Transcriptomic Analyses Reveal Insights into the Shared Regulatory Network of Phenolic Compounds and Steviol Glycosides in Stevia rebaudiana.

Stevia rebaudiana (Bertoni) is a highly valuable crop for the steviol glycoside content in its leaves, which are no-calorie sweeteners hundreds of times more potent than sucrose. The presence of health-promoting phenolic compounds, particularly flavonoids, in the leaf of S. rebaudiana adds further nutritional value to this crop. Although all these secondary metabolites are highly desirable in S. rebaudiana leaves, the genes regulating the biosynthesis of phenolic compounds and the shared gene network between the regulation of biosynthesis of steviol glycosides and phenolic compounds still need to be investigated in this species. To identify putative candidate genes involved in the synergistic regulation of steviol glycosides and phenolic compounds, four genotypes with different contents of these compounds were selected for a pairwise comparison RNA-seq analysis, yielding 1136 differentially expressed genes. Genes that highly correlate with both steviol glycosides and phenolic compound accumulation in the four genotypes of S. rebaudiana were identified using the weighted gene co-expression network analysis. The presence of UDP-glycosyltransferases 76G1, 76H1, 85C1, and 91A1, and several genes associated with the phenylpropanoid pathway, including peroxidase, caffeoyl-CoA O-methyltransferase, and malonyl-coenzyme A:anthocyanin 3-O-glucoside-6″-O-malonyltransferase, along with 21 transcription factors like SCL3, WRK11, and MYB111, implied an extensive and synergistic regulatory network involved in enhancing the production of such compounds in S. rebaudiana leaves. In conclusion, this work identified a variety of putative candidate genes involved in the biosynthesis and regulation of particular steviol glycosides and phenolic compounds that will be useful in gene editing strategies for increasing and steering the production of such compounds in S. rebaudiana as well as in other species.

Characterisation of LTR-Retrotransposons of Stevia rebaudiana and Their Use for the Analysis of Genetic Variability.

Stevia rebaudiana is one of the most important crops belonging to the Asteraceae family. Stevia is cultivated all over the world as it represents a valid natural alternative to artificial sweeteners thanks to its leaves, which produce steviol glycosides that have high sweetening power and reduced caloric value. In this work, the stevia genome sequence was used to isolate and characterise full-length long-terminal repeat retrotransposons (LTR-REs), which account for more than half of the genome. The Gypsy retrotransposons were twice as abundant as the Copia ones. A disproportionate abundance of elements belonging to the Chromovirus/Tekay lineage was observed among the Gypsy elements. Only the SIRE and Angela lineages represented significant portions of the genome among the Copia elements. The dynamics with which LTR-REs colonised the stevia genome were also estimated; all isolated full-length elements turned out to be relatively young, with a proliferation peak around 1-2 million years ago. However, a different analysis conducted by comparing sequences encoding retrotranscriptase showed the occurrence of an older period in which there was a lot of LTR-RE proliferation. Finally, a group of isolated full-length elements belonging to the lineage Angela was used to analyse the genetic variability in 25 accessions of S. rebaudiana using the Inter-Retrotransposon Amplified Polymorphism (IRAP) protocol. The obtained fingerprints highlighted a high degree of genetic variability and were used to study the genomic structures of the different accessions. It was hypothesised that there are four ancestral subpopulations at the root of the analysed accessions, which all turned out to be admixed. Overall, these data may be useful for genome sequence annotations and for evaluating genetic variability in this species, which may be useful in stevia breeding.

Protective effects of Stevia rebaudiana extracts on beta cells in lipotoxic conditions.

