Spatial and evolutionary predictability of phytochemical diversity.

To cope with environmental challenges, plants produce a wide diversity of phytochemicals, which are also the source of numerous medicines. Despite decades of research in chemical ecology, we still lack an understanding of the organization of plant chemical diversity across species and ecosystems. To address this challenge, we hypothesized that molecular diversity is not only related to species diversity, but also constrained by trophic, climatic, and topographical factors. We screened the metabolome of 416 vascular plant species encompassing the entire alpine elevation range and four alpine bioclimatic regions in order to characterize their phytochemical diversity. We show that by coupling phylogenetic information, topographic, edaphic, and climatic variables, we predict phytochemical diversity, and its inherent composition, of plant communities throughout landscape. Spatial mapping of phytochemical diversity further revealed that plant assemblages found in low to midelevation habitats, with more alkaline soils, possessed greater phytochemical diversity, whereas alpine habitats possessed higher phytochemical endemism. Altogether, we present a general tool that can be used for predicting hotspots of phytochemical diversity in the landscape, independently of plant species taxonomic identity. Such an approach offers promising perspectives in both drug discovery programs and conservation efforts worldwide.

Integrated Transcriptome and Metabolome Analysis Reveals Key Metabolites Involved in Camellia oleifera Defense against Anthracnose.

Camellia oleifera (Ca. oleifera) is a woody tree species cultivated for the production of edible oil from its seed. The growth and yield of tea-oil trees are severely affected by anthracnose (caused by Colletotrichum gloeosporioides). In this study, the transcriptomic and metabolomic analyses were performed to detect the key transcripts and metabolites associated with differences in the susceptibility between anthracnose-resistant (ChangLin150) and susceptible (ChangLin102) varieties of Ca. oleifera. In total, 5001 differentially expressed genes (DEGs) were obtained, of which 479 DEGs were common between the susceptible and resistant varieties and further analyzed. KEGG enrichment analysis showed that these DEGs were significantly enriched in tyrosine metabolism, phenylpropanoid biosynthesis, flavonoid biosynthesis and isoquinoline alkaloid biosynthesis pathways. Furthermore, 68 differentially accumulated metabolites (DAMs) were detected, including flavonoids, such as epicatechin, phenethyl caffeate and procyanidin B2. Comparison of the DEGs and DAMs revealed that epicatechin, procyanidin B2 and arachidonic acid (peroxide free) are potentially important. The expression patterns of genes involved in flavonoid biosynthesis were confirmed by qRT-PCR. These results suggested that flavonoid biosynthesis might play an important role in the fight against anthracnose. This study provides valuable molecular information about the response of Ca. oleifera to Co. gloeosporioides infection and will aid the selection of resistant varieties using marker-assisted breeding.

Regeneration of Phytochemicals by Structure-Driven Organization of Microbial Biosynthetic Steps.

Medicinal phytochemicals, such as artemisinin and taxol, have impacted the world, and hypericin might do so if its availability issue could be addressed. Hypericin is the hallmark component of Saint John's wort (Hypericum perforatum L.), an approved depression alleviator documented in the US, European, and British pharmacopoeias with its additional effectiveness against diverse cancers and viruses. However, the academia-to-industry transition of hypericin remain hampered by its low in planta abundance, unfeasible bulk chemical synthesis, and unclear biosynthetic mechanism. Here, we present a strategy consisting of the hypericin-structure-centered modification and reorganization of microbial biosynthetic steps in the repurposed cells that have been tamed to enable the designed consecutive reactions to afford hypericin (43.1 mg L-1 ), without acquiring its biosynthetic knowledge in native plants. The study provides a synthetic biology route to hypericin and establishes a platform for biosustainable access to medicinal phytochemicals.

Biosynthesis and heterologous production of furanocoumarins: perspectives and current challenges.

Covering: up to October 2020 Furanocoumarins are plant secondary metabolites used to treat several skin disorders, such as psoriasis and vitiligo, and also with other potential therapeutic activities. Furanocoumarins are extracted from plants where they accumulate in low amounts over long growth periods. In addition, their extraction and purification are difficult in an environmentally unfriendly and expensive process. Hence, new sustainable and greener production schemes able to overcome such limitations ought to be developed. While the heterologous production of simple coumarins has been demonstrated, the biosynthesis of more complex furanocoumarins remains greatly unexplored. Although several important steps of the pathway have been elucidated in the last decade, the complete pathway has not been completely unravelled. In this paper, we review the natural conversion of amino acids into furanocoumarins, as well as the heterologous expression of each enzyme of the pathway. We also explore the challenges that need to be addressed so that their heterologous production can become a viable alternative.

