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The fruits of Coccinia grandis (L.) Voigt have been used to treat several aliments in traditional medicine whereas Coccinia rehmannii Cogn. fruits are consumed as food due to their high starch content. Coccinia rehmannii are also known to contain vast amounts of indispensable bioactive compounds that are used in various cosmetics, foods, and medicines. Among these compounds are hydroxycinnamic acids (HCAs) which are usually conjugated/esterified to quinic acid (QA), tartaric acid, citric acid, shikimic acid and sugars and are collectively referred to as chlorogenic acids (CGAs). Over the years, metabolite profiling has been used in plant taxonomy and species classification. Herein, UPLC-qTOF-MS/MS semi-targeted approached was use to profile HCA conjugates from two closely related plant species i.e. C. grandis and C. rehmannii leaves in order to distinguish them from each other. Examination of their relative metabolite contents showed that the two closely related Coccinia species utilise contrasting means of HCA conjugations as means of metabolite diversification strategy. C. grandis was found to conjugate its HCA derivatives to a QA, whilst C. rehmannii conjugates its HCA derivatives to mainly flavonoid glycosides and to QA, even though present in low concentration in this plant. These results shows that plants from the same genus can utilise different chemical diversification means for storage of their metabolites and that processes such as conjugation and isomerization can be exploited as chemotaxonomic markers. Future studies can also be designed to look at the pharmacological potency of these plants as a function of their contrasting chemical differences.

This study investigated the phytochemical constituents and evaluated the total phenolic content, it also determined the antioxidant and antibacterial activities of the ethanol (EtOH) extract of the whole plant of Dicliptera verticillata. The phytochemical study was carried out using repeated silica gel column chromatography followed by Sephadex LH-20 to purify the extract which afforded three known compounds: lutein (1), shikimic acid (2) and guaiacylglycerol (3). The structures of these compounds were identified after analyses of their NMR and MS data and comparison of these data with those reported in the literature. The antioxidant and antibacterial activities were evaluated by the 2,2-diphenyl-1-picrylhydrazyl radical assay and the broth macrodilution method, respectively. The EtOH extract showed moderate antioxidant activity (63.9%; IC50 40.16 μg/mL), weak total phenolic content and had a bactericidal effect against both Gram-positive (Staphylococcus aureus) and Gram-negative bacteria (Klebsiella pneumonia and Escherichia coli). This is the first report on the isolation of compounds (1–3), as well as the total phenolic content and the antibacterial and antioxidant activities of the EtOH extract of D. verticillata.

Two undescribed shikimic acid derivatives named shikimerans B and C (1 and 2), one new natural compound shikimeran D (3), as well as five known compounds 4−8 were obtained from the EtOAc extract of the endophytic fungus Chaetomium sp. FZ-4. Their structures were determined by the NMR spectroscopy and HRESIMS examination, while the absolute configurations of compounds 1−3 were clarified based on the electronic circular dichroism (ECD) experiment. These new compounds enriched the structural diversity of shikimic acid analogues and also expanded the chemical diversity of the metabolites of Chaetomium sp..

Background Illicium griffithii is an aromatic medicinal tree species that has been listed in the IUCN Red List as an endangered species. Dried seed pods of I. griffithii have a good market potential in the spices and pharmaceutical industries. Fruits are the potential source of shikimic acid and used for the production of oseltamivir (a drug against bird flu). However, in recent years, unscientific harvesting and rampant exploitation of the species has caused a negative and adverse effect on its natural population. Proper knowledge of genetic diversity and population structure is crucial to understand the population dynamics, adaptation, and evolutionary pattern of a particular species for conservation. It was from this view point that the present study was undertaken so as to compare the various types of DNA-based molecular markers namely RAPD, ISSR, DAMD, and SCoT by their efficiency and SPAR approach to evaluate the genetic diversity of I. griffithii as well as to analyze population genetic structure for conservation purpose. Result A total of 250 discernible bands were generated with 246 bands (98.40 %) being polymorphic in nature. All the primers in combination gave a mean polymorphic information content (PIC) of 0.81 and Rp value (resolving power) of 4.32. Nei’s, Gst, and AMOVA analysis showed similar values of genetic differentiation among populations (Gst = 0.396, FST = 0.30, respectively), revealing a low level of genetic differentiation among the eight sampled populations. I. griffithii with an estimated gene flow value of Nm = 0.761 was significantly low among populations. Clustering pattern obtained with Bayesian structure and PCoA diagram revealed that intermixing of genetic material across populations is only possible when the populations lie close to each other. This is further validated with UPGMA clustering method where a positive correlation of genetic variability with geographical distance among closely related populations could be clearly seen. Conclusion The result aids in the identification, collection, and preservation of diverse germplasm of I. griffithii from Arunachal Pradesh and Meghalaya of Northeast India. This would further help in understanding the population structure and genetic diversity among other Illicium species in order to formulate effective conservation strategies for the improvement of this endangered taxa.

Chemical control has become less effective for the invasive weed species horseweed (Conyza canadensis) found in tangerine (Citrus reticulate) orchards of Korea. The dose–response assays of the biotypes of C. canadensis collected in 2017 from the Jeju province of Korea demonstrated that the amount of glyphosate needed for 50% growth reduction (GR50) in the glyphosate-resistant (GR) biotype was up to 100 times greater than that in the glyphosate-susceptible (GS) biotype (0.328 kg a.e. ha−1). However, the mechanism of resistance in the GR populations of C. canadensis in Korea is unknown. Therefore, this study aimed to determine the mechanism of resistance to glyphosate in the Korean C. canadensis populations. Shikimic acid accumulation and 5-enolpyruvyl shikimate-3-phosphate synthase (EPSPS) gene expression were analyzed in a GR and a GS biotype of C. canadensis. The GR biotype showed less shikimic acid accumulation than the GS biotype. A target point mutation in the sequence encoding the Pro 106 of EPSPS1 was not found after sequencing the GR biotype. Moreover, the expression level of EPSPS1 was not altered significantly following application of glyphosate to the GS and GR biotypes, whereas the levels of M10 and M11 ABC transporter genes which play an important role in plant growth and nutrition, and the interaction of the plant with its environment were found to be slightly higher in the glyphosate-treated GS and GR biotypes than the untreated control plants. Our results show that M10 and M11 are likely to be involved in the glyphosate resistance mechanism in C. canadensis. Consequently, non-chemical control methods should be implemented to manage or slow the spread of glyphosate resistance in C. canadensis.

To improve the production of cephalotaxine and explore its biosynthesis pathway in the suspension cells of Cephalotaxus mannii, the cell suspension cultures were separately treated with elicitors, including coronatine (0.5, 1.0, 2.0 μmol/L) and methyl jasmonate (100 μmol/L), and then analyzed their transcriptome differences. The results showed that coronatine could improve the content of cephalotaxine in suspension cells of C. mannii. The highest product (6.75 mg/L) appeared in the coronatine treatment of 1.0 μmol/L, which was higher than that of methyl jasmonate treatment (4.29 mg/L) and control (3.14 mg/L). The key enzyme of the shikimic acid pathway, 3-deoxy-d-arabino-heptulosonate-7-phosphate synthase (DS) activity increased after 24 h of adding elicitors above. Transcriptome analysis indicated that the phenylpropane pathway, shikimic acid pathway, glucose metabolism process, phenylalanine, and tyrosine synthesis pathway were involved in the synthesis of cephalotaxine. In addition, glucose-6-phosphate dehydrogenase (G6PDH), hexokinase, phenylalanine ammonialyase (PAL), and Cytochrome P450, etc. were found out to be closely related to the cephalotaxine accumulation. It could be concluded that coronatine promoted the synthesis of cephalotaxine by affecting the expression of some enzymes, especially DS in the synthesis pathway of cephalotaxine. Coronatine (1.0 μmol/L) led a yield of 6.75 mg/L cephalotaxine, which was 2.15 times of the control. The transcriptome analysis indicated DS played an important role in cephalotaxine biosynthesis.

