Activities of photosystem II and antioxidant enzymes in chickpea (<Emphasis Type="Italic">Cicer arietinum</Emphasis> L<Emphasis Type="Italic">.</Emphasis>) cultivars exposed to chilling temperatures

This study was carried out to determine the effect of chilling on both cold-acclimated and non-acclimated chickpea (Cicer arietinum L.) cultivars (Gökçe and Can?tez 87). Chickpea seedlings grown in soil culture for 12 days were subjected to chilling temperatures (2 and 4°C for 12 days) after maintaining in cold-acclimation (10°C, 7 days) or non-acclimation (25°C, 7 days) periods. The lowest values of growth parameters were obtained with cold-acclimated plants, whereas non-acclimated plants exhibited the lowest water content values, especially at 2°C. There was no effect of cold-acclimation period on chlorophyll fluorescence parameters. Plants subjected to chilling temperatures after cold-acclimation were more tolerant with respect to chlorophyll fluorescence parameters, and Gökçe had better photosystem II (PSII) photochemical activity. In the chilling treatments, total chlorophyll (a + b) content reduced, especially at 2°C, while anthocyanin and flavonoid contents increased to a greater extent in Gökçe and carotenoid content of the cultivars did not change. Malondialdehyde (MDA) content was higher for Can?tez 87, mostly at 2°C, while proline accumulation was greater for Gökçe. The cold-acclimation period led to a remarkable increase in antioxidant enzyme activities of both cultivars. The superoxide dismutase (SOD) activity was much higher in Gökçe for both chilling temperatures and the ascorbate peroxidase (APX) activity increased only in the cold-acclimated 4°C treatments. Similarly, with APX activity, the glutathione reductase (GR) and peroxidase (POD) activities of cultivars were higher in cold-acclimated plants at both the chilling temperatures, mostly in Gökçe. The results of this study indicate that cold-acclimation increased the cultivars ability to withstand the chilling temperatures. The lower MDA content and higher antioxidant and photochemical activities in Gökçe indicated an enhanced chilling tolerance capacity of this cultivar to protect the plant from oxidative damage.     

Effect of auxin physiological analogues on rapeseed (Brassica napus) cold hardening, seed yield and quality

The effect of the auxin physiological analogues analogues 1-[2-chloroethoxycarbonylmethyl]-4-naphthalenesulfonic acid calcium salt (TA-12) and 1-[2-dimethylaminoethoxicarbonylmethyl]naphthalene chlormethylate (TA-14) TA-14 on different winter rapeseed cultivars were studied with regard to their autumnal growth, cold hardening, accumulation of the stress-protective metabolites proline and saccharide in plant organs: apical bud and root collum, winter survival and productivity formation. The test cultivars were the very early ‘Libea’ medium-resistant to wintering, the medium-early ‘Sunday’ resistant to wintering, the medium–early ‘Valesca’ less than medium resistant to wintering, and the early ‘Hornet’ (hybrid) tolerant to stress growth conditions. During the period of cold hardening in natural field conditions, the test compounds TA-12 (2 mM) and TA-14 (4 mM), applied to different winter rapeseed cultivars at the 4th–5th leaf stage, stimulate accumulation proline and saccharides (sucrose and glucose) in the root collum and apical bud tissues, influence plants acclimation to cold, overwintering and productivity formation. Compounds TA-12 and especially TA-14 produced a stable effect on seed and crude fat yield in cvs. ‘Hornet’, ‘Sunday’ and ‘Libea’. The genotypic peculiarities of a cultivar and the meteorological conditions of the plant vegetation period were the factors that mostly determined fatty acid content in seed oil.     

