Publications by authors named "Wagner Campos Otoni"

26 Publications

  • Page 1 of 1

Transcriptome Analysis of (Cactaceae) Reveals Metabolic Changes During Shoot Organogenesis Induction.

Front Plant Sci 2021 20;12:697556. Epub 2021 Aug 20.

Plant Biology Department/Laboratory of Plant Tissue Culture II-BIOAGRO, Federal University of Viçosa (UFV), Viçosa, Brazil.

is an endangered cactus highly valued for its ornamental properties. shoot production of this species provides a sustainable alternative to overharvesting from the wild; however, its propagation could be improved if the genetic regulation underlying its developmental processes were known. The present study generated transcriptome data, describing shoot organogenesis induction in . Total RNA was extracted from explants before (control) and after shoot organogenesis induction (treated). A total of 14,478 unigenes (average length, 520 bases) were obtained using Illumina HiSeq 3000 (Illumina Inc., San Diego, CA, USA) sequencing and transcriptome assembly. Filtering for differential expression yielded 2,058 unigenes. Pairwise comparison of treated vs. control genes revealed that 1,241 (60.3%) unigenes exhibited no significant change, 226 (11%) were downregulated, and 591 (28.7%) were upregulated. Based on database analysis, more transcription factor families and unigenes appeared to be upregulated in the treated samples than in controls. Expression of () and () genes, both of which were upregulated in treated samples, was further validated by real-time quantitative PCR (RT-qPCR). Differences in gene expression patterns between control and treated samples indicate substantial changes in the primary and secondary metabolism of after the induction of shoot organogenesis. These results help to clarify the molecular genetics and functional genomic aspects underlying propagation in the Cactaceae family.
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http://dx.doi.org/10.3389/fpls.2021.697556DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8417902PMC
August 2021

Shaping the root system: the interplay between miRNA regulatory hubs and phytohormones.

J Exp Bot 2021 10;72(20):6822-6835

Laboratory of Molecular Genetics of Plant Development, Department of Biological Sciences, Luiz de Queiroz College of Agriculture, University of Sao Paulo, Piracicaba, Sao Paulo, Brazil.

The root system commonly lies underground, where it provides anchorage for the aerial organs, as well as nutrients and water. Both endogenous and environmental cues contribute to the establishment of the root system. Among the endogenous cues, microRNAs (miRNAs), transcription factors, and phytohormones modulate root architecture. miRNAs belong to a subset of endogenous hairpin-derived small RNAs that post-transcriptionally control target gene expression, mostly transcription factors, comprising the miRNA regulatory hubs. Phytohormones are signaling molecules involved in most developmental processes. Some miRNAs and targets participate in more than one hormonal pathway, thereby providing new bridges in plant hormonal crosstalk. Unraveling the intricate network of molecular mechanisms underlying the establishment of root systems is a central aspect in the development of novel strategies for plant breeding to increase yield and optimize agricultural land use. In this review, we summarize recent findings describing the molecular mechanisms associated with the interplay between miRNA regulatory hubs and phytohormones to ensure the establishment of a proper root system. We focus on post-embryonic growth and development of primary, lateral, and adventitious roots. In addition, we discuss novel insights for future research on the interaction between miRNAs and phytohormones in root architecture.
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http://dx.doi.org/10.1093/jxb/erab299DOI Listing
October 2021

Sapucaia nut: Morphophysiology, minerals content, methodological validation in image analysis, phenotypic and molecular diversity in Lecythis pisonis Cambess.

Food Res Int 2020 11 4;137:109383. Epub 2020 Jun 4.

Federal University of Espírito Santo/UFES, Center of Agricultural Sciences and Engineers, Alto Universitário, s/n, 29500-000 Alegre, ES, Brazil. Electronic address:

Sapucaia or Lecythis pisonis Cambess. is an ornamental tree that produces edible, tasty, and nutritious nuts, and can be used for timber production. Sapucaia has potential as a functional food, owing to optimal nutritional and particularly Se levels. The present study sought to characterize the seeds of 21 sapucaia matrices from forest remnants of a neotropical hotspot (Atlantic Forest) and farms in two Brazilian states. Biometrics, germination, vigor, lignin content in the teguments, minerals content, and phenotypic and molecular diversity were analyzed. The seeds of matrices 16 and 21 were the most vigorous. Nuts from matrices 21 and 17 contained the highest amounts of Se. Matrices 5, 8, and 21 were the most phenotypically distant; whereas matrix 21 was the most genetically distant. Importantly, we validated a new non-destructive and efficient X-ray-based methodology for internal and densitometric analysis of sapucaia seeds, and demonstrated a high genetic divergence among matrices.
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http://dx.doi.org/10.1016/j.foodres.2020.109383DOI Listing
November 2020

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

J Ethnopharmacol 2021 Mar 18;267:113599. Epub 2020 Nov 18.

