Biological Networks Underlying Abiotic Stress Tolerance in Temperate Crops--A Proteomic Perspective.

Abiotic stress factors, especially low temperatures, drought, and salinity, represent the major constraints limiting agricultural production in temperate climate. Under the conditions of global climate change, the risk of damaging effects of abiotic stresses on crop production increases. Plant stress response represents an active process aimed at an establishment of novel homeostasis under altered environmental conditions. Proteins play a crucial role in plant stress response since they are directly involved in shaping the final phenotype. In the review, results of proteomic studies focused on stress response of major crops grown in temperate climate including cereals: common wheat (Triticum aestivum), durum wheat (Triticum durum), barley (Hordeum vulgare), maize (Zea mays); leguminous plants: alfalfa (Medicago sativa), soybean (Glycine max), common bean (Phaseolus vulgaris), pea (Pisum sativum); oilseed rape (Brassica napus); potato (Solanum tuberosum); tobacco (Nicotiana tabaccum); tomato (Lycopersicon esculentum); and others, to a wide range of abiotic stresses (cold, drought, salinity, heat, imbalances in mineral nutrition and heavy metals) are summarized. The dynamics of changes in various protein functional groups including signaling and regulatory proteins, transcription factors, proteins involved in protein metabolism, amino acid metabolism, metabolism of several stress-related compounds, proteins with chaperone and protective functions as well as structural proteins (cell wall components, cytoskeleton) are briefly overviewed. Attention is paid to the differences found between differentially tolerant genotypes. In addition, proteomic studies aimed at proteomic investigation of multiple stress factors are discussed. In conclusion, contribution of proteomic studies to understanding the complexity of crop response to abiotic stresses as well as possibilities to identify and utilize protein markers in crop breeding processes are discussed.

Proteome analysis of cold response in spring and winter wheat (Triticum aestivum) crowns reveals similarities in stress adaptation and differences in regulatory processes between the growth habits.

A proteomic response to cold treatment (4 °C) has been studied in crowns of a frost-tolerant winter wheat cultivar Samanta and a frost-sensitive spring wheat cultivar Sandra after short-term (3 days) and long-term (21 days) cold treatments. Densitometric analysis of 2-D differential in gel electrophoresis (2D-DIGE) gels has resulted in the detection of 386 differentially abundant protein spots, which reveal at least a two-fold change between experimental variants. Of these, 58 representative protein spots have been selected for MALDI-TOF/TOF identification, and 36 proteins have been identified. The identified proteins with an increased relative abundance upon cold in both growth habits include proteins involved in carbohydrate catabolism (glycolysis enzymes), redox metabolism (thioredoxin-dependent peroxidase), chaperones, as well as defense-related proteins (protein revealing similarity to thaumatin). Proteins exhibiting a cold-induced increase in the winter cultivar include proteins involved in regulation of stress response and development (germin E, lectin VER2), while proteins showing a cold-induced increase in the spring cultivar include proteins involved in restoration of cell division and plant growth (eIF5A2, glycine-rich RNA-binding protein, adenine phosphoribosyltransferase). These results provide new insights into cold acclimation in spring and winter wheat at the proteome level and enrich our previous work aimed at phytohormone dynamics in the same plant material.

The choice of reference gene set for assessing gene expression in barley (Hordeum vulgare L.) under low temperature and drought stress.

Drought and low temperature are the two most significant causes of abiotic stress in agricultural crops and, therefore, they pose considerable challenges in plant science. Hence, it is crucial to study response mechanisms and to select genes for identification signaling pathways that lead from stimulus to response. The assessment of gene expression is often attempted using real-time RT-PCR (qRT-PCR), a technique which requires a careful choice of reference gene(s) for normalization purpose. Here, we report a comparison of 13 potential reference genes for studying gene expression in the leaf and crown of barley seedlings subjected to low temperature or drought stress. All three currently available software packages designed to identify reference genes from qRT-PCR data (GeNorm, NormFinder and BestKeeper) were used to identify informative sets of up to three reference genes. Interestingly, the data obtained from the separate treatment of leaf and crown have led to the recommendations that HSP70 and S-AMD (and possibly HSP90) to be used as the reference genes for low-temperature stressed leaves, HSP90 and EF1α for low-temperature stressed crowns, cyclophilin and ADP-RF (and possibly ACT) for drought-stressed leaves, and EF1α and S-AMD for drought-stressed crowns. Our results have demonstrated that the gene expression can be highly tissue- or organ-specific in barley and have confirmed that reference gene choice is essential in qRT-PCR. The findings can also serve as guidelines for the selection of reference genes under different stress conditions and lay foundation for more accurate and widespread use of qRT-PCR in barley gene analysis.

