Morphophysiological and molecular aspects of the in vitro development of Pfaffia glomerata (Spreng.) Pedersen as influenced by light

A Pfaffia glomerata apresenta potenciais propriedades farmacológicas e medicinais, como anabolizante, analgésica, anti-inflamatória, antimutagênica, afrodisíaca, sedativa, antidiabética e de tônico muscular. As propriedades nessa espécie se devem principalmente à produção de um metabólito secundário, o fitoecdisteroide 20-hidroxiecdisona (20E). Contudo sua via biossintética e suas funções em plantas ainda não estão totalmente elucidadas. A luz (qualidade e quantidade) afeta inúmeros processos morfofisiológicos em plantas cultivadas in vitro, como crescimento e desenvolvimento, fotossíntese, morfogênese, produção de metabólitos secundários, entre outros. Diante disso, objetivou-se avaliar a influência da qualidade e quantidade de luz na morfofisiologia e na produção de 20E em dois acessos (Ac 22 e Ac 43) de Pfaffia glomerata cultivadas in vitro. Para tal, foram executados dois experimentos. Primeiro foi analisado a influência de diferentes níveis de irradiâncias (65, 130 e 200 μmol m -2 s -1 ) e segundo a influência de diferentes proporções de vermelho (V) e azul (A) (1V:1A; 1V:3A e 3V:1A). Em ambos os experimentos foram utilizados explantes com um segmento nodal, os quais foram inoculados meio de Murashige e Skoog (MS) sem adição de fitorreguladores, sendo as culturas foram mantidas a 25 ± 2 ° C e sob fotoperíodo de 16 h de luz. Observou-se que tanto a quantidade, quanto a qualidade da luz no cultivo in vitro de Pffafia glomerata alteram o teor e a produção de 20E. No entanto, não houve relação direta do conteúdo de 20E com a expressão relativa do gene Phantom, envolvido possivelmente na biossíntese de 20E. Além disso, as características morfofisiológicas, assim como a produção de 20E, se mostraram genótipo-dependentes em ambos os experimentos. O aumento da irradiância promove o crescimento e o desenvolvimento in vitro de P. glomerata, aumentando também a eficiência fotossintética, o que refletiu no maior acúmulo de biomassa nas plantas. A qualidade de luz também leva a modificações na morfofisiologia de P. glomerata cultivada in vitro. Visto que, a qualidade espectral 1V:3A influenciou na eficiência fotossintética e na atividade das enzimas do estresse oxidativo. Já a igual proporção de luz (1V:1A) favoreceu o desenvolvimento morfoanatômico das plantas e a produção de 20E. Este trabalho fornece novas perspectivas a respeito dos efeitos da luz no ambiente in vitro e pode auxiliar na otimização de protocolos de bioprospecção e propagação em larga escala de 20E em P. glomerata.Pfaffia glomerata has potential pharmacological and medicinal properties, such as anabolic, analgesic, anti-inflammatory, antimutagenic, aphrodisiac, sedative, antidiabetic and muscle tonics. The properties in this species are mainly due to the production of a secondary metabolite, the phytoecdysteroid 20-hydroxyecysone (20E). However its biosynthetic pathway and its functions in plants are not yet fully elucidated. Light (quality and quantity) affects numerous morphophysiological processes in plants grown in vitro, such as growth and development, photosynthesis, morphogenesis, production of secondary metabolites and others. The aim of this study was to evaluate the influence of quality and quantity of light on morphophysiology and 20E production in two accessions (Ac22 and Ac43) of Pfaffia glomerata grown in vitro. For this, two experiments were performed. In the first experiment, the influence of different levels of irradiance (65, 130 and 200 μmol m -2 s -1 ) and in second experiment different proportions of red (R) and blue (B) LED lights (1R:1B; 1R:3B and 3R: 1B) in two accessions (Ac22 and Ac43) of P. glomerata were evaluated. In both experiments explants with a single nodal segment were used. The explants were cultured in Murashige and Skoog (MS) medium lacking phytoregulators and the cultures were maintained at 25 ± 2 ° C and photoperiod of 16 h of light. We observed that both the amount and the quality of light in the in vitro culture of Pfafia glomerata alter the content and the production of 20E. However, there was no direct relationship of the 20E content with the relative expression of the Phantom gene, possibly involved in 20E biosynthesis. In addition, the morphophysiological characteristics as well as the production of 20E were shown to be genotype-dependent in both experiments. The increase in irradiance levels promotes the in vitro growth and development of P. glomerata, also increasing the photosynthetic efficiency, which reflected into a greater accumulation of biomass in the plants. The light quality also leads to modifications in the morphology of P. glomerata cultivated in vitro. Remarkably, the spectral quality 1R:3B influenced the photosynthetic efficiency and the activity of oxidative stress enzymes, whereas the proportion of light (1R:1B) favored the morfoanatomic development of the plants and the production of 20E. This work provides new perspectives on the effects of light in the in vitro environment and may aid in the optimization of bioprospecting protocols and large-scale propagation of 20E in P. glomerata

