Fresh or frozen plant material was homogenized using a 'Magic Bullet' homogenizer (Homeland Housewares, Los Angeles, CA, USA) for shoots and roots and a 'SK mill' for cultured cells and proline concentration was determined by using the method of Bates et al. (1973).
Ten grams of plant material was homogenized in 20 mL of HPLC grade methanol using an Ultra-Turrax tissue homogenizer (Takmar, Cincinnati, OH, USA) at moderate speed (setting of 60) for 30 s.
50 mg of frozen plant material was homogenized in a 2 mL Eppendorf tube, and 1 ml methanol (100%) containing two internal standards (1µg/mL ampicillin and 1µg/mL chloramphenicol) were added.
One gram of material was homogenized at 0°C in 10 mL buffer I (containing 5 mM EDTA, 1 mM MgCl2, 1 mM MnCl2, 2 mM NaNO3, 0.5 mM K2HPO4 and 0.1 M sucrose).
For each approach, 1 g infectious hamster brain material was homogenized in 10 ml PBS (phosphate buffered saline) and added to 20 g of standard German sandy loom soil (Borstel).
A 250 g sample of seedling material was homogenized in 1000 ml buffer (0.5 M sucrose, 50 mM Tris-HCl, 10 mM EDTA, 1% bovine serum albumin, 5 mmol/L β-mercaptoethanol, 1.5% polyvinyl-pyrrolidone, pH 7.5).
Frozen Arabidopsis leaf material was homogenized using a ball mill, pre-cooled in liquid N2 to avoid thawing, instead of using a mortar as mortar-ground material was insufficiently filtered through a 20 µm nylon net (used instead of quartz wool (data not shown)).
Approximately 10 15 mg frozen plant material was homogenized in a 2 mL Eppendorf tube, and 1 ml metabolite extraction buffer (methanol/chloroform/water; 2.5∶1∶0.5; v/v/v) and 10 µl internal standard D-sorbitol-13C6 (200 µg/mL in H2O) was added subsequently.
