Rapid freeze acclimation of Poncirus trifoliata seedlings exposed to 10 degrees C and long days

Poncirus trifoliata (L.) Raf. seeds were germinated in perlite under intermittent mist at about 25 degrees C and natural daylight in a greenhouse. Two-week-old seedlings were then transferred into a growth chamber at 25 degrees C and 16-hour daylength for 1 week. Tissue samples were collected at 0, 6, 24, 168, and 504 hours after temperature equilibration at 10 degrees C. Freezing tolerance at -6.7 degrees C, as determined by electrolyte leakage, and stem (leaves attached) water potential (psi x), measured using a pressure chamber, was recorded for a subset of seedlings for each time interval. Red coloration (apparently anthocyanin) developed at the petiole leaflet junction and buds after 48 hours at 10 degrees C and gradually occurred throughout the leaves during further exposure. Complementary DNA clones for phenylalanine ammonia lyase (PAL), 4-coumarate: coA ligase (4CL), and chalcone synthase (CHS) were used to probe RNA isolated from the leaves. No increase in steady-state messenger RNA level was detected. Increases in freeze hardiness occurred within 6 hours in the leaves, and continued for up to 1 week. Water potential initially decreased from -0.6 to -2.0 MPa after 6 hours, then returned to -0.6 MPa after 1 week. Thus, Poncirus trifoliata seedlings freeze-acclimate significantly after only 6 hours at 10 degrees C