AIMS: Stevia rebaudiana Bertoni leaf extracts have gained increasing attention for their potential protection against type 2 diabetes. In this study, we have evaluated the possible beneficial effects of Stevia rebaudiana leaf extracts on beta-cells exposed to lipotoxicity and explored some of the possible mechanisms involved. METHODS: Extracts, deriving from six different chemotypes (ST1 to ST6), were characterized in terms of steviol glycosides, total phenols, flavonoids, and antioxidant activity. INS-1E beta cells and human pancreatic islets were incubated 24 h with 0.5 mM palmitate with or without varying concentrations of extracts. Beta-cell/islet cell features were analyzed by MTT assay, activated caspase 3/7 measurement, and/or nucleosome quantification. In addition, the proteome of INS-1E cells was assessed by bi-dimensional electrophoresis (2-DE). RESULTS: The extracts differed in terms of antioxidant activity and stevioside content. As expected, 24 h exposure to palmitate resulted in a significant decrease of INS-1E cell metabolic activity, which was counteracted by all the Stevia extracts at 200 µg/ml. However, varying stevioside only concentrations were not able to protect palmitate-exposed cells. ST3 extract was also tested with human islets, showing an anti-apoptotic effect. Proteome analysis showed several changes in INS-1E beta-cells exposed to ST3, mainly at the endoplasmic reticulum and mitochondrial levels. CONCLUSIONS: Stevia rebaudiana leaf extracts have beneficial effects on beta cells exposed to lipotoxicity; this effect does not seem to be mediated by stevioside alone (suggesting a major role of the leaf phytocomplex as a whole) and might be due to actions on the endoplasmic reticulum and the mitochondrion.

Flaxseed and Camelina Meals as Potential Sources of Health-Beneficial Compounds.

Seed meals and cakes, deriving from minor oilseed crops, represent interesting co-products for the presence of a high content of proteins and bioactive compounds that could be successfully explored as valuable plant-derived feedstocks for food and non-food purposes. In this contest, flaxseed (Linum usitatissimum L.) and camelina (Camelina sativa (L.) Crantz) are becoming increasingly important in the health food market as functional foods and cosmetic ingredients. Thus, this study aimed to evaluate the effect of genetic characteristics and cultivation sites on the chemical features of seed meals deriving from two flaxseed varieties (Sideral and Buenos Aires) and a camelina cultivar (Italia), cultivated in Central and Northern Italy (Pisa and Bologna). The content of total phenols and flavonoids, seed oil, proteins and fatty acids have been evaluated, together with the chemical profiles of flaxseed and camelina meals. In addition, radical-scavenging activity has been investigated. All the examined seed meals resulted as rich in bioactive compounds. In particular, flaxseed meal is a good source of the lignan secoisolariciresinol diglucoside (SDG) and hydroxycinnamic acid glucosides, while camelina meal contains glucosinolates and quercetin glycosides. Furthermore, all extracts exhibited a very strong radical-scavenging activity, that make these plant-derived products interesting sources for food or cosmetic ingredients with health outcomes.

Health-Promoting Compounds in Stevia: The Effect of Mycorrhizal Symbiosis, Phosphorus Supply and Harvest Time.

This work aimed to establish the synergic role of arbuscular mycorrhizal fungi (AMF) symbiosis, phosphorus (P) fertilization and harvest time on the contents of stevia secondary metabolites. Consequently, steviol glycosides (SVglys) concentration and profile, total phenols and flavonoids as well as antioxidant assays, have been assessed in inoculated and no-inoculated plants, grown with or without P supply and collected at different growth stages(69, 89 and 123 days after transplanting).The obtained results suggest that the synthesis of stevia secondary metabolites is induced and/or modulated by all the investigated variability factors. In particular, AMF symbiosis promoted total SVglys content and positively influenced the concentration of some minor compounds (steviolbioside, dulcoside A and rebaudioside B), indicating a clear effect of mycorrhizal inoculation on SVglys biosynthetic pathway. Interestingly, only the mycorrhizal plants were able to synthesize rebaudioside B. In addition, P supply provided the highest levels of total phenols and flavonoids at leaf level, together with the maximum in vitro antioxidant activities (FRAP and ORAC). Finally, the harvest time carried out during the full vegetative phase enhanced the entire composition of the phytocomplex (steviolbioside, dulcoside A, stevioside, rebaudioside A, B, C. total phenols and flavonoids). Moreover, polyphenols and SVglys appeared to be the main contributors to the in vitro antioxidant capacity, while only total phenols mostly contributed to the cellular antioxidant activity (CAA). These findings provide original information about the role played by AMF in association with P supply, in modulating the accumulation of bioactive compounds during stevia growth. At the cultivation level, the control of these preharvest factors, together with the most appropriate harvest time, can be used as tools for improving the nutraceutical value of raw material, with particular attention to its exploitation as functional ingredient for food and dietary supplements and cosmetics.

Flaxseed Cake as a Tool for the Improvement of Nutraceutical and Sensorial Features of Sourdough Bread.

