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Engineering a Reverse C4 Photosynthetic Pathway in C3 species Arabidopsis thaliana

Sun, 17/07/2022 - 16:34
Engineering a Reverse C4 Photosynthetic Pathway in C3 species Arabidopsis thaliana Di, Zhengao C4 photosynthesis is a specialised carbon concentrating mechanism that achieves higher rates of photosynthesis. Engineering C4 photosynthesis into C3 plants could improve agronomic features of staple C3 crops. Introducing the biochemistry of C4 photosynthesis is considered a key step in engineering the C3-to-C4 transition. Herein, a reverse C4 photosynthesis system that aimed to install C4 biochemistry in the opposite cell types from those used by C4 species was investigated. This approach was hypothesised to replicate the spatial patterning of the C4 biochemical pathway but make use of the native leaf anatomy of C3 species. The main objective of this thesis was to test if introducing a reverse C4 photosynthetic pathway in C3 Arabidopsis thaliana could improve its photosynthetic efficiency. Two subtypes of the C4 biochemical pathway, the PEPCK and NAD-ME subtypes were tested. A. thaliana genes most suitable for assembling these subtypes were selected based on homology to C4 genes and relative transcript abundance. Transgenic plants carrying a reverse PEPCK C4 pathway displayed no differences in carbon isotope composition or Photosystem II activity, and in a genetic background with less RuBisCO showed lower CO2 assimilation. Genes for a reverse NAD-ME C4 pathway were assembled into two large multigene constructs containing 9 and 10 transcription units. Homozygous lines carrying each construct were obtained and showed overexpression of most transgenes. Although plants carrying bundle sheath genes of the pathway showed no visible phenotypes and those carrying mesophyll genes showed retarded growth, the latter phenotype seemed to be alleviated in F1 crosses containing genes encoding a full reverse NAD-ME C4 pathway. Work is also reported that aimed to explore two alternative approaches to accelerate the main objective of C4 engineering. First, clustering enzymes of a metabolic pathway via protein scaffold was tested. Three enzymes for the biosynthesis of betalain were transiently expressed in tobacco in the presence of scaffold proteins of various structures. However, betalain production was inhibited. Varying the number of domains on scaffold proteins or attaching them to the vacuole membrane did not generate significant impact on pathway efficiency. Second, a microdroplet-based cell-free protein expression system was built to mimic plant metabolic pathways in vitro. Fluorescent proteins and enzymes could be directly synthesised from DNA templates in microdroplets. Fluorescence corresponding to betalains was observed when part of the pathway was expressed and substrate provided. A potential future application of this system was to mimic multicellular physiological processes such as the C4 biochemical pathway.

Understanding the development, evolution, and function of bullseye pigmentation patterns in Hibiscus trionum

Wed, 29/06/2022 - 10:22
Understanding the development, evolution, and function of bullseye pigmentation patterns in Hibiscus trionum Fairnie, Alice Colourful spot, stripe and ring patterns decorate the corolla of many flowering plants and fulfil important biotic and abiotic functions. These petal patterns are created by spatial differences in pigmentation, cell shape and texture of the adaxial petal epidermis. The mechanisms controlling formation and evolution of these patterns, and their exact role in plant-animal communication are not well understood. My PhD adds to current understanding of petal patterns by investigating the development, evolution, and function of the bullseye pigmentation pattern on Hibiscus trionum petals. The bi-coloured bullseye pattern of H.trionum is created from contrast in cell shape, cuticle texture, and pigmentation between the bottom and top of the petal. The bottom petal appears shiny and purple because cells in this region are elongated with a striated cuticle, and are pigmented with anthocyanins. A minimal regulatory network restricting anthocyanin pigment production to the bottom of Hibiscus trionum petal was identified. The network relies on two MYB regulators: HtCREAM1 which represses anthocyanin production in the top of the petal; HtBERRY1 which promotes anthocyanin biosynthesis in the bottom of the petal. This minimal network is a starting point to understand the molecular mechanisms both creating, and creating diversity, in petal patterns. Natural variation in the bullseye pattern in close relatives of H.trionum from Australia and New Zealand is in part due to four independent restrictions of anthocyanin pigmentation in the petal bottom. Preliminary results suggest restriction of pigmentation in H.richardsonii, sister-species to H.trionum, results from mutation in the regulatory region and the coding sequence of the BERRY1 homolog of H. richardsonii. Natural variation in the bullseye pigmentation pattern could reflect a function in plant-pollinator communication. Buff-tailed bumblebees were found to discriminate between, and prefer, artificial flowers with H.trionum-like bullseye patterns to H.richardsonii-like bullseye patterns.

