MOLECULAR AND FUNCTIONAL CHARACTERIZATION OF GENES INVOLVED IN SAPONIN BIOSYNTHESIS IN MEDICAGO SPP.

Saponins are a large group of plant secondary metabolites including triterpenoids and steroids glycosylated with one or more sugar chains (Hostettmann and Marston, 1995). In the Medicago genus saponins are a complex mixture of triterpenic glycosides showing a broad spectrum of biological (antifungal, insecticidal, phytotoxic, allelopathic and hemolytic) and pharmacological properties (anticholesterolemic, anti-cancer adjuvant) (Tava and Avato, 2006). In spite of their role in plant defence mechanisms, their importance as antimicrobial compounds and their possible benefits for human health, knowledge of the genetic control of saponin biosynthesis is still lagging behind. Triterpenic saponins are derived from \uf062-amyrin that is converted to various aglycones by a series of oxidative reactions catalyzed by cytochromes P450; finally glycosyltransferases (GTs) convert aglycones into saponins. In M. truncatula at least three genes encoding early enzymes of the \uf062-amyrin skeleton formation have been functionally characterized (Suzuki et al., 2002). However, in M. truncatula no gene involved in the oxidative reactions of \uf062-amyrin has yet been isolated. In our laboratory, we have used M. truncatula mutant collections to search mutations affecting saponin biosynthesis. In an activation-tagging mutant collection of M. truncatula (Porceddu et al., 2008), one plant lacking hemolytic saponins was evidenced (lha-1 mutant); the T-DNA tagged locus was identified by inverse-PCR and the putative gene implied in this mutation turned out to be a cytochrome P450 named CYP716A12. A second collection, obtained by EMS-mutagenesis (Porceddu et al., 2008), was screened by TILLING analysis and two mutants for the CYP716A12 gene were identified (lha-2 and lha-3). Both these mutants lacked hemolytic saponins, although they accumulated soyasaponins. In addition, all lha mutants lacked sapogenin precursors of hemolytic saponins suggesting that the biosynthetic pathway was blocked at an early and common step. In order to validate the correspondence between the CYP716A12 gene and the lha phenotype, a vector carrying a full-length CDS of CYP716A12 was stably transformed into lha-1 mutant plants and restoration of the biosynthetic pathway of hemolytic saponins was confirmed by GC-MS analyses. This result confirmed the knock-out of CYP716A12 was responsible for the block in hemolytic sapogenin pathway. Objectives of this Ph.D project were: i) investigating the role of cytochrome CYP716A12 in saponin biosynthesis by heterologous expression in yeast (Saccharomyces cerevisiae), ii) studying gene expression levels and sapogenin content in M. truncatula plants at different developmental stages and iii) identifying the orthologue of CYP716A12 in M. sativa. To elucidate the function of CYP716A12, the gene was expressed in yeast (S. cerevisiae); first, the CYP716A12 coding sequence was introduced in a commercial yeast strain. In vivo and in vitro microsome assays were performed using \uf062-amyrin and oleanolic acid as possible substrates of the cytochrome, but no modifications of the supplemented substrates were evidenced. These negative results may be explained by the low efficiency of yeast NADPH-P450 reductase (Pompon et al., 1996) that has been suggested to be important for efficient electron transfer to cytochromes P450. For this reason a new experiment was performed using yeast strains WR and WAT11, overexpressing S. cerevisiae and Arabidopsis thaliana P450 reductase respectively. An in vivo assay was performed, but GC analysis did not show any additional peak except those of the substrates. Enzymatic activities in microsomes (in vitro assay) were then tested by supplying \uf062-amyrin, and erythrodiol as substrates; GC-MS analysis revealed the substrate transformation to yield oleanolic acid. These results indicate that CYP716A12 mainly catalyzes the sequential three-step oxidation at the C-28 position necessary to transform \uf062-amyrin into oleanolic acid. To identify which substrate could restore the saponin pathway in lha-1 mutant, plant microsomal fractions were used. Using \uf062-amyrin and oleanolic acid as substrate, wild-type microsomes produced all sapogenins, while mutant microsomes yielded non-hemolytic sapogenins only. In contrast, when supplemented with hederagenin, neither wild-type nor lha mutant microsomes restored the saponin pathway. These contrasting results may reflect the difficulty in setting up an efficient and stable plant microsome preparation. To investigate the expression profiles of the CYP716A12 gene, quantitative PCR analyses were performed on a wild-type line. Leaf stem and root samples were collected at three biological stages (vegetative growth, early flowering, and early pod setting). Roots displayed highest and most stable expression across stages, while in leaves a significant increase in expression appeared in the reproductive stages with a maximum at flowering. The same plants were examined for sapogenin content by GC/FID analysis. Hemolytic sapogenin content was significantly influenced by both phenological stages and plant organs. Finally, in order to transfer the new findings in hemolytic saponin biosynthesis in crop species, the ORF of the orthologous gene in M. sativa was isolated using the same primers utilized in M. truncatula. In summary, results of this Ph.D work revealed that CYP716A12 is a multifunctional oxidase catalyzing the sequential three-step oxidation at C-28 position necessary to transform \uf062-amyrin into oleanolic acid. CYP716A12 can use different substrates and plays a key role in hemolytic saponin biosynthesis as its disruption prevents the formation of any aglycone with an oxygenated group in C-28 and consequently the presence of triterpenes glycosylated in this position. In contrast to other transcripts involved in triterpenic saponin pathway, generally restricted to root system, CYP716A12 is expressed in different plant organs and developmental stages. Building upon this work, we plan to use the WAT11 yeast expression system to investigate the role of CYP716A12 cytochrome from M. sativa, and perform TILLING analysis on other candidate P450 genes in order to identify and characterize new functions possibly involved in saponin biosynthesis

