Engineer SUberin biopolyester production in potato to Improve stress Tolerance – SUIT

Suberin is a tissue sealing polyester deposited at the plant environment interphase. In peri-derms of aerial plant organs and in the root exodermis and endodermis, suberin functions as a barrier preventing uncontrolled water and nutrient loss and pathogen attack. In addition, con-trolled suberization occurs in response to wounding, salt or drought stress. In potato these suberin functions are not only crucial for growth dependent yield, but also for post harvest stor-age properties of tubers. Despite these important functions our understanding of the enzymes and genes involved in suberization is surprisingly limited. Recently, reverse genetics ap-proaches in Arabidopsis and potato identified the first suberin involved genes. These proof of concept studies demonstrated that reductions in predominant suberin monomers result in incre-ased water permeability of potato periderms. Arabidopsis enhanced suberin mutants exhibited an increased tolerance to salt and drought stress conditions. From these studies it can be postu-lated that targeted engineering of suberization can modify barrier properties of suberized tissues leading to improved yield stability and stress tolerance in crop plants. This project aims are an increased knowledge of the metabolic pathways and the underlying molecular attributes involved in suberin formation, and the production of plants with engineered suberin to improve performance of crops under unfavourable environmental conditions. To reach this goals a com-plementary approach combining bioinformatics, molecular genetics, recombinant protein bio-chemistry, physiology, and analytical chemistry is proposed with the following main objectives (MO):. MO 1. Discover and validate key suberin genes by reverse genetics in plant models (Arabidopsis and potato) A bioinformatics approach considering tissue specificity, environmental conditions, coexpres-sion with known suberin involved genes and potential functions in processes expected in suberization (e.g. fatty acid modification) will generate a comprehensive list of suberin candidate genes (SG) from Arabidopsis. Expected 80-120 good SG will be short listed to 20-30 by detailed expression profiling. GC-MS based compositional suberin profiling in corresponding knock out and overexpression lines will verify involvement in suberization. For expected 15-25 verified Arabidopsis putative orthologues will be obtained by homology based cloning. Tissue-specific expression profiling of isolated potato SG will be used to verify the specificity for suberizing tis-sue. Sequencing genomic 5'-regions of SG will identify suberin regulatory elements and enables tissuespecific expression strategies. MO 2. Use heterologous expression systems to characterize the catalytic properties of key enzymes in suberin biosynthesis SG, especially Cytochrome P450 oxygenases involved in w-oxidation of the predominant suberin monomers, w-hydroxyacids and a,w-diacids, will be introduced in engineered yeast and baculovirus expression systems. Catalytic properties will be determined testing collections of potential substrates e.g. fatty acids, acyl-CoAs and acyl-glycerols. This will indicate in vivo substratesand help selecting additional SG to optimize substrate availability in co-overexpression strategies. MO 3.Elucidate the role of SG in suberin macromolecular structure SG characterized in MO 1will be cloned into optimized expression vectors, enabling ectopic overexpression, RNAi silencing and expression in suberizing peridermal tissues. Large scale transformation generate approximately 600 transgenic potato lines for the selection of 3-5 stably expressing lines from 5-10 SG over expression construct, 5-10 SG silencing constructs and 5-10 Promoter:Reporter constructs. The latter will be investigated for expression in suberizing tissue. Isolated tuber periderms from SG-transgenic potato will be analytically characterized regarding monomer composition, molecular connectivity and macromolecular structure, using mass spectrometry techniques (GC-MS, ESI-MS/MS), FTIR and 13C NMR. The tubers of expected 5-15 SG-trangenic potatos with quantitatively or qualitatively modified suberin will be investigated for alterations in suberin ultra structure using electron microscopy. MO 4. Determine the impact of compositional components and macromolecular structure on barrier properties Tuber periderms from transgenic potatoes - characterized inMO 3 as being affected in suberization - will be investigated for modified features such as function as an efficient permeation barrier. The permeability will be determined for water and isotopic model tracers. Intact tubers will be used to investigate the effect of suberin modification on post harvest food loss during storage and upon pathogen attack. MO 5. Targeted modification of SG to engineer suberin for improved stress tolerance. The performance of whole potato plants and Arabidopsis with modified root suberin (MO 1 and MO 3) will be tested under unfavourable environmental conditions. These studies will uncover coherences between the composition, macromolecular- and ultra structure of suberin and its barrier characteristics as well as suberin characteristics and tolerance to biotic and abiotic stresses.