Phytochemical Analysis and Bioactivity Screening of Primary and Secondary Metabolic Products of Medicinal Plants in the Valleys of Medina Region Saudi Arabia

Medicinal plants are highly valued for their active compounds. These plants can be used in various fields and preservation of these plants in their environment. The present study aimed to screen medicinal plants used in traditional medicine in Medina valleys for the presence of metabolites, and to answer the following question: is the ethnomedicinal importance of medicinal plants used in Medina valleys conform to their primary and secondary metabolite content. Eight plants (Pulicaria incise, Heliotropium arbainense, Commicarpus grandiflorus, Rumex vesicarius, Senna alexandrina, Rhazya stricta, Withania somnifera and Asphodelus fistulosus) were collected from the Medina valleys and were biochemically analyzed to determine the different compounds after leaves extraction analyzed statistically to clarify the content of primary compounds. The chemical compounds in the most active fraction were determined using quantitative phytochemical and gas chromatography-mass spectrometry (GC/MS) analytical methods, comparing the mass spectra of the GC/MS identified compounds with those of the Center of Excellence in Environmental Studies (CEES) database library. The result showed 16 aroma compounds representing the GC/MS analysis revealed the presence of various compounds like 4,4-Dimethyl octane, 5H-1-Pyrindine and 1,3- Cyclopentadiene, 1,2,5,5-tetramethyl- in the ethanolic extract of Pulicaria incisa. The most prevalent plants were Pulicaria incisa, Senna alexandrina and Heliotropium arbainense the study plants have high content of protein. There is a need to focus phytochemical screening on ethnobotanical studies to complete research into traditional medicine which leads to the discovery of new drugs.

Sugarcane leaves-derived polyphenols alleviate metabolic syndrome and modulate gut microbiota of ob/ob mice

Sugarcane leaves-derived polyphenols(SLP)have been demonstrated to have diverse health-promoting benefits,but the mechanism of action has not been fully elucidated.This study aimed to investigate the anti-metabolic disease effects of SLP and the underlying mechanisms in mice.In the current study,we prepared the SLP mainly consisting of three flavonoid glycosides,three phenol derivatives,and two lignans including one new compound,and further demonstrated that SLP reduced body weight gain and fat accumulation,improved glucose and lipid metabolism disorders,ameliorated hepatic steatosis,and regulated short-chain fatty acids(SCFAs)production and secondary bile acids metabolism in ob/ob mice.Notably,SLP largely altered the gut microbiota composition,especially enriching the commensal bacteria Akkermansia muciniphila and Bacteroides acidifaciens.Oral gavage with the above two strains ameliorated metabolic syndrome(MetS),regulated secondary bile acid metabolism,and increased the production of SCFAs in high-fat diet(HFD)-induced obese mice.These results demonstrated that SLP could be used as a prebiotic to attenuate MetS via regulating gut microbiota composition and further activating the secondary bile acids-mediated gut-adipose axis.

Recent Advances on Key Enzyme Genes in Metabolic Pathways Related to the Synthesis of Aroma Compounds in Tobacco

This article summarized three main kinds of metabolic pathways related to the synthesis of aroma compounds in plants, concluded the roles and expres- sion patterns of key enzyme genes catalyzing the formation of major intermediate products in phenylpropanoid metabolic pathway, isoprene metabolic pathway and alkaloid biosynthetic pathway respectively, highlighted the latest developments of these key enzyme genes in tobacco, and accordingly proposed that in-depth study at the protein level and analysis of metabolic network interaction should be carried out in tobacco besides the expression regulation and transgenic crop improvement at the genetic level. Based on the above analysis, further improvement of tobacco aroma quality through metabolic engineering and its application prospect in agricultural production were prospected.

Effects of Waterlogging Stress on the Physiological Characteristics and Secondary Metabolites of Herbaceous Peony (<i>Paeonia lactiflora</i>Pall.)

