Impact of Heavy Metals on Cold Acclimation of Salix viminalis Roots.

In nature, plants are exposed to a range of climatic conditions. Those negatively impacting plant growth and survival are called abiotic stresses. Although abiotic stresses have been extensively studied separately, little is known about their interactions. Here, we investigate the impact of long-term mild metal exposure on the cold acclimation of Salix viminalis roots using physiological, transcriptomic, and proteomic approaches. We found that, while metal exposure significantly affected plant morphology and physiology, it did not impede cold acclimation. Cold acclimation alone increased glutathione content and glutathione reductase activity. It also resulted in the increase in transcripts and proteins belonging to the heat-shock proteins and related to the energy metabolism. Exposure to metals decreased antioxidant capacity but increased catalase and superoxide dismutase activity. It also resulted in the overexpression of transcripts and proteins related to metal homeostasis, protein folding, and the antioxidant machinery. The simultaneous exposure to both stressors resulted in effects that were not the simple addition of the effects of both stressors taken separately. At the antioxidant level, the response to both stressors was like the response to metals alone. While this should have led to a reduction of frost tolerance, this was not observed. The impact of the simultaneous exposure to metals and cold acclimation on the transcriptome was unique, while at the proteomic level the cold acclimation component seemed to be dominant. Some genes and proteins displayed positive interaction patterns. These genes and proteins were related to the mitigation and reparation of oxidative damage, sugar catabolism, and the production of lignans, trehalose, and raffinose. Interestingly, none of these genes and proteins belonged to the traditional ROS homeostasis system. These results highlight the importance of the under-studied role of lignans and the ROS damage repair and removal system in plants simultaneously exposed to multiple stressors.

Characterization of MdMYB68, a suberin master regulator in russeted apples.

Introduction: Apple russeting is mainly due to the accumulation of suberin in the cell wall in response to defects and damages in the cuticle layer. Over the last decades, massive efforts have been done to better understand the complex interplay between pathways involved in the suberization process in model plants. However, the regulation mechanisms which orchestrate this complex process are still under investigation. Our previous studies highlighted a number of transcription factor candidates from the Myeloblastosis (MYB) transcription factor family which might regulate suberization in russeted or suberized apple fruit skin. Among these, we identified MdMYB68, which was co-expressed with number of well-known key suberin biosynthesis genes. Method: To validate the MdMYB68 function, we conducted an heterologous transient expression in Nicotiana benthamiana combined with whole gene expression profiling analysis (RNA-Seq), quantification of lipids and cell wall monosaccharides, and microscopy. Results: MdMYB68 overexpression is able to trigger the expression of the whole suberin biosynthesis pathway. The lipid content analysis confirmed that MdMYB68 regulates the deposition of suberin in cell walls. Furthermore, we also investigated the alteration of the non-lipid cell wall components and showed that MdMYB68 triggers a massive modification of hemicelluloses and pectins. These results were finally supported by the microscopy. Discussion: Once again, we demonstrated that the heterologous transient expression in N. benthamiana coupled with RNA-seq is a powerful and efficient tool to investigate the function of suberin related transcription factors. Here, we suggest MdMYB68 as a new regulator of the aliphatic and aromatic suberin deposition in apple fruit, and further describe, for the first time, rearrangements occurring in the carbohydrate cell wall matrix, preparing this suberin deposition.

MdMYB52 regulates lignin biosynthesis upon the suberization process in apple.

Our previous studies, comparing russeted vs. waxy apple skin, highlighted a MYeloBlastosys (Myb) transcription factor (MdMYB52), which displayed a correlation with genes associated to the suberization process. The present article aims to assess its role and function in the suberization process. Phylogenetic analyses and research against Arabidopsis thaliana MYBs database were first performed and the tissue specific expression of MdMYB52 was investigated using RT-qPCR. The function of MdMYB52 was further investigated using Agrobacterium-mediated transient overexpression in Nicotiana benthamiana leaves. An RNA-Seq analysis was performed to highlight differentially regulated genes in response MdMYB52. Transcriptomic data were supported by analytical chemistry and microscopy. A massive decreased expression of photosynthetic and primary metabolism pathways was observed with a concomitant increased expression of genes associated with phenylpropanoid and lignin biosynthesis, cell wall modification and senescence. Interestingly key genes involved in the synthesis of suberin phenolic components were observed. The analytical chemistry displayed a strong increase in the lignin content in the cell walls during MdMYB52 expression. More specifically, an enrichment in G-Unit lignin residues was observed, supporting transcriptomic data as well as previous work describing the suberin phenolic domain as a G-unit enriched lignin-like polymer. The time-course qPCR analysis revealed that the observed stress response, might be explain by this lignin biosynthesis and by a possible programmed senescence triggered by MdMYB52. The present work supports a crucial regulatory role for MdMYB52 in the biosynthesis of the suberin phenolic domain and possibly in the fate of suberized cells in russeted apple skins.