Overexpression of Osmyb4 enhances compatible solute accumulation and increases stress tolerance of arabidopsis thaliana

In this paper, we report the metabolic and molecular changes in response to cold and drought induced in Osmyb4 transgenic Arabidopsis thaliana compared with the wildtype (WT). The rice Osmyb4 gene codes for a transcription factor (Myb4) induced by cold treatment and, in Arabidopsis transgenic plants, improves cold and freezing tolerance [Vannini C, Locatelli F, Bracale M, Magnani E, Marsoni M, Osnato M, Mattana M, Baldoni E, Coraggio I (2004) Plant J 37: 115-127]. Here, we report the ability of Myb4 to induce also drought tolerance in Arabidopsis transgenic plants. By the use of nuclear magnetic resonance (NMR) and enzymatic assays, we showed that several compatible solutes (glucose, fructose, sucrose, proline, glycine betaine and sinapoyl malate) accumulate in higher amount in Osmyb4-overexpressing plants with respect to the WT, both under normal and stress conditions. Considering proline, we also found that in transgenic plants the levels of the mRNAs coding for \u3941- pyrroline-5-carboxylate synthase (EC not assigned) and for \u3941- pyrroline-5-carboxylate dehydrogenase (EC 1.5.1.12) were higher and lower, respectively. The constitutive activation of several stress-inducible pathways and different kinetics in the accumulation of several metabolites, in Myb4 transgenic plants, may represent an advantage to prepare plants to face the stress condition. Moreover, these results taken together suggest that Myb4 integrates the activation of multiple components of stress response

CYP72A67 catalyses a key oxidative step in Medicago truncatula hemolytic saponin biosynthesis

