A Study on the Use of the Phyto-Courier Technology in Tobacco Leaves Infected by Agrobacterium tumefaciens.

Climate change results in exceptional environmental conditions and drives the migration of pathogens to which local plants are not adapted. Biotic stress disrupts plants' metabolism, fitness, and performance, ultimately impacting their productivity. It is therefore necessary to develop strategies for improving plant resistance by promoting stress responsiveness and resilience in an environmentally friendly and sustainable way. The aim of this study was to investigate whether priming tobacco plants with a formulation containing silicon-stabilised hybrid lipid nanoparticles functionalised with quercetin (referred to as GS3 phyto-courier) can protect against biotic stress triggered by Agrobacterium tumefaciens leaf infiltration. Tobacco leaves were primed via infiltration or spraying with the GS3 phyto-courier, as well as with a buffer (B) and free quercetin (Q) solution serving as controls prior to the biotic stress. Leaves were then sampled four days after bacterial infiltration for gene expression analysis and microscopy. The investigated genes increased in expression after stress, both in leaves treated with the phyto-courier and control solutions. A trend towards lower values was observed in the presence of the GS3 phyto-courier for genes encoding chitinases and pathogenesis-related proteins. Agroinfiltrated leaves sprayed with GS3 confirmed the significant lower expression of the pathogenesis-related gene PR-1a and showed higher expression of peroxidase and serine threonine kinase. Microscopy revealed swelling of the chloroplasts in the parenchyma of stressed leaves treated with B; however, GS3 preserved the chloroplasts' mean area under stress. Furthermore, the UV spectrum of free Q solution and of quercetin freshly extracted from GS3 revealed a different spectral signature with higher values of maximum absorbance (Amax) of the flavonoid in the latter, suggesting that the silicon-stabilised hybrid lipid nanoparticles protect quercetin against oxidative degradation.

Molecular insights into plant desiccation tolerance: transcriptomics, proteomics and targeted metabolite profiling in Craterostigma plantagineum.

The resurrection plant Craterostigma plantagineum possesses an extraordinary capacity to survive long-term desiccation. To enhance our understanding of this phenomenon, complementary transcriptome, soluble proteome and targeted metabolite profiling was carried out on leaves collected from different stages during a dehydration and rehydration cycle. A total of 7348 contigs, 611 proteins and 39 metabolites were differentially abundant across the different sampling points. Dynamic changes in transcript, protein and metabolite levels revealed a unique signature characterizing each stage. An overall low correlation between transcript and protein abundance suggests a prominent role for post-transcriptional modification in metabolic reprogramming to prepare plants for desiccation and recovery. The integrative analysis of all three data sets was performed with an emphasis on photosynthesis, photorespiration, energy metabolism and amino acid metabolism. The results revealed a set of precise changes that modulate primary metabolism to confer plasticity to metabolic pathways, thus optimizing plant performance under stress. The maintenance of cyclic electron flow and photorespiration, and the switch from C3 to crassulacean acid metabolism photosynthesis, may contribute to partially sustain photosynthesis and minimize oxidative damage during dehydration. Transcripts with a delayed translation, ATP-independent bypasses, alternative respiratory pathway and 4-aminobutyric acid shunt may all play a role in energy management, together conferring bioenergetic advantages to meet energy demands upon rehydration. This study provides a high-resolution map of the changes occurring in primary metabolism during dehydration and rehydration and enriches our understanding of the molecular mechanisms underpinning plant desiccation tolerance. The data sets provided here will ultimately inspire biotechnological strategies for drought tolerance improvement in crops.

Biochemical and cytological interactions between callose synthase and microtubules in the tobacco pollen tube.

