The case of tryptamine and serotonin in plants: a mysterious precursor for an illustrious metabolite.

Indolamines are tryptophan-derived specialized metabolites belonging to the huge and ubiquitous indole alkaloids group. Serotonin and melatonin are the best-characterized members of this family, given their many hormonal and physiological roles in animals. Following their discovery in plants, the study of plant indolamines has flourished and their involvement in important processes, including stress responses, growth and development, and reproduction, has been proposed, leading to their classification as a new category of phytohormones. However, the complex indolamine puzzle is far from resolved, particularly the biological roles of tryptamine, the early serotonin precursor representing the central hub of many downstream indole alkaloids. Tryptophan decarboxylase, which catalyzes the synthesis of tryptamine, strictly regulates the flux of carbon and nitrogen from the tryptophan pool into the indolamine pathway. Furthermore, tryptamine accumulates to high levels in the reproductive organs of many plant species and therefore cannot be classed as a mere intermediate but rather as an end product with potentially important functions in fruits and seeds. This review summarizes current knowledge on the role of tryptamine and its close relative serotonin, emphasizing the need for a clear understanding of the functions of, and mutual relations between, these indolamines and their biosynthesis pathways in plants.

Optimization of a Sustainable Protocol for the Extraction of Anthocyanins as Textile Dyes from Plant Materials.

Anthocyanins are the largest group of polyphenolic pigments in the plant kingdom. These non-toxic, water-soluble compounds are responsible for the pink, red, purple, violet, and blue colors of fruits, vegetables, and flowers. Anthocyanins are widely used in the production of food, cosmetic and textile products, in the latter case to replace synthetic dyes with natural and sustainable alternatives. Here, we describe an environmentally benign method for the extraction of anthocyanins from red chicory and their characterization by HPLC-DAD and UPLC-MS. The protocol does not require hazardous solvents or chemicals and relies on a simple and scalable procedure that can be applied to red chicory waste streams for anthocyanin extraction. The extracted anthocyanins were characterized for stability over time and for their textile dyeing properties, achieving good values for washing fastness and, as expected, a pink-to-green color change that is reversible and can therefore be exploited in the fashion industry.

Untargeted and Targeted Metabolomics and Tryptophan Decarboxylase In Vivo Characterization Provide Novel Insight on the Development of Kiwifruits (Actinidia deliciosa).

Kiwifruit (Actinidia deliciosa cv. Hayward) is a commercially important crop with highly nutritional green fleshy fruits. The post-harvest maturation of the fruits is well characterized, but little is known about the metabolic changes that occur during fruit development. Here we used untargeted metabolomics to characterize the non-volatile metabolite profile of kiwifruits collected at different time points after anthesis, revealing profound metabolic changes before the onset of ripening including the depletion of many classes of phenolic compounds. In contrast, the phytohormone abscisic acid accumulated during development and ripening, along with two indolamines (serotonin and its precursor tryptamine), and these were monitored in greater detail by targeted metabolomics. The role of indolamines in kiwifruit development is completely unknown, so we also characterized the identity of genes encoding tryptophan decarboxylase in A. deliciosa and its close relative A. chinensis to provide insight into the corresponding biological processes. Our results indicate that abscisic acid and indolamines fulfill unrecognized functions in the development and ripening of kiwifruits.

Disclosing the Molecular Basis of the Postharvest Life of Berry in Different Grapevine Genotypes.

The molecular events that characterize postripening grapevine berries have rarely been investigated and are poorly defined. In particular, a detailed definition of changes occurring during the postharvest dehydration, a process undertaken to make some particularly special wine styles, would be of great interest for both winemakers and plant biologists. We report an exhaustive survey of transcriptomic and metabolomic responses in berries representing six grapevine genotypes subjected to postharvest dehydration under identical controlled conditions. The modulation of phenylpropanoid metabolism clearly distinguished the behavior of genotypes, with stilbene accumulation as the major metabolic event, although the transient accumulation/depletion of anthocyanins and flavonols was the prevalent variation in genotypes that do not accumulate stilbenes. The modulation of genes related to phenylpropanoid/stilbene metabolism highlighted the distinct metabolomic plasticity of genotypes, allowing for the identification of candidate structural and regulatory genes. In addition to genotype-specific responses, a core set of genes was consistently modulated in all genotypes, representing the common features of berries undergoing dehydration and/or commencing senescence. This included genes controlling ethylene and auxin metabolism as well as genes involved in oxidative and osmotic stress, defense responses, anaerobic respiration, and cell wall and carbohydrate metabolism. Several transcription factors were identified that may control these shared processes in the postharvest berry. Changes representing both common and genotype-specific responses to postharvest conditions shed light on the cellular processes taking place in harvested berries stored under dehydrating conditions for several months.

