Quantification of ATP in Plant Seedlings

ATP content was determined using the ATP assay kit (Invitrogen) essentially as described [10 (link)], with minor modifications. Entire 3-week-old seedlings from WT and OE lines were ground in liquid nitrogen, and 200 mg of tissue powder homogenized in a 4:2:1 methanol/chloroform/water mixture (1 ml). Samples were vortexed, kept on ice for 30 min and vigorously homogenized for additional 15 min. After the addition of water (1 ml), the samples were centrifuged at 12.200 x g for 5 min for phase separation and subsequently dried in a centrifugal vacuum concentrator. The pellet was resuspended in 10 mM phosphate buffer (pH 7.4). Measurement of ATP content was carried out on a GloMax-Multi Detection System (Promega). The obtained data were subjected to ANOVA (t test), and considered significantly different at the P < 0.05 probability level.