Production of active compounds against Phytophthora cinnamomi by Phlomis purpurea, Metabolite and transcript profiling

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A metabolome analysis and the immunity of Phlomis purpurea against Phytophthora cinnamomi

Publication . Neves, Dina; Figueiredo, Andreia; Maia, Marisa; Laczko, Endre; Pais, Maria Salomé; Cravador, Alfredo

Phlomis purpurea grows spontaneously in the southern Iberian Peninsula, namely in cork oak (Quercus suber) forests. In a previous transcriptome analysis, we reported on its immunity against Phytophthora cinnamomi. However, little is known about the involvement of secondary metabolites in the P. purpurea defense response. It is known, though, that root exudates are toxic to this pathogen. To understand the involvement of secondary metabolites in the defense of P. purpurea, a metabolome analysis was performed using the leaves and roots of plants challenged with the pathogen for over 72 h. The putatively identified compounds were constitutively produced. Alkaloids, fatty acids, flavonoids, glucosinolates, polyketides, prenol lipids, phenylpropanoids, sterols, and terpenoids were differentially produced in these leaves and roots along the experiment timescale. It must be emphasized that the constitutive production of taurine in leaves and its increase soon after challenging suggests its role in P. purpurea immunity against the stress imposed by the oomycete. The rapid increase in secondary metabolite production by this plant species accounts for a concerted action of multiple compounds and genes on the innate protection of Phlomis purpurea against Phytophthora cinnamomi. The combination of the metabolome with the transcriptome data previously disclosed confirms the mentioned innate immunity of this plant against a devastating pathogen. It suggests its potential as an antagonist in phytopathogens’ biological control. Its application in green forestry/agriculture is therefore possible.

2023-05-09Journal article

Open access

Isolamento e purificação do nortriterpeno phlomispurpentaolone a partir de raízes de Phlomis purpurea e determinação da sua atividade citotóxica nas linhas celulares HeLa e L929

Publication . Teixeira, Rúben Filipe Corvo; Cravador, A.

O cancro constitui, atualmente, uma das principais preocupações em saúde, não só ao nível do incremento do arsenal terapêutico antineoplásico disponível, através da incansável procura de fármacos mais potentes e eficazes, como também ao nível socioeconómico, na tentativa de fazer chegar essas terapêuticas a todos os doentes oncológicos. Ao longo dos séculos, as plantas revelaram-se preciosas fontes de novas moléculas, que estruturalmente modificadas no laboratório, ou não, resultaram por vezes na descoberta de novos e potentes fármacos. Estudos recentes em espécies vegetais do género Phlomis, mostraram que produzem numerosos nortriterpenos, alguns com propriedades antiproliferativas. A marioila (Phlomis purpurea L) é uma herbácea, da família das Lamiaceae, existente no Sul de Portugal e de onde se pode extrair phlomispurpentaolone, um nortriterpeno pouco caracterizado farmacologicamente, até à data. Neste trabalho, foi extraído, isolado e purificado phlomispurpentaolone a partir das raízes secundárias de P. purpurea. Foi determinada a atividade ótica deste nortriterpeno, para a qual foi registado um valor de +3.77, a λ= 589.3 nm. Foram, também, realizados ensaios de citotoxicidade em duas linhas de células tumorais – HeLa e L929 – com o intuito de avaliar uma possível atividade antineoplásica daquele composto. Para a determinação da concentração inibitória média (IC50) das células HeLa, foi utilizado o método Thiazolyl Blue Tetrazolium Bromide, com posterior leitura das absorvâncias a 570 nm. Recorrendo ao software Graphpad, foi traçada a curva de toxicidade de phlomispurpentaolone e foi verificado que o valor de IC50 relativo às células HeLa era de 22.63 ±1.07 μM. A partir do ensaio preliminar foi estimado um valor de IC50, nas células L929, compreendido entre 50 e 100 μM. Em relação às células HeLa, o resultado obtido é toxicologicamente promissor, na medida em que é legítimo perspetivar uma potencialização da sua atividade Isolamento e purificação do nortriterpeno phlomispurpentaolone a partir de raízes de Phlomis purpurea e determinação da sua atividade citotóxica nas linhas celulares HeLa e L929 antiproliferativa otimizando a sua purificação e a modificação controlada da sua estrutura química.

