Related Biosynthetic Pathways Research Articles

B-350 The application of MALDI-TOF imaging to biomarker discovery in a mouse model of papillomavirus associated tumorigenesis

Abstract Background Human papillomavirus (HPV) can cause infection and dysplasia in cutaneous epithelium of immunodeficient patients. An animal model has been developed to study HPV-associated tumorigenesis using Nude mouse (Foxn1nu) and mouse papillomavirus (MmuPV1). This study applies MALDI-TOF imaging to compare the expression pattern of small molecules between viral-infected dysplastic mouse skin and mock-infected control tissue. Three sets of molecules showing distinct patterns are identified in correlation with the upregulation of an enzyme in related biosynthetic pathway. Methods Nude mice were wounded on tail and inoculated with MmPV1 suspension (1×109 viral DNA genomes per infected site) or PBS 24 hours later for treatment and control group, respectively. Tail skins were harvested 4 weeks post-infection and processed for cryosectioning. Tissue sections were analyzed using timsTOF flex (Bruker) under negative ion mode with 9-aminoacridine as the matrix. Viral infection (MmuPV1-L1 antigen) and enzyme expression (3'-phosphoadenosine 5'-phosphosulfate synthetase 1, PAPSS1) were assessed by immunofluorescence on adjacent sections. Results Three peaks demonstrated distinct distribution patterns in mouse skin. One peak at m/Z = 465 was identified as cholesterol sulfate (CS) by tandem MS and comparing with the standard. CS was enriched in keratinocytes, hair follicles, and blood vessels in normal skins, and it increased in dysplastic skins with enhanced angiogenesis. The combined peaks at m/Z = 327 & 329 were tentatively identified as docosahexaenoic acid (DHA) and docosapentaenoic acid (DPA). These long chain polyunsaturated fatty acids (LC-PUFAs) were present in the dermis as foci partially co-localized with CS, and they also increased in virus-infected skins. The peak at m/Z = 426 may represent a combination of ADP and APS. Although these two isobars cannot be separated by tandem MS, APS was revealed by the presence of sulfur isotopes at expected natural abundance (34S and 32S). They were present at a low level in the epidermis of normal skins but significantly increased after virus infection. APS and ADP were closely associated with the proliferating cells, and the signal intensity also increased with the degree of tissue dysplasia. As a single factor, APS & ADP demonstrated a fair differential power for MmuPV1-induced dysplastic skin and normal control (AUC of ROC curve = 0.75). The bifunctional enzyme PAPSS1 catalyzes the conversion of ATP to 3’-phosphonato-5’-adenyl sulfate (PAPS) via APS as an intermediate. Interestingly, immunofluorescence showed the expression of PAPSS1 also increased significantly with the severity of tissue dysplasia and MmuPV1 infection. Conclusions MALDI-TOF imaging reveals three sets of molecules with distinct patterns in mouse skin, and their levels are increased during MmuPV1-induced epithelium dysplasia. As a precursor of the common sulfate donor (PAPS), increased APS and ADP in the epidermis of virus-infected skins indicate an upregulation of sulfation pathway and adenine metabolism. This finding is supported by enhanced expression of enzyme PAPSS1 and an increased sulfation reaction product, CS. In addition, DHA and DPA are also increased in the dermis of virus-infected skins, implying a disturbance in LC-PUFA metabolism. This study demonstrates combined use of MALDI-TOF imaging and immunostaining in the search of novel biomarkers of HPV-associated tumorigenesis.

Genetic and molecular regulation of chilling requirements in pear: breeding for climate change resilience.

