Frost tolerance and extracellular ice formation in needles of Pinus radiata

Conifers are some of the most frost tolerant tree species. Freezing can damage tissues that have not acclimated to low temperatures. During the freezing process, extracellular ice may form, drawing water out of cells with negative consequences from this dehydration. cryo-Scanning Electron Microscopy (cryo-SEM) can be used to visualize this ice formation and allow a better understanding of frost tolerance. Acclimated and un-acclimated needles of Pinus radiata were subjected to freezing treatments (at a rate of 2 ^oC h^-1), tested for electrolyte leakage and sampled for cryo-SEM analysis. Half maximal electrolyte leakage occurred at -4 and -12 ^oC for un-acclimated and acclimated needles, respectively. During freezing, mesophyll cells shrink, exhibiting cytorrhysis, while extracellular ice fills gas spaces of the mesophyll tissue. Mesophyll cells from acclimated needles recover their structure after thawing while un-acclimated mesophyll show significant dehydration damage. In hydraulic tissue, transfusion tracheids expand as ice accumulates in them during freezing. This behavior was reversible, and may play an important role in the water relations of needles during freeze/thaw events. To our knowledge, this is the first report of cryo-SEM images from conifer needles. This technique may help unravel the mystery of frost tolerance and how conifers can inhabit extremely cold environments such as those found in alpine and boreal forests.