Bud primordia of Picea abies, despite a frozen shoot, stay ice free down to −50 °C by a mechanism termed supercooling whose biophysical and biochemical requirements are poorly understood.Bud architecture was assessed by 3D—reconstruction, supercooling and freezing patterns by infrared video thermography, freeze dehydration and extraorgan freezing by water potential measurements, and cell‐specific chemical patterns by Raman microscopy and mass spectrometry imaging.A bowl‐like ice barrier tissue insulates primordia from entrance by intrinsic ice. Water repellent and densely packed bud scales prevent extrinsic ice penetration. At −18 °C, break‐down of supercooling was triggered by intrinsic ice nucleators whereas the ice barrier remained active. Temperature‐dependent freeze dehydration (−0.1 MPa K−1) caused accumulation of extraorgan ice masses that by rupture of the shoot, pith tissue are accommodated in large voids. The barrier tissue has exceptionally pectin‐rich cell walls and intercellular spaces, and the cell lumina were lined or filled with proteins, especially near the primordium. Primordial cells close to the barrier accumulate di, tri and tetrasaccharides.Bud architecture efficiently prevents ice penetration, but ice nucleators become active inside the primordium below a temperature threshold. Biochemical patterns indicate a complex cellular interplay enabling supercooling and the necessity for cell‐specific biochemical analysis.

Main conclusion An extremely high resolution infrared camera demonstrated various freezing events in wheat under natural conditions. Many of those events shed light on years of misunderstanding regarding freezing in small grains.Infrared thermography has enhanced our knowledge of ice nucleation and propagation in plants through visualization of the freezing process. The majority of infrared analyses have been conducted under controlled conditions and often on individual organs instead of whole plants. In the present study, high-definition (1280 × 720 pixel resolution) infrared thermography was used under natural conditions to visualize the freezing process of wheat plants during freezing events in 2016 and 2017. Plants within plots were found to freeze one at a time throughout the night and in an apparently random manner. Leaves on each plant also froze one at a time in an age-dependent pattern with oldest leaves freezing first. Contrary to a common assumption that freezing begins in the upper parts of leaves; freezing began at the base of the plant and spread upwards. The high resolution camera used was able to verify that a two stage sequence of freezing began within vascular bundles. Neither of the two stages was lethal to leaves, but a third stage was demonstrated at colder temperatures that was lethal and was likely a result of dehydration stress; this stage of freezing was not detectable by infrared. These results underscore the complexity of the freezing process in small grains and indicate that comprehensive observational studies are essential to identifying and selecting freezing tolerance traits in grain crops.

Freezing triggers extracellular ice formation leading to cell dehydration and deformation during a freeze-thaw cycle. Many plant species increase their freezing tolerance during This article is protected by copyright. All rights reserved. exposure to low, non-freezing temperatures, a process termed cold acclimation. In addition, exposure to mild freezing temperatures after cold acclimation evokes a further increase in freezing tolerance (sub-zero acclimation). Previous transcriptome and proteome analyses indicate that cell wall remodeling may be particularly important for sub-zero acclimation. In the present study, we used a combination of immunohistochemical, chemical and spectroscopic analyses to characterize the cell walls of Arabidopsis thaliana and characterized a mutant in the XTH19 gene, encoding a xyloglucan endotransglucosylase/hydrolase (XTH). The mutant showed reduced freezing tolerance after both cold and sub-zero acclimation, compared to the Col-0 wild type, which was associated with differences in cell wall composition and structure. Most strikingly, immunohistochemistry in combination with 3D reconstruction of centers of rosette indicated that epitopes of the xyloglucan-specific antibody LM25 were highly abundant in the vasculature of Col-0 plants after sub-zero acclimation but absent in the XTH19 mutant. Taken together, our data shed new light on the potential roles of cell wall remodeling for the increased freezing tolerance observed after low temperature acclimation.

The crown is the below ground portion of the stem of a grass which contains meristematic cells that give rise to new shoots and roots following winter. To better understand mechanisms of survival from freezing, a histological analysis was performed on rye, wheat, barley and oat plants that had been frozen, thawed and allowed to resume growth under controlled conditions. Extensive tissue disruption and abnormal cell structure was noticed in the center of the crown of all 4 species with relatively normal cells on the outside edge of the crown. A unique visual response was found in oat in the shape of a ring of cells that stained red with Safranin. A tetrazolium analysis indicated that tissues immediately inside this ring were dead and those outside were alive. Fluorescence microscopy revealed that the barrier fluoresced with excitation between 405 and 445 nm. Three dimensional reconstruction of a cross sectional series of images indicated that the red staining cells took on a somewhat spherical shape with regions of no staining where roots entered the crown. Characterizing changes in plants recovering from freezing will help determine the genetic basis for mechanisms involved in this important aspect of winter hardiness.