Flaxseed has been recently studied for the formulation of healthy functional foods that are also useful for the prevention of chronic diseases. In this context, the production of sourdough bread fortified with different percentages of flaxseed cake was performed and the interactions among the bioactive compounds derived from both sourdough and flaxseed cake were investigated. The organoleptic properties as well as nutraceutical and chemical characteristics regarding pH, ethanol, lactic and acetic acid content, fatty acids profile, the concentration of total polyphenols, antioxidant capacity, and aroma volatile organic compounds were determined to evaluate the efficacy of leavening in the different matrices in comparison with the traditional bread. The results obtained demonstrated that flaxseed cake-enriched sourdough bread can represent a potential vehicle for bioactive compounds with the possibility of obtaining high-quality products with improved nutritional profiles and desired health attributes. Furthermore, the bread obtained with the addition of 7.5% of flaxseed cake was individuated as the best formulation to produce sourdough bread fortified with flaxseed cake by the overlap between three series of information coming from physical-chemical, nutritional, and sensorial analyses. In conclusion, in the operating conditions adopted, the use of flaxseed cake could represent a viable alternative for the production of fortified bread based on sourdough technology.

Evaluation of Chemical Composition of Two Linseed Varieties as Sources of Health-Beneficial Substances.

Linseed (Linum usitatissimum L.) is becoming more and more important in the health food market as a functional food, since its seeds and oil represent a rich source of bioactive compounds. Its chemical composition is strongly correlated with, and dependent on, genetic characteristics. The aim of this study was to evaluate the variation in seed yield, oil content, fatty acid composition and secondary metabolite profiles between a low-linolenic linseed variety, belonging to the Solin-type group (Solal), and a high-linolenic traditional one (Bethune), cultivated, both as spring crops, in open field conditions of Central Italy. The achieved results pointed out the different behavior of the two varieties in terms of growth cycle, oil content, and some important yield components, such as capsule number per plant and thousand seed weight. There were also significant differences in seed composition regarding total phenols, total flavonoids, antioxidant activities as well as in carotenoid, tocopherol, and tocotrienol profiles between the two varieties. In particular, Solal was characterized by the greatest contents of oil, phenols, flavonoids, α- and δ- tocotrienol, together with the highest antioxidant activity. Bethune, on the contrary, showed the highest amounts of carotenoids (lutein and ß-carotene). These results indicate a clear effect of the genetic characteristics on the biosynthesis of these secondary metabolites and, consequently, on the related antioxidant activity. Our findings suggest that the mutation process, responsible for the selection of the low-linolenic cultivar, is able to modify the biosynthetic pathways of carotenoids and phenolics.

Effect of Nitrogen Fertilization and Harvest Time on Steviol Glycosides, Flavonoid Composition, and Antioxidant Properties in Stevia rebaudiana Bertoni.

This work investigated the effect of nitrogen fertilization and harvest time on the flavonoid composition and antioxidant properties of Stevia rebaudiana leaves. At the same time, changes in stevioside (Stev) and rebaudioside A (RebA) contents were recorded. A pot trial under open air conditions was set up, testing five N rates and three harvest times. The results showed that, by using an adequate N rate and choosing an appropriate harvest time, it was possible to significantly increase and optimize the bioactive compound levels. In particular, higher RebA, RebA/Stev ratio, total phenols and flavonoids, luteolin-7-O-glucoside, and apigenin-7-O-glucoside levels and antioxidant capacity were recorded by supplying 150 kg N ha(-1). Reduced or increased N availability in comparison with N150 had no consistent effect on Stevia phytochemicals content. Significant correlations were also found between stevioside and some of the flavonoids, indicating a possible role of flavonoids in the stevioside metabolic pathway, which deserves more investigations.

Opposing effects of external gibberellin and Daminozide on Stevia growth and metabolites.

