Plant age-dependent dynamics of annatto pigment (bixin) biosynthesis in Bixa orellana.

Age affects the production of secondary metabolites, but how developmental cues regulate secondary metabolism remains poorly understood. The achiote tree (Bixa orellana L.) is a source of bixin, an apocarotenoid used in diverse industries worldwide. Understanding how age-dependent mechanisms control bixin biosynthesis is of great interest for plant biology and for economic reasons. Here we overexpressed miRNA156 (miR156) in B. orellana to comprehensively study the effects of the miR156-SQUAMOSA PROMOTER BINDING PROTEIN-LIKE (SPL) module on age-dependent bixin biosynthesis in leaves. Overexpression of miR156 in annatto plants (miR156ox) reduced BoSPL transcript levels, impacted leaf ontogeny, lessened bixin production, and increased abscisic acid levels. Modulation of expression of BoCCD4-4 and BoCCD1, key genes in carotenoid biosynthesis, was associated with diverting the carbon flux from bixin to abscisic acid in miR156ox leaves. Proteomic analyses revealed an overall low accumulation of most secondary metabolite-related enzymes in miR156ox leaves, suggesting that miR156-targeted BoSPLs may be required to activate several secondary metabolic pathways. Our findings suggest that the conserved BomiR156-BoSPL module is deployed to regulate leaf dynamics of bixin biosynthesis, and may create novel opportunities to fine-tune bixin output in B. orellana breeding programs.

Functional Fruit Trees from the Atlantic and Amazon Forests: Selection of Potential Chestnut Trees Rich in Antioxidants, Nutrients, and Fatty Acids.

The Amazon rainforest and the biodiversity hotspot of the Atlantic Forest are home to fruit trees that produce functional foods, which are still underutilized. The present study aimed to select potential functional nut donor trees from two Brazilian chestnuts, by evaluating the nutritional and antioxidant composition of the nuts and the fatty acid profile of the oil. The nutritional characteristics, antioxidants, oil fatty acid profile, and X-ray densitometry of the nuts were evaluated, as well as the characterization of leaf and soil nutrients for each parent tree. The nut oil was evaluated through Brix (%), mass (g), yield (%), and the fatty acid profile. For L. pisonis, the most nutritious nuts were produced by L. pisonis tree 4 (N > P > K > Mg > Ca > Zn > Fe) and L. pisonis tree 6 (P > Ca > Mg > Mn > Zn > Cu > Fe), and for the species L. lanceolata, L. lanceolata tree 6 (N > P > Ca > Mg > Zn > Fe > Cu) and L. lanceolata tree 2 (P > K > Mg > Zn > Cu). In L. pisonis, the highest production of anthocyanins, DPPH, total phenolics, and flavonoids was obtained from the nuts of L. pisonis tree 4 as well as for L. lanceolata, from L. lanceolata tree 1, except for flavonoids. The Brix of the oil from the nuts of both species showed no difference between the trees and the fatty acid profile with a similar amount between saturated (48-65%) and unsaturated (34-57%) fatty acids. Both species have nuts rich in nutrients and antioxidant compounds and can be considered unconventional functional foods. The data collected in the present study confirm that the nuts of these species can replace other foods as a source of selenium.

Physiological, epigenetic, and proteomic responses in Pfaffia glomerata growth in vitro under salt stress and 5-azacytidine.

Plants adjust their complex molecular, biochemical, and metabolic processes to overcome salt stress. Here, we investigated the proteomic and epigenetic alterations involved in the morphophysiological responses of Pfaffia glomerata, a medicinal plant, to salt stress and the demethylating agent 5-azacytidine (5-azaC). Moreover, we investigated how these changes affected the biosynthesis of 20-hydroxyecdysone (20-E), a pharmacologically important specialized metabolite. Plants were cultivated in vitro for 40 days in Murashige and Skoog medium supplemented with NaCl (50 mM), 5-azaC (25 µM), and NaCl + 5-azaC. Compared with the control (medium only), the treatments reduced growth, photosynthetic rates, and photosynthetic pigment content, with increase in sucrose, total amino acids, and proline contents, but a reduction in starch and protein. Comparative proteomic analysis revealed 282 common differentially accumulated proteins involved in 87 metabolic pathways, most of them related to amino acid and carbohydrate metabolism, and specialized metabolism. 5-azaC and NaCl + 5-azaC lowered global DNA methylation levels and 20-E content, suggesting that 20-E biosynthesis may be regulated by epigenetic mechanisms. Moreover, downregulation of a key protein in jasmonate biosynthesis indicates the fundamental role of this hormone in the 20-E biosynthesis. Taken together, our results highlight possible regulatory proteins and epigenetic changes related to salt stress tolerance and 20-E biosynthesis in P. glomerata, paving the way for future studies of the mechanisms involved in this regulation.