Genetics and Biochemistry, Institute of Biotechnology, Federal University of Uberlândia, 38408-100, Uberlândia, MG, Brazil. Electronic address:

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).
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http://dx.doi.org/10.1016/j.jep.2020.113599DOI Listing
March 2021

Anatomy, Flow Cytometry, and X-Ray Tomography Reveal Tissue Organization and Ploidy Distribution in Long-Term Cultures of Species.

Front Plant Sci 2020 31;11:1314. Epub 2020 Aug 31.

Laboratory of Plant Tissue Culture II-BIOAGRO, Plant Biology Department, Federal University of Viçosa (UFV), Viçosa, Brazil.

Cacti have a highly specialized stem that enables survival during extended dry periods. Despite the ornamental value of cacti and the fact that stems represent the main source of explants in tissue culture, there are no studies on their morpho-anatomical and cytological characteristics in . The present study seeks to address the occurrence of cells with mixed ploidy level in cacti tissues. Specifically, we aim to understand how stem tissue is organized, how mixoploidy is distributed when present, and whether detected patterns of ploidy change after long periods of culture. To analyze tissue organization, and plants that had been germinated and cultivated were analyzed for stem structure using toluidine blue, Xylidine Ponceau, Periodic Acid Schiff, ruthenium red, and acid floroglucin. To investigate patterns of ploidy, apical, medial, and basal zones of the stem, as well as, periphery, cortex, and stele (vascular tissue and pith) regions of the stem and root apexes from four- and ten-year old cultured were analyzed by flow cytometry. X-ray micro-computed tomography (XRµCT) was performed with fragments of stems from both species. The scarcity of support elements (, sclereids and fibers) indicates that epidermis, hypodermis, and wide-band tracheids present in cortical vascular bundles and stele, as well as water stored in aquifer parenchyma cells along the cortex, provide mechanical support to the stem. Parenchyma cells increase in volume with a four-fold increase in ploidy. and exhibit the same pattern of cell ploidy irrespective of topophysical region or age, but there is a marked difference in ploidy between the stem periphery (epidermis and hypodermis), cortex, stele, and roots. Mixoploidy in is not related to the age of the culture, but is a developmental trait, whereby endocycles promote cell differentiation to accumulate valuable water.
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http://dx.doi.org/10.3389/fpls.2020.01314DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7488924PMC
August 2020

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

Protoplasma 2021 Jan 25;258(1):151-167. Epub 2020 Sep 25.

Departamento de Biologia Vegetal/BIOAGRO, Universidade Federal de Viçosa, Viçosa, MG, 36570-900, Brazil.

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 s 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 s than at 65 μmol m s, 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 s 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.
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http://dx.doi.org/10.1007/s00709-020-01558-1DOI Listing
January 2021

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

J Ethnopharmacol 2020 Oct 27;261:113182. Epub 2020 Jul 27.

Department of General Biology, Federal University of Viçosa, Peter Henry Rolfs Avenue S/n, 36570-900, Viçosa, MG, Brazil.

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 17β-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.
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http://dx.doi.org/10.1016/j.jep.2020.113182DOI Listing
October 2020

Leaf development stages and ontogenetic changes in passionfruit (Passiflora edulis Sims.) are detected by narrowband spectral signal.

J Photochem Photobiol B 2020 Aug 10;209:111931. Epub 2020 Jun 10.

