Exploring the Effect of Methyl Jasmonate on the Expression of microRNAs Involved in Biosynthesis of Active Compounds of Rosemary Cell Suspension Cultures through RNA-Sequencing

Abstract

Our aim in the experiment was to study the effects of methyl jasmonates (MeJA) on the active compounds of rosemary suspension cells, the metabolites' change of contents under different concentrations of MeJA, including 0 (CK), 10 (M10), 50 (M50) and 100 μM MeJA (M100). The results demonstrated that MeJA treatments promoted the accumulation of rosmarinic acid (RA), carnosic acid (CA), flavonoids, jasmonate (JA), gibberellin (GA), and auxin (IAA); but reduced the accumulations of abscisic acid (ABA), salicylic acid (SA), and aspartate (Asp). In addition, 50 and 100 μM MeJA promoted the accumulation of alanine (Ala) and glutamate (Glu), and 50 μM MeJA promoted the accumulation of linoleic acid and alpha-linolenic acid in rosemary suspension cells. Comparative RNA-sequencing analysis of different concentrations of MeJA showed that a total of 30, 61, and 39 miRNAs were differentially expressed in the comparisons of CKvsM10, CKvsM50, CKvsM100, respectively. The analysis of the target genes of the differentially expressed miRNAs showed that plant hormone signal transduction, linoleic acid, and alpha-linolenic acid metabolism-related genes were significantly enriched. In addition, we found that miR160a-5p target ARF, miR171d_1 and miR171f_3 target DELLA, miR171b-3p target ETR, and miR156a target BRI1, which played a key role in rosemary suspension cells under MeJA treatments. qRT-PCR of 12 differentially expressed miRNAs and their target genes showed a high correlation between the RNA-seq and the qRT-PCR result. Amplification culture of rosemary suspension cells in a 5 L stirred bioreactor showed that cell biomass accumulation in the bioreactor was less than that in the shake flask under the same conditions, and the whole cultivation period was extended to 14 d. Taken together, MeJA promoted the synthesis of the active compounds in rosemary suspension cells in a wide concentration range via concentration-dependent differential expression patterns. This study provided an overall view of the miRNAs responding to MeJA in rosemary.

Keywords: MeJA; RNA-seq; Rosmarinus officinalis L.; active compounds; miRNAs; stirred bioreactor; suspension cells.

Figure 1

Content of physiological and biochemical indicators in rosemary suspension cells under different concentrations of MeJA for 48 h. (A) rosmarinic acid (mg RA/g DW); (B) carnosic acid (mg CA/g DW); (C), flavonoids (mg flavonoids/g DW); (D) gibberellin (pmol GA/g FW); (E), abscisic acid (ng ABA/g FW); (F) auxin (μmol IAA/g FW); (G) jasmonate (pmol JA /g FW); (H) salicylic acid (μg SA/g FW); (I) alanine (μg Ala/g FW); (J) glutamate (μg Glu/g FW); (K) aspartate (μg Asp/g FW); (L) linoleic acid (mg/g DW); (M) alpha-linolenic acid (mg/g DW). Different letters above the bars respectively indicate a significant difference (p < 0.05) from CK (0) among the CK and MeJA treatment groups. Error bars represent SDs (n = 3).

Figure 2

Length distribution of small RNA sequences in the small RNA libraries. Four sRNA libraries 0 (CK), 10 (M10), 50 (M50), and 100 μM MeJA (M100).

Figure 3

Numbers of miRNA members in each family in rosemary.

Figure 4

Differently expressed miRNAs in CKvsM10, CKvsM50, and CKvsM100. (A) the numbers of miRNAs up or downregulated in the CKvsM10, CKvsM50, and CKvsM100 (>1.5-fold and p < 0.05); (B) A Venn diagram representing the unique and common regulated miRNAs in the CKvsM10, CKvsM50, and CKvsM100; (C) Differentially expressed known miRNAs in response to MeJA. From the red to the blue corresponds to the numerical value of log2(TPM) from the high to the low; (D) Differentially expressed novel miRNAs in response to MeJA. From the red to the blue corresponds to the numerical value of log2(TPM) from the high to the low.

Figure 5

The top 20 KEGG pathways enriched by target genes of differentially expressed miRNAs in the six comparisons. Red indicates significant enrichment and gray indicates no significant enrichment.

Figure 6

Network of MeJA-responsive miRNAs and their targets. Network analysis was performed using the Cytoscape network platform. (A) The interaction network in CKvsM10; (B) The interaction network in CKvsM50; (C) The interaction network in CKvsM50; Colored triangle nodes represented miRNAs, and circular nodes represented mRNAs. Solid lines indicated interaction associations between miRNAs and mRNAs.

Figure 6

Network of MeJA-responsive miRNAs and their targets. Network analysis was performed using the Cytoscape network platform. (A) The interaction network in CKvsM10; (B) The interaction network in CKvsM50; (C) The interaction network in CKvsM50; Colored triangle nodes represented miRNAs, and circular nodes represented mRNAs. Solid lines indicated interaction associations between miRNAs and mRNAs.

Figure 7

qRT-PCR verification of miRNAs and target genes in rosemary suspension cells under different concentrations of MeJA.

Figure 8

Amplification culture of rosemary suspension cells in 5 L stirring bioreactor. (A) Changes in the cell growth between the stirred bioreactor and shake flask; (B) The effects of MeJA on the rosemary suspension cells in 5 L stirred bioreactor. Different letters above the bars respectively indicate a significant difference (p < 0.05) among the CK and MeJA treatment.

Figure 9

Model depicting the regulatory miRNA-mediated mechanisms of metabolite biosynthesis under MeJA. Differentially expressed miRNAs and their target genes of plant hormone signal transduction; lipoic acid metabolism; alpha-linolenic acid metabolism; alanine, aspartate and glutamate metabolism; and phenylpropanoid and terpenoids biosynthesis pathways which played an essential beneficial in rosemary suspension cells responding to MeJA. The picture shows rosemary cell culture system. The parameters, miRNAs, and target genes named in red indicate the difference of level and expression under different concentrations of MeJA.