Facile Fabrication of Anthocyanin-Nanocellulose Hydrogel Indicator Label for Intelligent Evaluation of Minced Pork Freshness.

In order to develop a reliable and rapid method for meat freshness detection, nanocellulose (TOCNF) prepared via the TEMPO (2,2,6,6-tetramethylpiperidine oxidation) oxidation method was used as raw material to prepare hydrogels using Zn2+ coordination and binding. Physicochemical properties such as water absorption and porosity were analyzed. It was further used to select suitable hydrogels for the preparation of indication labels after anthocyanin adsorption, and it was applied in the freshness detection of fresh minced pork. Five percent TOCNF (w/w) aqueous solution was homogenized by high shear for 4 min, and 20% (w/w) zinc chloride solution was added to it, so that the concentration of zinc ions could reach 0.25 mol/L. After standing for 24 h, the hydrogel was obtained with good water absorption and a porous three-dimensional network structure. The activation energies of volatile base nitrogen (TVBN) and anthocyanin indicating label color changes were 59.231 kJ/mol and 69.453 kJ/mol, respectively. The difference between the two is within 25 kJ/mol, so the prepared indicator label can accurately visualize the shelf life of fresh pork.

Hippocampal pituitary adenylate cyclase-activating polypeptide mediates rapid antidepressant-like effects of Yueju pill.

Yueju pill, a classic Chinese Medicine formulated, was recently found to produce rapid antidepressant-like effects in a PKA-CREB signaling-dependent manner. In our study, we found that the Yueju pill induced a remarkable increase in PACAP. The intracerebroventricular injection of PACAP agonist induced a rapid antidepressant-like effect; conversely, the intrahippocampal infusion of a PACAP antagonist reversed the antidepressant response of the Yueju pill. Mice with hippocampal PACAP knockdown via viral-mediated RNAi displayed depression-like behavior. PACAP knockdown also blunted the antidepressant effect of the Yueju pill. PACAP knockdown resulted in down-regulated CREB and expression of the synaptic protein PSD95 at both baselines and after administration of the Yueju pill. However, administration of the Yueju pill in the knockdown mice promoted PACAP and PKA levels. Chronically stressed mice showed deficient hippocampal PACAP-PKA-CREB signaling and depression-like behavior, which were reversed by a single dose of the Yueju pill. In this study, we demonstrated that the up-regulation of PACAP induced activating of PKA-CREB signaling would play a part in the rapid antidepressant-like effects of the Yueju pill. We also identified iridoids fraction of Gardenia jasminoides Ellis (GJ-IF), a vital component of the Yueju pill, was identified to recapitulate rapid antidepressant-like behavior through increased hippocampal PACAP expression of the Yueju pill. The promotion of hippocampal PACAP may collectively represent a novel mechanism of rapid antidepressant-like effect.

Maize Transcription Factor ZmHsf28 Positively Regulates Plant Drought Tolerance.

Identification of central genes governing plant drought tolerance is fundamental to molecular breeding and crop improvement. Here, maize transcription factor ZmHsf28 is identified as a positive regulator of plant drought responses. ZmHsf28 exhibited inducible gene expression in response to drought and other abiotic stresses. Overexpression of ZmHsf28 diminished drought effects in Arabidopsis and maize. Gene silencing of ZmHsf28 via the technology of virus-induced gene silencing (VIGS) impaired maize drought tolerance. Overexpression of ZmHsf28 increased jasmonate (JA) and abscisic acid (ABA) production in transgenic maize and Arabidopsis by more than two times compared to wild-type plants under drought conditions, while it decreased reactive oxygen species (ROS) accumulation and elevated stomatal sensitivity significantly. Transcriptomic analysis revealed extensive gene regulation by ZmHsf28 with upregulation of JA and ABA biosynthesis genes, ROS scavenging genes, and other drought related genes. ABA treatment promoted ZmHsf28 regulation of downstream target genes. Specifically, electrophoretic mobility shift assays (EMSA) and yeast one-hybrid (Y1H) assay indicated that ZmHsf28 directly bound to the target gene promoters to regulate their gene expression. Taken together, our work provided new and solid evidence that ZmHsf28 improves drought tolerance both in the monocot maize and the dicot Arabidopsis through the implication of JA and ABA signaling and other signaling pathways, shedding light on molecular breeding for drought tolerance in maize and other crops.

