Light-induced LncRNA Regulates The Accumulation Of Anthocyanins In Apple Fruits

Apple is one of the most important fruits in the world.A large amount of data transcriptome analysis showed that eukaryotic genomes up to 90%of the DNA is transcribed,and the transcript was only 1-2%of the ability of the encoded protein.This suggests that most of the RNA molecules produced by most eukaryotic genomes have no protein coding potential.These RNA molecules are known as non-coding RNA.Long non-coding RNA(LncRNA)is a non-coding RNA of longer than 200 nucleotides.With the emergence of new sequencing technologies,thousands of lncRNAs have been identified in several plant species,such as Oryza sativa,Arabidopsis thaliana,Zea mays,Solanum lycopersicum,Triticum aestivum,Malus domestica,Anemone vitifolia etc.A large number of studies have proved that plant LncRNA plays an important role in seed development,light morphogenesis,fruit development,microRNA silencing,miRNA precursor synthesis,and biological and abiotic stress responses.In this study,we try to explore the molecular mechanism of lncRNA regulation of fruit coloration in the late stage of fruit ripening.1.Take the bagged 'Fuji' apple 1 month before the harvest period.The harvested apples were transferred from their bags under low light to an incubator for light treatments(15000 lux,20?)for 8 days.Fruit peels were collected at 0,3,5,8 days to conduct HPLC(High pressure liquid chromatography),gene expression and sequencing analysis.HPLC results showed that the the anthocyanin content increased with the light treatment.2.To characterize lncRNA,miRNA and mRNA expression profiles associated with anthocyanin biosynthesis,the RNA-seq was performed on 12 apple peel libraries(0 days,3 days,5 days and 8 days after initiated light treatment).Weighted network co-expression analysis(WGCNA)were performed by the combine of transcriptome analysis and HPLC content.was performed.The binding between lncRNA and miRNA was predicted using psMimic,psRobot software,and the cleavage between miRNA and mRNA was predicted using psRNATarget software.The ternary regulatory pathway of lncRNA-miRNA-mRNA(MLNC3.2/4.6-miRNA156a-SPL2-like/SPL33)was constructed by binding assay in tobacco leaves.3.The relationship between the miRNA156a and SPL2-like/SPL33 was verified by the RACE(Rapid amplification of cDNA ends)assay.The localization of MLNC3.2/4.6 was detected by in situ hybridization assay and the results showed that the two lncRNAs were localized in the cytoplasm.Transient expression in apple fruit and stable transformation of apple callus showed that overexpression of the eTMs and SPLs promoted anthocyanin accumulation,with the opposite results in eTM and SPLs-silenced fruit.4.To investigate the effect of light quality on lncRNA expression,we examined the anthocyanin content and related gene expression in apple peels under white light,red light,blue light or UV-B treatments with debagged(140 days after bloom,before full ripeness)apple fruit.The results showed white and blue light may participate in anthocyanin accumulation by inducing the interaction of miR156a with MLNC3.2 and MLNC4.6,which function as eTMs by blocking the cleavage of SPL TFs by miR156a the miR156a-MLNC3.2 and MLNC4.6-SPL2-like and SPL33 network is not involved in anthocyanin accumulation in response to UV-B treatment.These results indicate that MLNC3.2 and MLNC4.6 function as eTMs for miR156a and prevent cleavage of SPL2-like and SPL33 by miR156a during light-induced anthocyanin biosynthesis.Our study provides fundamental insights into lncRNA involvement in the anthocyanin biosynthetic pathway in apple fruit.