Pyruvate-kinase isoenzymes from zygotic and microspore-derived embryos of Brassica napus : Developmental profiles and subunit composition.

Polyclonal antibodies against castor-oil seed cytosolic and leucoplastic pyruvate kinases (PKc and PKp, respectively; EC 2.7.1.40) were utilized to examine the subunit compositions and developmental profiles of canola (Brassica napus L. cv. Topas) PKc and PKp over 6 d of seed germination and 35 d of culture of microspore-derived embryos. The PKc from germinating seeds appears to be composed of a single type of 56-kDa subunit, whereas the enzyme from cultured embryos contains equal proportions of immunologically related 57- and 56-kDa subunits. The PKp was immunologically undetectable in germinating seeds, while the enzyme from cultured embryos consisted of immunologically related 64- and 58-kDa subunits in a ratio of about 1∶2, respectively. The large increase in PK activity that occurs between the second and fourth days of seed gemination is based upon de-novo synthesis of PKc. Between 7 and 14 d of culture of microspore-derived embryos, the levels of PKp and PK maximal activity increased approx. 3- and 2.5-fold, respectively. These increases were coincident with an approximately fourfold rise in the in-vivo pyruvate: phosphoenolpyruvate concentration ratio. Conversely, PKc was not only far less abundant relative to PKp, but its level remained constant over 35 d of microspore-embryo culture. Developing non-zygotic (microspore-derived) embryos strongly resembled ripening zygotic (seed) embryos in terms of PK specific activity as well as relative amounts and subunit compositions of PKc and PKp. The results indicate that the synthesis of PK isoenzymes in B. napus seeds is highly regulated and that this regulation follows a preset developmental program.