Centaurium erythraea, Rafn, Rafn

Matekalo, Dragana, Skorić, Marijana, Nikolić, Tijana, Novaković, Lazar, Lukić, Milana, Božunović, Jelena, Aničić, Neda, Filipović, Biljana & Mišić, Danijela, 2018, Organ-specific and genotype-dependent constitutive biosynthesis of secoiridoid glucosides in Centaurium erythraea Rafn, and its elicitation with methyl jasmonate, Phytochemistry 155, pp. 69-82 : 76-78

2.5. MeJA elicitation of SG biosynthesis in C. erythraea leaves

Secoiridoid glucoside-related defence exists constitutively in C. erythraea plants, but it could also be enhanced upon insect herbivory, pathogen attack, or elicitation. The exogenous application of methyl jasmonate (MeJA) may elicit a response similar to stress, and was used in the present study to stimulate SG accumulation and expression of SG biosynthesis associated genes. Jasmonate and its derivatives are known as plant hormones with a significant role in a diverse set of physiological and developmental processes ( Pauwels et al., 2009; Wasternack, 2007). These plant-specific signalling molecules are widely used for elicitation purposes and for inducing a massive reprogramming of gene expression which leads to an increased production of specialized metabolites. Exogenous application of jasmonates, particularly MeJA, is reported to exert dramatic effects on different biosynthetic pathways, and to positively stimulate the biosynthesis and accumulation of terpenoids, alkaloids, phenolic and polyphenolic compounds by triggering the expression of key genes and/or transcriptional factors ( Ahmad et al., 2016; Cao et al., 2016; Fonseca et al., 2009). Accumulation of SG and the expression of the biosynthesis genes is known to be influenced by MeJA treatment in G. macrophylla ( Cao et al., 2016; Hua et al., 2014), and Swertia mussotii ( Wang et al., 2010) . Boroduske et al. (2016) have previously examined MeJA elicitation effects on C. erythraea shoots, showing that 14-day MeJA treatment reduced 3 content in shoots, while 4 and 5 content was not significantly affected. Several factors could have accounted for such results and the absence of MeJA-elicitation effects, including MeJA dosage, treatment duration, high variability among different genotypes, etc. In another study MeJA treatment induced xanthone accumulation in centaury ( Beerhues and Berger, 1995). In the present study the durations of 250 μM MeJA treatments were 5 and 10 days, and in order to exclude the influence of genotype on phenotype variability, plants used in experiments were of the same genetic background, i.e. of the same genotype. Initial screening of ten C. erythraea genotypes originating form locality Palja (P1-P10), highlighted P2 genotype as a SG low-productive one ( Fig. S2 ). Clonal propagation of P2 was performed through liquid root culture, which enabled efficient and large scale multiplication of shoots ( Fig. 7 A ). Three-months-old rooted shoots, grown on solid ½ MS medium, were transferred onto solid ½ MS medium supplemented with 250 μM MeJA, to find out whether the production of SG in C. erythraea was increased upon 5- and 10-day-long MeJA treatment.

Three-month-old shoots of P2, a low productive genotype, are generally characterized by the almost equal amounts of 3 and 4 (around 5.60 mg 100 mg −1 FW), significant amounts of 2 (2.42 mg 100 mg −1 FW), and considerably lower amounts of 5 (1.04 mg 100 mg −1 FW) and especially of 1 (0.05 mg 100 mg −1 FW). These amounts slightly increase (3, 4 and 5) or decrease (1 and 2) in non-treated plants, after 10 days of experiment, indicating developmental regulation of SG biosynthesis and accumulation. Regardless of the duration of the MeJA treatment (5 or 10 days), an increase in the accumulation of 4, 5 and 1 in P2 shoots was observed ( Fig. 8 ). No significant difference between non-treated and MeJA-treated plants in the amount of 3 was recorded after 5 and 10 days, while the amount of 2 was even slightly decreased upon treatments with MeJA. Compound 3 is presumed to be the first compound arising from 2 in the SG biosynthetic route, and is efficiently converted to 4 and 5. Results indicate that MeJA increases metabolic flux through the SG biosynthetic pathway leading to the production of 3, 4 and 5. Therefore, it is not surprising that the amounts of 1 and 2, the intermediates in the biosynthetic route, in both treated and non-treated plants, remain relatively low. Calculated ratios of 1 / 2 and 2 / 3 further corroborate the highly efficient conversion of 2 to 3 ( Fig. 8 ).

