Cytosolic invertases and the control of plant growth.

Plants convert carbon dioxide from the air into sugars in their leaves in the process of photosynthesis. These sugars are exported from the leaf to all of the other parts of the plant (roots, stems, flower, seeds etc), where they are converted into plant components such as starch, fats, proteins and fibre. Our project concerns the initial steps in this process. At the moment we do not understand what these steps are, or what controls how much of the sugar is converted to starch, to fats, to protein, and to fibre. We have recently been studying plants that lack enzymes that may be involved in the initial steps. This has provided new and surprising information: loss of some of the enzymes has almost no effect on the plant whereas loss of others (in particular a sort of enzyme called neutral invertase) slows plant growth so the plant is very stunted. In this project we want to understand fully the family of neutral invertase enzymes: what they do, how they work and where they exist in the plant cell. In particular, we want to know why the neutral invertases are necessary for normal growth. As well as providing new information about crop productivity, our results will have implications for understanding how much sugar is stored in plants and how much is converted to useful materials such as fibre. This balance affects how we use our crops, and gives us new opportunities to improve them for our food and for the manufacture of 'green' products and biofuels.

The aim of this project is to discover why cytosolic isoforms of invertase are essential for plant growth. The project stems from our recent, unexpected discovery that cytosolic invertases are required for growth of Arabidopsis and Lotus plants, but sucrose synthases are not. In this project we will use the two species in three comparative, complementary, approaches: 1) characterisation of the properties of the cytosolic invertases, and localisation studies for each isoform. 2) examination of the phenotypes of mutants lacking isoforms of this enzyme by (a) comparative microarray analysis of Arabidopsis and Lotus mutants to identify transcriptional changes associated with the common phenotype, (b) analysis of changes in activities and protein levels of sucrose metabolising enzymes and (c) analysis of changes in levels of key sucrose catabolites and cell wall components. 3) tests of hypotheses concerning the severe consequences for growth of the loss of cytosolic invertases. The two alternatives are that loss of cytosolic invertase starves the plant of carbon required for normal growth and development, or that its loss causes incorrect sensing/signalling of carbon availability bringing about massive growth and developmental abnormalities. We will distinguish between these 'starvation' and 'signalling' hypotheses by examining the extent of complementation of the phenotype through expression of heterologous sucrose-metabolising enzymes, and by exploring the consequences of the mutations for sugar sensing via hexokinase, trehalose phosphate metabolism and phosphoinositide signalling. Further information will come from an untargetted approach in which the Arabidopsis mutant will be re-mutagenised and screened for faster-growing lines. This work will provide new information on sugar signalling and carbon partitioning and give significant insights into factors that control and limit plant productivity, of potential value in the development of high-yielding crops.

Who will benefit from this research? 1. Academics and researchers in all fields of plant research. 2. Annotators in genomics and metabolomics, database and germplasm curators. 3. UK and international science base. 4. Agro-industry inc. biotechnologists and plant breeders seeking to increase plant productivity and/or harvest index; metabolic engineers. 5. Agricultural community and advisors. 6. Postdoctoral researcher. 7. Public. How will they benefit from this research? 1. Researchers will receive comprehensive, new information about sucrose breakdown and its importance in plant growth, about the integration of carbon supply with carbon demand and growth rate; about the properties of novel sucrose hydrolases; and about gene function in Arabidopsis and Lotus. 2. Researchers will have access at the point of publication to microarray and other phenotypic characterisations, and novel plant material in which primary metabolism has been altered in known ways. 3. The research will have a major impact on understanding of plant primary metabolism and enhance the UK's international standing in plant science. We are not aware of similar efforts on this vital topic elsewhere in the world. 4. Agro-industry will receive information that underpins rational approaches to increase stored sugar or fibre content for food, fuel and feedstocks, improve forage quality, engineer nitrogen fixation into non-legumes, and discover new enzymic tools for metabolic engineering. 5. The agricultural community will benefit from sustainable crop improvements enabled by our research (see 4). 6. The postdoctoral researcher will receive a wide training in plant integrative biology including 'at the bench' and 'informatics' approaches, access to professional skills and wider training courses, and the opportunity to work in labs in Germany. 7. Our research findings relate to issues of public interest including food quality and sustainable crop production. They can potentially benefit consumers by contributing to the development of fresher, more nutritious food produced in a more sustainable manner What will be done to ensure they benefit from this research? 1. Publish results in high-impact journals in a timely fashion, with open access where possible. Present research results at UK and international meetings and institutions 2. Submit data to relevant international depositories. Notify new/corrected gene and enzyme annotations to community databases e.g. Aracyc (http://www.arabidopsis.org/biocyc/index.jsp). Access to materials will be via NASC for Arabidopsis lines or RevGenUK for Lotus. 3. Exploit existing contacts with other UK and international academics with relevant research interests as soon as any exploitable results/materials are generated e.g. BBSRC Sustainable Bioenergy Centre 4. Make contacts with industrialists, supermarkets and relevant umbrella organisations; recognise and protect intellectual property to ensure wise and fruitful exploitation. 5. Use results as part of our regular engagement with the agricultural community through our outreach programmes and Defra genetic improvement networks. 6. Provide information and mentoring to ensure uptake of JIC's tailor-made postdoctoral training scheme. We will also encourage the researcher to participate in all aspects of the dissemination of research results, and to gain wider understanding of the implications and applications of the research. 7. Use results as part of our regular engagement with non-academic audiences, e.g. local interest groups, schools, local and national shows, science showcases, media.