Edible plants for oral delivery of biopharmaceuticals.

Molecular farming is the use of plants for the production of high value recombinant proteins. Over the last 25 years, molecular farming has achieved the inexpensive, scalable and safe production of pharmaceutical proteins using a range of strategies. One of the most promising approaches is the use of edible plant organs expressing biopharmaceuticals for direct oral delivery. This approach has proven to be efficacious in several clinical vaccination and tolerance induction trials as well as multiple preclinical studies for disease prevention. The production of oral biopharmaceuticals in edible plant tissues could revolutionize the pharmaceutical industry by reducing the cost of production systems based on fermentation, and also eliminating expensive downstream purification, cold storage and transportation costs. This review considers the unique features that make plants ideal as platforms for the oral delivery of protein-based therapeutics and describes recent developments in the production of plant derived biopharmaceuticals for oral administration.

Heterologous expression of moss light-harvesting complex stress-related 1 (LHCSR1), the chlorophyll a-xanthophyll pigment-protein complex catalyzing non-photochemical quenching, in Nicotiana sp.

Oxygenic photosynthetic organisms evolved mechanisms for thermal dissipation of energy absorbed in excess to prevent formation of reactive oxygen species. The major and fastest component, called non-photochemical quenching, occurs within the photosystem II antenna system by the action of two essential light-harvesting complex (LHC)-like proteins, photosystem II subunit S (PSBS) in plants and light-harvesting complex stress-related (LHCSR) in green algae and diatoms. In the evolutionary intermediate Physcomitrella patens, a moss, both gene products are active. These proteins, which are present in low amounts, are difficult to purify, preventing structural and functional analysis. Here, we report on the overexpression of the LHCSR1 protein from P. patens in the heterologous systems Nicotiana benthamiana and Nicotiana tabacum using transient and stable nuclear transformation. We show that the protein accumulated in both heterologous systems is in its mature form, localizes in the chloroplast thylakoid membranes, and is correctly folded with chlorophyll a and xanthophylls but without chlorophyll b, an essential chromophore for plants and algal LHC proteins. Finally, we show that recombinant LHCSR1 is active in quenching in vivo, implying that the recombinant protein obtained is a good material for future structural and functional studies.

The dynamic loss and gain of introns during the evolution of the Brassicaceae.

Sequence comparison allows the detailed analysis of evolution at the nucleotide and amino acid levels, but much less information is known about the structural evolution of genes, i.e. how the number, length and distribution of introns change over time. We constructed a parsimonious model for the evolutionary rate of intron loss (IL) and intron gain (IG) within the Brassicaceae and found that IL/IG has been highly dynamic, with substantial differences between and even within lineages. The divergence of the Brassicaceae lineages I and II marked a dramatic change in the IL rate, with the common ancestor of lineage I losing introns three times more rapidly than the common ancestor of lineage II. Our data also indicate a subsequent declining trend in the rate of IL, although in Arabidopsis thaliana introns continue to be lost at approximately the ancestral rate. Variations in the rate of IL/IG within lineage II have been even more remarkable. Brassica rapa appears to have lost introns approximately 15 times more rapidly than the common ancestor of B. rapa and Schenkiella parvula, and approximately 25 times more rapidly than its sister species Eutrema salsugineum. Microhomology was detected at the splice sites of several dynamic introns suggesting that the non-homologous end-joining and double-strand break repair is a common pathway underlying IL/IG in these species. We also detected molecular signatures typical of mRNA-mediated IL, but only in B. rapa.

Early transcriptomic response to Fe supply in Fe-deficient tomato plants is strongly influenced by the nature of the chelating agent.

