L’Hér. (Myrtaceae) is one of the economically most important and widely cultivated trees for wood crop purposes worldwide. Climatic changes together with the constant need to expand plantations to areas that do not always provide optimal conditions for plant growth highlight the need to assess the impact of abiotic stresses on eucalypt trees. We aimed to unveil the drought effect on the leaf metabolome of commercial clones with differential phenotypic response to this stress. For this, seedlings of 13 clones were grown at well-watered (WW) and water-deficit (WD) conditions and their leaf extracts were subjected to comparative analysis using ultra-high performance liquid chromatography coupled to mass spectrometry (UPLC-MS) and nuclear magnetic resonance spectroscopy (NMR). UPLC-MS and NMR analyses led to the annotation of over 100 molecular features of classes such as cyclitols, phenolics, flavonoids, formylated phloroglucinol compounds (FPCs) and fatty acids. Multivariate data analysis was employed for specimens\' classifications and markers identification from both platforms. The results obtained in this work allowed us to classify clones differing in drought tolerance. Classification models were validated using an extra subset of samples. Tolerant plants exposed to water deficit accumulated arginine, gallic acid derivatives, caffeic acid and tannins at higher levels. In contrast, stressed drought-sensitive clones were characterised by a significant reduction in glucose, inositol and shikimic acid levels. These changes in contrasting drought response eucalypt pave ways for differential outcomes of tolerant and susceptible phenotypes. Under optimal growth conditions, all clones were rich in FPCs. These results can be used for early screening of tolerant clones and to improve our understanding of the role of these biomarkers in Eucalyptus tolerance to drought stress.

Cis-(+)-12-oxophytodienoic acid (cis-(+)-OPDA) is a bioactive jasmonate, a precursor of jasmonic acid, which also displays signaling activity on its own. Modulation of cis-(+)-OPDA actions may be carried out via biotransformation leading to metabolites of various functions. This work introduces a methodology for the synthesis of racemic cis-OPDA conjugates with amino acids (OPDA-aa) and their deuterium-labeled analogs, which enables the unambiguous identification and accurate quantification of these compounds in plants. We have developed a highly sensitive liquid chromatography-tandem mass spectrometry-based method for the reliable determination of seven OPDA-aa (OPDA-Alanine, OPDA-Aspartate, OPDA-Glutamate, OPDA-Glycine, OPDA-Isoleucine, OPDA-Phenylalanine, and OPDA-Valine) from minute amount of plant material. The extraction from 10 mg of fresh plant tissue by 10% aqueous methanol followed by single-step sample clean-up on hydrophilic–lipophilic balanced columns prior to final analysis was optimized. The method was validated in terms of accuracy and precision, and the method parameters such as process efficiency, recovery and matrix effects were evaluated. In mechanically wounded 30-day-old Arabidopsis thaliana leaves, five endogenous (+)-OPDA-aa were identified and their endogenous levels were estimated. The time-course accumulation revealed a peak 60 min after the wounding, roughly corresponding to the accumulation of cis-(+)-OPDA. Our synthetic and analytical methodologies will support studies on cis-(+)-OPDA conjugation with amino acids and research into the biological significance of these metabolites in plants.

Seven undescribed dammarane-type triterpenoids, together with ten known compounds, were isolated from the stems of Ziziphus glaziovii Warm (= Sarcomphalus glaziovii (Warm.) Hauenschild). The structures were fully assigned by means of uni- and bidimensional NMR and HR-ESI-MS experiments. Extract, fractions and also isolated compounds were evaluated for their antibacterial (against Bacillus subtilis and Aliivibrio fischeri), cytotoxic (against PC-3 and HT-29 human cancer cell lines), anthelmintic (against Caenorhabditis elegans) and antifungal (against Septoria triciti, Botrytis cinerea and Phytopthoria infestans) activities. The methanolic crude extract exhibited substantial antibacterial and cytotoxic activity. The known triterpenes epigouanic acid and alphitolic acid were the most active compounds against B. subtilis, with IC50 of 12 and 22 μM, respectively. The isolated compounds presented up to a concentration of 10 μM none or only weak effects in the cytotoxicity assays. No anthelminthic and antifungal activities were observed.

