MeJA Treatment Research Articles

Comparative transcriptome analysis of shoot and root tissue of Bacopa monnieri identifies potential genes related to triterpenoid saponin biosynthesis

BackgroundBacopa monnieri commonly known as Brahmi is utilized in Ayurveda to improve memory and many other human health benefits. Bacosides enriched standardized extract of Bacopa monnieri is being marketed as a memory enhancing agent. In spite of its well known pharmacological properties it is not much studied in terms of transcripts involved in biosynthetic pathway and its regulation that controls the secondary metabolic pathway in this plant. The aim of this study was to identify the potential transcripts and provide a framework of identified transcripts involved in bacosides production through transcriptome assembly.ResultsWe performed comparative transcriptome analysis of shoot and root tissue of Bacopa monnieri in two independent biological replicate and obtained 22.48 million and 22.0 million high quality processed reads in shoot and root respectively. After de novo assembly and quantitative assessment total 26,412 genes got annotated in root and 18,500 genes annotated in shoot sample. Quality of raw reads was determined by using SeqQC-V2.2. Assembled sequences were annotated using BLASTX against public database such as NR or UniProt. Searching against the KEGG pathway database indicated that 37,918 unigenes from root and 35,130 unigenes from shoot were mapped to 133 KEGG pathways. Based on the DGE data we found that most of the transcript related to CYP450s and UDP-glucosyltransferases were specifically upregulated in shoot tissue as compared to root tissue. Finally, we have selected 43 transcripts related to secondary metabolism including transcription factor families which are differentially expressed in shoot and root tissues were validated by qRT-PCR and their expression level were monitored after MeJA treatment and wounding for 1, 3 and 5 h.ConclusionsThis study not only represents the first de novo transcriptome analysis of Bacopa monnieri but also provides information about the identification, expression and differential tissues specific distribution of transcripts related to triterpenoid sapogenin which is one of the most important pharmacologically active secondary metabolite present in Bacopa monnieri. The identified transcripts in this study will establish a foundation for future studies related to carrying out the metabolic engineering for increasing the bacosides biosynthesis and its regulation for human health benefits.

Transcriptome Analysis of Genes Associated with the Artemisinin Biosynthesis by Jasmonic Acid Treatment under the Light in Artemisia annua.

Artemisinin is a sesquiterpene lactone endoperoxide extracted from a traditional Chinese medicinal plant Artemisia annua. Artemisinin-based combination therapies (ACTs) are recommended as the best treatment of malaria by the World Health Organization (WHO). Both the phytohormone jasmonic acid (JA) and light promote artemisinin biosynthesis in A. annua. Interestingly, we found that the increase of artemisinin biosynthesis by JA was dependent on light. However, the relationship between the two signal pathways mediated by JA and light remains unclear. Here, we collected the A. annua seedlings of 24 h continuous light (Light), 24 h dark treatment (Dark), 4 h MeJA treatment under the continuous light conditions (Light-MeJA-4h) and 4 h MeJA treatment under the dark conditions (Dark-MeJA-4h) and performed the transcriptome sequencing using Illumina HiSeq 4000 System. A total of 266.7 million clean data were produced and assembled into 185,653 unigenes, with an average length of 537 bp. Among them, 59,490 unigenes were annotated and classified based on the public information. Differential expression analyses were performed between Light and Dark, Light and Light-MeJA-4h, Dark and Dark-MeJA-4h, Light-MeJA-4h, and Dark-MeJA-4h, respectively. Furthermore, transcription factor (TF) analysis revealed that 1588 TFs were identified and divided into 55 TF families, with 284 TFs down-regulated in the Dark relative to Light and 96 TFs up-regulated in the Light-MeJA-4h relative to Light. 8 TFs were selected as candidates for regulating the artemisinin biosynthesis and one of them was validated to be involved in artemisinin transcriptional regulation by Dual-Luciferase (Dual-LUC) assay. The transcriptome data shown in our study offered a comprehensive transcriptional expression pattern influenced by the MeJA and light in A. annua seedling, which will serve as a valuable resource for further studies on transcriptional regulation mechanisms underlying artemisinin biosynthesis.

