Abstract
In mature seeds of Brassica napus three major and three minor myrosinase isoenzymes were identified earlier. These myrosinases are known to be encoded by at least two different families of myrosinase genes, denoted MA and MB. In the work described in this paper the presence of different myrosinase isoenzymes in embryos, seedlings, and vegetative mature tissues of B. napus was studied and related to the expression of myrosinase MA and MB genes in the same tissues to facilitate future functional studies of these enzymes. In developing seeds, myrosinases of 75, 73, 70, 68, 66, and 65 kD were present. During seedling development there was a turnover of the myrosinase pool such that in 5-d-old seedlings the 75-, 70-, 66-, and 65-kD myrosinases were present, with the 70- and 75-kD myrosinases predominating. In 21-d-old seedlings the same myrosinases were present, but the 66- and 65-kD myrosinase species were most abundant. At flowering the mature organs of the plant contained only a 72-kD myrosinase. MA genes were expressed only in developing seeds, whereas MB genes were most highly expressed in seeds, seedling cotyledons, young leaves, and to a lesser extent other organs of the mature plant. During embryogenesis of B. napus, myrosinase MA and MB gene transcripts started to accumulate approximately 20 d after pollination and reached their highest level approximately 15 d later. MB transcripts accumulated to about 3 times the amount of MA transcripts. In situ hybridization analysis of B. napus embryos showed that MA transcripts were present predominatly in myrosin cells in the axis, whereas MB genes were expressed in myrosin cells of the entire embryo. The embryo axiz contained 75-, 70-, and 65-kD myrosinases, whereas the cotyledons contained mainly 70- and 65-kD myrosinases. Amino acid sequencing revealed the 75-kD myrosinase to be encoded by the MA gene family. The high degree of cell and tissue specificity of the expression of myrosinase genes suggests that studies of their transcription should provide interesting information concerning a complex type of gene regulation.