For the first time, the pigment composition of basidiocarps from the Chilean mushroom Cortinarius pyromyxa was studied under various aspects like phylogeny, chemistry and antibiotic activity. A molecular biological study supports the monotypic position of C. pyromyxa in subgenus Myxacium, genus Cortinarius. Four undescribed diterpenoids, named pyromyxones A-D, were isolated from fruiting bodies of C. pyromyxa. Their chemical structures were elucidated based on comprehensive one- and two-dimensional NMR spectroscopic analysis, ESI-HRMS measurements, as well as X-ray crystallography. In addition, the absolute configurations of pyromyxones A-D were established with the aid of JH,H, NOESY spectra and quantum chemical CD calculation. The pyromyxones A-D possess the undescribed nor-guanacastane skeleton. Tested pyromyxones A, B, and D exhibit only weak activity against gram-positive Bacillus subtilis and gram-negative Aliivibrio fischeri as well as the phytopathogenic fungi Botrytis cinerea, Septoria tritici and Phytophthora infestans.

In plant terpene biosynthesis, oxidation of the hydrocarbon backbone produced by terpene synthases is typically carried out by cytochrome P450 oxygenases (CYPs). The modifications introduced by CYPs include hydroxylations, sequential oxidations at one position and ring rearrangements and closures. These reactions significantly expand the structural diversity of terpenoids, but also provide anchoring points for further decorations by various transferases. In recent years, there has been a significant increase in reports of CYPs involved in plant terpene pathways. Plant diterpenes represent an important class of metabolites that includes hormones and a number of industrially relevant compounds such as pharmaceutical, aroma or food ingredients. In this review, we provide a comprehensive survey on CYPs reported to be involved in plant diterpene biosynthesis to date. A phylogenetic analysis showed that only few CYP clans are represented in diterpene biosynthesis, namely CYP71, CYP85 and CYP72. Remarkably few CYP families and subfamilies within those clans are involved, indicating specific expansion of these clades in plant diterpene biosynthesis. Nonetheless, the evolutionary trajectory of CYPs of specialized diterpene biosynthesis is diverse. Some are recently derived from gibberellin biosynthesis, while others have a more ancient history with recent expansions in specific plant families. Among diterpenoids, labdane-related diterpenoids represent a dominant class. The availability of CYPs from diverse plant species able to catalyze oxidations in specific regions of the labdane-related backbones provides opportunities for combinatorial biosynthesis to produce novel diterpene compounds that can be screened for biological activities of interest.

Phenylpropanoids are a class of plant natural products that have many biological functions, including stress defence. In barley, phenylpropanoids have been described as having protective properties against excess UV-B radiation and have been linked to resistance to pathogens. Although the phenylpropanoid composition of barley has recently been addressed in more detail, the biosynthesis and regulation of this pathway have not been fully established. Barley introgression lines, such as the S42IL-population offer a set of genetically diverse plants that enable the correlation of metabolic data to distinct genetic regions on the barley genome and, subsequently, identification of relevant genes.The phenylpropanoid profiles of the first and third leaf of barley seedlings in Scarlett and four members of the S42IL-population were obtained by LC-MS. Comparison of the leaf profiles revealed a change in the glycosylation pattern of the flavone-6-C-glucoside isovitexin in the elite cultivar Scarlett. The change was characterized by the stepwise decrease in isovitexin-7-O-glucoside (saponarin) and an increase in isovitexin-2″-O-β-D-glucoside content.The lines S42IL-101-, -177 and -178 were completely devoid of isovitexin-2″-O-β-D-glucoside. Parallel glucosyltransferase assays were consistent with the observed metabolic patterns. The genetic region responsible for this metabolic effect was located on chromosome 1H between 0.21 and 15.08 cM, encompassing 505 gene candidates in the genome of the sequenced cultivar Morex. Only one of these genes displayed sequence similarity with glucosyltransferases of plant secondary metabolism that possessed the characteristic PSPG motif.

In the highly aluminum-resistant tree Eucalyptus camaldulensis, hydrolyzable tannins are proposed to play a role in internal detoxification of aluminum, which is a major factor inhibiting plant growth on acid soils. To understand and modulate the molecular mechanisms of aluminum detoxification by hydrolyzable tannins, the biosynthetic genes need to be identified. In this study, we identified and characterized genes encoding UDP-glucose:gallate glucosyltransferase, which catalyzes the formation of 1-O-galloyl-β-d-glucose (β-glucogallin), the precursor of hydrolyzable tannins. By homology-based cloning, seven full-length candidate cDNAs were isolated from E. camaldulensis and expressed in Escherichia coli as recombinant N-terminal His-tagged proteins. Phylogenetic analysis classified four of these as UDP glycosyltransferase (UGT) 84A subfamily proteins (UGT84A25a, -b, UGT84A26a, -b) and the other three as UGT84J subfamily proteins (UGT84J3, -4, -5). In vitro enzyme assays showed that the UGT84A proteins catalyzed esterification of UDP–glucose and gallic acid to form 1-O-galloyl-β-d-glucose, whereas the UGT84J proteins were inactive. Further analyses with UGT84A25a and −26a indicated that they also formed 1-O-glucose esters of other structurally related hydroxybenzoic and hydroxycinnamic acids with a preference for hydroxybenzoic acids. The UGT84A genes were expressed in leaves, stems, and roots of E. camaldulensis, regardless of aluminum stress. Taken together, our results suggest that the UGT84A subfamily enzymes of E. camaldulensis are responsible for constitutive production of 1-O-galloyl-β-d-glucose, which is the first step of hydrolyzable tannin biosynthesis.

