a set of interlinked resources and techniques to develop Marchantia as testbed for bioengineering in plants. Marchantia is a liverwort, a simple plant with an open form of development that allows direct visualization of gene expression and dynamics of cellular growth in living tissues. We describe new techniques for simple and efficient axenic propagation and maintenance of Marchantia lines with no requirement for glasshouse facilities. Marchantia plants spontaneously produce clonal propagules within a few weeks of regeneration, and lines can be amplified million-fold in a single generation by induction of the sexual phase of growth, crossing and harvesting of progeny spores. The plant has a simple morphology and genome with reduced gene redundancy, and the dominant phase of its life cycle is haploid, making genetic analysis easier. We have built robust Loop assembly vector systems for nuclear and chloroplast transformation and genome editing. These have provided the basis for building and testing a modular library of standardized DNA elements with highly desirable properties. We have screened transcriptomic data to identify a range of candidate genes, extracted putative promoter sequences, and tested them in vivo to identify new constitutive promoter elements. The resources have been combined into a toolkit for plant bioengineering that is accessible for laboratories without access to traditional facilities for plant biology research. The toolkit is being made available under the terms of the OpenMTA and will facilitate the establishment of common standards and the use of this simple plant as testbed for synthetic biology.

synthetic biology↗