Herbaceous peony is an ornamental plant with medicinal properties. Waterlogging can affect its yield and quality as it grows and matures. In this study, we subjected “Taohuafeixue”, “Yangfeichuyu” and “Hongxiuqiu” herbaceous peony varieties to a simulated waterlogging stress treatment and investigated the effects of waterlogging on their physiological characteristics and the secondary metabolite contents in their leaves and roots. Short-term waterlogging caused the leaves to turn yellow or red and the roots to turn black. The stele and the cell wall of the endothelial cells thickened, and the cortical cells enlarged. Waterlogging did not significantly change plant height, leaf length, and leaf area;however, it significantly decreased the root-shoot ratio of “Yangfeichuyu” and “Hongxiuqiu” varieties. The activity of antioxidant enzymes and the content of osmotic regulators increased under waterlogging. After short-term waterlogging stress treatment, the content of paeoniflorin and albiflorin increased in the roots of “Taohuafeixue” and “Yangfeichuyu”, and the content of benzoylpaeoniflorin increased in the root of “Hongxiuqiu”. The content of gallic acid and total flavonoids increased in the leaves of “Taohuafeixue” and “Yangfeichuyu”. After the waterlogging, paeoniflorin and benzoylpaeoniflorin increased in the autumn root of “Hongxiuqiu”. This study expands our knowledge about the medicinal properties of herbaceous peony and informs about its production and cultivation under waterlogged conditions.

Effects of phenolic acids on soil nitrogen mineralization over successive rotations in Chinese fir plantations

Phenolic acids are secondary metabolites of plants that significantly affect nutrient cycling processes.To investigate such effects,the soil available nitrogen(N)content,phenolic acid content,and net N mineralization rate in three successive rotations of Chinese fir plantations in subtropical China were investigated.Net N mineralization and nitrification rates in soils treated with phenolic acids were measured in an ex situ experiment.Compared with first-rotation plantations(FCP),the contents of total soil nitrogen and nitrate in second(SCP)-and third-rotation plantations(TCP)decreased,and that of soil ammonium increased.Soil net N mineralization rates in the second-and third-rotation plantations also increased by 17.8%and 39.9%,respectively.In contrast,soil net nitrification rates decreased by 18.0%and 25.0%,respectively.The concentrations of total phenolic acids in the FCP soils(123.22±6.02 nmol g^-1)were 3.0%and 17.9%higher than in the SCP(119.68±11.69 nmol g^-1)and TCP(104.51±8.57 nmol g^-1,respectively).The total content of phenolic acids was significantly correlated with the rates of net soil N mineralization and net nitrification.The ex situ experiment showed that the net N mineralization rates in soils treated with high(HCPA,0.07 mg N kg^-1 day^-1)and low(LCPA,0.18 mg N kg^-1 day^-1)concentrations of phenolic acids significantly decreased by 78.6%and 42.6%,respectively,comparing with that in control(0.32 mg N kg^-1 day^-1).Soil net nitrification rates under HCPA and LCPA were significantly higher than that of the control.The results suggested that low contents of phenolic acids in soil over successive rotations increased soil net N mineralization rates and decreased net nitrification rates,leading to consequent reductions in the nitrate content and enhancement of the ammonium content,then resulting in enhancing the conservation of soil N of successive rotations in Chinese fir plantation.

A study on JA-and BTH-induced resistance of Rosa rugosa‘Plena’to powdery mildew(Sphaerotheca pannosa)

Different concentrations of jasmonic acid(JA)and benzothiadiazole(BTH) were sprayed on 2-year-old Rosa rugosa‘Plena’ seedlings. The induced resistance of JA and BTH to Sphaerotheca pannosa(Wallr.) and the changes of their related physiological indices were investigated. Results showed that JA and BTH treatments had inhibitory impacts on S. pannosa infection. The optimal concentration of JA and BTH was 0.5 mmol/L for the disease-resistance induction of the leaves, its inductive effect was up to 66.36% for BTH and 54.49% for JA. Our results confirmed that exogenous JA and BTH significantly improved R. rugose ‘Plena’ resistance to S. pannosa. When treated with JA and BTH, activities of the three defense enzymes(POD, PPO, and PAL) increased significantly.Contents of total phenolics, flavonoids, and lignin also increased significantly. It is inferred from these results that exogenous JA and BTH could improve the resistance of R.rugose ‘Plena’ to S. pannosa through enhancing activities of the defensive enzymes and accumulation of secondary metabolites in the leaves.