Pear (Pyrus spp.) is a deciduous fruit tree that requires exposure to sufficient chilling hours during the winter to establish dormancy, followed by favorable heat conditions during the spring for normal vegetative and floral budbreak. In contrast to most temperate woody species, apples and pears of the Rosaceae family are insensitive to photoperiod, and low temperature is the major factor that induces growth cessation and dormancy. Most European pear (Pyrus Communis L.) cultivars need to be grown in regions with high chilling unit (CU) accumulation to ensure early vegetative budbreak. Adequate vegetative budbreak time will ensure suitable metabolite accumulation, such as sugars, to support fruit set and vegetative development, providing the necessary metabolites for optimal fruit set and development. Many regions that were suitable for pear production suffer from a reduction in CU accumulation. According to climate prediction models, many temperate regions currently suitable for pear cultivation will experience a similar accumulation of CUs as observed in Mediterranean regions. Consequently, the Mediterranean region can serve as a suitable location for conducting pear breeding trials aimed at developing cultivars that will thrive in temperate regions in the decades to come. Due to recent climatic changes, bud dormancy attracts more attention, and several studies have been carried out aiming to discover the genetic and physiological factors associated with dormancy in deciduous fruit trees, including pears, along with their related biosynthetic pathways. In this review, current knowledge of the genetic mechanisms associated with bud dormancy in European pear and other Pyrus species is summarized, along with metabolites and physiological factors affecting dormancy establishment and release and chilling requirement determination. The genetic and physiological insights gained into the factors regulating pear dormancy phase transition and determining chilling requirements can accelerate the development of new pear cultivars better suited to both current and predicted future climatic conditions.

Evolutionarily related host and microbial pathways regulate fat desaturation in C. elegans

Fatty acid desaturation is central to metazoan lipid metabolism and provides building blocks of membrane lipids and precursors of diverse signaling molecules. Nutritional conditions and associated microbiota regulate desaturase expression, but the underlying mechanisms have remained unclear. Here, we show that endogenous and microbiota-dependent small molecule signals promote lipid desaturation via the nuclear receptor NHR-49/PPARα in C. elegans. Untargeted metabolomics of a β-oxidation mutant, acdh-11, in which expression of the stearoyl-CoA desaturase FAT-7/SCD1 is constitutively increased, revealed accumulation of a β-cyclopropyl fatty acid, becyp#1, that potently activates fat-7 expression via NHR-49. Biosynthesis of becyp#1 is strictly dependent on expression of cyclopropane synthase by associated bacteria, e.g., E. coli. Screening for structurally related endogenous metabolites revealed a β-methyl fatty acid, bemeth#1, which mimics the activity of microbiota-dependent becyp#1 but is derived from a methyltransferase, fcmt-1, that is conserved across Nematoda and likely originates from bacterial cyclopropane synthase via ancient horizontal gene transfer. Activation of fat-7 expression by these structurally similar metabolites is controlled by distinct mechanisms, as microbiota-dependent becyp#1 is metabolized by a dedicated β-oxidation pathway, while the endogenous bemeth#1 is metabolized via α-oxidation. Collectively, we demonstrate that evolutionarily related biosynthetic pathways in metazoan host and associated microbiota converge on NHR-49/PPARα to regulate fat desaturation.

The arachidonic acid metabolome reveals elevation of prostaglandin E2 biosynthesis in colorectal cancer.

Arachidonic acid metabolites are a family of bioactive lipids derived from membrane phospholipids. They are involved in cancer progression, but arachidonic acid metabolite profiles and their related biosynthetic pathways remain uncertain in colorectal cancer (CRC). To compare the arachidonic acid metabolite profiles between CRC patients and healthy controls, quantification was performed using a liquid chromatography-mass spectrometry-based analysis of serum and tissue samples. Metabolomics analysis delineated the distinct oxidized lipids in CRC patients and healthy controls. Prostaglandin (PGE2)-derived metabolites were increased, suggesting that the PGE2 biosynthetic pathway was upregulated in CRC. The qRT-PCR and immunohistochemistry analyses showed that the expression level of PGE2 synthases, the key protein of PGE2 biosynthesis, was upregulated in CRC and positively correlated with the CD68+ macrophage density and CRC development. Our study indicates that the PGE2 biosynthetic pathway is associated with macrophage infiltration and progression of CRC tumors.

Two Novel Iboga-Type and an Oxindole Glucuronide Alkaloid from Tabernaemontana peduncularis Disclose Related Biosynthetic Pathways to Tabernaemontana divaricata.