A 20-hydroxyecdysone-enriched fraction from Pfaffia glomerata (Spreng.) pedersen roots alleviates stress, anxiety, and depression in mice.

ETHNOPHARMACOLOGICAL RELEVANCE: Pfaffia glomerata roots are widely used in Brazil to treat various pathological conditions, particularly psychological disorders. 20-hydroxyecdysone, a phytosteroid present in the plant, can promote greater body resistance against exogenous and endogenous stressors. The objective of this study was to evaluate the possible neuroprotective effect of a 20-hydroxyecdysone-enriched fraction (20E-EF), obtained from P. glomerata roots, in an acute murine stress model. MATERIAL AND METHODS: The 20E-EF was obtained by partitioning the methanol extract from P. glomerata roots with dichloromethane. Mice were treated by gavage with three doses of 20E-EF (3, 10, and 30 mg/kg) and parameters of stress, anxiety, and depression were evaluated. Biomarkers of oxidative stress (enzymes, antioxidant profile, and oxidized molecules) were evaluated in the cortex, striatum (basal ganglia), and hippocampus of animals treated with 30 mg/kg of 20E-EF. RESULTS: Mass spectrometry revealed that 20E was the main compound in the dichloromethane fraction. At a dose of 30 mg/kg, 20E-EF reduced stress, anxiety, and depression, while stimulating antioxidant enzymes (catalase, superoxide dismutase, and glutathione peroxidase), promoting antioxidant activity (antioxidant capacity, sulfhydryl groups, and reduced glutathione), and reducing oxidative markers (lipid peroxidation). In addition, 20E increased the concentration of NO in the striatum, possibly improving memory function and antioxidant activity. CONCLUSION: A 30 mg/kg dose of 20E-EF was able to reduce stress, anxiety, and depression, in addition to maintaining antioxidant defenses of the cortex and striatum. These findings open new perspectives for understanding the therapeutic properties of P. glomerata and the underlying mechanism(s).

Irradiance-driven 20-hydroxyecdysone production and morphophysiological changes in Pfaffia glomerata plants grown in vitro.

Pfaffia glomerata possesses potential pharmacological and medicinal properties, mainly owing to the secondary metabolite 20-hydroxyecdysone (20E). Increasing production of biomass and 20E is important for industrial purposes. This study aimed to evaluate the influence of irradiance on plant morphology and production of 20E in P. glomerata grown in vitro. Nodal segments of accessions 22 and 43 (Ac22 and Ac43) were inoculated in culture medium containing MS salts and vitamins. Cultures were maintained at 25 ± 2 °C under a 16-h photoperiod and subjected to irradiance treatments of 65, 130, and 200 µmol m-2 s-1 by fluorescent lamps. After 30 days, growth parameters, pigment content, stomatal density, in vitro photosynthesis, metabolites content, and morphoanatomy were assessed. Notably, Ac22 plants exhibited 10-fold higher 20E production when cultivated at 200 µmol m-2 s-1 than at 65 µmol m-2 s-1, evidencing the importance of light quantity for the accumulation of this metabolite. 20E production was twice as high in Ac22 as in Ac43 plants although both accessions responded positively to higher irradiance. Growth under 200 µmol m-2 s-1 stimulated photosynthesis and consequent biomass accumulation, but lowered carotenoids and anthocyanins. Furthermore, increasing irradiance enhanced the number of palisade and spongy parenchyma cells, enhancing the overall growth of P. glomerata. Graphical abstract.