In order to gain an insight into the molecular background of SG overaccumulation caused by MeJA in C. erythraea leaves , and to recognize the major MeJA-responsive genes, we profiled the expression of SG biosynthesis genes by quantitative PCR. Results of relative expressions revealed that nine out of 13 transcripts had higher expression levels in C. erythraea plants grown for 5 days on medium with MeJA compared to non-treated plants ( Fig. 9 ). An increase in G8O, 7DLGT, and CPR1 relative expression was most notable, but for 8HGO, 7DLH2, IO and SLS it was also significant. Relative expression of LAMT and GPPS, on the other hand, was decreased. No significant elevation in gene expression was recorded in C. erythraea plants following 10 days of MeJA treatment ( Fig. 9 ). Downregulation of LAMT after 5 and 10 days is followed by the significant reduction of its product (1) in leaves, which implies that MeJA-mediated changes in the gene expression level precede changes at the product/compound level. Decrease in 1 amount might, at least partially, result from increased SLS gene expression and activity. As previously confirmed in C. roseus, SLS uses 1 as a substrate to produce 2, by direct cleavage of the cyclopentane ring of 1 via a radical or a hydride process ( Inoue et al., 1981). Interestingly, the expression of SLS was enhanced upon MeJA treatments, while the amount of 2 was decreased, which suggests an efficient conversion of 2 into 3, the next compound in the proposed biosynthetic route leading subsequently to 4 and further to 5. Lowered 2 / 3 ratio in MeJA elicited plants ( Fig. 8 ) further supported this presumption. Although genes involved in SG biosynthetic pathway downstream from 2, leading to the synthesis of 3, 4 and 5, are not fully characterized yet, and are therefore not analysed within the present study, an increase in 4 and 5 amounts in leaves by elicitation with MeJA suggests that the expression and activities of these putative enzymes might also be upregulated by MeJA. Further studies are needed in order to confirm this hypothesis.

Our findings are in accordance with previous studies on some Gentianaceae species. MeJA treatment elicited 5 biosynthesis in onemonth old seedlings of Gentiana macrophylla , whereby the biggest increase was detected after 5 days ( Cao et al., 2016). RNA-Seq analysis of G. macrophylla plants treated for 5 days with MeJA and of non-treated plants indicated that 5206 genes were differentially expressed. qPCR analysis confirmed that 8HGO and GES were upregulated, CPR2 was downregulated, while two putative genes encoding for G8O have shown different expression patterns: G10H1 was up-, and G10H2 was downregulated ( Cao et al., 2016). According to Hua et al. (2014) in G. macrophylla plants, after six days of MeJA application both putative genes encoding for G8O, as well as for CPR2 have shown elevated expression, and followed a trend that paralleled the accumulation of secoiridoids under MeJA. In seedlings of Swertia mussotii, MeJA treatment also increased the transcription of G8O, which was followed by an increase in 4 content ( Wang et al., 2010). In seedlings and cell suspensions of C. roseus treated with MeJA, Miettinen et al. (2014) reported induction of SG-pathway genes, although GES/G8O and LAMT/SLS exhibited different induction characteristics. Relative expression levels of CrGES displayed continuing increase over time, reaching maximum 8 h after MeJA treatment and showing a steady decline after 12 h ( Kumar et al., 2015). Actually, according to the literature, several terpenoid-pathway genes such as GPPS, G8O, TDC, STR, D4H, DAT, and transcriptional regulators like ORCA 2 and ORCA 3 are induced in response to MeJA, leading to elevated accumulation of terpenoids ( Peebles et al., 2009; van der Fits and Memelink, 2000; Zhao et al., 2013).

Considering the observed GPPS downregulation upon MeJA treatment ( Fig. 9 ), it should be emphasized that putative CeGPPS analysed within the present study corresponds to homomeric GPPS. Similarly, reverse trend of GPPS expression and SG accumulation was already shown in previous section ( Figs. 3 and 4 ). Rai et al. (2013) reported that MeJA treatment of C. roseus leaves significantly induced the expression of only CrGPPS.SSU, while the expression of CrGPPS.LSU and of homomeric CrGPPS was not affected, ultimately suggesting that involvement of only heteromeric GPPS with CrGPPS.SSU regulates GPP supply for MIA biosynthesis. It is worth noting, though, that no candidates for either CrGPPS.SSU or CrGPPS.LSU were retrieved in the C. erythraea transcriptome.

Interestingly, LAMT was downregulated in both low-productive organs (roots) and in MeJA-treated leaves, which implies that this enzyme is not a limiting factor determining SG biosynthesis and accumulation in C. erythraea . Similarly to the previous two experiments, the expression patterns of G8O, 8HGO and 7DLGT followed a trend of the higher accumulation of SG in C. erythraea plants treated with MeJA, thereby indicating possible rate-determining roles for those genes in SG biosynthesis.

In our experiment, 5-day MeJA treatment significantly altered CPR1 expression but did not affect the expression of CPR2. This is in accordance with findings of Schwarz et al. (2009) who isolated two CPR isoforms and showed their expression to be differentially regulated by MeJA treatment. While the increase of CPR2 mRNA level was strongly induced after 6- and 9-h MeJA-treatment of cell cultures, the CPR1 expression level did not change after this elicitation ( Schwarz et al., 2009). Different experimental set-ups of the two studies which include different growth conditions, plant material (cell cultures vs. shoots), MeJA dosage (100 μM MeJA vs. 250 μM), MeJA treatment duration, etc., must be taken into consideration. Although the expression of CeCPR2 was not significantly altered by MeJA after 5 and 10 days of the treatment, the possibility that this gene overexpression occurs earlier should not be neglected. The CeCPR2 association with P450s involved in some other biosynthetic routes not analysed within the present study (e.g. xanthones and other phenolics) is also possible.

