The regulation of pheophorbide a oxygenase (PaO) in the 'green seed' problem in canola and during leaf senescence.

摘要

Brassica napus, canola, is an important oil seed crop grown extensively in North America and northern Europe with annual yields exceeding seven million metric tons. Under normal field conditions, canola seeds produce chloroplasts during early seed development and then catabolize the photosynthetic machinery during seed maturation, producing seeds that are essentially free of chlorophyll. However an early frost can disrupt the normal pattern of chlorophyll degradation in canola seed development, resulting in green seed at harvest and significantly devaluing the crop. Candidate steps in the chlorophyll degradation pathway that may be disrupted by freezing conditions have been identified. Pheophorbide a oxygenase (PaO) is considered to be a key enzyme in regulating chlorophyll degradation. Pheophorbide a, the substrate of PaO, and other chlorophyll catabolites from canola seeds accumulated after exposure to freezing conditions. Freezing was found to interfere with the induction of PaO activity that normally occurs in the later phases of canola seed development when chlorophyll is supposed to be cleared from the seed. Moreover, it is demonstrated that the regulation of PaO activity is largely post-translational and it is at this level where freezing interferes with PaO activation in canola seeds. Detached Arabidopsis and canola leaves which senesce and degrade chlorophyll rapidly in darkness over a period of six days were used as a model system to further study the regulation of PaO. The levels of PaO transcript, protein and enzymatic activity in the leaves all increased dramatically during senescence but by different amounts. As was observed during seed development, the increase in PaO activity during leaf senescence was greater than could be explained by the increase in protein level. It was shown that PaO is phosphorylated and that the level of phosphorylation increased with activity implicating PaO phosphorylation as a posttranslational control mechanism. Additionally, two PaO genes, BnPaO1 and BnPaO2, were identified from Brassica napus leaves and both of which are highly homologous to AtPaO. Both BnPaO genes were highly induced in canola leaves during senescence. However, the expression of BnPaO2 was measured in seeds, while BnPaO1 transcripts were not detected at anytime during seed development.