Morfofisiologia e produção de 20-hidroxiecdisona em Pfaffia glomerata (Spreng.) Pedersen em resposta à atmosfera enriquecida com CO2 e estresse hídrico

Anthropogenic activities have been catalyzing climate changes, owing to increased atmospheric carbon dioxide concentration ([CO 2 ]). In respect to CO 2 , its general effects on the regulation of plant growth, development and metabolism are widely explored, especially in agricultural crops. However, the changes in medicinal species under elevated [CO 2 ] are still poorly understood. Pfaffia glomerata (Spreng.) Pedersen, a plant species native to Brazil and popularly known as Brazilian ginseng, stands out by the production of 20-hydroxyecdysone (20E), a phytoecdysteroid molecule with proven therapeutic and nutraceutical activities in mammals. Previous findings from our research group revealed that in vitro CO 2 -enrichment led to physiological changes in P. glomerata. We hypothesize that CO 2 -enrichment affects the production of primary and secondary metabolites, such as 20E, in P. glomerata. Therefore, the objective here was to evaluate the effect of the CO 2 -enriched atmosphere on morphophysiological, biochemical, structural, molecular aspects, as well as in water deficit tolerance responses. For this, two independent experiments were conducted in open top chambers (OTCs). For both experiments, clonally micropropagation-derived plantlets from the in vitro germplasm bank were used. In experiment I, we assessed the influence of two CO 2 concentrations: ambient (a[CO 2 ], ± 400 µmol mol -1 CO 2 ) and elevated (e[CO 2 ] ± 800 µmol mol -1 CO 2 ). After approximately 21 days of cultivation in OTC, our data showed that e[CO 2 ] increased photosynthesis (A N ), and water use efficiency (WUE), biomass accumulation, photosynthetic pigments, carbohydrate content, expression of genes of the lignin biosynthetic pathway, and induced changes in the proteomic profile. On the other hand, e[CO 2 ] promoted reduction in total protein, amino acids and 20E contents. In experiment II, the physiological and biochemical responses of plants exposed to combined e[CO 2 ] and under water deficit were analyzed. During the first 14 days of cultivation, all plants were regularly irrigated to maintain soil water at field capacity. Then, the plants were subjected for 21 days to the following treatments: (I) a[CO 2 ] and well-watered (a[CO 2 ]WW); (II) e[CO 2 ] and well- watered (e[CO 2 ]WW); (III) a[CO 2 ] and drought stressed (a[CO 2 ]D); and (IV) e[CO 2 ] and drought stressed (e[CO 2 ]D). Our data demonstrate that e[CO 2 ] mitigates drought impacts by reducing stomatal conductance (g S ), improving WUE, and promoting less negative water potentials. Although the reduction in gs decreased water loss by evapotranspiration, it negatively affected A N , and consequently plant growth. Hyperspectral analysis corroborates these findings and is a great non-destructive tool to analyse the effects of drought on plants. We found that P. glomerata exhibits different drought response mechanisms depending on [CO 2 ]. Plants exposed to a[CO 2 ]D increased root/shoot ratio, stem rustification, non- enzymatic antioxidant system by increased anthocyanin content and proteins of ascorbate and glutathione metabolism. Conversely, plants under e[CO 2 ]D invested strongly in osmoregulatory metabolites (e.g. soluble sugars and amino acids). We found that e[CO 2 ] associated to drought promoted changes in the proteomic profile, dramatically affecting the accumulation of stress response proteins. On the other hand, we show that drought positively affected the activity of antioxidant enzymes (CAT and SOD) and 20E content in both [CO 2 ] in P. glomerata plants. However, only in plants exposed to e[CO 2 ]D was there an increase in 20E production, overcoming the biomass limitation caused by this stress. Here, we report for the first time, the up-regulation of cytochrome P450 CYP72A219-like protein in plants grown in the combination e[CO 2 ]D. Therefore, we hypothesize a relationship of this protein with 20E biosynthesis and hypothesize possible ROS signaling (indirectly by increased CAT and SOD) under abiotic stress condition. These data provide relevant information in elucidating the pathway of 20E biosynthesis, as well as enabling biotechnological strategies to increase the production of this metabolite in P. glomerata plants. Keywords: Brazilian ginseng. 