Departamento de Biologia Vegetal, Universidade Federal de Viçosa, Viçosa, MG, Brazil. Electronic address:

During shoot development, leaves undergo various ontogenetic changes, including variation in size, shape, and geometry. Passiflora edulis (passionfruit) is a heteroblastic species, which means that it experiences conspicuous changes throughout development, enabling a morphological distinction between the juvenile and adult vegetative phases. Quantification of heteroblasty requires a practical, inexpensive, reliable, and non-destructive method, such as remote sensing. Moreover, relationships among ontogenetic changes and spectral signal at leaf level can be scaled up to support precision agriculture in passion fruit crops. In the present study, we used laboratory spectroscopic measurements (400-2500 nm) and narrowband vegetation indexes (or hyperspectral vegetation indexes - HVIs) to evaluate ontogenetic changes related to development and aging in P. edulis leaves. We also assessed leaf pigment concentration to further support the application of biochemical-related narrowband indexes. We report that 30-d-old leaves can be discriminated into developmental stages through their spectral signals. MSI (Moisture Stress Index) and NDVI (Normalized Difference Vegetation Index ρ750) contribute most to the variation of age (15 to 30-d-old leaves) and developmental stage (phytomer positions along the plant axis) in passionfruit leaves. PRI (Photochemical Reflectance Index) played an important role in detecting age and development alterations, including heteroblasty. A biochemical and spectral comparison of pigments revealed that spectroscopy offered potential for diagnosing phenology in P. edulis, as some narrowband indexes correlated strongly with chlorophylls and carotenoids content. Narrowband vegetation indexes are found to be a suitable tool for monitoring passionfruit crops.
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http://dx.doi.org/10.1016/j.jphotobiol.2020.111931DOI Listing
August 2020

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

J Photochem Photobiol B 2020 Jan 24;203:111761. Epub 2019 Dec 24.

Departamento de Biologia Vegetal/BIOAGRO, Universidade Federal de Viçosa, Viçosa, MG, Brazil. Electronic address:

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.
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http://dx.doi.org/10.1016/j.jphotobiol.2019.111761DOI Listing
January 2020

miR156-targeted SPL10 controls Arabidopsis root meristem activity and root-derived de novo shoot regeneration via cytokinin responses.

J Exp Bot 2020 01;71(3):934-950

Laboratory of Molecular Genetics of Plant Development, Department of Biological Sciences, Escola Superior de Agricultura 'Luiz de Queiroz', University of Sao Paulo, Piracicaba, Sao Paulo, Brazil.

Root growth is modulated by different factors, including phytohormones, transcription factors, and microRNAs (miRNAs). MicroRNA156 and its targets, the SQUAMOSA PROMOTER BINDING PROTEIN-LIKE (SPL) genes, define an age-dependent pathway that controls several developmental processes, including lateral root emergence. However, it remains unclear whether miR156-regulated SPLs control root meristem activity and root-derived de novo shoot regeneration. Here, we show that MIR156 and SPL genes have opposing expression patterns during the progression of primary root (PR) growth in Arabidopsis, suggesting that age cues may modulate root development. Plants with high miR156 levels display reduced meristem size, resulting in shorter primary root (PRs). Conversely, plants with reduced miR156 levels show higher meristem activity. Importantly, loss of function of SPL10 decreases meristem activity, while SPL10 de-repression increases it. Meristem activity is regulated by SPL10 probably through the reduction of cytokinin responses, via the modulation of type-B ARABIDOPSIS RESPONSE REGULATOR1(ARR1) expression. We also show that SPL10 de-repression in the PRs abolishes de novo shoot regenerative capacity by attenuating cytokinin responses. Our results reveal a cooperative regulation of root meristem activity and root-derived de novo shoot regeneration by integrating age cues with cytokinin responses via miR156-targeted SPL10.
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http://dx.doi.org/10.1093/jxb/erz475DOI Listing
January 2020

Irradiance and light quality affect two annatto (Bixa orellana L.) cultivars with contrasting bixin production.

J Photochem Photobiol B 2019 Aug 4;197:111549. Epub 2019 Jul 4.

