Overexpression of Arabidopsis COP1 results in partial suppression of light-mediated development: evidence for a light-inactivable repressor of photomorphogenesis.

Arabidopsis seedlings are genetically endowed with the capability to follow two distinct developmental programs: photomorphogenesis in the light and skotomorphogenesis in darkness. The regulatory protein CONSTITUTIVE PHOTO-MORPHOGENIC1 (COP1) has been postulated to act as a repressor of photomorphogenesis in the dark because loss-of-function mutations of COP1 result in dark-grown seedlings phenocopying the light-grown wild-type seedlings. In this study, we tested this working model by overexpressing COP1 in the plant and examining its inhibitory effects on photomorphogenic development. Stable transgenic Arabidopsis lines overexpressing COP1 were generated through Agrobacterium-mediated transformation. Overexpression was achieved using either the strong cauliflower mosaic virus 35S RNA promoter or additional copies of the wild-type gene. Analysis of these transgenic lines demonstrated that higher levels of COP1 can inhibit aspects of photomorphogenic seedling development mediated by either phytochromes or a blue light receptor, and the extent of inhibition correlated quantitatively with the vivo COP1 levels. This result provides direct evidence that COP1 acts as a molecular repressor of photomorphogenic development and that multiple photoreceptors can independently mediate the light inactivation of COP1. It also suggests that a controlled inactivation of COP1 may provide a basis for the ability of plants to respond quantitatively to changing light signals, such as fluence rate and photoperiod.     

Cloning and characterization of hydrogen uptake genes from Rhizobium leguminosarum.

A gene library of genomic DNA from the hydrogen uptake (Hup)-positive strain 128C53 of Rhizobium leguminosarum was constructed by using the broad-host-range mobilizable cosmid vector pLAFR1. The resulting recombinant cosmids contained insert DNA averaging 21 kilobase pairs (kb) in length. Two clones from the above gene library were identified by colony hybridization with DNA sequences from plasmid pHU1 containing hup genes of Bradyhizobium japonicum. The corresponding recombinant cosmids, pAL618 and pAL704, were isolated, and a region of about 28 kb containing the sequences homologous to B. japonicum hup-specific DNA was physically mapped. Further hybridization analysis with three fragments from pHU1 (5.9-kb HindIII, 2.9-kb EcoRI, and 5.0-kb EcoRI) showed that the overall arrangement of the R. leguminosarum hup-specific region closely parallels that of B. japonicum. The presence of functional hup genes within the isolated cosmid DNA was demonstrated by site-directed Tn5 mutagenesis of the 128C53 genome and analysis of the Hup phenotype of the Tn5 insertion strains in symbiosis with peas. Transposon Tn5 insertions at six different sites spanning 11 kb of pAL618 completely suppressed the hydrogenase activity of the pea bacteroids.