Inositol 1,4,5-trisphosphate formation in leaves of winter oilseed rape plants in response to freezing, tissue water potential aed abscisic acid

Time courses of formation of inositol 1,4,5-trisphosphate (IP₃) were followed in the leaves of non-acclimated and cold (2°C)-acclimated winter oilseed rape ( Brassica napus L. var. oleifera ) plants, subjected to different freezing temperatures or to polyethylene glycol 8000 (PEG) and abscisic acid (ABA) treatments. Changes in water potential (Ψ_w) and in ABA level in the frost- and PEG-treated tissues were also determined. Results obtained indicate that temperatures sligthly higher than LT₅₀ induced a transient and substantial increase in IP₃ level, both in non-acclimated and cold-acclimated tissues. At comparable freezing temperature (–5°C) the response of cold-acclimated leaves was lower than that of non-acclimated ones. The PEG-depedent decrease in Ψ_w to –0.9 MPa or ABA (0.1 m M ) treatment gave rise to a transient increase in IP₃ content in non-acclimated tissues only. Collectively, the data indicate that cold acclimation of plants may lead to lower cell responsiveness to the factors studied in terms of induction of IP₃ formation. Changes in the IP₃ content, observed in the present experiments, support our previous suggestion that non-killing freezing temperatures may induce the phosphoinositide pathway, both in non-acclimated and cold-acclimated tissues. Lowering of tissue water potential to some threshold value or a high exogenous ABA supply may mimic the freezing-dependent reaction in the non-acclimated leaves.     

The influence of cold acclimation on antioxidative enzymes and antioxidants in sensitive and tolerant barley cultivars

In order to better understand the role of cold acclimation in alleviating freezing injury, two barley cultivars with different cold tolerance, i.e. a sensitive cv. Chumai 1 and a tolerant cv. Mo 103, were used. The freezing treatment increased leaf soluble protein content more in the tolerant cultivar than in the sensitive one. Cold acclimation increased H₂O₂ content of the two cultivars during freezing treatment, especially in Mo 103. Glutathione and ascorbate contents during freezing and recovery were significantly higher in cold-acclimated plants than in non-acclimated ones. Activities of peroxidase, ascorbate peroxidase and glutathione reductase were also higher in cold-acclimated plants than non-acclimated plants during freezing treatment. However, there was no significant difference between cold-acclimated plants and the control plants in catalase activity. It may be assumed that cold acclimation induced H₂O₂ production, which in turn enhanced activities of antioxidative enzymes and synthesis of antioxidants, resulting in alleviation of oxidative stress caused by freezing.     

磷脂酶Dδ参与植物的低温驯化过程

对经低温驯化和未经低温驯化的磷脂酶Dδ（PLDδ）基因敲除突变体与野生型植株进行冻害胁迫处理后,比较2种基因型植株的抗冻性。结果发现,经低温驯化的PLDδ敲除突变体的抗冻性明显低于野生型,而未经低温驯化的PLD礅除突变体与野生型的抗冻性没有显著差异,表明PLDδ参与植物的低温驯化过程。对PLDδ的作用途径进行分析,发现PLDδ在低温驯化过程中不参与抗氧化酶活性的调节,对脯氨酸和可溶性糖的积累起负调节作用,但是参与低温信号转导物质ABA诱导抗冻性的过程。     

Heterosis in the freezing tolerance, and sugar and flavonoid contents of crosses between Arabidopsis thaliana accessions of widely varying freezing tolerance

Heterosis is defined as the increased vigour of hybrids in comparison to their parents. We investigated 24 F(1) hybrid lines of Arabidopsis thaliana generated by reciprocally crossing either C24 or Col with six other parental accessions (Can, Co, Cvi, Ler, Rsch, Te) that differ widely in their freezing tolerance. The crosses differed in the degree of heterosis for freezing tolerance, both in the non-acclimated state and after a 14 d cold acclimation period. Crosses with C24 showed more heterosis than crosses with Col, and heterosis was stronger in acclimated than in non-acclimated plants. Leaf content of soluble sugars and proline showed more deviation from mid-parent values in crosses involving C24 than in those involving Col, and deviations were larger in acclimated than in non-acclimated plants. There were significant correlations between the content of different sugars and leaf freezing tolerance, as well as between heterosis effects in freezing tolerance and sugar content. Flavonoid content and composition varied between accessions, and between non-acclimated and acclimated plants. In the crosses, large deviations from the mid-parent values in the contents of different flavonols occurred, and there were strikingly strong correlations between both flavonol content and freezing tolerance, and between heterosis effects in freezing tolerance and flavonol content.     