In Brassica napus, suppression of the key biosynthetic enzyme UDP-glucose:sinapic acid glucosyltransferase (UGT84A9) inhibits the biosynthesis of sinapine (sinapoylcholine), the major phenolic component of seeds. Based on the accumulation kinetics of a total of 158 compounds (110 secondary and 48 primary metabolites), we investigated how suppression of the major sink pathway of sinapic acid impacts the metabolome of developing seeds and seedlings. In UGT84A9-suppressing (UGT84A9i) lines massive alterations became evident in late stages of seed development affecting the accumulation levels of 58 secondary and 7 primary metabolites. UGT84A9i seeds were characterized by decreased amounts of various hydroxycinnamic acid (HCA) esters, and increased formation of sinapic and syringic acid glycosides. This indicates glycosylation and β-oxidation as metabolic detoxification strategies to bypass intracellular accumulation of sinapic acid. In addition, a net loss of sinapic acid upon UGT84A9 suppression may point to a feedback regulation of HCA biosynthesis. Surprisingly, suppression of UGT84A9 under control of the seed-specific NAPINC promoter was maintained in cotyledons during the first two weeks of seedling development and associated with a reduced and delayed transformation of sinapine into sinapoylmalate. The lack of sinapoylmalate did not interfere with plant fitness under UV-B stress. Increased UV-B radiation triggered the accumulation of quercetin conjugates whereas the sinapoylmalate level was not affected.

In search for new or chemo-taxonomically relevant bioactive compounds from chemically unexplored Hypericum species, four previously undescribed natural products, named peplidiforones A–D were isolated and characterized from Hypericum peplidifolium A. Rich., together with six known compounds. The structures of all compounds were elucidated by extensive 1D- and 2D-NMR experiments, high resolution mass spectrometric analyses (HR-MS), and by comparison with data reported in the literature. Seven of these compounds are phenyl polyketides while three are acylphloroglucinol type compounds. Peplidiforone C, which possesses an unusual carbon skeleton consisting of a furan ring substituted by a 2,2-dimethylbut-3-enoyl moiety, is the first example of a prenylated furan derivative isolated from the genus Hypericum. The cytotoxicity, antifungal, and anti-herpes simplex virus type 1 (HSV-1) activities of extracts and compounds are described.

Jasmonates (JAs) are plant hormones that integrate external stress stimuli with physiological responses. (+)-7-iso-JA-L-Ile is the natural JA ligand of COI1, a component of a known JA receptor. The upstream JA biosynthetic precursor cis-(+)-12-oxo-phytodienoic acid (cis-(+)-OPDA) has been reported to act independently of COI1 as an essential signal in several stress-induced and developmental processes. Wound-induced increases in the endogenous levels of JA/JA-Ile are accompanied by two to tenfold increases in the concentration of OPDA, but its means of perception and metabolism are unknown. To screen for putative OPDA metabolites, vegetative tissues of flowering Arabidopsis thaliana were extracted with 25% aqueous methanol (v/v), purified by single-step reversed-phase polymer-based solid-phase extraction, and analyzed by high throughput mass spectrometry. This enabled the detection and quantitation of a low abundant OPDA analog of the biologically active (+)-7-iso-JA-L-Ile in plant tissue samples. Levels of the newly identified compound and the related phytohormones JA, JA-Ile and cis-(+)-OPDA were monitored in wounded leaves of flowering Arabidopsis lines (Col-0 and Ws) and compared to the levels observed in Arabidopsis mutants deficient in the biosynthesis of JA (dde2-2, opr3) and JA-Ile (jar1). The observed cis-(+)-OPDA-Ile levels varied widely, raising questions concerning its role in Arabidopsis stress responses.