20-hydroxyecdysone. Phytoecdysteroids. Water stress. CO 2 enrichment. Secondary metabolism.Atividades antrópicas têm vindo a provocar alterações climáticas, tais como o aumento da concentração atmosférica de dióxido de carbono ([CO 2 ]). Em relação ao CO 2 , os seus efeitos gerais na regulação do crescimento, desenvolvimento e metabolismo das plantas são amplamente explorados, especialmente nas culturas agrícolas. No entanto, essas alterações nas espécies medicinais sob [CO 2 ] elevado ainda são mal compreendidas. Entre as espécies de interesse fitoterápico, destaca-se a Pfaffia glomerata (Spreng.) Pedersen, planta nativa do Brasil e popularmente conhecida como ginseng-brasileiro. As suas propriedades medicinais devem-se principalmente à produção do metabólito secundário 20-hidroxiecdisona (20E), um fitoecdisteroide com comprovadas atividades terapêutica e nutracêutica em mamíferos. Estudos do nosso grupo de pesquisa têm revelado que o enriquecimento da [CO 2 ] in vitro leva à alterações fisiológicas em P. glomerata. Levantamos a hipótese de que o enriquecimento da [CO 2 ] afeta a produção de metabólitos primários e secundários, como o 20E, na espécie-alvo. Portanto, avaliamos o efeito da elevada [CO 2 ] (e[CO 2 ]) nos aspectos morfofisiológicos, bioquímicos, estruturais e moleculares, bem como nas respostas de tolerância ao estresse hídrico. Para tal, foram realizados dois experimentos independentes em câmaras de topo aberto (CTA). Para ambos os experimentos, foram utilizadas plântulas derivadas da micropropagação clonal a partir do banco de germoplasma in vitro. No experimento I, foi analisado a influência de duas concentrações de CO 2 : ambiente (a[CO 2 ]) ± 400 µmol mol -1 ) e elevada (e[CO 2 ]) ± 800 µmol mol -1 ). Após aproximadamente 21 dias de cultivo em OTC, os nossos dados mostraram que a e[CO 2 ] afetou positivamente a fotossíntese (A N ) e uso eficiente da água (WUE), o acúmulo de biomassa, os pigmentos fotossintéticos, o conteúdo de carboidratos, genes da rota de biossíntese de lignina, além de promover alterações no perfil proteômico. Por outro lado, a e[CO 2 ] promoveu redução no teor de proteínas totais, de alguns aminoácidos e do conteúdo de 20E. No experimento II, foram analisadas as respostas fisiológicas e bioquímicas de plantas expostas a e[CO 2 ] e sob estresse hídrico. Durante os 14 dias iniciais do experimento, todas as plantas foram regularmente irrigadas para manter o conteúdo de água do solo próximo da capacidade do campo. Posteriormente, as plantas foram submetidas por 20 dias aos seguintes tratamentos: (I) a[CO 2 ] e sob irrigação regular (a[CO 2 ]I); (II) e[CO 2 ] e sob irrigação regular (e[CO 2 ]I); (III) a[CO 2 ] e sob estresse hídrico (a[CO 2 ]S); e (IV) e[CO 2 ] e sob estresse hídrico (e[CO 2 ]S);. Os nossos dados demonstraram que a e[CO 2 ] alivia os impactos da seca através da redução da condutância estomática (g S ), melhorando o WUE e promovendo potenciais hídricos menos negativos. Embora a redução da gs tenha favorecido a economia de água, ela afetou negativamente a A N , e consequentemente o crescimento das plantas. A análise hiperespectral corrobora com estes achados, sendo uma ótima ferramenta não destrutiva para analisar os efeitos da seca nas plantas. Descobrimos que P. glomerata apresenta diferentes mecanismos de resposta à seca dependendo da [CO 2 ]. Plantas expostas a a[CO 2 ]S investiram no aumento da razão raiz/parte aérea, na rustificação do caule, no sistema antioxidante não enzimático, pelo aumento no teor de antocianina e proteínas do metabolismo do ascorbato e glutationa. Enquanto que as plantas sob e[CO 2 ]S investiram em metabólitos osmorreguladores (e.g. açúcares solúveis e aminoácidos). Verificamos que a e[CO 2 ] promoveu alterações no perfil proteômico, afetando drasticamente o acúmulo de proteínas de resposta ao estresse. Por outro lado, mostramos que a seca promoveu aumento na atividade de enzimas antioxidantes (CAT e SOD) e o conteúdo 20E em ambas as [CO 2 ] em plantas de P. glomerata. Porém, somente nas plantas expostas a e[CO 2 ]S houve aumento da produção de 20E, superando a limitação de biomassa causada por esse estresse. Aqui, relatamos pela primeira vez, a regulação positiva da proteína cytochrome P450 CYP72A219-like em plantas cultivadas na combinação e[CO 2 ] e seca. Portanto, acreditamos em uma relação dessa proteína com a biossíntese de 20E e supomos uma possível sinalização de ROS (indiretamente pelo aumento de CAT e SOD) na produção de 20E em condição de estresse abiótico. Por fim, nossos dados fornecem informações importantes na elucidação da rota de biossíntese de 20E, além de possibilitar estratégias biotecnológicas para aumentar a produção desse metabólito em plantas de P. glomerata. Palavras-chave: Ginseng-brasileiro. 20-hidroxiecdisona. Fitoecdisteroides. Estresse hídrico. enriquecimento de CO 2 . Metabolismo secundário.Coordenação de Aperfeiçoamento de Pessoal de Nível Superio