Pfaffia glomerata hydroalcoholic extract stimulates penile tissue in adult Swiss mice.

ETHNOPHARMACOLOGICAL RELEVANCE: Roots of Pfaffia glomerata are used in traditional medicine as aphrodisiacs and sexual stimulants. AIM OF THE STUDY: The aim of this study was to evaluate the action of the hydroalcoholic extract from the roots of Pfaffia glomerata on the Leydig cells, cavernous bodies and other penile constituents, as well as on serum testosterone and 17ß-estradiol levels of adult mice. MATERIALS AND METHODS: Mature male Swiss mice were divided into 6 groups: control (water), sildenafil citrate, 3 groups receiving daily doses of P. glomerata extract (100, 200 and 400 mg/kg) and one group receiving intermittent doses of P. glomerata (200 mg/kg/3-3d). RESULTS: The proportions of blood vessels, lymphatic space and estradiol levels were increased. On the other hand, reduction of testosterone levels due to Leydig cells death was observed. As for penile parameters, volumetric proportions of cavernous bodies, collagen and nitric oxide were increased, while smooth muscle content was decreased. CONCLUSIONS: Despite that the long term intake of P. glomerata extract was related to a stimulant action, reduction on Leydig cell viability induced decreased testosterone production.

Blue and red light affects morphogenesis and 20-hydroxyecdisone content of in vitro Pfaffia glomerata accessions.

The combination of different colors from light-emitting diodes (LEDs) may influence growth and production of secondary metabolites in plants. In the present study, the effect of light quality on morphophysiology and content of 20-hydroxyecdysone (20E), a phytoecdysteroid, was evaluated in accessions of an endangered medicinal species, Pfaffia glomerata, grown in vitro. Two accessions (Ac22 and Ac43) were cultured in vitro under three different ratios of red (R) and blue (B) LEDs: (i) 1R:1B, (ii) 1R:3B, and (iii) 3R:1B. An equal ratio of red and blue light (1R:1B) increased biomass accumulation, anthocyanin content, and 20E production (by 30-40%). Moreover, 1R:1B treatment increased the size of vascular bundles and vessel elements, as well as strengthened xylem lignification and thickening of the cell wall of shoots. The 1R:3B treatment induced the highest photosynthetic and electron transport rates and enhanced the activity of oxidative stress-related enzymes. Total Chl content, Chl/Car ratio, and NPQ varied more by accession type than by light source. Spectral quality affected primary metabolism differently in each accession. Specifically, in Ac22 plants, fructose content was higher under 1R:1B and 1R:3B treatments, whereas starch accumulation was higher under 1R:3B, and sucrose under 3R:1B. In Ac43 plants, sugars were not influenced by light spectral quality, but starch content was higher under 3R:1B conditions. In conclusion, red and blue LEDs enhance biomass and 20E production in P. glomerata grown in vitro.

Accessions of Brazilian ginseng (Pfaffia glomerata) with contrasting anthocyanin content behave differently in growth, antioxidative defense, and 20-hydroxyecdysone levels under UV-B radiation.

Ultraviolet-B (UV-B) radiation is an elicitor of secondary metabolites in plant tissue culture, but the effects on 20-hydroxyecdysone (20E) are still unclear. The 20E may show biotechnological, pharmacological, medical, and agrochemical applicability. Here, we use Pfaffia glomerata, a medically important species, to understand the impacts of UV-B radiation on their physiological performance, the expression of key genes involved in the 20E biosynthesis, and the 20E content. Two accessions (A22 and A43) of plants 20 days old grown in vitro were exposed to 0 (control), 2 (6.84 kJ m-2), and 4 (13.84 kJ m-2) h UV-B radiation for 20 consecutive days. Our data showed that UV-B reduced glucose concentration in A22 and A43 under 4 h of exposure (29 and 30%, respectively), while sucrose concentration increased (32 and 57%, respectively). UV-B also differentially impacted the accessions (A22 and A43), where the A22 under 4 h of UV-B had reduced total dry weight (8%) and electron transport rate (31%); in contrast, A43 did not change. Also, only A22 had increased POD activity under 4 h of UV-B (66%), as well as increased gene expression of the 20E pathway and the 20E content under 2 and 4 h of UV-B in leaves (28 and 21%, respectively) and roots (16 and 13%, respectively). This differential performance to UV-B can be explained by the contrasting anthocyanin contents. Notably, A43 displayed 56% more anthocyanin to the former, a possible defense against UV-B. In conclusion, UV-B radiation is a potential elicitor for increasing 20E content in P. glomerata grown in vitro.

