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Identification of esterase isoenzymes related to cell differentiation in Armoracia lapathifolia Gilib. tissue culture by gel electrophoresis and mass spectrometry techniques

Biochemical markers, like peroxidase, acid phosphatase and other enzymes, may be useful in predicting developmental events, which are caused by modification of gene expression patterns. Esterases, a group of enzymes that hydrolyse ester bonds, are present in many isoforms in plant and animal cells. Isoesterases have also been studied as markers of embryogenic potential and of embryo development. Some isoesterases are related to tissue transformation of different plant species by the Ti plasmid of Agrobacterium tumefaciens. The goal of the present study was to isolate and identify esterases related to cell differentiation in horseradish (Armoracia lapathifolia Gilib.) tissues propagated in vitro. Crown-gall tumours were induced on the leaf fragments with a wild octopine strain B6S3 of A. tumefaciens. Two lines of transformed tissues were established: unorganised tumour without any morphogenic capacity and teratoma capable of shoot formation. The esterase isoenzyme patterns of horseradish leaf, teratoma and tumour tissues were compared. Isoenzymes were separated electrophoretically in polyacrylamide gels (8-18 % native gels and 12.5 % SDS gels) and by isoelectric focusing (IEF, pH 3-9). Two esterase substrates, 1- and 2-naphthylacetate, were applied for isoenzyme detection. Esterases from horseradish tissue were purified by classical chromatography techniques – ion exchange and size exclusion. After separation by IEF, proteins active towards esterase substrates were excised from gels and subjected to in-gel digestion with trypsin. Analysis of tryptic peptides with mass spectrometry (MS) and MS/MS of certain peptides, combined with database searching (databases SwissProt and NCBInr, via programs mMass and Mascot), allowed identification of several horseradish esterases.

esterase; Armoracia lapathifolia; protein identification; mass spectrometry

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