Transcriptional reprogramming strategies and miRNA-mediated regulation networks of Taxus media induced into callus cells from tissues

Abstract

Background: Taxus cells are a potential sustainable and environment-friendly source of taxol, but they have low survival ratios and slow grow rates. Despite these limitations, Taxus callus cells induced through 6 months of culture contain more taxol than their parent tissues. In this work, we utilized 6-month-old Taxus media calli to investigate their regulatory mechanisms of taxol biosynthesis by applying multiomics technologies. Our results provide insights into the adaptation strategies of T. media by transcriptional reprogramming when induced into calli from parent tissues.

Results: Seven out of 12 known taxol, most of flavonoid and phenylpropanoid biosynthesis genes were significantly upregulated in callus cells relative to that in the parent tissue, thus indicating that secondary metabolism is significantly strengthened. The expression of genes involved in pathways metabolizing biological materials, such as amino acids and sugars, also dramatically increased because all nutrients are supplied from the medium. The expression level of 94.1% genes involved in photosynthesis significantly decreased. These results reveal that callus cells undergo transcriptional reprogramming and transition into heterotrophs. Interestingly, common defense and immune activities, such as "plant-pathogen interaction" and salicylic acid- and jasmonic acid-signaling transduction, were repressed in calli. Thus, it's an intelligent adaption strategy to use secondary metabolites as a cost-effective defense system. MiRNA- and degradome-sequencing results showed the involvement of a precise regulatory network in the miRNA-mediated transcriptional reprogramming of calli. MiRNAs act as direct regulators to enhance the metabolism of biological substances and repress defense activities. Given that only 17 genes of secondary metabolite biosynthesis were effectively regulated, miRNAs are likely to play intermediate roles in the biosynthesis of secondary metabolites by regulating transcriptional factors (TFs), such as ERF, WRKY, and SPL.

Conclusion: Our results suggest that increasing the biosynthesis of taxol and other secondary metabolites is an active regulatory measure of calli to adapt to heterotrophic culture, and this alteration mainly involved direct and indirect miRNA-induced transcriptional reprogramming. These results expand our understanding of the relationships among the metabolism of biological substances, the biosynthesis of secondary metabolites, and defense systems. They also provide a series of candidate miRNAs and transcription factors for taxol biosynthesis.

Keywords: Plant defenses; Taxol biosynthesis; Taxus callus; Transcription factors; miRNA.

Fig. 1

Transcriptional alterations in callus cells Total six samples from two groups were high-throughput sequenced. Six samples were validated by Pearson correlation analysis (a), and they were obviously separated into two groups, callus cells and tissues. All differentially expressed genes were annotated with KEGG database, and the significant DE pathways were showed in (b). These DEGs were analyzed by Mapman3.6.0, and a metabolism overview were showed in (c)

Fig. 2

Function annotation of miRNA and degraded targets. Degraded DE genes were annotated to be involved in nearly all differentially expressed pathways (a). Moreover, the DE degraded genes significantly enriched in 19 pathways (b). Among the opposite expressed miRNAs and targets, there were six miRNAs and six degraded targets found to have the most targets and be targeted respectively (c). The DE miRNAs and their DE targets mostly enriched in 5 pathways, including “Plant-Pathogen Interaction”, “Plant Hormone signaling transduction”, “Ascorbate and aldarate metabolism”, “Starch and sucrose metabolism” and “Aminoacyl-tRNA biosynthesis”. FC was short for foldchange

Fig. 3

Taxol biosynthesis in callus cells and tissues Taxol biosynthesis was significantly upregulated in callus cells. a Taxol biosynthesis pathways. Genes/pathways in red indicated they were upregulated, blues were downregulated, darks had no differences. And bold gens mean they were targeted by miRNAs. MEP and MVA are short for Non-mevalonate pathway and Mevalonate pathway. Solid arrows mean the enzymatic step were certificated, while dotted arrows mean there were several unknown steps. b Taxanes content in callus cells and tissues. DBIII: 10-Deacetylbaccatin III, BIII: baccatin III, EDT: 10-deacetyl taxol. c Box-plot of expression values of taxol biosynthesis genes. Ns mean the different was not significantly in callus and tissues. TS (TASY): taxadiene synthase, DBAT: 10-deacetylbaccatin III-10-O-acetyl transferaseferase, PAM: phenylalanine ammonia-lyase, T5H: taxadiene 5-alpha hydroxylase, TAT: taxadienol acetyl transferase, T10H: 5-alpha-taxadienol-10-beta-hydroxylase, T13H: 13-alpha-hydroxylase gene, DBBT: taxane 2-alpha-O-benzoyltransferase, DBTNBT: 3′-N-debenzoyltaxol N-benzoyltransferase, BAPT: phenylpropanoyltransferase

Fig. 4

Annotation of degraded targets of 10 miRNAs. These 10 miRNAs, which targeted to taxol biosynthesis genes, degraded 226 genes totally. And these degraded targets were functional annotated with GO (a) and KEGG (b), they were significantly involved in the pathways that callus cells mainly transcriptional reprogrammed. The pre-miRNA of Pc-5p-97202_13, which targeted to taxol biosynthesis genes was predicted to form a stable hairpin in structure (c)

Fig. 5

Alterations of defense activities and biosynthesis of secondary metabolites in callus cells. Callus cells altered the cellular responses (a), responses of Biotic- and Abiotic-stress were dominantly changed. “Plant-pathogen interaction” and “Plant hormone signaling transduction” (b) were crucial pathways of biotic- and abiotic-stress responses, genes of them were dominantly downregulated. Biosynthesis of secondary metabolites were mostly upregulated (c)

Fig. 6

Transcription factors were important targets of miRNAs. More than half of TF families were significantly degraded by miRNAs (a), and several TFs were degraded by more than one miRNAs so that degraded fragments were much more than the DE TFs, suggesting these TFs played crucial roles in transcriptional reprogramming in callus cells (b)

Fig. 7

Candidate TFs and miRNAs related to regulation of taxol biosynthesis. Previously, transcriptional profiles during long-term subculture process were detected. Thus, integrative analysis of these expression profiles with Callus&Tissues were helpful to identify the key transcription factors. All DE TFs were analyzed their expression patterns comparing with 12 known taxol biosynthesis genes (a), and the co-expression relation values of candidate TFs were showed in (b). The candidate miRNAs and co-expression values were showed in (c)

Fig. 8

miRNA regulation models during Callus formation and long-term subculture. The regulation model of miRNA on transcriptional reprogramming in Callus cells. All the pathways in Figure were significantly differentially expressed, and the thickness of the arrows indicated the regulation strength by miRNAs. Some pathways also significantly changed but they were not regulated by miRNA, such as isoflavonoid biosynthesis. The pathways in green indicated most DEGs of these pathways were downregulated, red indicated most DEGs were upregulated, black means the upregulated genes of these pathways were as much as the downregulated ones. Most pathways of secondary metabolism were barely regulated by miRNAs directly, probably the regulation model was a “miRNA-TF-enzyme genes” mode