The SoyaGen Project: Putting Genomics to Work for Soybean Breeders.

The SoyaGen project was a collaborative endeavor involving Canadian soybean researchers and breeders from academia and the private sector as well as international collaborators. Its aims were to develop genomics-derived solutions to real-world challenges faced by breeders. Based on the needs expressed by the stakeholders, the research efforts were focused on maximizing realized yield through optimization of maturity and improved disease resistance. The main deliverables related to molecular breeding in soybean will be reviewed here. These include: (1) SNP datasets capturing the genetic diversity within cultivated soybean (both within a worldwide collection of > 1,000 soybean accessions and a subset of 102 short-season accessions (MG0 and earlier) directly relevant to this group); (2) SNP markers for selecting favorable alleles at key maturity genes as well as loci associated with increased resistance to key pathogens and pests (Phytophthora sojae, Heterodera glycines, Sclerotinia sclerotiorum); (3) diagnostic tools to facilitate the identification and mapping of specific pathotypes of P. sojae; and (4) a genomic prediction approach to identify the most promising combinations of parents. As a result of this fruitful collaboration, breeders have gained new tools and approaches to implement molecular, genomics-informed breeding strategies. We believe these tools and approaches are broadly applicable to soybean breeding efforts around the world.

Identification of Novel Candidate Genes Involved in Apple Cuticle Integrity and Russeting-Associated Triterpene Synthesis Using Metabolomic, Proteomic, and Transcriptomic Data.

Apple russeting develops on the fruit surface when skin integrity has been lost. It induces a modification of fruit wax composition, including its triterpene profile. In the present work, we studied two closely related apple varieties, 'Reinette grise du Canada' and 'Reinette blanche du Canada', which display russeted and non-russeted skin phenotypes, respectively, during fruit development. To better understand the molecular events associated with russeting and the differential triterpene composition, metabolomics data were generated using liquid chromatography coupled to high-resolution mass spectrometry (LC-HRMS) and combined with proteomic and transcriptomic data. Our results indicated lower expression of genes linked to cuticle biosynthesis (cutin and wax) in russet apple throughout fruit development, along with an alteration of the specialized metabolism pathways, including triterpene and phenylpropanoid. We identified a lipid transfer protein (LTP3) as a novel player in cuticle formation, possibly involved in the transport of both cutin and wax components in apple skin. Metabolomic data highlighted for the first time a large diversity of triterpene-hydroxycinnamates in russeted tissues, accumulation of which was highly correlated with suberin-related genes, including some enzymes belonging to the BAHD (HXXXD-motif) acyltransferase family. Overall, this study increases our understanding about the crosstalk between triterpene and suberin pathways.

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.

The Cell Wall Proteome of Craterostigma plantagineum Cell Cultures Habituated to Dichlobenil and Isoxaben.

The remarkable desiccation tolerance of the vegetative tissues in the resurrection species Craterostigma plantagineum (Hochst.) is favored by its unique cell wall folding mechanism that allows the ordered and reversible shrinking of the cells without damaging neither the cell wall nor the underlying plasma membrane. The ability to withstand extreme drought is also maintained in abscisic acid pre-treated calli, which can be cultured both on solid and in liquid culture media. Cell wall research has greatly advanced, thanks to the use of inhibitors affecting the biosynthesis of e.g., cellulose, since they allowed the identification of the compensatory mechanisms underlying habituation. Considering the innate cell wall plasticity of C. plantagineum, the goal of this investigation was to understand whether habituation to the cellulose biosynthesis inhibitors dichlobenil and isoxaben entailed or not identical mechanisms as known for non-resurrection species and to decipher the cell wall proteome of habituated cells. The results showed that exposure of C. plantagineum calli/cells triggered abnormal phenotypes, as reported in non-resurrection species. Additionally, the data demonstrated that it was possible to habituate Craterostigma cells to dichlobenil and isoxaben and that gene expression and protein abundance did not follow the same trend. Shotgun and gel-based proteomics revealed a common set of proteins induced upon habituation, but also identified candidates solely induced by habituation to one of the two inhibitors. Finally, it is hypothesized that alterations in auxin levels are responsible for the increased abundance of cell wall-related proteins upon habituation.

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.

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.

Differential regulation of triterpene biosynthesis induced by an early failure in cuticle formation in apple.