Steviol glycosides (SVglys) and gibberellins are originated from the shared biosynthesis pathway in Stevia (Stevia rebaudiana Bertoni). In this research, two experiments were conducted to study the opposing effects of external gibberellin (GA3) and Daminozide (a gibberellin inhibitor) on Stevia growth and metabolites. Results showed that GA3 significantly increased the stem length and stem dry weight in Stevia. Total soluble sugar content increased while the SVglys biosynthesis was decreased by external GA3 applying in Stevia leaves. In another experiment, the stem length was reduced by Daminozide spraying on Stevia shoots. The Daminozide did not affect the total SVglys content, while in 30 ppm concentration, significantly increased the soluble sugar production in Stevia leaves. Although the gibberellins biosynthesis pathway has previously invigorated in Stevia leaf, the Stevia response to external gibberellins implying on high precision regulation of gibberellins biosynthesis in Stevia and announces that Stevia is able to kept endogenous gibberellins in a low quantity away from SVglys production. Moreover, the assumption that the internal gibberellins were destroyed by Daminozide, lack of Daminozide effects on SVglys production suggests that gibberellins biosynthesis could not act as a competitive factor for SVglys production in Stevia leaves.

Stevia rebaudiana Bertoni as a source of bioactive compounds: the effect of harvest time, experimental site and crop age on steviol glycoside content and antioxidant properties.

BACKGROUND: This study was aimed at identifying the effect of harvest time, experimental site and crop age on the no-calorie sweetener steviol glycosides (SG) and on the antioxidant properties of stevia leaf extracts. The experiment was conducted over two growing seasons at two sites in the northeastern plain of Italy. RESULTS: The results showed that all analysed factors played an important role in defining the SG profile and the antioxidant properties of stevia extracts. A high level of phenols (78.24 mg GAE g⁻¹ DW by Folin-Ciocalteu method) and high antioxidant activity (812.6 µmol Fe²âº g⁻¹ DW by FRAP assay) were observed. The inhibition of DPPH free radicals was evaluated and an IC50 mean value of 250 µg mL⁻¹ was obtained. Significant relationships among the total antioxidant capacity and the analysed compounds were found. CONCLUSION: The results showed the possibility of obtaining, in the tested environments, very high SG yields thanks to the long-day conditions during the spring/summer season. The harvest time played a key role in determining the stevia quality, influencing the rebaudioside A/stevioside ratio. The strong antioxidant properties make very interesting the possibility of using stevia extracts to improve functional food properties.

Light quality influences indigo precursors production and seed germination in Isatis tinctoria L. and Isatis indigotica Fort.

Isatis tinctoria L. and Isatis indigotica Fort. are biennial herbaceous plants belonging to the family of Cruciferae that are used as a source of natural indigo and show several morphological and genetic differences. Production of indigo (indigotin) precursors, indican (indoxyl beta-D glucoside) and isatan B (indoxyl ketogluconate), together with seed germination ability were compared in Isatis tinctoria and Isatis indigotica grown under six different light conditions (darkness, white, red, far red, blue, yellow light) at 25 degrees C. Light quality influenced both germination and production of indigo precursors in the two Isatis species. Different responsiveness to far red and blue light was observed. Indeed, a detrimental effect on germination by blue and far red light was found in I. tinctoria only. Different amounts of isatan B were produced under red and far red light in the two Isatis species. In I. tinctoria, the level of main indigo precursor isatan B was maximal under red light and minimal under far red light. Whereas in I. indigotica far red light promoted a large accumulation of isatan B. The photon fluence rate dependency for white and yellow light responses showed that the accumulation of indigo precursors was differently influenced in the two Isatis species. In particular, both white and yellow light enhanced above 40 micromol m(-2) s(-1) the production of isatan B in I. indigotica while only white light showed a photon fluence dependency in I. tinctoria. These results suggest a different role played by the labile and stable phytochrome species (phyA and phyB) in the isatan B production in I. tinctoria and I. indigotica. I. indigotica, whose germination percentage was not influenced by light quality, demonstrated higher germination capability compared with I. tinctoria. In fact, I. tinctoria showed high frequency of germination in darkness and under light sources that establish high phytochrome photoequilibrium (red, white and yellow light). Germination in I. tinctoria was negatively affected by far red and blue light. I. indigotica seeds appear to be indifferent to canopy-like light (far red). Our results provide further insights on the distinct behaviour of I. tinctoria and I. indigotica that belong to two different genetic clusters and different original environments.

Environmental factors affecting productivity, indican content, and indigo yield in Polygonum tinctorium Ait., a subtropical crop grown under temperate conditions.

