How to make known quantities of volatile MDA for use in gene expression studies

Support material for Weber et al., Plant J. (2004)

The basic protocol was published previously in Volleweider et al., Plant J. 24, 467-476 (2000).

MDA is produced from malondialdehyde tetrabutylammonium salt (Fluka # 63278). The MDA salt (0.15g) is treated with 1M HCl (0.5 ml) in a closed glass vial with two tubes connected. The inlet tube bubbles N2 through the solution, the outlet tube (with volatile MDA transported in the N2 gas flow) channels volatiles through an anhydrous MgSO4 trap (which removes water) into a collector falcon tube containing EtOH (3 ml) on ice. After 30 min the EtOH from the collector tube is recovered and can be concentrated under a stream of nitrogen. This MDA stock can be stored at –80°C ( max. 3 weeks).

The MDA concentration in the EtOH from the collector tube is determined spectrophotometrically. The molar extinction coefficient of MDA in 10mM Na3PO4 is 31500 at 267nm (Esterbauer et al. (1991) Free radical Biology and Medicine 11, 81-128).

If 1µl of the MDA stock is measured in a cuvette with a 1 cm light path containing 0.999ml Na3PO4 (10 mM), the concentration of MDA in the stock can be calculated by: cMDA [µmol/µl] = A267/31.5.

Example :

You have 100 µl of MDA stock. The absorption at 267nm of 1 µl of a 1:10 dilution of your stock in 1ml Na3PO4 is 0.500.
cMDA=0.500/31.5
cMDA=0.0159 µmol/µl of the dilution; 0.0159 x 10= 0.159 µmol/µl in the original stock.
The total quantity of MDA is 0.158 x 100= 15.9 µmol

For treating plants with volatile MDA, plants are kept in airtight containers in the light and the desired amount of MDA is pipetted from the EtOH stock on cotton wicks in the containers, from where the compound will go into the gas phase. Always use an equivalent amount of ethanol for the control. A good control compound for MDA is pyruvaldehyde which has the same atomic formula as MDA but has different chemical activity and is not as strong inducer of gene expression. For Arabidopsis, 4 micromoles per Liter MDA gives a very strong response, affecting gene expression within less than 1 hour. We routinely look at expressed genes 3 hours after treatment.