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 ...

University of Saskatechewan

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.

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

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.

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

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.

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

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.

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

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.

Lawrence V. Gusta

Department of Plant Science

University of Saskatechewan

The Use of High-resolution Infrared Thermography (HRIT) for the Study of Ice Nucleation and Ice Propagation in Plants

Lawrence V. Gusta

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University of Saskatechewan

The Use of High-resolution Infrared Thermography (HRIT) for the Study of Ice Nucleation and Ice Propagation in Plants

Department of Plant Science