FaMYB123 interacts with FabHLH3 to regulate the late steps of anthocyanin and flavonol biosynthesis during ripening.

In this work, we identified and functionally characterized the strawberry (Fragaria × ananassa) R2R3 MYB transcription factor FaMYB123. As in most genes associated with organoleptic properties of ripe fruit, FaMYB123 expression is ripening-related, receptacle-specific, and antagonistically regulated by ABA and auxin. Knockdown of FaMYB123 expression by RNAi in ripe strawberry fruit receptacles downregulated the expression of enzymes involved in the late steps of anthocyanin/flavonoid biosynthesis. Transgenic fruits showed a parallel decrease in the contents of total anthocyanin and flavonoid, especially malonyl derivatives of pelargonidin and cyanidins. The decrease was concomitant with accumulation of proanthocyanin, propelargonidins, and other condensed tannins associated mainly with green receptacles. Potential coregulation between FaMYB123 and FaMYB10, which may act on different sets of genes for the enzymes involved in anthocyanin production, was explored. FaMYB123 and FabHLH3 were found to interact and to be involved in the transcriptional activation of FaMT1, a gene responsible for the malonylation of anthocyanin components during ripening. Taken together, these results demonstrate that FaMYB123 regulates the late steps of the flavonoid pathway in a specific manner. In this study, a new function for an R2R3 MYB transcription factor, regulating the expression of a gene that encodes a malonyltransferase, has been elucidated.

Polyphenolic Extract (PE) from Olive Oil Exerts a Potent Immunomodulatory Effect and Prevents Graft-versus-Host Disease in a Mouse Model.

Polyphenols are a group of chemical substances found in plants, with immunomodulatory, antiproliferative, and anti-inflammatory properties that might be useful in the prophylaxis and treatment of graft-versus-host disease (GVHD). Polyphenolic extract (PE) obtained from extra virgin olive oil (EVOO) decreased the activation and proliferation of activated T cells. In addition, a decreased production of proinflammatory cytokines was observed upon exposure to PE. Western blot assays showed a marked inhibition of Akt phosphorylation and nuclear translocation of NF-κB in activated T cells. In a murine model of acute GVHD, we observed that mice that received a diet supplemented in PE (600 ppm) presented a higher survival rate and lower risk of developing GVHD when compared with the group that received a control diet. Histopathologic examination showed a significantly lower gut involvement in mice receiving PE, with a decrease in proinflammatory cytokines (IL-2, IL-17, and TNF-α) in serum and the reestablishment of butyrate concentration in the gut. In conclusion, PE obtained from EVOO exerted a potent immunomodulatory effect, reducing the activation and proliferation of activated T cells and the production of proinflammatory cytokines. In a murine model of acute GVHD, a PE-supplemented diet reduced the incidence and severity of the disease and increased survival after transplantation.

An atypical HLH transcriptional regulator plays a novel and important role in strawberry ripened receptacle.

BACKGROUND: In soft fruits, the differential expression of many genes during development and ripening is responsible for changing their organoleptic properties. In strawberry fruit, although some genes involved in the metabolic regulation of the ripening process have been functionally characterized, some of the most studied genes correspond to transcription factors. High throughput transcriptomics analyses performed in strawberry red receptacle (Fragaria x ananassa) allowed us to identify a ripening-related gene that codes an atypical HLH (FaPRE1) with high sequence homology with the PACLOBUTRAZOL RESISTANCE (PRE) genes. PRE genes are atypical bHLH proteins characterized by the lack of a DNA-binding domain and whose function has been linked to the regulation of cell elongation processes. RESULTS: FaPRE1 sequence analysis indicates that this gene belongs to the subfamily of atypical bHLHs that also includes ILI-1 from rice, SlPRE2 from tomato and AtPRE1 from Arabidopsis, which are involved in transcriptional regulatory processes as repressors, through the blockage by heterodimerization of bHLH transcription factors. FaPRE1 presented a transcriptional model characteristic of a ripening-related gene with receptacle-specific expression, being repressed by auxins and activated by abscisic acid (ABA). However, its expression was not affected by gibberellic acid (GA3). On the other hand, the transitory silencing of FaPRE1 transcription by agroinfiltration in receptacle produced the down-regulation of a group of genes related to the ripening process while inducing the transcription of genes involved in receptacle growth and development. CONCLUSIONS: In summary, this work presents for the first time experimental data that support an important novel function for the atypical HLH FaPRE1 during the strawberry fruit ripening. We hypothesize that FaPRE1 modulates antagonistically the transcription of genes related to both receptacle growth and ripening. Thus, FaPRE1 would repress the expression of receptacle growth promoting genes in the ripened receptacle, while it would activate the expression of those genes related to the receptacle ripening process.

