Hairy roots of broccoli were treated with 0, 1, 5, 10, and 15 mmol/L MeJA for 12 h,The results shown that under the concentration of 10 mmol/L MeJA, the total yields of GRA and SF were 5049.27 µg/flask and 2549.34 µg/flask, respectively, which were 2.79 and 2.22-fold of the control(Fig. 1-A. B). Under the induction of 10 mmol/L MeJA concentration, the total yields of hairy roots GRA and SF reached the maximum.
Summed up the above the optimal concentration of MeJA was selected to be 10 mmol/L. On the basis, was added to the hairy roots of broccoli grown 18 d was treated with MeJA a concentration of 10 mmol/L treatment 0, 3, 6, 9, and 12 h, and 0-hour treatment was used as a control. As shown in Fig. 2, the yields of GRA and SF firstly increased then decreased between 3 to 12 h. The yield of GRA and SF reached the highest in 9 h, which were 2.27-fold and 1.74-fold of 0 h, respectively. Therefore, it can be preliminarily concluded that the yield of GRA and SF in broccoli hairy roots treated with 10 mmol/L MeJA reached the maximum at 9 h. At the same time, GRA yield in hairy roots was significantly higher than in medium, while SF yield in medium was significantly higher than in hairy roots (Fig. 2-A.B).
MeJA treatment of plants can stimulate the defense response mechanism in plants, thus affected the synthesis and metabolism of plant metabolites (Baenas et al., 2014). Zang et al (2015) found that treatment MeJA significantly increased the GLS content in hairy roots of turnips. In this study, when MeJA concentration reached 10 mmol/L, the production of GRA and SF in hairy roots reached the maximum. The yields of GRA in medium were lower than that in hairy roots under treatment with MeJA, but the yields of SF in medium were 10 ~ 100-fold of that in hairy roots. There was a difference in the yields of GRA and SF in the suspension culture system of hairy roots in the liquid medium. The reason may be that GRA exists in the vacuole of cells, and SF was produced after the vacuole was broken and contacted with MYR (Makoto et al., 2017). SF was easier to release to medium than GRA with vacuolar protection under the action of shear force generated by the rotation speed of the incubator shaker. Cacao et al (2012) reported that added MeJA to the suspension culture system of silymarin, which increased the amount of silymarin in the liquid medium by 2-fold. It was suggested that MeJA could increase the released number of metabolites to the medium. Probably because the induction of jasmonates makes the plant cell membrane extremely vulnerable to stress (Luo et al., 2010), which affects the permeability of the cell membrane and releases metabolites into the culture medium. The release mechanism of GRA and SF in hairy roots of broccoli treatment by MeJA was yet to be further studied.
3.2 MeJA treatment RNA sequencing of broccoli hairy root transcriptome
Based on the transcriptome sequencing results, 9 samples were selected for this study, and each sample produced approximately 4,214,000 to 6,308,800 paired end reads (Table 2). The GC content of the 9 library sequence data was between 47.47 ~ 48.12%, and the Q30% value (average mass fraction > 30) was 95%, Sequence read rates were all above 90%. The results showed that the quality and accuracy of the sequencing data could be analyzed in the next step.
3.3 Identification of differential expression genes (DEGs) in broccoli hairy roots treatment by MeJA
MeJA-treatment differentially expressed genes of hairy roots of broccoli were identified from the Illumina transcriptome. Broccoli hairy roots were treatment by MeJA for 0, 3 and 9 h. Compared 0 h sample with 3 h sample, a total of 5,757 differential genes (including 1,084 up-regulated genes and 4,673 down-regulated genes) were identified, compared 0 h sample with 6 h sample comparisons identified total differential genes 14,200 (1,392 up-regulated genes, 12,808 down-regulated genes), compared 0 h sample with 9 h sample comparisons identified total differential genes 13,235 (1,419 up-regulated genes, 11,816 down-regulated genes), compared 0 h sample with 12 h sample comparisons identified total differential genes 15,876 (1,540 up-regulated genes, 14,336 down-regulated genes) (Fig. 3A)(q values ≤ 0.001, the gene abundance is 10.) a total of 4,400 genes were regulated simultaneously by MeJA at 0, 3, 6, 9 and 12 h, including 574 up-regulated and 3,826 down-regulated DEGs (Fig. 3B,C).
3.4 GO and KEGG pathway enrichment analysis
In order to understand the gene regulation mechanism of MeJA on GRA and SF anabolic pathways in broccoli hairy roots, GO enrichment analysis was performed on all screened up-regulated and down-regulated DEGs in four groups (0 h and 3 h, 0 h and 6 h, 0 h and 9 h, 0 h and 12 h). As shown in Fig. 4, raised and lowered DEGs from “Cellular components” “Molecular function” and “Biological process” three functional groups were identified 36 and 47 classifications. Up-regulated and down-regulated DEGs enriched common 6 categories in the "Molecular Function" functional group, but the order was different. Up-regulated and down-regulated DEGs in the process of “biological” functional groups were enriched in 18 and 23 categories, in addition to order difference, more down-regulated DEGs enriched in negative regulation of the process of “negative regulation of biological process”, “biological adhesion”, “behavior”, “cell killing” and “locomotion” five categories. However, there were significant differences in the enrichment of up-regulated and down-regulated DEGs in the functional group of “cell component”, and two more classifications were enriched in the down-regulated DEGs than the up-regulated DEGs, which indicated that exogenous MeJA had a greater effect on the “biological process” and “cell component” group.
