Identification of two loci involved in phytochrome expression in Nicotiana plumbaginifolia and lethality of the corresponding double mutant

Four Nicotiana plumbaginifolia mutants exhibiting long hypocotyls and chlorotic cotyledons under white light, have been isolated from M2 seeds following mutagenesis with ethyl methane sulphonate. In each of these mutants, this partly etiolated in white light (pew) phenotype is due to a recessive nuclear mutation at a single locus. Complementation analysis indicates that three mutants, dap5, ems28 and ems3-6-34, belong to a single complementation group called pew1, while dap1 defines the pew2 locus. The mutants at pew1 contain normal levels of immunochemically detectable apoprotein of the phytochrome that is relatively abundant in etiolated seedlings, but are deficient in spectrophotometrically detectable phytochrome, whether seedlings are grown in darkness or light. Moreover, biliverdin, a precursor of the phytochrome chromophore, restores light-regulated responses in pew1 mutants and increases their level of photoreversible phytochrome when grown in darkness. These results indicate that the pew1 locus may be involved in chromophore biosynthesis. The mutant at the pew2 locus displays no photoreversible phytochrome in etiolated seedlings, but does contain normal levels of photoreversible phytochrome when grown in the light. Biliverdin had little effect on light-regulated responses in this mutant. In addition, biliverdin did not alter the level of phytochrome in etiolated seedlings. These observations lead us to propose that this mutant could be affected in the phyA gene itself. We have also obtained the homozygous double mutant at the pew1 and pew2 loci. This double mutant is lethal at an early stage of development, consistent with a critical role for phytochrome in early development of higher plants.