Peptide Brush Polymers for Efficient Delivery of a Gene Editing Protein to Stem Cells.

The scarcity of effective means to deliver functional proteins to living cells is a central problem in biotechnology and medicine. Herein, we report the efficient delivery of an active DNA-modifying enzyme to human stem cells through high-density cell penetrating peptide brush polymers. Cre recombinase is mixed with a fluorophore-tagged polymer carrier and then applied directly to induced pluripotent stem cells or HEK293T cells. This results in efficient delivery of Cre protein as measured by activation of a genomically integrated Cre-mediated recombination reporter. We observed that brush polymer formulations utilizing cell penetrating peptides promoted Cre delivery but oligopeptides alone or oligopeptides displayed on nanoparticles did not. Overall, we report the efficient delivery of a genome-modifying enzyme to stem cells that may be generalizable to other, difficult-to-transduce cell types.

Amino Acid Degradations Produced by Lipid Oxidation Products.

Differently to amino acid degradations produced by carbohydrate-derived reactive carbonyls, amino acid degradations produced by lipid oxidation products are lesser known in spite of being lipid oxidation a major source of reactive carbonyls in food. This article analyzes the conversion of amino acids into Strecker aldehydes, α-keto acids, and amines produced by lipid-derived free radicals and carbonyl compounds, as well as the role of lipid oxidation products on the reactions suffered by these compounds: the formation of Strecker aldehydes and other aldehydes from α-keto acids; the formation of Strecker aldehydes and olefins from amines; the formation of shorter aldehydes from Strecker aldehydes; and the addition reactions suffered by the olefins produced from the amines. The relationships among all these reactions and the effect of reaction conditions on them are discussed. This knowledge should contribute to better control food processing in order to favor the formation of desirable beneficial compounds and to inhibit the production of compounds with deleterious properties.

Formation of phenazines, phenoxazines, and benzoxazoles in the browning reactions of o-quinones.

Quinone-induced browning is widely produced in foods and is mostly considered a consequence of quinone/nucleophile reactions. However, even in the absence of amino acids or proteins, o-quinones develop browning. In an attempt to better understand the reaction pathways involved in this browning development, this study describes the reactions of 4-methyl-1,2-benzoquinone with alcohols, ammonia, and short chain aldehydes. These reaction mixtures developed browning at 37 °C and the main produced compounds were isolated by semipreparative HPLC and characterized by NMR and MS as phenazines, phenoxazines, and benzoxazoles. A reaction pathway that explains the formation of all these compounds is proposed. The formation of phenazines is responsible, at least partially, for the produced browning, and the formation of benzoxazoles inhibits such browning. Browning development seems to be a consequence of a competition among the reactions of formation of phenazines, phenoxazines, and benzoxazoles, which appear to be produced from a single intermediate.

Suppressing the rhamnogalacturonan lyase gene FaRGLyase1 preserves RGI pectin degradation and enhances strawberry fruit firmness.

