Low temperature acclimation and treatment with exogenous abscisic acid induce common polypeptides in Arabidopsis thaliana (L.) Heynh

Summary Exogenously applied abscisic acid (ABA) induced frost hardening of Arabidopsis thaliana (L.) Heynh. The freezing tolerance of A. thaliana plantlets treated with ABA (15 mg/l) at a non-acclimating temperature (20 °C) appeared to increase even more rapidly than following a low temperature (4 °C) acclimation. Analysis of in vivo-labelled soluble proteins by two-dimensional gel electrophoresis revealed several low temperature — or ABA — induced proteins, which where not produced in non-acclimated plants. A subset of these proteins was induced by both low temperature and ABA treatments, suggesting that they might be directly involved in the frost hardening process in A. thaliana.     

The manipulation of polar head group composition of phospholipids in the wheat Miranovskaja 808 affects frost tolerance

Caryopses of the frost-resistant cultivar of the wheat Triticum aestivum L., Miranovskaja 808, were germinated and grown in the presence of various concentrations of choline chloride. Changes in the composition of leaf total phospholipids and leaf total fatty acids at two extreme temperatures (25°C and 2°C) as well as changes in frost resistance were followed. A choline chloride concentration-dependent accumulation of phosphatidyl choline was observed in the leaves. Seedlings grown at 2°C accumulated more phosphatidyl choline at each choline chloride concentration than those grown at 25°C. There was an inverse relationship between the contents of phosphatidyl choline and phosphatidic acid in the leaves. Neither the temperature nor choline chloride seemed to affect fatty-acid composition. Modification of polar-head group composition of phospholipids affected frost tolerance: Seedlings grown in the presence of 15 mM choline chloride at 25°C exhibited a freezing resistance equal to that of hardened controls. The data indicate that the polar-head group composition of membrane phospholipids in plants can be easily manipulated and point to the importance of phosphatidyl choline in cold adaptation processes.     

Abscisic acid and mefluidide in the regulation of cold hardiness development in Solanum tuberosum L. potato

Plants of Solanum tuberosum L. potato do not cold acclimate when exposed to low temperature such as 5°C, day/night. When ABA (45 M) was added to the culture medium, stem-cultured plantlets of S. tuberosum, cv. Red Pontiac, either grown at 20°C/15°C, day/night, or at 5°C, increased in cold hardiness from –2°C (killing temperature) to –4.5°C. The increase in cold hardiness could be inhibited in both temperature regimes if cycloheximide (70 M) was added to the culture medium at the inception of ABA treatment. Cycloheximide did not inhibit cold hardiness development, however, when it was added to the culture medium 3 days after ABA treatment.When pot-grown plants were foliar sprayed with mefluidide (50 M), ABA content increased from 10 nmol to 30 nmol g^–1 dry weight and plants increased in cold hardiness from –2°C to about –3.5°C. The increases in free ABA and cold hardiness occurred only in plants grown at 20°C/15°C; neither ABA nor cold hardiness increased in plants grown at 5°C.The results suggest that an increase in ABA and a subsequent de novo synthesis of proteins are required for the development of cold hardiness in S. tuberosum regardless of temperature regime, and that the inability to synthesize ABA at low temperature, rather than protein synthesis, appears to be the reason why S. tuberosum does not cold acclimate.     

Oryzalin-Induced Changes in Water Status and Cytoskeleton Proteins of Winter Wheat Seedlings upon Cold Acclimation and ABA Treatment