Waxy apple cuticles predominantly accumulate ursane-type triterpenes, but the profile shifts with the induction of skin russeting towards lupane-type triterpenes. We previously characterised several key enzymes in the ursane-type and lupane-type triterpene pathways, but this switch in triterpene metabolism associated with loss of cuticle integrity is not fully understood. To analyse the relationship between triterpene biosynthesis and russeting, we used microscopy, RNA-sequencing and metabolite profiling during apple fruit development. We compared the skin of three genetically-close clones of 'Golden Delicious' (with waxy, partially russeted and fully russeted skin). We identified a unique molecular profile for the russet clone, including low transcript abundance of multiple cuticle-specific metabolic pathways in the early stages of fruit development. Using correlation analyses between gene transcription and metabolite concentration we found MYB transcription factors strongly associated with lupane-type triterpene biosynthesis. We showed how their transcription changed with the onset of cuticle cracking followed by russeting and that one factor, MYB66, was able to bind the promoter of the oxidosqualene cyclase OSC5, to drive the production of lupeol derivatives. These results provide insights into the breakdown of cuticle integrity leading to russet and how this drives MYB-regulated changes to triterpene biosynthesis.

Handling wine pomace: The importance of drying to preserve phenolic profile and antioxidant capacity for product valorization.

Four different wine grape pomaces (GP) (Vitis vinifera) varieties, Auxerrois, Pinot Blanc, Gamay and Pinot Noir, and obtained from white, rosé or red wine vinification, were considered for possible valorization in food supplement industry. Stabilization of GP by drying is paramount prior to further processing in the valorization chain, as GP might suffer spoilage over time. The objectives of this work were therefore to: evaluate the effect of microbiological spoilage and drying on the polyphenol profile and antioxidant capacity of GP; define a drying procedure by comparing kinetics of freeze-drying (FD) and vacuum oven (VO) (at 60 °C and 40 °C). Microbiological spoilage led to significant losses (P < 0.01) of antioxidant capacity (40% to 87%) and total phenolic content (70% to 90%), while drying had no significant effect. FD and VO at 60 °C drying kinetics exhibited similar drying curves, and a dry weight (DW) plateau was reached by 48 hr. In contrast VO at 40 °C required 170 hr to reach similar DW values, pointing out the importance of temperature when opting for VO technology. Antioxidant capacity of GP extracts did not differ between drying methods. Interestingly, GPs from white and rosé wines (AUX, PB, and GAM) had up to 3.5 times higher content (P < 0.001) of total polyphenols compared to PN, obtained from red wine. These results reinforce the importance of drying of GP as a pretreatment, which otherwise could result in significant product degradation. Additionally, we propose white and rosé GP as more interesting sources for valorization, with higher phenolic content, compared to red wine GP.

Molecular investigation of Tuscan sweet cherries sampled over three years: gene expression analysis coupled to metabolomics and proteomics.

Sweet cherry (Prunus avium L.) is a stone fruit widely consumed and appreciated for its organoleptic properties, as well as its nutraceutical potential. We here investigated the characteristics of six non-commercial Tuscan varieties of sweet cherry maintained at the Regional Germplasm Bank of the CNR-IBE in Follonica (Italy) and sampled ca. 60 days post-anthesis over three consecutive years (2016-2017-2018). We adopted an approach merging genotyping and targeted gene expression profiling with metabolomics. To complement the data, a study of the soluble proteomes was also performed on two varieties showing the highest content of flavonoids. Metabolomics identified the presence of flavanols and proanthocyanidins in highest abundance in the varieties Morellona and Crognola, while gene expression revealed that some differences were present in genes involved in the phenylpropanoid pathway during the 3 years and among the varieties. Finally, proteomics on Morellona and Crognola showed variations in proteins involved in stress response, primary metabolism and cell wall expansion. To the best of our knowledge, this is the first multi-pronged study focused on Tuscan sweet cherry varieties providing insights into the differential abundance of genes, proteins and metabolites.

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.

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.

De novo transcriptome assembly of textile hemp from datasets on hypocotyls and adult plants.

We here provide an updated de novo transcriptome of the hemp textile variety Santhica 27. The assembly was performed by merging the reads obtained previously on a time-series relative to the hypocotyl development and on bast fibers isolated from internodes of adult plants at different heights with those obtained from a newly performed transcriptome study on the hypocotyl in response to jasmonic acid treatment. More specifically, hypocotyls aged 15 days were treated with jasmonic acid and collected 3 and 5 days after the application of the plant growth regulator. RNA-Seq was then performed on the treated hypocotyls. The transcriptome reported here will be a useful resource for those scientists engaged in the study of bast fiber development, as well as cell wall biosynthesis in textile hemp. The transcriptome is also useful for molecular studies relative to the synthesis of secondary metabolites, such as phenolic compounds (e.g. flavonoids) and lignans/lignanamides.

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.