Phytochemical investigation of the two Tabernaemontana species (Apocynaceae) T. peduncularis Wall. and T. divaricata (L.) R.Br. ex Roem. & Schult. indicated closely related biosynthetic pathways leading to lipophilic and hydrophilic alkaloids. In total, 18 specialized metabolites comprising indole-derived alkaloid aglycones, three oxindole-derived alkaloid glycosides, and two iridoid glucosides could be identified in the studied species. Among the alkaloids, the two Iboga-type alkaloids 3,7-coronaridine isoindolenine, coronaridine 3,4-iminium and a javaniside derivative bearing a glucuronic acid, named javanuronic acid, could be described by spectroscopic and spectrometric methods for the first time. A docking experiment using alpha-fold was performed to generate a protein model of the enzyme 7-deoxyloganetic acid glucosyl transferase. Performed bioassays exhibited a growth reduction of neonate Spodoptera littoralis larvae and reduced cell viability of HepG2 cells of the extracts containing Iboga alkaloids, whilst the javaniside derivatives containing hydrophilic fraction did not show any effects. These findings indicate a high flexibility in the formation of bioactive indole alkaloid aglycones by Tabernaemontana species and also evidence similar accumulation trends in both species as well as indicate that biosynthetic routes leading to oxindole alkaloids like javanisides are more widespread than reported. Furthermore, the incorporation of the three novel compounds into potential biosynthetic pathways is discussed.

A Bacterial Cytochrome P450 Enzyme Catalyzes Multistep Oxidation Reactions in Pyrroindomycin Biosynthesis

Comprehensive SummaryCytochrome P450 enzymes (P450s) belong to a large family of oxidative hemeproteins and catalyze highly diverse oxygenation reactions that are involved in the biosynthesis of various natural products. Here, we report a multifunctional cytochrome P450 enzyme, PyrE2, which catalyzes the regioselective, successive 6‐electron oxidation of an inert methyl group to produce a carboxyl product through formation of the hydroxyl and aldehyde intermediates in pyrroindomycin biosynthesis. The time‐course biotransformation was characterized by the presence of the hydroxyl and aldehyde intermediates, the lag of the formation of the carboxyl product, and the subsequent loss of both intermediates, indicating that each 2‐electron oxidation exhibits the distributive mechanism that requires substrate binding and product releasing. Bioinformatics analysis shows that the homologs of pyrE2 are common in the gene clusters of the spirotetronates varying in the oxidative state of the corresponding exocyclic carbon, indicating the generality and diversity of P450‐catalyzed oxygenation in related biosynthetic pathways.

Unusual peptide-binding proteins guide pyrroloindoline alkaloid formation in crocagin biosynthesis

Ribosomally synthesized and post-translationally modified peptide natural products have provided many highly unusual scaffolds. This includes the intriguing alkaloids crocagins, which possess a tetracyclic core structure and whose biosynthesis has remained enigmatic. Here we use in vitro experiments to demonstrate that three proteins, CgnB, CgnC and CgnE, are sufficient for the production of the hallmark tetracyclic crocagin core from the precursor peptide CgnA. The crystal structures of the homologues CgnB and CgnE reveal them to be the founding members of a peptide-binding protein family and allow us to rationalize their distinct functions. We further show that the hydrolase CgnD liberates the crocagin core scaffold, which is subsequently N-methylated by CgnL. These insights allow us to propose a biosynthetic scheme for crocagins. Bioinformatic analyses based on these data led to the discovery of related biosynthetic pathways that may provide access to a structurally diverse family of peptide-derived pyrroloindoline alkaloids.

Engineered biosynthesis of plant polyketides by type III polyketide synthases in microorganisms.

Plant specialized metabolites occupy unique therapeutic niches in human medicine. A large family of plant specialized metabolites, namely plant polyketides, exhibit diverse and remarkable pharmaceutical properties and thereby great biomanufacturing potential. A growing body of studies has focused on plant polyketide synthesis using plant type III polyketide synthases (PKSs), such as flavonoids, stilbenes, benzalacetones, curcuminoids, chromones, acridones, xanthones, and pyrones. Microbial expression of plant type III PKSs and related biosynthetic pathways in workhorse microorganisms, such as Saccharomyces cerevisiae, Escherichia coli, and Yarrowia lipolytica, have led to the complete biosynthesis of multiple plant polyketides, such as flavonoids and stilbenes, from simple carbohydrates using different metabolic engineering approaches. Additionally, advanced biosynthesis techniques led to the biosynthesis of novel and complex plant polyketides synthesized by diversified type III PKSs. This review will summarize efforts in the past 10 years in type III PKS-catalyzed natural product biosynthesis in microorganisms, especially the complete biosynthesis strategies and achievements.

Solution Structure and Conformational Dynamics of a Doublet Acyl Carrier Protein from Prodigiosin Biosynthesis.

