Biosynthesis of Piceatannol from Resveratrol in Grapevine Can Be Mediated by Cresolase-Dependent Ortho-Hydroxylation Activity of Polyphenol Oxidase.

Piceatannol is a naturally occurring hydroxylated analogue of the stilbene phytoalexin resveratrol that can be found in grape fruit and derived products. Piceatannol has aroused great interest as it has been shown to surpass some human health-beneficial properties of resveratrol including antioxidant activity, several pharmacological activities and also bioavailability. The plant biosynthetic pathway of piceatannol is still poorly understood, which is a bottleneck for the development of both plant defence and bioproduction strategies. Cell cultures of Vitis vinifera cv. Gamay, when elicited with dimethyl-ß-cyclodextrin (MBCD) and methyl jasmonate (MeJA), lead to large increases in the accumulation of resveratrol, and after 120 h of elicitation, piceatannol is also detected due to the regiospecific hydroxylation of resveratrol. Therefore, an ortho-hydroxylase must participate in the biosynthesis of piceatannol. Herein, three possible types of resveratrol hydroxylation enzymatic reactions have been tested, specifically, a reaction catalyzed by an NADPH-dependent cytochrome, P450 hydroxylase, a 2-oxoglutarate-dependent dioxygenase and ortho-hydroxylation, similar to polyphenol oxidase (PPO) cresolase activity. Compared with P450 hydoxylase and the dioxygenase activities, PPO displayed the highest specific activity detected either in the crude extract, the particulate or the soluble fraction obtained from cell cultures elicited with MBCD and MeJA for 120 h. The overall yield of PPO activity present in the crude extract (107.42 EU) was distributed mostly in the soluble fraction (66.15 EU) rather than in the particulate fraction (3.71 EU). Thus, partial purification of the soluble fraction by precipitation with ammonium sulphate, dialysis and ion exchange chromatography was carried out. The soluble fraction precipitated with 80% ammonium sulphate and the chromatographic fractions also showed high levels of PPO activity, and the presence of the PPO protein was confirmed by Western blot and LC-MS/MS. In addition, a kinetic characterization of the cresolase activity of partially purified PPO was carried out for the resveratrol substrate, including Vmax and Km parameters. The Km value was 118.35 ± 49.84 µM, and the Vmax value was 2.18 ± 0.46 µmol min-1 mg-1.

Prenylated Flavonoids of the Moraceae Family: A Comprehensive Review of Their Biological Activities.

Prenylated flavonoids (PFs) are natural flavonoids with a prenylated side chain attached to the flavonoid skeleton. They have great potential for biological activities such as anti-diabetic, anti-cancer, antimicrobial, antioxidant, anti-inflammatory, enzyme inhibition, and anti-Alzheimer's effects. Medicinal chemists have recently paid increasing attention to PFs, which have become vital for developing new therapeutic agents. PFs have quickly developed through isolation and semi- or full synthesis, proving their high value in medicinal chemistry research. This review comprehensively summarizes the research progress of PFs, including natural PFs from the Moraceae family and their pharmacological activities. This information provides a basis for the selective design and optimization of multifunctional PF derivatives to treat multifactorial diseases.

High variability of perezone content in rhizomes of Acourtia cordata wild plants, environmental factors related, and proteomic analysis.

With the aim of exploring the source of the high variability observed in the production of perezone, in Acourtia cordata wild plants, we analyze the influence of soil parameters and phenotypic characteristics on its perezone content. Perezone is a sesquiterpene quinone responsible for several pharmacological effects and the A. cordata plants are the natural source of this metabolite. The chemistry of perezone has been widely studied, however, no studies exist related to its production under natural conditions, nor to its biosynthesis and the environmental factors that affect the yield of this compound in wild plants. We also used a proteomic approach to detect differentially expressed proteins in wild plant rhizomes and compare the profiles of high vs. low perezone-producing plants. Our results show that in perezone-producing rhizomes, the presence of high concentrations of this compound could result from a positive response to the effects of some edaphic factors, such as total phosphorus (Pt), total nitrogen (Nt), ammonium (NH4), and organic matter (O. M.), but could also be due to a negative response to the soil pH value. Additionally, we identified 616 differentially expressed proteins between high and low perezone producers. According to the functional annotation of this comparison, the upregulated proteins were grouped in valine biosynthesis, breakdown of leucine and isoleucine, and secondary metabolism such as terpenoid biosynthesis. Downregulated proteins were grouped in basal metabolism processes, such as pyruvate and purine metabolism and glycolysis/gluconeogenesis. Our results suggest that soil parameters can impact the content of perezone in wild plants. Furthermore, we used proteomic resources to obtain data on the pathways expressed when A. cordata plants produce high and low concentrations of perezone. These data may be useful to further explore the possible relationship between perezone production and abiotic or biotic factors and the molecular mechanisms related to high and low perezone production.