Cell wall composition and transcriptomics in stem tissues of stinging nettle (Urtica dioica L.): Spotlight on a neglected fibre crop.

Stinging nettle (Urtica dioica L.) produces silky cellulosic fibres, as well as bioactive molecules. To improve the knowledge on nettle and enhance its opportunities of exploitation, a draft transcriptome of the "clone 13" (a fibre clone) is here presented. The transcriptome of whole internodes sampled at the top and middle of the stem is then compared with the core and cortical tissues sampled at the bottom. Young internodes show an enrichment in genes involved in the biosynthesis of phytohormones (auxins and jasmonic acid) and secondary metabolites (flavonoids). The core of internodes collected at the bottom of the stem is enriched in genes partaking in different aspects of secondary cell wall formation (cellulose, hemicellulose, lignin biosynthesis), while the cortical tissues reveal the presence of a C starvation signal probably due to the UDP-glucose demand necessary for the thickening phase of bast fibres. Cell wall analysis indicates a difference in rhamnogalacturonan structure/composition of mature bast fibres, as evidenced by the higher levels of galactose measured, as well as the occurrence of more water-soluble pectins in elongating internodes. The targeted quantification of phenolics shows that the middle internode and the cortical tissues at the bottom have higher contents than top internodes. Ultrastructural analyses reveal the presence of a gelatinous layer in bast fibres with a lamellar structure. The data presented will be an important resource and reference for future molecular studies on a neglected fibre crop.

Water Deficit-Responsive QTLs for Cell Wall Degradability and Composition in Maize at Silage Stage.

The use of lignocellulosic biomass for animal feed or biorefinery requires the optimization of its degradability. Moreover, biomass crops need to be better adapted to the changing climate and in particular to periods of drought. Although the negative impact of water deficit on biomass yield has often been mentioned, its impact on biomass quality has only been recently reported in a few species. In the present study, we combined the mapping power of a maize recombinant inbred line population with robust near infrared spectroscopy predictive equations to track the response to water deficit of traits associated with biomass quality. The population was cultivated under two contrasted water regimes over 3 consecutive years in the south of France and harvested at silage stage. We showed that cell wall degradability and ß-O-4-linked H lignin subunits were increased in response to water deficit, while lignin and p-coumaric acid contents were reduced. A mixed linear model was fitted to map quantitative trait loci (QTLs) for agronomical and cell wall-related traits. These QTLs were categorized as "constitutive" (QTL with an effect whatever the irrigation condition) or "responsive" (QTL involved in the response to water deficit) QTLs. Fifteen clusters of QTLs encompassed more than two third of the 213 constitutive QTLs and 13 clusters encompassed more than 60% of the 149 responsive QTLs. Interestingly, we showed that only half of the responsive QTLs co-localized with constitutive and yield QTLs, suggesting that specific genetic factors support biomass quality response to water deficit. Overall, our results demonstrate that water deficit favors cell wall degradability and that breeding of varieties that reconcile improved drought-tolerance and biomass degradability is possible.

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.

Identification of the laccase-like multicopper oxidase gene family of sweet cherry (Prunus avium L.) and expression analysis in six ancient Tuscan varieties.

Laccase-like multicopper oxidases (LMCOs) are versatile enzymes used as biocatalysts performing the oxidation of different substrates of industrial relevance, with or without the intervention of a mediator. They have attracted a lot of interest for biotechnological applications in light of their eco-friendliness: they indeed oxidize the substrate(s) by coupling the four electron reduction of the final acceptor, molecular oxygen (O2), to water. Plant LMCOs represent a still poorly studied, important class of oxidoreductases controlling e.g. the post-harvest quality of fruits and enabling the tailoring of designer energy crops. We here sought to identify the LMCOs in Prunus avium L., whose fruits are rich in bioactive molecules, but are also highly perishable. The goal was to analyze them using bioinformatics (phylogenetic and in silico structural analyses) and to perform a targeted expression study on a subset of genes in six ancient varieties from Tuscany, all threatened by genetic erosion. These sweet cherry varieties contain higher amount of bioactive molecules, as compared to commercial counterparts. The results shown demonstrate strikingly different gene expression patterns in the six ancient varieties ('Benedetta', 'Carlotta', 'Crognola', 'Maggiola', 'Morellona', 'Moscatella') belonging to the Tuscan Regional Bank of Germplasm, as compared to a widely used commercial one ('Durone'). The motivation of this study is the economic importance of P. avium and the involvement of LMCOs in post-harvest fruit parameters, like color. The results presented pave the way to follow-up researches on LMCOs of sweet cherry exploring post-harvest fruit parameters (e.g. anthocyanin stability responsible for pericarp browning and the preservation of the appealing red color), as well as developmental processes, like stony pit formation.