The acyl carrier protein (ACP) is an indispensable component of both fatty acid and polyketide synthases and is primarily responsible for delivering acyl intermediates to enzymatic partners. At present, increasing numbers of multidomain ACPs have been discovered with roles in molecular recognition of trans-acting enzymatic partners as well as increasing metabolic flux. Further structural information is required to provide insight into their function, yet to date, the only high-resolution structure of this class to be determined is that of the doublet ACP (two continuous ACP domains) from mupirocin synthase. Here we report the solution nuclear magnetic resonance (NMR) structure of the doublet ACP domains from PigH (PigH ACP1-ACP2), which is an enzyme that catalyzes the formation of the bipyrrolic intermediate of prodigiosin, a potent anticancer compound with a variety of biological activities. The PigH ACP1-ACP2 structure shows each ACP domain consists of three conserved helices connected by a linker that is partially restricted by interactions with the ACP1 domain. Analysis of the holo (4'-phosphopantetheine, 4'-PP) form of PigH ACP1-ACP2 by NMR revealed conformational exchange found predominantly in the ACP2 domain reflecting the inherent plasticity of this ACP. Furthermore, ensemble models obtained from SAXS data reveal two distinct conformers, bent and extended, of both apo (unmodified) and holo PigH ACP1-ACP2 mediated by the central linker. The bent conformer appears to be a result of linker-ACP1 interactions detected by NMR and might be important for intradomain communication during the biosynthesis. These results provide new insights into the behavior of the interdomain linker of multiple ACP domains that may modulate protein-protein interactions. This is likely to become an increasingly important consideration for metabolic engineering in prodigiosin and other related biosynthetic pathways.

Effects of methyl jasmonate on the monoterpenes of Muscat Hamburg grapes and wine.

The importance of monoterpenes in grape and wine aroma has compelled researchers to focus on developing methods to increase their abundance. Recent research has revealed that exogenous elicitors can increase the contents of these compounds. This study determined the effects of methyl jasmonate (MeJA) preharvest treatments on the monoterpene profiles of Muscat Hamburg grapes and wine. A total of 27 monoterpenes were identified for Muscat Hamburg grapes and wine. The contents of most of the monoterpenes (free and glycosylated forms) in the grapes and wine increased in response to MeJA. An analysis of the expression of the genes in the terpenoid biosynthesis pathway indicated that the related biosynthetic pathways were activated by MeJA. The transcript levels of some genes were consistent with monoterpene production, including VviCSLinNer, VviGwbOciF, VviPNRLin, VviGT14 and VviUGT85A1L1. The developmental expression patterns of the VviPP2B1 and VviMYB24 transcription factor genes were positively correlated with monoterpene accumulation in ripening grapes. Our results suggest that MeJA may be useful for improving the aroma quality of grapes and wines.

Assigning the Origin of Microbial Natural Products by Chemical Space Map and Machine Learning.

Microbial natural products (NPs) are an important source of drugs, however, their structural diversity remains poorly understood. Here we used our recently reported MinHashed Atom Pair fingerprint with diameter of four bonds (MAP4), a fingerprint suitable for molecules across very different sizes, to analyze the Natural Products Atlas (NPAtlas), a database of 25,523 NPs of bacterial or fungal origin. To visualize NPAtlas by MAP4 similarity, we used the dimensionality reduction method tree map (TMAP). The resulting interactive map organizes molecules by physico-chemical properties and compound families such as peptides and glycosides. Remarkably, the map separates bacterial and fungal NPs from one another, revealing that these two compound families are intrinsically different despite their related biosynthetic pathways. We used these differences to train a machine learning model capable of distinguishing between NPs of bacterial or fungal origin.

