Scopoletin, the main component of clinical drugs and the functional component of health products, is highly abundant in noni fruit (Morinda citrifolia). Multiple enzyme genes regulate scopoletin accumulation. In the present study, differentially expressed genes of noni were analyzed by RNA sequencing (RNA-Seq) and the full-length genes by isoform-sequencing (Iso-Seq) to find the critical genes in the scopoletin accumulation mechanism pathway. A total of 32,682 full-length nonchimeric reads (FLNC) were obtained, out of which 16,620 non-redundant transcripts were validated. Based on KEGG (Kyoto Encyclopedia of Genes and Genomes) annotation and differential expression analysis, two differentially expressed genes, caffeic acid 3-O-methyltransferase (COMT) and caffeoyl-CoA O-methyltransferase (CCoAOMT), were found in the scopoletin accumulation pathway of noni. Real-time quantitative polymerase chain reaction (q-PCR), phylogenetic tree analysis, gene expression analysis, and the change in scopoletin content confirmed that these two proteins are important in this pathway. Based on these results, the current study supposed that COMT and CCoAOMT play a significant role in the accumulation of scopoletin in noni fruit, and COMT (gene number: gene 7446, gene 8422, and gene 6794) and CCoAOMT (gene number: gene 12,084) were more significant. These results provide the importance of COMT and CCoAOMT and a basis for further understanding the accumulation mechanism of scopoletin in noni.

The increasing popularity of Morinda citrifolia has many medical and health benefits because of its rich polysaccharides (PSC) and polyphenols (PPN). It has become popular to brew the dry M. citrifolia fruit slice as tea in some regions of China. In this study, optimize the extraction parameters of M. citrifolia fruit tea polysaccharides and polyphenols using response surface methodology. The results indicated the highest PSC yield of 17% at 46 °C for 11 min and the ratio of water/M. citrifolia fruit powder was 78 mL/g. The optimum extraction of PPN was at 95 °C for 10 min and the ratio of water/M. citrifolia fruit powder 90 mL/g, with 8.93% yield. Using dry M. citrifolia fruit slices as a tea is reported for the first time. Based on the results, the maximum level of PSC can be obtained under condition by infusing about four dried M. citrifolia fruit slice with average thickness and size in warm boiled water for 11 min, taking a 300 mL cup (300 mL of water) for example. The maximum level of PPN can be obtained by adding three slices of dried M. citrifolia fruit slice to boiled water for 10 min. Considering the powder used in our study, the further pulverization of cutting into powder is more conducive to material precipitation. This study provides a scientific basis for obtaining PSC and PPN from dry M. citrifolia fruit slice tea by brewing.

The relationship of the genes of PAL (phenylalanine ammonia-lyase) and scopoletin was explored to reveal the key gene involved in PAL activity, PAL expression and scopoletin accumulation in noni (Morinda citrifolia L.) leaves and fruit. Our results display how the enzyme activity of PAL in noni fruit and leaves increases gradually with the leaves maturity, and exogenous ethephon induces PAL activity in noni leaves, which is consistent with the scopoletin accumulation law. Based on our previous transcriptome data, three PAL fragments were retrieved, and their full lengths were cloned via RACE; named as McPAL1, McPAL2 and McPAL3. The analysis of qRT-PCR showed that McPAL3 is expressed in the leaves and mature fruit, McPAL2 in the leaves only and McPAL1 mainly in the young stage of fruit. Ethephon induced the expression of McPAL2 and McPAL3 in the leaves, and McPAL1 and McPAL3 in the fruit. In noni fruit treated with ethephon, the expression pattern of McPAL3 was consistent with the trend of the scopoletin content and total PAL activity. We conclude that McPAL3 is the key gene involved in the accumulation of scopoletin, which can be induced by ethylene (ETH).