The Jasmonate-Activated Transcription Factor MdMYC2 Regulates ETHYLENE RESPONSE FACTOR and Ethylene Biosynthetic Genes to Promote Ethylene Biosynthesis during Apple Fruit Ripening.

The plant hormone ethylene is critical for ripening in climacteric fruits, including apple (Malus domestica). Jasmonate (JA) promotes ethylene biosynthesis in apple fruit, but the underlying molecular mechanism is unclear. Here, we found that JA-induced ethylene production in apple fruit is dependent on the expression of MdACS1, an ACC synthase gene involved in ethylene biosynthesis. The expression of MdMYC2, encoding a transcription factor involved in the JA signaling pathway, was enhanced by MeJA treatment in apple fruits, and MdMYC2 directly bound to the promoters of both MdACS1 and the ACC oxidase gene MdACO1 and enhanced their transcription. Furthermore, MdMYC2 bound to the promoter of MdERF3, encoding a transcription factor involved in the ethylene-signaling pathway, thereby activating MdACS1 transcription. We also found that MdMYC2 interacted with MdERF2, a suppressor of MdERF3 and MdACS1 This protein interaction prevented MdERF2 from interacting with MdERF3 and from binding to the MdACS1 promoter, leading to increased transcription of MdACS1 Collectively, these results indicate that JA promotes ethylene biosynthesis through the regulation of MdERFs and ethylene biosynthetic genes by MdMYC2.

Cloning and functional analysis of the SiRLK35 gene in Setaria italic L.

Receptor like protein kinases (RLKs) play vital roles in both plant development and stress conditions. Using drought-treated "Yugu 1" as materials, a drought-responsive RLK gene, SiRLK35, was isolated through iTRAQ analysis. In this study, the further analyses of the gene functions were carried out. First, full-length SiRLK35 was amplified by PCR using the cDNA of foxtail millet seedlings as a template. The expression patterns of SiRLK35 under NaCl, PEG, ABA, GA and MeJA treatment were analyzed by quantitative real-time PCR (qRT-PCR), and we found that the expression of SiRLK35 could be induced under different treatments, especially under NaCl treatment. Second, the prokaryotic expression plasmid of SiRLK35 was constructed, and the salt resistance of SiRLK35 was detected by the bacterial plaque growth method. And we uncovered that the growth and tolerance of SiRLK35-containing Escherichia coli strains were in better conditions than control under the NaCl stress. Lastly, pCambia1301P-SiRLK35 was constructed and transformed into rice to obtain transgenic plants. The tolerance of transgenic rice plants to salt stress was higher than that of controls through physiological analysis. We propose that SiRLK35 may participate in salt and stress resistance processes, which could provide potential theoretical foundation for the stress resistance varieties cultivation and breeding of foxtail millet.

Characterization of the polyphenol oxidase gene family reveals a novel microRNA involved in posttranscriptional regulation of PPOs in Salvia miltiorrhiza

Salvia miltiorrhiza is a well-known material of traditional Chinese medicine. Understanding the regulatory mechanisms of phenolic acid biosynthesis and metabolism are important for S. miltiorrhiza quality improvement. We report here that S. miltiorrhiza contains 19 polyphenol oxidases (PPOs), forming the largest PPO gene family in plant species to our knowledge. Analysis of gene structures and sequence features revealed the conservation and divergence of SmPPOs. SmPPOs were differentially expressed in plant tissues and eight of them were predominantly expressed in phloem and xylem, indicating that some SmPPOs are functionally redundant, whereas the others are associated with different physiological processes. Expression patterns of eighteen SmPPOs were significantly altered under MeJA treatment, and twelve were yeast extract and Ag+-responsive, suggesting the majority of SmPPOs are stress-responsive. Analysis of high-throughput small RNA sequences and degradome data showed that miR1444-mediated regulation of PPOs existing in P. trichocarpa is absent from S. miltiorrhiza. Instead, a subset of SmPPOs was posttranscriptionally regulated by a novel miRNA, termed Smi-miR12112. It indicates the specificity and significance of miRNA-mediated regulation of PPOs. The results shed light on the regulation of SmPPO expression and suggest the complexity of SmPPO-associated phenolic acid biosynthesis and metabolism.

