The aim of this proposal is to investigate factors controlling the chemical composition of tomato fruit, a crop of major economic importance worldwide. Both the flavour and nutritional quality of tomatoes are determined by the chemicals that accumulate during fruit ripening, yet we have only a limited understanding of how this process is controlled. In mature fruit, the cells are dominated by a compartment called the central vacuole, which contains most of the sap in fleshy fruit. This compartment can occupy as much as 95 % of the cell's volume, the remaining 5 % being taken up by the cell cytoplasm and outlying cell wall. As the tomato fruit grows, chemicals such as sugars, organic acids and amino acids are produced in the cytoplasm. They are then removed from their site of synthesis by transport into the central vacuole across the bounding membrane surrounding this compartment, called the tonoplast. But this traffic is not all one-way. As the fruit ripens, some solutes leave the vacuole to be re-metabolized in the cytoplasm, with other solutes moving back into the vacuole to compensate. Thus, the composition of the mature fruit is a complex outcome of metabolic events in the cytoplasm combined with transport of solutes across the tonoplast membrane. Whereas the pathways of basic metabolism in fruit cells are well understood, we have much less knowledge of the transport proteins that reside in the tonoplast membrane. In fact, we have indirect evidence that these proteins may play a much more important role in determining fruit composition than previously suspected. As the first part of this project, therefore, we shall isolate the tonoplast membrane from tomato fruit at defined stages during their development and analyse its protein content by mass spectrometry. This will provide a valuable inventory of proteins residing in the tonoplast membrane, and of their changes in abundance during the ripening process. By correlating these changes with the chemical composition of the fruit, we should obtain the first clues as to which tonoplast proteins are important in regulating transport across the vacuolar membrane. In another strand of the project, we will use a genetic approach to obtain independent information on factors controlling fruit composition. A powerful resource for this purpose is provided by the natural genetic variation found between cultivated tomatoes and their close relatives in the wild. Indeed, several of these species are sufficiently closely related that they can be hybridized. By analysing the characteristics of the progeny of such crosses (e.g. with respect to fruit composition), it is possible to make deductions about which genes may be contributing to particular traits. Using this approach, we will investigate whether any of the genes correlated with differences in fruit composition encode likely tonoplast membrane proteins. If they do, we will cross-reference this list against the information on tonoplast proteins obtained by mass spectrometry. This will allow us to focus on a limited number of the most promising candidates for more detailed characterization. In the final part of the project, we will test the function of the selected candidate proteins directly to determine, first, whether they indeed reside in the tonoplast membrane in intact cells, and second, what solutes they are capable of transporting into and out of the vacuole. We will focus on candidate transporters of organic acids and amino acids, as these are important determinants of fruit flavour and acidity that have been little investigated to date. The combination of the protein identification and genetic approaches promises to yield important new information on the factors determining fruit composition. This will also be valuable for directing future breeding strategies towards the selection of new elite lines with improved fruit traits, without the need for intervention using genetic modification techniques.

Doctoral Training Partnerships: a range of postgraduate training is funded by the Research Councils. For information on current funding routes, see the common terminology at https://www.ukri.org/apply-for-funding/how-we-fund-studentships/. Training grants may be to one organisation or to a consortia of research organisations. This portal will show the lead organisation only.