BACKGROUND: It is well known that in the rhizosphere soluble Fe sources available for plants are mainly represented by a mixture of complexes between the micronutrient and organic ligands such as carboxylates and phytosiderophores (PS) released by roots, as well as fractions of humified organic matter. The use by roots of these three natural Fe sources (Fe-citrate, Fe-PS and Fe complexed to water-extractable humic substances, Fe-WEHS) have been already studied at physiological level but the knowledge about the transcriptomic aspects is still lacking. RESULTS: The (59)Fe concentration recorded after 24 h in tissues of tomato Fe-deficient plants supplied with (59)Fe complexed to WEHS reached values about 2 times higher than those measured in response to the supply with Fe-citrate and Fe-PS. However, after 1 h no differences among the three Fe-chelates were observed considering the (59)Fe concentration and the root Fe(III) reduction activity. A large-scale transcriptional analysis of root tissue after 1 h of Fe supply showed that Fe-WEHS modulated only two transcripts leaving the transcriptome substantially identical to Fe-deficient plants. On the other hand, Fe-citrate and Fe-PS affected 728 and 408 transcripts, respectively, having 289 a similar transcriptional behaviour in response to both Fe sources. CONCLUSIONS: The root transcriptional response to the Fe supply depends on the nature of chelating agents (WEHS, citrate and PS). The supply of Fe-citrate and Fe-PS showed not only a fast back regulation of molecular mechanisms modulated by Fe deficiency but also specific responses due to the uptake of the chelating molecule. Plants fed with Fe-WEHS did not show relevant changes in the root transcriptome with respect to the Fe-deficient plants, indicating that roots did not sense the restored cellular Fe accumulation.

Functional diversification of grapevine MYB5a and MYB5b in the control of flavonoid biosynthesis in a petunia anthocyanin regulatory mutant.

Flavonoids play a key role in grapevine physiology and also contribute substantially to the quality of berries and wines. VvMYB5a and VvMYB5b are R2R3-MYB transcription factors previously proposed to control the spatiotemporal expression of flavonoid structural genes during berry development. We investigated the functions of these two proteins in detail by heterologous expression in a petunia an2 mutant, which has negligible anthocyanin levels in the petals because it lacks the MYB protein PhAN2. We also expressed VvMYBA1, the grapevine ortholog of petunia PhAN2, in the same genetic background. The anthocyanin profiles induced by expressing these transgenes in the petals revealed that VvMYBA1 is the functional ortholog of PhAN2 and that, unlike VvMYB5a, VvMYB5b can partially complement the an2 mutation. Transcriptomic analysis of petals by microarray hybridization and quantitative PCR confirmed that VvMYB5b up-regulates a subset of anthocyanin structural genes, whereas VvMYB5a has a more limited impact on the expression of genes related to anthocyanin biosynthesis. Furthermore, we identified additional specific and common targets of these two regulators, related to vacuolar acidification and membrane remodeling. Taken together, these data provide insight into the role of VvMYB5a and VvMYB5b in flavonoid biosynthesis and provide evidence for additional regulatory roles in distinct pathways.

Pomegranate ( Punica granatum L.) expresses several nsLTP isoforms characterized by different immunoglobulin E-binding properties.

BACKGROUND: Pomegranate allergy is associated with sensitization to non-specific lipid transfer proteins (nsLTPs). Our aim was to identify and characterize the non-specific nsLTPs expressed in pomegranate at the molecular level and to study their allergenic properties in terms of immunoglobulin E (IgE)-binding and cross-reactivity with peach nsLTP (Pru p 3). METHODS: A non-equilibrium two-dimensional (2-D) electrophoretic approach based on acid-urea PAGE and sodium dodecyl sulfate PAGE was set up to separate pomegranate nsLTPs. Their immunoreactivity was tested by immunoblotting carried out with anti-Pru p 3 polyclonal antibodies and sera from pomegranate-allergic patients. For final identification, pomegranate nsLTPs were purified by chromatography and subjected to trypsin digestion and mass spectrometry (MS) analysis. For this purpose, the sequences obtained by cDNA cloning of three pomegranate nsLTPs were integrated in the database that was subsequently searched for MS data interpretation. RESULTS: Four nsLTPs were identified by 2-D immunoblotting. The detected proteins showed different IgE-binding capacity and partial cross-reactivity with Pru p 3. cDNA cloning and MS analyses led to the identification of three nsLTP isoforms with 66-68% amino acid sequence identity named Pun g 1.0101, Pun g 1.0201 and Pun g 1.0301. CONCLUSIONS: By 2-D electrophoresis, we could separate different nsLTP isoforms possessing different IgE-binding properties, which might reflect peculiar allergenic potencies. The contribution of Pru p 3 to prime sensitization is not central as in other plant nsLTPs.