Background Outbreaks of insect pests in paddy fields cause heavy losses in global rice yield annually, a threat projected to be aggravated by ongoing climate warming. Although significant progress has been made in the screening and cloning of insect resistance genes in rice germplasm and their introgression into modern cultivars, improved rice resistance is only effective against either chewing or phloem-feeding insects. Results In this study, the results from standard and modified seedbox screening, settlement preference and honeydew excretion tests consistently showed that Qingliu, a previously known leaffolder-resistant rice variety, is also moderately resistant to brown planthopper (BPH). High-throughput RNA sequencing showed a higher number of differentially expressed genes (DEGs) at the infestation site, with 2720 DEGs in leaves vs 181 DEGs in sheaths for leaffolder herbivory and 450 DEGs in sheaths vs 212 DEGs in leaves for BPH infestation. The leaf-specific transcriptome revealed that Qingliu responds to leaffolder feeding by activating jasmonic acid biosynthesis genes and genes regulating the shikimate and phenylpropanoid pathways that are essential for the biosynthesis of salicylic acid, melatonin, flavonoids and lignin defensive compounds. The sheath-specific transcriptome revealed that Qingliu responds to BPH infestation by inducing salicylic acid-responsive genes and those controlling cellular signaling cascades. Taken together these genes could play a role in triggering defense mechanisms such as cell wall modifications and cuticular wax formation. Conclusions This study highlighted the key defensive responses of a rarely observed rice variety Qingliu that has resistance to attacks by two different feeding guilds of herbivores. The leaffolders are leaf-feeder while the BPHs are phloem feeders, consequently Qingliu is considered to have dual resistance. Although the defense responses of Qingliu to both insect pest types appear largely dissimilar, the phenylpropanoid pathway (or more specifically phenylalanine ammonia-lyase genes) could be a convergent upstream pathway. However, this possibility requires further studies. This information is valuable for breeding programs aiming to generate broad spectrum insect resistance in rice cultivars.

Background: Pistacia lentiscus L. (Lentisk) an evergreen tree or shrub that is ubiquitous across the semi-arid areas of the Mediterranean and native to all Mediterranean islands. All parts of Lentisk, including resin, leaves, gum, fruits, and aerial parts, have been used as valuable remedies for various diseases since antiquity. Objective: This paper highlights a novel source for the isolation of pure shikimic acid from the Lentisk ethyl acetate extract, which could provide an alternative and renewable solution for shikimic acid in the future. Methods: Characterisation by melting point, elemental analysis, FTIR, HRMS, 1H-NMR, DEPT( 135)-NMR, and 13C-NMR spectroscopy confirmed the purity of shikimic acid. The antioxidant activity of Lentisk extract and shikimic acid was performed by DPPH free radical scavenging activity assay. Results: The results prove that Lentisk is an alternative and renewable source of shikimic acid. With regard to activity, the shikimic acid does not act as the lead compound in the antioxidant activity of Lentisk extract. Conclusion: Lentisk is a novel alternative and renewable source of shikimic acid.

UV radiation is one of the main contributors to skin photoaging by promoting the accumulation of cellular senescence, which in turn induces a proinflammatory and tissue-degrading state that favors skin aging. The members of the sirtuin family of NAD + -dependent enzymes play an anti-senescence role and their activation suggests a promising approach for preventing UV-induced senescence in the treatment of skin aging. A two-step screening designed to identify compounds able to protect cells from UV-induced senescence through sirtuin activation identified shikimic acid (SA), a metabolic intermediate in many organisms, as a bona-fide candidate. The protective effects of SA against senescence were dependent on specific activation of SIRT1 as the effect was abrogated by the SIRT1 inhibitor EX-527. Upon UV irradiation SA induced S-phase accumulation and a decrease in p16 INK4A expression but did not protect against DNA damage or increased polyploidies. In contrast, SA reverted misfolded protein accumulation upon senescence, an effect that was abrogated by EX-527. Consistently, SA induced an increase in the levels of the chaperone BiP, resulting in a downregulation of unfolded protein response (UPR) signaling and UPR-dependent autophagy, avoiding their abnormal hyperactivation during senescence. SA did not directly activate SIRT1 in vitro, suggesting that SIRT1 is a downstream effector of SA signaling specifically in the response to cellular senescence. Our study not only uncovers a shikimic acid/SIRT1 signaling pathway that prevents cellular senescence, but also reinforces the role of sirtuins as key regulators of cell proteostasis.

The plant shikimate pathway directs bulk carbon flow toward biosynthesis of aromatic amino acids (AAAs, i.e. tyrosine, phenylalanine, and tryptophan) and numerous aromatic phytochemicals. The microbial shikimate pathway is feedback inhibited by AAAs at the first enzyme, 3-deoxy-d-arabino-heptulosonate 7-phosphate synthase (DHS). However, AAAs generally do not inhibit DHS activities from plant extracts and how plants regulate the shikimate pathway remains elusive. Here, we characterized recombinant Arabidopsis thaliana DHSs (AthDHSs) and found that tyrosine and tryptophan inhibit AthDHS2, but not AthDHS1 or AthDHS3. Mixing AthDHS2 with AthDHS1 or 3 attenuated its inhibition. The AAA and phenylpropanoid pathway intermediates chorismate and caffeate, respectively, strongly inhibited all AthDHSs, while the arogenate intermediate counteracted the AthDHS1 or 3 inhibition by chorismate. AAAs inhibited DHS activity in young seedlings, where AthDHS2 is highly expressed, but not in mature leaves, where AthDHS1 is predominantly expressed. Arabidopsis dhs1 and dhs3 knockout mutants were hypersensitive to tyrosine and tryptophan, respectively, while dhs2 was resistant to tyrosine-mediated growth inhibition. dhs1 and dhs3 also had reduced anthocyanin accumulation under high light stress. These findings reveal the highly complex regulation of the entry reaction of the plant shikimate pathway and lay the foundation for efforts to control the production of AAAs and diverse aromatic natural products in plants.

Oseltamivir phosphate (OSP), marketed under the brand name of Tamiflu®, is a potent inhibitor of viral neuraminidase, a glycoside hydrolase enzyme essential for viral replication. OSP is also important to control influenza virus (H1N1) and is produced mainly from shikimic acid (SA) extracted from plants. The main source of SA is from seeds of I. verum. Alternatives to meet the worldwide demand are necessary. One of this is Cryptomeria japonica. In this study, we found a content of 56.36 mg.kg-1 of SA in dried biomass for C. japonica, while for I.verum, the traditional source, was 96.57 mg.kg-1. Due to the fact of C. japonica is a tree, exploited for commercial production, it can be a renewable and reliable source SA for OSP production.

Resveratrol is a plant secondary metabolite with multiple health-beneficial properties. Microbial production of resveratrol in model microorganisms requires extensive engineering to reach commercially viable levels. Here, we explored the potential of the non-conventional yeast Yarrowia lipolytica to produce resveratrol and several other shikimate pathway-derived metabolites (p-coumaric acid, cis,cis-muconic acid, and salicylic acid). The Y. lipolytica strain expressing a heterologous pathway produced 52.1 ± 1.2 mg/L resveratrol in a small-scale cultivation. The titer increased to 409.0 ± 1.2 mg/L when the strain was further engineered with feedback-insensitive alleles of the key genes in the shikimate pathway and with five additional copies of the heterologous biosynthetic genes. In controlled fed-batch bioreactor, the strain produced 12.4 ± 0.3 g/L resveratrol, the highest reported titer to date for de novo resveratrol production, with a yield on glucose of 54.4 ± 1.6 mg/g and a productivity of 0.14 ± 0.01 g/L/h. The study showed that Y. lipolytica is an attractive host organism for the production of resveratrol and possibly other shikimate-pathway derived metabolites., Highlights • Oleaginous yeast Y. lipolytica was engineered for production of aromatic compounds. • High resveratrol production required increased activities of Aro4p and Aro7p. • Multiple integration of resveratrol biosynthetic genes improved production. • Fed-batch fermentation enabled de novo production of 12.4 g/L resveratrol.

Chorismate mutase (CM), an essential enzyme at the branch-point of the shikimate pathway, is required for the biosynthesis of phenylalanine and tyrosine in bacteria, archaea, plants, and fungi. MtCM, the CM from Mycobacterium tuberculosis, has less than 1% of the catalytic efficiency of a typical natural CM and requires complex formation with 3-deoxy-d-arabino-heptulosonate 7-phosphate synthase for high activity. To explore the full potential of MtCM for catalyzing its native reaction, we applied diverse iterative cycles of mutagenesis and selection, thereby raising kcat/Km 270-fold to 5 × 105 m−1s−1, which is even higher than for the complex. Moreover, the evolutionarily optimized autonomous MtCM, which had 11 of its 90 amino acids exchanged, was stabilized compared with its progenitor, as indicated by a 9 °C increase in melting temperature. The 1.5 Å crystal structure of the top-evolved MtCM variant reveals the molecular underpinnings of this activity boost. Some acquired residues (e.g. Pro52 and Asp55) are conserved in naturally efficient CMs, but most of them lie beyond the active site. Our evolutionary trajectories reached a plateau at the level of the best natural enzymes, suggesting that we have exhausted the potential of MtCM. Taken together, these findings show that the scaffold of MtCM, which naturally evolved for mediocrity to enable inter-enzyme allosteric regulation of the shikimate pathway, is inherently capable of high activity. ISSN:0021-9258 ISSN:1083-351X

Biosynthesis of certain secondary metabolites, as a relief response to the severe environmental stress of the mediterranean summer, is a major reaction of the plants thriving in areas with mediterranean-type climate. Some of these compounds are of great importance to both humans and the environment. Tracing of certain secondary metabolites, after the exposure of Olea europaea L. individuals to stressing environmental conditions, is the aim of this investigation. Our two-day cycle, detailed data indicate that soon after the summer mid-day severe environmental stress, a threefold increase (+ 256%) in reactive oxygen species accumulation is recorded in the leaves while the absorbance of the photosynthetic pigments is drastically reduced (up to − 60%). Then, the secondary metabolic pathway of the shikimic acid is activated and the expression level of l-Dopa decarboxylase—an enzyme common to plants and animals, bisconnected to this pathway—is rapidly increased in order to metabolize the highly toxic l-Dopa or the amino acid l-tyrosine, to dopamine or tyramine, respectively. These reactions promote, at a certain period of the 24-h cycle, the biosynthesis of oleuropein (41% increase), an antioxidant phenolic compound possessing a wide range of important pharmacological properties. By the evening twilight, when the environmental conditions turn to mild, the cell functions reset to normal.

