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DEPARTMENT OF PLANT BIOLOGY FACULTY OF LIFE SCIENCES UNIVERSITY OF COPENHAGEN Proteome analysis of stored carrots grown in different cropping systems for evaluating changes in susceptibility to liquorice rot during storage Sébastien LOUARN 1, Dan Funck JENSEN 1, Birgit JENSEN 1, Ole NØRREGAARD JENSEN 2, Arkadiusz NAWROCKI 2 1 Department of plant biology, Faculty of Life Sciences, University of Copenhagen; 2 Department of Biochemistry and Molecular Biology, University of Southern Denmark. Introduction Liquorice rot of carrot, caused by Mycocentrospora acerina, has been reported to cause post harvest losses of up to or more than 50% in cold stored roots. The fungus is soil borne and over winters in soil as chlamydospores. The spores are brought into the storage with soil adhering to the roots. Carrots resist disease development at the beginning of storage. There is evidence that this is due to chemical defence mechanisms. The resistance decreases during storage, leading to disease development at the later stages of storage. It is hypothesised that specific proteins are important for this resistance and that resistance is affected by cultural practices before harvest, as well as by post harvest storage conditions. Aim The objective of the present project is to monitor the proteome changes in carrots from different cropping systems throughout the storage period and to identify proteins, which are playing key roles in post harvest resistance to liquorice rot. Table 1: Different cropping systems (conventional and organic) used. Overall approach of the project The proteome analysis will be based on comparison of one conventional cropping system and three organic cultural practices. The different cropping systems are described in table 1. Llittle is know about the proteome of carrots and M. acerina and therefore methods must be improved. In a pilot study, carrots from two of the systems (Conventional and Organic 3) were inoculated with clamydospores of M. acerina and subsequently stored at 4 C (Fig. 1). Carrots with and without artificial wounds were included. Infected tissue was sampled 8 weeks after inoculation, frozen in liquid nitrogen and freeze dried. Samples of fungal material from a pure culture of M. acerina grown on V 8 medium were also freeze dried. The proteins were extracted using a lysis buffer containing urea, then the samples were cleaned in an ethanol: acetone solution. Fig 1: Wounded carrot roots infected by M. acerina. Results and future plans Symptoms only developed on wounded carrots after two months of storage. The spores of M. acerina were still viable on non-wounded roots but did not penetrate the roots. During the first protein extraction experiment, only few weak protein spots were observed on the two dimensional gels, and the protein samples from infected tissues were dark brown colored. The efficiency of the protein extraction was improved by increasing the incubation period of the samples in the lysis buffer. The brown coloration from infected tissues was decreased by adding an extra cleaning step. Some proteins have been identified in both pure fungal culture and infected tissues (Fig 2). The analysis of the 2 D gels is in progress. Infection experiments will be repeated and samples taken at different time points during storage to reveal protein changes and how they relate to changes in susceptibility to liquorices rot. Protein changes will be identified on the gels and identified by mass spectrometry. Bioinformatic analysis will be carried out to interpret the changes in the proteome. a b Fig 2: 2 -dimensional electrophoreses gels of proteins from M. acerina pure culture (a) and from infected carrot root tissues (b). The arrows show similar protein spots found in both samples.