Characterization of genes responsive to aerobic conditions by transcriptomic and genomic analyses of upland rice

The adaptation to aerobic environments enables upland rice to produce a sustainable grain yield under suboptimal conditions. In order to understand the molecular mechanisms involved in providing the adaptation of upland rice to aerobic environments, we previously developed introgression lines (ILs) with the irrigated rice variety Minghui63 (MH63) as the recipient parent and the upland rice variety Luyin46 (LY46) as the donor parent for subsequent identification of the relevant genes. In this study, IL-U135 was analyzed in detail because of its adaptation to aerobic conditions. Transcriptome profiling revealed 20 and 8 differentially expressed genes (DEGs) between IL-U135 and MH63 under anaerobic and aerobic conditions, respectively. In contrast, 306 and 188 DEGs were identified between LY46 and MH63 under anaerobic and aerobic conditions, respectively. Gene ontology (GO) analysis indicated that the adaptation of upland rice to aerobic environments is intimately associated with CLV3/ESR-related (CLE) signal transduction pathways, CLAVATA1 kinase activity, and salicylic acid related metabolism biosynthetic pathways. The IL-U135, LY46 and MH63 genomes were resequenced to map the genes responsible for adaptations to aerobic conditions. One the basis of an integrated analysis of the transcriptomic and genomic profiles, we propose that genes encoding an NBS-LRR protein (LOC_Os11g10570) and an ATP-binding protein (LOC_Os11g31480) may contribute to the adaptation of upland rice to aerobic environments. Our results may provide new insights into the molecular mechanism underlying the adaptation of rice to aerobic conditions.

Drought stress modulates cuticular wax composition of the grape berry.

Drought events are a major challenge for many horticultural crops, including grapes, which are often cultivated in dry and warm climates. It is not understood how the cuticle contributes to the grape berry response to water deficit (WD); furthermore, the cuticular waxes and the related biosynthetic pathways are poorly characterized in this fruit. In this study, we identified candidate wax-related genes from the grapevine genome by phylogenetic and transcriptomic analyses. Developmental and stress response expression patterns of these candidates were characterized across pre-existing RNA sequencing data sets and confirmed a high responsiveness of the pathway to environmental stresses. We then characterized the developmental and WD-induced changes in berry cuticular wax composition, and quantified differences in berry transpiration. Cuticular aliphatic wax content was modulated during development and an increase was observed under WD, with wax esters being strongly up-regulated. These compositional changes were related to up-regulated candidate genes of the aliphatic wax biosynthetic pathway, including CER10, CER2, CER3, CER1, CER4, and WSD1. The effect of WD on berry transpiration was not significant. This study indicates that changes in cuticular wax amount and composition are part of the metabolic response of the grape berry to WD, but these changes do not reduce berry transpiration.

Developmental Variation in Fruit Polyphenol Content and Related Gene Expression of a Red-Fruited versus a White-Fruited Fragaria vesca Genotype

Two cultivars of F. vesca, red-fruited Baron Solemacher (BS) and white-fruited Pineapple Crush (PC), were studied to compare and contrast the quantitative accumulation of major polyphenols and related biosynthetic pathway gene expression patterns during fruit development and ripening. Developing PC fruit showed higher levels of hydroxycinnamic acids in green stages and a greater accumulation of ellagitannins in ripe fruit in comparison to BS. In addition to anthocyanin, red BS fruit had greater levels of flavan-3-ols when ripe than PC. Expression patterns of key structural genes and transcription factors of the phenylpropanoid/flavonoid biosynthetic pathway, an abscisic acid (ABA) biosynthetic gene, and a putative ABA receptor gene that may regulate the pathway, were also analyzed during fruit development and ripening to determine which genes exhibited differences in expression and when such differences were first evident. Expression of all pathway genes differed between the red BS and white PC at one or more times during development, most notably at ripening when phenylalanine ammonia lyase 1 (PAL1), chalcone synthase (CHS), flavanone-3′-hydroxylase (F3′H), dihydroflavonol 4-reductase (DFR), anthocyanidin synthase (ANS), and UDP:flavonoid-O-glucosyltransferase 1 (UFGT1) were significantly upregulated in the red BS fruit. The transcription factors MYB1 and MYB10 did not differ substantially between red and white fruit except at ripening, when both the putative repressor MYB1 and promoter MYB10 were upregulated in red BS but not white PC fruit. The expression of ABA-related gene 9-cis-epoxycarotenoid dioxygenase 1 (NCED1) was higher in red BS fruit but only in the early green stages of development. Thus, a multigenic effect at several points in the phenylpropanoid/flavonoid biosynthetic pathway due to lack of MYB10 upregulation may have resulted in white PC fruit.