A tissue culture independent in-planta transformation technique is an alternate option to generate transgenics in crop cultivars recalcitrant for regeneration. The T0 plants generated by in-planta technique are chimeric hence very large number of T1 seeds needs to be screened to identify the putative transformants. Besides, low transformation efficiency and stable integration is another lacuna, because the T0 seedling explants are not subjected for selection. To overcome these constraints, a modified in-planta technique was developed in this study to generate gain-in-function activation tagged mutants in the background of a recalcitrant rice genotype, KMP-153. A binary vector was developed with three glyphosate selectable marker genes and 4X CaMV 35S enhancer elements for developing rice plants. Simultaneous expression of Glyphosate insensitive CP4-EPSPS with two other glyphosate degrading enzymes, PsAKR1 (Aldo–Keto reductases) and GO (Glycine Oxidase) substantially improved tolerance up to 100 ppm of glyphosate in the seedling bioassay system. Selecting the seedling explants and T1 generation seeds at higher concentration (100 ppm) of glyphosate resulted in recovering fewer transformants but with stable integration and expression of all the transgenes compared to those screened at lower levels of glyphosate. Further, T2 generation plants from these transgenic lines grown in contained nethouse conditions were tolerant when sprayed up to 1500 ppm concentrations of glyphosate. These transgenics showed substantial lower levels of shikimic acid compared to wild type plants treated with glyphosate signifies the expression of the transgenes and less inhibition of shikimic acid pathway by glyphosate. The proteins extracted from the transgenic plants showed significant degradation of glyphosate. The stable integration was confirmed through flanking sequence information and event specific PCR information. Our results clearly demonstrate that the modified in-planta technique is a potential transformation protocol to generate stable transformants in rice cultivars.

Siniperca chuatsi is currently one of the most important economic farmed freshwater fish in China. The aim of this study was to evaluate the metabolic profile of recirculating ponds aquaculture system (RAS)-farmed S. chuatsi. Gas Chromatography-Mass Spectrophotometry (GC-MS) metabolomic platform was used to comprehensively analyze the effects of recirculating ponds aquaculture system (RAS) on the Mandarin fish S. chuatsi metabolism. Database searching and statistical analysis revealed that there were altogether 335 metabolites quantified (similarity > 0) and 205 metabolites were identified by mass spectrum matching with a spectral similarity > 700. Among the 335 metabolites quantified, 33 metabolites were significantly different (VIP > 1 and p S. chuatsi farmed in RAS. RAS can provide comprehensive benefits to the effects of Siniperca chuatsi metabolism, which suggest RAS is an efficient, economic, and environmentally friendly farming system compared to pond system.

Grouper iridovirus causes high mortality rates in cultured groupers, and effective treatment for grouper iridovirus infection is urgently required. Illicium verum Hook. f. is a well-known medicinal plant with a variety of biological activities. The aim of this study was to analyse the use of I. verum extracts to treat grouper iridovirus infection. The safe working concentration of each I. verum extract was identified both in vitro and in vivo as follows: I. verum aqueous extract (IVAE) ≤ 500 μg/ml; I. verum ethanol extract (IVEE) ≤ 250 μg/ml; shikimic acid (SKA) ≤ 250 μg/ml; trans-anethole (TAT) ≤ 800 μg/ml; 3,4-dihydroxybenzoic acid (DDBA) ≤ 400 μg/ml; and quercetin (QCE) ≤ 50 μg/ml. The inhibitory activity of each I. verum extract against grouper iridovirus infection was analysed using aptamer (Q2)-based fluorescent molecular probe (Q2-AFMP) and RT-qPCR. All of the I. verum extracts displayed dose-dependent antiviral activities against grouper iridovirus. Based on the achieved per cent inhibition, IVAE, IVEE, DDBA and QCE were associated with the greatest antiviral activity (all > 90%). Together, our results indicate that I. verum extracts have effective antiviral properties, making it an excellent potential source material for the development of effective treatment for grouper iridovirus infection.

Hairy fleabane (Conyza bonariensis (L.) Cronq.) is an annual weed which occurs typically on waste land and crops such as orchards and vineyards. It is considered one of the worst weeds worldwide, including Greece, because it has developed resistance to glyphosate. Glyphosate is a systemic, non-selective herbicide and it is the world’s biggest selling for weed control. Salicylic acid is a phenolic growth regulator, providing protection against biotic and abiotic stress of plants. In the present study, it is investigated the effect of a synthetic compound which is a chemical analog to salicylic acid (benzo (1,2,3) - thiadiazole −7-carbothioic acid S - methyl ester -BTH), in fleabane resistant and susceptible biotype, on glyphosate resistance. Some plants from resistant and susceptible biotype were sprayed with glyphosate, while others were sprayed with both BTH and glyphosate. The proline content, hydrogen peroxide (H2O2) content and malondialdehyde (MDA) content in fleabane leaves were then determined on the 1st, 6th and 10th days following treatment. The concentration of shikimic acid and photosynthetic activity was determined on the 3rd day after treatment. The results showed that the BTH caused protection against glyphosate damage in both fleabane biotypes, decreasing the hydrogen peroxide and MDA content and increasing the proline content. However, BTH did not affect the concentration of shikimic acid. Moreover, resistant and susceptible biotype were affected differently by BTH application. Considering all the above, we can conclude that BTH induced the defense in plants, in both biotypes, after the glyphosate application. Furthermore, susceptible biotype was affected more by BTH application than resistant biotype. Although, the defense mechanisms in susceptible biotype were induced more profoundly after BTH application compared to resistant biotype, susceptible biotype showed slow activation of defense mechanisms, thus it could not survive possibly due to the fact that glyphosate action had already caused detrimental effects on plants.

The present experiment was conducted to evaluate the influence of Lamiaceae herbs companion planting on growth and secondary metabolites changes in tomato plants. Furthermore, the free amino acid changes in tomato due to companion planting were also evaluated using tomato-basil companionship as a model. Four-week-old seedlings of tomato were grown in a pot containing autoclaved commercial soil with basil, peppermint and hyssop as a companion plant separately in different density. Four weeks after companion planting, tomato plants under 1:1 companionship with the herbs showed significant increase in dry weights of shoots compared to control. Higher density of the herbs on the other hand expressed a growth suppression on tomato possibly due to nutrient competition. By the LC-MS analysis, shikimic acid and apigenin were identified as the major secondary compounds found in tomato plants and 1:1 companionship with basil seemed to have a positive influence on their content in tomato shoots. On the other hand, in case of peppermint and hyssop, the increase was observed in all parts of tomato plants. In addition, promotion in several free amino acid contents was also observed in tomato plants with basil companion planting compared to control. Thus, tomato plants grown with herb companion planting in 1:1 ratio seems to have a positive impact on growth of tomato. This positive influence might be related to the increase in some secondary metabolites and changes in the free amino acids observed in this study.

Traditional Chinese Medicine uses plant-based ingredients, many of which have proven to have therapeutic effects through modern pharmacological studies. Such plant metabolites are also found in important cash crops such as tobacco (Nicotiana tabacum L.), the cultivation and processing of which produces waste leaves. We hypothesized that these waste leaves contained active compounds and when extracted could be converted into a useful resource. We, therefore, investigated the constitution of two tobacco cultivars (Basma and K326) using ultra-high-performance liquid chromatography tandem quadrupole time of flight mass spectrometry combined with non-targeted metabolomics. A total of 107 known Traditional Chinese Medicine active ingredients were detected, some of which are widely used in medicine, chemical synthesis, perfumes, and pesticide production. A total of 70 ingredients were present in the two cultivars, at significantly different levels (P 1). Among them, nicotine, shikimic acid, caffeic acid, scopoletin, quinic acid, 3-caffeoylquinic acid, and isoferulic acid were potentially valuable metabolites. Waste leaves generated in the production of Basma and K326 cultivars have potential as resources for production of these metabolites, but special attention should be paid to the varietal influence.

