Abstract
We have successfully developed a system to produce full-length plant phytochrome assembled with phytochromobilin in Pichia pastoris by co-expressing apophytochromes and chromophore biosynthetic genes, heme oxygenase (HY1) and phytochromobilin synthase (HY2) from Arabidopsis. Affinity-purified phytochrome proteins from Pichia cells displayed zinc fluorescence indicating chromophore attachment. Spectroscopic analyses showed absorbance maximum peaks identical to in vitro reconstituted phytochromobilin-assembled phytochromes, suggesting that the co-expression system is effective to generate holo-phytochromes. Moreover, mitochondria localization of the phytochromobilin biosynthetic genes increased the efficiency of holophytochrome biosynthesis. Therefore, this system provides an excellent source of holophytochromes, including oat phytochrome A and Arabidopsis phytochrome B.
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