Low temperature effects on growth and actin cytoskeleton organisation in suspension cells of winter oilseed rape

Rhodamine-phalloidin staining of winter oilseed rape suspension cells revealed that the structure of actin cytoskeleton changes with the phase of cell growth. In small, 4-day-old cells, entering the exponential phase of growth, a dense and uniformly distributed cortical microfilament networks was seen. In six-day-old vacuolated cells, which reached the stationary phase of growth, the actin cytoskeleton was composed of thicker microfilament cables in irregular arrangements. In cells acclimated in cold for 7 days a dense, uniformly distributed and cortical microfilament network was still seen. The fine microfilament network was sensitive to extracellular freezing since the structures underwent depolymerization at −3 °C (in the presence of extracellular ice), both in non-acclimated and cold-acclimated cells. The thicker transvacuolar cables in cells of the stationary growth phase resisted freezing to −7 °C. Acclimation of suspensions at 2 °C resulted in slowing down growth of cells and in the increased freezing tolerance of cells as indicated by a decrease of LT₅₀ from −11 °C to −17.5^o or to −25 °C when determined 7 or 20 days after the beginning of the cold treatment, respectively. Freezing tolerance of non-acclimated cells decreased from −11 °C to −8 °C during subculture, showing a transient increase to −17 °C on the day 6. Results indicate that the arrangement of actin microfilaments and their sensitivity to freezing-induced depolymerization depends on the phase of cell growth rather than on cell acclimation status. Possible mechanisms involved in the freezing-induced depolymerization of actin microfilaments are discussed. This revised version was published online in June 2006 with corrections to the Cover Date.     

磷脂酶Dδ参与植物的低温驯化过程

对经低温驯化和未经低温驯化的磷脂酶Dδ (PLDδ)基因敲除突变体与野生型植株进行冻害胁迫处理后, 比较2种基因型植株的抗冻性。结果发现, 经低温驯化的PLDδ敲除突变体的抗冻性明显低于野生型, 而未经低温驯化的PLDδ敲除突变体与野生型的抗冻性没有显著差异, 表明PLDδ参与植物的低温驯化过程。对PLDδ的作用途径进行分析, 发现PLDδ在低温驯化过程中不参与抗氧化酶活性的调节, 对脯氨酸和可溶性糖的积累起负调节作用, 但是参与低温信号转导物质ABA诱导抗冻性的过程。     

Relationship between Freezing Tolerance of Root-Tip Cells and Cold Stability of Microtubules in Rye (Secale cereale L. cv Puma)

The response of cortical microtubules to low temperature and freezing was assessed for root tips of cold-acclimated and non-acclimated winter rye (Secale cereale L. cv Puma) seedlings using indirect immunofluorescence microscopy with antitubulin antibodies. Roots cooled to 0 or −3°C were fixed for immunofluorescence microscopy at these temperatures or after an additional hour at 4°C. Typical arrays of cortical microtubules were present in root-tip cells of seedlings exposed to the cold-acclimation treatment of 4°C for 2 days. Microtubules in these cold-acclimated cells were more easily depolymerized by a 0°C treatment than microtubules in root-tip cells of nonacclimated, 22°C-grown seedlings. Microtubules were still present in some cells of both nonacclimated and cold-acclimated roots at 0 and −3°C; however, the number of microtubules in these cells was lower than in controls. Microtubules remaining during the −3°C freeze were shorter than microtubules in unfrozen control cells. Repolymerization of microtubules after both the 0 and −3°C treatments occurred within 1 h. Root tips of nonacclimated seedlings had an LT-50 of −9°C. Cold acclimation lowered this value to −14°C. Treatment of 22°C-grown seedlings for 24 h with the microtubule-stabilizing drug taxol caused a decrease in the freezing tolerance of root tips, indicated by a LT-50 of −3°C. Treatment with D-secotaxol, an analog of taxol that was less effective in stabilizing microtubules, did not alter the freezing tolerance. We interpret these data to indicate that a degree of depolymerization of microtubules is necessary for realization of maximum freezing tolerance in root-tip cells of rye.     