OsWRKY10 extensively activates multiple rice diterpenoid phytoalexin biosynthesis genes to enhance rice blast resistance.

Phytoalexin is the main chemical weapon against pathogens in plants. Rice (Oryza sativa L.) produces a number of phytoalexins to defend against pathogens, most of which belong to the class of diterpenoid phytoalexins. Three biosynthetic gene clusters (BGCs) and a few non-BGC genes are responsible for rice diterpenoid phytoalexin biosynthesis. The corresponding regulatory mechanism of these phytoalexins in response to pathogen challenges still remains unclear. Here we identified a transcription factor, OsWRKY10, which positively regulates rice diterpenoid phytoalexin biosynthesis. Knockout mutants of OsWRKY10 obtained by CRISPR/Cas9 technology are more susceptible to Magnaporthe oryzae infection, while overexpression of OsWRKY10 enhances resistance to rice blast. Further analysis revealed that overexpression of OsWRKY10 increases accumulation of multiple rice diterpenoid phytoalexins and expression of genes in three BGCs and non-BGC genes in response to M. oryzae infection. Knockout of OsWRKY10 impairs upregulation of rice diterpenoid phytoalexin biosynthesis gene expression by blast pathogen and CuCl2 treatment. OsWRKY10 directly binds to the W-boxes or W-box-like elements (WLEs) of rice diterpenoid phytoalexin biosynthesis gene promoters to regulate gene expression. This study identified an extensive regulator (OsWRKY10) with broad transcriptional regulatory effects on rice diterpenoid phytoalexin biosynthesis genes, providing insight into the regulation of chemical defense to improve disease resistance in rice.

ZmEREB92 interacts with ZmMYC2 to activate maize terpenoid phytoalexin biosynthesis upon Fusarium graminearum infection through jasmonic acid/ethylene signaling.

Maize (Zea mays) terpenoid phytoalexins (MTPs) induced by multiple fungi display extensive antimicrobial activities, yet how maize precisely regulates MTP accumulation upon pathogen infection remains elusive. In this study, pretreatment with jasmonic acid (JA)/ethylene (ET)-related inhibitors significantly reduced Fusarium graminearum-induced MTP accumulation and resulted in enhanced susceptibility to F. graminearum, indicating the involvement of JA/ET in MTP regulatory network. ZmEREB92 positively regulated MTP biosynthetic gene (MBG) expression by correlation analysis. Knockout of ZmEREB92 significantly compromised maize resistance to F. graminearum with delayed induction of MBGs and attenuated MTP accumulation. The activation of ZmEREB92 on MBGs is dependent on the interaction with ZmMYC2, which directly binds to MBG promoters. ZmJAZ14 interacts both with ZmEREB92 and with ZmMYC2 in a competitive manner to negatively regulate MBG expression. Altogether, our findings illustrate the regulatory mechanism for JA/ET-mediated MTP accumulation upon F. graminearum infection with the involvement of ZmEREB92, ZmMYC2, and ZmJAZ14, which provides new insights into maize disease responses.

The Rapid and Long-Lasting Antidepressant Effects of Iridoid Fraction in Gardenia Jasminoides J.Ellis Are Dependent on Activating PKA-CREB Signaling Pathway.

Lag periods of therapeutic efficacy cause poor compliance of patients, which has made solutions for rapid antidepressants the most urgent need in the depression study field at present. We have identified through our previous studies the rapid antidepressant effects of the traditional herb Gardenia jasminoides J.Ellis [Rubiaceae] (GJ) and its standardized fractions. Through screening different fractions of GJ, we decided to place our focus on the iridoid fraction of GJ (GJ-IF). Methods: 1. Tail suspension test (TST), forced swimming test (FST), and novelty suppressed-feeding test (NSFT) were performed in sequence on mice after GJ-IF administration. 2. Mice in the model group were under chronic unpredictable mild stress (CUMS) for 3 w. After GJ-IF treatment, mice were placed in an open field test (OFT), Sucrose preference test (SPT), NSFT, TST, and FST. 3. Western Blot was performed to examine the expression of brain-derived neurotrophic factor (BDNF), Synapsin 1, cyclic-AMP dependent protein kinase A (PKA), phosphorylated cyclic-AMP responsive element-binding protein (p-CREB), and cAMP response element-binding protein (CREB). 4. Mice in the test group were administrated with GJ-IF after intraperitoneal injection of PKA blocker H89. Results: 1. GJ-IF treatment significantly reduced the immobility time of TST at 1 d and FST at 26 h. 2. GJ-IF reversed the deficits induced by 3 w CUMS in SPT, TST, FST, and NSFT at 1 d and 26 h. The antidepressant effects of a single dose of iridoid fraction could also last for at least 14 d. 3. The results of molecule studies suggested that a single dose of GJ-IF activated p-CREB at 2 h and the PKA-CREB pathway at 1 d. The expression of BDNF did not significantly change from 30 min to 1 d after GJ-IF administration. 4. Blockade of PKA-CREB signaling pathway reversed the antidepressant effects of GJ-IF at 1 d, but not 30 min and 2 h. Conclusion: GJ-IF is the crucial component in the rapid antidepressant of GJ. Rapid and sustained antidepressant effects of GJ-IF were dependent on activating the PKA-CREB signaling pathway.