Co-enzyme A (CoA) ligation of hydroxycinnamic acids by 4-coumaric acid:CoA ligase (4CL) is a critical step in the biosynthesis of monolignols. Perturbation of 4CL activity significantly impacts the lignin content of diverse plant species. InPopulus trichocarpa, two well-studied xylem-specific Ptr4CLs (Ptr4CL3 and Ptr4CL5) catalyze the CoA ligation of 4-coumaric acid to 4-coumaroyl-CoA and caffeic acid to caffeoyl-CoA. Subsequently, two 4-hydroxycinnamoyl-CoA:shikimic acid hydroxycinnamoyl transferases (PtrHCT1 and PtrHCT6) mediate the conversion of 4-coumaroyl-CoA to caffeoyl-CoA. Here, we show that the CoA ligation of 4-coumaric and caffeic acids is modulated by Ptr4CL/PtrHCT protein complexes. Downregulation ofPtrHCTsreduced Ptr4CL activities in the stem-differentiating xylem (SDX) of transgenicP. trichocarpa. The Ptr4CL/PtrHCT interactions were then validatedin vivousing biomolecular fluorescence complementation (BiFC) and protein pull-down assays inP. trichocarpaSDX extracts. Enzyme activity assays using recombinant proteins of Ptr4CL and PtrHCT showed elevated CoA ligation activity for Ptr4CL when supplemented with PtrHCT. Numerical analyses based on an evolutionary computation of the CoA ligation activity estimated the stoichiometry of the protein complex to consist of one Ptr4CL and two PtrHCTs, which was experimentally confirmed by chemical cross-linking using SDX plant protein extracts and recombinant proteins. Based on these results, we propose that Ptr4CL/PtrHCT complexes modulate the metabolic flux of CoA ligation for monolignol biosynthesis during wood formation inP. trichocarpa.

In the original article, there was an error. The words "and T. catappa" should be removed. A correction has been made to Materials and Methods Section, under subsection Data processing, Paragraph Number: 3: "The 1H NMR and J-res projection spectra were previously stacked and then pre-processing according the following steps: baseline correction with the Whittaker Smoother option; apodization with Gaussian function of 1 GB [Hz]; normalization by the largest peak and smoothing Savitzky-Golay method. The processed NMR data were prepared using bin width of 0.04 ppm and the bin intensities method was average sum. Afterwards, the spectral data were saved with the peak intensities. Chemical shifts (δ) values were established between 0.0 and 10.0 ppm for S. marginata samples data and 0.0–13.0 ppm for B. intermedia." In the original article, there was an error. The words "and T. catappa" should be removed. A correction has been made to Discussion Section, Paragraph Number 1: "The analyses conducted for each set of samples using LC-HRMS and NMR showed that the extracts prepared from the leaves of the plant species B. intermedia and S. marginata had a comparable metabolite profile." In the original article, there weres errors. The words "and 90 compounds in the extracts of T. catappa" should be removed, and the reference to "Supplementary Table S7" should be “Supplementary Table S5." A correction has been made to Discussion Section, Paragraph Number 3: "The dereplication of the ESI-HRMS data using the macro database containing the Natural Products Dictionary (DNP) allowed a rapid and effective annotation of 68 compounds in the extracts of B. intermedia and 81 compounds in the extracts of S. marginata (see Supplementary Tables S4, S5)." In the original article, there was an error. The reference to "Supplementary Table S5" should be "Supplementary Table S4." A correction has been made to Discussion Section, Paragraph Number 4: "As mentioned previously in the results section, the metabolite profile of the hydroethanolic extracts of B. intermedia revealed that this plant is a rich source of cinnamic acids, phenolic acids derived from galloyl quinic and shikimic acid, proanthocyanidins, glycosylated flavonoids, triterpenes and other phenols (Supplementary Table S4)." In the original article, there was an error. A reference to "Supplementary Table S6" should be "Supplementary Table S5." A correction has been made to Discussion Section, Paragraph Number 8: "The extracts of S. marginata showed mainly exhibited compounds derived from saponins, glycosylated flavonoids, catechins and oligomeric proanthocyanidins (Supplementary Table S5). The detection of saponins in S. marginata in this study helped to confirm the findings reported in the literature which pointed out that saponins are the major class of compounds in this plant species (Heredia-Vieira et al., 2015). Precisely, the results of our study showed that triterpenic saponins were prevalent in the extracts of S. marginata; these compounds contained aglycones of oleanolic acid (olean-12-en-28-oic), hederagenin (3,23-dihydroxyolean-12-en-28-acid) and gypsogenin (3 hydroxy-23-oxo-olean-12-en-28-acid). The presence of glycosylated flavonoids was detected in three groups: C-glycosylated, C,O-glycosylated and O-glycosylated flavonoids (Supplementary Table S5). The compounds tetrastigma B (m/z 561.1603 [M+H]+, Rt = 9.89) and cassiaocidentalin A (m/z 561.1602 [M+H]+, Rt = 10.37) are examples of C,O glycosylated flavonoids isomers with an interglycosidic linkage between a deoxyhexose and 6-dideoxyhexose sugars (Heredia-Vieira et al., 2015). Two types of proanthocyanidin compounds were found in S. marginata; these included B-type and A-type proanthocyanidins (Supplementary Table S5). B-type proanthocyanidins are characterized by a single interflavan linkage between monomeric units which can be formed by the equivalent units of (epi)catechin. A-type proanthocyanidins, on the other hand, contain double interflavan bonds." The authors apologize for these errors and state that this does not change the scientific conclusions of the article in any way. The original article has been updated.

Flux balance analysis (FBA) using genome-scale metabolic model (GSM) is a useful method for improving the bio-production of useful compounds. However, FBA often does not impose important constraints such as nutrients uptakes, by-products excretions and gases (oxygen and carbon dioxide) transfers. Furthermore, important information on metabolic engineering such as enzyme amounts, activities, and characteristics caused by gene expression and enzyme sequences is basically not included in GSM. Therefore, simple FBA is often not sufficient to search for metabolic manipulation strategies that are useful for improving the production of target compounds. In this study, we proposed a method using literature and enzyme search to complement the FBA-based metabolic manipulation strategies. As a case study, this method was applied to shikimic acid production by Corynebacterium glutamicum to verify its usefulness. As unique strategies in literature-mining, overexpression of the transcriptional regulator SugR and gene disruption related to by-products productions were complemented. In the search for alternative enzyme sequences, it was suggested that those candidates are searched for from various species based on features captured by deep learning, which are not simply homologous to amino acid sequences of the base enzymes.

Of the six-glyphosate resistant weed species reported in Mexico, five were found in citrus groves. Here, the glyphosate susceptibility level and resistance mechanisms were evaluated in saltmarsh aster (Aster squamatus), a weed that also occurs in Mexican citrus groves. The R population accumulated 4.5-fold less shikimic acid than S population. S plants hardly survived at 125 g ae ha−1 while most of the R plants that were treated with 1000 g ae ha−1, which suffered a strong growth arrest, showed a vigorous regrowth from the third week after treatment. Further, 5-enolpyruvylshikimate-3-phosphate basal and enzymatic activities did not diverge between populations, suggesting the absence of target-site resistance mechanisms. At 96 h after treatment, R plants absorbed ~18% less glyphosate and maintained 63% of the 14C-glyphsoate absorbed in the treated leaf in comparison to S plants. R plants metabolized twice as much (72%) glyphosate to amino methyl phosphonic acid and glyoxylate as the S plants. Three non-target mechanisms, reduced absorption and translocation and increased metabolism, confer glyphosate resistance saltmarsh aster. This is the first case of glyphosate resistance recorded for A. squamatus in the world.