Phytochemical, Antiplasmodial, Cytotoxic and Antimicrobial Evaluation of a Southeast Brazilian Brown Propolis Produced by Apis mellifera Bees.

Seven phenolic compounds (ferulic acid, caffeic acid, 4-methoxycinnamic acid, 3,4-dimethoxycinnamic acid, 3-hydroxy-4-methoxybenzaldehyde, 3-methoxy-4-hydroxypropiophenone and 1-O,2-O-digalloyl-6-O-trans-p-coumaroyl-ß-D-glucopyranoside), a flavanonol (7-O-methylaromadendrin), two lignans (pinoresinol and matairesinol) and six diterpenic acids/alcohol (19-acetoxy-13-hydroxyabda-8(17),14-diene, totarol, 7-oxodehydroabietic acid, dehydroabietic acid, communic acid and isopimaric acid) were isolated from the hydroalcoholic extract of a Brazilian Brown Propolis and characterized by NMR spectral data analysis. The volatile fraction of brown propolis was characterized by CG-MS, composed mainly of monoterpenes and sesquiterpenes, being the major α-pinene (18.4 %) and ß-pinene (10.3 %). This propolis chemical profile indicates that Pinus spp., Eucalyptus spp. and Araucaria angustifolia might be its primary plants source. The brown propolis displayed significant activity against Plasmodium falciparum D6 and W2 strains with IC50 of 5.3 and 9.7 µg/mL, respectively. The volatile fraction was also active with IC50 of 22.5 and 41.8 µg/mL, respectively. Among the compounds, 1-O,2-O-digalloyl-6-O-trans-p-coumaroyl-ß-D-glucopyranoside showed IC50 of 3.1 and 1.0 µg/mL against D6 and W2 strains, respectively, while communic acid showed an IC50 of 4.0 µg/mL against W2 strain. Cytotoxicity was determined on four tumor cell lines (SK-MEL, KB, BT-549, and SK-OV-3) and two normal renal cell lines (LLC-PK1 and VERO). Matairesinol, 7-O-methylaromadendrin, and isopimaric acid showed an IC50 range of 1.8-0.78 µg/mL, 7.3-100 µg/mL, and 17-18 µg/mL, respectively, against the tumor cell lines but they were not cytotoxic against normal cell lines. The crude extract of brown propolis displayed antimicrobial activity against C. neoformans, methicillin-resistant Staphylococcus aureus, and P. aeruginosa at 29.9 µg/mL, 178.9 µg/mL, and 160.7 µg/mL, respectively. The volatile fraction inhibited the growth of C. neoformans at 53.0 µg/mL. The compounds 3-hydroxy-4-methoxybenzaldehyde, 3-methoxy-4-hydroxypropiophenone and 7-oxodehydroabietic acid were active against C. neoformans, and caffeic and communic acids were active against methicillin-resistant Staphylococcus aureus.

Actinidia arguta Leaf as a Donor of Potentially Healthful Bioactive Compounds: Implications of Cultivar, Time of Sampling and Soil N Level.

The aim of this study was to assess the enzymatic and non-enzymatic antioxidant status of kiwiberry (Actinidia arguta) leaf under different N regimes tested three times in field conditions during the 2015 growing season in two cultivars ('Weiki' and 'Geneva'). Leaf total antioxidant capacity using ABTS, DPPH and FRAP tests was evaluated in the years 2015 to 2017, which experienced different weather conditions. Both cultivars exhibited a significant fall in leaf L-ascorbic acid (L-AA) and reduced glutathione (GSH) as well as global content of these compounds during the growing season, while total phenolic contents slightly ('Weiki') or significantly ('Geneva') increased. There was a large fluctuation in antioxidative enzyme activity during the season. The correlation between individual antioxidants and trolox equivalent antioxidant capacity (TEAC) depended on the plant development phase. The study revealed two peaks of an increase in TEAC at the start and end of the growing season. Leaf L-AA, global phenolics, APX, CAT and TEAC depended on the N level, but thiol compounds were not affected. Over the three years, TEAC decreased as soil N fertility increased, and the strength of the N effect was year dependent. The relationship between leaf N content and ABTS and FRAP tests was highly negative. The antioxidant properties of kiwiberry leaves were found to be closely related to the plant development phase and affected by soil N fertility.

