The role of pheophorbide a oxygenase expression and activity in the canola green seed problem

Under normal field growth conditions, canola (Brassica napus) seeds produce chloroplasts during early seed development and then catabolize the photosynthetic machinery during seed maturation, producing mature seeds at harvest that are essentially free of chlorophyll (Chl). However, frost exposure early in canola seed development disrupts the normal programming of Chl degradation, resulting in green seed at harvest and thereby significantly devaluing the crop. Pheophorbide a oxygenase (PaO), a key control point in the overall regulation of Chl degradation, was affected by freezing. Pheophorbide a, the substrate of PaO, accumulated during late stages of maturation in seeds that had been exposed to freezing during early seed development. Freezing interfered with the induction of PaO activity that normally occurs in the later phases of canola seed development when Chl should be cleared from the seed. Moreover, we found that the induction of PaO activity in canola seed was largely posttranslationally controlled and it was at this level that freezing interfered with PaO activation. The increased accumulation of PaO transcript and protein levels during seed development was not altered by the freezing episode, and the increase in PaO protein was small compared to the increase in PaO activity. We found that PaO could be phosphorylated and that phosphorylation decreased with increasing activity, implicating PaO dephosphorylation as an important posttranslational control mechanism for this enzyme. Two PaO genes, BnPaO1 and BnPaO2, were identified in senescing canola leaves and during early seed development, but only BnPaO2 was expressed in maturing, degreening seeds.