Plant rhamnogalacturonan lyases (RGLyases) cleave the backbone of rhamnogalacturonan I (RGI), the "hairy" pectin and polymer of the disaccharide rhamnose (Rha)-galacturonic acid (GalA) with arabinan, galactan or arabinogalactan side chains. It has been suggested that RGLyases could participate in remodeling cell walls during fruit softening, but clear evidence has not been reported. To investigate the role of RGLyases in strawberry softening, a genome-wide analysis of RGLyase genes in the genus Fragaria was performed. Seventeen genes encoding RGLyases with functional domains were identified in Fragaria × ananassa. FaRGLyase1 was the most expressed in the ripe receptacle of cv. Chandler. Transgenic strawberry plants expressing an RNAi sequence of FaRGLyase1 were obtained. Three transgenic lines yielded ripe fruits firmer than controls without other fruit quality parameters being significantly affected. The highest increase in firmness achieved was close to 32%. Cell walls were isolated from ripe fruits of two selected lines. The amount of water-soluble and chelated pectins was higher in transgenic lines than in the control. A carbohydrate microarray study showed a higher abundance of RGI epitopes in pectin fractions and in the cellulose-enriched fraction obtained from transgenic lines. Sixty-seven genes were differentially expressed in transgenic ripe fruits when compared with controls. These genes were involved in various physiological processes, including cell wall remodeling, ion homeostasis, lipid metabolism, protein degradation, stress response, and defense. The transcriptomic changes observed in FaRGLyase1 plants suggest that senescence was delayed in transgenic fruits.

The strawberry (Fragariaxananassa) fruit-specific rhamnogalacturonate lyase 1 (FaRGLyase1) gene encodes an enzyme involved in the degradation of cell-wall middle lamellae.

Pectins are essential components of primary plant cell walls and middle lamellae, and are related to the consistency of the fruit and its textural changes during ripening. In fact, strawberries become soft as the middle lamellae of cortical parenchyma cells are extensively degraded during ripening, leading to the observed short post-harvest shelf life. Using a custom-made oligonucleotide-based strawberry microarray platform, a putative rhamnogalacturonate lyase gene (FaRGlyase1) was identified. Bioinformatic analysis of the FaRGlyase1 sequence allowed the identification of a conserved rhamnogalacturonate lyase domain, which was also present in other putative RGlyase sequences deposited in the databases. Expression of FaRGlyase1 occurred mainly in the receptacle, concurrently with ripening, and it was positively regulated by abscisic acid and negatively by auxins. FaRGLyase1 gene expression was transiently silenced by injecting live Agrobacterium cells harbouring RNA interference constructs into fruit receptacles. Light and electron microscopy analyses of these transiently silenced fruits revealed that this gene is involved in the degradation of pectins present in the middle lamella region between parenchymatic cells. In addition, genetic linkage association analyses in a strawberry-segregating population showed that FaRGLyase1 is linked to a quantitative trait loci linkage group related to fruit hardness and firmness. The results showed that FaRGlyase1 could play an important role in the fruit ripening-related softening process that reduces strawberry firmness and post-harvest life.

Ketone-phenol reactions and the promotion of aromatizations by food phenolics.

Heating of either 3,5-heptadien-2-one or 2,6-heptanedione in the presence of ammonia produced 2,6-dimethylpyridine, and also 3-methylcyclohex-2-en-1-one for the second ketone. When phenolics were present, inhibition of pyridine formation was only observed in mixtures of 3,5-heptadien-2-one and resorcinol. This inhibition was due to the formation of ketone-resorcinol adducts, which were isolated and identified by nuclear magnetic resonance (NMR) and mass spectrometry (MS) as 2,4-dimethyl-5,6-dihydro-4H-2,6-methanobenzo[d][1,3]dioxocin-9-ol and 1-(7-hydroxy-4-methylchroman-2-yl)propan-2-one. The other assayed phenolics increased pyridine formation. This increase was mainly observed in the presence of oxygen, at slightly basic pH values, depended on time, temperature, and the phenolic concentration, and had an activation energy of 56.8 kJ/mol for the formation of 2,6-dimethylpyridine from 2,6-heptanedione in the presence of orcinol. This increase was a consequence of the promotion by phenolics of a required aromatization step in the pyridine formation pathway. This phenolic function needs to be considered when phenolics are added to food products.

Carbonyl-trapping by phenolics and the inhibition of the formation of carcinogenic heterocyclic aromatic amines with the structure of aminoimidazoazaarene in beef patties.

