Growth and morphology were affected by various 5-Az concentrations
Growth and morphological changes in S. miltiorrhiza hairy roots with different concentrations of 5-Az are shown in Fig. 1. The fresh weight and dry weight were measured after 28 days of treatment, and more significant growth retardation of hairy roots was shown for treatment with higher dosages of 5-Az. The fresh weight of hairy roots decreased by approximately 40% when the medium contained 12.5 and 25 μM 5-Az. The mass of hairy roots decreased by more than half when the medium contained greater than 50 μM 5-azacytidine.
Liposoluble active component production was increased by HPLC analysis
The active components in S. miltiorrhiza hairy roots were measured after treatment with different concentrations of 5-Az for 28 days, and the contents of liposoluble components increased significantly during the treatment period. However, 5-Az treatment did not show any effect or even decrease the production of water-soluble components in S. miltiorrhiza hairy roots. As illustrated in Fig. 2, the amounts of liposoluble components were not affected when the concentration of 5-Az was below 25 μM. In particular, the production of liposoluble components, including dihydrotanshinone I, tanshinone I, cryptotanshinone, tanshinone IIA and tanshinone IIB, was increased by 1.5 to 5 times compared with that of the control groups when the 5-Az concentration was greater than 50 μM (Fig. 2-A~E). In contrast to the liposoluble components, 5-Az decreased the production of water-soluble components in hairy roots at all concentrations. The contents of rosmarinic acid and salvianolic acid B declined by approximately 50% and 20% compared with the control groups, respectively (Fig. 2-F, G).
The regulation of gene expression was affected by 5-Az (via RT-PCR)
Gene expression in the biosynthesis pathway of secondary metabolites in S. miltiorrhiza was measured on different days with or without 5-Az treatment. 5-Az enhanced gene expression in the tanshinone-related compounds biosynthesis pathway but decreased most gene expression in the phenolic compounds biosynthesis pathway.
Two biosynthesis pathways are involved in the production of hydrophilic phenolic acids, namely, the phenylpropanoid pathway and tyrosine-derived pathway. In the phenylpropanoid pathway, the gene expression of PAL, C4H and 4CL1 declined by approximately 30%, 20% and 50%, respectively, when S. miltiorrhiza hairy roots were treated with 5-Az for 4 days. On day 7, the levels of PAL and C4H were upregulated to 38% and 24%, respectively, but 4CL1 gene expression still declined by approximately 60%. On day 14, there was little difference in the gene expression of PAL, C4H and 4CL1 compared with the control group. In addition, the gene expression level of HPPR in the tyrosine-derived pathway fluctuated from 0.7- to 1.3-fold during the 5-Az treatment period. The gene expression of TAT was decreased by approximately half on day 1 but increased more than 2-fold from day 4 to day 14 compared with the control group (Fig. 3).
The mevalonate (MVA) pathway in the cytosol and the methylerythritol phosphate (MEP) pathway in plastids are the two biosynthesis pathways that contribute to the accumulation of tanshinones in S. miltiorrhiza. As shown in Fig. 4, the level of HMGR in the MVA pathway was measured during the experiment and showed that 5-Az slightly increased its gene expression level by approximately 16% on day 1, but it gradually declined by approximately 30% over time. Moreover, most genes involved in the MEP pathway, including DXS1, DXS2, DXR, GGPPS, CPS and KSL, showed an upward trend when the hairy roots were cultured with 5-Az within 4 days. The gene expression of DXS2 was upregulated approximately 2.7-fold on day 1 and day 4 but dropped to 1.2-fold on day 7, and there was no difference on day 14 compared with the control group. DXR expression showed a similar trend to that of DXS2. The expression level increased by approximately 44% on day 1 and day 4 and then showed no difference after day 7. Positive regulation of the expression of GGPPS and CPS was also observed. The gene expression of GGPPS increased approximately 1.5-fold on day 1 and day 4, suddenly dropped to 0.7-fold on day 7 and was enhanced again to 1.4-fold on day 14. In contrast, the expression level of CPS was upregulated approximately 1.4-fold on day 1, declined to 1.1-fold on day 4, and then increased up to 1.5-fold on day 14. In contrast to the above-mentioned trend, the relative expression levels of DXS1 and KSL in the MEP pathway showed negative regulation. DXS1 was downregulated by approximately 30% on day 1 and day 4, and there was no difference after day 7. The relative expression of KSL also decreased by approximately 20% on average during the experiment.
DNA methylation was changed by 5-Az
The ratio of methylation levels in genomic DNA was measured on different days with or without 5-Az treatment. The results indicated that there was no significant difference in the methylation ratio on day 1 and day 4, which was only approximately 5% less than that of the control group. However, the DNA methylation level changed sharply after day 4. The ratio decreased by approximately 84% on day 7, while it increased by 27% compared with the control group on day 14 (Fig. 5). Therefore, 5-Az indeed exerted its inhibitory activity to influence the methylation ratio in genomic DNA.
5-Az alters DNA methylation at potential transcription factor binding sites
Based on the sequence information from Szymczyk et al. (2016), several putative transcription factors (TFs) might bind to specific motifs on the CPS promoter and enhance CPS gene expression. To prove that 5-Az might change the DNA methylation patterns in the promoter region, the methylation level of each cytosine on the CPS promotor was investigated. According to our experimental results, samples were collected on day 7 and processed by bisulfite conversion. Then, specific primers were designed to amplify the promoter region. PCR products were analyzed by next-generation sequencing (NGS), and the results were investigated using PLANTPAN 3.0 26 for potential TF binding sites. (Fig. 6).
Our results indicated that the methylation level of cytosine on the CPS promoter was altered during 5-Az treatment compared with the control group. One hundred forty-five cytosines were detected in our amplicons, among which fifty-one showed decreased methylation levels during 5-Az treatment. The regions of approximately 1468 bp to 1704 bp and 791 bp to 803 bp showed a greater alteration in demethylation levels compared with another region; moreover, cytosines at 803 bp, 1470 bp, 1655 bp, 1661 bp, and 1668 bp showed decreased methylation levels to 23.07, 25.01, 40.92, 30.78, and 26.14%, respectively (Table S1).
According to the analysis results, each cytosine might be recognized as having different TF binding, and thirty-six different TFs were identified at the demethylation sites, including alpha-amylase, AP2, ARR-B, AT-hook, B3, bet_v_1, bHLH, bZIP, C2H2, C3H, dehydhydrin, Dof, ERF, G2-like, GATA, GRF, HD-ZIP, homeodomain, LEA_5, MADS box, MIKC, M-type, MYB, MYB/SANT, NAC, NAM, NF-Y, RAV, SBP, TALE, TCP, tify, trihelix, WOX, WRKY, and ZF-HD. Among these TFs, NF-Y and MYB were frequently found at demethylation sites (Table 2).