engleski

THE STRESS REGULATED ASR PROTEIN CAN BE DETECTED IN IN VITRO GROWN TISSUES OF THE CACTUS Mammillaria gracilis

Abscisic acid-, Stress-, and Ripening-induced (ASR) proteins are plant specific, low molecular weight, heat-stable proteins that have a high hydrophilicity. ASR proteins were shown to possess transcription factor and chaperon-like activities. They are involved in regulation of sugar, branched amino acids and cell wall metabolism, and in plant tolerance to drought and salinity. The ASR protein family is widespread in the plant kingdom ; ASR genes were cloned from a number of gymnosperm, monocotyledon and dicotyledon plant species. However, the model plant Arabidopsis as well as other species from the Brassicaceae family lack ASR genes, suggesting that ASR proteins are not ubiquitous to all plant species. Cacti plants are highly tolerant to water stress. Thus, it was interesting to find out if they encode ASR proteins. We analyzed two in vitro-grown tissues (callus and tumor), from the cactus M. gracilis Pfeiff. in order to reveal if the ASR-like proteins can be found in the member of the Cactaceae family. ASR proteins are acid soluble, and they can be effectively purified to homogeneity on Ni-NTA-agarose, since they contain an authentic pentahistidine sequence close to their N-termini. Therefore, we took advantage of ASR histidine-rich tract to purify the ASR-like protein from cactus callus and tumor tissues. Eluted proteins from Ni-NTA-agarose, were separated by SDS-PAGE. One gel was Coomassie stained and the proteins on the other gel were subsequently transferred onto the nitrocellulose membrane and treated with anti-ASR1 antibody. Protein bands which reacted with anti-ASR1 antibody on the membrane were excised from the gels and subjected to mass spectrometry analysis after which they were identified as ABA- and ripening-induced protein. Once we uncovered ASR-like protein presence in cactus tissues, our aim was to determine what impact abiotic stress has to ASR-like protein expression. For that purpose, both callus and tumor were subjected to short-term salt- and mannitol-induced osmotic stress and further analyzed. It was determined that salinity and osmotic stress in cactus tissue culture caused induction in ASR-like protein expression. ASR-like protein appeared as 15 kDa protein in both control tissues. In response to NaCl and mannitol, one additional band of around 30 kDa appeared. The 30 kDa protein was visible in callus tissue treated with mannitol and particularly pronounced in tumor tissue treated with both NaCl and mannitol.

abiotic stress; ASR protein; cactus; osmotic stress; salinity

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