Cloning, subcellular localization, and heterologous expression of ApNAC1 gene from Andrographis paniculata

Andrographis paniculata is widely used as medicinal herb in China for a long time and andrographolide is its main medicinal constituent. To investigate the underlying andrographolide biosynthesis mechanisms, RNA-seq for A. paniculata leaves with MeJA treatment was performed. In A. paniculata transcriptomic data, the expression pattern of one member of NAC transcription factor family (ApNAC1) matched with andrographolide accumulation. The coding sequence of ApNAC1 was cloned by RT-PCR, and GenBank accession number was KY196416. The analysis of bioinformatics showed that the gene encodes a peptide of 323 amino acids, with a predicted relative molecular weight of 35.9 kDa and isoelectric point of 6.14. To confirm the subcellular localization, ApNAC1-GFP was transiently expressed in A. paniculata protoplast. The results indicated that ApNAC1 is a nucleus-localized protein. The analysis of real-time quantitative PCR revealed that ApNAC1 gene predominantly expresses in leaves. Compared with control sample, its expression abundance sharply increased with methyl jasmonate treatment. Based on its expression pattern, ApNAC1 gene might involve in andrographolide biosynthesis. ApNAC1 was heterologously expressed in Escherichia coli and recombinant protein was purified by Ni-NTA agarose. Further study will help us to understand the function of ApNAC1 in andrographolide biosynthesis.

How to induce defense responses in wild plant populations? Using bilberry (Vaccinium myrtillus) as example.

Inducible plant defense is a beneficial strategy for plants, which imply that plants should allocate resources from growth and reproduction to defense when herbivores attack. Plant ecologist has often studied defense responses in wild populations by biomass clipping experiments, whereas laboratory and greenhouse experiments in addition apply chemical elicitors to induce defense responses. To investigate whether field ecologists could benefit from methods used in laboratory and greenhouse studies, we established a randomized block‐design in a pine‐bilberry forest in Western Norway. We tested whether we could activate defense responses in bilberry (Vaccinium myrtillus) by nine different treatments using clipping (leaf tissue or branch removal) with or without chemical treatment by methyljasmonate (MeJA). We subsequently measured consequences of induced defenses through vegetative growth and insect herbivory during one growing season. Our results showed that only MeJA‐treated plants showed consistent defense responses through suppressed vegetative growth and reduced herbivory by leaf‐chewing insects, suggesting an allocation of resources from growth to defense. Leaf tissue removal reduced insect herbivory equal to the effect of the MeJa treatments, but had no negative impact on growth. Branch removal did not reduce insect herbivory or vegetative growth. MeJa treatment and clipping combined did not give an additional defense response. In this study, we investigated how to induce defense responses in wild plant populations under natural field conditions. Our results show that using the chemical elicitor MeJA, with or without biomass clipping, may be a better method to induce defense response in field experiments than clipping of leaves or branches that often has been used in ecological field studies.

Overexpression of Heteromeric GhACCase Subunits Enhanced Oil Accumulation in Upland Cotton

Acetyl-CoA carboxylase (ACCase) catalyzes acetyl-CoA to produce malonyl-CoA, which is the essential first step in the biosynthesis of de novo fatty acids. The heteromeric ACCase of higher plants, such as upland cotton, is composed of four subunits: biotin carboxyl carrier protein (BCCP), biotin carboxylase (BC), α-subunit of carboxyltransferase (CTα), and β-subunit of carboxyltransferase (CTβ). In this study, four subunits encoding heteromeric ACCase were amplified and the structures were analyzed further. The result showed that 7, 16, 10, and 1 exon(s) were identified from GhBCCP1, GhBC1, GhCTα2, and GhCTβ, respectively. The expression pattern analysis showed that the transcripts of these four subunits were ubiquitous in all the tested tissues and may have a positive correlation between the four subunits and oil accumulation. The four subunits coordinate to enhance oil content. The expression patterns and levels varied under ABA, MeJA treatments, and cold stress. Furthermore, under the control of a seed-specific AGP promoter, overexpression of the four subunits separately in Upland cotton demonstrated that GhBCCP1 transgenic plants showed a significant increase of 21.92% in oil content in cotton seeds, while about a 17% increase was noted for GhBC1 and GhCTβ, respectively. So overexpression of each subunit of heteromeric GhACCase may effectively increase seed oil content in Upland cotton.