KEY MESSAGE: The article concerns the association between callose synthase and cytoskeleton by biochemical and ultrastructural analyses in the pollen tube. Results confirmed this association and immunogold labeling showed a colocalization. Callose is a cell wall polysaccharide involved in fundamental biological processes, from plant development to the response to abiotic and biotic stress. To gain insight into the deposition pattern of callose, it is important to know how the enzyme callose synthase is regulated through the interaction with the vesicle-cytoskeletal system. Actin filaments likely determine the long-range distribution of callose synthase through transport vesicles but the spatial/biochemical relationships between callose synthase and microtubules are poorly understood, although experimental evidence supports the association between callose synthase and tubulin. In this manuscript, we further investigated the association between callose synthase and microtubules through biochemical and ultrastructural analyses in the pollen tube model system, where callose is an essential component of the cell wall. Results by native 2-D electrophoresis, isolation of callose synthase complex and far-western blot confirmed that callose synthase is associated with tubulin and can therefore interface with cortical microtubules. In contrast, actin and sucrose synthase were not permanently associated with callose synthase. Immunogold labeling showed colocalization between the enzyme and microtubules, occasionally mediated by vesicles. Overall, the data indicate that pollen tube callose synthase exerts its activity in cooperation with the microtubular cytoskeleton.

Histochemical Techniques in Plant Science: More Than Meets the Eye.

Histochemistry is an essential analytical tool interfacing extensively with plant science. The literature is indeed constellated with examples showing its use to decipher specific physiological and developmental processes, as well as to study plant cell structures. Plant cell structures are translucent unless they are stained. Histochemistry allows the identification and localization, at the cellular level, of biomolecules and organelles in different types of cells and tissues, based on the use of specific staining reactions and imaging. Histochemical techniques are also widely used for the in vivo localization of promoters in specific tissues, as well as to identify specific cell wall components such as lignin and polysaccharides. Histochemistry also enables the study of plant reactions to environmental constraints, e.g. the production of reactive oxygen species (ROS) can be traced by applying histochemical staining techniques. The possibility of detecting ROS and localizing them at the cellular level is vital in establishing the mechanisms involved in the sensitivity and tolerance to different stress conditions in plants. This review comprehensively highlights the additional value of histochemistry as a complementary technique to high-throughput approaches for the study of the plant response to environmental constraints. Moreover, here we have provided an extensive survey of the available plant histochemical staining methods used for the localization of metals, minerals, secondary metabolites, cell wall components, and the detection of ROS production in plant cells. The use of recent technological advances like CRISPR/Cas9-based genome-editing for histological application is also addressed. This review also surveys the available literature data on histochemical techniques used to study the response of plants to abiotic stresses and to identify the effects at the tissue and cell levels.

Silicon induces adventitious root formation in rice under arsenate stress with involvement of nitric oxide and indole-3-acetic acid.

Arsenic (As) negatively affects plant development. This study evaluates how the application of silicon (Si) can favor the formation of adventitious roots in rice under arsenate stress (AsV) as a mechanism to mitigate its negative effects. The simultaneous application of AsV and Si up-regulated the expression of genes involved in nitric oxide (NO) metabolism, cell cycle progression, auxin (IAA, indole-3-acetic acid) biosynthesis and transport, and Si uptake which accompanied adventitious root formation. Furthermore, Si triggered the expression and activity of enzymes involved in ascorbate recycling. Treatment with L-NAME (NG-nitro L-arginine methyl ester), an inhibitor of NO generation, significantly suppressed adventitious root formation, even in the presence of Si; however, supplying NO in the growth media rescued its effects. Our data suggest that both NO and IAA are essential for Si-mediated adventitious root formation under AsV stress. Interestingly, TIBA (2,3,5-triiodobenzoic acid), a polar auxin transport inhibitor, suppressed adventitious root formation even in the presence of Si and SNP (sodium nitroprusside, an NO donor), suggesting that Si is involved in a mechanism whereby a cellular signal is triggered and that first requires NO formation, followed by IAA biosynthesis.

Silicon-induced mitigatory effects in salt-stressed hemp leaves.