Fostering Populations of Arbuscular Mycorrhizal Fungi Through Cover Crop Choices and Soil Management

Wed, 15/06/2022 - 10:58
Fostering Populations of Arbuscular Mycorrhizal Fungi Through Cover Crop Choices and Soil Management Crane, George Over 70% of land plants, including many key agricultural crops, form a beneficial, symbiotic relationship with arbuscular mycorrhizal (AM) fungi. This has triggered interest in the potential role of these fungi in sustain-able food production for an increasing population. However, it is known that many common farming practices can negatively influence both the diversity, and abundance of the AM fungi. It is therefore desirable to identify farming practices or amendments that can foster these fungal populations to increase crop and soil benefits, including yield. Cover cropping, the growing of non-food crops outside of regular crop produc-tion for the role of protecting and improving soil, has also been suggest-ed to influence both the diversity and abundance of AM fungi. A large-scale analysis of AM fungal diversity in UK agriculture provided a framework for further analysis of how cover crops, and soil amendments influence AM fungal communities. Replicated trials in both glasshouse and field conditions have shown evidence that multiple iterations of cover crops can increase the extent to which plants are colonised by AM fungi, although this had no measurable impact on yield. In the same trial, it was shown that long term application of nitrogen fertiliser influenced AM fungal community composition, but this observation was not made in a shorter validation experiment conducted at the field scale. In a sepa-rate trial, addition of a commercial AM fungal inoculum had little impact on the AM fungal community, crop growth, or yield in field conditions, further suggesting that multiple iterations of soil amendments are re-quired to cause measurable, long-term shifts in AM fungal diversity and benefit.

Modelling the impact of Phytophthora austrocedri on UK populations of native juniper (Juniperus communis s. l.)

Tue, 24/05/2022 - 18:08
Modelling the impact of Phytophthora austrocedri on UK populations of native juniper (Juniperus communis s. l.) Donald, Flora Introductions of non-native plant pests and pathogens are increasing, negatively impacting natural environments. Mitigation requires knowledge of the drivers of pathogen introduction, establishment and spread, rarely available when pathogens first emerge in novel settings. This thesis uses multi-scale ecological modelling to understand environmental and land management factors driving patterns in infection and impact of the newly discovered oomycete pathogen, Phytophthora austrocedri, on juniper (Juniperus communis). I first surveyed potential abiotic and biotic drivers of disease severity across three, geographically separate juniper populations with different infection histories. In all populations, disease severity increased with increasing soil moisture. Associated plant species that could be used to locate microsites at higher risk of infection were also identified. Change in infection intensity during a four-year period was then mapped across a single juniper population and related to environmental factors underpinning the presence and density of juniper and driving P. austrocedri spread. Colonisations usually occurred within a ~500m radius of previously symptomatic trees, with infrequent dispersal beyond 1km, potentially mediated by livestock and deer. By compiling a novel dataset, I revealed larger, more frequent supplementary juniper planting events increased the likelihood of P. austrocedri presence. Stakeholders managing, monitoring, and growing juniper then participated in a survey investigating how practitioners consider disease risks and whether these processes could be better supported by decision tools. Lastly, a machine learning model and risk map was developed that predicted juniper populations in northern England and central Scotland are at highest risk of infection due to acidic soil pH and increased roe deer density. My research demonstrates how incorporating a wider range of abiotic and biotic drivers, exploring scale dependence, and integrating stakeholder knowledge can improve the predictive accuracy of host-pathogen-environment models. The results are used to recommend strategies (e.g. reductions in grazing pressure, natural juniper regeneration and heightened on-site biosecurity) to mitigate the serious threat posed by the pathogen to UK biodiversity and habitat restoration goals.