Enhancement of polysaccharides accumulation in Dendrobium officinale by exogenously applied methyl jasmonate

The accumulation of polysaccharides, activities of sucrose metabolism enzymes, and the expression of sucrose biosynthetic genes in Dendrobium officinale were significantly affected by exogenous methyl jasmonate (MeJA). Application of MeJA increased the content of polysaccharides and the highest polysaccharide production occurred in the samples treated with 200 μM MeJa. The MeJA application influenced polysaccharide biosynthesis rather than degradation because the activities of sucrose metabolism enzymes and the expressions of sucrose biosynthetic genes were upregulated by MeJA. Interestingly, low MeJA concentrations promoted accumulation of dendrobium polysaccharides, while high MeJA amounts played an inhibitory role. The content of major constituent of polysaccharides, glucose and mannose, also increased after MeJa treatment.

Identification and Characterization of Chalcone Synthase Gene Family Members in Nicotiana tabacum

Chalcone synthase (CHS, EC 2.3.1.74) is a member of the plant polyketide synthase superfamily; it catalyzes the first committed step in the flavonoid biosynthetic pathway. In this study, we identified and characterized five CHS superfamily genes from Nicotiana tabacum. Our phylogenetic analysis suggested that the five tobacco CHS genes have diverged into two subgroups, including the classical CHS genes for NtCHS1–4 and CHS-Like genes for NtCHS5. Solanaceae CHSs diverged into different subfamilies prior to the divergence of the genera. We used RNA-seq data to explore the spatial and temporal expression patterns of the NtCHS1–5 genes. NtCHS1–4 were widely expressed in pigmented or/and non-pigmented vegetative tissues and floral organs. NtCHS5 was expressed strongly in the calyx and corolla organs at the full-bloom stage. Quantitative RT-PCR analysis was performed; it verified our RNA-seq results. Further, the expression of NtCHS1 and NtCHS3 increased markedly under drought and high salt stress. NtCHS2 expression and NtCHS4 expression were obviously induced by ABA and MeJA treatment, and NtCHS4 expression was specifically induced by 2,4-D. NtCHS5 expression was not induced by any of these treatments. Our results represent new data about the differentiation of gene expression and environmental responsiveness in the expression of CHS superfamily genes in Nicotiana tabacum.

Effects of MeJA on Arabidopsis metabolome under endogenous JA deficiency.

Jasmonates (JAs) play important roles in plant growth, development and defense. Comprehensive metabolomics profiling of plants under JA treatment provides insights into the interaction and regulation network of plant hormones. Here we applied high resolution mass spectrometry based metabolomics approach on Arabidopsis wild type and JA synthesis deficiency mutant opr3. The effects of exogenous MeJA treatment on the metabolites of opr3 were investigated. More than 10000 ion signals were detected and more than 2000 signals showed significant variation in different genotypes and treatment groups. Multivariate statistic analyses (PCA and PLS-DA) were performed and a differential compound library containing 174 metabolites with high resolution precursor ion-product ions pairs was obtained. Classification and pathway analysis of 109 identified compounds in this library showed that glucosinolates and tryptophan metabolism, amino acids and small peptides metabolism, lipid metabolism, especially fatty acyls metabolism, were impacted by endogenous JA deficiency and exogenous MeJA treatment. These results were further verified by quantitative reverse transcription PCR (RT-qPCR) analysis of 21 related genes involved in the metabolism of glucosinolates, tryptophan and α-linolenic acid pathways. The results would greatly enhance our understanding of the biological functions of JA.

Occurrence of jasmonates during cystocarp development in the red alga Grateloupia imbricata.

