Exploring the influence of the plant lateral root gravitropic set point angle on architecture in soils using X-Ray Computed Tomography.

When we look at a growing plant what we observe are the above ground tissues comprised of leaves stems and flowers. What is not immediately obvious is that all of the above ground growth is absolutely dependent on the root system which is hidden in the soil. The root system is responsible for accessing and taking up the water and essential minerals necessary for healthy growth as well as anchoring the plant in the soil. The importance of these resources to the plant is highlighted by the need for agricultural crops to be given additional water and nutrients in the form of costly fertilizers. However, the supplies of fertilizers are not infinite and already there are worrying decreases in the stocks of rock phosphate. Added to this is the threat of climate change which is likely to lead to increased problems with both short term and long term droughts. The world's population is ever increasing leading to growing concerns about food security. Put simply this raises the question of whether enough high quality nutritious food can be produced in the future to feed the world's population. This need is placing increasing demands on the available agricultural land. In order to increase agricultural output it will be necessary to increase crop productivity. A major target to achieve this goal is to improve the efficiency of plants roots to colonise the soil and increase uptake of water and nutrients. This will provide greater drought tolerance and reduce the need for fertilizer application. A major problem in studying plant roots is the fact that they are generally hidden from view within the soil. Thus to see the root system it is necessary to remove the soil and thus lose the 3-dimensional organisation of the root system. Plant scientists have therefore used clear agar based growth media to cultivate plants in the laboratory allowing the roots to be easily visualised. However this system does not accurately mimic the natural growing environment and questions whether the results obtained and really relevant for the natural world. This project will develop a technique known as X-ray Computed Tomography (CT) as a way to visualise the root system in situ in the soil. This will allow the 3-D organisation to be observed giving a direct way to assess the volume of soil colonised by a plant root system. The necessary high-technology equipment necessary to carry out this aspect of the project is available within the newly formed CT center at the University of Southampton. The plant root system is made of a primary root which grows downwards and which forms lateral branches, elaborating the network. Initially these lateral branches grow horizontally away from the primary root before altering their direction of growth downwards. The initial angle of growth (known as the Gravitropic Setpoint Angle or GSA) of the lateral roots and the length achieved before reorienting their growth downwards are key determinants in the 3-D architecture of the root system and hence the volume of colonised soil. A few Arabidopsis mutants including rhd3 have been identified which display alterations in the GSA demonstrating that it is under genetic control. Work will be carried out to identify and characterise further Arabidopsis mutants affected in their root GSA. Gaining a better understanding of the genetic and environmental factors which influence the growth characteristics of the lateral roots and hence the overall architecture of the root system will be invaluable in attempts to improve crop productivity in the future. The techniques and results obtained in this study will be applicable and transferable to a wide range of different plants including those such as wheat and oil seed rape which form a staple part of our diets.

Understanding about the genetic and developmental control of the formation of root systems has come from plants cultured in vitro on agar-based media. While this approach has undoubtedly been successful, the question remains about how relevant this information is for plants growing in the natural soil environment. The challenge is to be able to visualise the root system in its entirety whilst maintaining the 3-D organisation when growing in an opaque soil. To tackle this problem X-ray Computed Tomography (CT) will be used to image roots growing in soil. Although X-ray CT has been used for a number of years it is only recently that high resolution machines have been developed to allow fine roots to be detected. Despite this there are still challenges in resolving the roots from other organic materials, moisture and air pockets that are found in soils. Within this project we will develop and optimize scanning protocols to achieve the highest signal to noise ratio for non-destructive high-resolution in-situ imaging of complete root architecture in soil. This work coupled with development of 3-D CT reconstruction algorithms will contribute directly to produce root tracing methodology to detect full root system architecture. Work will be carried out to identify Arabidopsis mutants with an altered lateral root gravitropic setpoint angle (GSA) using an agar plate based in vitro assay. The lateral root GSA is a major determinant of root system architecture and hence a plant's ability to access nutrients and water. Mutants identified in the in vitro screen will be grown on soil and the root architecture determined using X-ray CT. This will generate a set of mutants that will be the basis for further studies to identify the mutated genes, identify homologs in crop species and ultimately to try and develop varieties with improved nutrient uptake properties.

New Arabidopsis mutants will be identified that are affected in the lateral root Gravitropic Setpoint Angle (GSA). This will allow the key genes controlling the lateral root GSA to be cloned providing plant biotechnologists and plant breeders with tools to enable them to generate improved crop varieties in the future. By optimising lateral root growth it will be possible to reduce the requirement for application of fertilizers to agricultural crops and also to increase tolerance to drought stress. This will therefore ultimately benefit society as a whole by improving food security, delivering nutritious foodstuffs and reducing environmental impacts of fertilizer use. The process of identifying the key genes, characterisation and transfer to commercial crops is a relatively long process but it is possible that this could be achieved to deliver improved varieties within 5-10 years provided funding is maintained. Plants are increasingly being used for the purpose of bioremediation to clean up contaminated soils. This process relies on the plants being able to grow on the contaminated soil and to take up the contaminant thereby removing it from the environment. There is considerable scope to improve this process either by increasing the uptake potential of the roots or the transport and storage capacity of the plant. The outputs from this project will provide the potential to alter the architecture of the root system of a wide range of plants such as perennial grasses which can be used for soil bioremediation. For example, a contaminant may be localised to the upper few centimetres of a soil and so a plant that has an extensive shallow root system would be optimal. By understanding more about the control of the direction of lateral roots it should be possible to engineer plants to explore different depths of the soil. The ability to engineer or breed crop plants which develop an extensive lateral root system close to the surface may be beneficial in efficiently utilising the available nutrient supply thereby reducing the need for fertilizer application. This would provide benefit to farmers and well as consumers in the form of reduced costs. The project will also further develop X-Ray Computed Tomography scanning as a way to visualise root development in soils. Although this approach has been in use for several years it is only recently that machines with high enough sensitivity have been developed to allow fine roots to be imaged. Within this study work will be undertaken to improve the CT scanning approach. This may involve the use and development of contrast agents as well as methodologies for distinguishing the roots from the organic matter, air pockets and moisture in the soil. Publication of the work will allow any improvements in the CT scanning technique to be communicated to other research groups in both the academic and industrial sectors. The research technician who will be employed on this project will gain experience in a range of different techniques encompassing both engineering and biology. CT scanning is becoming an increasingly used technique to image a wide range of materials such as metals, stone, foodstuffs and living material such as plants. Thus the skills gained by the researcher will be applicable to a number of different academic and commercial settings. Additionally experience will be gained in plant tissue culture, and plant physiology thus creating a broad set of skills thereby increasing employability.