Transcriptional Responses and Gentiopicroside Biosynthesis in Methyl Jasmonate-Treated <i>Gentiana macrophylla</i> Seedlings

<div><p><i>Gentiana macrophylla</i>, a medicinal plant with significant pharmacological properties, contains the bioactive compound gentiopicroside. Methyl jasmonate (MeJA) is an effective elicitor for enhancing the production of such compounds. However, little is known about MeJA-mediated biosynthesis of gentiopicroside. We investigated this phenomenon as well as gene expression profiles to determine the molecular mechanisms for MeJA-mediated gentiopicroside biosynthesis and regulation in <i>G</i>. <i>macrophylla</i>. Our HPLC results showed that <i>Gentiana macrophylla</i> seedlings exposed to MeJA had significantly higher concentrations of gentiopicroside when compared with control plants. We used RNA sequencing to compare transcriptional profiles in seedlings treated for 5 d with either 0 μmol L^-1 MeJA (C) or 250 μmol L^-1 MeJA (M5) and detected differentially expressed genes (DEGs). In total, 77,482 unique sequences were obtained from approximately 34 million reads. Of these, 48,466 (57.46%) sequences were annotated based on BLASTs performed against public databases. We identified 5,206 DEGs between the C and M5 samples, including genes related to the α-lenolenic acid degradation pathway, JA signaling pathway, and gentiopicroside biosynthesis. Expression of numerous enzyme genes in the glycolysis pathway was significantly up-regulated. Many genes encoding transcription factors (e.g. ERF, bHLH, MYB, and WRKY) also responded to MeJA elicitation. Rapid acceleration of the glycolysis pathway that supplies precursors for IPP biosynthesis and up-regulates the expression of enzyme genes in that IPP pathway are probably most responsible for MeJA stimulation of gentiopicroside synthesis. Our qRT-PCR results showed that the expression profiles of 12 gentiopicroside biosynthesis genes were consistent with the RNA-Seq data. These results increase our understanding about how the gentiopicroside biosynthesis pathway in <i>G</i>. <i>macrophylla</i> responds to MeJA.</p></div

Secondary metabolism pathways in <i>Gentiana macrophylla</i> sequencing.

<p>Secondary metabolism pathways in <i>Gentiana macrophylla</i> sequencing.</p

DEGs involved in gentiopicroside biosynthesis pathway in M5 samples compared with C samples.

<p>DXS, 1-deoxy-D-xylulose-5-phosphate synthase; DXR, deoxylulose 5-phosphate reductoisomerase; MCT, 2-C-methyl-D-erythritol 4-phosphate cytidylyltransferase; CMK, 4-(cytidine -5’-diphospho)- 2-C-methyl-D- erythritol kinase; MCS, 2-C-methyl-D-erythritol-2,4- cyclodiphosphate synthase; HDS, 1-hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate synthase; HDR, 1-hydroxy-2-methyl -2-(E)-butenyl 4-diphosphate reductase; IDI, isopentenyl diphosphate isomerase; AACT, acetyl-CoA C-acetyltransferase; HMGS, 3-hydroxy-3-methylglutaryl-coenzyme A synthase; HMGR, 3-hydroxy-3- methylglutaryl-coenzyme A reductase; MK, mevalonate kinase; PMK, 5-phosphomevelonate kinase; MVD, mevalonate-5-pyrophosphate decarboxylase; GPPS, geranyl diphosphate synthase; GES, geraniol synthase; G10H, geraniol 8-oxidase/geraniol 10-hydroxylase; CPR, cytochrome P450 reductase; 8/10HGO, 8-hydroxygeraniol oxidoreductase; IS, iridoid synthase; IO, iridoid oxidase; 7-DLGT, 7-deoxyloganetic acid glucosyl transferase; 7-DLH, 7-deoxyloganic acid hydroxylase; LAMT, loganic acid O-methyltransferase; SLS, secologanin synthase. Red squares, putative encoding genes are up-regulated; blue squares, putative encoding genes are down-regulated.</p

Effects of MeJA application (250 μmol L^-1) on gentiopicroside biosynthesis in <i>Gentiana macrophylla</i> seedlings.

<p>(A). Chromatogram of extraction from seedlings not exposed to MeJA. (B). Chromatogram of extraction from seedlings after 5 d of MeJA treatment. (C). Chromatogram of gentiopicroside standard. (D). Changes over time in gentiopicroside concentrations in treated seedlings relative to levels in untreated control (C) plants.</p

qRT-PCR validation of 12 unigenes involved in gentiopicroside biosynthesis in <i>G</i>. <i>macrophylla</i> seedlings.

<p>DXS, 1-deoxy-D-xylulose-5-phosphate synthase; DXR, deoxylulose 5-phosphate reductoisomerase; GES, geraniol synthase; G10H, geraniol 8-oxidase/geraniol 10-hydroxylase; 8/10HGO, 8/10-hydroxygeraniol oxidoreductase; HMGR, 3-hydroxy-3-methylglutaryl-coenzyme A reductase; HMGS, 3-hydroxy-3-methylglutaryl-coenzyme A synthase; MVD, mevalonate-5- pyrophosphate decarboxylase; CPR, cytochrome P450 reductase; HDS, 1-hydroxy-2-methyl-2- (E)-butenyl 4-diphosphate synthase; HDR, 1-hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate reductase.</p

COG classifications of assembled unigenes.

<p>COG classifications of assembled unigenes.</p

DEGs involved in alpha-linolenic acid metabolism in M5 samples compared with C samples.

<p>3.1.1.4, secretory phospholipase A2; <a href="http://www.kegg.jp/dbget-bin/www_bget?ec:1.13.11.12" target="_blank">1.13.11.12</a>, lipoxygenase; <a href="http://www.kegg.jp/dbget-bin/www_bget?ec:4.2.1.92" target="_blank">4.2.1.92</a>, allene oxide synthase; 1.1.1.1, alcohol dehydrogenase; <a href="http://www.kegg.jp/dbget-bin/www_bget?ec:5.3.99.6" target="_blank">5.3.99.6</a>, allene oxide cyclase; <a href="http://www.kegg.jp/dbget-bin/www_bget?ec:1.3.1.42" target="_blank">1.3.1.42</a>, 12-oxophytodienoic acid reductase; OPCL1, OPC-8:0 CoA ligase 1; ACX, acyl-CoA oxidase; <a href="http://www.kegg.jp/dbget-bin/www_bget?ec:2.3.1.16" target="_blank">2.3.1.16</a>, acetyl-CoA acyltransferase 1; <a href="http://www.kegg.jp/dbget-bin/www_bget?ec:2.1.1.141" target="_blank">2.1.1.141</a>, jasmonate O-methyltransferase. Green box, putative encoding gene is down-regulated; red boxes, putative encoding genes are up-regulated; blue boxes, some putative encoding genes are up-regulated while others are down-regulated.</p

DEGs up-regulated in JA biosynthesis and JA signaling pathway in M5 samples compared with expression in C samples.

<p>DEGs up-regulated in JA biosynthesis and JA signaling pathway in M5 samples compared with expression in C samples.</p