Overexpression of IbPAL1 promotes chlorogenic acid biosynthesis in sweetpotato

Sweetpotato[Ipomoea batatas(L.)Lam.],a food crop with both nutritional and medicinal uses,plays essential roles in food security and health-promoting.Chlorogenic acid(CGA),a polyphenol displaying several bioactivities,is distributed in all edible parts of sweetpotato.However,little is known about the specific metabolism of CGA in sweetpotato.In this study,IbPAL1,which encodes an endoplasmic reticulum-localized phenylalanine ammonia lyase(PAL),was isolated and characterized in sweetpotato.CGA accumulation was positively associated with the expression pattern of IbPAL1 in a tissue-specific manner,as further demonstrated by overexpression of IbPAL1.Overexpression of IbPAL1 promoted CGA accumulation and biosynthetic pathway genes expression in leaves,stimulated secondary xylem cell expansion in stems,and inhibited storage root formation.Our results support a potential role for IbPAL1 in sweetpotato CGA biosynthesis and establish a theoretical foundation for detailed mechanism research and nutrient improvement in sweetpotato breeding programs.

Elevated CO2 offsets the alteration of foliar chemicals(n-icosane,geranyl acetate,and elixene)induced by elevated O3 in three taxa of O3-tolerant eucalypts

Eucalypts are important forest resources in southwestern China,and may be tolerant to elevated ground-level ozone(O3)concentrations that can negatively affect plant growth.High CO2 may offset O3-induced effects by providing excess carbon to produce secondary metabolites or by inducing stomatal closure.Here,the effects of elevated CO2 and O3 on leaf secondary metabolites and other defense chemicals were studied by exposing seedlings of Eucalyptus globulus,E.grandis,and E.camaldulensis×E.deglupta to a factorial combination of two levels of O3(<10 nmol mol^(−1)and 60 nmol mol^(−1))and CO2(ambient:370μmol mol^(−1)and 600μmol mol^(−1))in open-top field chambers.GC-profiles of leaf extracts illustrated the effect of elevated O3 and the countering effect of high CO2 on compounds in leaf epicuticular wax and essential oils,i.e.,n-icosane,geranyl acetate and elixene,compounds known as a first-line defense against insect herbivores.n-Icosane may be involved in tolerance mechanisms of E.grandis and the hybrid,while geranyl acetate and elixene in the tolerance of E.globulus.Elevated O3 and CO2,singly or in combination,affected only leaf physiology but not biomass of various organs.Elevated CO2 impacted several leaf traits,including stomatal conductance,leaf mass per area,carbon,lignin,n-icosane,geranyl acetate and elixene.Limited effects of elevated O3 on leaf physiology(nitrogen,n-icosane,geranyl acetate,elixene)were commonly offset by elevated CO2.We conclude that E.globulus,E.grandis and the hybrid were tolerant to these O3 and CO2 treatments,and n-icosane,geranyl acetate and elixene may be major players in tolerance mechanisms of the tested species.

2-Oxoglutarate-dependent dioxygenases in plants:biosynthesis,mechanism and evolution

2-Oxoglutarate(2OG)-dependent dioxygenases(2-ODDs)are omnipresent iron-containing non-heme enzymes that catalyze various oxidation-reduction reactions in plant growth and development,nucleic acid modification and secondary metabolism.We systematically summarized recent research on the oxidative modifications of plant 2-ODDs and related enzymes,their vital importance in the biosynthesis of plant special metabolites,and their catalytic specificity/flexibility,and discussed the potential of 2-ODD as a new approach for the identification of pivotal genes and the elucidation of biosynthetic pathway.