Salinity-induced modifications on growth, physiology and 20-hydroxyecdysone levels in Brazilian-ginseng [Pfaffia glomerata (Spreng.) Pedersen].

- Salinity is a major threat to agriculture. However, depending on the concentration of soluble salts in soil, increased secondary metabolite levels can occur with no major damages to plant growth and development. The phytoecdysteroid (PE) 20-hydroxyecdysone (20E) is a secondary metabolite with biotechnological, medicinal, pharmaceutical and agrochemical applicability. Here, we characterize the responses (growth and physiology) of Pfaffia glomerata under different NaCl concentrations and examine the production of 20E as affected by salinity. Forty-day-old plants grown in greenhouse were exposed to 0, 120, 240, 360 or 480 mM of NaCl for 11 days. Moderate salinity (i.e., 120 mM of NaCl) led to increased 20E concentrations in leaves (47%) relative to the control with no significant effect on photosynthesis and biomass accumulation, thus allowing improved 20E contents on a per whole-plant basis. In contrast, plants under high salinity (i.e., 240-480 mM of NaCl) displayed similar 20E concentrations in leaves compared to the control, but with marked impairments to biomass accumulation and photosynthetic performance (coupled with decreased sucrose and starch levels) in parallel to nutritional imbalance. High salinity also strongly increased salicylic acid levels, antioxidant enzyme activities, and osmoregulatory status. Regardless of stress severity, 20E production was accompanied by the upregulation of Spook and Phantom genes. Our findings suggest that P. glomerata cultivation in moderate salinity soils can be considered as a suitable agricultural option to increase 20E levels, since metabolic and structural complexity that makes its artificial synthesis very difficult.

Hydroalcoholic extract of Pfaffia glomerata alters the organization of the seminiferous tubules by modulating the oxidative state and the microstructural reorganization of the mice testes.

ETHNOPHARMACOLOGICAL USAGES: Leaves and roots of Pfaffia glomerata areused as aphrodisiacs, tranquilizers and antirheumatics. Due to the lack of experimental scientific data, studies are necessary to identify its medicinal properties. AIMS: The present study aimed to evaluate the effect of hydroalcoholic root extract of P. glomerata (Brazilian ginseng extract - BGE) on testicular parenchyma, and evaluate possible harmful effects through testicular oxidative stress analysis. MATERIALS AND METHODS: Adult mice were divided into 6 groups: control (water), sildenafil citrate, BGE (100, 200 and 400 mg/kg/day), and BGE (200 mg/kg every three days). RESULTS: The treatment reduced the volumetric proportions of seminiferous tubules and epithelium, the number of Sertoli cells, and increased hydrogen peroxide levels, without affecting sperm production. It also caused cell death and changes in the frequency of stages of the seminiferous epithelium cycles. The 100 mg/kg dose responds in a similar way to sildenafil citrate, promoting changes in the gonadal structure, but with efficient response to contain the damage. CONCLUSIONS: Doses of 200 mg/kg, continuous or discontinuous, induced an increase in testicular nitric oxide, as well as sildenafil citrate, showing be efficient as aphrodisiac, but promotes cell death regardless of the form of administration.