Polygonum tinctorium Ait. is a herbaceous subtropical annual plant, belonging to the family Polygonaceae. Within the cells of its leaves P. tinctorium accumulates large amounts of a colorless glycoside, indican (indoxyl beta-d-glucoside), from which the blue dye indigo is synthesized. P. tinctorium is well-known in Japan, where it had been cultivated to produce natural indigo for textile dyeing, whereas it represents a potentially interesting new crop in Europe. To better understand the effects of environmental parameters on P. tinctorium crop production and indigo yield, field experiments were carried out in central Italy under temperate climate. Three lines were tested during the 2001 and 2002 growing seasons, and plant/leaf yields as well as indican contents were evaluated. The results showed that P. tinctorium grown in temperate climate conditions can be harvested three times a year. Yields of 82 and 120 t ha(-1) of fresh plant yield were obtained in 2001 and 2002, respectively. The contrasting weather conditions between the two years significantly affected biomass production, which was higher in the 2002 season, characterized by wet weather conditions. The cycle length from sowing to the last harvest was accomplished in 229-238 days when plants had accumulated 2017-2018 degrees C. Green leaves accounted for 40-45% by weight of fresh plant tissue and contained 11-20 g kg(-1) indican. The three lines did not significantly differ in the main productive parameters or in fresh leaf indican content (14.1 g kg(-1) mean value). Photosynthetic active radiation influences indican leaf production according to the model y = 0.0004x + 8.566 (P < 0.01, correlation coefficient = 0.818). Indican content ranged from 12 to 25 g kg(-1) of fresh leaves with PAR daily values from 10000 to 40000 mEinstein m(-2) (recorded in May and at the end of July-beginning of August, respectively). The results indicate that in nonlimiting rainfall conditions a very high indican content and a potentially high indigo yield can be obtained by cultivating P. tinctorium in this pioneer geographical area.

A new HPLC-ELSD method to quantify indican in Polygonum tinctorium L. and to evaluate beta-glucosidase hydrolysis of indican for indigo production.

A method to quantify the indigo precursor indican (indoxyl-beta-D-glucoside) in Polygonum tinctorium L. has been developed. Plant material was extracted in deionized water, and indican was identified and quantified using high performance liquid chromatography (HPLC) coupled to an evaporative light scattering detector (ELSD). Results confirmed that with this method it is possible to measure indican content in a short time, obtaining reliable and reproducible data. Using this method, leaf indican content was quantified every 15 days during the growing season (from May to October) in P. tinctorium crops grown in a field experiment in Central Italy. Results showed that indican increased along the growing season until flowering and was positively affected by photosynthetic active radiation (PAR). Indican is naturally hydrolyzed by native beta-glucosidase to indoxyl and glucose, the indoxyl yielding indigo. The activity of two enzymes, sweet almond beta-glucosidase and Novarom G preparation, were compared with P. tinctorium native beta-glucosidase to evaluate indigo production. Results showed that the ability to promote indigo formation increased as follows: almond beta-glucosidase

Essential oils from Mediterranean lamiaceae as weed germination inhibitors.

The essential oils obtained from rosemary (Rosmarinus officinalis L.), thyme (Thymus vulgaris L.), and savory (Satureja montana L.) and the four monoterpenes that are their major constituents have been analyzed by GC and GC-MS and tested for their allelopathic properties on the seeds of three different annual weeds (Chenopodium album, Portulaca oleracea, and Echinochloa crus-galli) and three crops (Raphanus sativus, Capsicum annuum, and Lactuca sativa), with the aim to evaluate in vitro their potential as germination inhibitors. The essential oil composition varied with the species, thymol being the main constituent (44%) of thyme and carvacrol (57%) that of savory oil. Differences in essential oil composition were observed within two different rosemary ecotypes, type A, with alpha-pinene (37%) and 1,8-cineole (23%), and type B, characterized by a 2-fold content of 1,8-cineole (47%). This latest essential oil inhibited completely the germination of weeds while concurrently displaying little effect on pepper. The other two oils showed less selective action. S. montana essential oil, with 57% carvacrol, is the most active compound, completely inhibiting germination both of crops and weeds. Borneol, one of the main constituents of the oil of rosemary type B, showed an activity comparable to that of the whole oil. Crop and weed seeds treated with 1,8-cineole showed germination values that were not significantly different from controls, even if a slowing of the germination process expressed in terms of a significant increase in mean germination time was observed. Monoterpene compounds also present in the essential oils mainly represented the volatile fraction released from the crops and their residues into the soil.