Global-Local Spatial-Temporal Residual Correlation Network for Urban Traffic Status Prediction.

The recent proposed Spatial-Temporal Residual Network (ST-ResNet) model is an effective tool to extract both spatial and temporal characteristics and has been successfully applied to urban traffic status prediction. However, the ST-ResNet model only extracts the local spatial characteristics and ignores the very important global spatial characteristics. In this paper, a novel Global-Local Spatial-Temporal Residual Correlation Network (GL-STRCN) model is proposed for urban traffic status prediction to further improve the prediction accuracy of the existing ST-ResNet model. The GL-STRCN model firstly applies Pearson's correlation coefficient method to extract high correlation series. Then, considering both global and local spatial properties, two components consisting of 2D convolution and residual operation are used to capture spatial features. After that, based on Long Short-Term Memory (LSTM) or Gated Recurrent Unit (GRU), a novel long-term temporal feature extraction component is proposed to capture temporal features. Finally, the spatial and temporal features are aggregated together in a weighted way for final prediction. Experiments have also been performed using two datasets from TaxiCD and PEMS-BAY. The results indicated that the proposed model produces a better prediction performance compared with the results based on other baseline solutions, e.g., CNN, ST-ResNet, GL-TCN, and DGLSTNet.

Maize WRKY Transcription Factor ZmWRKY79 Positively Regulates Drought Tolerance through Elevating ABA Biosynthesis.

Drought stress causes heavy damages to crop growth and productivity under global climatic changes. Transcription factors have been extensively studied in many crops to play important roles in plant growth and defense. However, there is a scarcity of studies regarding WRKY transcription factors regulating drought responses in maize crops. Previously, ZmWRKY79 was identified as the regulator of maize phytoalexin biosynthesis with inducible expression under different elicitation. Here, we elucidated the function of ZmWRKY79 in drought stress through regulating ABA biosynthesis. The overexpression of ZmWRKY79 in Arabidopsis improved the survival rate under drought stress, which was accompanied by more lateral roots, lower stomatal aperture, and water loss. ROS scavenging was also boosted by ZmWRKY79 to result in less H2O2 and MDA accumulation and increased antioxidant enzyme activities. Further analysis detected more ABA production in ZmWRKY79 overexpression lines under drought stress, which was consistent with up-regulated ABA biosynthetic gene expression by RNA-seq analysis. ZmWRKY79 was observed to target ZmAAO3 genes in maize protoplast through acting on the specific W-boxes of the corresponding gene promoters. Virus-induced gene silencing of ZmWRKY79 in maize resulted in compromised drought tolerance with more H2O2 accumulation and weaker root system architecture. Together, this study substantiates the role of ZmWRKY79 in the drought-tolerance mechanism through regulating ABA biosynthesis, suggesting its broad functions not only as the regulator in phytoalexin biosynthesis against pathogen infection but also playing the positive role in abiotic stress response, which provides a WRKY candidate gene to improve drought tolerance for maize and other crop plants.

Rice contains a biosynthetic gene cluster associated with production of the casbane-type diterpenoid phytoalexin ent-10-oxodepressin.