In this study, we highlight the effects of methyl jasmonate (MeJa) on cystocarp development in the red macroscopic alga Grateloupia imbricata. In G.imbricata, jasmonate release is related to the reproductive state, as fertile thalli (i.e., those that have cystocarps) released significant amounts of this volatilecompound (1.27±0.20mM·mg fw-1 ·h-1 ) compared with infertile thalli (0.95±0.12mM·mg fw-1 ·h-1 ). Treating G.imbricata thalli with MeJa revealed a significant increase in cystocarp number (1.5±0.27 cystocarps·mm-2 ), which was ~7.5-fold greater than in untreated thalli (0.2±0.07 cystocarps·mm-2 ). Maturation was completed within 48h with MeJa treatment, a shortening of the typical >3-week maturation period, and included the opening of cystocarps and the presence of dehiscent cavities. Release rates of jasmonates after exogenous MeJa treatment were also modified based on the cystocarp maturation level. All of these effects were reduced in the presence of phenidone, which blocks MeJa production, indicating that the MeJa action is genuine. The effects of MeJa during cystocarp maturation were not replicated by derivatives of reactive oxygen species from the same jasmonic acid biosynthetic pathway, asthe activities of scavenger enzymes and lipid peroxidation were unchanged between infertile and fertile thalli. Therefore, a reactive oxygen species-based mechanism is not involved during cystocarp development. We conclude that MeJa has an independent function as a growth regulator during G.imbricata reproduction.

Overexpression of a garlic nuclear factor Y (NF-Y) B gene, AsNF-YB3, affects seed germination and plant growth in transgenic tobacco

Nuclear factor Y (NF-Y) is a ubiquitous transcription factor found in many eukaryotic organisms. NF-Y consists of three distinct subunits, namely, NF-YA, NF-YB, and NF-YC. In this study, a NF-YB gene, AsNF-YB3, containing the full-length coding region was isolated from garlic. A sequence alignment and phylogenetic analysis showed that AsNF-YB3 clustered with TaNF-YB2 and AtNF-YB3. AsNF-YB3 was expressed in various organs, including the roots, cloves and leaves. A real-time RT-PCR analysis revealed that GA3, ABA and MeJA treatment upregulated the expression of AsNF-YB3 transcript. Overexpressing AsNF-YB3 in tobacco plants significantly accelerated germination compared to the wild type. Moreover, the total soluble sugar, fructose and sucrose levels were significantly higher in transgenic tobacco lines that constitutively overexpressed AsNF-YB3, suggesting that AsNF-YB3 might be involved in carbohydrate metabolism. Transgenic tobacco plants that overexpressed AsNF-YB3 displayed enhanced root elongation and shoot growth. The overexpression of AsNF-YB3 also strongly promoted early flowering in tobacco. The results of this study in tobacco suggest that garlic AsNF-YB3 likely plays an important role as a plant growth regulator.

In silicoanalysis of MeJA-induced comparative transcriptomes inBrassica oleraceaeL.var. capitata

Brassica oleraceae var capitata is a member of the Brassicaceae family and is widely used as an horticultural crop. In the present study, transcriptome analysis of B. oleraceae L. var capitata was done for the first time using eight-week old seedlings treated with MeJA, versus mock-treated samples. The complete transcripts for both samples were obtained using the GS-FLX sequencer. Overall, we obtained 275,570 and 266,457 reads from seedlings treated with or without MeJA, respectively. All the obtained reads were annotated using biological databases and functionally classified using gene ontology (GO), the Kyoto Encyclopedia of Genes and Genomics (KEGG). By using GO analyses, putative transcripts were examined in terms of biotic and abiotic stresses, cellular component organization, biogenesis, and secondary metabolic processes. The KEGG pathways for most of the transcripts were involved in carbohydrate metabolism, energy metabolism, and secondary metabolite synthesis. In order to double the sequenced data, we randomly chose two putative genes involved in terpene biosynthetic pathways and studied their transcript patterns under MeJA treatment. This study will provide us a platform to further characterize the genes in B. oleracea var capitata.