The cyst nematodes Heterodera schachtii and Heterodera trifolii, whose major hosts are sugar beet and clover, respectively, damage a broad range of plants, resulting in significant economic losses. Nematodes synthesize metabolites for organismal development and social communication. We performed metabolic profiling of H. schachtii and H. trifolii in the egg, juvenile 2 (J2), and female stages. In all, 392 peaks were analyzed by capillary electrophoresis time-of-flight mass spectrometry, which revealed a lot of similarities among metabolomes. Aromatic amino acid metabolism, carbohydrate metabolism, choline metabolism, methionine salvage pathway, glutamate metabolism, urea cycle, glycolysis, gluconeogenesis, coenzyme metabolism, purine metabolism, pyrimidine metabolism, and tricarboxylic acid (TCA) cycle for energy conversion (β-oxidation and branched-chain amino acid metabolism) energy storage were involved in all stages studied. The egg and female stages synthesized higher levels of metabolites compared to the J2 stage. The key metabolites detected were glycerol, guanosine, hydroxyproline, citric acid, phosphorylcholine, and the essential amino acids Phe, Leu, Ser, and Val. Metabolites, such as hydroxyproline, acetylcholine, serotonin, glutathione, and glutathione disulfide, which are associated with growth and reproduction, mobility, and neurotransmission, predominated in the J2 stage. Other metabolites, such as SAM, 3PSer, 3-ureidopropionic acid, CTP, UDP, UTP, 3-hydroxy-3-methylglutaric acid, 2-amino-2-(hydroxymethyl-1,3-propanediol, 2-hydroxy-4-methylvaleric acid, Gly Asp, glucuronic acid-3 + galacturonic acid-3 Ser-Glu, citrulline, and γ-Glu-Asn, were highly detected in the egg stage. Meanwhile, nicotinamide, 3-PG, F6P, Cys, ADP-Ribose, Ru5P, S7P, IMP, DAP, diethanolamine, p-Hydroxybenzoic acid, and γ-Glu-Arg_divalent were unique to the J2 stage. Formiminoglutamic acid, nicotinaminde riboside + XC0089, putrescine, thiamine 2,3-dihydroxybenzoic acid, 3-methyladenine, caffeic acid, ferulic acid, m-hydrobenzoic acid, o- and p-coumaric acid, and shikimic acid were specific to the female stage. Overall, highly similar identities and quantities of metabolites between the corresponding stages of the two species of nematode were observed. Our results will be a valuable resource for further studies of physiological changes related to the development of nematodes and nematode–plant interactions.

The widespread evolution of glyphosate-resistant (GR) Bassia scoparia in the U.S. Great Plains poses a serious threat to the long-term sustainability of GR sugar beet. Glyphosate resistance in B. scoparia is due to an increase in the EPSPS (5-enolpyruvyl-shikimate-3-phosphate) gene copy number. The variation in EPSPS gene copies among individuals from within a single GR B. scoparia population indicated a differential response to glyphosate selection. With the continued use of glyphosate in GR sugar beet, the effect of increasing glyphosate rates (applied as single or sequential applications) on the fitness of GR B. scoparia individuals with variable EPSPS gene copies was tested under field conditions. The variation in EPSPS gene copy number and total glyphosate rate (single or sequential applications) did not influence any of the reproductive traits of GR B. scoparia, except seed production. Sequential applications of glyphosate with a total rate of 2214 g ae ha−1 or higher prevented seed production in B. scoparia plants with 2–4 (low levels of resistance) and 5–6 (moderate levels of resistance) EPSPS gene copies. Timely sequential applications of glyphosate (full recommended rates) can potentially slow down the evolution of GR B. scoparia with low to moderate levels of resistance (2–6 EPSPS gene copies), but any survivors (highly-resistant individuals with ≥ 8 EPSPS gene copies) need to be mechanically removed before flowering from GR sugar beet fields. This research warrants the need to adopt ecologically based, multi-tactic strategies to reduce exposure of B. scoparia to glyphosate in GR sugar beet.

Rattail fescue (Vulpia myuros (L.) C.C. Gmel.) is a self-pollinating winter annual grassy weed of winter annual crops. The problems with V. myuros are mostly associated with no-till cropping systems where glyphosate application before sowing or emergence of the crop is the most important control measure. Ineffective V. myuros control has been reported following glyphosate applications. Experiments were performed to study the effectiveness of glyphosate on V. myuros, and determine the causes of the lower performance of glyphosate on V. myuros compared to other grass weeds. Estimated GR50 values demonstrated that V. myuros was less susceptible to glyphosate than Apera spica-venti regardless of the growth stage. Within each species, glyphosate efficacy at different growth stages was closely related to spray retention. However, the low susceptibility to glyphosate in V. myuros was not caused by lower retention as previously suggested. A significantly lower shikimic acid accumulation in V. myuros compared to A. spica-venti was associated with a higher activity of the EPSPS enzyme in V. myuros. Nevertheless, the relative responses in EPSPS activity to different glyphosate concentrations were similar in the two grass species, which indicate that EPSPS from V. myuros is as susceptible to glyphosate as EPSPS from A. spica-venti suggesting no alternation in the binding site of EPSPS. The results from the current study indicate that V. myuros is less susceptible to glyphosate compared to A. spica-venti, and the low susceptibility of V. myuros is caused by an increased EPSPS enzyme activity.

Induction of resistance to pathogen is associated with the colonization of root by Trichoderma spp. has been attributed as one of the major mechanisms contributing to pathogenic invasion. The present study sheds light on the defense network of brinjal plant bioprimed with Trichoderma spp. challenged with Sclerotinia sclerotiorum. Plants treated with dual inoculation of Trichoderma harzianum and Trichoderma asperellum triggered further synthesis of TPC under S. sclerotiorum challenge with maximum increment recorded at 72 hours. In consortium treated and pathogen challenged plants, a higher amount of shikimic acid was observed at 72 hours, whereas other phenolics showed little differences among the treatments. The consortium treatment showed significantly higher defense related enzymes (Phenylalanine Ammonia Lyase, Peroxidase and Polyphenol Oxidase) activity than other treatments. The study signifies how Trichoderma spp. reprograms the host's defense network to provide robust protection against S. sclerotiorum. In the present case, overall protection was provided to the brinjal plants against the attack of S. sclerotiorum.

The recent emergence of extensively drug-resistant tuberculosis has become a cause of concern for the management of tuberculosis globally. Shikimic acid pathway seems to be a potential and favorable target for the drug design of new anti-infective agents. This work aims to change the focus from traditional cell approaches to the target-based design of novel thiadiazolyl-azetidinone derivatives with Shikimate kinase as the drug target for anti-tubercular activity. Thiadiazole and azetidinone derivatives were methodically reprised to design a series of 3-chloro-4-(aryl)-1-(5-sulfanyl-1,3,4-thiadiazol-2-yl)azetidin-2-one derivatives (AZ1-AZ12). Molecular docking studies were performed on a crystal model of Mycobacterium tuberculosis Shikimate kinase (MtSK) using Vlife MDS 4.4 suite to evaluate their anti-tubercular potential. Further, drug-likeness properties and ADMET prediction were performed by molinspiration and admetSAR software to better describe the designed molecules as prospective candidates. 3-chloro-4-(4-nitrophenyl)-1-(5-sulfanyl-1,3,4-thiadiazol-2-yl)azetidin-2-one (AZ3), was found to be have better dock score when compared with the natural substrate, Shikimate. Docking studies confirmed that the molecules showed significant binding in the active site region of Shikimate kinase. Strong hydrogen bonding and hydrophobic interactions with amino acid residues and other parameters further explicate their effectiveness for inhibition of MtSK. Also, the physicochemical properties and drug scores for the designed compounds obtained by in silico studies were found to be satisfactory, signifying the overall potential of the designed molecules to be drug candidates. Thus these molecules could be explored as a lead for further anti-tubercular studies with Mycobacterium tuberculosis Shikimate kinase as the drug target.

To prevent the spread of anthracnose in strawberry plants and characterize the metabolic changes occurring during plant–pathogen interactions, we developed a method for the early diagnosis of disease based on an analysis of the metabolome by gas chromatography-mass spectrometry. An examination of the metabolic profile revealed 189 and 202 total ion chromatogram peaks for the control and inoculated plants, respectively. A partial least squares discriminant analysis (PLS-DA) model was conducted for the reliable and accurate discrimination between healthy and diseased strawberry plants, even in the absence of disease symptoms (e.g., early stages of infection). ANOVA (analysis of variance) and orthogonal partial least squares analysis (OPLS) identified 20 metabolites as tentative biomarkers of Colletotrichum theobromicola infection (e.g., citric acid, d-xylose, erythrose, galactose, gallic acid, malic acid, methyl α-galactopyranoside, phosphate, and shikimic acid). At least some of these potential biomarkers may be applicable for the early diagnosis of anthracnose in strawberry plants. Moreover, these metabolites may be useful for characterizing pathogen infections and plant defense responses. This study confirms the utility of metabolomics research for developing diagnostic tools and clarifying the mechanism underlying plant–pathogen interactions. Furthermore, the data presented herein may be relevant for developing new methods for preventing anthracnose in strawberry seedlings cultivated under field conditions.

l-Tyrosine serves as a common precursor for multiple valuable secondary metabolites. Synthesis of this aromatic amino acid in Bacillus licheniformis occurs via the shikimate pathway, but the underlying mechanisms involving metabolic regulation remain unclear. In this work, improved l-tyrosine accumulation was achieved in B. licheniformis via co-overexpression of aroGfbr and tyrAfbr from Escherichia coli to yield strain 45A12, and the l-tyrosine titer increased to 1005 mg/L with controlled glucose feeding. Quantitative RT-PCR results indicated that aroA, encoding DAHP synthase, and aroK, encoding shikimate kinase, were feedback-repressed by the end product l-tyrosine in the modified strain. Therefore, the native aroK was first expressed with multiple copies to yield strain 45A13, which could accumulate 1201 mg/L l-tyrosine. Compared with strain 45A12, the expression of aroB and aroF in strain 45A13 was upregulated by 21% and 27%, respectively, which may also have resulted in the improvement of l-tyrosine production. Furthermore, supplementation with 5 g/L shikimate enhanced the l-tyrosine titers of 45A12 and 45A13 by 29.1% and 24.0%, respectively. However, the yield of l-tyrosine per unit of shikimate decreased from 0.365 to 0.198 mol/mol after aroK overexpression in strain 45A12, which suggested that the gene product was also involved in uncharacterized pathways. This study provides a good starting point for further modification to achieve industrial-scale production of l-tyrosine using B. licheniformis, a generally recognized as safe workhorse.