Efficient Production of δ-Guaiene, an Aroma Sesquiterpene Compound Accumulated in Agarwood, by Mevalonate Pathway-Engineered Escherichia coli Cells

Mevalonate pathway for isoprenoid biosynthesis was constructed in Escherichia coli cells by the transformation with a gene cluster isolated from Streptomyces sp., and farnesyl diphosphate synthase and δ-guaiene synthase genes were coexpressed in this strain. This transformant was capable of liberating an appreciable amount of δ-guaiene, an aroma sesquiterpene compound accumulated in agarwood, and its concentration was elevated to more than 30 μg/ml culture by the incubation with mevalonolactone as an isoprene precursor in a nutrient-enriched Terrific broth. Coexpression of type 1 isopentenyl diphosphate isomerase plus acetoacetyl-CoA ligase genes also enhanced δ-guaiene production, and the concentration of the compound was approximately 38 - 42 μg/ml culture in the presence of mevalonolactone or lithium acetoacetate. These results clearly indicate that mevalonate pathway-engineered E. coli cells showed an appreciable δ-guaiene producing activity in the en- riched medium in the presence of appropriate isoprene precursors.

Germplasm resources and secondary metabolism regulation in Reishi mushroom(Ganoderma lucidum)

Ganoderma lucidum is a valuable medical macrofungus with a myriad of diverse secondary metabolites,in which triterpenoids are the major constituents.This paper introduced the germplasm resources of genus Ganoderma from textual research,its distribution and identification at the molecular level.Also we overviewed G.lucidum in the components,the biological activities and biosynthetic pathways of ganoderic acid,aiming to provide scientific evidence for the development and utilization of G.lucidum germplasm resources and the biosynthesis of ganoderic acid.

Genetic manipulation and tools in myxobacteria for the exploitation of secondary metabolism

Myxobacteria are famous for their capacity for social behavior and natural product biosynthesis.The unique sociality of myxobacteria is not only an intriguing scientific topic but also the main limiting factor for their ma-nipulation.After more than half a century of research,a series of genetic techniques for myxobacteria have been developed,rendering these mysterious bacteria manipulable.Here,we review the advances in genetic manipu-lation of myxobacteria,with a particular focus on the exploitation of secondary metabolism.We emphasize the necessity and urgency of constructing the myxobacterial chassis for synthetic biology research and the exploita-tion of untapped secondary metabolism.

Global regulation of fungal secondary metabolism in Trichoderma reesei by the transcription factor Ypr1,as revealed by transcriptome analysis

Trichoderma reesei Rut-C-30 is a well-known robust producer of cellulolytic enzymes,which are used to degrade lignocellulosic biomass for the sustainable production of biofuels and biochemicals.However,studies of its sec-ondary metabolism and regulation remain scarce.Ypr1 was previously described as a regulator of the biosynthesis of the yellow pigment sorbicillin(a bioactive agent with great pharmaceutical interest)in T.reesei and several other fungi.However,the manner in which this regulator affects global gene transcription has not been explored.In this study,we report the effect of Ypr1 on the regulation of both the secondary and primary metabolism of T.reesei Rut-C30.A global gene transcription profile was obtained using a comparative transcriptomic analysis of the wild-type strain T.reesei Rut-C-30 and its ypr1 deletion mutant.The results of this analysis suggest that,in addition to its role in regulating sorbicillin and the major extracellular(hemi)cellulases,Ypr1 also affects the transcription of genes encoding several other secondary metabolites.Although the primary metabolism of T.reeseiΔypr1 became less active compared with that of T.reesei Rut-C-30,several gene clusters involved in its secondary metabolism were activated,such as the gene clusters for the biosynthesis of specific polyketides and non-ribosomal peptides,together with the“sorbicillinoid-cellulase”super cluster,indicating that specific secondary metabolites and cellulases may be co-regulated in T.reesei Rut-C-30.The results presented in this study may benefit the development of genetic engineering strategies for the production of sorbicillin by T.reesei Rut-C-30,and provide insights for enhancing sorbicillin production in other filamentous fungal producers.

