Integrated Analysis of Transcriptomic and Proteomics Data Reveals the Induction Effects of Rotenoid Biosynthesis of Mirabilis himalaica Caused by UV-B Radiation

Abstract

Mirabilis himalaica (Edgew.) Heimerl is one of the most important genuine medicinal plants in Tibet, in which the special plateau habitat has been associated with its excellent medicinal quality and efficacy. However, the mechanisms by which environmental factors affect biosynthesis of secondary metabolic components remain unclear in this species. In this study, RNA sequencing and iTRAQ (isobaric Tags for Relative and Absolute Quantification) techniques were used to investigate the critical molecular "events" of rotenoid biosynthesis responding to UV-B radiation, a typical plateau ecological factor presented in native environment-grown M. himalaica plants. A total of 3641 differentially expressed genes (DEGs) and 106 differentially expressed proteins (DEPs) were identified in M. himalaica between UV-B treatment and control check (CK). Comprehensive analysis of protein and transcript data sets resulted in 14 and 7 DEGs from the plant hormone signal transduction and phosphatidylinositol signaling system pathways, respectively, being significantly enriched. The result showed that the plant hormone signal transduction and phosphatidylinositol signaling system might be the key metabolic strategy of UV-B radiation to improve the biosynthesis of rotenoid in M. himalaica. At same time, most of the DEGs were associated with auxin and calcium signaling, inferring that they might drive the downstream transmission of these signal transduction pathways. Regarding those pathways, two chalcone synthase enzymes, which play key roles in the biosynthesis of rotenoid that were thought as the representative medicinal component of M. himalaica, were significantly upregulated in UV-B radiation. This study provides a theoretical basis for further exploration of the adaptation mechanism of M. himalaica to UV-B radiation, and references for cultivation standardization.

Keywords: Mirabilis himalaica; UV-B radiation; phosphatidylinositol signaling system; plant hormone signal transduction; rotenoid biosynthesis.

Figure 1

Comparison of physiological indices of the roots of M. himalaica from the UV-B radiation and control check (CK) groups. The abscissa is the treatment time for M. himalaica from pre-treatment (Day 0) to Day 30 after treatment. The ordinate indicates the different enzyme activities or substance content. CAT: catalase; SOD: superoxide dismutase; GSH: glutathione; Pro: proline; POD: peroxidase; MDA: malondialdehyde. The “**” represented the significant difference level between treatments (p-value < 0.01).

Figure 2

Analysis of rotenoid content in the roots of M. himalaica compared UV-B radiation with (CK). The “**” represented the significant difference level between treatments (p-value < 0.01).

Figure 3

Annotation of genes in various databases and statistics of homologous genes. (A) Annotation of M. himalaica transcriptome library assembly genes in different databases. (B) Statistics on the number of homologous genes between M. himalaica and other species.

Figure 4

Functional annotations of transcribed conserved domain (CD)-containing sequences and differentially expressed volcano plots between UV-B radiation and CK. (A) Kyoto Encyclopedia of Genes and Genomes (KEGG) classification of transcribed CD-containing sequences in M. himalaica. (B) Differentially expressed volcano plots between UV-B radiation and CK. In this study, the MARS (MA-plot-based method with Random sampling model) model in the DEGseq v1.20.0 package (http://bioconductor.org/packages/release/bioc/html/DEGseq.html) was used to confirm whether there is a difference between different treatments based on the criteria both |fold change| > 2 and p-value < 0.05. (C) Go functional annotation of different expression unigenes between UV-B radiation and CK.

Figure 5

Gene Ontology (GO) function annotation of differentially expressed proteins between different treatments and differential expression analysis of major GO terms. (A–C) GO function annotation of differentially expressed proteins between different treatments, (A) biological process ontology; (B) molecular function ontology; (C) cellular components Ontology; and (D) Differential expression of major GO terms in different ontology between treatments according to the criteria of |Fold change| > 1.2 and p-value < 0.05, which is used to determine whether there is a difference in the expression levels of proteins between different treatments.

Figure 6

qRT-PCR verification of the key unigenes responding to UV-B radiation.

Figure 7

Key molecular pathways for M. himalaica response to UV-B radiation.

Figure 8

Experimental processing settings and operational procedures. (A) Artificial climate chamber used for the experiment; (B) Experimental processing settings, the number indicates the days of UV-B radiation; and (C) Samples collection and experimental procedures.