Glyphosate is a highly efficient, broad-spectrum nonspecific herbicide that inhibits the 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS)-mediated pathway of shikimic acid. The screening of glyphosate-resistant EPSPS gene is a major means for the development of new genetically modified glyphosate-resistant transgenic crop. Currently, the main commercialized glyphosate-resistant soybean contains glyphosate-resistant gene CP4-EPSPS. In this study, a G10-EPSPS gene was reported providing glyphosate resistance in Zhongdou 32. Here, G10-EPSPS gene was introduced into soybeans through Agrobacterium-mediated soybean cotyledon node. PCR, Southern blotting, semi-quantitative RT-PCR, qRT-PCR, and Western blotting were used, and the results revealed that G10-EPSPS had been integrated into the soybean genome and could be expressed steadily at both mRNA and protein levels. In addition, glyphosate resistance analysis showed that the growth of transgenic soybean had not been affected by concentrations of 900 and 2700 g a.e. ha−1 of glyphosate. All the results indicated that G10-EPSPS could provide high glyphosate resistance in soybeans and be applied in production of glyphosate-resistant soybean.

Blue mould caused by Penicillium expansum is one of the important diseases of apple fruit during storage. Phenylpropanoid pathway is an important induction mechanism that can utilize downstream metabolites of shikimate pathway to synthesize a series of secondary metabolites. Apple fruit (cv. Fuji) were treated with sodium nitroprusside (SNP) to study its effect on blue mould, shikimate and phenylpropanoid pathways. The results showed that 1.0 mmol L−1 SNP significantly inhibited lesion development of apple fruit inoculated with P. expansum. The results also indicated that SNP enhanced MdDHQS, MdSKDH, MdSK and MdEPSPS genes expressions, increased shikimic acid, tryptophan, tyrosine and phenylalanine contents in apple fruit. The activities of phenylalanine ammonialyase, 4-coumarate: coenzyme A, ligase, cinnamate 4-hydroxylase, lignin, total phenolic compounds and flavonoids contents in apple fruit were also increased by SNP treatment. These results suggest that SNP might modulate shikimate and phenylpropanoid pathways to enhance disease resistance of apple fruit.

Summary Trichomes are storage compartments for specialized metabolites in many plant species. In trichome, plant primary metabolism is significantly changed, providing substrates for downstream secondary metabolism. However, little is known of how plants coordinate trichome formation and primary metabolism regulation. In this report, tomato (Solanum lycopersicum) trichome regulator SlMIXTA‐like is indicated as a metabolic regulation gene by mGWAS analysis. Overexpression of SlMIXTA‐like in tomato fruit enhances trichome formation. In addition, SlMIXTA‐like can directly bind to the promoter region of gene encoding 3‐deoxy‐7‐phosphoheptulonate synthase (SlDAHPS) to activate its expression. Induction of SlDAHPS expression enhances shikimate pathway activities and provides substrates for downstream secondary metabolism. Our data provide direct evidence that trichome regulator can directly manipulate primary metabolism, in which way plants can coordinate metabolic regulation and the formation of storage compartments for specialized metabolites. The newly identified SlMIXTA‐like can be used for future metabolic engineering.

Cerrado is a Brazilian savannah ecosystem that has a high diversity of flora and fauna and originally covered 2 million km2, but in recent years more than 50% of its area has been converted to grain crops and pastures. The intensive grain production in Cerrado has been accompanied by an increase in pesticide and herbicide use, glyphosate is the most common. This may have implications for biodiversity loss that are still unknown. Seeking this understanding, we assessed biochemical changes in two native Cerrado plants species subjected to glyphosate. The aim of this study was to identify biomarkers in Pouteria torta and Alibertia edulis treated with glyphosate and evaluate their use as phytoindicators of the herbicide on the Cerrado vegetation. The plants were challenged with glyphosate [Roundup Transorb®, containing 480 g L−1 acid equivalent (a. e.)] in two independent experiments. In both, glyphosate was used at 0, 200, 400, and 800 g a. e. ha−1. The activities of the enzymes phenylalanine ammonia lyase (PAL), peroxidase (POX), polyphenol oxidase (PPO), and 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS), as well as the concentrations of shikimic acid, phenolic compounds, amino acids, proteins, and malondialdehyde (MDA) were evaluated at 1 and 13 and at 1 and 72 days post-application in P. torta and A. edulis leaves, respectively. Glyphosate had reduced EPSPS activity and blocks the shikimate pathway in P. torta and A. edulis. Thus, this biochemical changes can be biomarkers of glyphosate in both native Cerrado plants. The activity of the PAL and PPO enzymes also showed this potential. Glyphosate caused leaf chlorosis and necrosis in P. torta, in addition, the concentrations of phenolic compounds, proteins, and amino acids were altered only in this species. Thus, concentrations of these cell compounds can be biomarkers of glyphosate stress in susceptible plants. The herbicide did not have the same effects on A. edulis. The changes in P. torta leaves occurred more quickly than in A. edulis. Thus, A. edulis plants may be used as a glyphosate biosensor. P. torta were susceptible and can be a phytoindicator of the herbicide. Its high sensitivity is useful for monitoring the initial effects of glyphosate on native Cerrado vegetation.

Candicidin is one of the frequent antibiotics for its high antifungal activity, but the productivity is still extremely low. Introduction of adpA into Streptomyces ZYJ-6 could improve candicidin productivity significantly and achieved 9338 μg/mL, which was the highest value ever reported in the literature. Combined analyses of transcriptional levels, metabolic flux and metabolomics indicate that para-aminobenzoic acid and the first step of shikimic acid metabolism were not the bottleneck for the candicidin production in the control. However, methylmalonyl-CoA played a central role in the candicidin production and the gene methB responsible for the biosynthesis of methylmalonyl-CoA might be the candidate gene target for further improving the production of candicidin.

The oil palm tree (Elaeis guineensis) from the family Arecaceae is a high oil-producing agricultural crop. A significant amount of vegetation liquor is discarded during the palm oil milling process amounting to 90 million tons per year around the world. This water-soluble extract is rich in phenolic compounds known as Oil Palm Phenolics (OPP). Several phenolic acids including the three isomers of caffeoylshikimic acid (CFA), p-hydroxybenzoic acid (PHBA), protocatechuic acid (PCA) and hydroxytyrosol are among the primary active ingredients in the OPP. Previous investigations have reported several positive pharmacological potentials by OPP such as neuroprotective and atheroprotective effects, anti-tumor and reduction in Aβ deposition in Alzheimer's disease model. In the current review, the pharmacological potential for CFA, PHBA, PCA and hydroxytyrosol is carefully reviewed and evaluated.

This study aimed to quantify the content of bioactive constituents of European bladdernut (Staphylea pinnata L.) seed kernels, which have been poorly examined chemically, even though they have a long history of use for food. Chlorophylls, carotenoids, tocopherols, ascorbic acid, phenolics, fatty acids, sugars and organic acids were analysed. European bladdernut kernels contain appreciable amounts of bioactive constituents. The high contents of tocopherols, carotenoids and chlorophyll make bladdernut kernels a very promising food source in comparison to other nuts. The phenolic content was lower than in other nut species. The content of total fatty acids was up to 419 g/kg fresh weight, and the ratio n-6/n-3 was 10/1. The major fatty acid was linoleic acid (45% of total fatty acids). No significant difference was found between wild and cultivated (ornamental) plants, except for shikimic acid, which was significantly higher in cultivated bladdernut. The chemical composition of the kernels makes bladdernut one of the most interesting sources of health promoting substances among seeds and nuts. The results of this study show that the utilisation of E. bladdernut seeds for food uses is worth encouraging. However, more research is needed to investigate potential antinutritive substances in bladdernut kernels.