Departamento de Biologia Vegetal/Laboratório de Cultura de Tecidos/BIOAGRO, Universidade Federal de Viçosa, 36570-900 Viçosa, MG, Brazil. Electronic address:

Light is a key factor influencing growth and development in plants. Specific irradiance and light quality can improve development and production of secondary compounds such as carotenoids during plant tissue culture. Bixin and norbixin, two apocarotenoids obtained from the seeds of Bixa orellana L. (annatto), are used as natural dyes in various industries. While annatto tissue culture has been successful, the effect of light in this species remains poorly understood. Here, we analyze for the first time the effect of irradiance regime (50, 150, 50 + 150, 200, 50 + 200 μmol m s) and light spectral quality (fluorescent, white, blue/red LED) on in vitro development of apexes and bixin content in two contrasting bixin-producing varieties of B. orellana, namely 'Piave Vermelha' and 'UESB74'. The number of leaves per plant, stomatal density, leaf area, leaf expansion, chlorophylls and carotenoids content, malondialdehyde and bixin content were analyzed in the leaves of both cultivars. 'Piave Vermelha' produced 1.6-fold more bixin than 'UESB74'. Stomata cells of both cultivars had a paracytic arrangement with peltate trichomes along the adaxial and abaxial leaf surfaces. 'Piave Vermelha' preferred blue/red LED light; whereas fluorescent light was optimal for 'UESB74'. Under fluorescent light, an irradiance of 50 μmol m s is indicated for both cultivars. LED light increased bixin content only in 'Piave Vermelha', suggesting that the dye biosynthetic pathway is genotype-dependent. The present findings suggest the possibility of using light to modulate the bixin biosynthetic pathway.
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http://dx.doi.org/10.1016/j.jphotobiol.2019.111549DOI Listing
August 2019

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

Protoplasma 2019 Nov 17;256(6):1557-1571. Epub 2019 Jun 17.

Laboratório de Cultura de Tecidos Vegetais (LCTII), Departamento de Biologia Vegetal/BIOAGRO, Universidade Federal de Viçosa, Campus Universitário, Avenida Peter Henry Rolfs s/n, Viçosa, MG, 36570-900, Brazil.

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), and 4 (13.84 kJ m) 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.
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http://dx.doi.org/10.1007/s00709-019-01400-3DOI Listing
November 2019

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

Plant Physiol Biochem 2019 Jul 3;140:43-54. Epub 2019 May 3.

Departamento de Biologia Vegetal/BIOAGRO, Universidade Federal de Viçosa, Viçosa, MG, Brazil. Electronic address:

- 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.
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http://dx.doi.org/10.1016/j.plaphy.2019.05.002DOI Listing
July 2019

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.

J Ethnopharmacol 2019 Apr 31;233:179-189. Epub 2018 Dec 31.

Department of General Biology, Federal University of Viçosa, Viçosa/MG, Brazil. Electronic address:

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.
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http://dx.doi.org/10.1016/j.jep.2018.12.047DOI Listing
April 2019

De novo assembly and transcriptome of Pfaffia glomerata uncovers the role of photoautotrophy and the P450 family genes in 20-hydroxyecdysone production.

Protoplasma 2019 May 25;256(3):601-614. Epub 2018 Oct 25.

Departamento de Biologia Vegetal/BIOAGRO, Universidade Federal de Viçosa, Viçosa, MG, Brazil.

Pfaffia glomerata is a medically important species because it produces the phytoecdysteroid 20-hydroxyecdysone (20-E). However, there has been no ready-to-use transcriptome data available in the literature for this plant. Here, we present de novo transcriptome sequencing of RNA from P. glomerata in order to investigate the 20-E production as well as to understand the biochemical pathway of secondary metabolites in this non-model species. We then analyze the effect of photoautotrophy on the production of 20-E genes phylogenetically identified followed by expression analysis. For this, total messenger RNA (mRNA) from leaves, stems, roots, and flowers was used to construct indexed mRNA libraries. Based on the similarity searches against plant non-redundant protein database, gene ontology, and eukaryotic orthologous groups, 164,439 transcripts were annotated. In addition, the effect of photoautotrophy in two genes putatively involved in the 20-E synthesis pathway was analyzed. The Phantom gene (CYP76C), a precursor of the route, showed increased expression in P. glomerata plants cultured under photoautotrophic conditions. This was accompanied by increased production of this metabolite indicating a putative involvement in 20-E synthesis. This work reveals that several genes in the P. glomerata transcriptome are related to secondary metabolism and stresses, that genes of the P450 family participate in the 20-E biosynthesis route, and that plants cultured under photoautotrophic conditions promote an upregulated Phantom gene and enhance the productivity of 20-E. The data will be used for future investigations of the 20-E synthesis pathway in P. glomerata while offering a better understanding of the metabolism of the species.
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http://dx.doi.org/10.1007/s00709-018-1322-1DOI Listing
May 2019

Genetic, evolutionary and phylogenetic aspects of the plastome of annatto (Bixa orellana L.), the Amazonian commercial species of natural dyes.