Supplementary Far-Red and Blue Lights Influence the Biomass and Phytochemical Profiles of Two Lettuce Cultivars in Plant Factory.

Three different LED spectra (W: White light; WFR: W + far-red light; WB: W + blue light) with similar photosynthetic photon flux density (PPFD) were designed to explore the effects of supplementary far-red and blue lights on leaf color, biomass and phytochemicals of two cultivars of red-leaf lettuce ("Yanzhi" and "Red Butter") in an artificial lighting plant factory. Lettuce plants under WB had redder leaf color and significantly higher contents of pigments, such as chlorophyll a, chlorophyll b, chlorophyll (a + b) and anthocyanins. The accumulation of health-promoting compounds, such as vitamin C, vitamin A, total phenolic compounds, total flavonoids and anthocyanins in the two lettuce cultivars were obviously enhanced by WB. Lettuce under WFR showed remarkable increase in fresh weight and dry weight; meanwhile, significant decreases of pigments, total phenolic compounds, total flavonoids and vitamin C were found. Thus, in the plant factory system, the application of WB can improve the coloration and quality of red leaf lettuce while WFR was encouraged for the purpose of elevating the yield of lettuce.

Combination of Selenium and UVA Radiation Affects Growth and Phytochemicals of Broccoli Microgreens.

Addition of selenium or application of ultraviolet A (UVA) radiation for crop production could be an effective way of producing phytochemical-rich food. This study was conducted to investigate the effects of selenium and UVA radiation, as well as their combination on growth and phytochemical contents in broccoli microgreens. There were three treatments: Se (100 µmol/L Na2SeO3), UVA (40 µmol/m2/s) and Se + UVA (with application of Se and UVA). The control (CK) was Se spraying-free and UVA radiation-free. Although treatment with Se or/and UVA inhibited plant growth of broccoli microgreens, results showed that phytochemical contents increased. Broccoli microgreens under the Se treatment had higher contents of total soluble sugars, total phenolic compounds, total flavonoids, ascorbic acid, Fe, and organic Se and had lower Zn content. The UVA treatment increased the contents of total chlorophylls, total soluble proteins, total phenolic compounds, and FRAP. However, the Se + UVA treatment displayed the most remarkable effect on the contents of total anthocyanins, glucoraphanin, total aliphatic glucosinolates, and total glucosinolates; here, significant interactions between Se and UVA were observed. This study provides valuable insights into the combinational selenium and UVA for improving the phytochemicals of microgreens grown in an artificial lighting plant factory.

The genome of the medicinal plant Andrographis paniculata provides insight into the biosynthesis of the bioactive diterpenoid neoandrographolide.

Andrographis paniculata is a herbaceous dicot plant widely used for its anti-inflammatory and anti-viral properties across its distribution in China, India and other Southeast Asian countries. A. paniculata was used as a crucial therapeutic treatment during the influenza epidemic of 1919 in India, and is still used for the treatment of infectious disease in China. A. paniculata produces large quantities of the anti-inflammatory diterpenoid lactones andrographolide and neoandrographolide, and their analogs, which are touted to be the next generation of natural anti-inflammatory medicines for lung diseases, hepatitis, neurodegenerative disorders, autoimmune disorders and inflammatory skin diseases. Here, we report a chromosome-scale A. paniculata genome sequence of 269 Mb that was assembled by Illumina short reads, PacBio long reads and high-confidence (Hi-C) data. Gene annotation predicted 25 428 protein-coding genes. In order to decipher the genetic underpinning of diterpenoid biosynthesis, transcriptome data from seedlings elicited with methyl jasmonate were also obtained, which enabled the identification of genes encoding diterpenoid synthases, cytochrome P450 monooxygenases, 2-oxoglutarate-dependent dioxygenases and UDP-dependent glycosyltransferases potentially involved in diterpenoid lactone biosynthesis. We further carried out functional characterization of pairs of class-I and -II diterpene synthases, revealing the ability to produce diversified labdane-related diterpene scaffolds. In addition, a glycosyltransferase able to catalyze O-linked glucosylation of andrograpanin, yielding the major active product neoandrographolide, was also identified. Thus, our results demonstrate the utility of the combined genomic and transcriptomic data set generated here for the investigation of the production of the bioactive diterpenoid lactone constituents of the important medicinal herb A. paniculata.

