Lawrence V. Gusta

Plant Cold Acclimation: The Role of Abscisic Acid

The freezing tolerance or cold acclimation of plants is enhanced over a period of time by temperatures below 10°C and by a short photoperiod in certain species of trees and grasses. During this process, freezing tolerance increases 2–8°C in spring annuals, 10–30°C in winter annuals, and 20–200°C in tree species. Gene upregulation and downregulation have been demonstrated to be involved in response to environmental cues such as low temperature. Evidence suggests ABA can substitute for the low temperature stimulus, provided there is also an adequate supply of sugars. Evidence also suggests there may be ABA-dependent and ABA-independent pathways involved in the acclimation process. This review summarizes the role of ABA in cold acclimation from both a historical and recent perspective. It is concluded that it is highly unlikely that ABA regulates all the genes associated with cold acclimation; however, it definitely regulates many of the genes associated with an increase in freezing tolerance.

The Effect of Water, Sugars, and Proteins on the Pattern of Ice Nucleation and Propagation in Acclimated and Nonacclimated Canola Leaves

Infrared video thermography was used to observe ice nucleation temperatures, patterns of ice formation, and freezing rates in nonacclimated and cold acclimated leaves of a spring (cv Quest) and a winter (cv Express) canola (Brassica napus). Distinctly different freezing patterns were observed, and the effect of water content, sugars, and soluble proteins on the freezing process was characterized. When freezing was initiated at a warm subzero temperature, ice growth rapidly spread throughout nonacclimated leaves. In contrast, acclimated leaves initiated freezing in a horseshoe pattern beginning at the uppermost edge followed by a slow progression of ice formation across the leaf. However, when acclimated leaves, either previously killed by a slow freeze (2°C h−1) or by direct submersion in liquid nitrogen, were refrozen their freezing pattern was similar to nonacclimated leaves. A novel technique was developed using filter paper strips to determine the effects of both sugars and proteins on the rate of freezing of cell extracts. Cell sap from nonacclimated leaves froze 3-fold faster than extracts from acclimated leaves. The rate of freezing in leaves was strongly dependent upon the osmotic potential of the leaves. Simple sugars had a much greater effect on freezing rate than proteins. Nonacclimated leaves containing high water content did not supercool as much as acclimated leaves. Additionally, wetted leaves did not supercool as much as nonwetted leaves. As expected, cell solutes depressed the nucleation temperature of leaves. The use of infrared thermography has revealed that the freezing process in plants is a complex process, reminding us that many aspects of freezing tolerance occur at a whole plant level involving aspects of plant structure and metabolites rather than just the expression of specific genes alone.

Abscisic acid-induced heat tolerance in Bromus inermis leyss cell-suspension cultures : heat-stable, abscisic acid-responsive polypeptides in combination with sucrose confer enhanced thermostability

Increased heat tolerance is most often associated with the synthesis of heat-shock proteins following pre-exposure to a nonlethal heat treatment. In this study, a bromegrass (Bromus inermis Leyss cv Manchar) cell suspension cultured in a medium containing 75 [mu]M abscisic acid (ABA) without prior heat treatment had a 87% survival rate, as determined by regrowth analysis, following exposure to 42.5[deg]C for 120 min. In contrast, less than 1% of the control cells survived this heat treatment. The heat tolerance provided by treatment with 75 [mu]M ABA was first evidenced after 4 d of culture and reached a maximum tolerance after 11 d of culture. Preincubation with sucrose partially increased the heat tolerance of control cells and rendered ABA-treated cells tolerant to 45[deg]C for 120 min (a completely lethal heat treatment for control cells). Comparative two-dimensional polyacrylamide gel electrophoresis of cellular protein isolated from heat-tolerant cells identified 43 ABA-responsive proteins of which 26 were heat stable (did not coagulate and remained soluble after 30 min at 90[deg]C). Eight heat-stable, ABA-responsive proteins ranging from 23 to 45 kD had similar N-terminal sequences. The ABA-responsive (43-20 kD), but none of the control heat-stable, proteins cross-reacted to varying degrees with a polyclonal antibody directed against a conserved, lysine-rich dehydrin sequence. A group of 20- to 30-kD heat-stable, ABA-responsive proteins cross-reacted with both the anti-dehydrin antibody and an antibody directed against a cold-responsive winter wheat protein (Wcs 120). In ABA-treated cells, there was a positive correlation between heat- and pH-induced coagulation of a cell-free homogenate and the heat tolerance of these cells. At 50[deg]C, control homogenates coagulated after 8 min, whereas cellular fractions from ABA-treated cells showed only marginal coagulation after 15 min. In protection assays, addition of heat-stable, ABA-responsive polypeptides to control fractions reduced the heat-induced coagulation of cell-free homogenates. Sucrose (8%) alone and control, heat-stable fractions enhanced the thermostability of control fractions, but the most protection was conferred by ABA-responsive, heat-stable proteins in combination with sucrose. These data suggest that stress-tolerance mechanisms may develop as a result of cooperative interactions between stress proteins and cell osmolytes, e.g. sucrose. Hypotheses are discussed implicating the role of these proteins and osmolytes in preventing coagulation and denaturation of cellular proteins and membranes.

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.