The effect of oryzalin (a specific inhibitor of tubulin polymerization in plant cells) on water retention by the leaves and roots of winter wheat (Triticum aestivum L.) seedlings was studied. The cultivars differing in their frost resistance were compared after their acclimation to low temperature (3°C for 3 or 7 days) and after treatment with ABA. In control untreated plants, oryzalin reduced the water-retaining capacity (WRC) of leaves and roots. Both hardening and ABA lowered the effect of the inhibitor on WRC in leaves, whereas their effects on water retention by roots were opposite, i.e., hardening weakened and ABA intensified the effect of oryzalin. Oryzalin-induced reduction of WRC decreased in the following sequence of cultivars: weakly frost resistant moderately frost resistant highly frost resistant. It was more pronounced in the leaves than in the roots, the latter being characterized by the lower WRC and lower frost resistance. After three-day-long hardening of plants, an additive effect of hypothermia and ABA on oryzalin-induced decrease in WRC of leaves and the lack of such effect in the roots were observed. The immunochemical analysis of the composition and content of cytoskeletal proteins with Western blotting showed that in the leaves the actin/tubulin ratio was higher than in the roots. The treatment of nonacclimated plants with ABA lowered the content of - and -tubulins and actin in roots but did not affect the level of actin in leaves. Hardening negated the effects of ABA on cytoskeletal proteins. Oryzalin produced the greatest inhibitory effect on WRC and an increase in frost resistance in ABA-treated plants in the experiments with leaves of the weakly frost resistant cultivar before and after hardening. Organ- and cultivar-specific and ABA-mediated dependence of WRC on cytoskeletal proteins and microtubules and microfilaments formed by them is supposed to result from their effect on the state of intracellular water and water permeability of the plasma membrane. In the course of cold acclimation of plants and upon their treatment with ABA, this dependence was more distinctly expressed in leaves than in roots, and especially in the plants of the weakly frost resistant cultivar.     

Comparison of Barley Response to Short-Term Cold or Dehydration

Abscisic acid (ABA) content and relative water content (RWC) in second fully expanded leaves of cold hardened plants and in dehydrated leaves of freezing tolerant barley (Hordeum vulgare L. cv. Lunet) were compared. ABA content and RWC in leaves did not change during the first day of cold hardening. On the contrary, dehydration of leaves led to a decrease of RWC and to an increase of ABA content.     

Effects of abscisic acid,low temperature,and plant age on cytoskeleton and phosphorylated proteins

The effects of exogenous abscisic acid (ABA), low temperature, and seedling age on the content of tubulin, actin, and phosphorylated proteins and the structural organization of microtubules (MTs) in cells of different tissues and organs of winter wheat cultivars contrasting in cold hardiness were studied by immunocytochemical methods using monoclonal (against - and -tubulin and actin) and polyclonal (phosphothreonine) antibodies. The leaves and roots of five- and nine- day-old seedlings of three cultivars were characterized by unequal proportion of actin/tubulin proteins. ABA decreased the content of the cytoskeleton and the 60-kD phosphorylated proteins, thus promoting a decrease in the number of MTs and occurrence of a less branched network of weakly fluorescent tubulin components in the cells of the root differentiating zone (which is most responsible for the development of cold hardiness in wheat). Although the cold acclimation of plants (3°C, 7 days) did not change the level of tubulin and actin proteins, it evoked the spatial aggregation of MT, leading to formation of a dense network of tubulin cytoskeleton comprised of thick bundles of intensively fluorescent MTs. In the case of a combined action of the studied factors, low temperatures abolished the hormone effect described above, evoking an increase in the content of the cytoskeletal and 60-kD phosphorylated proteins and MT structures. We suggest that the ABA-induced decrease in the levels of proteins and MTs occurs at the initial stages of plant cold acclimation (3°C, 2-3 days). It may be the signal that triggers the processes of low-temperature adaptation. As the duration of cold acclimation increased (3°C, 7 days), the role of ABA in the formation of plant tolerance decreased. Apparently, in this case other hormone-independent mechanisms of frost hardiness development are triggered, in which the role of the cytoskeleton components and cytoskeleton-associated proteins increases.     

Ultrastructural and protein changes in cell suspension cultures of peach associated with low temperature-induced cold acclimation and abscisic acid treatment

Cell suspension cultures were initiated from callus derived from xylem tissues of peach [Prunus persica (L.) Batsch]. Cold acclimation was induced (LT₅₀ of-13°C) in cell suspensions at 3°C in the dark for 10 days. Freezing tolerance returned to the level of nonacclimated cells (LT₅₀ of –4.5°C) when cold-acclimated cells were transferred to 24°C (in dark) for 3 days. Addition of 75 M abscisic acid (ABA) to the growth medium failed to induce cold acclimation after cells were cultured for 5 days at 24°C. Microvacuolation, cytoplasmic augmentation and disappearance of starch grains were observed in cells that were cold-acclimated by exposure to low temperature. Similar ultrastructural alterations were not observed in ABA-treated cells. Several qualitative and quantitative changes in proteins were noted during both cold acclimation and ABA treatment. Both the ultrastructural and protein changes observed during cold acclimation were reversed during deacclimation. The relationship of these changes to cold acclimation in peach cell-cultures is discussed.Abbreviations ABA abscisic acid - 2,4-d 2,4-dichlorophenoxyacetic acid - IBA indole-3-butyric acid - Ms Murashige & Skoog - PMSF phenylmethylsulfonyl fluoride - LT₅₀ or Freezing Tolerance temperature that resulted in 50% decrease in TTC reduction - TTC 2,3,5-triphenyltetrazolium chloride     

