Characterization of transplastomic tobacco plants with a plastid localized barley 4-hydroxyphenylpyruvate dioxygenase.

The 4-hydroxyphenylpyruvate dioxygenase (HPD) is the only enzyme of the biosynthetic pathway of tocopherols and tocotrienols localized outside of plastids. In order to investigate whether a plastid localized HPD could give rise to an increase in tocopherol levels of tobacco leaves and seeds, in the present study the HPD enzyme was transferred to the plastid by introducing a cDNA specific for the barley hpd gene into the plastome of tobacco. The expression of the hpd gene expression cassette was demonstrated by accumulation of the hpd gene-specific transcript and by a higher resistance of the transgenic seedlings towards the HPD specific inhibitor sulcotrione. The alpha-tocopherol content was increased in leaves of the transplastomic plants, whereas the transplastomic seeds contained a significantly increased gamma-tocochromanol level. With respect to enhanced accumulation of the tocopherol content in plants tissue, overexpression of the hpd gene in plastids did not prove to be advantageous in comparison to transgenic plants expressing high HPD level in the cytoplasm. It is hypothesized that homogentisate synthesized in plastids will have to pass the envelope membrane in order to be accessible to the following enzymes of the tocopherol biosynthetic pathway.