Freezing tolerance, cold acclimation and oxidative stress in potato. Paraquat tolerance is related to acclimation but is a poor indicator of freezing tolerance

Potato is a species commonly cultivated in temperate areas where the growing season may be interrupted by frosts, resulting in loss of yield. Cultivated potato, Solanum tuberosum, is freezing sensitive, but it has several freezing-tolerant wild potato relatives, one of which is S. commersonii. Our study was aimed to resolve the relationship between enhanced freezing tolerance, acclimation capacity and capacity to tolerate active oxygen species. To be able to characterize freezing tolerant ideotypes, a potato population (S1), which segregates in freezing tolerance, acclimation capacity and capacity to tolerate superoxide radicals, was produced by selfing a somatic hybrid between a freezing-tolerant Solanum commersonii (LT₅₀=-4.6°C) and -sensitive S. tuberosum (LT₅₀=-3.0°C). The distribution of non-acclimated freezing tolerance (NA-freezing tolerance) of the S1 population varied between the parental lines and we were able to identify genotypes, having significantly high or low NA-freezing tolerance. When a population of 25 genotypes was tested both for NA-freezing and paraquat (PQ) tolerance, no correlation was found between these two traits (R = 0.02). However, the most NA-freezing tolerant genotypes were also among the most PQ tolerant plants. Simultaneously, one of the NA-freezing sensitive genotypes (2022) (LT₅₀=-3.0°C) was observed to be PQ tolerant. These conflicting results may reflect a significant, but not obligatory, role of superoxide scavenging mechanisms in the NA-freezing tolerance of S. commersonii. The freezing tolerance after cold acclimation (CA-freezing tolerance) and the acclimation capacity (AC) was measured after acclimation for 7 days at 4/2°C. Lack of correlation between NA-freezing tolerance and AC (R =-0.05) in the S1 population points to independent genetic control of NA-freezing tolerance and AC in Solanum sp. Increased freezing tolerance after cold acclimation was clearly related to PQ tolerance of all S1 genotypes, especially those having good acclimation capacity. The rapid loss of improved PQ tolerance under deacclimation conditions confirmed the close relationship between the process of cold acclimation and enhanced PQ tolerance. Here, we report an increased PQ tolerance in cold-acclimated plants compared to non-acclimated controls. However, we concluded that high PQ tolerance is not a good indicator of actual freezing tolerance and should not be used as a selectable marker for the identification of a freezing-tolerant genotype.     

Transgenic creeping bentgrass plants expressing a <Emphasis Type="Italic">Picea wilsonii</Emphasis> dehydrin gene (<Emphasis Type="Italic">PicW</Emphasis>) demonstrate improved freezing tolerance

Agrostis stolonifera L. ‘Penn A-4’ is a common creeping bentgrass species that is widely used in urban landscaping and golf courses. To prolong the green stage of this grass, a dehydrin gene PicW isolated from Wilson’s spruce (Picea wilsonii) was transformed into plants of ‘Penn A-4’ cultivar via a straightforward stolon node infection system. A putative transgenic plant was obtained and its tolerance to low-temperature stress was evaluated. When the transgenic line was subjected to a freezing (??5 °C) treatment, it showed better viability and more robust physiology than wild type, as evidenced by higher soluble sugar and proline contents, and lower relative electrical conductivity and malondialdehyde content. The transgenic line also showed tolerance to a chilling treatment (5 °C), although its performance was not significantly different from that of wild-type plants. Overall, the research here clearly revealed the explicit role of PicW in increasing freezing tolerance of grass at the whole-plant level, and demonstrated that the straightforward stolon node transformation method could be well used to genetically modify turfgrass. The obtained transgenic line might be as genetic resource for breeding program and practiced to grow in cold temperate zones.     