Enhanced drought and heat stress tolerance of tobacco plants with ectopically enhanced cytokinin oxidase/dehydrogenase gene expression.

Responses to drought, heat, and combined stress were compared in tobacco (Nicotiana tabacum L.) plants ectopically expressing the cytokinin oxidase/dehydrogenase CKX1 gene of Arabidopsis thaliana L. under the control of either the predominantly root-expressed WRKY6 promoter or the constitutive 35S promoter, and in the wild type. WRKY6:CKX1 plants exhibited high CKX activity in the roots under control conditions. Under stress, the activity of the WRKY6 promoter was down-regulated and the concomitantly reduced cytokinin degradation coincided with raised bioactive cytokinin levels during the early phase of the stress response, which might contribute to enhanced stress tolerance of this genotype. Constitutive expression of CKX1 resulted in an enlarged root system, a stunted, dwarf shoot phenotype, and a low basal level of expression of the dehydration marker gene ERD10B. The high drought tolerance of this genotype was associated with a relatively moderate drop in leaf water potential and a significant decrease in leaf osmotic potential. Basal expression of the proline biosynthetic gene P5CSA was raised. Both wild-type and WRKY6:CKX1 plants responded to heat stress by transient elevation of stomatal conductance, which correlated with an enhanced abscisic acid catabolism. 35S:CKX1 transgenic plants exhibited a small and delayed stomatal response. Nevertheless, they maintained a lower leaf temperature than the other genotypes. Heat shock applied to drought-stressed plants exaggerated the negative stress effects, probably due to the additional water loss caused by a transient stimulation of transpiration. The results indicate that modulation of cytokinin levels may positively affect plant responses to abiotic stress through a variety of physiological mechanisms.

Proteins involved in distinct phases of cold hardening process in frost resistant winter barley (Hordeum vulgare L.) cv Luxor.

Winter barley is an economically important cereal crop grown in higher latitudes and altitudes where low temperatures represent an important environmental constraint limiting crop productivity. In this study changes in proteome of leaves and crowns in a frost tolerant winter barley cv. Luxor in relation to short and long term periods of cold followed by a brief frost treatment were studied in order to disclose proteins responsible for the cold hardening process in distinct plant tissues. The mentioned changes have been monitored using two dimensional difference gel electrophoresis (2D-DIGE) with subsequent peptide-mapping protein identification. Regarding approximately 600-700 distinct protein spots detected on 2D gels, there has been found at least a two-fold change after exposure to low temperatures in about 10% of proteins in leaves and 13% of proteins in crowns. Protein and nitrogen metabolic processes have been influenced by low temperature to a similar extent in both tissues while catabolism, carbohydrate metabolism and proteins involved in stress response have been more affected in crowns than in leaves. The range of changes in protein abundance was generally higher in leaves and chloroplast proteins were frequently affected which suggests a priority to protect photosynthetic apparatus. Overall, our data proved existence of slightly different response strategies to low temperature stress in crowns and leaves, i.e., tissues with different biological role. Moreover, there have been found several proteins with large increase in accumulation, e.g., 33 kDa oxygen evolving protein of photosystem II in leaves and "enhanced disease susceptibility 1" in crowns; these proteins might have potential to indicate an enhanced level of frost tolerance in barley.

Proteomics of wheat and barley cereals in response to environmental stresses: Current state and future challenges.

Wheat and barley genera represent a wide range of genotypes from Triticeae group grown around the globe. The broad plasticity of Triticeae phenotypes mirrors the robustness of their genomes revealing a high level of gene homeology. Publication and annotation of the reference genome sequences for spring barley Morex and Chinese Spring wheat represents an important milestone enabling the researchers to precisely identify and annotate nearly all proteins. Due to the broad range of environments used for wheat and barley cultivation and their economical importance, proteomic studies focused on their responses to environmental stresses including combined stress treatments. Most of the Triticeae stress proteomics studies are comparative ones aimed at determination of differentially abundant proteins (DAPs) between two or more genotypes with contrasting stress tolerance. Studies focused on subcellular fractions and protein posttranslational modifications (PTMs) are still relatively rare although PTMs play a crucial role in modulation of protein biological function. Functional and interactomics studies are needed although gene homeology and the resulting protein functional redundancy practically excludes the utilization of knock-out mutants. The alternatives could represent either gene overexpression in a heterologous system such as A. thaliana or transient posttranscriptional gene silencing using RNAi. Publication of complete reference genome sequences together with novel technological approaches such as pQTL mapping boost the Triticeae proteomics studies not only to provide data but also to contribute to designing novel genotypes with improved adaptations to ever changing environments.

