Cold and exogenous calcium alter Allium fistulosum cell wall pectin to depress intracellular freezing temperatures

De-methyl esterification of homogalacturonan and subsequent cross-linking with Ca (2+) is hypothesized to enhance the freezing survival of cold acclimated plants by reducing the porosity of primary cell walls. To test this theory, we collected leaf epidermal peels from non- (23/18 degrees C) and cold acclimated (2 weeks at 12/4 degrees C) Japanese bunching onion (Allium fistulosum L.). Cold acclimation enhances the temperature at which half the cells survived freezing injury by 8 degrees C (LT50 = -20 degrees C) and reduces tissue permeability by 70-fold as compared with non-acclimated epidermal cells. These effects were associated with greater activity of pectin methylesterase (PME) and a reduction in the methyl esterification of homogalacturonan. Non-acclimated plants treated with 50 mM CaCl2 accumulated higher concentrations of galacturonic acid, Ca (2+) in the cell wall and a lower number of visible cell wall pores compared to levels observed in cold acclimated plants. Using cryo-microscopy, we observed that a 50 mM CaCl2 treatment did not lower the LT50 of non-acclimated cells but reduced the lethal intracellular ice nucleation to temperatures observed in cold acclimated epidermal cells. We postulate the PME-homogalacturonan-mediated reduction in cell wall porosity is integral to intracellular freezing avoidance strategies in cold acclimated herbaceous cells.