Saponin determination, expression analysis and functional characterization of saponin biosynthetic genes in Chenopodium quinoa leaves

Quinoa (Chenopodium quinoa Willd.) is a highly nutritious pseudocereal with an outstanding protein, vitamin, mineral and nutraceutical content. The leaves, flowers and seed coat of quinoa contain triterpenoid saponins, which impart bitterness to the grain and make them unpalatable without postharvest removal of the saponins. In this study, we quantified saponin content in quinoa leaves from Ecuadorian sweet and bitter genotypes and assessed the expression of saponin biosynthetic genes in leaf samples elicited with methyl jasmonate. We found saponin accumulation in leaves after MeJA treatment in both ecotypes tested. As no reference genes were available to perform qPCR in quinoa, we mined publicly available RNA-Seq data for orthologs of 22 genes known to be stably expressed in Arabidopsis thaliana using geNorm, NormFinder and BestKeeper algorithms. The quinoa ortholog of At2g28390 (Monensin Sensitivity 1, MON1) was stably expressed and chosen as a suitable reference gene for qPCR analysis. Candidate saponin biosynthesis genes were screened in the quinoa RNA-Seq data and subsequent functional characterization in yeast led to the identification of CqbAS1, CqCYP716A78 and CqCYP716A79. These genes were found to be induced by MeJA, suggesting this phytohormone might also modulate saponin biosynthesis in quinoa leaves. Knowledge of the saponin biosynthesis and its regulation in quinoa may aid the further development of sweet cultivars that do not require postharvest processing.

Overexpression of Poplar PtrWRKY89 in Transgenic Arabidopsis Leads to a Reduction of Disease Resistance by Regulating Defense-Related Genes in Salicylate- and Jasmonate-Dependent Signaling.

The plant hormones jasmonic acid (JA) and salicylic acid (SA) play key roles in plant defenses against pathogens and several WRKY transcription factors have been shown to have a role in SA/JA crosstalk. In a previous study, overexpression of the poplar WRKY gene PtrWRKY89 enhanced resistance to pathogens in transgenic poplars. In this study, the promoter of PtrWRKY89 (ProPtrWRKY89) was isolated and used to drive GUS reporter gene. High GUS activity was observed in old leaves of transgenic Arabidopsis containing ProPtrWRKY89-GUS construct and GUS expression was extremely induced by SA solution and SA+MeJA mixture but not by MeJA treatment. Subcellular localization and transactivation assays showed that PtrWRKY89 acted as a transcription activator in the nucleus. Constitutive expression of PtrWRKY89 in Arabidopsis resulted in more susceptible to Pseudomonas syringae and Botrytis cinerea compared to wild-type plants. Quantitative real-time PCR (qRT-PCR) analysis confirmed that marker genes of SA and JA pathways were down-regulated in transgenic Arabidopsis after pathogen inoculations. Overall, our results indicated that PtrWRKY89 modulates a cross talk in resistance to P. syringe and B. cinerea by negatively regulating both SA and JA pathways in Arabidopsis.

A Novel Non-specific Lipid Transfer Protein Gene from Sugarcane (NsLTPs), Obviously Responded to Abiotic Stresses and Signaling Molecules of SA and MeJA

Non-specific lipid transfer proteins (NsLTPs) are soluble, small, basic proteins in plant, which have been reported to be involved in plant physiological functions such as the catalyzing transfer of phospholipids and play an important role in plant defense and stress responses. In this study, a member of NsLTP family gene (ScNsLTP, Acc. No. KR259657) was isolated from a full-length cDNA library of sugarcane stalk. The cDNA of ScNsLTP was 671 bp long and contained a 312 bp open reading frame (ORF), which can encode a protein of 103 amino acid residues with molecular weight of 10.66 kDa. The ScNsLTP transcript levels in sugarcane seedlings decreased in response to SA, whereas it increased under MeJA treatment, suggesting an antagonistic regulatory mechanism between the signaling molecules of SA and MeJA. The transcript levels of ScNsLTP were obviously up-regulated under chilling and PEG stresses, implying that the ScNsLTP gene was involved in response to abiotic stresses and playing a positive role in adaption to low temperature and drought stresses. These results provide important information for further functional studies of plant NsLTPs gene.