Factors Affecting the Bioproduction of Resveratrol by Grapevine Cell Cultures under Elicitation.

Here we present a study of the characterization and optimization of the production of trans-Resveratrol (t-R) in grape (Vitis vinifera cv. Gamay) cell cultures elicited with methyl jasmonate (MeJA) and dimethyl-ß-cyclodextrin (DIMEB). The aim of this study was to determine the influence of a number of factors of the grapevine cell culture on t-R production level in 250 mL shaken flasks that would enable the better control of this bioproduction system when it is upscaled to a 2 L stirred bioreactor. The factors included the optimal growth phase for elicitation, the concentration of elicitors and of biomass, the order of addition of elicitors, and the illumination regime and ageing of cells. We found out that the optimal biomass density for the production of t-R was 19% (w/v) with an optimal ratio of 0.5 g DIMEB/g biomass. The most productive concentrations of the elicitors tested were 50 mM DIMEB and 100 µM MeJA, reaching maximum values of 4.18 mg·mL-1 and 16.3 mg·g biomass-1 of t-R concentration and specific production, respectively. We found that the order of elicitor addition matters since, as compared with the simultaneous addition of both elicitors, the addition of MeJA 48 h before DIMEB results in ca. 40% less t-R production, whilst there is no significant difference when MeJA is added 48 h after DIMEB. Upon upscaling, the better conditions tested for t-R production were aeration at 1.7 vol/vol/min without agitation, 24 °C, and 30 g·L-1 sucrose, achieving production rates similar to those obtained in shaken flasks.

Rational Design of Resveratrol O-methyltransferase for the Production of Pinostilbene.

Pinostilbene is a monomethyl ether analog of the well-known nutraceutical resveratrol. Both compounds have health-promoting properties, but the latter undergoes rapid metabolization and has low bioavailability. O-methylation improves the stability and bioavailability of resveratrol. In plants, these reactions are performed by O-methyltransferases (OMTs). Few efficient OMTs that monomethylate resveratrol to yield pinostilbene have been described so far. Here, we report the engineering of a resveratrol OMT from Vitis vinifera (VvROMT), which has the highest catalytic efficiency in di-methylating resveratrol to yield pterostilbene. In the absence of a crystal structure, we constructed a three-dimensional protein model of VvROMT and identified four critical binding site residues by applying different in silico approaches. We performed point mutations in these positions generating W20A, F24A, F311A, and F318A variants, which greatly reduced resveratrol's enzymatic conversion. Then, we rationally designed eight variants through comparison of the binding site residues with other stilbene OMTs. We successfully modified the native substrate selectivity of VvROMT. Variant L117F/F311W showed the highest conversion to pinostilbene, and variant L117F presented an overall increase in enzymatic activity. Our results suggest that VvROMT has potential for the tailor-made production of stilbenes.

Phytostilbenes as agrochemicals: biosynthesis, bioactivity, metabolic engineering and biotechnology.

Covering: 1976 to 2020. Although constituting a limited chemical family, phytostilbenes represent an emblematic group of molecules among natural compounds. Ever since their discovery as antifungal compounds in plants and their ascribed role in human health and disease, phytostilbenes have never ceased to arouse interest for researchers, leading to a huge development of the literature in this field. Owing to this, the number of references to this class of compounds has reached the tens of thousands. The objective of this article is thus to offer an overview of the different aspects of these compounds through a large bibliography analysis of more than 500 articles. All the aspects regarding phytostilbenes will be covered including their chemistry and biochemistry, regulation of their biosynthesis, biological activities in plants, molecular engineering of stilbene pathways in plants and microbes as well as their biotechnological production by plant cell systems.

