Exposure of six wheat (Triticum aestivum L.) and one rye (Secale cereale L.) cultivar to 40% relative humidity for 24 hours induced the same degree of freezing tolerance in seedling epicotyls as did cold conditioning for 4 weeks at 2°C.Frost hardiness varietal relationships were the same in desiccationstressed and cold-hardened seedfings. Drought stress could, therefore, be used as a rapid and simple method for inducing frost hardiness in seedling shoots in replacement of cold conditioning.Desiccation stress is now recognized as able to induce freezing tolerance in winter wheat and rye (5, 11) and in other plants (2,7,9). However, the degree of freezing tolerance conferred by this treatment is not as great as is that by conventional cold-conditioning. Recently, it was shown that freezing tolerance can be increased by changing the conditions of the drought to a 24 h, 40%o RH treatment ofendosperm-dependent seedlings at room temperature (12), enabling us to obtain a high level of frost hardiness in seedling shoots of rye. Under these conditions, tissues do not reach equilibrium with respect to the imposed water stress, and, hence, different degrees of freezing tolerance are induced in different parts of the seedling, maximum freezing tolerance being induced in shoots. Questions arise as to whether freezing tolerance induced by desiccation is the same as that induced in the cold and if the same varietal relationship of freezing tolerance will be observed in desiccation-stressed shoots as is seen in the cold-hardened ones. If so, it will be possible to induce freezing tolerance in cereal varieties by drought instead of by cold, which will be much less demanding of time and equipment.In this work, we report the degree of freezing tolerance induced by drought and cold in six varieties of wheat and one of rye and the correlation between the hardiness resulting from the two stresses. (to be unhardened or desiccation-stressed) or for 4 weeks at 2°C (to be cold-hardened). Seedlings were desiccation-stressed by placing them for 24 h at 21°C over H2SO4 solution of 40%o RH (12) and were reimbibed by immersion in tap water for 16 h. MATERIALS AND METHODSFrost Hardiness Evaluation. Seedlings were surface-dried by blotting over filter paper and were frozen in groups of five in Petri dishes. Cooling rates were 1°C/h from 0 to -21°C, and 3°C/h from -21 to -30°C. For lower temperatures, the seedlings were transferred directly from -30°C to the desired temperature. Samples were seeded with ice crystals at -3°C to avoid supercooling. Frozen samples were thawed at 2°C for 1 h. Survival of shoots or of excised epicotyls was then assessed by the following methods: (a) vital staining with neutral red followed by observation of protoplasmic streaming; (b) detection of ability to plasmolyze and deplasmolyze in salt solution (6); (c) greening in White's solution (13) + 1% Agar-Agar after 2 days at room temperature or in White's solution + 0.2% sucrose after 2 weeks at 2°C (epicotyls developing into a leaf); (d) measuring the rel...

The degrees of freezing tolerance acquired by winter wheat (Triticum aestivium L.) and rye (Secake cereale L. cv Puma) were similar following a 4-week cold conditioning and a 24-hour desiccation stress. Soluble proteins were extracted from shoots of cold-conditioned or desiccation-stressed seedlings and electrophoresed on sodium dodecyl sulfate-polyacrylamide gels. Quantitative changes in the electrophoretic patterns of the soluble proteins of the different cultivars grown in different environments were detected, but the changes were not equivalent following cold conditioning and desiccation stress. The abundance of two polypeptide bands showed a significant increase correlated to the degree of freezing tolerance and, hence, the polypeptides in these bands may play a role in the development of freezing tolerance.The plasma membrane has been suggested as the primary site of frost and plasmolysis dehydration injury of plant cells (7,16,18,19). The mechanism by which the membrane of hardy cells tolerates more freezing and dehydration is not fully known but could be related to the general observation that metabolites accumulate in plant cells during hardening (17), a condition called augmentation of protoplasm (16).The amount of soluble proteins increases in cold and desiccation-hardened plants (2,4,15) and soluble proteins during cold hardening also undergo changes in electrophoretic mobility (1,5,6,(9)(10)(11)(12)14). Soluble protein fractions with apparent mol wt of 240,000 and 115,000 in wheat (14) NaOCl for 3 min and imbibed at 21 or at 2'C (cold conditioned) for 6 h. Seeds were germinated on moist fiter paper in the dark for 2.0 to 2.5 d at 24°C or for 4 weeks at 2°C (cold-conditioned). Seedlings were desiccation stressed by placing them for 24 h at 21°C over H2SO4 solution of 40% RH (3) and were reimbibed by immersion in aerated tap water for 24 h at 9°C.Evaluation of Freezing Tolerance. Water was removed from the surface of seedlings by blotting with filter paper. Seedlings were frozen in groups of 25 in Petri dishes. Cooling rates were 1°C/h from 0 to -21°C and 3°C/h from -21 to -30°C. Some of the seedlings were further cooled to -40°C for 1 h and put on Dry Ice for 1 h additional. Samples were seeded with ice crystals at -3°C to avoid supercooling. Frozen samples were thawed at 2°C for 1 h. Survival of excised epicotyls was assessed by the method we described in a previous paper (3).Soluble Protein Extraction and Determination. For each preparation of soluble proteins, 20 shoots were cut from fresh seedlings and weighed. Proteins were extracted by homogenization of the tissues in 0.1 M Tris-HCl (pH 7.0) at 0°C with a Polytron homogenizer and determined according to Siminovitch et al. (16).Electrophoresis and Calculation of Relative Abundance of Polypeptides. TCA-precipitated soluble proteins were solubilized in the final sample buffer of Laemmli (13) and electrophoresed on 1% SDS-10% polyacrylamide discs (13). Gels were stained with Coomassie blue (8) and gel scans were done at 570 nm on a compute...