Silicon, a quasi-essential element for plants, improves vigour and resilience under stress. Recently, studies on textile hemp (Cannabis sativa L.) showed its genetic predisposition to uptake silicic acid and accumulate it as silica in epidermal leaf cells and trichomes. Here, microscopy, silicon quantification and gene expression analysis of candidate genes involved in salt stress were performed in hemp to investigate whether the metalloid protects against salinity. The results obtained with microscopy reveal that silicon treatment ameliorated the symptoms of salinity in older fan leaves, where the xylem tissue showed vessels with a wider lumen. In younger ones, it was difficult to assess any mitigation of stress symptoms after silicon application. At the gene level, salinity with and without silicon induced the expression of a putative Si efflux transporter gene 2 (low silicon 2, Lsi2). The addition of the metalloid did not result in any statistically significant changes in the expression of genes involved in stress response, although a trend towards a decrease was observed. In conclusion, our results show that hemp stress symptoms can be alleviated in older leaves by silicon application, that the metalloid is accumulated in fan leaves and highlight one putative rice Lsi2 orthologue as responsive to salinity.

Purification and Biochemical Characterization of Sucrose synthase from the Stem of Nettle (Urtica dioica L.).

Sucrose synthase is a key enzyme in sucrose metabolism as it saves an important part of sucrose energy in the uridine-5'-diphosphate glucose (UDP-glucose) molecule. As such it is also involved in the synthesis of fundamental molecules such as callose and cellulose, the latter being present in all cell walls of plant cells and therefore also in the gelatinous cell walls of sclerenchyma cells such as bast fibers. Given the importance of these cells in plants of economic interest such as hemp, flax and nettle, in this work we have studied the occurrence of Sucrose synthase in nettle stems by analyzing its distribution between the cytosol, membranes and cell wall. We have therefore developed a purification protocol that can allow the analysis of various characteristics of the enzyme. In nettle, Sucrose synthase is encoded by different genes and each form of the enzyme could be subjected to different post-translational modifications. Therefore, by two-dimensional electrophoresis analysis, we have also traced the phosphorylation profile of Sucrose synthase isoforms in the various cell compartments. This information paves the way for further investigation of Sucrose synthase in plants such as nettle, which is both economically important, but also difficult to study.

Transcriptomic Changes in Internode Explants of Stinging Nettle during Callogenesis.

Callogenesis, the process during which explants derived from differentiated plant tissues are subjected to a trans-differentiation step characterized by the proliferation of a mass of cells, is fundamental to indirect organogenesis and the establishment of cell suspension cultures. Therefore, understanding how callogenesis takes place is helpful to plant tissue culture, as well as to plant biotechnology and bioprocess engineering. The common herbaceous plant stinging nettle (Urtica dioica L.) is a species producing cellulosic fibres (the bast fibres) and a whole array of phytochemicals for pharmacological, nutraceutical and cosmeceutical use. Thus, it is of interest as a potential multi-purpose plant. In this study, callogenesis in internode explants of a nettle fibre clone (clone 13) was studied using RNA-Seq to understand which gene ontologies predominate at different time points. Callogenesis was induced with the plant growth regulators α-napthaleneacetic acid (NAA) and 6-benzyl aminopurine (BAP) after having determined their optimal concentrations. The process was studied over a period of 34 days, a time point at which a well-visible callus mass developed on the explants. The bioinformatic analysis of the transcriptomic dataset revealed specific gene ontologies characterizing each of the four time points investigated (0, 1, 10 and 34 days). The results show that, while the advanced stage of callogenesis is characterized by the iron deficiency response triggered by the high levels of reactive oxygen species accumulated by the proliferating cell mass, the intermediate and early phases are dominated by ontologies related to the immune response and cell wall loosening, respectively.

Plant Extracellular Vesicles and Nanovesicles: Focus on Secondary Metabolites, Proteins and Lipids with Perspectives on Their Potential and Sources.