Comparison of viability tests for assessing cross-adaptation to freezing, heat and salt stresses induced by abscisic acid in bromegrass (Bromus inermis Leyss) suspension cultured cells

Several viability assays were compared to determine the most sensitive and appropriate method for estimating the freezing, heat and salt tolerance of Bromus inermis Leyss cells cultured with or without 75 μM abscisic acid (ABA) for 4–7 days at 25°C. The sensitivity and reliability of individual viability tests depended on the type of stress applied and degree of injury. Regrowth, amino acid (AA) leakage and fluorescein diacetate (FDA) staining all gave comparable estimates of freezing tolerance. Triphenyl-tetrazolium chloride (TTC) reduction assays slightly overestimated freezing tolerance, but was most convenient. Polypeptide leakage from freeze-thawed cells, as determined by SDS-polyacrylamide electrophoresis (SDS-PAGE), revealed that protein leakage only occurred in ABA-treated cells frozen to −21°C or colder in contrast to control cells which leaked proteins following freezing to −5°C. The most sensitive test for assessing heat tolerance was regrowth, followed by FDA staining, when TTC. TTC tests overestimated heat tolerance compared with the other tests. The degree of overestimation was greater for heat tolerance estimates than for freezing tolerance estimates. It was partially improved by washing the cells prior to TTC assays. AA leakage tests were not appropriate for assessing heat tolerance, due to the erroneously high values of A280 readings obtained. For estimating salt tolerance, TTC assays were most convenient and were in close agreement with regrowth measurements whereas FDA staining tended to overestimate it. In general, while regrowth was most sensitive and reliable, TTC was most convenient. All viability assays consistently showed that ABA induced cross-adaptation to freezing, heat and salt stresses in bromegrass cells without a prior exposure to any of these stresses.

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.

Winter Flounder Antifreeze Protein Improves the Cold Hardiness of Plant Tissues

Summary Exposure of plant tissues to the winter flounder antifreeze protein (AFP) has revealed three novel properties by which plant cold hardiness may be improved. Firstly, vacuum infiltration of the protein into leaves of potato, canola ( Brassica napus ) and Arabidopsis thaliana resulted in a significant depression of the spontaneous freezing temperature relative to water infiltrated controls. In the case of canola, the freezing temperature was decreased by an average of 1.8 °C. These results demonstrated the ability of the AFP to function as an anti-nucleator in plant tissues. Secondly, exposure of suspension cultured cells of bromegrass to the antifreeze protein resulted in a reduction in the amount of freezable water frozen at any given temperature. This showed that the protein could act as a cryoprotectant. Thirdly, the antifreeze protein decreased the rate of ice crystal formation. These results demonstrate the feasibility of improving the cold hardiness of plants by introduction of the antifreeze protein gene.

Metabolism of (+)-abscisic acid to (+)-7′-hydroxyabscisic acid by bromegrass cell cultures

Abstract 7′-Hydroxyabscisic acid was isolated from the medium of smooth bromegrass ( Bromus inermis Leyss.) cell suspension cultures supplied with either (±)- or natural (+)-abscisic acid, and the identity of the metabolite confirmed by 1 H NMR. Analysis of the methyl esters of the metabolite by chiral HPLC showed only the (+) enantiomer of 7′- hydroxyabscisic acid to be present in the medium of cultures fed (+)-abscisic acid. In cultures treated with (±)-abscisic acid, the (−) enantiomer of the metabolite is formed from the (−)-abscisic acid component of the racemic mixture supplied.

Plant cold hardiness: from the laboratory to the field.

Section One The Freezing Process Section Two Molecular Basis for the Acquisition of Freezing Tolerance Section Three Linkage Between Developmental Arrest and Cold Hardiness Section Four Genetic Basis of Superior Cold Tolerance Section Five Impact of Global Climate Change on Plants Section Six From the Lab to the Field: Bridging the Gap Section Seven Photosynthesis and Signalling Section Eight Systems Biology.

Effects of Abscisic Acid Metabolites and Analogs on Freezing Tolerance and Gene Expression in Bromegrass (Bromus inermis Leyss) Cell Cultures.

Optical isomers and racemic mixtures of abscisic acid (ABA) and the ABA metabolites abscisyl alcohol (ABA alc), abscisyl aldehyde (ABA ald), phaseic acid (PA), and 7[prime]hydroxyABA (7[prime]OHABA) were studied to determine their effects on freezing tolerance and gene expression in bromegrass (Bromus inermis Leyss) cell-suspension cultures. A dihydroABA analog (DHABA) series that cannot be converted to PA was also investigated. Racemic ABA, (+)-ABA, ([plus or minus])-DHABA, and (+)-DHABA were the most active in inducing freezing tolerance, (-)-ABA, ([plus or minus])-7[prime]OHBA, (-)-DHABA, ([plus or minus])-ABA ald, and ([plus or minus])-ABA alc had a moderate effect, and PA was inactive. If the relative cellular water content decreased below 82%, dehydrin gene expression increased. Except for (-)-ABA, increased expression of dehydrin genes and increased accumulation of responsive to ABA (RAB) proteins were linked to increased levels of frost tolerance. PA had no effect on the induction of RAB proteins; however, ([plus or minus])- and (+)-DHABA were both active, which suggests that PA is not involved in freezing tolerance. Both (+)-ABA and (-)-ABA induced dehydrin genes and the accumulation of RAB proteins to similar levels, but (-)-ABA was less effective than (+)-ABA at increasing freezing tolerance. The (-)-DHABA analog was inactive, implying that the ring double bond is necessary in the (-) isomers for activating an ABA response.