2015-11-25Master thesis

Open access

Structure, anti-Phytophthora and anti-tumor activities of a nortriterpenoid from the rhizome of Phlomis purpurea (Lamiaceae)

Publication . Mateus, Maria Da Conceição; Neves, Dina; Dacunha, Bruno; Laczko, Endre; Maia, Cristiana; Teixeira, Rúben; Cravador, Alfredo

To investigate bioactive compounds potentially involved in the biotic interactions exhibited by Phlomis purpurea (Lamiaceae) in rhizospheres infested with Phytophthora cinnamomi, the plant rhizome was chemically analysed. The nortriterpenoid (175)-2 alpha,3 alpha,11 alpha,23,24-pentahydroxy-19(18 -> 17)-abeo-28-norolean-12-en-18-one, was isolated and its structure was elucidated by comprehensive spectroscopic analysis, chiefly using 2D NMR experiments, and X-ray analysis. It was shown to be exuded by roots and to exhibit anti-Phytophthora and antitumor activities. (C) 2016 Published by Elsevier Ltd.

2016-07Journal article

Restricted access

De novo assembly of Phlomis purpurea after challenging with Phytophthora cinnamomi

Publication . Baldé, Aladje; Neves, Dina; García-Breijo, Francisco J; Pais, Maria S; Cravador, A.

Background Phlomis plants are a source of biological active substances with potential applications in the control of phytopathogens. Phlomis purpurea (Lamiaceae) is autochthonous of southern Iberian Peninsula and Morocco and was found to be resistant to Phytophthora cinnamomi. Phlomis purpurea has revealed antagonistic effect in the rhizosphere of Quercus suber and Q. ilex against P. cinnamomi. Phlomis purpurea roots produce bioactive compounds exhibiting antitumor and anti-Phytophthora activities with potential to protect susceptible plants. Although these important capacities of P. purpurea have been demonstrated, there is no transcriptomic or genomic information available in public databases that could bring insights on the genes underlying this anti-oomycete activity. Results Using Illumina technology we obtained a de novo assembly of P. purpurea transcriptome and differential transcript abundance to identify putative defence related genes in challenged versus non-challenged plants. A total of 1,272,600,000 reads from 18 cDNA libraries were merged and assembled into 215,739 transcript contigs. BLASTX alignment to Nr NCBI database identified 124,386 unique annotated transcripts (57.7%) with significant hits. Functional annotation identified 83,550 out of 124,386 unique transcripts, which were mapped to 141 pathways. 39% of unigenes were assigned GO terms. Their functions cover biological processes, cellular component and molecular functions. Genes associated with response to stimuli, cellular and primary metabolic processes, catalytic and transporter functions were among those identified. Differential transcript abundance analysis using DESeq revealed significant differences among libraries depending on post-challenge times. Comparative cyto-histological studies of P. purpurea roots challenged with P. cinnamomi zoospores and controls revealed specific morphological features (exodermal strips and epi-cuticular layer), that may provide a constitutive efficient barrier against pathogen penetration. Genes involved in cutin biosynthesis and in exodermal Casparian strips formation were up-regulated. Conclusions The de novo assembly of transcriptome using short reads for a non-model plant, P. purpurea, revealed many unique transcripts useful for further gene expression, biological function, genomics and functional genomics studies. The data presented suggest a combination of a constitutive resistance and an increased transcriptional response from P. purpurea when challenged with the pathogen. This knowledge opens new perspectives for the understanding of defence responses underlying pathogenic oomycete/plant interaction upon challenge with P. cinnamomi.

2017-09-06Journal article

Open access

A metabolome analysis and the immunity of Phlomis purpurea against Phytophthora cinnamomi

Publication . Neves, Dina; Figueiredo, Andreia; Maia, Marisa; Laczko, Endre; Pais, Maria Salomé; Cravador, Alfredo

Phlomis purpurea grows spontaneously in the southern Iberian Peninsula, namely in cork oak (Quercus suber) forests. In a previous transcriptome analysis, we reported on its immunity against Phytophthora cinnamomi. However, little is known about the involvement of secondary metabolites in the P. purpurea defense response. It is known, though, that root exudates are toxic to this pathogen. To understand the involvement of secondary metabolites in the defense of P. purpurea, a metabolome analysis was performed using the leaves and roots of plants challenged with the pathogen for over 72 h. The putatively identified compounds were constitutively produced. Alkaloids, fatty acids, flavonoids, glucosinolates, polyketides, prenol lipids, phenylpropanoids, sterols, and terpenoids were differentially produced in these leaves and roots along the experiment timescale. It must be emphasized that the constitutive production of taurine in leaves and its increase soon after challenging suggests its role in P. purpurea immunity against the stress imposed by the oomycete. The rapid increase in secondary metabolite production by this plant species accounts for a concerted action of multiple compounds and genes on the innate protection of Phlomis purpurea against Phytophthora cinnamomi. The combination of the metabolome with the transcriptome data previously disclosed confirms the mentioned innate immunity of this plant against a devastating pathogen. It suggests its potential as an antagonist in phytopathogens’ biological control. Its application in green forestry/agriculture is therefore possible.

2023-05-09Journal article

Open access