Investigating the role of the circadian clock in the physiology of wheat

Sat, 14/05/2022 - 06:07
Investigating the role of the circadian clock in the physiology of wheat Taylor, Laura The circadian clock is an endogenous timing mechanism which acts to synchronise internal processes with daily and seasonal cycles in light and temperature. In Arabidopsis, the components which comprise the circadian oscillator and output pathways are well defined. Photoperiod dependent flowering, metabolism, growth and defence signalling networks receive information regarding the timing of the day/night cycle from the circadian clock. Many circadian output pathways are targets for crop improvement, with variation at circadian loci selected for during domestication and breeding. Investigations into how the circadian clock controls agronomically important traits may identify novel targets for breeders looking to create new elite wheat cultivars. The first aim of this thesis was to identify naturally occurring variation in circadian clock genes across wheat cultivars important in modern breeding efforts. In Arabidopsis, LUX ARRYTHMO (LUX) and EARLY FLOWERING 3 (ELF3) are circadian clock genes which are co- expressed at dusk, forming a transcriptional repressor complex which restricts the expression of day phased circadian clock genes. Deletions, which may result in a null allele, were identified in LUX along with presence absence variation (PAV) for ELF3 across different wheat cultivars. Sequence analysis of ELF3 and LUX promoter regions identified enrichment of motifs associated with morning and evening phased expression respectively, supporting previous studies which indicate that the phasing of LUX and ELF3 expression is not conserved between Arabidopsis and wheat. I present further tools to investigate the diel turnover of LUX and ELF3 proteins. The second aim of this thesis was to study the effect of perturbation to the circadian clock on circadian output pathways. Predicted loss of function of the circadian clock gene GIGANTEA (TtGIab) results in circadian arrythmia in chlorophyll fluorescence parameters and late flowering in long and short day controlled environment conditions. No differences in flowering time were observed in the field. Perturbation to the circadian clock through loss of functional ELF3 (TtELF3ab) and TtGIab resulted in mismanagement of diel carbohydrate turnover. The implications for growth and biomass accumulation are discussed. This thesis demonstrates that the wheat flowering and carbohydrate turnover pathways are controlled by the circadian clock. Utilising variation at circadian loci may provide novel targets for breeders looking to manipulate these systems for improved crop performance.

Research data supporting "Investigating the role of the circadian clock in the physiology of wheat".

Sat, 14/05/2022 - 06:07
Research data supporting "Investigating the role of the circadian clock in the physiology of wheat". Taylor, Laura; Hannah, Matthew Electronic Tables: In Table 1 the circadian clock gene ID's previously determined using the wheat genome assembly TGAC (Wittern, L. M., 2019, are listed followed by updated IWGSC RefSeq v1.1 gene identifiers. Table 2-4 detail wheat orthologs to Arabidopsis circadian clock genes as determined by Plaza v5, Plaza v6 and Ensembl Compara respectively. In Table 2 and 3 the Evidence(s) column refers to the different method used by Plaza to determine if a gene is an ortholog. A 'Y" letter (i.e. YES) indicates that a gene was identified as an ortholog by that particular method. Table 5 outlines the results of analysis using GMAP. The IWGSC RefSeq v1.1 circadian clock gene sequences were mapped to the genome sequences of different wheat cultivars (row 1) and summary statistics were calculated. Sequence alignments: Sequence alignments for circadian clock genes LUX, ELF3, GI and LHY were created between IWGS Refseqv1.1 and wheat cultivars important in modern breeding efforts. Each alignment is preceded by a sequence overview. The height of the pink bar graphically represents the level of conservation between the sequences. For more information see methods section of Chapter 3 of the associated manuscript.

Understanding C4 Photosynthesis Through Quantitative Genetics

Wed, 04/05/2022 - 19:53
Understanding C4 Photosynthesis Through Quantitative Genetics Simpson, Conor Photosynthesis is fundamental to all life on Earth. In terrestrial plants it relies exclusively on RuBisCO to fix atmospheric CO2 into the sugar-generating Calvin-Benson-Bassham cycle. RuBisCO however, binds with oxygen preferentially under favourable conditions which has led to the evolution of carbon concentrating mechanisms such as C4 photosynthesis. While being present in just 3% of global flora, the C4 cycle is responsible for around a quarter of primary productivity. This increased efficiency if engineered into C3 species such as rice would dramatically enhance yield. This objective is challenging as it involves introducing incompletely understood traits into C3 leaves including complex changes to their biochemistry, cell biology and anatomy. Quantitative genetics offers an under-explored route to identify regulators of these processes. Taking advantage of the natural variation present for C4 characteristics in the dicotyledon Gynandropsis gynandra, this thesis aims to carry out linkage mapping and heritability estimates of C4 traits. Additionally, this thesis describes the generation of a Multi-Parental Advanced Generation Inter-Cross population and tools for high-throughput phenotyping of vein density, a trait integral to the evolution of the C4 syndrome. Resources presented in this thesis can be used by the C4 community to compliment knowledge gained through functional genomics so that improvements can be made in our understanding of C4 photosynthesis.