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.

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.

Transcriptome Analysis Reveals Differentially Expressed Genes That Regulate Biosynthesis of the Active Compounds with Methyl Jasmonate in Rosemary Suspension Cells.

To study the effects of Methyl jasmonates (MeJA) on rosemary suspension cells, the antioxidant enzymes' change of activities under different concentrations of MeJA, including 0 (CK), 10 (M10), 50 (M50) and 100 µM MeJA (M100). The results demonstrated that MeJA treatments increased the activities of phenylalanine ammonla-lyase (PAL), superoxide dismutase (SOD), peroxidase (POD), catalase (CAT) and polyphenol oxidase (PPO) and reduced the contents of hydrogen peroxide (H2O2) and malondialdehyde (MDA), thus accelerating the ROS scavenging. Comparative transcriptome analysis of different concentrations of MeJA showed that a total of 7836, 6797 and 8310 genes were differentially expressed in the comparisons of CKvsM10, CKvsM50, CKvsM100, respectively. The analysis of differentially expressed genes (DEGs) showed phenylpropanoid biosynthesis, vitamin B6, ascorbate and aldarate metabolism-related genes were significantly enriched. The transcripts of flavonoid and terpenoid metabolism pathways and plant hormone signal transduction, especially the jasmonic acid (JA) signal-related genes, were differentially expressed in CKvsM50 and CKvsM100 comparisons. In addition, the transcription factors (TFs), e.g., MYC2, DELLA, MYB111 played a key role in rosemary suspension cells under MeJA treatments. qRT-PCR of eleven DEGs showed a high correlation between the RNA-seq and the qRT-PCR result. Taken together, MeJA alleviated peroxidative damage of the rosemary suspension cells in a wide concentration range via concentration-dependent differential expression patterns. This study provided a transcriptome sequence resource responding to MeJA and a valuable resource for the genetic and genomic studies of the active compounds engineering in rosemary.

Exploration of the Effect of Blue Light on Functional Metabolite Accumulation in Longan Embryonic Calli via RNA Sequencing.

Light is an important factor that affects the synthesis of functional metabolites in longan embryogenic calli (ECs). However, analysis of the effect of light on functional metabolites in longan ECs via RNA sequencing has rarely been reported and their light regulation network is unclear. The contents of various functional metabolites as well as the enzymatic activities of superoxide dismutase and peroxidase and the level of H2O2 in longan ECs were significantly higher under blue light treatment than under the other treatments (dark, white). In this study, we sequenced three mRNA libraries constructed from longan ECs subjected to different treatments. A total of 4463, 1639 and 1806 genes were differentially expressed in the dark versus blue (DB), dark versus white (DW) and white versus blue (WB) combinations, respectively. According to GO and KEGG analyses, most of the differentially expressed genes (DEGs) identified were involved in transmembrane transport, taurine and hypotaurine metabolism, calcium transport and so forth. Mapman analysis revealed that more DEGs were identified in each DB combination pathway than in DW combination pathways, indicating that blue light exerts a significantly stronger regulatory effect on longan EC metabolism than the other treatments. Based on previous research and transcriptome data mining, a blue light signaling network of genes that affect longan functional metabolites was constructed and HY5, PIF4 and MYC2 were shown to be the key regulatory genes in the network. The results of this study demonstrate that the expression levels of phase-specific genes vary with changes in longan EC functional metabolites.

Exploration of the effect of blue light on microRNAs involved in the accumulation of functional metabolites of longan embryonic calli through RNA-sequencing.

BACKGROUND: The regulation of functional metabolites under light by structural genes and regulatory genes is understood but the roles of microRNAs in this pathway have rarely been reported and their regulation network is not yet clear. RESULTS: Blue light was most conducive to promoting the synthesis of some functional metabolites in longan embryonic callus (ECs). In this study, we sequenced three small RNA libraries of constructed longan ECs under different light qualities (dark, blue, and white). A total of 29 and 22 miRNAs were differentially expressed in the dark versus blue (DB) and dark versus white (DW) combinations, respectively. According to Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis, most of the differentially expressed miRNA target genes were involved in plant hormone signal transduction, mitogen-activated protein kinase (MAPK) signaling, biosynthesis of unsaturated fatty acids, and so on. Cytoscape analysis of the target genes of miRNAs indicated that miR396b-5p and miR5139 had the most target genes in DB. Moreover, this study also found that miR171f_3 targeted DELLA, miR390e targeted BRI1, miR396b-5p targeted EBF1/2 and EIN3; these miRNAs participated in the blue light signaling network through their target genes and regulated the accumulation of longan functional metabolites. CONCLUSIONS: The results of the study revealed that the expressions of phase-specific miRNAs vary with the change of functional metabolites in longan ECs. This study provides new insights into the molecular mechanisms that allow light to influence plant metabolism. © 2018 Society of Chemical Industry.

[Research on ursolic acid production of Eriobotrya japonica cell suspension culture in WAVE bioreactor].

Through scale-up cultivation of Eriobotrya japonica suspension cells using WAVE bioreactor, the cell growth and ursolic acid (UA) accumulation were studied. The comparison test was carried out in the flask and the reactor with cell dry weight (DW) and UA content as evaluation indexes. The culture medium, DW and UA content were compared in 1 L and 5 L working volumes of bioreactor. The orthogonal test with main actors of inoculation amount, speed and angle of rotation was developed to find the optimal combination, in 1 L working volume of bioreactor. DW of the cell growth and the UA content in bioreactor were higher than those of the shaker by 105.5% and 27.65% respectively. In bioreactor, the dynamic changes of elements in the fluid culture, the dry weight of the cell growth and the UA content in 1 L and 5 L working volumes were similar. Inoculation of 80 g, rotational speed of 26 r · min(-1), and angle of 6 ° was the optimal combination, and the cell biomass of 19.01 g · L(-1) and the UA content of 27.750 mg · g(-1) were achieved after 100 h cultivation in 1 L working volume of bioreactor. WAVE Bioreactor is more suitable than flasks for the E. japonica cell suspension culture, and culture parameters can be achieved from 1 L to 5 L amplification.