The influence of OsAUX1 on root system architecture and phosphorus uptake in rice (Oryza sativa)

Rice (Oryza sativa L.) provides up to 50% of the total calories consumed in countries such as India, Madagascar and Nigeria. As a crop, rice can require significant fertiliser inputs to maintain the required yields. Additionally, climate change has increased the need for rice varieties with improved drought resistance, tolerance to pests and more efficient acquisition of nutrients from soil. One major fertiliser input for rice is phosphate; reducing phosphorus (P) fertiliser use would have environmental and economic implications. Root traits linked to P acquisition in crops include shallow root angle, lateral root proliferation and increases in root hair length and density. Two T-DNA knockout alleles with reduced gravitropic response, Osaux1-1 and Osaux1-2, were used to investigate the influence of shallow root angle on P uptake. OsAUX1 is a rice ortholog for the Arabidopsis thaliana gene AUX1, which controls lateral root growth and gravitropic response. The wildtype and mutant rice plants were grown in soil and non-destructively imaged using X-ray micro Computed Tomography (X-ray CT). In Chapter Three, visualisation of rice roots in soil using X-ray CT was optimised by determining the ideal soil moisture content that would produce the best images. Water in soils has a similar X- ray attenuation density to that of plant roots and can influence segmentation of roots from soil in X-ray CT images. It was found that soil at nominal field capacity (ca. 3 days of drainage) produced the best contrast between soil fractions (organic matter, minerals and pore space) and root material. In Chapter Four, the impact of X-ray dose on root growth was quantified because the experimental design included repeated scanning of the same sample (Chapter Five). It was found that even under repeated scanning, the X-ray doses involved in this work (ca. 15 Gy per sample) did not significantly affect the root architecture and overall plant growth in rice cultivars used. In Chapter Five, Osaux1-1 and Osaux1-2 retained the agravitropic phenotype that was observed on agar-based systems when plants were grown in loamy sand soil. However, when subject to various soil P concentrations and distributions (Chapter Six), Osaux1-1 had similar gravitropic response and P uptake as wildtype. It was unclear what role gravitropism and topsoil foraging played in P uptake for these rice cultivars, if any. OsAUX1 could be linked to P uptake as well as responses to soil P concentration and distribution. Under uniformly low soil P wildtype had a shallower root system distribution than Osaux1-1. Of most interest were the results when sufficient soil P was sequestered to the top 4 cm of the soil column and low P was maintained in the bottom 6 cm. Under these conditions, wildtype took up more overall P, had almost twice the biomass, twice the total root length and twice the surface area when compared to Osaux1-1. This provides evidence that OsAUX1 can be linked to adaptation to P stress and distribution of P in soil through control of fine root characteristics and not necessarily its impact on gravitropic response. Chapter Seven describes the investigation into the impact of OsAUX1 on sub-architectural effects of the root system that could influence P uptake. It was determined that OsAUX1 was involved in root hair density and elongation under varying P availability for agar grown plants. In comparison to wildtype, Osaux1-1 had significant variation in root hair phenotype that seemed unrelated to a P stress response. In flooded environments, root hairs influence the potential for root:soil contact that is integral to P uptake in rice paddies which have reduced soil conditions and mass water flow that can transport plant available soluble P. This reinforces the potential for an interaction between OsAUX1 and P uptake in paddy rice

The influence of OsAUX1 on root system architecture and phosphorus uptake in rice (Oryza sativa)