Comparative analysis of different biofactories for the production of a major diabetes autoantigen.

The 65-kDa isoform of human glutamic acid decarboxylase (hGAD65) is a major diabetes autoantigen that can be used for the diagnosis and (more recently) the treatment of autoimmune diabetes. We previously reported that a catalytically-inactive version (hGAD65mut) accumulated to tenfold higher levels than its active counterpart in transgenic tobacco plants, providing a safe and less expensive source of the protein compared to mammalian production platforms. Here we show that hGAD65mut is also produced at higher levels than hGAD65 by transient expression in Nicotiana benthamiana (using either the pK7WG2 or MagnICON vectors), in insect cells using baculovirus vectors, and in bacterial cells using an inducible-expression system, although the latter system is unsuitable because hGAD65mut accumulates within inclusion bodies. The most productive of these platforms was the MagnICON system, which achieved yields of 78.8 µg/g fresh leaf weight (FLW) but this was substantially less than the best-performing elite transgenic tobacco plants, which reached 114.3 µg/g FLW after six generations of self-crossing. The transgenic system was found to be the most productive and cost-effective although the breeding process took 3 years to complete. The MagnICON system was less productive overall, but generated large amounts of protein in a few days. Both plant-based systems were therefore advantageous over the baculovirus-based production platform in our hands.

Temperature and solar irradiation effects on secondary metabolism during ripening of field-grown everbearing strawberries.

The garden strawberry (Fragaria x ananassa Duch.) is cultivated and consumed worldwide because of the pleasant flavor and health-promoting phytochemicals of its false fruits. Monocrop cultivars produce fully ripe strawberries in about one month post-anthesis throughout the spring, while everbearing cultivars undergo additional strawberry production in autumn. In this work, we evaluated the impact of different season-dependent environmental conditions on the ripening program of an everbearing field-gown strawberry variety from autumn 2015 to spring 2016. We combined ad hoc sampling and environmental data collection with LC-MS-based untargeted metabolomics to dissect the effects of cumulative temperature and solar irradiation on fruit quality parameters and secondary metabolism during ripening. Different dynamics in specific sub-groups of metabolites were observed in strawberries experiencing distinct amounts of cumulative temperature and solar irradiation during spring and autumn. The integration of statistical analyses on collected data revealed that solar irradiation mainly affected fruit fresh weight and organic acid levels, whereas temperature had a more selective effect on the accumulation of specific flavonols, anthocyanins, and soluble sugar. These findings are of suitable interest to design further approaches for the study of the complex interactions among environmental conditions and ripening in strawberries grown in a real-world scenario.

A Novel Strategy for Glioblastoma Treatment by Natural Bioactive Molecules Showed a Highly Effective Anti-Cancer Potential.

Glioblastoma (GBM) is a severe form of brain tumor that has a high fatality rate. It grows aggressively and most of the time results in resistance to traditional treatments like chemo- and radiotherapy and surgery. Biodiversity, beyond representing a big resource for human well-being, provides several natural compounds that have shown great potential as anticancer drugs. Many of them are being extensively researched and significantly slow GBM progression by reducing the proliferation rate, migration, and inflammation and also by modulating oxidative stress. Here, the use of some natural compounds, such as Allium lusitanicum, Succisa pratensis, and Dianthus superbus, was explored to tackle GBM; they showed their impact on cell number reduction, which was partially given by cell cycle quiescence. Furthermore, a reduced cell migration ability was reported, accomplished by morphological cytoskeleton changes, which even highlighted a mesenchymal-epithelial transition. Furthermore, metabolic studies showed an induced cell oxidative stress modulation and a massive metabolic rearrangement. Therefore, a new therapeutic option was suggested to overcome the limitations of conventional treatments and thereby improve patient outcomes.

Bioprospecting of Artemisia genus: from artemisinin to other potentially bioactive compounds.

