For details on experimental protocols, etc., see the
published paper (Welti et al., 2002, J. Biol. Chem. 277, 31994).
Treatments: Arabidopsis wild-type (Columbia ecotype) and PLD alpha-deficient plants (Fan, L., Zheng, S., and Wang, X. (1997) Plant Cell 9, 2183‑2196) were sown in Scotts Metromix soil. The pots were kept at 4ºC for 2 days, then moved to a growth chamber at 23ºC (day) and 19ºC (night) with a 12-hour day length, daytime fluorescent lighting at 120 µmol m-2s-1, and 58% relative humidity. For cold acclimation, 35-day-old, pre-flowering plants were placed at 4ºC for 3 days in a growth chamber with a light intensity of 30 µmol m-2s-1. Non-acclimated plants remained in the 23ºC growth chamber until they were harvested on the same day that the cold-acclimated plants were harvested. For freezing, a second set of cold-acclimated plants at 4ºC were subjected to a temperature drop from 4ºC to -2ºC at 3ºC per hour in the growth chamber. When the temperature reached -2ºC, ice crystals were placed on the soil to induce crystallization and prevent super-cooling. After 2 hours at -2ºC, the temperature was lowered to -8ºC at 1ºC per hour. After 2 hours at -8ºC, the plants were harvested for lipid analysis.
Sampling: Each sample for lipid profiling consisted of the above-ground rosette of two or three plants that were cut at the sampling time and transferred to 3 ml isopropanol with 0.01% butylated hydroxytoluene (BHT) at 75ºC, followed by extraction with chloroform/methanol. Five replicates were analyzed for each treatment/genotype combination.
Excel file: In the Excel file, one sheet contains the calculated lipid amount data from individual samples ("Individual data") and the other sheet contains averages for each treatment and genotype. Units = nmol/ mg dry weight remaining after extraction. The data were processed as described in the JBC paper. The red data in the "individual data" sheet were not included in the averages, because they were eliminated by the Q-test.
Graphic data: There are three sets of data in the graphs: the data from non-acclimated plants, cold-acclimated plants, and those treated at -8ºC. The cold-acclimated data is from the non-acclimation/cold-acclimation experiment. (The two sets of cold acclimation data are similar, with some notable differences in the levels of lysolipids.)
Abbreviations: "PLDa-def" is the designation for the strain in which PLD alpha is antisense-supressed, and "wt" is the designation for wild-type. "Non-cold-ac" or "Non-ac" = not cold-acclimated; “cold-ac" = cold acclimated; "-8" = treated at -8ºC. The lipid species are designated by "total carbon number: total number of double bonds