Carbohydrate content of Eucalyptus gunnii leaves along an annual cycle in the field and during induced frost-hardening in controlled conditions

The annual changes in frost hardiness were studied for three Eucalyptus gunnii genotypes. Frost resistance evaluated on leaf discs by the electrolyte leakage method reached a maximum in the coldest period and a minimum in summer demonstrating winter frost hardening. Genotype 634 exhibited a higher intrinsic resistance than the other genotypes both in the hardened and in the non-hardened stages. Plants of this genotype were also frost acclimated in controlled conditions by a progressive decrease of culture temperature (25 to 0 °C) but the degree of hardening appeared to be lower in these conditions. The carbohydrate patterns in leaves varied with acclimation. In controlled conditions the leaves of genotype 634 exhibited a rise in sucrose, fructose and raffinose concentration up to a temperature of 10 to 7 °C which subsequently decreased. In natural conditions a comparison of the three genotypes allowed us to correlate the higher intrinsic resistance of genotype 634 to a higher soluble sugar content. During acclimation fructose and raffinose changes were also correlated to an increase in cold resistance even though the kinetics of these changes differed in controlled and natural conditions. The starch content was very low in the various genotypes in the different conditions but oligosaccharides such as stachyose and possibly verbascose were detected. The results point out the relationships occurring between increased frost resistance and changes in fructose and raffinose concentration in E. gunnii leaves.     

Enhancement of frost tolerance in olive shoots in vitro by cold acclimation and sucrose increase in the culture medium

The evaluation of frost tolerance in olive shoots in vitro has been successfully accomplished. The behavior of in vitro shoots at freezing temperatures was comparable to that of intact plants. Cold acclimation was found to increase frost tolerance in cv. Moraiolo and the LT₅₀ was about 4 °C lower compared to nonacclimated shoots. Damage in acclimated shoots occurred at –15 °C, whereas control shoots were damaged at –10 °C. Olive shoots were unable to withstand freezing temperatures of –20 °C, even when acclimated. The effects of sucrose were also determined. 6% (w/v) sucrose in the medium conferred the highest frost tolerance in both acclimated and nonacclimated plants.     

Up-regulation of stress-inducible genes in tobacco and <Emphasis Type="Italic">Arabidopsis</Emphasis> cells in response to abiotic stresses and ABA treatment correlates with dynamic changes in histone H3 and H4 modifications

Animal cells react to mitogenic or stress stimuli by rapid up-regulation of immediate-early (IE) genes and a parallel increase in characteristic modifications of core histones: chromatin changes, collectively termed the nucleosomal response. With regard to plants little is known about the accompanying changes at the chromatin level. We have used tobacco BY-2 and Arabidopsis T87 cell lines to study the nucleosomal response of plant cells to high salinity, cold and exogenous abscisic acid (ABA). When in quiescent stage, both tobacco and Arabidopsis cells show the typical nucleosomal response to high salinity and cold stress, manifested by rapid transient up-regulation of histone H3 Ser-10 phosphorylation, immediately followed by transient up-regulation of H3 phosphoacetylation and histone H4 acetylation. For each of the studied stresses the observed nucleosomal response was strictly correlated with the induction of stress-type specific genes. The dynamics of histone modifications in BY-2 cells in response to exogenous ABA exhibited a more complex pattern than that evoked by the two abiotic stresses, probably due to superposition of the primary and secondary effects of ABA. A rapid increase in H3 Ser-10 phosphorylation was also observed in whole leaves subjected to high salinity; however, the rate of change in this modification was much slower than in cultured cells. Together, these results indicate that the quiescent BY-2 and T87 cell lines show a typical nucleosomal response to abiotic stresses and ABA treatment and may represent suitable models for the study of chromatin-mediated mechanisms of stress tolerance in plants.     

