Faculty Bibliography

Land plants comprise two large monophyletic lineages, the vascular plants and the bryophytes, which diverged from their most recent common ancestor approximately 480 million years ago. Of the three lineages of bryophytes, only the mosses and the liverworts are systematically investigated, while the hornworts are understudied. Despite their importance for understanding fundamental questions of land plant evolution, they only recently became amenable to experimental investigation, with Anthoceros agrestis being developed as a hornwort model system. Availability of a high-quality genome assembly and a recently developed genetic transformation technique makes A. agrestis an attractive model species for hornworts. Here we describe an updated and optimised transformation protocol for A. agrestis which can be successfully used to genetically modify one more strain of A. agrestis and three more hornwort species, Anthoceros punctatus, Leiosporoceros dussi and Phaeoceros carolinianus. The new transformation method is less laborious, faster and results in the generation of greatly increased numbers of transformants compared to the previous method. We have also developed a new selection marker for transformation. Finally, we report the development of a set of different cellular localisation signal peptides for hornworts providing new tools to better understand hornwort cell biology.

The rising population and the ongoing climate crisis call for improved means to monitor and optimise agriculture. A promising approach to tackle current challenges in food production is the early diagnosis of plant diseases through non-invasive methods, such as the detection of volatiles. However, current devices for detection of multiple volatiles are based on electronic noses, which are expensive, require complex circuit assembly, may involve metal oxides with heating elements, and cannot easily be adapted for some applications that require miniaturisation or limit front-end use of electronic components. To address these challenges, a low-cost optoelectronic nose using chemo-responsive colorimetric dyes drop-casted onto filter paper has been developed in the current work. The final sensors could be used for the quantitative detection of up to six plant volatiles through changes in colour intensities with a sub-ppm level limit of detection, one of the lowest limits of detection reported so far using colorimetric gas sensors. Sensor colouration could be analysed using a low-cost spectrometer and the results could be processed using a microcontroller. The measured volatiles could be used for the early detection of plant abiotic stress as early as two days after exposure to two different stresses: high salinity and starvation. This approach allowed a lowering of costs to GBP 1 per diagnostic sensing paper. Furthermore, the small size of the paper sensors allows for their use in confined settings, such as Petri dishes. This detection of abiotic stress could be easily achieved by exposing the devices to living plants for 1 h. This technology has the potential to be used for monitoring of plant development in field applications, early recognition of stress, implementation of preventative measures, and mitigation of harvest losses.

The direct quantification of plant biomarkers in sap is crucial to enhancing crop production. However, current approaches are inaccurate, involving the measurement of non-specific parameters such as colour intensity of leaves, or requiring highly invasive processes for the extraction of sap. In addition, these methods rely on bulky and expensive equipment, and they are time-consuming. The present work reports for the first time a low-cost sensing device that can be used for the simultaneous determination of sap K+ and pH in living plants by means of reverse iontophoresis. A screen-printed electrode was modified by deposition of a K+-selective membrane, achieving a super-Nernstian sensitivity of 70 mV Log[K+]−1 and a limit of detection within the micromolar level. In addition, the cathode material of the reverse iontophoresis device was modified by electrodeposition of RuOx particles. This electrode could be used for the direct extraction of ions from plant leaves and the amperometric determination of pH within the physiological range (pH 3−8), triggered by the selective reaction of RuOx with H+. A portable and low-cost (<£60) microcontroller-based device was additionally designed to enable its use in low-resource settings. The applicability of this system was demonstrated by measuring the changes in concentration of K+ and pH in tomato plants before and after watering with deionised water. These results represent a step forward in the design of affordable and non-invasive devices for the monitoring of key biomarkers in plants, with a plethora of applications in smart farming and precision agriculture among others.

The development of sensing devices for precision agriculture is crucial to boost crop yields and limit shortages in food productions due to the growing population. However, current approaches cannot provide direct information about the physiological status of the plants, reducing sensing accuracy. The development of implanted devices for plant monitoring represents a step forward in this field, enabling the direct assessment of key biomarkers in plants. However, available devices are expensive and cannot be used for long-term applications. The current work presents the application of ruthenium oxide-based nanofilms for the in vivo monitoring of pH in plants. The sensors were manufactured using the low-cost electrodeposition of RuO2 films, and the final device could be successfully incorporated for the monitoring of xylem sap pH for at least 10 h. RuO2 nanoparticles were chosen as the sensing material due to its biocompatibility and chemical stability. To reduce the noise rates and drift of the sensors, a protective layer consisting of a cellulose/PDMS hybrid material was deposited by an aerosol method (>GBP 50), involving off-the-shelf devices, leading to a good control of film thickness. Nanometrically thin films with a thickness of 80 nm and roughness below 3 nm were fabricated. This film led to a seven-fold decrease in drift while preserving the selectivity of the sensors towards H+ ions. The sensing devices were tested in vivo by implantation inside a tomato plant. Environmental parameters such as humidity and temperature were additionally monitored using a low-cost Wio Terminal device, and the data were sent wirelessly to an online server. The interactions between plant tissues and metal oxide-based sensors were finally studied, evidencing the formation of a lignified layer between the sensing film and xylem. Thus, this work reports for the first time a low-cost electrochemical sensor that can be used for the continuous monitoring of pH in xylem sap. This device can be easily modified to improve the long-term performance when implanted inside plant tissues, representing a step forward in the development of precision agriculture technologies.

