Accumulation of a High Molecular Weight Protein during Cold Hardening of Wheat (Triticum aestivum L.)

Electrophoretic patterns of soluble protein fractions from cold-tolerantwinter wheats (Triticum aestivum L. cv. Frederick and cv. Norstar)and cold-sensitive spring wheat (T. aestiaum L. cv. Glenlea)were analysed in hardened and unhardened plants. One and two-dimensionalgel electrophoresis analysis reveals that cold hardening conditionsinduce changes in the soluble protein patterns. The most importantis the accumulation of a high molecular weight protein in therange of 200 kDa. This protein accumulated at higher concentrationin cold-tolerant cultivars compared to the coldsensitive onesuggesting a correlation between the degree of freezing toleranceand the accumulation of this specific protein. In addition,the intensity of three protein bands (mol wt 48, 47 and 42 kDa)increased while that of five others (mol wt 93, 89, 80, 67 and63 kDa) decreased during hardening. These changes occured inthe three cultivars suggesting that they are part of the metabolicadjustments in response to low temperature rather than a specificchange associated with the development of cold hardiness. (Received April 23, 1987; Accepted June 5, 1987)     

Free and Bound Gibberellin Activities and Ent-kaurene Synthesis during Induction of Cold Hardiness in Black Locust Seedlings

The role of gibberellin in the development of cold hardiness in black locust (Robinia pseudoacacia L.) seedlings was investigated. Free and bound gibberellin activities were followed during induction of cold hardiness using ethyl acetate partitioning and pH variation, with subsequent paper chromatographic fractionation and gibberellin bioassay. While total gibberellin activity decreased during the induction of cold hardiness in black locust seedlings, no convincing evidence was found to support conversion of free gibberellin to a bound form. However, bound gibberellin activity did appear to be more stable than did free gibberellin activity during the final stages of cold hardening at freezing temperatures. Gibberellin synthesis was followed using ¹⁴C-mevalonate conversion to ent-kaurene in a cell-free extract of the tissue. Ent-kaurene synthesis decreased during cold hardening with no detectable synthesis in fully hardened seedlings. However, since growth cessation precedes development of cold hardiness, decreased gibberellin synthesis and corresponding trends in free and bound fractions might have been expected, and a cause and effect relationship is difficult to establish. Even so, a decline in one step in gibberellin synthesis and a greater stability of bound than free gibberelin activity are associated with induction of cold hardiness in black locust seedlings.     

Synthesis of Freezing Tolerance Proteins in Leaves, Crown, and Roots during Cold Acclimation of Wheat

Protein synthesis was studied in leaves, crown, and roots during cold hardening of freezing tolerant winter wheat (Triticum aestivum L. cv Fredrick and cv Norstar) and freezing sensitive spring wheat (T. aestivum L. cv Glenlea). The steady state and newly synthesized proteins, labeled with [³⁵S]methionine, were resolved by one- and two-dimensional polyacrylamide gels. The results showed that cold hardening induced important changes in the soluble protein patterns depending upon the tissue and cultivar freezing tolerance. At least eight new proteins were induced in hardened tissues. A 200 kilodalton (kD) (isoelectric point [pl] 6.85) protein was induced concomitantly in the leaves, crown, and roots. Two proteins were specifically induced in the leaves (both 36 kD, pl 5.55 and 5.70); three in the crown with M_r 150 (pl 5.30), 45 (pl 5.75), and 44 kD (pl > 6.80); and two others in the roots with M_r 64 (pl 6.20) and 52 kD (pl 5.55). In addition, 19 other proteins were synthesized at a modified rate (increased or decreased) in the leaves, 18 in the crown and 23 in the roots. Among the proteins induced or increased in hardened tissues, some were expressed at a higher level in the freezing tolerant cultivars than in the sensitive one, indicating a correlation between the synthesis and accumulation of these proteins and the degree of freezing tolerance. These proteins, suggested to be freezing tolerance proteins, may have an important role in the cellular adaptation to freezing.     