Rice (Oryza sativa L.) provides up to 50% of the total calories consumed in countries such as India, Madagascar and Nigeria. As a crop, rice can require significant fertiliser inputs to maintain the required yields. Additionally, climate change has increased the need for rice varieties with improved drought resistance, tolerance to pests and more efficient acquisition of nutrients from soil. One major fertiliser input for rice is phosphate; reducing phosphorus (P) fertiliser use would have environmental and economic implications. Root traits linked to P acquisition in crops include shallow root angle, lateral root proliferation and increases in root hair length and density. Two T-DNA knockout alleles with reduced gravitropic response, Osaux1-1 and Osaux1-2, were used to investigate the influence of shallow root angle on P uptake. OsAUX1 is a rice ortholog for the Arabidopsis thaliana gene AUX1, which controls lateral root growth and gravitropic response. The wildtype and mutant rice plants were grown in soil and non-destructively imaged using X-ray micro Computed Tomography (X-ray CT). In Chapter Three, visualisation of rice roots in soil using X-ray CT was optimised by determining the ideal soil moisture content that would produce the best images. Water in soils has a similar X- ray attenuation density to that of plant roots and can influence segmentation of roots from soil in X-ray CT images. It was found that soil at nominal field capacity (ca. 3 days of drainage) produced the best contrast between soil fractions (organic matter, minerals and pore space) and root material. In Chapter Four, the impact of X-ray dose on root growth was quantified because the experimental design included repeated scanning of the same sample (Chapter Five). It was found that even under repeated scanning, the X-ray doses involved in this work (ca. 15 Gy per sample) did not significantly affect the root architecture and overall plant growth in rice cultivars used. In Chapter Five, Osaux1-1 and Osaux1-2 retained the agravitropic phenotype that was observed on agar-based systems when plants were grown in loamy sand soil. However, when subject to various soil P concentrations and distributions (Chapter Six), Osaux1-1 had similar gravitropic response and P uptake as wildtype. It was unclear what role gravitropism and topsoil foraging played in P uptake for these rice cultivars, if any. OsAUX1 could be linked to P uptake as well as responses to soil P concentration and distribution. Under uniformly low soil P wildtype had a shallower root system distribution than Osaux1-1. Of most interest were the results when sufficient soil P was sequestered to the top 4 cm of the soil column and low P was maintained in the bottom 6 cm. Under these conditions, wildtype took up more overall P, had almost twice the biomass, twice the total root length and twice the surface area when compared to Osaux1-1. This provides evidence that OsAUX1 can be linked to adaptation to P stress and distribution of P in soil through control of fine root characteristics and not necessarily its impact on gravitropic response. Chapter Seven describes the investigation into the impact of OsAUX1 on sub-architectural effects of the root system that could influence P uptake. It was determined that OsAUX1 was involved in root hair density and elongation under varying P availability for agar grown plants. In comparison to wildtype, Osaux1-1 had significant variation in root hair phenotype that seemed unrelated to a P stress response. In flooded environments, root hairs influence the potential for root:soil contact that is integral to P uptake in rice paddies which have reduced soil conditions and mass water flow that can transport plant available soluble P. This reinforces the potential for an interaction between OsAUX1 and P uptake in paddy rice

Assessing spatiotemporal relationships between atmospheric nitrogen deposition and butterfly species records through statistical modelling

Atmospheric nitrogen deposition has been linked with an overall loss of plant species richness and homogenisation of semi-natural habitats both in GB and elsewhere. We expect that nitrogen-induced changes in plant communities will impact invertebrate species through the loss of reproductive habitat, food plants and suitable microclimatic conditions caused by the shifts in composition of plant communities. Prior to this thesis, no quantitative research had been undertaken to assess the potential effects of nitrogen on fauna in GB. Butterflies are often used as indicator species due to their sensitivity to environmental change, our comprehensive understanding of their ecology, and the existence of long-term datasets on their abundance and distribution. In this study, I analysed butterfly data from the UK Butterfly Monitoring Scheme alongside data on expected driver variables including nitrogen deposition, sulphur deposition, temperature, rainfall, land use intensity, and elevation. I performed a spatio-temporal analysis on the data for each species individually using generalised additive models to understand the complex and expected non-linear relationships between butterfly trends and their drivers. Model results were summarised to provide an overview of the total number of species exhibiting responses to nitrogen. In addition, results were summarised by trait groupings such as voltinism, host plant category, host plant specificity, and breeding habitat to summarise whether any trait groupings may be particularly strongly impacted by nitrogen pollution. In addition, I performed further detailed analysis on Lasiommata megera, the Wall Brown butterfly, which has been shown to be negatively impacted by nitrogen in studies undertaken elsewhere in Europe. I ran a similar spatio-temporal analysis to that mentioned above, but with the addition of two variables I hypothesised would be key drivers of L. megera: temperature in the previous September and elevation. The results for this additional analysis were presented separately. I demonstrated that individual butterfly species vary in their relationships with nitrogen deposition and highlighted both species-level and potential trait level responses. Nine butterfly species were negatively correlated with historic nitrogen deposition, and nine were negatively correlated with percentage change in nitrogen deposition at the site over time. Two species showed significant negative relationships with both historic nitrogen deposition and percentage change in nitrogen deposition over time: Fabriciana adippe (High Brown Fritillary) and Hipparchia semele (Grayling). These findings suggest that there is a strong correlative relationship between nitrogen deposition and the abundance of many butterfly species in GB. Other key drivers of change identified in this analysis were time, rainfall, and temperature in the current and previous year. I also demonstrated a strong relationship between abundance of L. megera and historic nitrogen deposition using the model with more detailed covariates. Initial summaries based on traits were inconclusive, not highlighting any particular trait groupings as being especially susceptible to the effects of nitrogen pollution. The results of this study present the first correlative link between nitrogen deposition and negative impacts on terrestrial fauna in GB. It reinforces the importance of continued efforts to reduce emissions to protect the natural environment. It also provides a basis for further field and lab-based work to be undertaken to better understand the causal mechanisms behind the observed relationships

