Dynamic metabolic and transcriptomic profiling of methyl jasmonate-treated hairy roots reveals synthetic characters and regulators of lignan biosynthesis in Isatis indigotica Fort

Abstract

A molecular description of lignan biosynthesis in Isatis indigotica displaying its synthetic characteristics and regulatory mechanism is of great importance for the improvement of the production of this class of active compounds. To discover the potential key catalytic steps and regulatory genes, I. indigotica hairy roots elicited by methyl jasmonate (MeJA) were used as a source of systematic variation for exploring the metabolic/transcriptional changes and candidate genes that might play key roles in lignan biosynthesis. The reprogramming modulated by MeJA was classified into three distinct phases, referred to as signal responding, transcriptional activation of metabolic pathways and accumulation of metabolites. Candidate genes were pooled according to the three phases and applied to co-expression network analysis. In total, 17 genes were identified as hub genes. 4CL3 was selected to validate its impact on lignan biosynthesis. RNAi of 4CL3 resulted in a significant reduction in lignan production. Taken together with its catalytic property, a major route of lignan biosynthesis in I. indigotica was highlighted, which was catalysed by 4CL3 via the esterization of caffeic acid. In conclusion, this study provides new insights into lignan biosynthesis as well as potential targets for metabolic engineering in I. indigotica.

Keywords: Isatis indigotica; co-expression network; lignan biosynthesis; metabolic profiling; transcriptomic profiling.

Figure 1

Transcriptomic and metabolic shifts modulated by MeJA in I. indigotica hairy roots. (a) PCA reveals transcriptomic shifts during a time course of 0–24 h. (b) PCA reveals metabolic shifts during a time course of 0–36 h. Plots with different colours indicate samples of different time points. Plots in red indicate control groups (0 h). Dark green plots, yellow plots, pink plots, black plots, blue plots and green plots indicated samples treated by MeJA for 1, 3, 6, 12, 24 and 36 h, respectively.

Figure 2

Transcriptomic and metabolic shifts in lignan metabolism as a result of MeJA induction. Heat maps show transcriptional and metabolic shift in samples of each time point. Dashed arrows indicate conversion catalysed by multiple steps. 4CL, 4‐coumarate: CoA ligase; C4H, cinnamate 4‐hydroxylase; CAD, cinnamyl alcohol dehydrogenase; CCoAoMT, caffeoyl‐CoA O‐methyltransferase; CCR, cinnamoyl‐CoA reductase; COMT, caffeic acid 3‐O‐methyltransferase; CS, chorismate synthase; DIR, dirgent; EPSPS, 5‐enolpyruvylshikimate‐3‐phosphate synthase; F5H, ferulate‐5‐hydroxylase; HCT, p‐hydroxycinnamoyl‐CoA shikimate quinate hydroxycinnamoyl transferase; PAL, phenylalanine ammonia‐lyase; PLR, pinoresinol/lariciresinol reductase; SDH, secoisolariciresinol dehydrogenase; SK, shikimate kinase.

Figure 3

Temporal patterns of different expressed genes in response to MeJA induction. (a) 26 expression patterns of all DEGs. Gene expression profiles of individual genes are depicted in red lines, and average expression profiles for each series are depicted in black lines. Each cluster is numbered according to their expression patterns. Line charts with background colour indicate significant expression difference of the cluster. (b) Cluster 18 represents the profile of 418 transcripts that are primarily involved in JA signalling. (c) Cluster 21 represents the profile of 341 transcripts that are primarily related to metabolic pathways.

Figure 4

The Pearson correlation network reveals lignan synthesis and regulation. Different colour of nodes presents TF genes (green), metabolic genes (grey) and metabolites (red). Size of each node indicates amount of correlated genes. Red edges represent positive correlations and blue edges represent negative correlations. Thickness of each edge represents the value of correlation coefficient for each correlated pairs. Hub genes are highlighted with thick edging.

Figure 5

Phenotyping of 4CL expression reduced RNAi hairy roots. qRT‐PCR detected transcription level of 4CL1, 4CL2 and 4CL3 in RNAi‐transgenic hairy roots of (a) 4CL1, (b) 4CL2 and (c) 4CL3 compared with that of controls (empty vector transgenic lines). (d) Content of lariciresinol in transgenic hairy roots. All transcripts and metabolites were detected for three biological replicates. Asterisks indicate statistically significant differences compared with control (Student's t‐test, *P < 0.05, **P < 0.01).

Figure 6

Specific synthesis route of lignan in I. indigotica. The putative route mainly leading to synthesis of lignans is highlighted by red background. Full names of the genes are given in Figure 2.