Modern Trends in the In Vitro Production and Use of Callus, Suspension Cells and Root Cultures of Medicinal Plants.

This paper studies modern methods of producing and using callus, suspension cells and root cultures of medicinal plants in vitro. A new solution for natural product production is the use of an alternative source of renewable, environmentally friendly raw materials: callus, suspension and root cultures of higher plants in vitro. The possibility of using hairy root cultures as producers of various biologically active substances is studied. It is proven that the application of the genetic engineering achievements that combine in vitro tissue culture and molecular biology methods was groundbreaking in terms of the intensification of the extraction process of compounds significant for the medical industry. It is established that of all the callus processing methods, suspension and root cultures in vitro, the Agrobacterium method is the most widely used in practice. The use of agrobacteria has advantages over the biolistic method since it increases the proportion of stable transformation events, can deliver large DNA segments and does not require special ballistic devices. As a result of the research, the most effective strains of agrobacteria are identified.

Production of Encecalin in Cell Cultures and Hairy Roots of Helianthella quinquenervis (Hook.) A. Gray.

Plant cell and organ cultures of Helianthella quinquenervis, a medicinal plant whose roots are used by the Tarahumara Indians of Chihuahua, Mexico, to relieve several ailments, were established to identify and quantify some chromenes with biological activity, such as encecalin, and to evaluate their potential for biotechnological production. Gas chromatography-mass spectrometry (GC-MS) analysis corroborated the presence of quantifiable amounts of encecalin in H. quinquenervis cell cultures (callus and cell suspensions). In addition, hairy roots were obtained through three transformation protocols (prick, 45-s sonication and co-culture), using wild type Agrobacterium rhizogenes A4. After three months, cocultivation achieved the highest percentage of transformation (66%), and a comparable production (FW) of encecalin (110 µg/g) than the sonication assay (120 µg/g), both giving far higher yields than the prick assay (19 µg/g). Stable integration of rolC and aux1 genes in the transformed roots was confirmed by polymerase chain reaction (PCR). Hairy roots from cocultivation (six months-old) accumulated as much as 1086 µg/g (FW) of encecalin, over three times higher than the cell suspension cultures. The production of encecalin varied with growth kinetics, being higher at the stationary phase. This is the first report of encecalin production in hairy roots of H. quinquenervis, demonstrating the potential for a future biotechnological production of chromenes.

Anthocyanin Pigments: Beyond Aesthetics.

Anthocyanins are polyphenol compounds that render various hues of pink, red, purple, and blue in flowers, vegetables, and fruits. Anthocyanins also play significant roles in plant propagation, ecophysiology, and plant defense mechanisms. Structurally, anthocyanins are anthocyanidins modified by sugars and acyl acids. Anthocyanin colors are susceptible to pH, light, temperatures, and metal ions. The stability of anthocyanins is controlled by various factors, including inter and intramolecular complexations. Chromatographic and spectrometric methods have been extensively used for the extraction, isolation, and identification of anthocyanins. Anthocyanins play a major role in the pharmaceutical; nutraceutical; and food coloring, flavoring, and preserving industries. Research in these areas has not satisfied the urge for natural and sustainable colors and supplemental products. The lability of anthocyanins under various formulated conditions is the primary reason for this delay. New gene editing technologies to modify anthocyanin structures in vivo and the structural modification of anthocyanin via semi-synthetic methods offer new opportunities in this area. This review focusses on the biogenetics of anthocyanins; their colors, structural modifications, and stability; their various applications in human health and welfare; and advances in the field.

[Biosynthetic pathways of Polygonatum cyrtonema polysaccharide and diosgenin based on its transcriptomic data].