Effects of diapause and cold-acclimation on the avoidance of freezing injury in fat body tissue of the rice stem borer, Chilo suppressalis Walker

Overwintering freeze-tolerant larvae of Chilo suppressalis can survive at -25 degrees C, but non-diapausing larvae cannot. We reported earlier that to prevent intracellular freezing, which causes death in overwintering larvae of the Saigoku ecotype distributed in southwestern Japan, water leaves and glycerol enters fat body cells through water channels during freezing. However, it is still unclear how diapause and low-temperature exposure are related to the acquisition of freeze tolerance. We compared the extent of tissue damage, accumulation of glycerol, and transport of glycerol and water in fat body tissues between cold-acclimated and non-acclimated non-diapausing and diapausing larvae. The tissue from cold-acclimated diapausing larvae could survive only when frozen in Grace's insect medium with 0.25 M glycerol at -20 degrees C. The protection provided by glycerol was offset by mercuric chloride, which is a water-channel inhibitor. Fat body tissue isolated from non-acclimated diapausing larvae was injured by freezing even though glycerol was added to the medium, but the level of freezing injury was significantly lower than in non-diapausing larvae. Radiotracer assays in cold-acclimated diapausing larvae showed that during freezing, water left the cells into the medium and glycerol entered the cells from the medium at the same time. Therefore, in Saigoku ecotype larvae of the rice stem borer, both diapause and cold-acclimation are essential to accumulate glycerol and activate aquaporin for the avoidance of freezing injury.     

Influence of soil temperature on root freezing tolerance of Scots pine (Pinus sylvestris L.) seedlings

The influence of soil temperature on the root freezing tolerance of one-year-old containerized Scots pine (Pinus sylvestris L.) seedlings was investigated. In addition, the TTC and electrolyte leakage methods were evaluated in terms of their suitability for use in detecting damage to roots caused by freezing. In mid-August, seedlings were placed in three thermostat-controlled soil beds in a greenhouse with an initial soil temperature of 14.3 °C. Soil temperature was lowered in two of the soil beds, resulting in temperatures of 10.7 and 5.3 °C respectively. Each soil temperature, i.e. 14.3, 10.7 and 5.3 °C was maintained for eight weeks. Starting in early September, damage to roots induced by artificial freezing was estimated biweekly by measuring electrolyte leakage, triphenyl tetrazolium chloride (TTC) reduction and potential root growth in a three-week cultivation test. In addition, the root freezing tolerance of seedlings placed outdoors was tested. Measurements showed that these seedlings were exposed to soil temperatures ranging from 13.0 °C in mid-August to 0.5 °C in November. Generally, the development of root freezing tolerance was more pronounced for seedlings exposed to lower (0.5 and 5.3 °C) soil temperatures compared with those exposed to higher (10.7 and 14.3 °C) ones. Root freezing tolerance was highest among the seedlings placed outdoors which were also exposed to the lowest soil temperatures registered in the study. To examine the effect of a temporary warm period, the soil temperature in one treatment was increased from 5.4 °C to 13.9 °C, maintained at the latter temperature for two weeks in October and then lowered to 5.7 °C. Root freezing tolerance was reduced by exposure to the warmer soil temperature. However, after four weeks at the colder soil temperature, the tolerance of the seedlings had returned to the level measured prior to exposure to the warm soil temperature. Methods based on the measurement of root electrolyte leakage and TTC reduction were both found to have limitations when used to detect root freezing damages in containerized seedlings. This revised version was published online in June 2006 with corrections to the Cover Date.     