Planta 2019 Feb 11;249(2):563-582. Epub 2018 Oct 11.

Laboratório de Fisiologia Molecular de Plantas, Departamento de Biologia Vegetal, Universidade Federal de Viçosa, Viçosa, MG, Brazil.

Main Conclusion: The plastome of B. orellana reveals specific evolutionary features, unique RNA editing sites, molecular markers and the position of Bixaceae within Malvales. Annatto (Bixa orellana L.) is a native species of tropical Americas with center of origin in Brazilian Amazonia. Its seeds accumulate the apocarotenoids, bixin and norbixin, which are only found in high content in this species. The seeds of B. orellana are commercially valued by the food industry because its dyes replace synthetic ones from the market due to potential carcinogenic risks. The increasing consumption of B. orellana seeds for dye extraction makes necessary the increase of productivity, which is possible accessing the genetic basis and searching for elite genotypes. The identification and characterization of molecular markers are essential to analyse the genetic diversity of natural populations and to establish suitable strategies for conservation, domestication, germplasm characterization and genetic breeding. Therefore, we sequenced and characterized in detail the plastome of B. orellana. The plastome of B. orellana is a circular DNA molecule of 159,708 bp with a typical quadripartite structure and 112 unique genes. Additionally, a total of 312 SSR loci were identified in the plastome of B. orellana. Moreover, we predicted in 23 genes a total of 57 RNA-editing sites of which 11 are unique for B. orellana. Furthermore, our plastid phylogenomic analyses, using the plastome sequences available in the plastid database belonging to species of order Malvales, indicate a closed relationship between Bixaceae and Malvaceae, which formed a sister group to Thymelaeaceae. Finally, our study provided useful data to be employed in several genetic and biotechnological approaches in B. orellana and related species of the family Bixaceae.
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http://dx.doi.org/10.1007/s00425-018-3023-6DOI Listing
February 2019

Cellular and Morpho-histological Foundations of In Vitro Plant Regeneration.

Methods Mol Biol 2018 ;1815:47-68

Laboratório de Cultura de Tecidos-LCT, Instituto de Biotecnologia Aplicada à Agropecuária-BIOAGRO/Departamento de Biologia Vegetal, Campus Universitário, Universidade Federal de Viçosa, Viçosa, MG, Brazil.

In vitro plant regeneration systems have turned into invaluable tools to plant biotechnology. Despite being poorly understood, the molecular mechanisms underlying the control of both morphogenetic pathways, de novo organogenesis and somatic embryogenesis, have been supported by recent findings involving proteome-, metabolome-, and transcriptome-based profiles. Notwithstanding, the integration of molecular data with structural aspects has been an important strategy of study attempting to elucidate the basis of the cell competence acquisition to further follow commitment and determination to specific a particular in vitro regeneration pathway. In that sense, morpho-histological tools have allowed to recognize cellular markers and patterns of gene expression at cellular level and this way have collaborated in the identification of the cell types with high regenerative capacity. This chapter ties together up those fundamental and important microscopy techniques that help to elucidate that regeneration occurs, most of the time, from epidermis or subepidermal cells and from the procambial cells (pericycle and vascular parenchyma). Important findings are discussed toward ultrastructural differences observed in the nuclear organization among pluripotent and totipotent cells, implying that regeneration occurs from two cellular mechanisms based on cellular reprogramming or reactivation.
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http://dx.doi.org/10.1007/978-1-4939-8594-4_3DOI Listing
March 2019

Morpho-histological, histochemical, and molecular evidences related to cellular reprogramming during somatic embryogenesis of the model grass Brachypodium distachyon.

Protoplasma 2017 Sep 13;254(5):2017-2034. Epub 2017 Mar 13.

Laboratório de Cultura de Tecidos/BIOAGRO, Departamento de Biologia Vegetal, Universidade Federal de Viçosa, Avenida P. H. Rolfs s/n, 36570-900, Viçosa, MG, Brazil.

