Ice segregation in the crown of winter cereals: Evidence for extraorgan and extratissue freezing.

Meaningful improvements in winter cereal cold hardiness requires a complete model of freezing behaviour in the critical crown organ. Magnetic resonance microimaging diffusion-weighted experiments provided evidence that cold acclimation decreased water content and mobility in the vascular transition zone (VTZ) and the intermediate zone in rye (Secale cereale L. Hazlet) compared with wheat (Triticum aestivum L. Norstar). Differential thermal analysis, ice nucleation, and localization studies identified three distinct exothermic events. A high-temperature exotherm (-3°C to -5°C) corresponded with ice formation and high ice-nucleating activity in the leaf sheath encapsulating the crown. A midtemperature exotherm (-6°C and -8°C) corresponded with cavity ice formation in the VTZ but an absence of ice in the shoot apical meristem (SAM). A low-temperature exotherm corresponded with SAM injury and the killing temperature in wheat (-21°C) and rye (-27°C). The SAM had lower ice-nucleating activity and freezing survival compared with the VTZ when frozen in vitro. The intermediate zone was hypothesized to act as a barrier to ice growth into the SAM. Higher cold hardiness of rye compared with wheat was associated with higher VTZ and intermediate zone desiccation resulting in the formation of ice barriers surrounding the SAM.

High-definition infrared thermography of ice nucleation and propagation in wheat under natural frost conditions and controlled freezing.

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.

The use of high-resolution infrared thermography (HRIT) for the study of ice nucleation and ice propagation in plants.

Freezing events that occur when plants are actively growing can be a lethal event, particularly if the plant has no freezing tolerance. Such frost events often have devastating effects on agricultural production and can also play an important role in shaping community structure in natural populations of plants, especially in alpine, sub-arctic, and arctic ecosystems. Therefore, a better understanding of the freezing process in plants can play an important role in the development of methods of frost protection and understanding mechanisms of freeze avoidance. Here, we describe a protocol to visualize the freezing process in plants using high-resolution infrared thermography (HRIT). The use of this technology allows one to determine the primary sites of ice formation in plants, how ice propagates, and the presence of ice barriers. Furthermore, it allows one to examine the role of extrinsic and intrinsic nucleators in determining the temperature at which plants freeze and evaluate the ability of various compounds to either affect the freezing process or increase freezing tolerance. The use of HRIT allows one to visualize the many adaptations that have evolved in plants, which directly or indirectly impact the freezing process and ultimately enables plants to survive frost events.

Understanding plant cold hardiness: an opinion.

How plants adapt to freezing temperatures and acclimate to survive the formation of ice within their tissues has been a subject of study for botanists and plant scientists since the latter part of the 19th century. In recent years, there has been an explosion of information on this topic and molecular biology has provided new and exciting opportunities to better understand the genes involved in cold adaptation, freezing response and environmental stress in general. Despite an exponential increase in our understanding of freezing tolerance, understanding cold hardiness in a manner that allows one to actually improve this trait in economically important crops has proved to be an elusive goal. This is partly because of the growing recognition of the complexity of cold adaptation. The ability of plants to adapt to and survive freezing temperatures has many facets, which are often species specific, and are the result of the response to many environmental cues, rather than just low temperature. This is perhaps underappreciated in the design of many controlled environment experiments resulting in data that reflects the response to the experimental conditions but may not reflect actual mechanisms of cold hardiness in the field. The information and opinions presented in this report are an attempt to illustrate the many facets of cold hardiness, emphasize the importance of context in conducting cold hardiness research, and pose, in our view, a few of the critical questions that still need to be addressed.

Varietal and chromosome 2H locus-specific frost tolerance in reproductive tissues of barley (Hordeum vulgare L.) detected using a frost simulation chamber.

Exposure of flowering cereal crops to frost can cause sterility and grain damage, resulting in significant losses. However, efforts to breed for improved low temperature tolerance in reproductive tissues (LTR tolerance) has been hampered by the variable nature of natural frost events and the confounding effects of heading time on frost-induced damage in these tissues. Here, we establish conditions for detection of LTR tolerance in barley under reproducible simulated frost conditions in a custom-built frost chamber. An ice nucleator spray was used to minimize potential effects arising from variation in naturally occurring extrinsic nucleation factors. Barley genotypes differing in their field tolerance could be distinguished. Additionally, an LTR tolerance quantitative trait locus (QTL) on the long arm of barley chromosome 2H could be detected in segregating families. In a recombinant family, the QTL was shown to be separable from the effects of the nearby flowering time locus Flt-2L. At a minimum temperature of -3.5 degrees C for 2 h, detection of the LTR tolerance locus was dependent on the presence of the nucleator spray, suggesting that the tolerance relates to freezing rather than chilling, and that it is not the result of plant-encoded variation in ice-nucleating properties of the tiller surface.