Proteomic analysis of Zanthoxylum nitidum seeds dormancy release: Influence of stratification and gibberellin

Zanthoxylum nitidum (Roxb.) DC. is one of the most important economic crops on account of its value as a Chinese herbal medicine (Zanthoxyli radix), health product and daily chemical raw materials. Zanthoxylum nitidum seeds are characterized to be an intermediate physiological dormancy that requires three months of cold stratification (4 °C) or gibberellin (GA) treatment (300 mg L−1, soaking for two days) to relieve seed dormancy and promote germination. Exploring the changes of proteins and related biosynthetic and metabolic pathways during seed dormancy release will contribute to expand our knowledge of this process. In this study, mature Zanthoxylum nitidum seeds from Pingyuan County, Guangdong province were treated with cold stratification and GA solution. The differently expressed proteins after treatments were characterized by iTRAQ coupled LC–MS/MS. In total, 484 proteins showed that at least 1.3-fold differences in abundance were detected after treatments; a majority of the differently abundant proteins are involved in the respiration-related metabolism processes, biosynthesis of amino acids and translation during the dormancy release of Zanthoxylum nitidum seeds. This study is the first to use the iTRAQ-based proteomics approach to analyse the proteome of Zanthoxylum nitidum seeds, pointing out the direction for our further research on the pathways and even the key genes and proteins associated with the dormancy release of Zanthoxylum nitidum seeds.

A Novel AP2/ERF Transcription Factor CR1 Regulates the Accumulation of Vindoline and Serpentine in Catharanthus roseus.

As one type of the most important alkaloids in the world, terpenoid indole alkaloids (TIAs) show a wide range of pharmaceutical activities that are beneficial for clinical treatments. Catharanthus roseus produces approximately 130 identified TIAs and is considered to be a model plant to study TIA biosynthesis. In order to increase the production of high medical value metabolites whose yields are extremely low in C. roseus, genetic engineering combined with transcriptional regulation has been applied in recent years. By using bioinformatics which is based on RNA sequencing (RNA-seq) data from methyl jasmonate (MeJA)-treated C. roseus as well as phylogenetic analysis, the present work aims to screen candidate genes that may be involved in the regulation of TIA biosynthesis, resulting in a novel AP2/ERF transcription factor, CR1 (Catharanthus roseus 1). Subsequently, virus-induced gene silencing (VIGS) of CR1 was carried out to identify the involvement of CR1 in the accumulations of several TIAs and quantitative real-time PCR (qRT-PCR) was then applied to detect the expression levels of 7 genes in the related biosynthetic pathway in silenced plants. The results show that all the 7 genes were upregulated in CR1-silenced plants. Furthermore, metabolite analyses indicate that silencing CR1 could increase the accumulations of vindoline and serpentine in C. roseus. These results suggest a novel negative regulator which may be involved in the TIAs biosynthetic pathway.

Cultivar Diversity of Grape Skin Polyphenol Composition and Changes in Response to Drought Investigated by LC-MS Based Metabolomics.

Phenolic compounds represent a large family of plant secondary metabolites, essential for the quality of grape and wine and playing a major role in plant defense against biotic and abiotic stresses. Phenolic composition is genetically driven and greatly affected by environmental factors, including water stress. A major challenge for breeding of grapevine cultivars adapted to climate change and with high potential for wine-making is to dissect the complex plant metabolic response involved in adaptation mechanisms. A targeted metabolomics approach based on ultra high-performance liquid chromatography coupled to triple quadrupole mass spectrometry (UHPLC-QqQ-MS) analysis in the Multiple Reaction Monitoring (MRM) mode has been developed for high throughput profiling of the phenolic composition of grape skins. This method enables rapid, selective, and sensitive quantification of 96 phenolic compounds (anthocyanins, phenolic acids, stilbenoids, flavonols, dihydroflavonols, flavan-3-ol monomers, and oligomers…), and of the constitutive units of proanthocyanidins (i.e., condensed tannins), giving access to detailed polyphenol composition. It was applied on the skins of mature grape berries from a core-collection of 279 Vitis vinifera cultivars grown with or without watering to assess the genetic variation for polyphenol composition and its modulation by irrigation, in two successive vintages (2014–2015). Distribution of berry weights and δ13C values showed that non irrigated vines were subjected to a marked water stress in 2014 and to a very limited one in 2015. Metabolomics analysis of the polyphenol composition and chemometrics analysis of this data demonstrated an influence of water stress on the biosynthesis of different polyphenol classes and cultivar differences in metabolic response to water deficit. Correlation networks gave insight on the relationships between the different polyphenol metabolites and related biosynthetic pathways. They also established patterns of polyphenol response to drought, with different molecular families affected either positively or negatively in the different cultivars, with potential impact on grape and wine quality.