Species from genus Artemisia are widely distributed throughout temperate regions of the northern hemisphere and many cultures have a long-standing traditional use of these plants as herbal remedies, liquors, cosmetics, spices, etc. Nowadays, the discovery of new plant-derived products to be used as food supplements or drugs has been pushed by the exploitation of bioprospection approaches. Often driven by the knowledge derived from the ethnobotanical use of plants, bioprospection explores the existing biodiversity through integration of modern omics techniques with targeted bioactivity assays. In this work we set up a bioprospection plan to investigate the phytochemical diversity and the potential bioactivity of five Artemisia species with recognized ethnobotanical tradition (A. absinthium, A. alba, A. annua, A. verlotiorum and A. vulgaris), growing wild in the natural areas of the Verona province. We characterized the specialized metabolomes of the species (including sesquiterpenoids from the artemisinin biosynthesis pathway) through an LC-MS based untargeted approach and, in order to identify potential bioactive metabolites, we correlated their composition with the in vitro antioxidant activity. We propose as potential bioactive compounds several isomers of caffeoyl and feruloyl quinic acid esters (e.g. dicaffeoylquinic acids, feruloylquinic acids and caffeoylferuloylquinic acids), which strongly characterize the most antioxidant species A. verlotiorum and A. annua. Morevoer, in this study we report for the first time the occurrence of sesquiterpenoids from the artemisinin biosynthesis pathway in the species A. alba.

Performance of plant-produced RBDs as SARS-CoV-2 diagnostic reagents: a tale of two plant platforms.

The COVID-19 pandemic has underscored the need for rapid and cost-effective diagnostic tools. Serological tests, particularly those measuring antibodies targeting the receptor-binding domain (RBD) of the virus, play a pivotal role in tracking infection dynamics and vaccine effectiveness. In this study, we aimed to develop a simple enzyme-linked immunosorbent assay (ELISA) for measuring RBD-specific antibodies, comparing two plant-based platforms for diagnostic reagent production. We chose to retain RBD in the endoplasmic reticulum (ER) to prevent potential immunoreactivity issues associated with plant-specific glycans. We produced ER-retained RBD in two plant systems: a stable transformation of BY-2 plant cell culture (BY2-RBD) and a transient transformation in Nicotiana benthamiana using the MagnICON system (NB-RBD). Both systems demonstrated their suitability, with varying yields and production timelines. The plant-made proteins revealed unexpected differences in N-glycan profiles, with BY2-RBD displaying oligo-mannosidic N-glycans and NB-RBD exhibiting a more complex glycan profile. This difference may be attributed to higher recombinant protein synthesis in the N. benthamiana system, potentially overloading the ER retention signal, causing some proteins to traffic to the Golgi apparatus. When used as diagnostic reagents in ELISA, BY2-RBD outperformed NB-RBD in terms of sensitivity, specificity, and correlation with a commercial kit. This discrepancy may be due to the distinct glycan profiles, as complex glycans on NB-RBD may impact immunoreactivity. In conclusion, our study highlights the potential of plant-based systems for rapid diagnostic reagent production during emergencies. However, transient expression systems, while offering shorter timelines, introduce higher heterogeneity in recombinant protein forms, necessitating careful consideration in serological test development.

The use of plants for the production of therapeutic human peptides.

Peptides have unique properties that make them useful drug candidates for diverse indications, including allergy, infectious disease and cancer. Some peptides are intrinsically bioactive, while others can be used to induce precise immune responses by defining a minimal immunogenic region. The limitations of peptides, such as metabolic instability, short half-life and low immunogenicity, can be addressed by strategies such as multimerization or fusion to carriers, to improve their pharmacological properties. The remaining major drawback is the cost of production using conventional chemical synthesis, which is also difficult to scale-up. Over the last 15 years, plants have been shown to produce bioactive and immunogenic peptides economically and with the potential for large-scale synthesis. The production of peptides in plants is usually achieved by the genetic fusion of the corresponding nucleotide sequence to that of a carrier protein, followed by stable nuclear or plastid transformation or transient expression using bacterial or viral vectors. Chimeric plant viruses or virus-like particles can also be used to display peptide antigens, allowing the production of polyvalent vaccine candidates. Here we review progress in the field of plant-derived peptides over the last 5 years, addressing new challenges for diverse pathologies.

Plant-Based Systems for Vaccine Production.

