Putrescine Biosynthesis Inhibition in Tomato by DFMA and DFMO Treatment

Materials and Reagents

1. Tomato seeds (Solanum lycopersicum Mill. cv. Ailsa Craig)
   
2. Vermiculite (Asfaltex SA, TERMITA^®)
   
3. Potassium hydroxide (KOH) (Scharlab, catalog number: PO0275 )
   
4. Potassium sulfate (K₂SO₄) (Scharlab, catalog number: PO0365 )
   
5. Ortho-Phosphoric acid (H₃PO₄) (Scharlab, catalog number: AC1100 )
   
6. Ammonium sulfate [(NH₄)₂SO₄] (Avantor Performance Materials, J.T.Baker, catalog number: 4628 )
   
7. Calcium sulfate dihydrate (CaSO₄·2H₂O) (Scharlab, catalog number: CA0285 )
   
8. Magnesium sulfate heptahydrate (MgSO₄·7H₂O) (Scharlab, catalog number: MA0085 )
   
9. Boric acid (H₃BO₃) (AppliChem, catalog number: 131015 )
   
10. Manganese(II) sulfate monohydrate (MnSO₄) (Scharlab, catalog number: MA0131 )
    
11. Zinc sulfate heptahydrate (ZnSO₄·7H₂O) (Scharlab, catalog number: CI0207 )
    
12. Copper(II) sulfate pentahydrate (CuSO₄·5H₂O) (Scharlab, catalog number: CO0101 )
    
13. Molybdenum trioxide (MoO₃) (Panreac, Vidrafoc, catalog number: 142791 )
    
14. Sequestrene (Fe 6%) (Syngenta)
    
15. 2-(N-Morpholino) ethanesulfonic acid sodium salt (MES sodium salt) (Sigma-Aldrich, catalog number: M3885 )
    
16. DL-α-(Difluoromethyl)arginine (DFMA) (Santa Cruz Biotechnology, catalog number: sc-211368 )
    
17. DL-α-(Difluoromethyl)ornithine hydrochloride (DFMO) (Santa Cruz Biotechnology, catalog number: sc-252762 )
    
18. Distilled water
    
19. Nutrient solution (see Recipes)
    
20. Inhibitors mix (see Recipes)
    

Equipment

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

Software

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

Procedure

1. Tomato growth conditions
   1. Tomato seeds (Solanum lycopersicum Mill. cv. Ailsa Craig) are germinated in pots (50 ml) with 100% vermiculite in a growth room under the following environmental conditions: light/dark cycle of 16/8 h, temperature of 24/18 °C, light intensity of 200 μmol m^-2 s^-1, and relative humidity of 60%.
      
   2. Seeds are irrigated twice a week with approximately 50 ml/pot of distilled water during 1 week.
      
   3. Seedlings are irrigated twice each week for 3 weeks with approximately 50 ml/pot of nutrient solution. N is supplied in the form of [(NH₄)₂SO₄] 0.33 g/L.
      
   4. The pH of the nutrient solution is adjusted to 6.0 with 1 mM KOH.
      
      
2. Application of inhibitor mix to tomato plants
   
   1. 20 uniform tomato plants grown as described above are divided into two groups (10 plants are not treated and 10 plants are treated with the inhibitor solution).
      
   2. 21-day-old tomato plants are treated with 2 ml of the inhibitor mix by soil drench (final concentration in each pot is DMFA 80 nM and DFMO 200 nM)
      
   3. 23-day-old tomato plants are treated with 4 ml of the inhibitor mix by soil drench (final concentration in each pot is DMFA 160 nM and DFMO 400 nM).
      
   4. 25-day-old and 27-day-old tomato plants are treated with 8 ml of the inhibitor mix (final concentration in each pot is DMFA 320 nM and DFMO 800 nM) (Figure 1). Inhibitors mix treatment does not produce changes in tomato growth (Figure 2).
      
   5. 24 h after the last treatment, the 3rd and 4th true leaves of treated or not treated tomato plants (Figure 3) are collected and stored at -80 °C.
      
   6. A pool of 10 plants is crushed in liquid N₂ in a mortar and 0.2 g is further used for putrescine extraction.
      
   7. Three biological replicated are realized.
      
      
3. Putrescine quantification
   
   1. Putrescine quantification is realized following the protocol of Sanchez-Lopez et al. (2009) by ion pair LC coupled with electrospray tandem mass spectrometry.
      
   2. Putrescine content is expressed as the average of three independent experiments (Fernández-Crespo et al., 2015).
      
      
      Figure 1. Application of inhibitor mix to tomato plants grown in vermiculite
      
      
      Figure 2. Tomato plants treated with inhibitor mix do not display changes in growth. A. Control and treated 21-day-old tomato plants; B. The same plants after one week of inhibitors treatment.
      
      
      Figure 3. Tomato plant with five true leaves. The arrows indicate the 3rd and 4th true leaf of tomato plants collected for putrescine quantification.
      

Data analysis

Statistical analysis is carried out using a one-way analysis of variance in the Statgraphics-plus software of Windows V.5 (Statistical Graphics Corp., Rockville, MD, USA). The putrescine content means are calculated and expressed with standard errors and compared using a Fisher’s least-significant difference test at the 95% confidence interval. The experiment is repeated three times.

Notes

Fernández-Crespo et al. (2015) demonstrated that tomato plants grown with nutrient solution containing NH₄⁺ 5 mM as a solely N source displayed high putrescine content compared with control plants, to which N was provided as NO₃^- form [(KNO₃ (4 mM) and Ca(NO₃)₂ (5 mM)]. Putrescine reduction (35.3%) by inhibitors application was tested in tomato plants grown with (NH₄)₂SO₄ as N source.

Recipes

1. Nutrient solution
   0.33 g/L (NH₄)₂SO₄
   0.35 g/L K₂SO₄
   0.07 ml/L H₃PO₄
   0.04 g/L MgSO₄·7H₂O
   0.54 g/L CaSO₄·2H₂O
   2.86 mg/L H₃BO₃
   2.2 mg/L ZnSO₄·7H₂O
   0.11 mg/L CuSO₄·5H₂O
   0.15 mg/L MnSO₄
   0.09 mg/L MoO₃
   6.7 mg/L sequestrene (6% Fe)
   217 mg/L MES sodium salt
   Adjust pH to 6.0 with 1 mM KOH
   
2. Inhibitors mix
   DL-α-(Difluoromethyl)arginine DFMA (2 mM)
   DL-α-(Difluoromethyl)ornithine hydrochloride DFMO (5 mM)
   Both compounds were dissolved in distilled water.
   Note: Both solutions (Nutrient solution and Inhibitors mix) do not need to be sterilized.
   

Acknowledgments

The work was supported by a grant from the Spanish Ministry of Science and Innovation (AGL2013-49023-C-2-R). This protocol has been modified from Mochou et al. (2008).

References

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