Assessment of the impacts of GABA and AChE targeting pesticides on freshwater invertebrate family richness in English rivers

Globally, riverine system biodiversity is threatened by a range of stressors, spanning pollution, sedimentation, alterations to water flow, and climate change. Pesticides have been associated with population level impacts on freshwater invertebrates for acute high-level exposures, but far less is known about the chronic impact of episodic exposure to specific classes of pesticides or their mixtures. Here we employed the use of the UK Environment Agency's monitoring datasets over 40 years (covering years 1980 to 2019) to assess the impacts of AChE (acetylcholinesterase) and GABA (gamma-aminobutyric acid) receptor targeting pesticides on invertebrate family richness at English river sites. Concentrations of AChE and GABA pesticides toxic to freshwater invertebrates occurred (measured) across 18 of the 66 river sites assessed. For one of the three river sites (all found in the Midlands region of England) where data recorded over the past 40 years were sufficient for robust modelling studies, both AChE and GABA pesticides associated with invertebrate family richness. Here, where AChE total pesticide concentrations were classified as high, 46 of 64 invertebrate families were absent, and where GABA total pesticide concentration were classified as high, 16 of 64 invertebrate families were absent. Using a combination of field evidence and laboratory toxicity thresholds for population relevant endpoints we identify families of invertebrates most at risk in the selected English rivers to AChE and GABA pesticides. We, furthermore, provide strong evidence that the absence of the invertebrate family Polycentropodidae (caddisfly) from one field site is due to exposure effects to AChE pesticides

Rice auxin influx carrier OsAUX1 facilitates root hair elongation in response to low external phosphate

Root traits such as root angle and hair length influence resource acquisition particularly for immobile nutrients like phosphorus (P). Here, we attempted to modify root angle in rice by disrupting the OsAUX1 auxin influx transporter gene in an effort to improve rice P acquisition efficiency. We show by X-ray microCT imaging that root angle is altered in the osaux1 mutant, causing preferential foraging in the top soil where P normally accumulates, yet surprisingly, P acquisition efficiency does not improve. Through closer investigation, we reveal that OsAUX1 also promotes root hair elongation in response to P limitation. Reporter studies reveal that auxin response increases in the root hair zone in low P environments. We demonstrate that OsAUX1 functions to mobilize auxin from the root apex to the differentiation zone where this signal promotes hair elongation when roots encounter low external P. We conclude that auxin and OsAUX1 play key roles in promoting root foraging for P in rice

Effects of X-ray dose on rhizosphere studies using X-ray computed tomography

X-ray Computed Tomography (CT) is a non-destructive imaging technique originally designed for diagnostic medicine, which was adopted for rhizosphere and soil science applications in the early 1980s. X-ray CT enables researchers to simultaneously visualise and quantify the heterogeneous soil matrix of mineral grains, organic matter, air-filled pores and water-filled pores. Additionally, X-ray CT allows visualisation of plant roots in situ without the need for traditional invasive methods such as root washing. However, one routinely unreported aspect of X-ray CT is the potential effect of X-ray dose on the soil-borne microorganisms and plants in rhizosphere investigations. Here we aimed to i) highlight the need for more consistent reporting of X-ray CT parameters for dose to sample, ii) to provide an overview of previously reported impacts of X-rays on soil microorganisms and plant roots and iii) present new data investigating the response of plant roots and microbial communities to X-ray exposure. Fewer than 5% of the 126 publications included in the literature review contained sufficient information to calculate dose and only 2.4% of the publications explicitly state an estimate of dose received by each sample. We conducted a study involving rice roots growing in soil, observing no significant difference between the numbers of root tips, root volume and total root length in scanned versus unscanned samples. In parallel, a soil microbe experiment scanning samples over a total of 24 weeks observed no significant difference between the scanned and unscanned microbial biomass values. We conclude from the literature review and our own experiments that X-ray CT does not impact plant growth or soil microbial populations when employing a low level of dose (<30 Gy). However, the call for higher throughput X-ray CT means that doses that biological samples receive are likely to increase and thus should be closely monitored