Synthetic Biology is the underpinning discipline for advances in the UK bioeconomy, a sector currently worth ~£200Bn GVA globally. It is a technology base that is revolutionising methods of working in the biotechnology sector and has been the subject of important Government Roadmaps and supported by significant UKRI investments through the Synthetic Biology for Growth programme. This is now leading to a vibrant translational landscape with many start-ups taking advantage of the rapidly evolving technology landscape and traditional industries seeking to embed new working practices. We have sought evidence from key industry leaders within the emerging technology space and received a clear and consistent response that there is a significant deficit of suitably trained PhDs that can bridge the gap between biological understanding and data science. Our vision is a CDT with an integrative training programme that covers experimentation, coding, data science and entrepreneurship applied to the design, realisation and optimisation of novel biological systems for diverse applications: BioDesign Engineers. It directly addresses the priority area 'Engineering for the Bioeconomy' and has the potential to underpin growth across many sectors of the bioeconomy including pharmaceutical, healthcare, chemical, energy, and food. This CDT will bring together three world-leading academic institutions, Imperial College London (Imperial), University of Manchester (UoM) and University College London (UCL) with a wide portfolio of industrial partners to create an integrated approach to training the next generation of visionary BioDesign Engineers. Our CDT will focus on providing an optimal training environment together with a rigorous interdisciplinary program of cohort-based training and research, so that students are equipped to address complex questions at the cutting edge of the field. It will provide the highly-skilled workforce required by this emerging industry and establish a network of future UK Bioindustry leaders. The joint location of the CDT in London and Manchester will provide a strong dynamic link between the SE England biotech cluster and the Northern Powerhouse. Our vision, which brings together a BioDesign perspective with Engineering expertise, can only be delivered by an outstanding and proven grouping of internationally renowned researchers. We have a supervisor pool of 66 world class researchers that span the associated disciplines and have a demonstrated commitment to interdisciplinary research and training. Furthermore, students will work directly with the London and Manchester DNA Foundries, embedding the next generation bioscience technologies and automation in their training and working practices. Cohort training will be delivered through a common first year MRes at Imperial College London, with students following a 3-month taught programme and a 9-month research project at one of the 3 participating institutions. Cohort and industry stakeholder engagement will be ensured through bespoke training and CDT activities that will take place every 6 months during the entire 4-year span of the programme and include multi-year group hackathons, training in responsible research and innovation, PhD research symposia, industry research days, and entrepreneurial skills training. Through this ambitious cohort-based training, we will deliver PhD-level BioDesign Engineers that can bridge the gap between rigorous engineering, efficient model-based design, in-depth cellular and biomolecular knowledge, high throughput automation and data science for the realisation and exploitation of engineered biological systems. This unique cohort-based training platform will create the next generation of visionaries and leaders needed to accelerate growth of the UK bioeconomy.

Doctoral Training Partnerships: a range of postgraduate training is funded by the Research Councils. For information on current funding routes, see the common terminology at https://www.ukri.org/apply-for-funding/how-we-fund-studentships/. Training grants may be to one organisation or to a consortia of research organisations. This portal will show the lead organisation only.

Insect pollinators provide a vital ecosystem service supporting crop pollination and reproduction in wild plants. Reported declines in pollinators threaten this service and could have serious implications for food security. A key crop dependent on insect pollination is apples, and the contribution of wild pollinators to UK apple production is worth an estimated £95M p.a. However, "pollination gaps" of more £6000/ha have been identified in some varieties, where desired yields and quality are not being achieved due to inadequate pollination. This presents a major opportunity for growers for increased production and profit through better pollination. The documented decline of pollinating insects also poses a significant risk to fruit production by negatively impacting on crop production. In response, top fruit growers have articulated the need to effectively manage pollination services by wild insects in a way that is cost effective in order to maintain production and quality in the face of continued environmental change. Our project will develop high quality science to address this need by designing and testing three pollinator management strategies in field scale trials in commercial apple orchards. These include establishing flower rich strips to provide food and shelter for pollinators, providing nesting habitat for ground nesting bees, and adapting the number and placement of 'polleniser' trees in orchards to increase levels of pollination. Apple trees are predominantly self-incompatible and require pollen from 'polleniser' trees to set fruit, so although they don't produce saleable fruit, pollenisers are planted in orchards. Currently the number of pollinisers planted is based on a rule of thumb of 1 polleniser for every 12 apples trees. As a first step in this project we will trial different numbers and arrangements of polliniser tree in study orchards and measure how this effects pollination and apple production in order to establish an optimum arrangement and ratio. Flower strips are known to benefit pollinators. In this project, for the first time, we will design bespoke flower strips specifically aimed at supporting known apple pollinators. We will design our flower strips to contain plants that are particularly good for ground nesting bees and bumblebees which have been identified as top pollinators in apple orchards. Furthermore, we will top our margins (i.e. use a high level cut to remove just the flowers) during apple flowering to push pollinators off the flower strips and onto the apple blossom thus maximising the benefit strips provide. The impact of these targeted flower strips on pollinators and apple production will be measured in field scale trials in commercial orchards. While the provision of floral resources for pollinators is a well-established approach for increasing pollinator numbers, provision of nesting sites has been widely overlooked and little is known about the effect this can have on pollination service. In this project we will create novel ground nesting bee nest sites in our study orchards and measure the impact these have on bee populations and their contribution to the pollination of apples. Findings from our field trials will be brought together, and the cost of interventions and the economic return in terms of long-term improvements in quality and production will be established. Our overarching aim is to understand the mechanistic basis of how these three interventions, individually and in combination, effect the value of pollination service contributions to production and profit, so that we can "engineer" the most effective in-orchard interventions. The costs and benefits of these approaches will be assessed to allow specific management recommendations to be made to growers that are practical to implement and provide proven economic returns to growers by supporting long-term stable pollination of apple orchards.