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.

Automated summary

Cold acclimated plants can enhance freezing survival by reducing cell wall porosity through de-methyl esterification of homogalacturonan and subsequent cross-linking with Ca(2+).