Factors contributing to enhanced freezing tolerance in wheat during frost hardening in the light

The interaction between light and temperature during the development of freezing tolerance was studied in winter wheat (Triticum aestivum L. var. Mv Emese). Ten-day-old plants were cold hardened at 5 degrees C for 12 days under normal (250 micromol m(-2)s(-1)) or low light (20 micromol m(-2)s(-1)) conditions. Some of the plants were kept at 20/18 degrees C for 12 days at high light intensity (500 micromol m(-2)s(-1)), which also increased the freezing tolerance of winter wheat. The freezing survival rate, the lipid composition, the antioxidant activity, and the salicylic acid content were investigated during frost hardening. The saturation level of hexadecanoic acid decreased not only in plants hardened at low temperature, but also, to a lesser extent, in plants kept under high light irradiation at normal growth temperature. The greatest induction of the enzymes glutathione reductase (EC 1.6.4.2.) and ascorbate peroxidase (EC 1.11.1.11.) occurred when the cold treatment was carried out in normal light, but high light intensity at normal, non-hardening temperature also increased the activity of these enzymes. The catalase (EC 1.11.1.6.) activity was also higher in plants grown at high light intensity than in the controls. The greatest level of induction in the activity of the guaiacol peroxidase (EC 1.11.1.7.) enzyme occurred under cold conditions with low light. The bound ortho-hydroxy-cinnamic acid increased by up to two orders of magnitude in plants that were cold hardened in normal light. Both high light intensity and low temperature hardening caused an increase in the free and bound salicylic acid content of the leaves. This increase was most pronounced in plants that were cold treated in normal light.     

Gene expression during cold and heat shock in wheat

Translatable messenger RNAs expression was compared in cold- and heat-stressed winter wheat (Triticum aestivum L. 'Fredrick' and 'Norstar') and spring wheat (T. aestivum L. 'Glenlea'). Polyadenylated RNA isolated from the crown and leaf tissues was translated in a wheat germ cell free system and the acidic and basic in vitro products were resolved by two-dimensional SDS-PAGE and autoradiography. The results showed that low temperature stress rapidly induced two groups of mRNAs. The first group was transient in nature and consists of 18 mRNAs that reached their highest levels of induction after 24 h of low temperature exposure and then decreased to undetectable levels. The second group consists of 53 mRNAs that were also induced or increased rapidly, but maintained their levels of expression during the 4 weeks required to induce freezing tolerance. Among those, at least 34 were expressed at higher levels in the freezing tolerant winter wheat compared with the less tolerant spring wheat. This suggests a possible relation between the expression of these mRNAs and the capacity of each genotype to develop freezing tolerance. In the case of heat shock, 50 mRNAs were induced or increased after 3 h at 40 degrees C. Among these, the expression of only six mRNAs was altered in a similar manner in the three genotypes by both treatments. The remaining mRNAs code for typical heat shock proteins which are different from those induced by low temperature. None of these mRNAs has been associated with the development of freezing tolerance. These results suggest that heat and cold stress are controlled by different genetic systems.     

Heat Tolerance of Cold Acclimated Puma Winter Rye Seedlings and the Effect of a Heat Shock on Freezing Tolerance

An increase in tolerance to one form of abiotic stress oftenresults in an increase in tolerance to another stress. The heattolerance of Puma rye (Secale cereale) was determined for seedlingseither not cold hardened or hardened under either controlledenvironmental or natural conditions. The heat tolerance wasdetermined either as a function of time at 42°C or the abilityto tolerate a maximum temperature. The seedlings were eithernot heat preconditioned or heat preconditioned before the heatstress. In all cases cold hardened seedlings were more heattolerant than non or partially cold hardened seedlings. Heatpreconditioning had no effect on the heat tolerance of naturallycold hardened seedlings. In contrast, seedlings cold hardenedin a controlled environment chamber, then heat preconditioned,were more heat tolerant than non preconditioned seedlings. Aheat shock of 36°C for 2 h increased the freezing toleranceof non hardened seedlings from –2.5°C to –4.5°C.Analysis of heat shock protein 70 (HSP70) gene expression indicatedthat the HSP70 gene was not induced by cold acclimation andtherefore not directly involved in the increased thermo toleranceobserved. A number of heat stable proteins, simple sugars andlong chain carbohydrate polymers accumulated during the coldacclimation process and may have a role in increasing heat toleranceas well as freezing tolerance. These data suggest cold hardeningincreases heat tolerance, however, a heat shock to non acclimatedseedlings only marginally increased the freezing tolerance ofPuma rye seedlings. ³Present address: Agriculture and Agri-Food Canada, 107 SciencePlace, Saskatoon SK S7N 0X2, Canada.     