The wild grass species Brachypodium distachyon (L.) has been proposed as a new model for temperate grasses. Among the biotechnological tools already developed for the species, an efficient induction protocol of somatic embryogenesis (SE) using immature zygotic embryos has provided the basis for genetic transformation studies. However, a systematic work to better understanding the basic cellular and molecular mechanisms that underlie the SE process of this grass species is still missing. Here, we present new insights at the morpho-histological, histochemical, and molecular aspects of B. distachyon SE pathway. Somatic embryos arose from embryogenic callus formed by cells derived from the protodermal-dividing cells of the scutellum. These protodermal cells showed typical meristematic features and high protein accumulation which were interpreted as the first observable steps towards the acquisition of a competent state. Starch content decreased along embryogenic callus differentiation supporting the idea that carbohydrate reserves are essential to morphogenetic processes. Interestingly, starch accumulation was also observed at late stages of SE process. Searches in databanks revealed three sequences available annotated as BdSERK, being two copies corresponding to SERK1 and one showing greater identity to SERK2. In silico analysis confirmed the presence of characteristic domains in a B. distachyon Somatic Embryogenesis Receptor Kinase genes candidates (BdSERKs), which suggests SERK functions are conserved in B. distachyon. In situ hybridization demonstrated the presence of transcripts of BdSERK1 in all development since globular until scutellar stages. The results reported in this study convey important information about the morphogenetic events in the embryogenic pathway which has been lacking in B. distachyon. This study also demonstrates that B. distachyon provides a useful model system for investigating the genetic regulation of SE in grass species.
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http://dx.doi.org/10.1007/s00709-017-1089-9DOI Listing
September 2017

Translationally controlled tumour protein (TCTP) from tomato and Nicotiana benthamiana is necessary for successful infection by a potyvirus.

Mol Plant Pathol 2017 06 27;18(5):672-683. Epub 2016 Jul 27.

Departamento de Microbiologia/BIOAGRO/National Institute of Science and Technology in Plant-Pest Interactions, Universidade Federal de Viçosa, Viçosa, MG, 36570-900, Brazil.

Translationally controlled tumour protein (TCTP) is a ubiquitously distributed protein in eukaryotes, involved in the regulation of several processes, including cell cycle progression, cell growth, stress protection, apoptosis and maintenance of genomic integrity. Its expression is induced during the early stages of tomato (Solanum lycopersicum) infection by the potyvirus Pepper yellow mosaic virus (PepYMV, a close relative of Potato virus Y). Tomato TCTP is a protein of 168 amino acids, which contains all the conserved domains of the TCTP family. To study the effects of TCTP silencing in PepYMV infection, Nicotiana benthamiana plants were silenced by virus-induced gene silencing (VIGS) and transgenic tomato plants silenced for TCTP were obtained. In the early stages of infection, both tomato and N. benthamiana silenced plants accumulated less virus than control plants. Transgenic tomato plants showed a drastic reduction in symptoms and no viral accumulation at 14 days post-inoculation. Subcellular localization of TCTP was determined in healthy and systemically infected N. benthamiana leaves. TCTP was observed in both the nuclei and cytoplasm of non-infected cells, but only in the cytoplasm of infected cells. Our results indicate that TCTP is a growth regulator necessary for successful PepYMV infection and that its localization is altered by the virus, probably to favour the establishment of virus infection. A network with putative interactions that may occur between TCTP and Arabidopsis thaliana proteins was built. This network brings together experimental data of interactions that occur in other eukaryotes and helps us to discuss the possibilities of TCTP involvement in viral infection.
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http://dx.doi.org/10.1111/mpp.12426DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6638207PMC
June 2017

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

J Ethnopharmacol 2016 Jan 25;177:81-4. Epub 2015 Nov 25.

Universidade Federal de Juiz de Fora, Instituto de Ciências Biológicas, Departamento de Biologia, Laboratório de Genética e Biotecnologia, CEP 36036-900 Juiz de Fora, MG, Brazil. Electronic address:

Ethnaopharmacologial Relevance: In South America, the β-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 β-ecdysone (a natural ecdysteroid found in plants) in Rodent Bone Marrow Micronuclei and Allium cepa Assays.

