Biology subjects

Patron, N. J.

Publications and source records attributed to Patron, N. J..

8 recordsLinked to original sources

bioRxiv2026

Evolutionary Rewiring of Root Cell-Type Nitrogen Responses

Nitrate induces widespread transcriptional reprogramming in plants, but how transcription factors coordinate these responses across cell types and evolve across species with divergent root patterning remains unclear. Using single-cell transcriptomics in Arabidopsis thaliana and Solanum lycopersicum (tomato) under varying nitrate conditions, we demonstrate that many transcriptional responses are cell type and developmental stage-specific. Arabidopsis NIN-LIKE PROTEIN 7 (NLP7) directly modulates both broadly expressed and cell-type-specific targets, but is not required to establish the cell type specific nature of these patterns. Comparative analysis reveals evolutionary shifts; key nitrogen programs are localized to different cell types in tomato, with the exodermis as a major regulatory hub. Identification of tomato NLP7a and NLP7b direct targets at cellular resolution supports their role in direct modulation of cell type N-transcriptional programs. This is evidenced by NLP7-controlled exodermal cell wall remodeling in low nitrate, highlighting functional conservation despite divergent cellular topographies.

plant biology↗

bioRxiv2025

Biosynthesis and Bioactivity of Anti-Inflammatory Triterpenoids in Calendula officinalis (pot marigold)

Plants have been central to traditional medicine for millennia, yet the precise metabolites responsible for their therapeutic properties often remain unidentified. Moreover, the low natural abundance and structural complexity of bioactive molecules can hinder their accessibility and chemical synthesis. Here, we investigate the widely reported anti-inflammatory properties of Calendula officinalis (pot marigold), an ancient medicinal herb. We confirm C16-hydroxylated triterpenoids as key contributors to the anti-inflammatory activity of C. officinalis floral extracts and uncover a novel mechanism by which they act in modulating interleukin 6 release. Through biosynthetic pathway elucidation, we demonstrate that the oxidosqualene synthase catalysing the first committed step emerged early in Asteraceae evolution and identify residues governing product specificity. Further, we functionally characterise cytochrome P450s and acyltransferases responsible for downstream modifications. By reconstructing the complete biosynthetic pathway in the heterologous plant chassis Nicotiana benthamiana, we provide a platform for production of the anti-inflammatory components. Our work highlights how integrated studies of bioactivity and biosynthesis can unlock the therapeutic potential of medicinal plants.

plant biology↗

bioRxiv2022

Tunable control of insect pheromone biosynthesis in Nicotiana benthamiana

Previous work has demonstrated that plants can be used as production platforms for molecules used in health, medicine, and agriculture. Production has been exemplified in both stable transgenic plants and using transient expression strategies. In particular, species of Nicotiana have been engineered to produce a range of useful molecules, including insect sex pheromones, which are valued for species-specific control of agricultural pests. To date, most studies have relied on strong constitutive expression of all pathway genes. However, work in microbes has demonstrated that yields can be improved by controlling and balancing gene expression. Synthetic regulatory elements that provide control over the timing and levels of gene expression are therefore useful for maximizing yields from heterologous biosynthetic pathways. In this study, we demonstrate the use of pathway engineering and synthetic genetic elements for controlling the timing and levels of production of Lepidopteran sex pheromones in Nicotiana benthamiana. We demonstrate that copper can be used as a low-cost molecule for tightly regulated inducible expression. Further, we show how construct architecture influences relative gene expression and, consequently, product yields in multigene constructs. We compare a number of synthetic orthogonal regulatory elements and demonstrate maximal yields from constructs in which expression is mediated by dCas9-based synthetic transcriptional activators. The approaches demonstrated here provide new insights into the heterologous reconstruction of metabolic pathways in plants.

synthetic biology↗

bioRxiv2021

Reconstitution of monoterpene indole alkaloid biosynthesis in Nicotiana benthamiana