Carcinogenic heterocyclic aromatic amines (HAAs) with the structure of aminoimidazoazaarene (PhIP, MeIQx, IQ, and MeIQ) are produced by reaction of creatin(in)e, ammonia, and reactive carbonyls (phenylacetaldehyde, acrolein, and crotonaldehyde). In an attempt to provide efficient methodologies for HAA reduction in beef patties, this study: identified phloroglucinol as the most efficient phenolic to reduce HAA formation (76-96% inhibition); isolated and characterized by NMR and MS phloroglucinol/phenylcetaldehyde and phloroglucinol/acrolein adducts; and determined by LC-MS/MS adduct formation in beef patties treated with phloroglucinol. Obtained results suggested that addition of trihydroxyphenols (including phloroglucinol) to beef patties should decrease HAA formation. This was confirmed by both immersing beef patties in apple (or pear) juice before cooking (>90% inhibition) and including wheat bran in patty recipe. All these results confirm the key role of reactive carbonyls in the formation of carcinogenic HAAs and propose carbonyl-trapping as a way for controlling HAA formation in food products.

Malondialdehyde trapping by food phenolics.

The reactions between malondialdehyde and 2,5-dimethylresorcinol, orcinol, olivetol, and alkylresocinols were studied in an attempt to investigate both if this lipid oxidation product is trapped by phenolics analogously to other reactive carbonyls and to elucidate the chemical structures of the produced adducts. After being formed, malondialdehyde is both partially fractionated to acetaldehyde and oligomerized into dimers and trimers. All these compounds react with phenolics producing three main kinds of derivatives: 5(or 7)-alkyl-7(or 5)-hydroxy-4-methyl-4H-chromene-3-carbaldehydes, 7-alkyl-9-hydroxy-6H-2,6-methanobenzo[d][1,3]dioxocine-5-carbaldehydes, and 4-(3-formylphenyl)-7-hydroxy-4H-chromene-3-carbaldehydes. A total of twenty-four adducts were isolated by semipreparative high-performance liquid chromatography (HPLC) and characterized by mono- and bi-dimensional nuclear magnetic resonance (NMR) spectroscopy and mass spectrometry (MS). Reaction pathways to explain the formation of all these compounds are proposed. Obtained results show that phenolics can trap malondialdehyde producing stable derivatives. The function(s) that such derivatives can play in foods remain(s) to be elucidated.

Quality and Nutritional Changes of Traditional Cupcakes in the Processing and Storage as a Result of Sunflower Oil Replacements with Refined Olive Pomace Oil.

Recent nutritional studies have shown that the regular consumption of olive pomace oil (OPO) contributes to cardiovascular and cardiometabolic disease prevention. OPO could be a healthier alternative to the polyunsaturated oils employed in a number of bakery foods. However, little is known about the quality and nutritional changes of OPO in these products, especially the amounts of its bioactive components that finally reach consumers. The aim of this research was to evaluate refined OPO as a substitute for sunflower oil (SO) in cupcakes specially manufactured with a 6-month shelf-life. The influence of processing and storage on lipid oxidative changes and the levels of OPO bioactive components was studied. OPO samples exhibited much higher resistance to oxidative degradation in the processing and especially after storage, which had a greater oxidative impact. OPO reduced considerably the levels of oxidised lipids. HPLC analysis showed hydroperoxide triglyceride concentrations of 0.25 (±0.03) mmol/kg fat against 10.90 (±0.7) mmol/kg in the control containing SO. Sterols, triterpenic alcohols and triterpenic acids remained unchanged, and only slight losses of squalene (8 wt%) and α-tocopherol (13 wt%) were observed in OPO after processing and storage, respectively. Therefore, OPO preserved its nutritional properties and improved the quality and nutritional value of the cupcakes.

Carbonyl-trapping abilities of 5-alkylresorcinols.