Cold acclimation and cold-regulated gene expression in ABA mutants of Arabidopsis thaliana

We have examined the cold-induced enhancement of freezing tolerance and expression of cold-regulated (cor) genes in Arabidopsis thaliana (L.) Heynh (Landsberg erecta) and abscisic acid (ABA)-deficient (aba) and ABA-insensitive (abi) mutants derived from it. The results indicate that the abi mutations had no apparent effect on freezing tolerance, while the aba mutations did: cold-acclimated aba mutants were markedly impaired in freezing tolerance compared to wild-type plants. In addition, it was observed that non-frozen leaves from both control and cold-treated aba mutant plants were more ion-leaky than those from corresponding wild-type plants. These data are consistent with previous observations indicating that ABA levels can affect freezing tolerance. Whether ABA has a direct role in the enhancement of freezing tolerance that occurs during cold acclimation, however, is uncertain. Several studies have suggested that ABA might mediate certain changes in gene expression that occur during cold acclimation. Our data indicate that the ABA-induced expression of three ABA-regulated Arabidopsis cor genes was unaffected in the abi2, abi3, and aba-1 mutants, but was dramatically impaired in the abi1 mutant. Cold-regulated expression of all three cor genes, however, was nearly the same in wild-type and abi1 mutant plants. These data suggest that the cold-regulated and ABA-regulated expression of the three cor genes may be mediated through independent control mechanisms.     

The effects of ABA and GA3 treatments on resistance to frost and high-light treatment in oilseed rape leaf discs

The experiments were performed to check the effects of exogenous ABA and gibberellin on photosynthetic apparatus and leaf resistance to freezing. In the experiment, two cultivars (winter and spring) of oilseed rape were used in the experiment. Discs, cut out from leaves of cold acclimated plants grown at 12 and 20 °C at similar PPFD levels, were immersed for 72 hours in growth regulator solutions. Some of discs were additionally subjected to high radiation. Independently on cultivar studied, the effects of growth regulator treatments were significant only in leaves developed at 20 °C. ABA treatment increased frost resistance, promoted photosynthetic activity measured in cold and inhibited expansion of leaf-disc area, whereas GA₃ evoked opposite effects. The treatment with growth regulators particularly affected the resistance of photosynthetic apparatus to high light. In this case ABA treatment decreased, whereas GA₃ increased photoinhibition of PSII. The outcomes may suggest that in the ABA-treated plants PSII is better protected against photoinduced inactivation both by the increase in effectiveness of photosynthetic dark reactions at high light/low temperature conditions, increased energy dissipation in xantophyll cycle and enhanced accumulation of anthocyanins. GA₃ treatment may affect the resistance to photoinhibition directly via decrease in anthocyanins contents and indirectly through increase of elongation growth rate in the tissue.     

The role of the photosynthetic apparatus in cold acclimation of <Emphasis Type="Italic">Lolium multiflorum</Emphasis>. Characteristics of novel genotypes low-sensitive to PSII over-reduction

During cold acclimation by higher plants, temperature perception via changes in redox state of Photosystem II (PSII) and subsequent acclimation of the photosynthetic apparatus to cold is very important for achieving freezing tolerance. These properties were studied in two groups (A and B) of the same backcross 3 (BC₃) progeny derived from a triploid hybrid of Festuca pratensis (2×) × Lolium multiflorum (4×) backcrossed three times onto diploid L. multiflorum cultivars. Leaves of Group A plants formed at 20°C at medium-low light were unable to acclimate their photosynthetic apparatus to cold. Compared to Group B, the Group A plants were also more frost sensitive. This acclimation ability correlated with the freezing tolerance of the plants. However, leaves of the same Group A plants developed at 20°C, but under higher-light conditions had increased ability to acclimate their photosynthetic apparatus to cold. It was concluded that Group A plants may have impaired PSII temperature perception, and this then resulted in their poor capability to cold acclimate.     