Materials And Methods: Three β-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 β-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 β-ecdysone. Therefore, the popular use of Pfaffia and others species containing β-ecdysone should be considered with caution.
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http://dx.doi.org/10.1016/j.jep.2015.11.038DOI Listing
January 2016

Cellular and molecular changes associated with competence acquisition during passion fruit somatic embryogenesis: ultrastructural characterization and analysis of SERK gene expression.

Protoplasma 2016 Mar 26;253(2):595-609. Epub 2015 May 26.

Departamento de Biologia Vegetal, Universidade Federal de Viçosa, Av. P.H. Rolfs, Viçosa, MG, 36570-900, Brazil.

The integration of cellular and molecular data is essential for understanding the mechanisms involved in the acquisition of competence by plant somatic cells and the cytological changes that underlie this process. In the present study, we investigated the dynamics and fate of Passiflora edulis Sims cotyledon explants that were committed to somatic embryogenesis by characterizing the associated ultrastructural events and analysing the expression of a putative P. edulis ortholog of the Somatic Embryogenesis Receptor-like Kinase (SERK) gene. Embryogenic calli were obtained from zygotic embryo explants cultured on Murashige and Skoog medium supplemented with 2,4-dichlorophenoxyacetic acid and 6-benzyladenine. Callus formation was initiated by the division of cells derived from the protodermal and subprotodermal cells on the abaxial side of the cotyledons. The isodiametric protodermal cells of the cotyledon explants adopted a columnar shape and became meristematic at the onset of PeSERK expression, which was not initially detected in explant cells. Therefore, we propose that these changes represent the first observable steps towards the acquisition of a competent state within this regeneration system. PeSERK expression was limited to the early stages of somatic embryogenesis; the expression of this gene was confined to proembryogenic zones and was absent in the embryos after the globular stage. Our data also demonstrated that the dynamics of the mobilization of reserve compounds correlated with the differentiation of the embryogenic callus.
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http://dx.doi.org/10.1007/s00709-015-0837-yDOI Listing
March 2016

Calcium oxalate crystals in eucalypt ectomycorrhizae: morphochemical characterization.

PLoS One 2013 2;8(7):e67685. Epub 2013 Jul 2.

Departamento de Microbiologia, Universidade Federal de Viçosa, Viçosa, Minas Gerais, Brazil.

Ectomycorrhizal fungi are ubiquitous in forest ecosystems, benefitting plants principally by increasing the uptake of water and nutrients such as calcium from the soil. Previous work has demonstrated accumulation of crystallites in eucalypt ectomycorrhizas, but detailed morphological and chemical characterization of these crystals has not been performed. In this work, cross sections of acetic acid-treated and cleared ectomycorrhizal fragments were visualized by polarized light microscopy to evaluate the location of crystals within cortical root cells. Ectomycorrhizal sections were also observed by scanning electron microscopy (SEM) coupled with energy dispersive x-ray (EDS) microprobe analysis. The predominant forms of crystals were crystal sand (granules) and concretions. Calcium, carbon and oxygen were detected by EDS as constituent elements and similar elemental profiles were observed between both crystal morphologies. All analyzed crystalline structures were characterized as calcium oxalate crystals. This is the first report of the stoichiometry and morphology of crystals occurring in eucalypt ectomycorrhizas in tropical soils. The data corroborates the role of ectomycorrhizae in the uptake and accumulation of calcium in the form of calcium oxalate crystals in hybrid eucalypt plants.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0067685PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3699605PMC
January 2014

Somatic embryogenesis of a wild passion fruit species Passiflora cincinnata Masters: histocytological and histochemical evidences.

Protoplasma 2012 Jul 17;249(3):747-58. Epub 2011 Sep 17.

Plant Biology Department, Plant Tissue Culture Laboratory/BIOAGRO, Federal University of Viçosa, University Campus, P.H. Rolfs Avenue, 36570-000, Viçosa, MG, Brazil.