Light quality in plant tissue culture: does it matter?

The primary issues regarding the lack of protocol reproducibility among laboratories are environmental factors. Light (quantity and particularly quality), is one of those main factors, and studies seldom present the spectral quality of the light sources used. With the advent of light-emitting diode (LED) technology, impressive progress has been made in environmental controls and morphogenetic responses, as directed by the light used in the culture shelves. A wide array of LED lights with different spectra are currently available and light is important in large-scale propagation, especially liquid bioreactor systems. LED technology continues to evolve rapidly and has created additional possibilities. This laboratory has dedicated extensive efforts to implement photoautotrophic propagation, and light is a key component of the system. This review presents relevant topics on the influence of light in various plant tissue culture-based techniques

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

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

Characterisation of microbial attack on archaeological bone

As part of an EU funded project to investigate the factors influencing bone preservation in the archaeological record, more than 250 bones from 41 archaeological sites in five countries spanning four climatic regions were studied for diagenetic alteration. Sites were selected to cover a range of environmental conditions and archaeological contexts. Microscopic and physical (mercury intrusion porosimetry) analyses of these bones revealed that the majority (68%) had suffered microbial attack. Furthermore, significant differences were found between animal and human bone in both the state of preservation and the type of microbial attack present. These differences in preservation might result from differences in early taphonomy of the bones. © 2003 Elsevier Science Ltd. All rights reserved