Dimethyl Labeling-Based Quantitative Proteomics of Recalcitrant Cocoa Pod Tissue.

Dimethyl labeling is a type of stable-isotope labeling suitable for creating isotopic variants of peptides and thus be utilized for quantitative proteomics experiments. Labeling is achieved through a reductive amination/alkylation reaction using the low-cost reagents formaldehyde and cyanoborohydride, resulting in dimethylation of free amine groups of Lys and N-termini. Availability of isotopomeric forms of these reagents allows for the generation of up to six different isotopic variants. Here we describe the application of dimethylation to create two isotopic variants, light and heavy, differing in 4 Da, to label the total tryptic digest peptides of cocoa pod extracted from healthy pods from cultivars susceptible and resistant to the fungal disease called "frosty pod" caused by Moniliophthora roreri.

Rosa hybrida orcinol O-methyl transferase-mediated production of pterostilbene in metabolically engineered grapevine cell cultures.

Stilbenes are naturally scarce high-added-value plant compounds with chemopreventive, pharmacological and cosmetic properties. Bioproduction strategies include engineering the metabolisms of bacterial, fungal and plant cell systems. Strikingly, one of the most effective strategies consists in the elicitation of wild grapevine cell cultures, which leads to vast stilbene resveratrol accumulation in the extracellular medium. The combination of both cell culture elicitation and metabolic engineering strategies to produce resveratrol analogs proved more efficient for the hydroxylated derivative piceatannol than for the dimethylated derivative pterostilbene, for which human hydroxylase HsCYP1B1- and grapevine O-methyltransferase VvROMT-transformed cell cultures were respectively used. Rose orcinol O-methyltransferase (OOMT) displays enzymatic properties, which makes it an appealing candidate to substitute VvROMT in the combined strategy to enhance the pterostilbene production level by engineered grapevine cells upon elicitation. Here we cloned a Rosa hybrida OOMT gene, and created a genetic construction suitable for Agrobacterium-mediated plant transformation. OOMT's ability to catalyze the conversion of resveratrol into pterostilbene was first assessed in vitro using protein extracts of agroinfiltrated N. benthamiana leaves and transformed grapevine callus. The grapevine cell cultures transformed with RhOOMT produced about 16 mg/L culture of pterostilbene and reached an extracellular distribution of up to 34% of total production at the best, which is by far the highest production reported to date in a plant system. A bonus large resveratrol production of ca. 1500-3000 mg/L was simultaneously obtained. Our results demonstrate a viable successful metabolic engineering strategy to produce pterostilbene, a resveratrol analog with enhanced pharmacological properties.

Targeted Quantification of Isoforms of a Thylakoid-Bound Protein: MRM Method Development.

Targeted mass spectrometric methods such as selected/multiple reaction monitoring (SRM/MRM) have found intense application in protein detection and quantification which competes with classical immunoaffinity techniques. It provides a universal procedure to develop a fast, highly specific, sensitive, accurate, and cheap methodology for targeted detection and quantification of proteins based on the direct analysis of their surrogate peptides typically generated by tryptic digestion. This methodology can be advantageously applied in the field of plant proteomics and particularly for non-model species since immunoreagents are scarcely available. Here, we describe the issues to take into consideration in order to develop a MRM method to detect and quantify isoforms of the thylakoid-bound protein polyphenol oxidase from the non-model and database underrepresented species Eriobotrya japonica Lindl.

A Tau Class Glutathione-S-Transferase is Involved in Trans-Resveratrol Transport Out of Grapevine Cells.