In an attempt to investigate the carbonyl-trapping abilities of 5-alkylresorcinols, this study describes the role of these compounds in inhibiting the formation of the 2,5-dialkylpyridines (5-ethyl-2-methylpyridine, 5-butyl-2-propylpyridine, and 5-hexyl-2-pentylpyridine) produced by 2-alkenals (crotonaldehyde, 2-hexenal, and 2-octenal) in the presence of ammonia. 5-Alkylresorcinols (as well as orcinol and olivetol) inhibited the formation of pyridines to an extend that depended on the 2-alkenal involved and the reaction conditions. This inhibition was consequence of the trapping of 2-alkenals by the phenolics. Thus, the major adducts produced between the C21:0 alkylresorcinol and crotonaldehyde were isolated and characterized by nuclear magnetic resonance (NMR) and mass spectrometry (MS). These results confirm that, in addition to their free radical scavenging abilities, 5-alkylresorcinols also trap reactive carbonyls. Because trapped carbonyls are involved in the formation of flavors and processing-induced antioxidants, 5-alkylresorcinols might be implied in some of the observed differences between whole and refined grain products.

Carbonyl Chemistry and the Formation of Heterocyclic Aromatic Amines with the Structure of Aminoimidazoazaarene.

Recent studies have shown that the formation of heterocyclic aromatic amines (HAAs) with the structure of aminoimidazoazaarene is produced by reaction of specific reactive carbonyls with ammonia and creatin(in)e. These carbonyl compounds, which are usually the limiting reagents, have multiple origins. Therefore, HAA formation cannot be considered to be produced as a consequence of a single process, such as the Maillard reaction, but of any process that generates the involved reactive carbonyls. In addition, inhibition of HAA formation should be related to the control of these reactive carbonyls: inhibiting their formation, using conditions that limit their reactivity, and promoting their trapping.

Strecker aldehydes and α-keto acids, produced by carbonyl-amine reactions, contribute to the formation of acrylamide.

The formation and disappearance of acrylamide in binary and ternary mixtures of asparagine (or acrylamide), carbonyl compounds and amino acid derivatives was studied in an attempt to understand the different reactions produced in mixtures of carbonyl compounds and amino acids. The carbonyl compounds assayed included glucose, 2,4-decadienal, mercaptopyruvic acid, phenylpyruvic acid, and phenylacetaldehyde. The assayed amino acids were cysteine, N-acetylcysteine, and phenylalanine, in addition to asparagine. All assayed carbonyl compounds were able to convert asparagine into acrylamide to various extents, and some of them were more reactive than glucose. In addition, they inhibited the Michael additions responsible for acrylamide disappearance. On the other hand, the addition of amino acids mostly resulted in decreases of acrylamide, although acrylamide also increased in some mixtures. Amino acids decreased acrylamide yield because of both their competence with asparagine for carbonyl compounds and their reaction with the produced acrylamide. However, carbonyl-amine reactions formed new carbonyl compounds, which increased acrylamide content. Therefore, asparagine degradation in the presence of amino acids is likely to be a balance between the decrease of degradation produced by the original carbonyl compounds and the increase of degradation due to the carbonyl compounds formed.

Formation of naphthoquinones and anthraquinones by carbonyl-hydroquinone/benzoquinone reactions: A potential route for the origin of 9,10-anthraquinone in tea.

The reaction of 2-alkenals (crotonaldehyde and 2-pentenal) with hydroquinones (hydroquinone and tert-butylhydroquinone) and benzoquinones (benzoquinone, methylbenzoquinone, and methoxybenzoquinone) was studied as a potential route for the endogenous formation of naphthoquinones and anthraquinones in foods. Polycyclic quinones were produced at a low water activity, within a wide pH range, and in the presence of air. 9,10-Anthraquinone formation had an activation energy of 46.1 ± 0.1 kJ·mol-1, and a reaction pathway for the formation of the different naphthoquinones and anthraquinones is proposed. These reactions also took place in tea, therefore suggesting that the common tea pollutant 9,10-anthraquinone is also a process-induced contaminant. In fact, when four commercial teas (from a total of eight studied teas) were heated at 60 °C for 72 h, they significantly (p < 0.05) increased the amount of this toxicant. Reduction of 9,10-anthraquinone formation in teas is suggested to be carried out by reducing/scavenging its precursors.

Identification of acrolein as the reactive carbonyl responsible for the formation of 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx).