Background Allelopathy in rice (Oryza sativa) is a chemically induced response that is elevated by the exogenous application of chemical compounds and barnyard grass root exudates. An in-depth understanding of the response mechanisms of rice to chemical induction is necessary for the identification of target genes for increasing the allelopathic potential of rice. However, no previous studies have evaluated the transcriptomic changes associated with allelopathy in rice in response to barnyard grass exudates treatment. Thus, the aim of the present study was to reveal differentially expressed genes (DEGs) in allelopathic and non-allelopathic rice seedlings treated with barnyard grass exudates to identify target allelopathy genes. Results The inhibitory effect of the culture solutions on the allelopathic rice accession PI312777 (PI) and the non-allelopathic rice accession Lemont (LE) significantly increased (P

In Egypt, alfalfa represts a suitable choice for forage cultivation expansion, since, the available land and water are oflower quality. The early seeding stage of alfalfa is the most val near able to weed competition. Research results regardingimprovement of alfalfa tolerance to glyphosate in Egypt is relatively scare. The recent study was an attempt to tracevariability in glyphosate tolerance of alfalfa germplasm. The improvement in tolerance due to recurrent selection was alsoconsidered. Alfalfa plant materials (Medicago sativa,L. ) used in that recent study were five base populations .Two cyclesof recurrent selection for Glyphosate tolerance were imposed on each base population. Evaluation of selected cycles (C1and C2) along with base populations (C0) was carried-out for each population as a split plot design with Glyphosatetreatment (+ and -) as main plots and populations (C0, C1 and C2) as a sub –plots. Glyphosate treated plots were evaluatedfor injury characters, i.e.; total chlorophyll, injury level, percent of death and shikimic acid content.Significant (p≥0.01) variations were detected among the studied alfalfa population. Also, the recorded values for allinjury indicator characters significantly varied among selection cycles. In the meantime, the interactions between thestudied population and selection cycles were significant (p ≥0.05). Chlorophyll content expressed as "spade" units was notchanged after one cycle of selection (30.22 and 29.15 spade for base population (C0) and first cycle of selection (C1) as anaverage over the studied population, respectively). While, the second cycle of selection resulted in significantly higherlevel of total chlorophyll content (34.51 spade). This might indicates that tolerance to glyphosate was associated to higherchlorophyll content. The value of injury level decreased in both of Siwa and Hasawi populations after the first cycle ofselection to glyphosate tolerance(-19.51 and -13.22%, respectively). Meanwhile, the progress of selection for glyphosatetolerance in alfalfa populations, gave a substantial decrease in value of injury level in all studied populations, except for,Siwa population that recorded an increase in value of injury level (-47.05, -44.44, -40.91 and -4.35 percent of therespective value recorded for injury level at the first cycle of selection in C.U.F101, Baladi 1, Sirivar and Hasawipopulations, respectively).Change in shikimic acid content represented by a decrease reached -35.05, -22.99, -5.49 and -4.26% in Siwa, Baldi 1, C.U.F 101 and Sirivar populations, respectively, after the first cycle of selection for glyphosatetolerance.Only Hasawi population recorded an increase in shikimic acid content reached +251.8% relative to base population.While, after the second cycle of selection an increase in shikimic acid content reached +63.60 and +4.688% for C.U.F101and Baldi1 populations relative to (C1). Sharing genes among tolerant individuals, increase the frequency of genesresponsible for tolerance, consequently expressing higher levels of cide- tolerance. The recent results assume thepossibility of obtaining glyphosate tolerant alfalfa population depending on frequent cycles of recurrent selection.

Research into plants plays an important role in evaluations of water pollution with pesticides. Lemna minor (common duckweed) is widely used as an indicator organism in environmental risk assessments. The aim of this study was to determine by biological Lemna test and chemical methods the effect of glyphosate (GlyPh) concentrations of 0–40 μM on duckweed, an important link in the food chain. There are no published data on glyphosate's effects on the activity of enzymes of the amine biosynthesis pathway: ornithine decarboxylase, S-adenosylmethionine decarboxylase, tyrosine decarboxylase, lysine decarboxylase and arginine decarboxylase, and the content of shikimic acid and glyphosate residues in the tissues of common duckweed. It was found that glyphosate was taken up by duckweed. In plants exposed to 3 μM of glyphosate for 7 days, glyphosate content exceeded the acceptable Maximum Residue Level (MRL) 10-fold. Glyphosate accumulation in plant tissues exerted toxic effects on duckweed by decreasing its growth and yield, inhibiting the synthesis of chlorophyll a and b and carotenoids, and decreasing the photochemical activity of photosystem II (PSII). However, glyphosate increased the concentration of shikimic acid in the tested plants. The activity of ornithine decarboxylase increased 4-fold in plants exposed to 20 μM of the herbicide. As a water pollutant, glyphosate increased the content of biogenic amines tyramine, putrescine, cadaverine, spermidine and spermine. The activity of peroxidase and catalase was highest in duckweed exposed to 20 μM and 7 μM of the herbicide, respectively. The predicted toxic units were calculated based on glyphosate content and the computed EC values. The mean effective concentration calculated for all morphological and biochemical parameters of duckweed was determined at EC10 = 1.55, EC25 = 3.36, EC50 = 6.62 and EC90 = 14.08 μM of glyphosate. The study demonstrated that glyphosate, the active ingredient of Roundup Ultra 360 SL herbicide, induces morphological and biochemical changes in non-target plants and exerts toxic effects on aquatic ecosystems even during short-term exposure.

Shikimate dehydrogenase (SDH) from Mycobacterium tuberculosis (MtbSDH), encoded by the aroE gene, is essential for viability of M. tuberculosis but absent from humans. Therefore, it is a potentially promising target for antituberculosis agent development. Molecular-level understanding of the interactions of MtbSDH with its 3-dehydroshikimate (DHS) substrate and NADPH cofactor will help in the design of novel and effective MtbSDH inhibitors. However, this is limited by the lack of relevant crystal structures for MtbSDH complexes. Here, molecular dynamics (MD) simulations were performed to generate these MtbSDH complexes and investigate interactions of MtbSDH with substrate and cofactor and the role of MtbSDH dynamics within these. The results indicate that, while structural rearrangements are not necessary for DHS binding, reorientation of individual side chains in the NADPH binding pocket is involved in ternary complex formation. The mechanistic roles for Lys69, Asp105, and Ala213 were investigated by generating Lys69Ala, Asp105Asn, and Ala213Leu mutants in silico and investigating their complexes with DHS and NADPH. Our results show that Lys69 plays a dual role, in positioning NADPH and in catalysis. Asp105 plays a crucial role in positioning both the ϵ-amino group of Lys69 and nicotinamide ring of NADPH for MtbSDH catalysis but makes no direct contribution to DHS binding. Ala213 is the selection key for NADPH binding with the nicotinamide ring in the proS, rather than proR, conformation in the MtbSDH complex. Our results identify three strategies for MtbSDH inhibition: prevention of MtbSDH binary and ternary complex formation by blocking DHS and NADPH binding (first and second strategies, respectively) and the prevention of MtbSDH complex formation with either DHS or NADPH by blocking both DHS and NADPH binding (third strategy). Further, based on this third strategy, we propose guidelines for the rational design of "hybrid" MtbSDH inhibitors able to bind in both the substrate (DHS) and cofactor (NADPH) pockets, providing a new avenue of exploration in the search for anti-TB therapeutics.

Glyphosate, currently the world's most extensively used herbicide was on the market for more than 20 years since its introduction in 1975 without reported evolution of resistant weed cases. Glyphosate is the only reported herbicide to inhibit 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS), and endogenous accumulation of shikimic acid has been used as a biomarker for the herbicide's activity in plants after spraying. Increased levels of shikimic acid indicate sensitivity or injury while a lack or limited accumulation of shikimic acid shows resistance to glyphosate. Conyza bonariensis seed germinates in flushes in South Africa and its emergence is staggered throughout the year under conducive temperature conditions. This variability in growth stages in the same field poses a challenge in chemical control. Glyphosate herbicide efficacy is affected by environmental conditions, particularly temperature. A glyphosate resistance screening study was carried out based on reports from farmers that C. bonariensis has become increasingly difficult to control especially in conservation tillage systems. This study has confirmed the occurrence of glyphosate-resistant C. bonariensis (hairy fleabane) cases in the western and southern Cape regions of South Africa. A glasshouse screening experiment of 24 biotypes indicates resistance levels ranging from 0.6 to 26.9-fold, with GR50 values of up to 3908.42 g.ae.ha− 1 being reported in a population collected from a vineyard in the Piketberg district of the western Cape. The influence of growth stage and temperature on glyphosate resistance in hairy fleabane was evaluated by using a shikimate assay. Results from this study have shown that response of hairy fleabane to glyphosate is influenced by the phenological stage, with sensitivity or injury decreasing with growth stage. The significant statistical tests verified the dependence of the shikimate pathway on temperature, with more shikimic acid accumulating at 15 °C compared to 27 °C across all biotypes, regardless of origin and glyphosate tolerance status. More tolerance to the herbicide was demonstrated under warm temperature conditions. Possible outcomes of applying glyphosate at the bolting stage and under warm temperature conditions are escapes and no control of susceptible and resistant plants, respectively.