Vitis vinifera cell cultures respond to pathogens and elicitors by synthesizing and extracellularly accumulating stilbenoid phytoalexins. Large amounts of trans-resveratrol (t-R) are produced when a cell culture is elicited with methylated cyclodextrins (MBCD), either alone or combined with methyl jasmonate (MeJA). t-R transport to the extracellular medium, which represents the apoplastic space, would place this antifungal defense right in the battlefield to efficiently fight against pathogen attack. Yet despite their physiological relevance, these transport pathways are mostly unknown. A broad hypothesis-free DIGE-based proteomic experiment of a temporal series of elicited grapevine cell cultures was performed to explore the expression profiles of t-R biosynthetic proteins and other co-expressing proteins potentially involved in such a cell response. A correlation between two tau class glutathione-S-transferases (GSTs) with several stilbene synthase and phenylalanine ammonia-lyase isoforms, and with the t-R metabolite itself, was found and further assessed by a qRT-PCR gene expression analysis. The best candidate, GSTU-2, was cloned from the cDNA of the MBCD + MeJA-elicited grapevine cells and used for Agrobacterium-mediated grapevine cell transformation. The non-elicited lines that overexpressed GSTU-2 displayed an extracellular t-R accumulating phenotype, but stabilization of t-R required the addition to culture medium of adsorbent compounds, e.g., PVP or ß-cyclodextrin. The wild-type cell cultures accumulated no t-R, not even in the presence of adsorbents. The transient expression of the GSTU-2-GFP fusion proteins in grapevine cells showed localisation in the plasma membrane, and the immunoprecipitation of HA-tagged GSTU-2 revealed its interaction with HIR, a plasma membrane-bound protein. These findings are consistent with a functional role in transport. This is the first report providing several pieces of experimental evidence for the involvement of a specific tau class GST in t-R transport to the extracellular medium.

Bioconversion of stilbenes in genetically engineered root and cell cultures of tobacco.

It is currently possible to transfer a biosynthetic pathway from a plant to another organism. This system has been exploited to transfer the metabolic richness of certain plant species to other plants or even to more simple metabolic organisms such as yeast or bacteria for the production of high added value plant compounds. Another application is to bioconvert substrates into scarcer or biologically more interesting compounds, such as piceatannol and pterostilbene. These two resveratrol-derived stilbenes, which have very promising pharmacological activities, are found in plants only in small amounts. By transferring the human cytochrome P450 hydroxylase 1B1 (HsCYP1B1) gene to tobacco hairy roots and cell cultures, we developed a system able to bioconvert exogenous t-resveratrol into piceatannol in quantities near to mg L-1. Similarly, after heterologous expression of resveratrol O-methyltransferase from Vitis vinifera (VvROMT) in tobacco hairy roots, the exogenous t-resveratrol was bioconverted into pterostilbene. We also observed that both bioconversions can take place in tobacco wild type hairy roots (pRiA4, without any transgene), showing that unspecific tobacco P450 hydroxylases and methyltransferases can perform the bioconversion of t-resveratrol to give the target compounds, albeit at a lower rate than transgenic roots.

A Focused Multiple Reaction Monitoring (MRM) Quantitative Method for Bioactive Grapevine Stilbenes by Ultra-High-Performance Liquid Chromatography Coupled to Triple-Quadrupole Mass Spectrometry (UHPLC-QqQ).

Grapevine stilbenes are a family of polyphenols which derive from trans-resveratrol having antifungal and antimicrobial properties, thus being considered as phytoalexins. In addition to their diverse bioactive properties in animal models, they highlight a strong potential in human health maintenance and promotion. Due to this relevance, highly-specific qualitative and quantitative methods of analysis are necessary to accurately analyze stilbenes in different matrices derived from grapevine. Here, we developed a rapid, sensitive, and specific analysis method using ultra-high-performance liquid chromatography coupled to triple-quadrupole mass spectrometry (UHPLC-QqQ) in MRM mode to detect and quantify five grapevine stilbenes, trans-resveratrol, trans-piceid, trans-piceatannol, trans-pterostilbene, and trans-ε-viniferin, whose interest in relation to human health is continuously growing. The method was optimized to minimize in-source fragmentation of piceid and to avoid co-elution of cis-piceid and trans-resveratrol, as both are detected with resveratrol transitions. The applicability of the developed method of stilbene analysis was tested successfully in different complex matrices including cellular extracts of Vitis vinifera cell cultures, reaction media of biotransformation assays, and red wine.

Silybum marianum cell cultures stably transformed with Vitis vinifera stilbene synthase accumulate t-resveratrol in the extracellular medium after elicitation with methyl jasmonate or methylated ß-cyclodextrins.