It has been reported that MeJA coordinately regulates CrG10H and CrCPR expression in C. roseus MP 183L suspension cells ( van der Fits and Memelink, 2000), and also induces the expression of two transcription factors involved in the regulation of TIA biosynthesis, ORCA 2 and ORCA 3. Moreover, ORCA 3 was also shown to be involved in the regulation of certain secoiridoid pathway gene expression, including CrCPR ( van der Fits and Memelink, 2000). As previously suggested, ORCA 3 does not influence CrG10H gene expression, indicating that other transcription factors could be involved in its regulation ( Zhou et al., 2010). However, CeCPR1, CeG8O, and CeSLS are obviously coordinately expressed in MeJA-treated C. erythraea plants, which strongly suggests that putative CeCPR1 is a reductase associated with CeG8O, and CeSLS.

Figures

Fig. 2. UHPLC-MS 2 SRM (Single Reaction Monitoring) chromatograms of targeted compounds (loganin, secologanin, sweroside, swertiamarin and gentiopicrin) in young leaves (red line), mature leaves (green line) and roots (orange line), and corresponding MS2 spectra. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Fig. 7. Low productive P2 C. erythraea genotype has been clonally propagated through in vitro root culture (A). Three-month-old shoots with developed roots were used in experiments to examine elicitation effect of 5-day (B and C) and 10-day (D and E) treatments with 250 μM methyl jasmonate (MeJA).

Fig. 8. UHPLC/qqqMS quantification of SG content in non-treated and MeJAtreated C. erythraea plants. The durations of 250 μM MeJA treatments were 5 and 10 days. Orange and light grey bars – control: green and dark grey bars – MeJA treatment. Marked with an orange rectangle are ratios of secologanin to loganin and sweroside. 1 – loganin; 2 – secologanin; 3 – sweroside; 4 – swertiamarin; 5 – gentiopicrin; FW – fresh weight. Error bars represent SE of three biological replicates. In all cases, asterisks denote statistically significant difference (P <0.05) according to post hoc Tukey's test of one way ANOVA. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Fig. 9. qRT-PCR analysis of putative SG biosynthesis genes in C. erythraea plants treated with 250 μM MeJA for either 5 or 10 days. Genes encoding putative key enzymes in the SG biosynthetic pathway: GPPS – geranyl diphosphate synthase, GES – geraniol synthase, G8O – geraniol-8-oxidase, 8HGO – 8-hydrohygeraniol oxidoreductase, IS – iridoid synthase, IO – iridoid oxidase,7DLGT – 7-deoxyloganetic acid glucosyltransferase, 7DLH1 and 7DLH2 – 7-deoxyloganic acid hydrolase, LAMT – loganic acid O-methyltransferase, SLS – secologanin synthase; CPR1 and CPR2 – two putative cytochrome P450 reductases. The relative gene expression was normalized against 18S rRNA gene as an internal control; values for untreated leaves (control) were set as a calibrator. Asterisks denote statistically significant difference (P <0.05) according to Student's t-test.

Fig. 3. UHPLC-MS/MS analysis of organspecific accumulation of SG in C. erythraea (young leaves – yl, mature leaves – ml and roots – r). SG concentration is expressed as mg per 100 mg of FW. Abbreviations: 1 – loganin; 2 – secologanin; 3 – sweroside; 4 – swertiamarin; 5 – gentiopicrin; FW- fresh weight. Error bars represent SE of five biological replicates. In all cases, bars with different letters are significantly different (P <0.05) according to post hoc Tukey's test of one way ANOVA.

Fig. 4. Relative gene expression patterns of 13 selected C. erythraea transcripts in young leaves – yl, mature leaves – ml and roots – r. Genes encoding putative key enzymes in the SG biosynthetic pathway: GPPS – geranyl diphosphate synthase, GES – geraniol synthase, G8O – geraniol-8-oxidase, 8HGO- 8-hydrohygeraniol oxidoreductase, IS – iridoid synthase, IO – iridoid oxidase,7DLGT – 7-deoxyloganetic acid glucosyltransferase, 7DLH1 and 7DLH2 – 7-deoxyloganic acid hydrolase, LAMT - loganic acid O – methyltransferase, SLS – secologanin synthase; genes encoding two putative cytochrome P450 reductases – CPR1 and CPR2 are indicated with green rectangle. The relative expressions of the genes were normalized against 18S rRNA gene as an internal control; r was set as a calibrator. Asterisks denote statistically significant difference (P <0.05) according to Student's ttest. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)