Effect of growth phase on survival of bromegrass suspension cells following cryopreservation and abiotic stresses.

BACKGROUND AND AIMS: Cryopreservation is a practical method of preserving plant cell cultures and their genetic integrity. It has long been believed that cryopreservation of plant cell cultures is best performed with cells at the late lag or early exponential growth phase. At these stages the cells are small and non-vacuolated. This belief was based on studies using conventional slow prefreezing protocols and survival determined with fluorescein diacetate staining or 2,3,5-triphenyltetrazolium chloride assays. This classical issue was revisited here to determine the optimum growth phase for cryopreserving a bromegrass (Bromus inermis) suspension culture using more recently developed protocols and regrowth assays for determination of survival. METHODS: Cells at different growth phases were cryopreserved using three protocols: slow prefreezing, rapid prefreezing and vitrification. Stage-dependent trends in cell osmolarity, water content and tolerance to freezing, heat and salt stresses were also determined. In all cases survival was assayed by regrowth of cells following the treatments. KEY RESULTS: Slow prefreezing and rapid prefreezing protocols resulted in higher cell survival compared with the vitrification method. For all the protocols used, the best regrowth was obtained using cells in the late exponential or early stationary phase, whereas lowest survival was obtained for cells in the late lag or early exponential phase. Cells at the late exponential phase were characterized by high water content and high osmolarity and were most tolerant to freezing, heat and salt stresses, whereas cells at the early exponential phase, characterized by low water content and low osmolarity, were least tolerant. CONCLUSIONS: The results are contrary to the classical concept which utilizes cells in the late lag or early exponential growth phase for cryopreservation. The optimal growth phase for cryopreservation may depend upon the species or cell culture being cryopreserved and requires re-investigation for each cell culture. Stage-dependent survival following cryopreservation was proportionally correlated with the levels of abiotic stress tolerance in bromegrass cells.

Identification and immunogold localization of a novel bromegrass (Bromus inermis Leyss) peroxisome channel protein induced by ABA, cold and drought stresses, and late embryogenesis.

A cDNA (BG-15) was isolated through differential screening of a cDNA library made from an ABA-treated bromegrass (Bromus inermis Leyss) suspension cell culture. The 819 bp pair cDNA encoded a 174 amino acid polypeptide with a calculated molecular mass of 18.08 kD and isolectric point of 7.50. The deduced amino acid sequences for the cDNA were 29.5% and 32.6% homologous to the known amino acid-selective channel proteins of the chloroplastic outer membrane in pea and barley, but were highly homologous (55.6% to 83.2%) to the putative membrane channel proteins from rice and Arabidopsis. Immunogold localization demonstrated that the channel protein encoded by this cDNA was present on the peroxisome membrane. High stringency southern analysis revealed that 1 to 2 copies of the peroxisomal channel protein (PCP) genes were present in the bromegrass genome. Northern and Western blots revealed that the PCP gene was responsive to both cold and drought stresses, and was rapidly induced by ABA (75 microM). The transcript of the PCP gene also accumulated during late embryogenesis, but declined rapidly during germination. Data taken together, responsiveness of the PCP to cold and drought stresses, and accumulation during late embryogenesis suggest this novel peroxisomal channel protein is associated with sugar and fatty acid metabolism through fatty acid import or succinate export from peroxisome during desiccation tolerance and energy metabolism.

A lipid transfer protein gene BG-14 is differentially regulated by abiotic stress, ABA, anisomycin, and sphingosine in bromegrass (Bromus inermis).

The objective was to investigate the expression of a lipid transfer protein gene (LTP) both in bromegrass (Bromus inermis) cells and seedlings after exposure to abiotic stresses, abscisic acid (ABA), anisomycin, and sphingosine. A full-length cDNA clone BG-14 isolated from bromegrass suspension cell culture encodes a polypeptide of 124 amino acids with typical LTP characteristics, such as a conserved arrangement of cysteine residues. During active stages of cold acclimation LTP expression was up-regulated, whereas at the final stage of cold acclimation LTP transcript level declined to pre-acclimation level. A severe drought stress induced the LTP gene; yet, LTP expression doubled 3 d after re-hydration. Both temperature and heat shock duration influence LTP induction; however temperature is the primary factor. Treatment with NaCl stimulated accumulation of LTP mRNA within 15 min and the transcripts remained at elevated levels for the duration of the salinity stress. Most interestingly, Northern blots showed LTP was rapidly induced not only by ABA, but also by anisomycin and sphingosine in suspension cell cultures. Of the three chemicals, ABA induced the most rapid and highest response in LTP expression as well as highest freezing tolerance, whereas sphingosine was the least active for both LTP expression and freezing tolerance.