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1.
MethodsX ; 7: 101098, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33102159

RESUMO

•This work describes a protocol for hairy root transformation of the medicinal crop legume fenugreek (Trigonella foenum-graecum L.). Hairy root plant transformation mediated by Agrobacterium rhizogenes is an established method for the rapid genetic transformation of various dicotyledonous plants. We have adapted a hairy root transformation protocol from the model legume Medicago truncatula for use in this metabolically rich non-model crop legume. Considering the great variety and abundance of phytochemicals in fenugreek and its established use in traditional medicine, we aim for this method to become a resource for metabolic pathway identification and for production of valuable specialised metabolites via metabolic engineering approaches.•Development rapid transformation (2.5-3 weeks) of fenugreek roots via A. rhizogenes.•Marker gene cassette with suitable promoter for visual detection of transformed fenugreek roots.

2.
Nat Chem Biol ; 16(7): 740-748, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32424305

RESUMO

Glycosylation is one of the most prevalent molecular modifications in nature. Single or multiple sugars can decorate a wide range of acceptors from proteins to lipids, cell wall glycans and small molecules, dramatically affecting their activity. Here, we discovered that by 'hijacking' an enzyme of the cellulose synthesis machinery involved in cell wall assembly, plants evolved cellulose synthase-like enzymes (Csls) and acquired the capacity to glucuronidate specialized metabolites, that is, triterpenoid saponins. Apparently, endoplasmic reticulum-membrane localization of Csls and of other pathway proteins was part of evolving a new glycosyltransferase function, as plant metabolite glycosyltransferases typically act in the cytosol. Discovery of glucuronic acid transferases across several plant orders uncovered the long-pursued enzymatic reaction in the production of a low-calorie sweetener from licorice roots. Our work opens the way for engineering potent saponins through microbial fermentation and plant-based systems.


Assuntos
Regulação da Expressão Gênica de Plantas , Glucosiltransferases/genética , Glicosiltransferases/genética , Proteínas de Plantas/genética , Saponinas/biossíntese , Spinacia oleracea/metabolismo , Terpenos/metabolismo , Beta vulgaris/genética , Beta vulgaris/metabolismo , Membrana Celular/metabolismo , Parede Celular/metabolismo , Celulose/metabolismo , Retículo Endoplasmático/metabolismo , Cromatografia Gasosa-Espectrometria de Massas , Glucosiltransferases/metabolismo , Ácido Glucurônico/metabolismo , Glicosilação , Glicosiltransferases/metabolismo , Glycyrrhiza/genética , Glycyrrhiza/metabolismo , Células Vegetais/metabolismo , Proteínas de Plantas/metabolismo , Raízes de Plantas/metabolismo , Spinacia oleracea/genética
3.
Plant Physiol Biochem ; 109: 452-466, 2016 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-27816826

RESUMO

Glucosinolates (GSLs) are a highly important group of secondary metabolites in the Caparalles order, both due to their significance in plant-biome interactions and to their chemoprotective properties. This study identified genes involved in all steps of aliphatic and indolic GSL biosynthesis in Eruca sativa, a cultivated plant closely related to Arabidopsis thaliana with agronomic and nutritional value. The impact of nitrogen (N) and sulfur (S) availability on GSL biosynthetic pathways at a transcriptional level, and on the final GSL content of plant leaf and root tissues, was investigated. N and S supply had a significant and interactive effect on the GSL content of leaves, in a structure-specific and tissue-dependent manner; the metabolites levels were significantly correlated with the relative expression of the genes involved in their biosynthesis. A more complex effect was observed in roots, where aliphatic and indolic GSLs and related biosynthetic genes responded differently to the various nutritional treatments suggesting that nitrogen and sulfur availability are important factors that control plant GSL content at a transcriptional level. The biological activity of extracts derived from these plants grown under the specific nutritional schemes was examined. N and S availability were found to significantly affect the cytotoxicity of E. sativa extracts on human cancer cells, supporting the notion that carefully designed nutritional schemes can promote the accumulation of chemoprotective substances in edible plants.


Assuntos
Brassicaceae/metabolismo , Glucosinolatos/biossíntese , Antineoplásicos Fitogênicos/isolamento & purificação , Antineoplásicos Fitogênicos/farmacologia , Brassicaceae/genética , Proliferação de Células/efeitos dos fármacos , Clonagem Molecular , Regulação da Expressão Gênica de Plantas , Genes de Plantas , Glucosinolatos/genética , Células HeLa , Células Hep G2 , Humanos , Células MCF-7 , Nitrogênio/metabolismo , Filogenia , Extratos Vegetais/isolamento & purificação , Extratos Vegetais/farmacologia , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Plantas Comestíveis/metabolismo , Estresse Fisiológico , Enxofre/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo
4.
Res Microbiol ; 157(4): 376-85, 2006 May.
Artigo em Inglês | MEDLINE | ID: mdl-16307869

RESUMO

The use of two-phase centrifugal decanters has been widely adopted in the olive oil extraction industry in order to reduce the huge quantities of wastewaters produced during the traditional three-phase extraction process. The resulting sludge-like byproduct, widely known as "alpeorujo", has a pH of 4-6, low water activity (a(w)) and high phytotoxicity. Addition of Ca(OH)(2) to alpeorujo, which is commonly performed at the olive oil mill to handle disposal problems related to acidic pH and odor emissions, creates an alkaline secondary waste (alkaline alpeorujo). Bacteria isolated from alkaline alpeorujo were cultured in order to investigate their physiological and phylogenetic characteristics. The bacterial population at neutral pH was estimated to be 6.0+/-0.4 x 10(7) cells g(-1) dw, while the bacterial population at pH 11 reached 2.1+/-0.3 x 10(5) cells g(-1) dw. Fourteen strains isolated from alkaline pH were halotolerant alkaliphiles, while seven isolates from neutral pH were moderate to extreme halotolerant or/and alkalitolerant bacteria. Based on 16S rRNA gene sequence analysis, four of the halotolerant alkaliphilic isolates showed 98.4-99.2% similarity to known sequences of Bacillus alcalophilus and Nesterenkonia lacusekhoensis, whereas ten isolates demonstrated low percentage similarities (94.4-96.9%) to the genera Idiomarina, Halomonas and Nesterenkonia. As concerns bacteria isolated from neutral pH, four isolates were associated with Corynebacterium, Novosphingobium, Serratia marcescens and Pseudomonas aeruginosa (98.3-99.9% similarities), while three isolates presented 96.5-97.2% sequence similarities to Rhodobacter, Pseudomonas and Ochrobactrum. At least six groups of isolates represent novel phylogenetic linkages among Bacteria.


Assuntos
Bactérias/classificação , Resíduos Industriais/análise , Óleos de Plantas/química , Bactérias/genética , Bactérias/metabolismo , Hidróxido de Cálcio , Microbiologia Ambiental , Concentração de Íons de Hidrogênio , Azeite de Oliva , Fenóis/metabolismo , Filogenia , RNA Ribossômico 16S/genética
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