Diterpenoids play important roles in rice microbial disease resistance as phytoalexins, as well as acting in allelopathy and abiotic stress responses. Recently, the casbane-type phytoalexin ent-10-oxodepressin was identified in rice, but its biosynthesis has not yet been elucidated. Here ent-10-oxodepressin biosynthesis was investigated via co-expression analysis and biochemical characterisation, with use of the CRISPR/Cas9 technology for genetic analysis. The results identified a biosynthetic gene cluster (BGC) on rice chromosome 7 (c7BGC), containing the relevant ent-casbene synthase (OsECBS), and four cytochrome P450 (CYP) genes from the CYP71Z subfamily. Three of these CYPs were shown to act on ent-casbene, with CYP71Z2 able to produce a keto group at carbon-5 (C5), while the closely related paralogues CYP71Z21 and CYP71Z22 both readily produce a keto group at C10. Together these C5 and C10 oxidases can elaborate ent-casbene to ent-10-oxodepressin (5,10-diketo-ent-casbene). OsECBS knockout lines no longer produce casbane-type diterpenoids and exhibit impaired resistance to the rice fungal blast pathogen Magnaporthe oryzae. Elucidation of ent-10-oxodepressin biosynthesis and the associated c7BGC provides not only a potential target for molecular breeding, but also, gives the intriguing parallels to the independently assembled BGCs for casbene-derived diterpenoids in the Euphorbiaceae, further insight into plant BGC evolution, as discussed here.

Maize transcription factor ZmEREB20 enhanced salt tolerance in transgenic Arabidopsis.

Soil salinity severely limits agricultural crop production worldwide. As one of the biggest plant specific transcription factor families, AP2/ERF members have been extensively studied to regulate plant growth, development and stress responses. However, the role of AP2/ERF family in maize salt tolerance remains largely unknown. In this study, we identified a maize AP2-ERF family member ZmEREB20 as a positive salinity responsive gene. Overexpression of ZmEREB20in Arabidopsis enhanced ABA sensitivity and resulted in delayed seed germination under salt stress through regulating ABA and GA related genes. ZmEREB20 overexpression lines also showed higher survival rates with elevated ROS scavenging toward high salinity. Furthermore, root hair growth inhibition by salt stress was markedly rescued in ZmEREB20 overexpression lines. Auxin transport inhibitor TIBA drastically enhanced root hair growth in ZmEREB20 overexpression Arabidopsis under salt stress, together with the increased expression of auxin-related genes, ion transporter genes and root hair growth genes by RNA-seq analysis. ZmEREB20 positively regulated salt tolerance through the molecular mechanism associated with hormone signaling, ROS scavenging and root hair plasticity, proving the potential target for crop breeding to improve salt resistance.

Direct formation of the sesquiterpeonid ether liguloxide by a terpene synthase in Senecio scandens.

KEY MESSAGE: SsLOS directly catalyzed formation of the sesquiterpenoid ether liguloxide in the medicinal plant Senecio scandens. Terpene synthases determine the diversity of terpene skeletons and corresponding terpenoid natural products. Oxygenated groups introduced in catalysis of terpene synthases are important for solubility, potential bioactivity and further elaboration of terpenoids. Here we identified one terpene synthase, SsLOS, in the Chinese medicinal plant Senecio scandens. SsLOS acted as the sesquiterpene synthase and utilized (E,E)-farnesyl diphosphate as the substrate to produce a blend of sesquiterpenoids. GC-MS analysis and NMR structure identification demonstrated that SsLOS directly produced the sesquiterpenoid ether, liguloxide, as well as its alcoholic isomer, 6-epi-guaia-2(3)-en-11-ol. Homology modeling and site-directed mutagenesis were combined to explore the catalytic mechanism of SsLOS. A few key residues were identified in the active site and hedycaryol was identified as the neutral intermediate of SsLOS catalysis. The plausible catalytic mechanism was proposed as well. Altogether, SsLOS was identified and characterized as the sesquiterpenoid ether synthase, which is the second terpenoid ether synthase after 1,8-cineol synthase, suggesting some insights for the universal mechanism of terpene synthases using the water molecule in the catalytic cavity.

Probing Enzymatic Structure and Function in the Dihydroxylating Sesquiterpene Synthase ZmEDS.