While human extracellular vesicles (EVs) have attracted a big deal of interest and have been extensively characterized over the last years, plant-derived EVs and nanovesicles have earned less attention and have remained poorly investigated. Although a series of investigations already revealed promising beneficial health effects and drug delivery properties, adequate (pre)clinical studies are rare. This fact might be caused by a lack of sources with appropriate qualities. Our study introduces plant cell suspension culture as a new and well controllable source for plant EVs. Plant cells, cultured in vitro, release EVs into the growth medium which could be harvested for pharmaceutical applications. In this investigation we characterized EVs and nanovesicles from distinct sources. Our findings regarding secondary metabolites indicate that these might not be packaged into EVs in an active manner but enriched in the membrane when lipophilic enough, since apparently lipophilic compounds were associated with nanovesicles while more hydrophilic structures were not consistently found. In addition, protein identification revealed a possible explanation for the mechanism of EV cell wall passage in plants, since cell wall hydrolases like 1,3-ß-glucosidases, pectinesterases, polygalacturonases, ß-galactosidases and ß-xylosidase/α-L-arabinofuranosidase 2-like are present in plant EVs and nanovesicles which might facilitate cell wall transition. Further on, the identified proteins indicate that plant cells secrete EVs using similar mechanisms as animal cells to release exosomes and microvesicles.

Computational Analysis of Thermal Adaptation in Extremophilic Chitinases: The Achilles' Heel in Protein Structure and Industrial Utilization.

Understanding protein stability is critical for the application of enzymes in biotechnological processes. The structural basis for the stability of thermally adapted chitinases has not yet been examined. In this study, the amino acid sequences and X-ray structures of psychrophilic, mesophilic, and hyperthermophilic chitinases were analyzed using computational and molecular dynamics (MD) simulation methods. From the findings, the key features associated with higher stability in mesophilic and thermophilic chitinases were fewer and/or shorter loops, oligomerization, and less flexible surface regions. No consistent trends were observed between stability and amino acid composition, structural features, or electrostatic interactions. Instead, unique elements affecting stability were identified in different chitinases. Notably, hyperthermostable chitinase had a much shorter surface loop compared to psychrophilic and mesophilic homologs, implying that the extended floppy surface region in cold-adapted and mesophilic chitinases may have acted as a "weak link" from where unfolding was initiated. MD simulations confirmed that the prevalence and flexibility of the loops adjacent to the active site were greater in low-temperature-adapted chitinases and may have led to the occlusion of the active site at higher temperatures compared to their thermostable homologs. Following this, loop "hot spots" for stabilizing and destabilizing mutations were also identified. This information is not only useful for the elucidation of the structure-stability relationship, but will be crucial for designing and engineering chitinases to have enhanced thermoactivity and to withstand harsh industrial processing conditions.

The Roots of Plant Frost Hardiness and Tolerance.

Frost stress severely affects agriculture and agroforestry worldwide. Although many studies about frost hardening and resistance have been published, most of them focused on the aboveground organs and only a minority specifically targets the roots. However, roots and aboveground tissues have different physiologies and stress response mechanisms. Climate models predict an increase in the magnitude and frequency of late-frost events, which, together with an observed loss of soil insulation, will greatly decrease plant primary production due to damage at the root level. Molecular and metabolic responses inducing root cold hardiness are complex. They involve a variety of processes related to modifications in cell wall composition, maintenance of the cellular homeostasis and the synthesis of primary and secondary metabolites. After a summary of the current climatic models, this review details the specificity of freezing stress at the root level and explores the strategies roots developed to cope with freezing stress. We then describe the level to which roots can be frost hardy, depending on their age, size category and species. After that, we compare the environmental signals inducing cold acclimation and frost hardening in the roots and aboveground organs. Subsequently, we discuss how roots sense cold at a cellular level and briefly describe the following signal transduction pathway, which leads to molecular and metabolic responses associated with frost hardening. Finally, the current options available to increase root frost tolerance are explored and promising lines of future research are discussed.

Identification of Callose Synthases in Stinging Nettle and Analysis of Their Expression in Different Tissues.

