Cloning and characterization of two chlorophyll synthesis genes expressed during tomato fruit development
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Abstract
Full-length clones that encode tomato (Lycopersicon esculentum Mill.) glutamate-1-semialdehyde-2,1-aminomutase (GSAAM) and 5-aminolevulinic acid dehydratase (ALAD) were isolated and characterized. Sequence analysis showed that tomato GSAAM and ALAD clones exhibited a high level of homology to corresponding clones found in other plant species. The primary structure of GSAAM predicted a 481-amino acid precursor that comprised a 46.7 kDa, 437-amino acid mature protein and a transit peptide of 44 amino acids. The primary structure of ALAD predicted a 430-amino acid precursor that comprised a 41.7 kDa, 383-amino acid mature protein and a 47-amino acid transit sequence. Southern analysis showed that GSAAM and ALAD genes are present in low copy number (1 to 2) in the tomato genome. Northern analysis showed that the abundance of GSAAM transcripts declined throughout tomato fruit development and ripening, whereas ALAD transcripts showed little or no change in abundance throughout this same time period. Expression of these two genes also differed at the protein level. GSAAM protein content decreased dramatically by 25 days postanthesis, and GSAAM protein was undetectable by day 45. Western analysis revealed the presence of two closely-spaced proteins that reacted with the ALAD antibody. The higher molecular weight band decreased in abundance throughout tomato fruit development and ripening, whereas the abundance of the lower molecular weight band remained constant. These results show that GSAAM, but not ALAD, may be regulated developmentally at the level of transcript abundance and that both GSAAM and ALAD may be regulated at one or more posttranscriptional levels.