Metabolic Engineering of Tropane Alkaloid Biosynthesis in Plants

Abstract: Over the past decade, the evolving commercial importance of so-called plant secondary metabolites has resulted in a great interest in secondary metabolism and, particularly, in the possibilities to enhance the yield of fine metabolites by means of genetic engineering. Plant alkaloids, which constitute one of the largest groups of natural products, provide many pharmacologically active compounds. Several genes in the tropane alkaloids biosynthesis pathways have been cloned, making the metabolic engineering of these alkaloids possible. The content of the target chemical scopolamine could be significantly increased by various approaches, such as introducing genes encoding the key biosynthetic enzymes or genes encoding regulatory proteins to overcome the specific rate-limiting steps. In addition, antisense genes have been used to block competitive pathways. These investigations have opened up new, promising perspectives for increased production in plants or plant cell culture. Recent achievements have been made in the metabolic engineering of plant tropane alkaloids and some new powerful strategies are reviewed in the present paper.

Molecular regulation of the key specialized metabolism pathways in medicinal plants

The basis of modern pharmacology is the human ability to exploit the production of specialized metabolites from medical plants,for example,terpenoids,alkaloids,and phenolic acids.However,in most cases,the availability of these valuable compounds is limited by cellular or organelle barriers or spatio-temporal accumulation patterns within different plant tissues.Transcription factors(TFs)regulate biosynthesis of these specialized metabolites by tightly controlling the expression of biosynthetic genes.Cutting-edge technologies and/or combining multiple strategies and approaches have been applied to elucidate the role of TFs.In this review,we focus on recent progress in the transcription regulation mechanism of representative high-value products and describe the transcriptional regulatory network,and future perspectives are discussed,which will help develop high-yield plant resources.

Transcriptional Regulation of Plant Secondary Metabolism

Plant secondary metabolites play critical roles in plant-environment interactions. They are synthesized in different organs or tissues at particular developmental stages, and in response to various environmental stimuli, both biotic and abiotic. Accordingly, corresponding genes are regulated at the transcriptional level by multiple transcription factors. Several families of transcription factors have been identified to participate in controlling the biosynthesis and accumulation of secondary metabolites. These regulators integrate internal （often developmental） and external signals, bind to corresponding cis-elements -- which are often in the promoter regions -- to activate or repress the expression of enzyme-coding genes, and some of them interact with other transcription factors to form a complex. In this review, we summarize recent research in these areas, with an emphasis on newly-identified transcription factors and their functions in metabolism regulation.

Transcriptome and Metabolic Profiling Provides Insigh into Betalain Biosynthesis and Evolution in Mirabilis jalapa

Betalains are tyrosine-derived pigments that occur solely in one plant order, the Caryophyllales, where they largely replace the anthocyanins in a mutually exclusive manner. In this study, we conducted multi-species transcriptome and metabolic profiling in Mirabilisjalapa and additional betalain-producing species to identify candidate genes possibly involved in betalain biosynthesis. Among the candidates identified, betalainrelated ytochrome P450 and glucosyltransferase-type genes, which catalyze tyrosine hydroxylation or （hydroxy）cinnamoyl-glucose formation, respectively, were further functionally characterized. We detected the expression of genes in the flavonoid/anthocyanin biosynthetic pathways as well as their metabolite intermediates in betalain-accumulating M. jalapa flowers, and found that the anthocyanin-related gene ANTHOCYANIDIN SYNTHASE （MjANS） is highly expressed in the betalain-accumulating petals. However, it appears that MjANS contains a significant deletion in a region spanning the corresponding enzyme active site. These findings provide novel insights into betalain biosynthesis and a possible explanation for how anthocyanins have been lost in this plant species. Our study also implies a complex, non-uniform history for the loss of anthocyanin production across betalain producers, previously assumed to be strictly due to diminished expression of anthocyanin-related genes.