Callose is an important biopolymer of ß-1,3-linked glucose units involved in different phases of plant development, reproduction and response to external stimuli. It is synthesized by glycosyltransferases (GTs) known as callose synthases (CalS) belonging to family 48 in the Carbohydrate-Active enZymes (CAZymes) database. These GTs are anchored to the plasma membrane via transmembrane domains. Several genes encoding CalS have been characterized in higher plants with 12 reported in the model organism Arabidopsis thaliana. Recently, the de novo transcriptome of a fibre-producing clone of stinging nettle (Urtica dioica L.) was published and here it is mined for CalS genes with the aim of identifying members differentially expressed in the core and cortical tissues of the stem. The goal is to understand whether specific CalS genes are associated with distinct developmental stages of the stem internodes (elongation, thickening). Nine genes, eight of which encoding full-length CalS, are identified in stinging nettle. The phylogenetic analysis with CalS proteins from other fibre crops, namely textile hemp and flax, reveals grouping into 6 clades. The expression profiles in nettle tissues (roots, leaves, stem internodes sampled at different heights) reveal differences that are most noteworthy in roots vs leaves. Two CalS are differentially expressed in the internodes sampled at the top and middle of the stem. Implications of their role in nettle stem tissue development are discussed.

Does long-term cadmium exposure influence the composition of pectic polysaccharides in the cell wall of Medicago sativa stems?

BACKGROUND: The heavy metal cadmium (Cd) accumulates in the environment due to anthropogenic influences. It is unessential and harmful to all life forms. The plant cell wall forms a physical barrier against environmental stress and changes in the cell wall structure have been observed upon Cd exposure. In the current study, changes in the cell wall composition and structure of Medicago sativa stems were investigated after long-term exposure to Cd. Liquid chromatography coupled to mass spectrometry (LC-MS) for quantitative protein analysis was complemented with targeted gene expression analysis and combined with analyses of the cell wall composition. RESULTS: Several proteins determining for the cell wall structure changed in abundance. Structural changes mainly appeared in the composition of pectic polysaccharides and data indicate an increased presence of xylogalacturonan in response to Cd. Although a higher abundance and enzymatic activity of pectin methylesterase was detected, the total pectin methylation was not affected. CONCLUSIONS: An increased abundance of xylogalacturonan might hinder Cd binding in the cell wall due to the methylation of its galacturonic acid backbone. Probably, the exclusion of Cd from the cell wall and apoplast limits the entry of the heavy metal into the symplast and is an important factor during tolerance acquisition.

Distribution of cell-wall polysaccharides and proteins during growth of the hemp hypocotyl.

MAIN CONCLUSION: The immuno-ultrastructural investigation localized cell-wall polysaccharides of bast fibers during hemp hypocotyl growth. Moreover, for the first time, the localization of a peroxidase and laccase is provided in textile hemp. In the hypocotyl of textile hemp, elongation and girth increase are separated in time. This organ is therefore ideal for time-course analyses. Here, we follow the ultrastructural rearrangement of cell-wall components during the development of the hemp hypocotyl. An expression analysis of genes involved in the biosynthesis of cellulose, the chief polysaccharide of bast fiber cell walls and xylan, the main hemicellulose of secondary cell walls, is also provided. The analysis shows a higher expression of cellulose and xylan-related genes at 15 and 20 days after sowing, as compared to 9 days. In the young hypocotyl, the cell walls of bast fibers show cellulose microfibrils that are not yet compacted to form a mature G-layer. Crystalline cellulose is detected abundantly in the S1-layer, together with unsubstituted/low-substituted xylan and, to a lesser extent, in the G-layer. The LM5 galactan epitope is confined to the walls of parenchymatic cells. LM6-specific arabinans are detected at the interface between the cytoplasm and the gelatinous cell wall of bast fibers. The class III peroxidase antibody shows localization in the G-layer only at older developmental stages. The laccase antibody shows a distinctive labelling of the G-layer region closest to the S1-layer; the signal becomes more homogeneous as the hypocotyl matures. The data provide important insights on the cell wall distribution of polysaccharide and protein components in bast fibers during the hypocotyl growth of textile hemp.

Identification of Jasmonic Acid Biosynthetic Genes in Sweet Cherry and Expression Analysis in Four Ancient Varieties from Tuscany.