Terpene synthases (TPSs) play a vital role in forming the complex hydrocarbon backbones that underlie terpenoid diversity. Notably, some TPSs can add water prior to terminating the catalyzed reaction, leading to hydroxyl groups, which are critical for biological activity. A particularly intriguing example of this is the maize (Zea mays) sesquiterpene TPS whose major product is eudesmanediol, ZmEDS. This production of dual hydroxyl groups is presumably enabled by protonation of the singly hydroxylated transient stable intermediate hedycaryol. To probe the enzymatic structure-function relationships underlying this unusual reaction, protein modeling and docking were used to direct mutagenesis of ZmEDS. Previously, an F303A mutant was shown to produce only hedycaryol, suggesting a role in protonation. Here this is shown to be dependent on the steric bulk positioning of hedycaryol, including a supporting role played by the nearby F299, rather than π-cation interaction. Among the additional residues investigated here, G411 at the conserved kink in helix G is of particular interest, as substitution of this leads to predominant production of the distinct (-)-valerianol, while substitution for the aliphatic I279 and V306 can lead to significant production of the alternative eudesmane-type diols 2,3-epi-cryptomeridiol and 3-epi-cryptomeridol, respectively. Altogether, nine residues that are important for this unusual reaction were investigated here, with the results not only emphasizing the importance of reactant positioning suggested by the stereospecificity observed among the various product types but also highlighting the potential role of the Mg2+-diphosphate complex as the general acid for the protonation-initiated (bi)cyclization of hedycaryol.

Transcriptional Factors Regulate Plant Stress Responses through Mediating Secondary Metabolism.

Plants are adapted to sense numerous stress stimuli and mount efficient defense responses by directing intricate signaling pathways. They respond to undesirable circumstances to produce stress-inducible phytochemicals that play indispensable roles in plant immunity. Extensive studies have been made to elucidate the underpinnings of defensive molecular mechanisms in various plant species. Transcriptional factors (TFs) are involved in plant defense regulations through acting as mediators by perceiving stress signals and directing downstream defense gene expression. The cross interactions of TFs and stress signaling crosstalk are decisive in determining accumulation of defense metabolites. Here, we collected the major TFs that are efficient in stress responses through regulating secondary metabolism for the direct cessation of stress factors. We focused on six major TF families including AP2/ERF, WRKY, bHLH, bZIP, MYB, and NAC. This review is the compilation of studies where researches were conducted to explore the roles of TFs in stress responses and the contribution of secondary metabolites in combating stress influences. Modulation of these TFs at transcriptional and post-transcriptional levels can facilitate molecular breeding and genetic improvement of crop plants regarding stress sensitivity and response through production of defensive compounds.

[Cloning and functional characterization of farnesyl diphosphate synthase in Senecio scandens].

As a secondary metabolite, sesquiterpenes are not only have important functions in plant defense and signaling, but also play potential roles in basic materials for pharmaceuticals, cosmetic and flavor. As a traditional Chinese herbal medicine, Senecio scandens exhibits effects of anti-inflammatory and immunosuppressive, as well as invigorating the blood and removing extravasated blood. Over 600 sesquiterpenes with diverse structures were isolated from S. scandens and related species in the same genus. To characterize sesquiterpenes synthesis, two FPS genes(SsFPS1 and SsFPS2) were identified in S. scandens through transcriptomic analysis. Bioinformatic analysis showed that both SsFPSs have conserved motifs for FPS function. Both SsFPSs exhibited constitutive gene expression in S. scandens tissues and SsFPS2 accumulated higher transcript in leaves and roots than SsFPS1. Meanwhile consistent with constitutive sesquiterpene accumulation in S.scandens tissues, most of these sesquiterpenes were detected in leaves and roots more than stems and flowers. Recombinant expression through Escherichia coli metabolic engineering, SsFPS1 or SsFPS2 was co-transformed with ZmTPS11(maize ß-macrocarpene synthase) into BL21 competent cells. The results showed that the content of ß-macrocarpene was increased by co-transformation with SsFPSs. It is demonstrated that SsFPS1 and SsFPS2 catalyzed E,E-FPP formation and provided FPP precursor for downstream sesquiterpene synthases. Characterization of SsFPSs provided the foundation for the exploration of biosynthesis of sesquiterpenoid with diverse structures and potential pharmaceutical values in S.scandens, and provide an important theoretical basis for the development of S. scandens abundant resources.

ZmMYC2 exhibits diverse functions and enhances JA signaling in transgenic Arabidopsis.

