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.

Effect of the dietary inclusion of Camelina sativa cake into quail diet on live performance, carcass traits, and meat quality.

The present research studied the effect of the dietary inclusion of 3 different camelina (Camelina sativa (L.) Crantz) cakes on the live performance, slaughter traits, and breast meat quality of broiler quails (Coturnix japonica). With this purpose, a total of 480 fifteen-day-old broiler quails of both sexes were allocated to 48 cages (12 replicates/treatment, 10 quails/replicate) and received 4 dietary treatments: a control diet (Control), and 3 diets containing 15% of 1 commercial cultivar (Calena), and 2 improved lines (Pearl: low linoleic acid; Alan: low glucosinolates). During the experiment, individual live weight (LW) and cage feed intake were recorded to calculate body weight gain (BWG) and feed conversion ratio (FCR). At 35 d of age, quails were slaughtered, and carcasses were weighed and dissected to compute yields. On breast (pectoralis major muscle) the measurements and analyses considered ultimate pH, L*a*b* color values, proximate composition, oxidative status, cooking loss and WBSF toughness. Broiler quails receiving diets with camelina cakes exhibited mortality and health status similar to the control. However, growth performance was impaired in camelina-fed groups, especially for Calena (P < 0.05). Overall LW and BWG were slightly lower, whereas feed intake was slightly higher in camelina-fed groups compared to Control (P > 0.05). Therefore, higher FCR was recorded for camelina-fed groups compared to Control (P = 0.0004). Moreover, breast meat from Calena treatment displayed higher water (P = 0.0170), and lower lipid (P = 0.0051) contents compared to those of the Control group, while protein and ash content remained unaffected. Heme-iron content and oxidative status of breast meat were not influenced by the dietary incorporation of camelina (P > 0.05). The research outcomes indicated that camelina cakes can be used as an alternative feed ingredient for broiler quails' diets, without compromising carcass yields and meat quality. However, as 15% dietary incorporation worsened live performance, the ideal camelina cake inclusion level should be thoroughly investigated as well as a parallel research effort into further reducing glucosinolates content of camelina.

Valorization of an Old Variety of Triticum aestivum: A Study of Its Suitability for Breadmaking Focusing on Sensory and Nutritional Quality.

"Avanzi 3-Grano 23" (G23) is an old variety of Triticum aestivum from the mountain areas of Lunigiana (north Tuscany, Italy), where traditional farming communities have contributed to its success and on-farm conservation. G23 flour, traditionally used for typical food products, is characterized by particular nutritional and sensory traits but has technological properties which limit its suitability for breadmaking. The aim of this work was to evaluate how to promote the use of G23 through the optimization of bread formulation by leveraging both flour blending and the leavening system. During the preliminary test, three different mixes of G23 flour and a strong flour (C) were tested in terms of their leavening power as a function of leavening agent (baker's yeast or sourdough). The selected M2 flour, composed of G23:C (1:1 w/w), was used for further breadmaking trials and 100% C flour was utilized as a control. The sourdough bread obtained with the M2 flour (SB-M2) showed an improved sensory profile compared with the related control (SB-C). Furthermore, SB-M2 exhibited the best aromatic (high content in aldehydes, pyrazines and carboxylic acids) and phytochemical profile (total polyphenols and flavonoids content and antioxidant activity). In contrast, the use of baker's yeast, although optimal from the point of view of breadmaking, did not result in the same levels of aromatic complexity because it tends to standardize the product without valorizing the sensory and nutritional qualities of the flour. In conclusion, in the experimental conditions adopted, this old wheat variety appears to be suitable for the production of sourdough bakery products.

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.

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.

GC-MS and HS-SPME-GC×GC-TOFMS Determination of the Volatile Composition of Essential Oils and Hydrosols (By-Products) from Four Eucalyptus Species Cultivated in Tuscany.

Essential oils are widely used as functional ingredients for potential multi-purpose functional uses. Hydrosols, co-products of the distillation of plant material, are used in food and cosmetic industries and in biological agriculture, but their volatile composition is poorly investigated. The volatile fractions of essential oils and hydrosols from four less-studied 1,8-cineol-rich Eucalyptus species (E. parvula L.A.S. Johnson & K.D. Hill, E. cinerea F. Muell, E. pulverulenta Sims and E. pulverulenta baby blue Sims), cultivated in Tuscany in a system of organic farming, were characterized by solvent dilution (essential oils) or extraction (hydrosols) followed by GC-MS and by HS-SPME-GC×GC-TOFMS analysis. GC-MS analysis showed that essential oils were mainly constituted by oxygenated monoterpenes, particularly 1,8-cineole, with monoterpenes hydrocarbons up to 10.8%. Relative differences in the abundance of minor terpenes as limonene, α-pinene, γ-terpinene, p-cymene, terpinen-4-ol, α-terpineol, and alloaromandrene were pointed out and seem to be suitable for differentiation among EOs of the four different Eucalyptus species. Hydrosols of these species were characterized for the first time: they were mainly constituted by oxygenated monoterpenes (97.6⁻98.9%), with 1,8-cineole up to 1.6 g/L, while monoterpene and sesquiterpene hydrocarbons were detected only in traces. HS-SPME-GC×GC-TOFMS analysis also allowed providing metabolic profiling of hydrosols for the direct comparison and visualization of volatile components, pointing out the potentially different uses of these products as functional ingredients in food, beverage, and cosmetic industries.