Flask sealing on in vitro seed germination and morphogenesis of two types of ornamental pepper explants / Tipo de vedação na germinação e na morfogênese in vitro de dois tipos de explante de pimenteira ornamental

ABSTRACT: The influence of flask sealing and explant source on the in vitro morphogenesis of pepper, were evaluated in Capsicum annuum. Seeds were sterilized and inoculated in Murashige and Skoog media supplemented with vitamins, myo-inositol, sucrose, and agar. Gas exchange was evaluated in the germination stage by comparing 3 flask-sealing systems: rigid polypropylene lids (PLs) without vents, PLs with 1 vent, and PLs with 2 vents covered with membranes. In the regeneration stage, cotyledon and hypocotyl segments were transferred to the organogenesis-inducing media, being the different sealing types also tested in a factorial scheme. Photosynthetic pigments, morphological and hystological analyses were conducted for each treatment. Plants maintained in glass flasks capped with vented lids showed more vigorous growth and differentiated anatomical structures. These treatments resulted in taller plants, higher numbers and more expanded leaves, higher fresh and dry weights, and an increase in photosynthetic pigments. Cultivation of C. annuum in flasks with reduced gas exchange was more effective on callus induction. During the regeneration stage, hypocotyls were more effective than cotyledons. Sealing type influenced the morphogenic responses of pepper, demonstrating that an increase in gas exchange has a positive effect on biomass production and acclimatization of the plantlets.

RESUMO: Objetivando avaliar a influência de trocas gasosas e da fonte de explante na morfogênese in vitro de pimenteira ornamental, sementes de Capsicum annuum foram desinfestadas e inoculadas em meio de Murashige e Skoog suplementado com vitaminas, mio-inositol, sacarose e ágar. O efeito das trocas gasosas foi avaliado na fase de germinação, utilizando três tipos de vedações nos frascos: tampas de polipropileno rígido (TPR) sem membrana (controle), TPR com um orifício (10mm de diâmetro) coberto com membrana e TPR com dois orifícios cobertos com membranas permeáveis à gases. Na fase de regeneração, segmentos de hipocótilo e cotilédones foram transferidos para o meio de indução de organogênese e os diferentes tipos de vedação foram novamente testados em esquema fatorial. Análises anatômicas de pigmentos fotossintéticos e de características morfológicas foram realizadas. Plântulas cultivadas em frascos com tampas com membranas apresentaram estruturas anatômicas diferenciadas e mais vigorosas. Esses tratamentos resultaram em plântulas maiores, com folhas mais expandidas, maior número de folhas, maior massa fresca e seca e maior quantidade de pigmentos fotossintéticos. O cultivo de C. annuum em frascos com menores trocas gasosas foi mais eficaz na indução de calos. Durante o estágio de regeneração, hipocótilos foram mais eficazes do que cotilédones. O tipo de vedação influenciou nas respostas morfogênicas em pimenteira, demonstrando que o aumento nas trocas gasosas tem um efeito positivo sobre a produção de biomassa e aclimatização das plântulas.

The phytoecdysteroid ß-ecdysone is genotoxic in Rodent Bone Marrow Micronuclei and Allium cepa L. Assays.

ETHNAOPHARMACOLOGIAL RELEVANCE: In South America, the ß-ecdysone ecdysteroid has been found in species of the genus Pfaffia Mart. Due to the similar morphology of its roots to the Panax ginseng C. A. Mey. (Korean ginseng), some species of this genus has been known as Brazilian ginseng and have been used as tonic and aphrodisiac, as well as for the treatment of diabetes and rheumatism. AIM OF THE STUDY: Here we report a cytogenotoxic evaluation of ß-ecdysone (a natural ecdysteroid found in plants) in Rodent Bone Marrow Micronuclei and Allium cepa Assays. MATERIALS AND METHODS: Three ß-ecdysone (pure) concentrations (based in human therapeutic dosage) were used in the Micronucleus Assay. The animals were treated during two consecutive days. Micronucleated cells were counted in 2000 polychromatic erythrocytes per animal. For A. cepa L. Assay, one ß-ecdysone concentration was analyzed. The onions bulbs were exposed for 24h. RESULTS: The Micronucleus Assay showed genotoxic effects for all treatments, expressed by an increase of micronucleated cells. In A. cepa L. Assay, cell abnormalities associated to the malfunction/non-formation of mitotic spindle (aneugenic effect) and chromosomal bridges (clastogenic effect) were observed. CONCLUSIONS: The results indicate a cytogenotoxic activity of ß-ecdysone. Therefore, the popular use of Pfaffia and others species containing ß-ecdysone should be considered with caution.