Sweet cherries are non-climacteric fruits whose early development is characterized by high levels of the phytohormone jasmonic acid (JA). Important parameters, such as firmness and susceptibility to cracking, can be affected by pre- and postharvest treatments of sweet cherries with JA. Despite the impact of JA on sweet cherry development and fruit characteristics, there are no studies (to the best of our knowledge) identifying the genes involved in the JA biosynthetic pathway in this species. We herein identify the sweet cherry members of the lipoxygenase family (13-LOX); allene oxide synthase, allene oxide cyclase and 12-oxo-phytodienoic acid reductase 3, as well as genes encoding the transcriptional master regulator MYC2. We analyze their expression pattern in four non-commercial Tuscan varieties ('Carlotta', 'Maggiola', 'Morellona', 'Crognola') having different levels of bioactives (namely phenolics). The highest differences are found in two genes encoding 13-LOX in the variety 'Maggiola' and one MYC2 isoform in 'Morellona'. No statistically-significant variations are instead present in the allene oxide synthase, allene oxide cyclase and 12-oxo-phytodienoic acid reductase 3. Our data pave the way to follow-up studies on the JA signaling pathway in these ancient varieties, for example in relation to development and post-harvest storage.

Impact of Nitrogen Nutrition on Cannabis sativa: An Update on the Current Knowledge and Future Prospects.

Nitrogen (N) availability represents one of the most critical factors affecting cultivated crops. N is indeed a crucial macronutrient influencing major aspects, from plant development to productivity and final yield of lignocellulosic biomass, as well as content of bioactive molecules. N metabolism is fundamental as it is at the crossroad between primary and secondary metabolic pathways: Besides affecting the synthesis of fundamental macromolecules, such as nucleic acids and proteins, N is needed for other types of molecules intervening in the response to exogenous stresses, e.g. alkaloids and glucosinolates. By partaking in the synthesis of phenylalanine, N also directly impacts a central plant metabolic 'hub'-the phenylpropanoid pathway-from which important classes of molecules are formed, notably monolignols, flavonoids and other types of polyphenols. In this review, an updated analysis is provided on the impact that N has on the multipurpose crop hemp (Cannabis sativa L.) due to its renewed interest as a multipurpose crop able to satisfy the needs of a bioeconomy. The hemp stalk provides both woody and cellulosic fibers used in construction and for biocomposites; different organs (leaves/flowers/roots) are sources of added-value secondary metabolites, namely cannabinoids, terpenes, flavonoids, and lignanamides. We survey the available literature data on the impact of N in hemp and highlight the importance of studying those genes responding to both N nutrition and abiotic stresses. Available hemp transcriptomic datasets obtained on plants subjected to salt and drought are here analyzed using Gene Ontology (GO) categories related to N metabolism. The ultimate goal is to shed light on interesting candidate genes that can be further studied in hemp varieties growing under different N feeding conditions and showing high biomass yield and secondary metabolite production, even under salinity and drought.

Selection of Appropriate Reference Genes for Gene Expression Analysis under Abiotic Stresses in Salix viminalis.

Salix viminalis is a fast growing willow species with potential as a plant used for biomass feedstock or for phytoremediation. However, few reference genes (RGs) for quantitative real-time polymerase chain reaction (qPCR) are available in S. viminalis, thereby limiting gene expression studies. Here, we investigated the expression stability of 14 candidate reference genes (RGs) across various organs exposed to five abiotic stresses (cold, heat, drought, salt, and poly-metals). Four RGs ranking algorithms, namely geNormPLUS, BestKeeper, NormFinder, and GrayNorm were applied to analyze the qPCR data and the outputs were merged into consensus lists with RankAggreg, a rank aggregation algorithm. In addition, the optimal RG combinations were determined with geNormPLUS and GrayNorm. The genes that were the most stable in the roots were TIP41 and CDC2. In the leaves, TIP41 was the most stable, followed by EF1b and ARI8, depending on the condition tested. Conversely, GAPDH and ß-TUB, two genes commonly used for qPCR data normalization were the least stable across all organs. Nevertheless, both geNormPLUS and GrayNorm recommended the use of a combination of genes rather than a single one. These results are valuable for research of transcriptomic responses in different S. viminalis organs.