The growing demand for t-resveratrol for industrial uses has generated considerable interest in its production. Heterologous resveratrol production in plant cell suspensions, apart from requiring the introduction of only one or two genes, has the advantage of high biomass yield and a short cultivation time, and thus could be an option for large-scale production. Silybum marianum is the source of the flavonolignan silymarin. Phenylpropanoid synthesis in cultures of this species can be activated by elicitation with methyl jasmonate and methylated ß-cyclodextrins, with products of the pathway (coniferyl alcohol and some isomers of the silymarin complex) being released into the medium. Given that stilbene synthase shares the same key precursors involved in flavonoid and /or monolignol biosynthesis, we explored the potential of metabolically engineered S. marianum cultures for t-resveratrol production. Cell suspensions were stably transformed with Vitis vinifera stilbene synthase 3 and the expression of the transgene led to extracellular t-resveratrol accumulation at the level of milligrams per litre under elicitation. Resveratrol synthesis occurred at the expense of coniferyl alcohol. Production of silymarin was less affected in the transgenic cultures, since the flavonoid pathway is limiting for its synthesis, due to the preferred supply of precursors for the monolignol branch. The fact that the expressed STS gene took excessively produced precursors of non-bioactive compounds (coniferyl alcohol), while keeping the metabolic flow for target secondary compounds (i.e. silymarin) unaltered, opens a way to extend the applications of plant cell cultures for the simultaneous production of both constitutive and foreign valuable metabolites.

Production of highly bioactive resveratrol analogues pterostilbene and piceatannol in metabolically engineered grapevine cell cultures.

Grapevine stilbenes, particularly trans-resveratrol, have a demonstrated pharmacological activity. Other natural stilbenes derived from resveratrol such as pterostilbene or piceatannol, display higher oral bioavailability and bioactivity than the parent compound, but are far less abundant in natural sources. Thus, to efficiently obtain these bioactive resveratrol derivatives, there is a need to develop new bioproduction systems. Grapevine cell cultures are able to produce large amounts of easily recoverable extracellular resveratrol when elicited with methylated cyclodextrins and methyl jasmonate. We devised this system as an interesting starting point of a metabolic engineering-based strategy to produce resveratrol derivatives using resveratrol-converting enzymes. Constitutive expression of either Vitis vinifera resveratrol O-methyltransferase (VvROMT) or human cytochrome P450 hydroxylase 1B1 (HsCYP1B1) led to pterostilbene or piceatannol, respectively, after the engineered cell cultures were treated with the aforementioned elicitors. Functionality of both gene products was first assessed in planta by Nicotiana benthamiana agroinfiltration assays, in which tobacco cells transiently expressed stilbene synthase and VvROMT or HsCYP1B1. Grapevine cell cultures transformed with VvROMT produced pterostilbene, which was detected in both intra- and extracellular compartments, at a level of micrograms per litre. Grapevine cell cultures transformed with HsCYP1B1 produced about 20 mg/L culture of piceatannol, displaying a sevenfold increase in relation to wild-type cultures, and reaching an extracellular distribution of up to 45% of total production. The results obtained demonstrate the feasibility of this novel system for the bioproduction of natural and more bioactive resveratrol derivatives and suggest new ways for the improvement of production yields.

Enhanced extracellular production of trans-resveratrol in Vitis vinifera suspension cultured cells by using cyclodextrins and coronatine.

In the present work the effect of cyclodextrin and coronatine on both trans-resveratrol production and the expression of stilbene biosynthetic genes in Vitis vinifera L. cv Monastrell suspension cultured cells were evaluated. The results showed the maximum level of trans-resveratrol produced by cells and secreted to the culture medium with 50 mM cyclodextrins and 1 µM coronatine. Since the levels of trans-resveratrol produced in the combined treatment were higher than the sum of the individual treatments, a synergistic effect between both elicitors was assumed. In addition, all the analysed genes were induced by cyclodextrins and/or coronatine. The expression of the phenylalanine ammonia lyase and stilbene synthase genes was greatly enhanced by coronatine although an increase in the amount of trans-resveratrol in the spent medium was not detected. Therefore, despite the fact that trans-resveratrol production is related with the expression of genes involved in the biosynthetic process, other factors may be involved, such as post-transcriptional and post-traductional regulation. The expression maximal levels of cinnamate 4-hydroxylase and 4-coumarate-CoA ligase genes were found with cyclodextrins alone or in combination with coronatine suggesting that the activity of these enzymes could be not only important for the formation of intermediates of trans-R biosynthesis but also for those intermediates involved in the biosynthesis of lignins and/or flavonoids.