Mitochondrial energy-dissipating systems (alternative oxidase,uncoupling proteins,and external NADH dehydrogenase) are involved in development of frost-resistance of winter wheat seedlings

Gene expression, protein synthesis, and activities of alternative oxidase (AOX), uncoupling proteins (UCP), adenine nucleotide translocator (ANT), and non-coupled NAD(P)H dehydrogenases (NDex, NDPex, and NDin) were studied in shoots of etiolated winter wheat (Triticum aestivum L.) seedlings after exposure to hardening low positive (2°C for 7 days) and freezing (?2°C for 2 days) temperatures. The cold hardening efficiently increased frost-resistance of the seedlings and decreased the generation of reactive oxygen species (ROS) during further cold shock. Functioning of mitochondrial energy-dissipating systems can represent a mechanism responsible for the decrease in ROS under these conditions. These systems are different in their response to the action of the hardening low positive and freezing temperatures. The functioning of the first system causes induction of AOX and UCP synthesis associated with an increase in electron transfer via AOX in the mitochondrial respiratory chain and also with an increase in the sensitivity of mitochondrial non-phosphorylating respiration to linoleic and palmitic acids. The increase in electron transfer via AOX upon exposure of seedlings to hardening freezing temperature is associated with retention of a high activity of NDex. It seems that NDex but not the NDPex and NDin can play an important role in maintaining the functional state of mitochondria in heterotrophic tissues of plants under the influence of freezing temperatures. The involvement of the mitochondrial energy-dissipating systems and their possible physiological role in the adaptation of winter crops to cold and frost are discussed.     

Adaptation of membrane fluidity of rye wheat seedlings according to temperature

Order parameters of chloroplast membrane lipids of rye wheat seedlings differing in cold hardiness were compared before after hardening. Seedlings grown at 25° exhibited similar membrane microviscosities. When hardened, the cultivars most resistant to freezing temperatures possessed the most fluid membranes, while those sensitive to cold were unable to alter them. Changes in linolenic acid levels alone cannot be responsible for the observed phenomena.     

Adaptive Changes in ATPase Activity in the Cells of Winter Wheat Seedlings during Cold Hardening

A cytochemical study of ATPase activity in the cells of cold hardened and nonhardened winter wheat (Triticum aestivum L. cv. Nongke No. 1) seedlings was carried out by electron microscopic observation of lead phosphate precipitation. ATPase activity associated with various cellular organelles was altered during cold hardening. (a) At 22°C, high plasmalemma ATPase activity was observed in both cold hardened and nonhardened tissues; at 5°C, high activity of plasmalemma ATPase was observed in hardened tissues, but not in unhardened tissues. (b) In nonhardened tissues, tonoplast and vacuoles did not exhibit high ATPase activity at either 22 or 5°C, while in hardened tissues high activity was observed at both temperatures. (c) At 5°C, ATPase activity of nucleoli and chromatin was decreased in hardened tissues, but not in nonhardened tissues. It is suggested that adaptive changes in ATPase activity associated with a particular cellular organelle or membrane may be associated with the development of frost resistance of winter wheat seedlings.     

Nitrate-induced polypeptides in membranes from corn seedling roots

The polypeptide composition of the membranes from corn (Zeamays L.) seedling roots upon nitrate induction was determinedby two-dimensional gel electrophoresis and silver-staining.The synthesis of five polypeptides (49, 48, 35, 33, and 32 kDa)in the tono-plast fraction and four polypeptides (50, 49, 38,and 33 kDa) in the plasma membrane fraction was induced by both2.5 mM Ca(NO₃)₂ and 5 mM KNO₃. Extensive washing of the membraneswith salt and NaOH demonstrated that three induced polypeptides(49, 48, and 35 kDa) in the tonoplast fraction and two inducedpolypeptides (49 and 33 kDa) in the plasma membrane fractionwere integral proteins. After incubation of seedlings in N-freemedium for 4 d, the 49 and 32 kDa polypeptides in the tonoplastfraction had disappeared. By the sixth day in N-free medium,the 35 kDa polypeptide had disappeared from the tonoplast fraction.The 50 kDa polypeptide of the plasma membrane fraction was nolonger detectable in seedlings incubated for 6 d in N-free medium.The size of the spots corresponding to the 33 kDa polypeptidesof both membrane fractions and to the 49 kDa polypeptide ofthe plasma membrane fraction was reduced following incubationof seedlings in N-free medium. The changes in nitrate-inducedpolypeptides in both membrane fractions following transfer toN-free medium correlated with a reduced capacity to take upnitrate in the treated seedlings. The results support the conclusionthat the nitrate-induced polypeptides may be involved in nitratetransport across the tonoplast and plasma membrane. Key words: Nitrate transport, induction, membrane peptides     