The characterization of cellular changes that occur during somatic embryogenesis is essential for understanding the factors involved in the transition of somatic cells into embryogenically competent cells and determination of cells and/or tissues involved. The present study describes the anatomical and ultrastructural events that lead to the formation of somatic embryos in the model system of the wild passion fruit (Passiflora cincinnata). Mature zygotic embryos were inoculated in Murashige and Skoog induction media supplemented with 2,4-dichlorophenoxyacetic acid and 6-benzyladenine. Zygotic embryo explants at different development stages were collected and processed by conventional methods for studies using light, scanning, and transmission electron microscopy (TEM). Histochemical tests were used to examine the mobilization of reserves. The differentiation of the somatic embryos began in the abaxial side of the cotyledon region. Protuberances were formed from the meristematic proliferation of the epidermal and mesophyll cells. These cells had large nuclei, dense cytoplasm with a predominance of mitochondria, and a few reserve compounds. The protuberances extended throughout the abaxial surface of the cotyledons. The ongoing differentiation of peripheral cells of these structures led to the formation of proembryogenic zones, which, in turn, dedifferentiated into somatic embryos of multicellular origin. In the initial stages of embryogenesis, the epidermal and mesophyll cells showed starch grains and less lipids and protein reserves than the starting explant. These results provide detailed information on anatomical and ultrastructural changes involved in the acquisition of embryogenic competence and embryo differentiation that has been lacking so far in Passiflora.
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http://dx.doi.org/10.1007/s00709-011-0318-xDOI Listing
July 2012

High-efficiency Agrobacterium-mediated transformation of citrus via sonication and vacuum infiltration.

Plant Cell Rep 2009 Mar 2;28(3):387-95. Epub 2008 Dec 2.

Plant Biology Department, Plant Tissue Culture Laboratory/BIOAGRO, Federal University of Viçosa, Viçosa, MG, Brazil.

An improved method for the Agrobacterium infiltration of epicotyl segments of 'Pineapple' sweet orange [Citrus sinensis (L.) Osbeck] and 'Swingle' citrumelo [Citrus paradisi Macf. X Poncirus trifoliata (L.) Raf.] was developed in order to increase transformation frequency. Sonication-assisted Agrobacterium-mediated transformation (SAAT), vacuum infiltration, and a combination of the two procedures were compared with conventional Agrobacterium-mediated inoculation method ('dipping' method). It was observed that the morphogenic potential of the epicotyl segments decreased as the duration of SAAT and vacuum treatments increased. Transient GUS expression was not affected by the different SAAT treatments, but it was significantly enhanced by the vacuum infiltration treatments. The highest transformation efficiencies were obtained when the explants were subjected to a combination of SAAT for 2 s followed by 10 min of vacuum infiltration. PCR and Southern blot analysis of the uidA gene were used to confirm the integration of the transgenes. The transformation frequencies achieved in this study (8.4% for 'Pineapple' sweet orange and 11.2% for 'Swingle' citrumelo) are the highest ones reported for both cultivars.
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http://dx.doi.org/10.1007/s00299-008-0646-2DOI Listing
March 2009

Statistical and image analysis of sister chromatid exchange in maize.

Hereditas 2004 ;141(3):318-22

Plant Cytogenetics Laboratory, General Biology Department, Federal University of Viçosa, Viçosa, MG-Brazil.

The present study reports the use of the fluorescence plus Giemsa (FPG) technique, image analysis and statistical methods to assess the sister chromatid exchanges (SCEs) frequency in maize. Roots derived from germinated maize seeds were treated with BrdU solution and fixed. The slides were prepared by enzymatic cellular dissociation, air-drying technique, stained with Hoechst 33258 fluorochrome, and incubated in salt solution. The chromosomes were irradiated with ultraviolet light and stained with Giemsa solution. The FPG technique associated with digital analysis system was used to measure the length of 597 BrdU-incorporated maize chromosomes and to identify 0.5243 SCE per chromosome. A range from 0 to 4 SCE events were classified and the chi-square test (chi2=1.586, P=0.662) showed a good fit to the hypothesis that the SCEs are independent and random events that follow Poisson distribution. The SCE frequencies in long and short chromosome arms corresponded to a mean value of 0.876 SCE microm(-1). Considering that the maize line used in this study contains 5.78 picogram (pg) DNA (2C value) in interphasic G0/G1 nuclei or 11.56 pg DNA (4C value) in metaphase, and that the DNA mean value corresponds to 0.578 pg/metaphasic chromosome, the analysis suggests an occurrence of approximately 0.9 SCE/pg DNA.
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http://dx.doi.org/10.1111/j.1601-5223.2004.01848.xDOI Listing
June 2006
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