Effect of methyl jasmonate on the aroma of Sangiovese grapes and wines

Methyl jasmonate (MeJA) was applied in a vineyard on leaves and grape clusters of cv Sangiovese to test its ability to stimulate the production of aromas and identify the main genes involved in the biosynthetic pathways switched on by the elicitor.MeJA application led to a delay in grape technological maturity and a significant increase in the concentration of several berry aroma classes (about twice the total aroma: from around 3 to 6μg/g of berry). Of these, monoterpenes showed the most significant increase. An analysis of the expression of terpenoid biosynthesis genes confirmed that the MeJA application activated the related biosynthetic pathway. The expression of all the TPS genes analyzedwas higher in samples treated with MeJA. Also the wines produced by microvinification of Sangiovese treated and untreated grapes showed a rise in the aroma concentration as in berries, with an important impact on longevity and sensorial characters of wines.

Altered metabolite accumulation in tomato fruits by coexpressing a feedback-insensitive AroG and the PhODO1 MYB-type transcription factor.

SummaryTargeted manipulation of phenylalanine (Phe) synthesis is a potentially powerful strategy to boost biologically and economically important metabolites, including phenylpropanoids, aromatic volatiles and other specialized plant metabolites. Here, we use two transgenes to significantly increase the levels of aromatic amino acids, tomato flavour‐associated volatiles and antioxidant phenylpropanoids. Overexpression of the petunia MYB transcript factor, ODORANT1 (ODO1), combined with expression of a feedback‐insensitive E. coli 3‐deoxy‐D‐arabino‐heptulosonate 7‐phosphate synthase (AroG), altered the levels of multiple primary and secondary metabolites in tomato fruit, boosting levels of multiple secondary metabolites. Our results indicate that coexpression of AroG and ODO1 has a dual effect on Phe and related biosynthetic pathways: (i) positively impacting tyrosine (Tyr) and antioxidant related metabolites, including ones derived from coumaric acid and ferulic acid; (ii) negatively impacting other downstream secondary metabolites of the Phe pathway, including kaempferol‐, naringenin‐ and quercetin‐derived metabolites, as well as aromatic volatiles. The metabolite profiles were distinct from those obtained with either single transgene. In addition to providing fruits that are increased in flavour and nutritional chemicals, coexpression of the two genes provides insights into regulation of branches of phenylpropanoid metabolic pathways.

Ectopic Expression of DREB Transcription Factor, AtDREB1A, Confers Tolerance to Drought in Transgenic Salvia miltiorrhiza.

Drought decreases crop productivity more than any other type of environmental stress. Transcription factors (TFs) play crucial roles in regulating plant abiotic stress responses. The Arabidopsis thaliana gene DREB1A/CBF3, encoding a stress-inducible TF, was introduced into Salvia miltiorrhiza Ectopic expression of AtDREB1A resulted in increased drought tolerance, and transgenic lines had higher relative water content and Chl content, and exhibited an increased photosynthetic rate when subjected to drought stress. AtDREB1A transgenic plants generally displayed lower malondialdehyde (MDA), but higher superoxide dismutase (SOD), catalase (CAT) and peroxidase (POD) activities under drought stress. In particular, plants with ectopic AtDREB1A expression under the control of the stress-induced RD29A promoter exhibited more tolerance to drought compared with p35S::AtDREB1A transgenic plants, without growth inhibition or phenotypic aberrations. Differential gene expression profiling of wild-type and pRD29A::AtDREB1A transgenic plants following drought stress revealed that the expression levels of various genes associated with the stress response, photosynthesis, signaling, carbohydrate metabolism and protein protection were substantially higher in transgenic plants. In addition, the amount of salvianolic acids and tanshinones was significantly elevated in AtDREB1A transgenic S. miltiorrhiza roots, and most of the genes in the related biosynthetic pathways were up-regulated. Together, these results demonstrated that inducing the expression of a TF can effectively regulate multiple genes in the stress response pathways and significantly improve the resistance of plants to abiotic stresses. Our results also suggest that genetic manipulation of a TF can improve production of valuable secondary metabolites by regulating genes in associated pathways.