Fig 1.
A solid arrow represents a known step and a dotted arrow represents an assumed step revised from Ma et al. and Gao et al [8,13]. Enzymes of the MEP pathway (2-C-methyl-D-erythritol 4-phosphate) are as follows: DXS, 1-deoxy-D-xylulose-5-phosphate synthase; DXR, 1-deoxy-D-xylulose-5-phosphate reductoisomerase; MCT, 2-C-methyl-D-erythritol 4-phosphate cytidylyltransferase; CMK, 4-diphosphocytidyl-2-Cmethyl-D-erythritol kinase; MDS, 2-Cmethyl- D-erythritol 2,4-cyclodiphosphate synthase; HDS, 4-hydroxy-3-methylbut-2-enyl diphosphate synthase; and HDR, 4-hydroxy-3- methylbut-2-enyl diphosphate reductase. Enzymes of the mevalonate (MVA) pathway are as follows: AACT, acetyl-CoA acetyltransferase; HMGS, 3-hydroxy-3-methylglutaryl-CoA synthase; HMGR, 3-hydroxy-3-methylglutaryl-CoA reductase; MVK, mevalonate kinase; PMK, 5-phosphomevalonate kinase; and PMD, 5-diphosphomevalonate decarboxylase. Isopentenyl diphosphate isomerase (IPPI) catalyses the isomerisation of dimethylallyl diphosphate (DMAPP) into isopentenyl diphosphate (IPP), whereas conversion of IPP to geranylgeranyl diphosphate (GGPP) is catalysed by geranylgeranyl diphosphate synthase (GGPPS). Copalyl diphosphate synthase (CPS), kaurene synthase-like (KSL) genes and the tanshinone biosynthesis pathway was inferred from previous studies identifying natural diterpenoids from S.miltiorrhiza [2,14]. These steps include a series of hydroxylation, dehydrogenation, and reduction reactions catalysed by cytochrome P450s, dehydrogenase, and reductase.
Fig 2.
The effects of low nitrogen level on the growth in S. miltiorrhiza roots at 45, 60, 75, and 90 days after transplanting (DAT).
(a) Phenotypes of S. miltiorrhiza under Full-N (Nf) and N-deficient conditions (Non-N(N0) and Low-N (Nl)) at the indicated times (Photographed by Li-Lan Lu). (b) Fresh weight of the roots under full-nitrogen (Nf), middle level-nitrogen (Nm), low-nitrogen (Nl), and non-N (N0) conditions at the indicated times. (c) Dry weight of the roots, (d) number of the roots. Values are presented as means ± SD, n = 5. Asterisks (*) indicate significant differences (P < 0.05, Student’s t-test).
Fig 3.
Low nitrogen influencing on the tanshinones in roots of S. miltiorrhiza at 45, 60, 75, and 90 days after transplanting (DAT), respectively.
Values are as means ±SD, n = 5. Asterisks represented significant differences between treatments, NS indicate no significant differences (Student’s t-test, P < 0.05).
Fig 4.
Number and distribution of up- and downregulated genes in the roots of S. miltiorrhiza after treatment with different N levels at 45, 60, and 75 days after transplanting (DAT).
(a) N0 vs. Nf. (b) Nl vs. Nf. (c) N0 vs.Nl.
Fig 5.
Venn diagram of the number of genes in the Nf-N0 (N0 compared to Nf), Nf-Nl, and Nl-N0 groups at 45, 60, and 75 days after transplanting (DAT).
(a) All differentially expressed genes (DEGs), (b) upregulated DEGs, (c) downregulated DEGs.
Fig 6.
Histogram of GO classification of differentially expressed genes (DEGs) in N0 vs. Nf, Nl vs. Nf, and N0 vs. Nl at 75 days after transplanting (DAT).
Blue represents all unigenes annotated in each subcategory, red represents all DEGs annotated in each subcategory. The right y-axis represents the number of genes annotated in each subcategory. The left y-axis represents the percentage of unigenes annotated or DEGs in the main category. (a, b) N0 vs. Nf, (c, d) Nl vs. Nf, (e, f) N0 vs. Nf.
Fig 7.
KEGG enrichment pathways in N0 vs. Nf, Nl vs. Nf, and N0 vs. Nl at 75 days after transplanting (DAT).
All differentially expressed genes, including upregulated and downregulated DEGs, were distributed in the KEGG pathways. The considerably changed pathways indicate the most abundant dots. The rich factor was the ratio of the number of DEGs in a pathway and the number of all genes involved in this pathway. The degree of gene enrichment was enhanced by increasing rich factor and decreasing (Q-value). The Q-value was the rectified by P-value (FDR). (a) All DEGs in N0 vs. Nf, (b) all DEGs in Nl vs. Nf, (c) all DEGS in N0 vs. Nl, (d) upregulated DEGs in N0 vs. Nf, (e) upregulated DEGs in Nl vs. Nf, (f) upregulated DEGs in N0 vs. Nl, (g) downregulated DEGs in N0 vs. Nf, (h) downregulated DEGs in Nl vs. Nf, (i) downregulated DEGs in N0 vs. Nl.
Fig 8.
Activities of glutamine synthetase (GS) and nitrate reductase (NR) in S. miltiorrhiza roots under normal nitrogen supply and nitrogen starvation.
Fig 9.
Transcription expressions (FPKM) of GS family genes (GS1, GS2), Nar1 and nitrate transporter genes NRT2 family (NRT2.1, NRT2.5, NRT2.6, NRT2.7) in S. miltiorrhiza roots under normal nitrogen supply and nitrogen Starvation.
Fig 10.
Proposed terpenoid biosynthesis pathways in S. miltiorrhiza (a). Heat map describing the expression profile of tanshinone biosynthesis-related genes from the MEP and MVA pathways in the roots of S. miltiorrhiza grown under low and full-nitrogen levels in N0, Nl, and Nf at 45, 60, and 75 days after transplanting (DAT) (b). qRT-PCR validation of differentially expressed genes in N0, Nl, and Nf at 45 (c), 60 (d), and 75 (e) DAT. Different letters represent significant differences between treatments (P < 0.05).