Cold-induced physiological and biochemical responses of three grapevine cultivars differing in cold tolerance

In this study the cold tolerance potential of three Vitis vinifera cultivars including ‘Red Sultana’, ‘White Sultana,’ and ‘Flame Seedless’ was evaluated under greenhouse condition. After 15 leaves stage in average, the grapevine plants were subjected to cold stress regimes (4, 0 and ? 4 °C) and compared with control plants (24 °C). A clear increase in leaf electrolyte leakage (EL), thiobarbituric acid reactive substances (TBARS), and H₂O₂ concentrations was observed with decreasing temperature from 4 to ? 4 °C in all grapevine cultivars. Chilled plants showed marked increases in their abscisic acid (ABA), soluble sugars, and proline contents in compared to control vines. Upon exposure to cold stress, the EL, TBARS, H₂O₂, and relative water content of ‘Red Sultana’ were found to be lower compared to ‘White Sultana’ and ‘Flame Seedless’. Under 0 °C condition, ‘Red Sultana’ had the highest superoxide dismutase, guaiacol peroxidase and catalase activities, which was approximately twofold higher than those of all other cultivars. Soluble sugars such as glucose, fructose, and sucrose increased from 4 to ? 4 °C. These increments were higher in ‘Red Sultana’ compared to other cultivars which was concomitant with higher accumulation of endogenous ABA concentration in this cultivar. Higher accumulation of ABA and soluble sugars in ‘Red Sultana’ confirmed the key roles of these compounds in cold tolerance which could be applied as a cold tolerance marker for early selection of grapevine cultivars with the aim to establish vineyards in cold winter regions.     

Suppression of phospholipase Dalpha1 induces freezing tolerance in Arabidopsis: response of cold-responsive genes and osmolyte accumulation

Phospholipase D (PLD; EC 3.1.4.4) plays an important role in membrane lipid hydrolysis and in mediation of plant responses to a wide range of stresses. PLDalpha1 abrogation through antisense suppression in Arabidopsis thaliana resulted in a significant increase in freezing tolerance of both non-acclimated and cold-acclimated plants. Although non-acclimated PLDalpha1-deficient plants did not show the activation of cold-responsive C-repeat/dehydration-responsive element binding factors (CBFs) and their target genes (COR47 and COR78), they did accumulate osmolytes to much higher levels than did the non-acclimated wild-type plants. However, a stronger expression of COR47 and COR78 in response to cold acclimation and to especially freezing was observed in PLDalpha1-deficient plants. Furthermore, a slower activation of CBF1 was observed in response to cold acclimation in these plants compared to the wild-type plants. Typically, cold acclimation resulted in a higher accumulation of osmolytes in PLDalpha1-deficient plants than in wild-type plants. Inhibition of PLD activity by using lysophosphatidylethanolamine (LPE) also increased freezing tolerance of Arabidopsis, albeit to a lesser extent than did the PLD antisense suppression. Exogenous LPE induced expression of COR15a and COR47 in the absence of cold stimulus. These results suggest that PLDalpha1 plays a key role in freezing tolerance of Arabidopsis by modulating the cold-responsive genes and accumulation of osmolytes.     

Characterization of antifreeze activity in Antarctic plants

Deschampsia antarctica and Colobanthus quitensis are the only vascular plants to have colonized the Maritime Antarctic, which is characterized by its permanently low temperature and frequent summer frosts. To understand how the plants survive freezing temperatures year-round, antifreeze activity was assayed in apoplastic extracts obtained from both non-acclimated and cold-acclimated Antarctic plants. By observing the shape of ice crystals grown in dilution series of the extracts, it was found that D. antarctica had antifreeze activity, but C. quitensis did not. D. antarctica exhibited antifreeze activity in the non-acclimated state and this activity increased after cold acclimation. The antifreeze activity in D. antarctica was labile to proteolysis and high temperature, active over a wide pH range, and associated with molecules greater than 10 kDa in molecular weight. These results show that D. antarctica produces antifreeze proteins that are secreted into the apoplast. When examined by SDS-PAGE, the apoplastic extracts from cold-acclimated D. antarctica exhibited 13 polypeptides. It is concluded that D. antarctica accumulates AFPs as part of its mechanism of freezing tolerance. Moreover, this is the first plant in which antifreeze activity has been observed to be constitutive.     

