通过DFMA 和 DFMO 抑制番茄中腐胺的生物合成

Emma Fernández-Crespo; Ana Isabel  González-Hernández; Loredana Scalschi; Eugenio Llorens; Pilar García-Agustín; Gemma Camañes

doi:10.21769/BioProtoc.1987

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Peer-reviewed

Putrescine Biosynthesis Inhibition in Tomato by DFMA and DFMO Treatment

通过DFMA 和 DFMO 抑制番茄中腐胺的生物合成

EF Emma Fernández-Crespo

AG Ana Isabel González-Hernández

Loredana Scalschi

Eugenio Llorens

PG Pilar García-Agustín

GC Gemma Camañes email

发布: 2016年11月05日第6卷第21期 DOI: 10.21769/BioProtoc.1987 浏览次数: 8454

评审: Arsalan DaudiWenrong HeAnonymous reviewer(s)

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Abstract

This protocol can be used to inhibit the biosynthesis of polyamines, specifically putrescine, in tomato plants grown with NH₄⁺ as a solely N source. In general, polyamines are positively charged small metabolites implicated in physiological processes, including organogenesis, embryogenesis, floral initiation and development, leaf senescence, pollen tube growth, fruit development and ripening and participate in the response to abiotic and biotic stresses (Tiburcio et al., 2014). Polyamines are synthesized from amino acids by decarboxylation of ornithine or arginine by ornithine decarboxylase (ODC) or arginine decarboxylase (ADC), respectively (Walters, 2003). Tomato plants grown with NH₄⁺ as the sole N source presented an increase of putrescine content in leaves (Fernández-Crespo et al., 2015). To assess the importance of putrescine accumulation, DL-α-(Difluoromethyl)arginine (DFMA) and DL-α-(Difluoromethyl)ornithine (DFMO), inhibitors of putrescine synthesis, were used as irreversible inhibitors of ADC and ODC enzymes, respectively (Fallon and Phillips, 1988), with the purpose of reducing cellular putrescine accumulation induced by NH₄⁺ nutrition.

The inhibitor solution containing 2 mM DFMA and 5 mM DFMO was applied directly to each pot during the week prior to sample collection. Putrescine content was reduced by 35.3% in tomato plants grown with NH₄⁺.

Background

The application of the inhibitors DFMA and DFMO was normally performed in MS medium and in vitro assays (Perez-Amador et al., 2002; Stes et al., 2011). However, we needed to test effectiveness of these inhibitors in vivo with the purpose to maintain natural growth conditions. Moschou et al. (2008) demonstrated the inhibition effect of DFMA and DFMO when applied in hydroponic cultures at 0.1 mM and 1 mM respectively. In this work, we used similar approaches with some modifications: the hydroponic culture was changed by vermiculite growing medium and the concentration applied for the inhibitors was modified.

Materials and Reagents

Tomato seeds (Solanum lycopersicum Mill. cv. Ailsa Craig)
Vermiculite (Asfaltex SA, TERMITA^®)
Potassium hydroxide (KOH) (Scharlab, catalog number: PO0275 )
Potassium sulfate (K₂SO₄) (Scharlab, catalog number: PO0365 )
Ortho-Phosphoric acid (H₃PO₄) (Scharlab, catalog number: AC1100 )
Ammonium sulfate [(NH₄)₂SO₄] (Avantor Performance Materials, J.T.Baker, catalog number: 4628 )
Calcium sulfate dihydrate (CaSO₄·2H₂O) (Scharlab, catalog number: CA0285 )
Magnesium sulfate heptahydrate (MgSO₄·7H₂O) (Scharlab, catalog number: MA0085 )
Boric acid (H₃BO₃) (AppliChem, catalog number: 131015 )
Manganese(II) sulfate monohydrate (MnSO₄) (Scharlab, catalog number: MA0131 )
Zinc sulfate heptahydrate (ZnSO₄·7H₂O) (Scharlab, catalog number: CI0207 )
Copper(II) sulfate pentahydrate (CuSO₄·5H₂O) (Scharlab, catalog number: CO0101 )
Molybdenum trioxide (MoO₃) (Panreac, Vidrafoc, catalog number: 142791 )
Sequestrene (Fe 6%) (Syngenta)
2-(N-Morpholino) ethanesulfonic acid sodium salt (MES sodium salt) (Sigma-Aldrich, catalog number: M3885 )
DL-α-(Difluoromethyl)arginine (DFMA) (Santa Cruz Biotechnology, catalog number: sc-211368 )
DL-α-(Difluoromethyl)ornithine hydrochloride (DFMO) (Santa Cruz Biotechnology, catalog number: sc-252762 )
Distilled water
Nutrient solution (see Recipes)
Inhibitors mix (see Recipes)

Equipment

Plant growth room (A.S.L Snijders)
Pots (50 ml) (Pöppelmann, model: Serie TO )
pH meter (HACH LANGE SPAIN, CRISON, model: GLP21 )

Software

Statgraphics-plus software of Windows V.5 (Statistical Graphics Corp., Rockville, MD, USA)

Procedure

English

中文翻译

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如何引用

Fernández-Crespo, E., González-Hernández, A. I., Scalschi, L., Llorens, E., García-Agustín, P. and Camañes, G. (2016). Putrescine Biosynthesis Inhibition in Tomato by DFMA and DFMO Treatment. Bio-protocol 6(21): e1987. DOI: 10.21769/BioProtoc.1987.