Induction of freezing tolerance in spinach is associated with the synthesis of cold acclimation induced proteins

Spinach (Spinacia oleracea L. cv Bloomsdale) seedlings cultured in vitro were used to study changes in protein synthesis during cold acclimation. Seedlings grown for 3 weeks postsowing on an inorganic-nutrient-agar medium were able to increase their freezing tolerance when grown at 5°C. During cold acclimation at 5°C and deacclimation at 25°C, the kinetics of freezing tolerance induction and loss were similar to that of soil-grown plants. Freezing tolerance increased after 1 day of cold acclimation and reached a maximum within 7 days. Upon deacclimation at 25°C, freezing tolerance declined within 1 day and was largely lost by the 7th day. Leaf proteins of intact plants grown at 5 and 25°C were in vivo radiolabeled, without wounding or injury, to high specific activities with [³⁵S]methionine. Leaf proteins were radiolabeled at 0, 1, 2, 3, 4, 7, and 14 days of cold acclimation and at 1, 3, and 7 days of deacclimation. Up to 500 labeled proteins were separated by two-dimensional gel electrophoresis and visualized by fluorography. A rapid and stable change in the protein synthesis pattern was observed when seedlings were transferred to the low temperature environment. Cold-acclimated leaves contained 22 polypeptides not found in nonacclimated leaves. Exposure to 5°C induced the synthesis of three high molecular weight cold acclimation proteins (CAPs) (M_r of about 160,000, 117,000, and 85,000) and greatly increased the synthesis of a fourth high molecular weight protein (M_r 79,000). These proteins were synthesized during day 1 and throughout the 14 day exposure to 5°C. During deacclimation, the synthesis of CAPs 160, 117, and 85 was greatly reduced by the first day of exposure to 25°C. However, CAP 79 was synthesized throughout the 7 day deacclimation treatment. Thus, the induction at low temperature and termination at warm temperature of the synthesis of CAPs 160, 117, and 85 was highly correlated with the induction and loss of freezing tolerance. Cold acclimation did not result in a general posttranslational modification of leaf proteins. Most of the observed changes in the two-dimensional gel patterns could be attributed to the de novo synthesis of proteins induced by low temperature. In spinach leaf tissue, heat shock altered the pattern of protein synthesis and induced the synthesis of several heat shock proteins (HSPs). One polypeptide synthesized in cold-acclimated leaves had a molecular weight and net charge (M_r 79,000, pI 4.8) similar to that of a HSP (M_r 83,000, pI 4.8). However, heat shock did not increase the freezing tolerance, and cold acclimation did not increase heat tolerance over that of nonacclimated plants, but heat-shocked leaf tissue was more tolerant to high temperatures than nonacclimated or cold-acclimated leaf tissue. When protein extracts from heat-shocked and cold-acclimated leaves were mixed and separated in the same two-dimensional gel, the CAP and HSP were shown to be two separate polypeptides with slightly different isoelectric points and molecular weights.     

Heat-Stable COR (Cold-Regulated) Proteins Associated with Freezing Tolerance in Spinach

While most soluble proteins are coagulated by heating at 100°Cfor 10 minutes, some highly hydrophilic COR (Cold-regulated)proteins remain soluble in aqueous solution (Lin et al. 1990).We report here changes in levels of heat-stable proteins andtheir mRNAs during cold acclimation of spinach (Spinacia oleraceaL.). We analyzed heat-stable proteins and the heat-stable translationproducts from poly(A)⁺RNA generated in a wheat germ system.Heat-stable COR proteins with molecular masses of 140 kDa and85 kDa (CORs 140 and 85), were detected in the leaves of cold-acclimatedplants. Increased levels of CORs 140 and 85 correlated withthe development of freezing tolerance during cold acclimation.Interestingly, CORs 140 and 85 accumulated specifically in theleaves and stems and not in the roots of the cold-acclimatedplants. Consistent with this observation, freezing tolerancewas also induced in leaves and stems, but not in roots. Thesedata strongly suggest that CORs 140 and 85 are closely associatedwith freezing tolerance. Accumulation of COR 85 was also inducedby exogenous ABA, drought, and wounding. The possible rolesof CORs 140 and 85 in plants acclimating to low temperatureis given attention. (Received June 11, 1992; Accepted September 1, 1992)     