Identification and expression analysis of cold-regulated genes from the cold-hardy Citrus relative Poncirus trifoliata (L.) Raf.

Citrus is a cold-sensitive genus and most commercially important varieties of citrus are susceptible to freezes. On the other hand, Poncirus trifoliata (L.) Raf. is an interfertile Citrus relative that can tolerate temperatures as low as −26°C when fully cold acclimated. Therefore, it has been used for improving cold tolerance in cold-sensitive commercial citrus rootstock varieties and in attempts to improve scion varieties. In this study, cDNA libraries were constructed from both 2-day cold-acclimated and from non-acclimated Poncirus seedlings using a subtractive hybridization method with the objective of identifying cold-regulated genes. A total of 192 randomly picked clones, 136 from the cold-induced library and 56 from the cold-repressed library, were sequenced. The majority of these clones showed sequence homology to previously identified cold-induced and/or environmental stress-regulated genes in Arabidopsis. In addition, some of them shared homology with cold and/or environmental stress-induced genes previously identified in other herbaceous and woody perennial plants and some showed no homology with sequences in GenBank. When these 192 cDNAs were analyzed by reverse northern blot with cold-acclimated and non-acclimated probes, 92 of the cDNAs displayed significantly increased expression, ranging from 2 to 49-fold, during cold acclimation; all 92 were from the cold-induced library. Surprisingly no clones displayed significantly repressed expression in response to cold. Analysis of a number of selected genes individually in northern blots of mRNA from cold-acclimated and non-acclimated plants largely confirmed the reverse northern analysis, verifying induction of expression of selected cDNAs in response to cold. The results showed that subtractive hybridization is an efficient method for identification of cold-induced genes in plants with limited sequence information available. This study also revealed that genes induced during cold acclimation of the cold-hardy citrus relative P. trifoliata are similar to those in Arabidopsis, indicating that similar pathways may be present and activated during cold acclimation in woody perennial plants.     

Free radical and freezing injury to cell membranes of winter wheat

The symptoms of injury in microsomal membranes isolated from crowns of seedlings of Triticum aestivum , L. cultivar Fredrick after a lethal freeze-thaw stress included an increased lipid phase transition temperature, loss of lipid phosphate (lipid-P), and increased free fatty acid levels. However, minimal changes in fatty acid saturation were observed, suggesting minimal amounts of lipid peroxidation. All of these injury symptoms, including the lack of lipid peroxidation, were simulated in vitro by treatment of isolated membranes with oxygen free radicals, generated from either xanthine oxidase (EC 1.1.3.22) or paraquat (l,r-dimethyl-4,4'-bipyridinium dichloride). Further evidence indicating a relationship between free radicals and freezing injury comes from the observation that both protoplasts and microsomal membranes isolated from wheat seedlings, that had been acclimated to induce freezing tolerance, also had increased tolerance of oxygen free radicals, and contained higher lipid-soluble antioxidant levels, than those from non-acclimated seedlings. Lipid-soluble antioxidants accumulated in the crown tissue of the wheat seedling during the acclimation period. Freezing stress accelerated the formation of oxygen free radicals. Membranes isolated from crowns after a freeze–thaw stress tended to produce higher levels of superoxide as shown by the reduction of Tiron (1,2-dihydroxy-l,3-benzenedisulfonic acid). In protoplasts, increased superoxide production coincided with lethal freezing injury. These results are discussed in terms of the possible involvement of oxygen free radicals in mediating aspects of freezing injury to cell membranes.