Molecular analysis of PHYA in wild-type and phytochrome A-deficient mutants of tomato

Tomato (Lycopersicon esculentum Mill., recently redesignated Solanum lycopersicum L.), an agronomically important crop plant, has been adopted as a model species complementary to Arabidopsis in which to characterize the phytochrome family. Here we describe the cloning and molecular characterization of the gene encoding the apoprotein of phytochrome A in wild-type tomato and in the far-red-light-insensitive (fri1 and fri2) tomato mutants. The physical organization of this gene is similar to that of other angiosperm phytochromes with the four exons of the coding region interrupted by three introns. The pool of transcripts is heterogeneous due to multiple transcription start sites and to three modes of alternative splicing of the 5' leader. The leader in each alternative transcript carries multiple upstream open reading frames of considerable length. At the genomic level, both fri mutants share an identical base substitution which changes a consensus AG/ to TG/ at the 3' end of the intron between exons 1 and 2. This mutation leads to aberrant processing of the resultant pre-mRNA. While most mature transcripts retain the mutated intron, both cryptic splicing and exon skipping were also detected. Cryptic splicing occurred both upstream and downstream from the wild-type splice site. These observations are consistent with the hypothesis that exon definition in splicing of plant pre-mRNAs plays a secondary role to that of intron definition. Analysis of the frequency with which potentially functional phytochrome A apoproteins might be produced indicates that both fri1 and fri2 have less than 1% of the wild-type phytochrome A level.