Characterization of epithio- and nitrile-specifier proteins: in vitro activities and in planta gene expression patterns
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- Institutt for biologi 
The glucosinolate-myrosinase system has long been considered as a strategy for host defensein Capparales plants. The degradation products from the myrosinase-catalyzed hydrolysis of glucosinolates have been shown to be toxic to a vast range of organisms. The toxicity, however, varies, and the isothiocyanates seem to be the compounds with highest toxicitypotential. But nevertheless, the model plant Arabidopsis thaliana possesses genes encoding specifier proteins, with abilities for promoting nitrile production in lieu of the isothiocyanates.This seems to be more advantageous for the herbivore than the plant, so the biologicalfunction of these proteins has become a debated issue. The potential role of five homologues proteins from A. thaliana were characterizedregarding their specifier activities, and transcription profiles in different tissues and growth stages. The activities of the epithio-specifier protein (ESP encoded by At1g54040), three nitrile-specifier proteins (NSP1-3 encoded by At5g48180, At2g33070 and At3g16400,respectively) and a supposed ancestral protein (ESP_like1 encoded by At3g07720) were characterized using purified, recombinant proteins expressed in Escherichia coli, myrosinase extracted from Sinapis alba, and three glucosinolates (2-propenyl glucosinolate, benzylglucosinolate and indole-3-ylmethyl glucosinolate). The in vitro activity assays were monitored by GC-MS and HPLC-UV. Except for the supposed ancestral protein, the tested ESP and NSPs possess specifier activities. The proportions of nitriles were dependent on the concentration of ferrous ions, buffer conditions, and possibly substrate specificity of both myrosinase and the specifier proteins. The transcription levels for the five genes were monitored in two ecotypes, Col-0 andMt-0 by quantitative real-time RT-PCR. Of the tested organs AtNSP1 and AtNSP2 seem to be transcribed at the highest levels in seeds, with high levels of AtNSP1 in siliques as well. InMt-0 AtNSP1 seems to be up-regulated in rosette leaves like AtESP, while only AtESP seemed to be up-regulated in rosette leaves in Col-0. The roots show predominantly expression ofAtNSP3 in both ecotypes. An elevation of AtESP in inflorescence was observed in Mt-0, but a decrease in Col-0 as compared to an early growth stage. AtESP_like1 seems to be stably expressed in Col-0, but elevated in rosette leaves, root and siliques in Mt-0.