In recent work, researchers from the Beijing Academy of Agriculture and Forestry Sciences and the Chinese Academy of Sciences characterized the molecular mechanism of color formation in an orange-fruited tomato inbred line, orange fruited tomato3 (oft3). Using high-performance liquid chromatography (HPLC), they found that oft3 fruit had a markedly reduced carotenoid content, as well as a higher β-carotene/lycopene ratio during ripening. Further genetic analysis through crossing experiments suggested that oft3 was controlled by a single recessive gene. Bulk segregant analysis by high-throughput sequencing (BSA-Seq) and fine mapping combined with genome sequence analysis identified SlIDI1, which harbored a 116-bp deletion, as the candidate gene for the oft3 locus. Functional complementation and CRISPR-Cas9 knockout experiments confirmed that SlIDI1 was the causal gene.
Next, the authors confirmed that SlIDI1 produced both long and short transcripts simultaneously by alternative transcription initiation and alternative splicing. Expression of a green fluorescent protein fusion revealed that the long isoform was mainly localized in plastids and that an N-terminal 59-amino acid extension sequence was responsible for its plastid targeting. Short transcripts were identified in leaves and fruit by 5′ RACE and in fruit by 3′ RACE; their corresponding proteins lacked transit peptides and/or putative peroxisome targeting sequences, respectively.
Click here to see more...