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Project 1 (Plant Physiology, University of Kaiserslautern) Impact of intracellular sugar homeostasis on plant yield and stress tolerance Sugars are of enormous importance for plant properties. They serve as precursors for nearly all primary metabolites, for starch and cellulose biosynthesis, as carbon and energy equivalents for storage organ development and they are required to raise tolerance against biotic and abiotic stress stimuli. In sum, knowledge on plant sugar metabolism is of superior importance to feed the increasing human population and to gain renewable sources for energy, biofuel and chemical feedstock. Due to their central position in metabolism and because of their critical functions in processes mentioned above, it does not surprise that nearly all types of organisms sense their intracellular sugar levels. In plants, this sensing machinery is located in the cell cytosol1 and it is mandatory to adjust both, physiological processes and gene-expression programs accordingly2. Sugars are transported via a large number of carrier proteins mediating movement of these solutes across the plasma membrane or across the membranes of various organelles. Most of these carriers which allow transport of mono- or disaccharides belong to the large group of Major Facilitator Superfamily (MFS) proteins3. However, recently first members of the so called SWEET proteins have also been found to mediate mono- and disaccharide transport across human- and plant cell membranes4. In plant cells the vacuole occupies up to 90 % of the cell volume5. This large volume makes this organelle uniquely suited to serve as a dynamic storage compartment for various solutes and sugars represent the most abundant molecule type in the vacuole. Recent evidence illustrated that especially the modification of vacuolar sugar transport influences plant properties in terms of storage-organ size, yield and abiotic stress tolerance6-9. So far, two types of vacuolar located sugar carriers have been shown to actively control intracellular sugar homeostasis, namely the TMT-type carriers and the carriers SWEET16 and 17. The vacuolar monosaccharide carrier TMT (tonoplast monosaccharide transporter) acts as a proton-driven importer of glucose and fructose10. It has been shown that this carrier is regulated on the level of gene expression and post-translationally via protein phosphorylation11. Interestingly, transgenic plants with increased TMT activity show a markedly increased seed size leading to an overall stimulation of seed yield of up to 20 %12. Due to the proton-antiport activity of TMT this type of carrier is unable to export previously imported monosaccharide under conditions of cellular sugar demand. Vacuolar export of fructose is mediated by SWEET16 and 17 and modification of both transporter activities affect seed germination and freezing tolerance13,14. First data indicate that the two vacuolar SWEET isoforms are unable to transport glucose under in vivo conditions, instead, this latter sugar is exported from the vacuole by the early-response to dehydration-like transporter6 (ERDL6, 15 ). So far, it is unknown how sucrose loading into the vacuole is catalyzed, while sucrose export seems to be mediated by the sucrose carrier SUT4 16 . In sum, we know how fructose and glucose enters the plant vacuole and we know how fructose, glucose and sucrose leave the vacuole into the cytosol. Given that TMT activity governs storage organ size, and given that the activities of ERDL6, SWEET16 and SWEET17 have been identified to influence plant properties like seed germination and cold tolerance it is challenging to further analyze the impact of cellular sugar homeostasis in much more detail. Since we have hand on all genes coding for the carriers involved we have accordingly, for the first time, the opportunity to modify cytosolic sugar levels and their composition in a predicted manner. The proposed PhD project will cover the creation of several Arabidopsis mutants with altered activity of selected vacuolar sugar transport proteins. These mutants will be analyzed in depth for e.g. seed size, seed yield and tolerance against abiotic stress stimuli like cold, drought, salt, or growth at limiting nitrogen availability. Sophisticated molecular and biochemical analyses like q-RT-PCR, metabolite quantification and flux measurements will be complemented from modern physiological techniques like quantification of photosynthesis, chlorophyll fluorescence and gas exchange measurements. Moreover, TMT proteins from all plant species exhibit a remarkable long hydrophilic central loop domain known to bind protein kinases. In cooperation with our canadian partners we aim to raise first detailed structural information on this unique loop domain. Reference List 1. Rolland, F., Moore, B. & Sheen, J. Sugar sensing and signaling in plants. Plant Cell 14, 185-205 (2002). 2. Hanson, J. & Smeekens, S. Sugar perception and signaling--an update. Curr. Opin. Plant Biol. 12, 562-567 (2009). 3. Büttner, M. & Sauer, N. Monsaccharide transporters in plants: structure, function and physiology. Biochim. Biophys. Acta 1465, 263-274 (2000). 4. Chen, L. Q. et al. Sugar transporters for intercellular exchange and nutrition of pathogens. Nature 468, 527-532 (2010). 5. Martinoia, E., Maeshima, M. & Neuhaus, H. E. Vacuolar transporters and their essential role in plant metabolism. Journal of Experimental Botany 58, 83-102 (2007). 6. Wingenter, K. et al. Increased activity of the vacuolar monosaccharide transporter TMT1 alters cellular sugar partitioning, sugar signalling and seed yield in Arabidopsis. PLANT PHYSIOLOGY 154, 665-677 (2010). 7. Chardon, F. et al. Leaf fructose content is controlled by the vacuolar transporter SWEET17 in Arabidopsis. Curr. Biol. 23, 697-702 (2013). 8. Poschet, G. et al. A novel Arabidopsis vacuolar glucose exporter is involved in cellular sugar homeostasis and affects the composition of seed storage compounds. Plant Physiol 157, 16641676 (2011). 9. Klemens, P. A. et al. Overexpression of a proton-coupled vacuolar glucose exporter impairs freezing tolerance and seed germination. New Phytol. in press, (2013). 10. Wormit, A. et al. Molecular identification and physiological characterization of a novel monosaccharide transporter from Arabidopsis involved in vacuolar sugar transport. Plant Cell 18, 3476-3490 (2006). 11. Wingenter, K. et al. A member of the mitogen-activated protein 3-kinase family is involved in the regulation of plant vacuolar glucose uptake. Plant J 68, 890-900 (2011). 12. Wingenter, K. et al. Increased activity of the vacuolar monosaccharide transporter TMT1 alters cellular sugar partitioning, sugar signalling and seed yield in Arabidopsis. PLANT PHYSIOLOGY 154, 665-677 (2010). 13. Chardon, F. et al. Leaf fructose content is controlled by the vacuolar transporter SWEET17 in Arabidopsis. Curr. Biol. 23, 697-702 (2013). 14. Klemens, P. A. et al. Overexpression of the vacuolar sugar carrier AtSWEET16 modifies germination, growth, and stress tolerance in Arabidopsis 1. Plant Physiol 163, 1338-1352 (2013). 15. Poschet, G. et al. A novel Arabidopsis vacuolar glucose exporter is involved in cellular sugar homeostasis and affects the composition of seed storage compounds. Plant Physiol 157, 16641676 (2011). 16. Schneider, S. et al. Vacuoles release sucrose via tonoplast-localised SUC4-type transporters. Plant Biol. (Stuttg) 14, 325-336 (2012).