First published online July 18, 2002; 10.1104/pp.003285
Plant Physiol, August 2002, Vol. 129, pp. 1592-1599
Complex Formation of Myrosinase Isoenzymes in Oilseed Rape Seeds
Are Dependent on the Presence of Myrosinase-Binding
Proteins1
Susanna
Eriksson,2
Erik
Andréasson,3
Barbara
Ekbom,
Georg
Granér,
Bo
Pontoppidan,4
Jan
Taipalensuu,5
Jiaming
Zhang,6
Lars
Rask, and
Johan
Meijer*
Department of Plant Biology, Genetics Center (S.E., E.A., G.G.,
B.P., J.T., J.Z., J.M.), and Department of Entomology (B.E.), Swedish
University of Agricultural Sciences, S-750 07 Uppsala, Sweden; and
Department of Medical Biochemistry and Microbiology, Biomedical Center,
Uppsala University, S-751 23 Uppsala, Sweden (L.R.)
The enzyme myrosinase (EC 3.2.3.1) degrades the secondary
compounds glucosinolates upon wounding and serves as a defense to
generalist pests in Capparales. Certain myrosinases are present in complexes together with other proteins such as myrosinase-binding proteins (MBP) in extracts of oilseed rape (Brassica
napus) seeds. Immunhistochemical analysis of wild-type seeds
showed that MBPs were present in most cells but not in the myrosin
cells, indicating that the complex formation observed in extracts is
initiated upon tissue disruption. To study the role of MBP in complex
formation and defense, oilseed rape antisense plants lacking the seed
MBPs were produced. Western blotting and immunohistochemical staining confirmed depletion of MBP in the transgenic seeds. The exclusive expression of myrosinase in idioblasts (myrosin cells) of the seed was
not affected by the down-regulation of MBP. Using size-exclusion chromatography, we have shown that myrosinases with subunit molecular masses of 62 to 70 kD were present as free dimers from the antisense seed extract, whereas in the wild type, they formed complexes. In
accordance with this, MBPs are necessary for myrosinase complex formation of the 62- to 70-kD myrosinases. The product formed from
sinalbin hydrolysis by myrosinase was the same whether MBP was present
or not. The performance of a common beetle generalist (Tenebrio
molitor) fed with seeds, herbivory by flea beetles
(Phyllotreta undulata) on cotyledons, or growth rate of
the Brassica fungal pathogens Alternaria
brassicae or Lepthosphaeria maculans in the presence of seed extracts were not affected by the down-regulation of
MBP, leaving the physiological function of this protein family open.
1
This work was supported by the Swedish
University of Agricultural Sciences, by the Nordic Joint Committee for
Agricultural Research, by the Swedish Research Council for Agriculture
and Forestry, and by the Foundation for Strategic Research.
2
Present address: AlphaHelix AB, Uppsala Science Park
SE-751 83 Uppsala, Sweden.
3
Present address: Department of Plant Physiology,
Institute of Molecular Biology, University of Copenhagen, Oester
Farimagsgade 2A DK-1353 Copenhagen, Denmark.
4
Present address: Amersham Biosciences, Björkgatan
30 SE-751 84 Uppsala, Sweden.
5
Present address: Länssjukhuset Ryhov, Department
of Laboratory Medicine, SE-551 85 Jönköping, Sweden.
6
Present address: Department of Botany, University of
Wyoming, P.O. Box 3165, Aven Nelson Building, Laramie, WY
82071-3165.
*
Corresponding author; e-mail johan.meijer{at}vbiol.slu.se; fax
46-18-673389.
© 2002 American Society of Plant Physiologists
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