On a Cold Night: Transcriptomics of Grapevine Flower Unveils Signal Transduction and Impacted Metabolism.

Low temperature is a critical environmental factor limiting plant productivity, especially in northern vineyards. To clarify the impact of this stress on grapevine flower, we used the Vitis array based on Roche-NimbleGen technology to investigate the gene expression of flowers submitted to a cold night. Our objectives were to identify modifications in the transcript levels after stress and during recovery. Consequently, our results confirmed some mechanisms known in grapes or other plants in response to cold stress, notably, (1) the pivotal role of calcium/calmodulin-mediated signaling; (2) the over-expression of sugar transporters and some genes involved in plant defense (especially in carbon metabolism), and (3) the down-regulation of genes encoding galactinol synthase (GOLS), pectate lyases, or polygalacturonases. We also identified some mechanisms not yet known to be involved in the response to cold stress, i.e., (1) the up-regulation of genes encoding G-type lectin S-receptor-like serine threonine-protein kinase, pathogen recognition receptor (PRR5), or heat-shock factors among others; (2) the down-regulation of Myeloblastosis (MYB)-related transcription factors and the Constans-like zinc finger family; and (3) the down-regulation of some genes encoding Pathogen-Related (PR)-proteins. Taken together, our results revealed interesting features and potentially valuable traits associated with stress responses in the grapevine flower. From a long-term perspective, our study provides useful starting points for future investigation.

Insights into Lignan Composition and Biosynthesis in Stinging Nettle (Urtica dioica L.).

Stinging nettle (Urtica dioica L.) has been used as herbal medicine to treat various ailments since ancient times. The biological activity of nettle is chiefly attributed to a large group of phenylpropanoid dimers, namely lignans. Despite the pharmacological importance of nettle lignans, there are no studies addressing lignan biosynthesis in this plant. We herein identified 14 genes encoding dirigent proteins (UdDIRs) and 3 pinoresinol-lariciresinol reductase genes (UdPLRs) in nettle, which are two gene families known to be associated with lignan biosynthesis. Expression profiling of these genes on different organs/tissues revealed a specific expression pattern. Particularly, UdDIR7, 12 and 13 displayed a remarkable high expression in the top internode, fibre tissues of bottom internodes and roots, respectively. The relatively high expression of UdPLR1 and UdPLR2 in the young internodes, core tissue of bottom internode and roots is consistent with the high accumulation of lariciresinol and secoisolariciresinol in these tissues. Lignan quantification showed a high abundance of pinoresinol in roots and pinoresinol diglucosides in young internodes and leaves. This study sheds light on lignan composition and biosynthesis in nettle, providing a good basis for further functional analysis of DIRs and PLRs and, ultimately, engineering lignan metabolism in planta and in cell cultures.

Tuscan Varieties of Sweet Cherry Are Rich Sources of Ursolic and Oleanolic Acid: Protein Modeling Coupled to Targeted Gene Expression and Metabolite Analyses.

The potential of six ancient Tuscan sweet cherry (Prunus avium L.) varieties as a source of health-promoting pentacyclic triterpenes is here evaluated by means of a targeted gene expression and metabolite analysis. By using a sequence homology criterion, we identify five oxidosqualene cyclase genes (OSCs) and three cytochrome P450s (CYP85s) that are putatively involved in the triterpene production pathway in sweet cherries. We performed 3D structure prediction and induced-fit docking using cation intermediates and reaction products for some OSCs to predict their function. We show that the Tuscan varieties have different amounts of ursolic and oleanolic acids and that these variations are related to different gene expression profiles. This study stresses the interest of valorizing ancient fruits as alternative sources of functional molecules with nutraceutical value. It also provides information on sweet cherry triterpene biosynthetic genes, which could be the object of follow-up functional studies.