Pinus gerardiana (Chilgoza) is a small to medium sized evergreen tree yielding a highly valuable edible nut. The present investigations were undertaken to assess the different isozymes in Chilgoza pine. Seven isozymes viz., MDH (Malate dehydrogenase), GDH (Glutamate dehydrogenase), SKDH (Shikimic acid dehydrogenase), 6PGDH (6-Phosphogluconate dehydrogenase), MNR (Menadione reductase), IDH (Isocitrate dehydrogenase) and ADH (Alcohol dehdrogenase) were studied. A total of sixteen gene loci were recorded for seven enzyme systems out of which 6 loci namely MDH- A, 6PGDH- A, SKDH- A, IDH- B, MNR- B and ADH- A were polymorphic whereas the remaining ten gene loci i.e. MDH- B, MDH- C, MDH- D, GDH- A, 6PGDH- B, SKDH- B, IDH- A, MNR- A, MNR- B and ADH- B showed no variation. The percentage of polymorphic loci and average number of alleles per locus are one of the useful criterions for comparing species of populations for genetic diversity.

Background The evolution of herbicide-resistant weeds is one of the most important concerns of global agriculture. Amaranthus hybridus L. is a competitive weed for summer crops in South America. In this article, we intend to unravel the molecular mechanisms by which an A. hybridus population from Argentina has become resistant to extraordinarily high levels of glyphosate. Results The glyphosate-resistant population (A) exhibited particularly high parameters of resistance (GR50 = 20 900 g ai ha-1 , Rf = 314), with all plants completing a normal life cycle even after 32X dose application. No shikimic acid accumulation was detected in the resistant plants at any of the glyphosate concentrations tested. Molecular and genetic analyses revealed a novel triple substitution (TAP-IVS: T102I, A103V, and P106S) in the 5-enol-pyruvylshikimate-3-phosphate synthase (EPSPS) enzyme of population A and an incipient increase on the epsps relative copy number but without effects on the epsps transcription levels. The novel mechanism was prevalent, with 48% and 52% of the individuals being homozygous and heterozygous for the triple substitution, respectively. In silico conformational studies revealed that TAP-IVS triple substitution would generate an EPSPS with a functional active site but with an increased restriction to glyphosate binding. Conclusion The prevalence of the TAP-IVS triple substitution as the sole mechanism detected in the highly glyphosate resistant population suggests the evolution of a new glyphosate resistance mechanism arising in A. hybridus. This is the first report of a naturally occurring EPSPS triple substitution and the first glyphosate target-site resistance mechanism described in A. hybridus. © 2018 Society of Chemical Industry.

One of the most widespread pathogens worldwide is methicillin-resistant Staphylococcus aureus, a bacterium that provokes severe life-threatening illnesses both in hospitals and in the community. The principal challenge lies in the resistance of MRSA to current treatments, which encourages the study of different molecular targets that could be used to develop new drugs against this infectious agent. With this goal, a detailed characterization of shikimate kinase from this microorganism (SaSK) is described. The results showed that SaSK has a Km of 0.153 and 224 µM for shikimate and ATP, respectively, and a global reaction rate of 13.4 µmol/min/mg; it is suggested that SaSK utilizes the Bi–Bi Ping Pong reaction mechanism. Furthermore, the physicochemical data indicated that SaSK is an unstable, hydrophilic, and acidic protein. Finally, structural information showed that SaSK presented folding that is typical of its homologous counterparts and contains the typical domains of this family of proteins. Amino acids that have been shown to be important for SaSK protein function are conserved. Therefore, this study provides fundamental information that may aid in the design of inhibitors that could be used to develop new antibacterial agents.

Summer leaves of the seasonally dimorphic Phlomis fruticosa were investigated concerning their mesophyll-cell fine structure, secondary metabolite accumulation, absorption of the photosynthetic pigments, ROS levels and L-Dopa DeCarboxylase (DDC) expression, after being exposed, in nature, to the routine, combined environmental stress (high temperature, high light intensity and high aridity) of the Mediterranean summer. Although the stressed summer leaves present rather unaffected tissue arrangement, ROS levels increase significantly after the severe stress of the midday, the photosynthetic pigments are dramatically reduced, the mesophyll cells accumulate large amounts of secondary metabolites and the presence of DDC is recorded at impressively high levels. Leaves appear to react to the severe oxidative stress through the activation of the shikimic acid pathway and the consequent accumulation of secondary metabolites. Among them, the toxic L-Dopa which, through the activity of the massively detected DDC, is converted to the catecholamine dopamine, a powerful, water-soluble antioxidant, which appears to be a “relief” compound produced whenever the leaves have to cope with the harmful effects of the oxidative stress.

Antimetabolites are small molecules that inhibit enzymes by mimicking physiological substrates. We report the discovery and structural elucidation of the antimetabolite 7-deoxy-sedoheptulose (7dSh). This unusual sugar inhibits the growth of various prototrophic organisms, including species of cyanobacteria, Saccharomyces, and Arabidopsis. We isolate bioactive 7dSh from culture supernatants of the cyanobacterium Synechococcus elongatus. A chemoenzymatic synthesis of 7dSh using S. elongatus transketolase as catalyst and 5-deoxy-d-ribose as substrate allows antimicrobial and herbicidal bioprofiling. Organisms treated with 7dSh accumulate 3-deoxy-d-arabino-heptulosonate 7-phosphate, which indicates that the molecular target is 3-dehydroquinate synthase, a key enzyme of the shikimate pathway, which is absent in humans and animals. The herbicidal activity of 7dSh is in the low micromolar range. No cytotoxic effects on mammalian cells have been observed. We propose that the in vivo inhibition of the shikimate pathway makes 7dSh a natural antimicrobial and herbicidal agent., Mother Nature is a valuable resource for the discovery of drug and agricultural chemicals. Here, the authors show that 7-deoxy-sedoheptulose produced by a cyanobacterium is an antimicrobial and herbicidal compound that acts through inhibition of 3-dehydroquniate synthase in the shikimate pathway.

This study investigated the impact of Shikimic Acid (SA) obtained from leaves of Artemisia absinthium on protein glycation in the retina of diabetic rats. The GC/MS analysis of A. absinthium showed that the most abundant bioactive compound was SA (C7H10O5) with a measured retention Index (RI) of 1960 compared to that of the reference sample (1712). Male albino rats were divided into two main groups, Group I (control) and Group II (diabetic); Group II was further divided into four subgroups: Group IIa (diabetic control), Group IIb (diabetic rats were given SA orally [50 mg/kg, body weight (bw)/day], Group IIc diabetic rats were given SA orally [100 mg/kg, bw/day], and Group IId (diabetic rats were given metformin orally [100 mg/kg, bw/day] as positive control). The data obtained suggested that SA reduced glucose and glycated hemoglobin levels. In addition, SA also decreased the formation of glucose-derived advanced glycation end products. Interestingly, SA showed interference with the release of inflammatory mediators in retina and possess antioxidant potential. In conclusion, SA protected the tissues from detrimental effects of hyperglycemia and enhanced antioxidant activity. SA could be a potential lead in the process of drug development in the future to prevent retinopathy in diabetic subjects.

3-Deoxy- d -arabino-heptulosonate 7-phosphate (DAHP) synthase catalyzes the condensation of phosphoenolpyruvate (PEP) with d -erythrose 4-phosphate (E4P) and plays an important role in regulating carbon flux toward aromatic amino acid biosynthesis in bacteria and plants. Sequence analysis of the DAHP synthases AroG1 and AroG2 from Bacillus methanolicus MGA3 suggested this thermophilic, methylotrophic bacterium possesses two type Iβ DAHP synthases. This study describes production of AroG1 and AroG2 in Escherichia coli as hexa-histidine fused proteins, which were purified by affinity chromatography. Treatment with TEV protease afforded native proteins for characterization and kinetic analysis. AroG1 and AroG2 are, respectively, 30.1 kDa and 40.0 kDa proteins. Both enzymes have maximal activity over a pH range of 6.3–7.2. The apparent kinetic parameters at 50 °C and pH 7.2 for AroG1 are KmPEP 1100 ± 100 μM, KmE4P 530 ± 100 μM, and kcat 10.3 ± 1.2 s−1. The kinetic parameters for AroG2 are KmPEP 90 ± 20 μM, KmE4P 130 ± 40 μM, and kcat 2.0 ± 0.2 s−1. At 50 °C AroG2 retains 50% of its activity after 96 min whereas AroG1 retains less than 5% of its activity after 10 min. AroG2, which contains an N-terminal regulatory domain, is inhibited by chorismate and prephenate but not l -phenylalanine, l -tyrosine, or l -tryptophan. AroG1 is not inhibited by any of the molecules examined. Understanding DAHP synthase regulation in B. methanolicus is a first step toward generating biocatalysts that exploit the target-rich aromatic amino acid biosynthetic pathway for synthesis of chemicals from methanol.