Effects of Cold Acclimation and Salicylic Acid on Changes in ACC and MACC Contents in Maize during Chilling

The effect of 0.5 mM salicylic acid (SA) pretreatment and of growing at hardening temperatures on chilling-induced changes in 1-aminocyclopropane-1-carboxylic acid (ACC) and malonyl 1-aminocyclopropane-1-carboxylic acid (MACC) was investigated in young maize (Zea mays L.) plants grown in hydroponic solution at 22/20 °C. Chilling at 5 °C caused an increase in ACC content;however, this increase was less pronounced in plants cold acclimated at 13/11 °C 4 d before the chilling treatment, and in those which were pretreated with SA for 1 d before the cold stress. Changes in MACC at low temperature showed no correlation with chilling tolerance in maize.     

Changes in abscisic acid and its glucose ester in Phaseolus vulgaris L. during chilling and water stress

Levels of free and conjugated abscisic acid (ABA) were determined in leaves and roots of intact bean (Phaseolus vulgaris L., cv. Mondragone) seedlings under chilling (3C) and drought as well as during recovery from stress. Abscisic acid-glucose ester (ABAGE) was the only conjugate releasing free ABA after alkaline hydrolysis of the crude aqueous extracts. During the first 20–30 h chilled plants rapidly dehydrated and wilted without any change in ABA and ABAGE levels. Subsequently, leaf and root ABA levels increased and plants regained turgor. ABAGE concentration showed a slight increase in leaves but not in roots. Upon recovery from chilling a transient, but significant, rise in leaf ABA content was observed, while no appreciable change in ABAGE was found. Drought triggered ABA accumulation in leaves and roots, while a rise in ABAGE content was detected only in leaf tissues. Recovery from stress caused a drop in ABA levels without a correspondent increase in ABAGE concentration. We conclude that ABAGE is not a source of free ABA during either chilling or water stress and that only a small proportion of the ABA produced under stress is metabolised to ABAGE during recovery.Abbreviations ABA = abscisic acid - ABAGE = abscisic acid-glucose ester - DW = dry weight - FW = fresh weight - RIA = radioimmunoassay - RWC = relative water content - w = water potential - o = osmotic potential - p = turgor potential     

Abscisic acid deficiency prevents development of freezing tolerance in Arabidopsis thaliana (L.) Heynh.

Summary Abscisic acid (ABA) has been implicated as a regulatory factor in plant cold acclimation. In the present work, the cold-acclimation properties of an ABA-deficient mutant (aba) of Arabidopsis thaliana (L.) Heynh. were analyzed. The mutant had apparently lost its capability to cold acclimate: the freezing tolerance of the mutant was not increased by low temperature treatment but stayed at the level of the nonacclimated wild type. The mutational defect could be complemented by the addition of exogenous ABA to the growth medium, restoring freezing tolerance close to the wild-type level. This suggests that ABA might have a central regulatory function in the development of freezing tolerance in plants. Cold acclimation has been previously correlated to the induction of a specific set of proteins that have been suggested to have a role in freezing tolerance. However, these proteins were also induced in the aba mutant by low temperature treatment.     

ABA and gibberellin-like substances during prehardening,cold acclimation,de- and reacclimation of oilseed rape

Previously published results showed that high relative reduction state of PSII (PSII excitation pressure) during both early seedling growth (prehardening) as well as cold deacclimation caused significant changes in growth pattern. The differences in elongation growth rate were related to the cold acclimation of photosynthetic apparatus and to frost resistance. To study changes in the hormonal balance connected with alterations in elongation growth rate observed during prehardening and deacclimation under different PSII excitation pressure (modulated by day-temperatures), endogenous concentration of ABA, GA₃ and GA-like substances (GAs) were analysed. Analyses were also performed during cold acclimation and reacclimation of plants characterized by different elongation growth rate triggered by prehardening or deacclimation under different day-temperatures. Growth under high PSII excitation pressure (prehardening) resulted in a significant increase in ABA and a considerable decrease in GAs contents. On the other hand, different ABA content played almost no role in controlling growth rate during cold deacclimation and subsequent reacclimation, when the induction of elongation growth was connected with the changes in concentration of GAs including GA₃. The possible role of ABA and GAs in controlling prehardening, cold acclimation and deacclimation is discussed.     

