Manipulation of growth and development in tobacco through targeted inactivation of the Phytochrome Chromophore

The photo-regulatory activity of the plant photoreceptor phytochrome requires covalent attachment of the plastid-synthesised linear tetrapyrrole, phytochromobilin. Constitutive expression of the mammalian enzyme biliverdin reductase (BVR) in plants inactivates chromophore biosynthesis, resulting in a loss of all phytochromes throughout development. Extensive molecular, biochemical and physiological analyses of tobacco plants {Nicotiana tabacum cv. Maryland Mammoth), expressing BVR targeted to both cytosol and plastid, have been used to investigate the consequences of total phytochrome deficiency on the growth and development of a short-day plant.Constitutive expression of BVR in tobacco resulted in reduced germination, elongated hypocotyls, smaller cotyledons and decreased chlorophyll content in light-grown seedlings. Impaired photomorphogenic responses under continuous far-red and red wavelengths are consistent with deficiencies in both phytochrome A and phytochrome B activities. Mature plants with plastid-targeted BVR displayed elongated intemodes, altered leaf morphology and reduced chlorophyll levels, consonant with deficiencies in multiple phytochromes. Photoperiodic sensitivity was reduced in transgenic plants with all lines flowering early in photoperiods above 10.5 hours. This result is consistent with an inhibitory role for phytochrome in controlling flowering in short-day plants. Cytosol-targeted BVR lines showed an intermediate phenotype between that of plastid-targeted BVR lines and wild-type plants, a result consistent with a partial reduction in photoactive holophytochrome. Targeting of BVR to plastids also resulted in perturbations of the tetrapyrrole pathway and altered plastid morphology. A severe protochlorophyllide deficiency was recorded in dark-grown seedlings, despite increased levels of ALA synthesis. Transcription of Mg-chelatase subunits was unaffected in these plants and feeding studies revealed no post-transcriptional impairment of Mg-chelatase activity. In contrast, a marked inhibition of ALA synthesis was observed in light-grown seedlings, an effect resulting in pronounced cotyledon bleaching. Decreased levels of chlorophyll a/b binding protein {CAB) transcript and reduced granal stacking were also observed. Altered expression patterns of tetrapyrrole synthesis enzymes were recorded in plastid-targeted BVR seedlings grown both in the dark and the light and are consistent with a role for tetrapyrroles in plastid-nuclear signalling