Exposure of seedlings of winter rye (Secale cereale L., cv. Puma) for 2 weeks or 24 hours to desiccation stress (40% relative humidity) at room temperature (21°C) in the dark induced degrees of freezing and drought tolerance in the plumules comparable to those produced by cold conditioning for 2 weeks at 3°C. The induction was associated with repression of growth and could not be produced in plumules excised from the seedlings indicating a requirement for translocation of nutrients from the endosperm. Rapid increase in osmotic pressure, soluble proteins, and phospholipids in plumules in association with the development of freezing and drought tolerance and the requirement ofendosperm suggested diversion ofnutrient from use in extension growth, to use in augmentation of protoplasm in plumule cells. Since cold acclimation slowed or arrested growth and is associated with augmentation of protoplasm, it is suggested that the common element in the induction of freezing tolerance by cold and drought is the necessity for producing a condition of augmented protoplasm and membranes in cells thus reinforcing a similar conclusion reached from seasonal studies on woody plants.A number of studies have established that freezing tolerance can be induced in plants by mere desiccation stress without exposure to low temperature (2,4,8,10,23,24). During the course of studies of cold hardening of Puma winter rye seedlings to determine the extent of correlation of their dehydration tolerance with freezing tolerance we observed that the resistance of the cells of the plumules to plasmolysis injury had increased during the desiccation stress. In accordance with correlations which have been established between freezing tolerance and plasmolysis injury resistance (14), it was expected that the freezing tolerance of these cells would also increase and this was confirmed in preliminary studies on rye and wheat (6,21 lings by desiccation stress in 40%o RH (21°C) in the dark and on processes and events related to this induction. We will attempt to show the significance of these results for the study of adaptation of plants to freezing. MATERIALS AND METHODSSeedlings. Winter rye (Secale cereale L., cv Puma) seedlings were used. Seed was washed with 2% NaOCl for 3 min and germinated by immersion of the seeds for 6 h in water and then spreading on moistened blotting paper in trays placed at 24°C. Sprouting was allowed to proceed for 2 days until the plumules had reached 5 to 7 mm.Desiccation Stress. For the desiccation stress, the seedlings were transferred to open Petri dishes placed in a desiccator on a perforated steel plate just above a solution of H2SO4, the concentration of which has been adjusted to produce an atmosphere of 40o RH. Desiccations were allowed to proceed for 2 weeks or 24 h in the dark at 21°C.Test of Viability. At the termination of the desiccation the seedlings were removed and reimbibed. Viability was assessed by regrowth of whole seedlings after planting in vermiculite or by regrowth of excised plumules incubate...

The photoreaction center from Rhodospirillum rubrum contains about 90% protein, 6% pigment, mere traces of lipids, and no cytochromes. It also contains at least 1 mol of ubiquinone and 1 iron atom per mol. Its three-component polypeptide chains were isolated by preparative electrophoresis, and their molar stoichiometry was established as 1:1:1. The amino acid composition of the photoreaction center from strain S1 and from its subunits is reported. The protein as a whole contains about 65% nonpolar residues, and the degree of hydrophobicity of its subunits is alpha less than beta less than gamma. The minimal molecular weight based on the extinction coefficient and on the amino acid content is 90 000. This corresponds to a half-cystine mole number of 6.

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