Reaction mixtures of reactive carbonyls and creatinine were submitted to high temperature and studied to identify the reactive carbonyl(s) responsible for 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx) formation. MeIQx was produced by reaction of acrolein and creatinine within a wide pH range and with an activation energy of 81.1 ± 1.4 kJ/mol. No additional reactants were required, although methylglyoxal, ammonia, and formaldehyde also participated in the reaction. Nevertheless, these additional reactants were produced in situ from either acrolein or creatinine. A reaction pathway that both explains the formation of MeIQx and is valid for the formation of other heterocyclic aromatic amines (HAAs) with the structure of quinoxaline is proposed. Obtained results demonstrate the key role of reactive carbonyls present in foods (the food carbonylome) on HAA formation. Because creatinine is ubiquitous in proteinaceous foods, the control of the food carbonylome seems to be the key point to control HAA formation in foods.

Why and How to Dig into Plant Metabolite-Protein Interactions.

Interaction between metabolites and proteins drives cellular regulatory processes within and between organisms. Recent reports highlight that numerous plant metabolites embrace multiple biological activities, beyond a sole role as substrates, products, or cofactors of enzymes, or as defense or growth-regulatory compounds. Though several technologies have been developed to identify and characterize metabolite-protein interactions, the systematic implementation of such methods in the plant field remains limited. Here, we discuss the plant metabolic space, with a specific focus on specialized metabolites and their roles, and review the technologies to study their interaction with proteins. We approach it both from a plant's perspective, to increase our understanding of plant metabolite-dependent regulatory networks, and from a human perspective, to empower agrochemical and drug discoveries.

Subfunctionalization of Paralog Transcription Factors Contributes to Regulation of Alkaloid Pathway Branch Choice in Catharanthus roseus.

Catharanthus roseus produces a diverse range of specialized metabolites of the monoterpenoid indole alkaloid (MIA) class in a heavily branched pathway. Recent great progress in identification of MIA biosynthesis genes revealed that the different pathway branch genes are expressed in a highly cell type- and organ-specific and stress-dependent manner. This implies a complex control by specific transcription factors (TFs), only partly revealed today. We generated and mined a comprehensive compendium of publicly available C. roseus transcriptome data for MIA pathway branch-specific TFs. Functional analysis was performed through extensive comparative gene expression analysis and profiling of over 40 MIA metabolites in the C. roseus flower petal expression system. We identified additional members of the known BIS and ORCA regulators. Further detailed study of the ORCA TFs suggests subfunctionalization of ORCA paralogs in terms of target gene-specific regulation and synergistic activity with the central jasmonate response regulator MYC2. Moreover, we identified specific amino acid residues within the ORCA DNA-binding domains that contribute to the differential regulation of some MIA pathway branches. Our results advance our understanding of TF paralog specificity for which, despite the common occurrence of closely related paralogs in many species, comparative studies are scarce.

The Intragenesis and Synthetic Biology Approach towards Accelerating Genetic Gains on Strawberry: Development of New Tools to Improve Fruit Quality and Resistance to Pathogens.

Under climate change, the spread of pests and pathogens into new environments has a dramatic effect on crop protection control. Strawberry (Fragaria spp.) is one the most profitable crops of the Rosaceae family worldwide, but more than 50 different genera of pathogens affect this species. Therefore, accelerating the improvement of fruit quality and pathogen resistance in strawberry represents an important objective for breeding and reducing the usage of pesticides. New genome sequencing data and bioinformatics tools has provided important resources to expand the use of synthetic biology-assisted intragenesis strategies as a powerful tool to accelerate genetic gains in strawberry. In this paper, we took advantage of these innovative approaches to create four RNAi intragenic silencing cassettes by combining specific strawberry new promoters and pathogen defense-related candidate DNA sequences to increase strawberry fruit quality and resistance by silencing their corresponding endogenous genes, mainly during fruit ripening stages, thus avoiding any unwanted effect on plant growth and development. Using a fruit transient assay, GUS expression was detected by the two synthetic FvAAT2 and FvDOF2 promoters, both by histochemical assay and qPCR analysis of GUS transcript levels, thus ensuring the ability of the same to drive the expression of the silencing cassettes in this strawberry tissue. The approaches described here represent valuable new tools for the rapid development of improved strawberry lines.