KEY MESSAGE: ZmMYC2 was identified as the key regulator of JA signaling in maize and exhibited diverse functions through binding to many gene promoters as well as enhanced JA signaling in transgenic Arabidopsis. The plant hormone jasmonate (JA) extensively coordinates plant growth, development and defensive responses. MYC2 is the master regulator of JA signaling and has been widely studied in many plant species. However, little is known about this transcription factor in maize. Here, we identified one maize transcription factor with amino acid identity of 47% to the well-studied Arabidopsis AtMYC2, named as ZmMYC2. Gene expression analysis demonstrated inducible expression patterns of ZmMYC2 in response to multiple plant hormone treatments, as well as biotic and abiotic stresses. The yeast two-hybrid assay indicated physical interaction among ZmMYC2 and JA signal repressors ZmJAZ14, ZmJAZ17, AtJAZ1 and AtJAZ9. ZmMYC2 overexpression in Arabidopsis myc2myc3myc4 restored the sensitivity to JA treatment, resulting in shorter root growth and inducible anthocyanin accumulation. Furthermore, overexpression of ZmMYC2 in Arabidopsis elevated resistance to Botrytis cinerea. Further ChIP-Seq analysis revealed diverse regulatory roles of ZmMYC2 in maize, especially in the signaling crosstalk between JA and auxin. Hence, we identified ZmMYC2 and characterized its roles in regulating JA-mediated growth, development and defense responses.

Yueju-Ganmaidazao Decoction confers rapid antidepressant-like effects and the involvement of suppression of NMDA/NO/cGMP signaling.

ETHNOPHARMACOLOGICAL RELEVANCE: Yueju-Ganmaidazao Decoction (YG) is a multiherbal medicine prescribed for treatment of mood disorder, consisting of two classical traditional Chinese herbal medicine Yueju and Ganmaidazao. Yueju and Ganmaidazao both are used for depression treatment. The combined decoction of Yueju and Ganmaidazao is prescribed to achieve optimal clinical outcomes by dealing with different symptoms of depression. Recent studies indicated ethanol extract of Yueju was capable to confer rapid antidepressant-like response. The antidepressant activity of YG decoction with fast-onset feature remains to be investigated. AIM OF THE STUDY: Rapid and safe antidepressant treatment is urgently needed. This study aimed to assess the rapid antidepressant-like activity of YG and the underlying mechanism, focusing on NMDA/NO/cGMP signaling. MATERIALS AND METHODS: The optimal doses for immediate and persistent antidepressant-like response were first screened using tail suspension test (TST) and forced swimming test (FST) post a single administration of YG. The rapid action was further confirmed by using the chronic mild stress (CMS) and learned helplessness (LH) paradigms. The expressions of NMDA receptor subunits were evaluated post stress and YG. The contributions of NMDA, NO, and cGMP signaling to the antidepressant effect of YG were investigated systematically using pharmacological interventions. RESULTS: The optimal dose for immediate and persistent antidepressant potential, evidenced with reduced immobility times in TST or FST from 30 min to 7 days, was determined. The rapid antidepressant-like effect was confirmed in CMS and LH paradigms, including instant normalization of sucrose preference behavior. The expression of NMDA subunit NR1 in the hippocampus was reduced from 30 min to 5 days post YG. In animals subjected to CMS and LH, hippocampal NR1 expression increased, reversed by YG. YG's antidepressant-like effect was blunted by pretreatment with the agonists along the signalings including NMDA (75 mg/kg), L-arginine (750 mg/kg) and sildenafil (5 mg/kg) in TST or FST. Conversely, administration of subeffective dose of individual antagonists, including MK-801 (0.05 mg/kg), 7-nitroindazole (30 mg/kg), methylene blue (10 mg/kg), in combination with a subeffective dose of YG, elicited antidepressant effects. CONCLUSION: YG conferred rapid antidepressant-like effects, and the antidepressant response was essentially dependent on suppression of NMDA/NO/cGMP signaling.

A Wheat ß-Patchoulene Synthase Confers Resistance Against Herbivory in Transgenic Arabidopsis.

Terpenoids play important roles in plant defense. Although some terpene synthases have been characterized, terpenoids and their biosynthesis in wheat (Triticumaestivum L.) still remain largely unknown. Here, we describe the identification of a terpene synthase gene in wheat. It encodes a sesquiterpene synthase that catalyzes ß-patchoulene formation with E,E-farnesyl diphosphate (FPP) as the substrate, thus named as TaPS. TaPS exhibits inducible expression in wheat in response to various elicitations. Particularly, alamethicin treatment strongly induces TaPS gene expression and ß-patchoulene accumulation in wheat. Overexpression of TaPS in Arabidopsis successfully produces ß-patchoulene, verifying the biochemical function of TaPS in planta. Furthermore, these transgenic Arabidopsis plants exhibit resistance against herbivory by repelling beet armyworm larvae feeding, thereby indicating anti-herbivory activity of ß-patchoulene. The catalytic mechanism of TaPS is also explored by homology modeling and site-directed mutagenesis. Two key amino acids are identified to act in protonation and stability of intermediates and product formation. Taken together, one wheat sesquiterpene synthase is identified as ß-patchoulene synthase. TaPS exhibits inducible gene expression and the sesquiterpene ß-patchoulene is involved in repelling insect infestation.