Polygonatum cyrtonema belongs to the plant family Liliaceae, and its dried rhizome is one of the sources of Chinese traditional medicine of Polygonati Rhizoma. It possesses the dual function as both medicine and food. Its main chemical components are polysaccharides and saponins. In order to understand the biosynthesis pathway of polysaccharides and diosgenin in P. cyrtonema, the corresponding transcriptomic data were obtained by extracting and sequencing the RNA of four parts of P. cyrtonema, namely, leaves, stems, rhizomes and roots. By adopting BGISEQ-500 sequencing platform, 42.03 Gb data were retrieved. Subsequently, the de novo assembly was carried out by Trinity software to obtain 137 233 transcripts, of which 68.13% of unigenes were annotated in seven databases including KEGG, GO, NR, NT, SwissProt, Pfam and KOG. Transcripts that may be involved in the biosynthesis of polysaccharides and diosgenin were analyzed by data mining. With help of qPCR, we validated expression data of four genes that were possibly involved in the biosynthesis of target metabolites. This experiment provides data for the study of biosynthetic pathways of P. cyrtonema secondary metabolites and the clarification of related structural gene functions.

Bioactivities, biosynthesis and biotechnological production of phenolic acids in Salvia miltiorrhiza.

Salvia miltiorrhiza (Danshen in Chinese), is a well-known traditional Chinese medicinal plant, which is used as not only human medicine but also health-promotion food. Danshen has been extensively used for the treatment of various cardiovascular and cerebrovascular diseases. As a major group of bioactive constituents from S. miltiorrhiza, water-soluble phenolic acids such as salvianolic acid B possessed good bioactivities including antioxidant, anti-inflammatory, anti-cancer and other health-promoting activities. It is of significance to improve the production of phenolic acids by modern biotechnology approaches to meet the increasing market demand. Significant progresses have been made in understanding the biosynthetic pathway and regulation mechanism of phenolic acids in S.miltiorrhiza, which will facilitate the process of targeted metabolic engineering or synthetic biology. Furthermore, multiple biotechnology methods such as in vitro culture, elicitation, hairy roots, endophytic fungi and bioreactors have been also used to obtain pharmaceutically active phenolic acids from S. miltiorrhiza. In this review, recent advances in bioactivities, biosynthetic pathway and biotechnological production of phenolic acid ingredients were summarized and future prospective was also discussed.

Dynamic metabolic solutions to the sessile life style of plants.

Covering: up to 2018 Plants are sessile organisms. To compensate for not being able to escape when challenged by unfavorable growth conditions, pests or herbivores, plants have perfected their metabolic plasticity by having developed the capacity for on demand synthesis of a plethora of phytochemicals to specifically respond to the challenges arising during plant ontogeny. Key steps in the biosynthesis of phytochemicals are catalyzed by membrane-bound cytochrome P450 enzymes which in plants constitute a superfamily. In planta, the P450s may be organized in dynamic enzyme clusters (metabolons) and the genes encoding the P450s and other enzymes in a specific pathway may be clustered. Metabolon formation facilitates transfer of substrates between sequential enzymes and therefore enables the plant to channel the flux of general metabolites towards biosynthesis of specific phytochemicals. In the plant cell, compartmentalization of the operation of specific biosynthetic pathways in specialized plastids serves to avoid undesired metabolic cross-talk and offers distinct storage sites for molar concentrations of specific phytochemicals. Liquid-liquid phase separation may lead to formation of dense biomolecular condensates within the cytoplasm or vacuole allowing swift activation of the stored phytochemicals as required upon pest or herbivore attack. The molecular grid behind plant plasticity offers an endless reservoir of functional modules, which may be utilized as a synthetic biology tool-box for engineering of novel biological systems based on rational design principles. In this review, we highlight some of the concepts used by plants to coordinate biosynthesis and storage of phytochemicals.

Phytochemical and biotechnological studies on Schisandra chinensis cultivar Sadova No. 1-a high utility medicinal plant.