The role of root phenes in nitrogen (N) acquisition and biomass production was evaluated in 10 contrasting natural accessions of Arabidopsis thaliana L. Seedlings were grown on vertical agar plates with two different nitrate supplies. The low N treatment increased the root to shoot biomass ratio and promoted the proliferation of lateral roots and root hairs. The cost of a larger root system did not impact shoot biomass. Greater biomass production could be achieved through increased root length or through specific root hair characteristics. A greater number of root hairs may provide a low-resistance pathway under elevated N conditions, while root hair length may enhance root zone exploration under low N conditions. The variability of N uptake and the expression levels of genes encoding nitrate transporters were measured. A positive correlation was found between root system size and high-affinity nitrate uptake, emphasizing the benefits of an exploratory root organ in N acquisition. The expression levels of NRT1.2/NPF4.6, NRT2.2, and NRT1.5/NPF7.3 negatively correlated with some root morphological traits. Such basic knowledge in Arabidopsis demonstrates the importance of root phenes to improve N acquisition and paves the way to design eudicot ideotypes.

Cell-free systems for gene expression have gained attention as platforms for the facile study of genetic circuits and as highly effective tools for teaching. Despite recent progress, the technology remains inaccessible for many in low- and middle-income countries due to the expensive reagents required for its manufacturing, as well as specialized equipment required for distribution and storage. To address these challenges, we deconstructed processes required for cell-free mixture preparation and developed a set of alternative low-cost strategies for easy production and sharing of extracts. First, we explored the stability of cell-free reactions dried through a low-cost device based on silica beads, as an alternative to commercial automated freeze dryers. Second, we report the positive effect of lactose as an additive for increasing protein synthesis in maltodextrin-based cell-free reactions using either circular or linear DNA templates. The modifications were used to produce active amounts of two high-value reagents: the isothermal polymerase Bst and the restriction enzyme BsaI. Third, we demonstrated the endogenous regeneration of nucleoside triphosphates and synthesis of pyruvate in cell-free systems (CFSs) based on phosphoenol pyruvate (PEP) and maltodextrin (MDX). We exploited this novel finding to demonstrate the use of a cell-free mixture completely free of any exogenous nucleotide triphosphates (NTPs) to generate high yields of sfGFP expression. Together, these modifications can produce desiccated extracts that are 203-424-fold cheaper than commercial versions. These improvements will facilitate wider use of CFS for research and education purposes.

Chloroplasts are attractive platforms for synthetic biology applications since they are capable of driving very high levels of transgene expression, if mRNA production and stability are properly regulated. However, plastid transformation is a slow process and currently limited to a few plant species. The liverwort Marchantia polymorpha is a simple model plant that allows rapid transformation studies; however, its potential for protein hyperexpression has not been fully exploited. This is partially due to the fact that chloroplast post-transcriptional regulation is poorly characterized in this plant. We have mapped patterns of transcription in Marchantia chloroplasts. Furthermore, we have obtained and compared sequences from 51 bryophyte species and identified putative sites for pentatricopeptide repeat protein binding that are thought to play important roles in mRNA stabilization. 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 homoplastic transformed plants. We have produced novel DNA tools for protein hyperexpression in this facile plant system that is a test-bed for chloroplast engineering.

The bryophyte Marchantia polymorpha , has attracted significant attention as a powerful experimental system for studying aspects of plant biology including synthetic biology applications. We describe an efficient and simple recursive Type IIS DNA assembly method for the generation of DNA constructs for chloroplast genome manipulation, and an optimized technique for Marchantia chloroplast genome transformation. The utility of the system was demonstrated by the expression of a chloroplast codon-optimized cyan fluorescent protein.

Cell-free gene expression systems have emerged as a promising platform for field-deployed biosensing and diagnostics. When combined with programmable toehold switch-based RNA sensors, these systems can be used to detect arbitrary RNAs and freeze-dried for room temperature transport to the point-of-need. These sensors, however, have been mainly implemented using reconstituted PURE cell-free protein expression systems that are difficult to source in the Global South due to their high commercial cost and cold-chain shipping requirements. Based on preliminary demonstrations of toehold sensors working on lysates, we describe the fast prototyping of RNA toehold switch-based sensors that can be produced locally and reduce the cost of sensors by two orders of magnitude. We demonstrate that these in-house cell lysates provide sensor performance comparable to commercial PURE cell-free systems. We further optimize these lysates with a CRISPRi strategy to enhance the stability of linear DNAs by knocking-down genes responsible for linear DNA degradation. This enables the direct use of PCR products for fast screening of new designs. As a proof-of-concept, we develop novel toehold sensors for the plant pathogen Potato Virus Y (PVY), which dramatically reduces the yield of this important staple crop. The local implementation of low-cost cell-free toehold sensors could enable biosensing capacity at the regional level and lead to more decentralized models for global surveillance of infectious disease.

During development, cells gain positional information through the interpretation of dynamic morphogen gradients. A proposed mechanism for interpreting opposing morphogen gradients is mutual inhibition of downstream transcription factors, but isolating the role of this specific motif within a natural network remains a challenge. Here, we engineer a synthetic morphogen-induced mutual inhibition circuit in E. coli populations and show that mutual inhibition alone is sufficient to produce stable domains of gene expression in response to dynamic morphogen gradients, provided the spatial average of the morphogens falls within the region of bistability at the single cell level. When we add sender devices, the resulting patterning circuit produces theoretically predicted self-organised gene expression domains in response to a single gradient. We develop computational models of our synthetic circuits parameterised to timecourse fluorescence data, providing both a theoretical and experimental framework for engineering morphogen-induced spatial patterning in cell populations.