High-throughput sequencing reveals miRNA effects on the primary and secondary production properties in long-term subcultured Taxus cells.

Meng Zhang,Chunhua Fu,Mingbo Lu,Longjiang Yu,Lin Nie,Yanshan Dong

doi:10.3389/fpls.2015.00604

Meng Zhang, Chunhua Fu + Show 4 more

Open Access

https://doi.org/10.3389/fpls.2015.00604

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Abstract

Plant-cell culture technology is a promising alternative for production of high-value secondary metabolites but is limited by the decreased metabolite production after long-term subculture. The goal of this study was to determine the effects of miRNAs on altered gene expression profiles during long-term subculture. Two Taxus cell lines, CA (subcultured for 10 years) and NA (subcultured for 6 months), were high-throughput sequenced at the mRNA and miRNA levels1. A total of 265 known (78.87% of 336) and 221 novel (79.78% of 277) miRNAs were differentially expressed. Furthermore, 67.17% of the known differentially expressed (DE) miRNAs (178) and 60.63% of the novel DE-miRNAs (134) were upregulated in NA. A total of 275 inverse-related miRNA/mRNA modules were identified by target prediction analysis. Functional annotation of the targets revealed that the high-ranking miRNA targets were those implicated in primary metabolism and abiotic or biotic signal transduction. For example, various genes for starch metabolism and oxidative phosphorylation were inversely related to the miRNA levels, thereby indicating that miRNAs have important roles in these pathways. Interestingly, only a few genes for secondary metabolism were inversely related to miRNA, thereby indicating that factors other than miRNA are present in the regulatory system. Moreover, miR8154 and miR5298b were upregulated miRNAs that targeted a mass of DE genes. The overexpression of these miRNAs in CA increased the genes of taxol, phenylpropanoid, and flavonoid biosynthesis, thereby suggesting their function as crucial factors that regulate the entire metabolic network during long-term subculture. Our current studies indicated that a positive conversion of production properties from secondary metabolism to primary metabolism occurred in long-term subcultured cells. miRNAs are important regulators in the upregulation of primary metabolism.

Highlights

Most of the valuable natural products from plants are present at very low concentrations
baccatin III (BIII) is an important precursor in taxane biosynthesis; notably, this compound could not be detected in CA, whereas the BIII content in NA was as high as 118 μg/g drought weight (DW)
All these results indicated that NA had stronger biosynthesis of secondary metabolites

Summary

Introduction

Most of the valuable natural products from plants are present at very low concentrations. Only 1 kg of taxol is present in a thousand century-old Taxus trees Direct isolation of these secondary metabolites from plant tissues was a rough way leading to an extinct exploration. The long-term subcultured cells frequently have epigenetic modifications (e.g., DNA methylation), and their cellular ploidy levels varied after long-term subculture (Baebler et al, 2005; Miguel and Marum, 2011). These observations implied that the conversion of longterm subcultures is related to a complicated and complex regulatory network. The previous reports could not explain the regulatory mechanisms in detail

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