The role of proteomics in progressing insights into plant secondary metabolism.

The development of omics has enabled the genome-wide exploration of all kinds of biological processes at the molecular level. Almost every field of plant biology has been analyzed at the genomic, transcriptomic and proteomic level. Here we focus on the particular contribution that proteomic technologies have made in progressing knowledge and characterising plant secondary metabolism (SM) pathways since early expectations were created 15 years ago. We analyzed how three major issues in the proteomic analysis of plant SM have been implemented in various research studies. These issues are: (i) the selection of a suitable plant material rich in secondary metabolites of interest, such as specialized tissues and organs, and in vitro cell cultures; (ii) the proteomic strategy to access target proteins, either a comprehensive or a differential analysis; (iii) the proteomic approach, represented by the hypothesis-free discovery proteomics and the hypothesis-driven targeted proteomics. We also examine to what extent the most-advanced technologies have been incorporated into proteomic research in plant SM and highlight some cutting edge techniques that would strongly benefit the progress made in this field.

RNA isolation from loquat and other recalcitrant woody plants with high quality and yield.

RNA isolation is difficult in plants that contain large amounts of polysaccharides and polyphenol compounds. To date, no commercial kit has been developed for the isolation of high-quality RNA from tissues with these characteristics, especially for fruit. The common protocols for RNA isolation are tedious and usually result in poor yields when applied to recalcitrant plant tissues. Here an efficient RNA isolation protocol based on cetyltrimethylammonium bromide (CTAB) and two successive precipitations with 10 M lithium chloride (LiCl) was developed specifically for loquat fruits, but it was proved to work efficiently in other tissues of loquat and woody plants. The RNA isolated by this improved protocol was not only of high purity and integrity (A260/A280 ratios ranged from 1.90 to 2.04 and A260/A230 ratios were>2.0) but also of high yield (up to 720 µg on average [coefficient of variation=21%] total RNA per gram fresh tissue). The protocol was tested on loquat fruit (different stages of development, postharvest, ripening, and bruising), leaf, root, flower, stem, and bud; quince fruit and root; grapevine cells in liquid culture; and rose petals. The RNA obtained with this method is amenable to enzymatic treatments and can be efficiently applied for research on gene characterization, expression, and function.

Development and validation of MRM methods to quantify protein isoforms of polyphenol oxidase in loquat fruits.

Multiple reaction monitoring (MRM) is emerging as a promising technique for the detection and quantification of protein biomarkers in complex biological samples. Compared to Western blotting or enzyme assays, its high sensitivity, specificity, accuracy, assay speed, and sample throughput represent a clear advantage for being the approach of choice for the analysis of proteins. MRM assays are capable of detecting and quantifying proteolytic peptides differing in mass unique to particular proteins, that is, proteotypic peptides, through which different protein isoforms can be distinguished. We have focused on polyphenol oxidase (PPO), a plant conspicuous enzyme encoded by a multigenic family in loquat (Eriobotrya japonica Lindl.) and other related species. PPO is responsible for both the protection of plants from biotic stress as a feeding deterrent for herbivore insects and the enzymatic browning of fruits and vegetables. The latter makes fruit more attractive to seed dispersal agents but is also a major cause of important economic losses in agriculture and food industry. An adequate management of PPO at plant breeding level would maximize the benefits and minimize the disadvantages of this enzyme, but it would require a precise knowledge of the biological role played by each isoform in the plant. Thus, for the functional study of the PPOs, we have cloned and overexpressed fragments of three PPO isoforms from loquat to develop MRM-based methods for the quantification of each isoform. The method was developed using an ion trap instrument and validated in a QQQ instrument. It resulted in the selection of at least two peptides for each isoform that can be monitored by at least three transitions. A combination of SDS-PAGE and MRM lead to detect two out of three monitored isoforms in different gel bands corresponding to different processing stages of PPO. The method was applied to determine the amount of the PPO2 isoform in protein extracts from fruit samples using external calibrants.