Characterization of tomato PHYB1 and identification of molecular defects in four mutant alleles

The structure of the gene encoding the apoprotein of phytochrome B1 (PHYB1) in tomato has been determined from genomic and cDNA sequences. In contrast to PHYA, PHYB1 lacks an intron upstream of the first ATG. A single transcr iption start site was found by 5' RACE at -116. Tomato PHYB1 spans 7 kb sta rting from the first ATG. The coding region is organized into four exons as for other angiosperm PHY. The deduced apoprotein consists of 1131 amino ac ids, with a molecular mass of 125.4 kDa. Tomato phytochrome B1 shares 78% a nd 74% identity with Arabidopsis phytochromes B and D, respectively. Along with the normally spliced full-length transcripts, sequences of reverse tra nscriptase-PCR clones revealed five types of alternative transcripts. Each type of alternative transcript was missing a considerable part of the codin g region, including the chromophore-binding site. The four putative PHYB1 mutants in tomato, which are temporarily I ed-light insensitive (tri), were each confirmed to have a mutation in PHYB1. Each m utation arose from a different, single-base substitution. Allele tri(1) is presumably a null because the mutation introduces a stop at codon 92. In tr i(3), val-238 is replaced by Phe. The importance of this valine residue is evidenced by the fact that the tri3 phenotype is as strong as that of tri(1 ) Alleles tri(2) and tri(4) encode proteins truncated at their C-termini. T he former lacks either 170 or 438 amino acids, depending upon which of two types of splicing occurs during transcript maturation, while the latter lac ks 225.