In the presented work, raw materials (fruits and leaves) and in vitro biomass of a highly productive Schisandra chinensis Sadova No. 1 cultivar (SchS) were evaluated for the production of therapeutically useful schisandra lignans (SL). In vitro cultures of SchS were initiated, followed by extensive optimization studies focused on maximizing secondary metabolite production, with the aim of establishing a sustainable source of SL. Different cultivation systems (agar, agitated, bioreactor), experiment times (10, 20, 30, 40, 50 and 60 days) and plant growth regulators (6-benzyladenine-BA and 1-naphthaleneacetic acid-NAA, from 0 to 3 mg/l) in Murashige-Skoog (MS) medium were tested. Moreover, an elicitation procedure was applied to bioreactor-grown microshoots in order to increase SL production. Validated HPLC-DAD protocol enabled to detect fourteen SL in the extracts from in vitro and in vivo materials. The main compounds in the in vitro cultures were as follows: schisandrin (max. 176.3 mg/100 g DW), angeloylgomisin Q (max. 85.1 mg/100 g DW), gomisin A (max. 71.4 mg/100 g DW) and angeloylgomisin H (max. 67.0 mg/100 g DW). The highest total SL content (490.3 mg/100 g DW) was obtained in extracts from the biomass of agar cultures cultivated for 30 days on the MS medium variant containing 3 mg/l BA and 1 mg/l NAA. This amount was 1.32 times lower than in fruit extracts (646.0 mg/100 g DW) and 2.04 times higher than in leaf extracts (240.7 mg/100 g DW). The study demonstrated that SchS is a rich source of SL, thus proving its value for medical, cosmetic and food industry.

Role of plant phytochemicals and microbial enzymes in biosynthesis of metallic nanoparticles.

Metal-based nanoparticles have gained tremendous popularity because of their interesting physical, biological, optical, and magnetic properties. These nanoparticles can be synthesized using a variety of different physical, chemical, and biological techniques. The biological means are largely preferred as it provides an environmentally benign, green, and cost-effective route for the biosynthesis of nanoparticles. These bioresources can act as a scaffold, thereby playing the role of reducing as well as capping agents in the biosynthesis of nanoparticles. Medicinal plants tend to have a complex phytochemical constituent such as alcohols, phenols, terpenes, alkaloids, saponins, and proteins, while microbes have key enzymes which can act as reducing as well as stabilizing agent for NP synthesis. However, the mechanism of biosynthesis is still highly debatable. Herein, the present review is directed to give an updated comprehensive overview towards the mechanistic aspects in the biosynthesis of nanoparticles via plants and microbes. Various biosynthetic pathways of secondary metabolites in plants and key enzyme production in microbes have been discussed in detail, along with the underlying mechanisms for biogenic NP synthesis.

Rice Secondary Metabolites: Structures, Roles, Biosynthesis, and Metabolic Regulation.

Rice (Oryza sativa L.) is an important food crop providing energy and nutrients for more than half of the world population. It produces vast amounts of secondary metabolites. At least 276 secondary metabolites from rice have been identified in the past 50 years. They mainly include phenolic acids, flavonoids, terpenoids, steroids, alkaloids, and their derivatives. These metabolites exhibit many physiological functions, such as regulatory effects on rice growth and development, disease-resistance promotion, anti-insect activity, and allelopathic effects, as well as various kinds of biological activities such as antimicrobial, antioxidant, cytotoxic, and anti-inflammatory properties. This review focuses on our knowledge of the structures, biological functions and activities, biosynthesis, and metabolic regulation of rice secondary metabolites. Some considerations about cheminformatics, metabolomics, genetic transformation, production, and applications related to the secondary metabolites from rice are also discussed.

Sulphated Flavonoids: Biosynthesis, Structures, and Biological Activities.

The great diversity of enzymatic reactions in plant secondary metabolism allows the continuous discovery of new natural compounds and derivatives. Flavonoids, for example, can be found as aglycone or as several sorts of glycosylated, acetylated, methylated, and sulphated derivatives. This review focuses on sulphated flavonoids, an uncommon group of flavonoid derivatives found in some plant families. This work presents a compilation of sulphated flavonoids and their natural sources reported in the literature. Biosynthetic aspects and biological activities have also been reviewed, showing that these particular kinds of natural compounds play an interesting role in plant metabolism, as well as being potential candidates for the development of new drugs.

Transporter and its engineering for secondary metabolites.

Secondary metabolites possess a lot of biological activities, and to achieve their functions, transmembrane transportation is crucial. Elucidation of their transport mechanisms in the cell is critical for discovering ways to improve the production. Here, we have summarized the recent progresses for representative secondary metabolite transporters and also the strategies for uncovering the transporter systems in plants and microbes. We have also discussed the transporter engineering strategies being utilized for improving the heterologous natural product production, which exhibits promising future under the guide of synthetic biology.