In the Medicago genus, triterpenic saponins are bioactive secondary metabolites constitutively synthesized in the aerial and subterranean parts of plants via the isoprenoid pathway. Exploitation of saponins as pharmaceutics, agrochemicals and in the food and cosmetic industries has raised interest in identifying the enzymes involved in their synthesis. We have identified a cytochrome P450 (CYP72A67) involved in hemolytic sapogenin biosynthesis by a reverse genetic TILLING approach in a Medicago truncatula ethylmethanesulfonate (EMS) mutagenized collection. Genetic and biochemical analyses, mutant complementation, and expression of the gene in a microsome yeast system showed that CYP72A67 is responsible for hydroxylation at the C-2 position downstreamof oleanolic acid synthesis. The affinity of CYP72A67 for substrates with different substitutions at multiple carbon positions was investigated in the same in vitro yeast system, and in relation to two other CYP450s (CYP72A68) responsible for the production of medicagenic acid, the main sapogenin in M. truncatula leaves and roots. Full sib mutant and wild-type plants were compared for their sapogenin profile, expression patterns of the genes involved in sapogenin synthesis, and response to inoculation with Sinorhizobium meliloti. The results obtained allowed us to revise the hemolytic sapogenin pathway in M. truncatula and contribute to highlighting the tissue specificities (leaves/roots) of sapogenin synthesis

Microalgal biostimulants and biofertilisers in crop productions

Microalgae are attracting the interest of agrochemical industries and farmers, due to their biostimulant and biofertiliser properties. Microalgal biostimulants (MBS) and biofertilisers (MBF) might be used in crop production to increase agricultural sustainability. Biostimulants are products derived from organic material that, applied in small quantities, are able to stimulate the growth and development of several crops under both optimal and stressful conditions. Biofertilisers are products containing living microorganisms or natural substances that are able to improve chemical and biological soil properties, stimulating plant growth, and restoring soil fertility. This review is aimed at reporting developments in the processing of MBS and MBF, summarising the biologically-active compounds, and examining the researches supporting the use of MBS and MBF for managing productivity and abiotic stresses in crop productions. Microalgae are used in agriculture in different applications, such as amendment, foliar application, and seed priming. MBS and MBF might be applied as an alternative technique, or used in conjunction with synthetic fertilisers, crop protection products and plant growth regulators, generating multiple benefits, such as enhanced rooting, higher crop yields and quality and tolerance to drought and salt. Worldwide, MBS and MBF remain largely unexploited, such that this study highlights some of the current researches and future development priorities

Levantamento farmacoepidemiológico de antibióticos dispensados em um bairro da zona leste de São José dos Campos/SP/ Pharmacoepidemiological Survey of Antibiotics Dispensed in a Neighborhouse in the East Zone of São José Dos Campos / SP

Levantamento farmacoepidemiológico de antibióticos dispensados em uma Drogaria de bairro da Zona Leste de São José dos Campos – SP, onde a coleta de dados foi realizada entre os meses de janeiro de 2017 a junho de 2019 através de um relatório de saída de medicamentos (SNGPC) previsto pela ANVISA – RDC 68/2014. Foram coletadas informações sobre os antibióticos mais dispensados para os usuários da comunidade, sendo Amoxicilina e a Azitromicina os de maior frequência. Foram inclusas todas as faixas etárias e diferentes formas farmacêuticas para posterior discussão dos dados obtidos. A finalidade deste estudo foi discutir sobre a importância do uso racional dos antibióticos a fim de prevenir efeitos adversos e resistências bacterianas

Medicamentos uterotônicos utilizados em hemorragia puerperal / Uterotonic drugs used in postpartum haemorrhage

Os medicamentos uterotônicos são fármacos que causam contração uterina e são a primeira linha de escolha no tratamento e prevenção de hemorragia pós-parto. A atonia uterina, quadro onde o útero não consegue manter contrações de forma suficiente, é a principal desencadeadora de hemorragias fatais. Todo hospital e centro obstétrico deve possuir protocolos para o uso de fármacos uterotônicos no pós-parto imediato. O objetivo deste trabalho foi realizar uma revisão bibliográfica sobre os principais medicamentos uterotônicos utilizados na prática obstétrica atualmente. Os dados foram coletados de literaturas e artigos científicospublicados nos últimos 10 anos. Espera-se que o resultado desse trabalho venha a oferecer uma nova fonte de pesquisa para estudantes e profissionais da área da saúde e demonstrar que os fármacos ocitocina, ergometrina e misoprostol são os mais usados atualmente no tratamento e prevenção de hemorragia pós-parto