Plant systems have been used as biofactories to produce recombinant proteins since 1983. The huge amount of data, collected so far in this framework, suggests that plants display several key advantages over existing traditional platforms when they are intended for therapeutic uses, including safety, scalability, and the speed in obtaining the final product.Here, we describe a method that could be applied for the expression and production of a candidate subunit vaccine in Nicotiana benthamiana plants by transient expression, defining all the protocols starting from plant cultivation to target recombinant protein purification.

Indolamine accumulation and TDC/T5H expression profiles reveal the complex and dynamic regulation of serotonin biosynthesis in tomato (Solanum lycopersicum L.).

Tryptamine and serotonin are indolamines that fulfill diverse biological functions in all kingdoms of life. Plants convert l-tryptophan into tryptamine and then serotonin via consecutive decarboxylation and hydroxylation reactions catalyzed by the enzymes tryptophan decarboxylase (TDC) and tryptamine 5-hydroxylase (T5H). Tryptamine and serotonin accumulate to high levels in the edible fruits and seeds of many plant species, but their biological roles in reproductive organs remain unclear and the metabolic pathways have not been characterized in detail. We identified three TDC genes and a single T5H gene in tomato (Solanum lycopersicum L.) by homology-based screening and confirmed their activity by heterologous expression in Nicotiana benthamiana. The co-analysis of targeted metabolomics and gene expression data revealed complex spatiotemporal gene expression and metabolite accumulation patterns that suggest the involvement of the serotonin pathway in multiple biological processes. Our data support a model in which SlTDC1 allows tryptamine to accumulate in fruits, SlTDC2 causes serotonin to accumulate in aerial vegetative organs, and SlTDC3 works with SlT5H to convert tryptamine into serotonin in the roots and fruits.

Non-food/feed seeds as biofactories for the high-yield production of recombinant pharmaceuticals.

We describe an attractive cloning system for the seed-specific expression of recombinant proteins using three non-food/feed crops. A vector designed for direct subcloning by Gateway® recombination was developed and tested in Arabidopsis, tobacco and petunia plants for the production of a chimeric form (GAD67/65) of the 65 kDa isoform of glutamic acid decarboxylase (GAD65). GAD65 is one of the major human autoantigens involved in type 1 diabetes (T1D). The murine anti-inflammatory cytokine interleukin-10 (IL-10) was expressed with the described system in Arabidopsis and tobacco, whereas proinsulin, another T1D major autoantigen, was expressed in Arabidopsis. The cost-effective production of these proteins in plants could allow the development of T1D prevention strategies based on the induction of immunological tolerance. The best yields were achieved in Arabidopsis seeds, where GAD67/65 reached 7.7% of total soluble protein (TSP), the highest levels ever reported for this protein in plants. IL-10 and proinsulin reached 0.70% and 0.007% of TSP, respectively, consistent with levels previously reported in other plants or tissues. This versatile cloning vector could be suitable for the high-throughput evaluation of expression levels and stability of many valuable and difficult to produce proteins.

Undifferentiated In Vitro Cultured Actinidia deliciosa as Cell Factory for the Production of Quercetin Glycosides.

Land plants produce a vast arsenal of specialized metabolites and many of them display interesting bioactivities in humans. Recently, flavonol quercetin gained great attention in the light of the COVID-19 pandemic because, in addition to the anti-inflammatory, antiviral and anti-cancer activity already described, it emerged as possible inhibitor of 3CLpro, the major protease of SARS-CoV-2 virus. Plant cell and tissue culture (PCTC) is an attractive platform for the biotechnological production of plant metabolites. This technology allows a large amount of water and agricultural land to be saved and, being free of contaminants in the process, it is suitable for scaling up the production in bioreactors. In a project aimed to generate and screen in vitro plant cells for the production of valuable specialized metabolites for commercial production, we generated various cell lines from Actinidia deliciosa (kiwi fruit tree) and Actinidia chinensis (gold kiwi fruit tree), that were able to produce relevant amounts of quercetin derivatives, mainly quercetin glycosides. Three cell lines from A. deliciosa were characterized by targeted and untargeted metabolomics. In standard growing conditions, they produce and accumulate up to 13.26 mg/100 g fresh weight (419.76 mg/100 g dry weight) of quercetin derivatives. To address future industrial applications, these cell lines should be entered into an acceleration program to further increase the amount of these metabolites by optimizing the culture conditions and elicitation.