Characterization of the gene encoding the apoprotein of phytochrome B2 in tomato, and identification of molecular lesions in two mutant alleles.

The structure of the gene encoding the apoprotein of tomato phytochrome B2 (PHYB2) has been determined from genomic and cDNA sequences. The coding region is organized into four exons, like almost every other angiosperm phytochrome (phy). The deduced phyB2 apoprotein (PHYB2) consists of 1121 amino acids, with 82, 74 and 70% identity to tomato PHYB1, Arabidopsis PHYB, and Arabidopsis PHYD, respectively. In order to facilitate the identification of new mutants, we constructed a double mutant that is deficient in phyA and phyB1. When grown in white light, this mutant becomes only slightly taller than wild type and is similar in phenotype to the monogenic phyB1-deficient mutant. This double mutant has been used as the parent line for mutagenesis with gamma radiation. Several recessive mutants with long hypocotyls and reduced anthocyanin content were selected under white light and screened for mutations in PHYB2, PHYE and PHYF. Two of the triple-mutant lines, designated 55H and 70F, had elongated hypocotyls and fruit trusses, and pale immature fruits. Both belong to the same complementation group and both were found to have defects in PHYB2. Line 70F was found by Northern analysis to have a slightly larger PHYB2 transcript. Part or all of the intron between the second and third exons was found to be retained following RT-PCR of PHYB2 mRNA from line 70F. Three base substitutions were detected near the donor splice site for this intron, including a change from the consensus /GT to /GA at the 5' end of this intron. In every case, the C-terminal 164 amino acids of PHYB2 were replaced by 59 nonsense amino acids followed by a stop codon. Sequencing of PHYB2 from 55H revealed a single-nucleotide deletion near the end of the third exon, resulting in one incorrect codon followed immediately by a stop codon. The predicted mutant apoprotein in 55H is 90 residues shorter than wild-type PHYB2.