Genome-wide association analysis identifies six new loci associated with forced vital capacity

Forced vital capacity (FVC), a spirometric measure of pulmonary function, reflects lung volume and is used to diagnose and monitor lung diseases. We performed genome-wide association study meta-analysis of FVC in 52,253 individuals from 26 studies and followed up the top associations in 32,917 additional individuals of European ancestry. We found six new regions associated at genome-wide significance (P < 5 × 10−8) with FVC in or near EFEMP1, BMP6, MIR129-2–HSD17B12, PRDM11, WWOX and KCNJ2. Two loci previously associated with spirometric measures (GSTCD and PTCH1) were related to FVC. Newly implicated regions were followed up in samples from African-American, Korean, Chinese and Hispanic individuals. We detected transcripts for all six newly implicated genes in human lung tissue. The new loci may inform mechanisms involved in lung development and the pathogenesis of restrictive lung disease

Genetic effects on gene expression across human tissues

Characterization of the molecular function of the human genome and its variation across individuals is essential for identifying the cellular mechanisms that underlie human genetic traits and diseases. The Genotype-Tissue Expression (GTEx) project aims to characterize variation in gene expression levels across individuals and diverse tissues of the human body, many of which are not easily accessible. Here we describe genetic effects on gene expression levels across 44 human tissues. We find that local genetic variation affects gene expression levels for the majority of genes, and we further identify inter-chromosomal genetic effects for 93 genes and 112 loci. On the basis of the identified genetic effects, we characterize patterns of tissue specificity, compare local and distal effects, and evaluate the functional properties of the genetic effects. We also demonstrate that multi-tissue, multi-individual data can be used to identify genes and pathways affected by human disease-associated variation, enabling a mechanistic interpretation of gene regulation and the genetic basis of diseas

Genetic effects on gene expression across human tissues

Characterization of the molecular function of the human genome and its variation across individuals is essential for identifying the cellular mechanisms that underlie human genetic traits and diseases. The Genotype-Tissue Expression (GTEx) project aims to characterize variation in gene expression levels across individuals and diverse tissues of the human body, many of which are not easily accessible. Here we describe genetic effects on gene expression levels across 44 human tissues. We find that local genetic variation affects gene expression levels for the majority of genes, and we further identify inter-chromosomal genetic effects for 93 genes and 112 loci. On the basis of the identified genetic effects, we characterize patterns of tissue specificity, compare local and distal effects, and evaluate the functional properties of the genetic effects. We also demonstrate that multi-tissue, multi-individual data can be used to identify genes and pathways affected by human disease-associated variation, enabling a mechanistic interpretation of gene regulation and the genetic basis of disease

Recovering complete plant root system architectures from soil via X-ray μ-computed tomography

Background: X-ray micro-Computed Tomography (μCT) offers the ability to visualise the three-dimensional structure of plant roots growing in their natural environment – soil. Recovery of root architecture descriptions from X-ray CT data is, however, challenging. The X-ray attenuation values of roots and soil overlap, and the attenuation values of root material vary. Any successful root identification method must both explicitly target root material and be able to adapt to local changes in root properties. RooTrak meets these requirements by combining the level set method with a visual tracking framework and has been shown to be capable of segmenting a variety of plant roots from soil in X-ray μCT images. The approach provides high quality root descriptions, but tracks root systems top to bottom and so omits upward-growing (plagiotropic) branches. Results: We present an extension to RooTrak which allows it to extract plagiotropic roots. An additional backwardlooking step revisits the previous image, marking possible upward-growing roots. These are then tracked, leading to efficient and more complete recovery of the root system. Results show clear improvement in root extraction, without which key architectural traits would be underestimated. Conclusions: The visual tracking framework adopted in RooTrak provides the focus and flexibility needed to separate roots from soil in X-ray CT imagery and can be extended to detect plagiotropic roots. The extended software tool produces more complete descriptions of plant root structure and supports more accurate computation of architectural traits. Keywords: Root systems architecture, 3D, X-ray Computed Tomography, Image analysis, Root phenotypin