Monoterpene indole alkaloids (MIAs) are a diverse class of plant natural products that include a number of medicinally significant compounds. We set out to reconstitute the pathway for strictosidine, a key intermediate of all MIAs, from central metabolism in Nicotiana benthamiana. A disadvantage of this host is that its rich background metabolism results in the derivatization of some heterologously produced molecules. We used transcriptomic analysis to identify glycosyltransferases that were upregulated in response to biosynthetic intermediates and produced plant lines with targeted mutations in the genes encoding them. Expression of the early MIA pathway in these lines produced a more favorable product profile. Strictosidine biosynthesis was successfully reconstituted, with the best yields obtained by the co-expression of 14 enzymes, of which a major latex protein-like enzyme (MLPL) from Nepeta (catmint) was critical for improving flux through the iridoid pathway. The removal of endogenous glycosyltransferases did not impact the yields of strictosidine, highlighting that the metabolic flux of the pathway enzymes to a stable biosynthetic intermediate minimizes the need to engineer the endogenous metabolism of the host. The production of strictosidine in planta expands the range of MIA products amenable to biological synthesis.

synthetic biology↗

bioRxiv2021

Biofoundry-assisted expression and characterisation of plant proteins

Many goals in synthetic biology, including the elucidation and refactoring of biosynthetic pathways and the engineering of regulatory circuits and networks, require knowledge of protein function. In plants, the prevalence of large gene families means it can be particularly challenging to link specific functions to individual proteins. However, protein characterisation has remained a technical bottleneck, often requiring significant effort to optimise expression and purification protocols. To leverage the ability of biofoundries to accelerate design-built-test-learn cycles, we present a workflow for automated DNA assembly and cell-free expression of plant proteins that accelerates optimisation and enables rapid progression to characterisation. First, we developed a phytobrick-compatible Golden Gate DNA assembly toolbox containing plasmid acceptors for cell-free expression using E. coli or wheat germ lysates as well as a set of N- and C-terminal tag parts for detection, purification, and improved expression/folding. We next optimised automated assembly of miniaturised cell-free reactions using an acoustic liquid handling platform and then compared tag configurations to identify those that increase expression. We additionally developed a luciferase-based system for rapid quantification that requires a minimal 11 aa tag and demonstrate facile removal of tags following synthesis. Finally, we show that several functional characterisation experiments can be performed with cell-free protein synthesis reactions without the need for protein purification. Together, the combination of automated assembly of DNA parts and cell-free expression reactions should significantly increase the throughput of experiments to test and understand plant protein function and enable the direct reuse of DNA parts in downstream plant engineering workflows.

synthetic biology↗

bioRxiv2020

Rational Design of Minimal Synthetic Promoters for Plants

ABSTRACTPromoters serve a critical role in establishing baseline transcriptional capacity through the recruitment of proteins, including transcription factors (TFs). Previously, a paucity of data for cis-regulatory elements in plants meant that it was challenging to determine which sequence elements in plant promoter sequences contributed to transcriptional function. In this study, we have identified functional elements in the promoters of plant genes and plant pathogens that utilise plant transcriptional machinery for gene expression. We have established a quantitative experimental system to investigate transcriptional function, investigating how identity, density and position contribute to regulatory function. We then identified permissive architectures for minimal synthetic plant promoters enabling computational design of a suite of synthetic promoters of different strengths. These have been used to regulate the relative expression of output genes in simple genetic devices. Competing Interest StatementThe authors have declared no competing interest.View Full Text

synthetic 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↗

bioRxiv2018

Comparison of efficiency and specificity of CRISPR-associated (Cas) nucleases in plants: An expanded toolkit for precision genome engineering

Molecular tools adapted from bacterial CRISPR (Clustered Regulatory Interspaced Short Palindromic Repeats) systems for adaptive immunity have become widely used for plant genome engineering, both to investigate gene functions and to engineer desirable traits. A number of different Cas (CRISPR-associated) nucleases are now used but, as most studies performed to date have engineered different targets using a variety of plant species and molecular tools, it has been difficult to draw conclusions about the comparative performance of different nucleases. Due to the time and effort required to regenerate engineered plants, efficiency is critical. In addition, there have been several reports of mutations at sequences with less than perfect identity to the target. While in some plant species it is possible to remove these so-called off-targets by backcrossing to a parental line, the specificity of genome engineering tools is important when targeting specific members of closely-related gene families, especially when recent paralogues are co-located in the genome and unlikely to segregate. Specificity is also important for species that take years to reach sexual maturity or that are clonally propagated. Here, we directly compare the efficiency and specificity of Cas nucleases from different bacterial species together with engineered variants of Cas9. We find that the nucleotide content correlates with efficiency and that Cas9 from Staphylococcus aureus is comparatively most efficient at inducing mutations. We also demonstrate that high-fidelity variants of Cas9 can reduce off-target mutations in plants. We present these molecular tools as standardised DNA parts to facilitate their re-use.

plant biology↗