The Metabolic Response of Arabidopsis Roots to Oxidative Stress is Distinct from that of Heterotrophic Cells in Culture and Highlights a Complex Relationship between the Levels of Transcripts, Metabolites, and Flux

Metabolic adjustments are a significant, but poorly understood, part of the response of plants to oxidative stress. In a previous study （Baxter et al., 2007）, the metabolic response of Arabidopsis cells in culture to induction of oxidative stress by menadione was characterized. An emergency survival strategy was uncovered in which anabolic primary metabolism was largely down-regulated in favour of catabolic and antioxidant metabolism. The response in whole plant tissues may be different and we have therefore investigated the response of Arabidopsis roots to menadione treatment, analyzing the transcriptome, metabolome and key metabolic fluxes with focus on primary as well as secondary metabolism. Using a redox-sensitive GFP, it was also shown that menadione causes redox perturbation, not just in the mitochondrion, but also in the cytosol and plastids of roots. In the first 30 min of treatment, the response was similar to the cell culture： there was a decrease in metabolites of the TCA cycle and amino acid biosynthesis and the transcriptomic response was dominated by up-regulation of DNA regulatory proteins. After 2 and 6 h of treatment, the response of the roots was different to the cell culture. Metabolite levels did not remain depressed, but instead recovered and, in the case of pyruvate, some amino acids and aliphatic glucosinolates showed a steady increase above control levels. However, no major changes in fluxes of central carbon metabolism were observed and metabolic transcripts changed largely independently of the corresponding metabolites. Together, the results suggest that root tissues can recover metabolic activity after oxidative inhibition and highlight potentially important roles for glycolysis and the oxidative pentose phosphate pathway.

Exploiting Natural Variation in Tomato to Define Pathway Structure and Metabolic Regulation of Fruit Polyphenolics in the Lycopersicum Complex

While the structures of plant primary metabolic pathways are generally well defined and highly conserved across species,those defining specialized metabolism are less well characterized and more highly variable across species.In this study,we investigated polyphenolic metabolism in the lycopersicum complex by characterizing the underlying biosynthetic and decorative reactions that constitute the metabolic network of polyphenols across eight different species of tomato.For this purpose,GC-MS-and LC-MS-based metabolomics of different tissues of Solatium lycopersicum and wild tomato species were carried out,in concert with the evaluation of cross-hybridized microarray data for MapMan-based transcriptomic analysis,and publicly available RNA-sequencing data for annotation of biosynthetic genes.The combined data were used to compile species-specific metabolic networks of polyphenolic metabolism,allowing the establishment of an entire pan-species biosynthetic framework as well as annotation of the functions of decoration enzymes involved in the formation of metabolic diversity of the flavonoid pathway.The combined results are discussed in the context of the current understanding of tomato flavonol biosynthesis as well as a global view of metabolic shifts during fruit ripening.Our results provide an example as to how large-scale biology approaches can be used for the definition and refinement of large specialized metabolism pathways.

Mapping theArabidopsis Metabolic Landscape by Untargeted Metabolomics at Different Environmental Conditions

Metabolic genome-wide association studies （mGWAS）, whereupon metabolite levels are regarded as traits, can help unravel the genetic basis of metabolic networks. A total of 309Arabidopsis accessions were grown under two independent environmental conditions （control and stress） and subjected to untargeted LC-MS- based metabolomic profiling; levels of the obtained hydrophilic metabolites were used in GWAS. Our two- condition-based GWAS for more than 3000 semi-polar metabolites resulted in the detection of 123 highly resolved metabolite quantitative trait loci （p ≤ 1.0E-08）, 24.39% of which were environment-specific. Interestingly, differently from natural variation in Arabidopsis primary metabolites, which tends to be controlled by a large number of small-effect loci, we found several major large-effect loci alongside a vast number of small-effect loci controlling variation of secondary metabolites. The two-condition-based GWAS was fol- lowed by integration with network-derived metabolite-transcript correlations using a time-course stress experiment. Through this integrative approach, we selected 70 key candidate associations between struc- tural genes and metabolites, and experimentally validated eight novel associations, two of them showing differential genetic regulation in the two environments studied. We demonstrate the power of combining large-scale untargeted metabolomics-based GWAS with time-course-derived networks both performed under different ablotic environments for identifying metabollte-gene associations, providing novel global insights into the metabolic landscape of Arabidopsis.