Transcription of lncRNA <i>ACoS-AS1</i> is essential to <i>trans</i>-splicing between <i>SlPsy1</i> and <i>ACoS-AS1</i> that causes yellow fruit in tomato

Phytoene synthase (PSY) has been considered as an important regulatory enzyme in carotenoids biosynthesis pathway. Previous study finds that the yellow fruit in <i>Solanum lycopersicum</i> var. <i>cerasiforme</i> accession PI 114490 is caused by loss-of-function of SlPSY1 due to <i>trans</i>-splicing between <i>SlPsy1</i> and an unknown gene transcribed from neighbour opposite strand DNA of <i>SlPsy1</i>. The genomic DNA sequences of <i>SlPsy1</i> between red and yellow-fruited tomato lines have one single-nucleotide polymorphism (SNP) in the fourth intron and one SSR in the intergenic region. In the current study, the cause of <i>trans</i>-splicing event was further investigated. The data showed that the previously defined unknown gene was a putative long non-coding RNA <i>ACoS-AS1</i> with three variants in many yellow-fruited tomato lines. The intronic SNP and intergenic SSR were tightly associated with <i>trans</i>-splicing event <i>SlPsy1-ACoS-AS1</i>. However, transgenic tomato lines carrying the genomic DNA of <i>SlPsy1</i> from PI 114490 did not generate transcripts of <i>ACoS-AS1</i>and <i>SlPsy1-ACoS-AS1</i> suggesting that only the intronic SNP could not cause the <i>trans</i>-splicing event. Over-expression of <i>SlPsy1-ACoS-AS1</i> in red-fruited tomato line M82 did not have any phenotype change while over-expression of wild type <i>SlPsy1</i> resulted in altered leaf colour. Sub-cellular localization analysis showed that SlPSY1-ACoS-AS1 could not enter plastids where SlPSY1 has its enzyme activity. Mutation of <i>ACoS-AS1</i> in PI 114490 generated by CRISPR/Cas9 techniques resulted in red fruits implying that <i>ACoS-AS1</i> was essential to <i>trans</i>-splicing event <i>SlPsy1-ACoS-AS1</i>. The results obtained here will extend knowledge to understand the mechanism of <i>trans</i>-splicing event <i>SlPsy1-ACoS-AS1</i> and provide additional information for the regulation of carotenoids biosynthesis.