Correlation between loss of turgor and accumulation of abscisic acid in detached leaves

Mature leaves of Phaseolus vulgaris L. (red kidney bean), Xanthium strumarium L. (cocklebur), and Gossypium hirsutum L. (cotton) were used to study accumulation of abscisic acid (ABA) during water stress. The water status of individual, detached leaves was monitored while the leaves slowly wilted, and samples were cut from the leaves as they lost water. The leaf sections were incubated at their respecitive water contents to allow ABA to build up or not. At least 8 h were required for a new steady-state level of ABA to be established. The samples from any one leaf covered a range of known water potentials (), osmotic pressures (), and turgor pressures (p). The and p values were calculated from pressure-volume curves, using a pressure bomb to measure the water potentials. Decreasing water potential had little effect on ABA levels in leaves at high turgor. Sensitivity of the production of ABA to changes in progressively increased as turgor approached zero. At p=1 bar, ABA content averaged 4 times the level found in fully turgid samples. Below p=1 bar, ABA content increased sharply to as much as 40 times the level found in unstressed samples. ABA levels rose steeply at different water potentials for different leaves, according to the at which turgor became zero. These differences were caused by the different osmotic pressures of the leaves that were used; must cqual - for turgor to be zero. Leaves vary in , not only among species, but also between plants of one and the same species depending on the growing conditions. A difference of 6 bars (calculated at =0) was found between the osmotic pressures of leaves from two groups of G. hirsutum plants; one group had previously experienced periodic water stress, and the other group had never been stressed. When individual leaves were subsequently wilted, the leaves from stress-conditioned plants required a lower water potential in order to accumulate ABA than did leaves from previously unstressed plants. On the basis of these results we suggest that turgor is the critical parameter of plant water relations which controls ABA production in water-stressed leaves.Abbreviations ABA abscisic acid - me-ABA abscisic-acid methyl ester - leaf water potential - osmotic pressure - p volumeaveraged turgor - volumetric modulus of elasticity     

Changes in abscisic acid levels in embryo axes of Norway maple and sycamore seeds during maturation and dehydration

Changes in the abscisic acid (ABA) levels in embryo axes of seeds, belonging to the orthodox (Norway maple — Acer platanoides L.) and recalcitrant (sycamore — Acer pseudoplatanus L.) categories, were investigated throughout maturation using an ELISA (enzyme-linked immunosorbent assay) test. Concentration of ABA in embryo axes substantially differed depending on species and sampling date. ABA was always higher in Norway maple except at the end of seed maturation when ABA content was similar in both species. During maturation ABA decreased in both species but the decline was more marked in Norway maple than in sycamore (11 vs. 3 fold). These species also differed in the pattern of ABA changes, which in sycamore embryo axes was very regular, while in Norway maple a sharp decrease was recorded after acquisition by the seeds of tolerance to desiccation. Dehydration of embryo axes of Norway maple caused a further significant decrease of ABA level. In contrast, in dehydrated sycamore embryo axes ABA content did not decrease, but slightly increased. The role of ABA in desiccation tolerance and dormancy of Norway maple and sycamore seeds is discussed.     

Variations in abscisic acid, indole-3-acetic acid and zeatin riboside concentrations in two Mediterranean shrubs subjected to water stress

Water stress induced an increase in endogenous concentrations of ABA in Lavandula stoechas L. plants to 13100 pmol ABA g^–1 FW, which may contribute to the maintenance of water relations between the second and the third day of water stress treatment. After the third day, a sharp decrease in ABA levels was observed to 2630 pmol ABA g^–1 FW, together with a decrease in water content and water potential and a loss of plant response to water stress. Water deficit did not induce an increase in endogenous ABA concentration, which remained at 514 pmol ABA g^–1 FW in Rosmarinus officinalis L., which is more sclerophyllous than L. stoechas. Nevertheless, the relative water content of Rosmarinus officinalis L. after seven days of water stress decreased more than 40% and reached values of –3.2 MPa. R. officinalis showed lower levels of ABA, but significantly higher levels of IAA and ZR than L. stoechas (4 times and 6 times respectively in well watered-plants). The increase in ABA levels is not a common mechanism in these two Mediterranean shrubs which survive under water stress conditions.Abbreviations ABA abscisic acid - d days of water stress treatment - DW dry weight - FW fresh weight - IAA indole-3-acetic acid - RP Reversed Phase - RWC relative water content - TW turgid weight - WC water content - ZR zeatin riboside - water potential