CYP71Z18 overexpression confers elevated blast resistance in transgenic rice.

KEY MESSAGE: CYP71Z18 exhibited plastic substrate specificity to catalyze oxidation of multiple rice diterpenes and elevated chemical defense against the blast fungus in transgenic rice. Diversified plant specialized metabolism relies on corresponding biosynthetic enzymes with differential substrate specificity. CYP71Z18 catalyzed formation of maize phytoalexins including zealexin A1, the sesquiterpenoid phytoalexin, and diterpenoid phytoalexin dolabralexin, indicating catalytic promiscuity on different terpene substrates. Here substrate specificity of CYP71Z18 was further explored through microbial metabolic engineering and it was identified to accept multiple rice diterpenes as substrates for oxidation. One CYP71Z18 enzymatic product derived from syn-pimaradiene was identified as 15,16-epoxy-syn-pimaradiene by NMR analysis, which was further elaborated by CYP99A3 to generate C19 hydroxylated product. 15,16-epoxy-syn-pimaradien-19-ol exhibited inhibitory effect on spore germination and appressorium formation of the blast pathogen Magnaporthe oryzae. Overexpression of CYP71Z18 in rice resulted in accumulation of several new diterpenoids, indicating promiscuous activity in planta. Transgenic rice also showed stronger resistance against M. oryzae infection, suggesting elevated chemical defense through changed diterpenoid metabolism by CYP71Z18 overexpression. This investigation sheds light on plant metabolic engineering using plastic substrate specificity of P450s to strengthen disease resistance and potentially provide abundant lead compounds.

[Functional characterization of SsNES responsible for nerolidol biosynthesis in Senecio scandens].

A short terpene synthase gene was obtained by screening the transcriptome data of Senecio scandens. The phylogenetic tree and sequence alignment putatively identified this gene as a nerolidol synthase gene, named SsNES(GenBank MH518312). Protein homology modeling indicated that SsNES contained a complete conserved domain and folded correctly. SsNES was cloned and successfully expressed in Escherichia coli as soluble protein. The biochemical function of SsNES was characterized by E. coli metabolic engineering, which showed that SsNES catalyzed formation of trans-nerolidol with(E, E)-farnesyl diphosphate as the substrate. Nerolidol was also detected in stems and leaves of S. scandens, indicating that SsNES might act as the nerolidol synthase in plant. RT-PCR analysis indicated that SsNES was mainly expressed in stem, flowers and leaves, and no expression was observed in roots. After the treatment of SA, MeJA or Ala, SsNES was induced significantly at 6 h, indicating involvement in the defense response of S. scandens. The identification of SsNES not only clarified biosynthesis of nerolidol in S. scandens, but also provided diversity of sesquiterpene synthase, as well as theoretical basis for disease and pest defense mediated by the terpene metabolites.

[Identification of stable reference gene by qRT-PCR in Senecio scandens].

As a traditional Chinese medicine, Senecio scandens is rich in important compounds such as flavonoid and sesquiterpenoid. Based on the transcriptome data of S. scandens, 15 candidate reference genes were selected including ABCT, ACT1, ACT2, ACT3, ACBP, ARF, ATPS, EF-H, EF-1α, ETIF, GAPDH, GTPB, MPS, UCE and 60S. Firstly, 9 candidate genes with relatively stable expressions such as ACT1, ACBP, ARF, ATPS, EF-1α, GAPDH, MPS, UCE and 60S were screened from different tissues of S. scandens by RT-PCR. Then, qRT-PCR was used to quantitatively analyze gene expression of these nine candidates in S. scandens with or without stress treatments. Further analysis of these gene expression data by geNorm and NormFinder showed that ACT1 exhibited the stablest expression in all samples and could serve as a reference gene for future study of S. scandens, and provide an endogenous control for gene expression analysis.