Identification and characterization of soluble and insoluble myrosinase isoenzymes in different organs of Sinapis alba.
Extraction of Sinapis alba seeds under native conditions solubilized 3 myrosinase isoforms, pool I, II and III, which could be separated by ion exchange chromatography. Sequencing of numerous peptides of the I and III isoforms showed that they belonged to the Myrosinase A (MA) family of myrosinases and that they were encoded by different genes. Western blot analysis of S. alba seed proteins, extracted with a sodium dodecyl sulphate-containing buffer, using an anti-myrosinase monoclonal antibody, showed the presence of two additional myrosinase isoforms with approximate molecular sizes of 62 and 59 kDa. These myrosinases, which only could be solubilized from seeds by inclusion of denaturing agents in the extraction buffer, were by sequence analysis identified as MB myrosinases. These isoenzymes or very similar forms were also present in seedling cotyledons. However, from this tissue, they could be extracted with non-denaturing buffers. In addition, cotyledons contained a 65-kDa MB myrosinase not found in seeds. In contrast, seedling cotyledons contained only minute amounts of pool I and no pool III MA myrosinases, emphasizing the tissue-specific expression of the corresponding gene families. Sequence analysis of myrosinase cDNAs generated cDNA by reversed transcription-polymerase chain reaction using degenerate primers with mRNA isolated from seeds, cotyledons and leaves confirmed the result that the MA isoforms were expressed only in seed tissue, while MB myrosinases were found in all tissues investigated. Furthermore, seed and leaf contained unique MB myrosinase transcripts, suggesting organ-specific expression of individual MB genes.
Uppsala Genetic Center, Department of Plant Biology, Swedish University of Agricultural Sciences, Box 7080, SE-750 07 Uppsala, Sweden; Department of Biochemistry and Molecular Biology, University of Calgary, 3330 Hospital Drive NW, Calgary, AB, T2N 4N1, Canada; Department of Medical Biochemistry and Microbiology, Biomedical Center, Uppsala University, Box 582, SE-751 23 Uppsala, Sweden.
Susanna
S
Eriksson
Bo
B
Ek
Jiaping
J
Xue
Lars
L
Rask
Johan
J
Meijer