Low-Temperature Effects on Photosynthesis and Correlation with Freezing Tolerance in Spring and Winter Cultivars of Wheat and Rye

Winter cultivars of rye (Secale cereale L., cv Musketeer) and wheat (Triticum aestivum L. cvs Kharkov and Monopol), but not a spring cultivar of wheat (Glenlea), grown at cold-hardening temperatures showed, at high irradiances, a higher proportion of oxidized to reduced primary, stable quinone receptor (QA) than did the same cultivars grown under nonhardening conditions. In addition, there was a positive correlation between the effects of low-growth temperature on this increased proportion of oxidized QA, and a concomitant increase in the capacity for photosynthesis, and LT50, the temperature at which 50% of the seedlings are killed, in cultivars showing different freezing tolerances. This suggests that low-temperature modulation of the photosynthetic apparatus may be an important factor during the induction of freezing resistance in cereals. Finally, the control of photosystem II photochemistry by nonphotochemical quenching of excitation energy was identical for nonhardened and cold-hardened winter rye. However, examination of measuring temperature effects per se revealed that, irrespective of growth temperature, nonphotochemical quenching exerted a stronger control on photosystem II photochemistry at 10[deg] C rather than at 20[deg] C.     

Altered Gene Expression during Auxin-Induced Root Development from Excised Mung Bean Seedlings

Changes in the pattern of protein synthesis and in the translatable mRNA population have been examined during auxin-induced root development from excised mung bean seedlings. Several proteins, predominantly of low molecular weight and high pI, as shown by two-dimensional polyacrylamide gel electrophoresis, are synthesized specifically by auxin-treated tissue. These auxin-induced proteins appear between 6 and 12 hours of auxin treatment, reach a maximum at 24 hours, and decline at 48 hours. Untreated seedlings (placed in Hoagland solution), known to produce small number of roots at the cut end probably due to endogeneous auxin accumulated at the cut end through basipetal transport, show low level synthesis of auxin-specific proteins. Antiauxin treatment that completely inhibits auxin-induced rooting also prevents the appearance of auxin-induced proteins. The induction of a group of three to four proteins appears to be specific to antiauxin treatment. In vitro translation of mRNA from auxin-treated tissue, but not of mRNA from antiauxin-treated tissue, yields several polypeptides of low molecular weight and high pI. Since the auxin-induced proteins precede root development and are synthesized transitorily, it is likely that they play some regulatory role during the initiation of root development. The result show that auxin-induced root formation involves altered gene expression.     

Variation of total soluble seminal root proteins of tetraploid wild and cultivated wheat induced at cold acclimation and freezing

The relationship between total soluble seminal root proteins induced at cold acclimation and freezing tolerance in tetraploid wild wheat Aegilops L. (Ae. biuncialis, Ae. cylindrica) and cultivated wheat Triticum turgitum L. (Firat-93, Harran-95) was investigated. Cold acclimation was performed at 0 °C for 7 days. Freezing tolerance was determined with survived roots after freezing treatments at −5 and/or −7 °C for 3, 6, 12 and 24 h. At −5°C, all tetraploid genotypes showed over 60% tolerance for 3 h. This effect was also present in wild wheat for 6 h, but was decreased in cultivated wheat to 30–35% tolerance for 6 h. Only Ae. biuncialis was able to show 52% tolerance just for 3 h freezing period at −7 °C. However, all the genotypes were not survived at −7 °C, for 6, 12 and 24 h. Cold acclimation induced greater amounts of new soluble seminal root proteins in tolerant Ae. biuncialis (29–104 kDa, pI 5.4–7.4) than in sensitive Harran-95 (29–66 kDa, pI 6.1–8.3). Synthesis and accumulation of these proteins may be related to degree of freezing tolerance of these genotypes.