At the same time, KEGG enrichment analysis was carried out on the up-regulated and down regulated DEGs selected from three groups (0 h and 3 h, 0 h and 6 h, 0 h and 9 h, 0 h and 12 h). As shown in Fig. 5, the top three in up-regulated enriched according to P value were “Alpha-Linolenic acid metabolism”, “Glutathione metabolism” and “Sulfur metabolism”. GLS is a sulfur-containing compounds (Kim et al., 2015), MeJA treatment hairy roots of broccoli promoted GLS metabolism. In addition, it is also enriched " endoplasmic reticulum protein processing". As a signaling molecule, MeJA required a large number of glycoproteins to transmit signals when it functioned in the hairy roots of broccoli. Galactose metabolism would produce a large amount of ATP (Lee et al., 2014), which would provide energy for the GRA biosynthesis in broccoli hairy roots and the conversion of GRA to SF. The increase of GRA production in broccoli hairy roots by MeJA may be due to the enhanced conversion of alanine to GRA, which needed further verification. The top three in down-regulated enrichment pathways of DEGs were “Phenylpropanoid biosynthesis”, “Metabolic pathways” and “Biosynthesis of secondary metabolites”, this indicated that the treatment of broccoli hairy roots with methyl jasmonate mainly affected the synthesis and metabolism of secondary metabolites.
3.5 Analysis of DEGs in the biosynthesis of GRA
To verify the results of RNA-Seq gene expression, qRT-PCR was used to detect the expression of 6 key genes and 3 key transcription factors in GLS synthesis of broccoli hairy roots after MeJA induction for 0,3,6,9 and 12 h. As shown in Fig. 6, the results of RNA-Seq and qRT-PCR matched well, which indicated the results of RNA-Seq were reliable. According to the results of transcriptome sequencing, 6 genes (FPKM > 10) were selected to regulate the biosynthesis of GLS in hairy roots of broccoli. BCAT2 and MAM1 played a major role in the process of side chain elongation, CYP79B1, CYP83B1 and UGT74B1 played a role in the process of core structure construction, and FMOGS-OX5 played a role in the process of side chain secondary modification. According to the Fig. 7, the GLS biosynthesis and transformation pathways containing key genes in hairy roots of broccoli was mapped in Fig. 8. As shown in Figs. 6, 7 and 8, the responses of BCAT2, MAM1, CYP79B1, CYP83B1, UGT74B1, FMOGS-OX5 and transcription factor MYB34, MYB51, MYB122 to MeJA were inconsistent. The six key genes that regulated GLS synthesis, MAM1, CYP79B1, UGT74B1, and FMOGS-OX5, were up-regulated at 0 and 3 h, and down-regulated at the rest of the time; BCAT2 was up-CYP83B1 regulated at 6, 9, 12 h, and at 0, 3 h expression was down-regulated. Lee et al (2018) found that BCAT4 transcripts were observed in phloem, and the accumulation of BCAT4 transcripts followed a circadian rhythm. Subcellular protein localization showed that BCAT4 was localized in vacuole. The results explained the reason for the low expression of BCAT4 gene in GRA synthesis process in this study which was the weakening of phloem and vacuole function of hairy root in liquid suspension culture system resulted in BCAT4 gene expression was low. In addition, the expression of BCAT2 was high in this study, but BCAT3 and BCAT4 were more reported in the process elongation of GLS synthesis amino acid side chain in Brassica (Lee et al., 2018; Sonderby et al., 2010), while BCAT2 has not been reported. Wu et al (2017) found that the expression of GLS gene in 11 tissues of cabbage was higher than that of MAM1 gene in root tissue. Wu et al (2019) found that the treatment of cabbage with MeJA caused up-regulation of CYP79B1 and CYP83B1. However, Kong et al (2016) found that the expression of FMOGS-OX5 in Arabidopsis treated with MeJA was up-regulated compared with that in the control, which was different from this study. The reason may be that there was a difference in gene expression during the secondary side-chain modification of GLS in hairy root of broccoli, Because there are differences in the secondary modification of amino acid side chains, most Brassica plants were very conservative, but there were some differences in some Brassica plants (Sonderby et al., 2010). Comprehensive analyzing data of GRA and SF yield and RNA-Seq, it can be found that MeJA had a promoting effect on GRA and SF production in broccoli hairy roots. The possible reason was that, broccoli hairy roots had a response process to exogenous MeJA treatment in the early stage, and metabolites were accumulated in the later stage, which was similar with the research results that Guo et al (2013) find that jasmonates not only increased the content of indole group GLS in Arabidopsis, but also promoted the accumulation of aliphatic GLS. For the cause of the accumulation of aliphatic GLS in broccoli hairy roots treated by MeJA, it needs to be further explored. transcription factors MYB34 and MYB122 were up-regulated at 3 h, and down-regulated at other time points, MYB51 was up-regulated at 0, 3 h, and down-regulated at 6, 9, 12 h. Deng et al (2020) revealed the reason why exogenous addition of MeJA increased the yield of Salvia miltiorrhiza hairy root from the transcription factor level, which provided a research idea for MeJA-induced broccoli hairy roots to increase GRA and SF production. Jeon et al (2017) treated watercress with MeJA and found that there were differences in the expression of GLS biosynthesis key genes in different tissues. In addition, Wu et al (2017) found that GLS biosynthesis genes in cabbage were more strongly expressed in roots, petioles and aging leaves than in other tissues. This also explains the difference between the GLS synthesis genes of broccoli hairy roots and those of other cruciferous plants.