Cross-talk between freezing response and signaling for regulatory transcriptions of MIR475b and its targets by miR475b promoter in Populus suaveolens.

MicroRNAs (miRNAs) are small, non-coding RNAs that play important roles in post-transcriptional regulation of their target genes, yet the transcriptional regulation of plant miRNAs by promoter is poorly understood. Here, we firstly clone pri-miR475b cDNA and its native promoter from P. suaveolens, and characterize Psu-MIR475b as class-II gene transcribed by RNA polymerase II. By 5′ deletion analysis of Psu-miR475b promoter in a series of promoter-GUS chimeric vectors, we functionally identify three positive regulatory regions and multiple cis-acting elements responsible for Psu-miR475b promoter activity in response to freezing stress and exogenous hormone treatment. Moreover, the Psu-miR475b promoter activity displays a tissue-specific manner, negatively regulated by freezing stress and positively by MeJA, SA or GA treatment. Importantly, we comparatively analyze the time-course transcriptional profiles of Psu-miR475b and its targets in Psu-miR475b over-expression transgenic plants controlled by Psu-miR475b-specific promoter or CaMV 35S constitutive promoter, and explore the regulatory mechanism of Psu-miR475b promoter controlling transcriptional expressions of Psu-MIR475b and its targets in response to freezing stress and exogenous hormone treatment. Our results reveal that Psu-miR475b promoter-mediated transcriptions of Psu-MIR475b and its targets in response to freezing stress may be involved in a cross-talk between freezing response and stress signaling process.

Isolation a P450 gene in Pinus armandi and its expression after inoculation of Leptographium qinlingensis and treatment with methyl jasmonate

In conifers, the cytochrome P450 monooxygenase of the CYP720B family plays an important role in the synthesis of diterpene resin acids, which are specialised metabolites of the oleoresin defence. We isolated CYP720B19 from Pinus armandi and found that it belongs to the CYP720B family. The CYP720B19 open reading frame of 1467 bp encodes a protein of 488 amino acid residues with high similarity to abietadienol/abietadienal oxidase. Differential expression of the CYP720B19 gene significantly changed when plants were inoculated with the fungus Leptographium qinlingensis, mechanically wounded, or treated with methyl jasmonate (MeJa) and Tween at 4 days and 8 days. Increased expression of the CYP720B19 gene after these treatments suggested that the gene was involved in pine defence against inoculation by L. qinlingensis, MeJa treatment and mechanical injury.

Gene-to-metabolite network for biosynthesis of lignans in MeJA-elicited Isatis indigotica hairy root cultures.

Root and leaf tissue of Isatis indigotica shows notable anti-viral efficacy, and are widely used as “Banlangen” and “Daqingye” in traditional Chinese medicine. The plants' pharmacological activity is attributed to phenylpropanoids, especially a group of lignan metabolites. However, the biosynthesis of lignans in I. indigotica remains opaque. This study describes the discovery and analysis of biosynthetic genes and AP2/ERF-type transcription factors involved in lignan biosynthesis in I. indigotica. MeJA treatment revealed differential expression of three genes involved in phenylpropanoid backbone biosynthesis (IiPAL, IiC4H, Ii4CL), five genes involved in lignan biosynthesis (IiCAD, IiC3H, IiCCR, IiDIR, and IiPLR), and 112 putative AP2/ERF transcription factors. In addition, four intermediates of lariciresinol biosynthesis were found to be induced. Based on these results, a canonical correlation analysis using Pearson's correlation coefficient was performed to construct gene-to-metabolite networks and identify putative key genes and rate-limiting reactions in lignan biosynthesis. Over-expression of IiC3H, identified as a key pathway gene, was used for metabolic engineering of I. indigotica hairy roots, and resulted in an increase in lariciresinol production. These findings illustrate the utility of canonical correlation analysis for the discovery and metabolic engineering of key metabolic genes in plants.