COR/LEA Proteins as Indicators of Frost Tolerance in Triticeae: A Comparison of Controlled versus Field Conditions.

Low temperatures in the autumn induce enhanced expression/relative accumulation of several cold-inducible transcripts/proteins with protective functions from Late-embryogenesis-abundant (LEA) superfamily including dehydrins. Several studies dealing with plants grown under controlled conditions revealed a correlation (significant quantitative relationship) between dehydrin transcript/protein relative accumulation and plant frost tolerance. However, to apply these results in breeding, field experiments are necessary. The aim of the review is to provide a summary of the studies dealing with the relationships between plant acquired frost tolerance and COR/LEA transcripts/proteins relative accumulation in cereals grown in controlled and field conditions. The impacts of cold acclimation and vernalisation processes on the ability of winter-type Triticeae to accumulate COR/LEA proteins are discussed. The factors determining dehydrin relative accumulation under controlled cold acclimation treatments versus field trials during winter seasons are discussed. In conclusion, it can be stated that dehydrins could be used as suitable indicators of winter survival in field-grown winter cereals but only in plant prior to the fulfilment of vernalisation requirement.

Relationship between WCS120 Protein Family Accumulation and Frost Tolerance in Wheat Cultivars Grown under Different Temperature Treatments.

Frost tolerance (FT) is generally acquired after exposure of plants to low, but non-freezing temperatures, where it is associated with the accumulation of COR proteins. The aim of the study was to reveal the effect of different temperature treatments (25, 17, 9 and 4 °C) on accumulation of cold-regulated dehydrins, dry weight content, and the development of FT in five wheat cultivars of different frost-tolerances in detail. The levels of cold-regulated dehydrins, WCS120 proteins in wheat were determined by immunoblot analysis, probed with an anti-dehydrin antibody. The lower the growth temperature: the higher the level of frost tolerance, dry weight content, and dehydrin accumulation, in all cultivars. There was a significant correlation between the level of induced FT and the accumulation of WCS120 proteins in cultivars grown at lower temperatures (9 and 4 °C). Moreover, the highly frost-tolerant wheat cultivars (as opposed to the lower-tolerant) accumulated higher levels of WCS120 proteins at 17 °C, a temperature at which it was not possible to differentiate between them via a frost test. Here, we demonstrated the possibility to distinguish differently frost-tolerant cultivars grown at different temperatures by the accumulation of different members of WCS120 family.

Plant Proteoforms Under Environmental Stress: Functional Proteins Arising From a Single Gene.

Proteins are directly involved in plant phenotypic response to ever changing environmental conditions. The ability to produce multiple mature functional proteins, i.e., proteoforms, from a single gene sequence represents an efficient tool ensuring the diversification of protein biological functions underlying the diversity of plant phenotypic responses to environmental stresses. Basically, two major kinds of proteoforms can be distinguished: protein isoforms, i.e., alterations at protein sequence level arising from posttranscriptional modifications of a single pre-mRNA by alternative splicing or editing, and protein posttranslational modifications (PTMs), i.e., enzymatically catalyzed or spontaneous modifications of certain amino acid residues resulting in altered biological functions (or loss of biological functions, such as in non-functional proteins that raised as a product of spontaneous protein modification by reactive molecular species, RMS). Modulation of protein final sequences resulting in different protein isoforms as well as modulation of chemical properties of key amino acid residues by different PTMs (such as phosphorylation, N- and O-glycosylation, methylation, acylation, S-glutathionylation, ubiquitinylation, sumoylation, and modifications by RMS), thus, represents an efficient means to ensure the flexible modulation of protein biological functions in response to ever changing environmental conditions. The aim of this review is to provide a basic overview of the structural and functional diversity of proteoforms derived from a single gene in the context of plant evolutional adaptations underlying plant responses to the variability of environmental stresses, i.e., adverse cues mobilizing plant adaptive mechanisms to diminish their harmful effects.

Relationship Between Dehydrin Accumulation and Winter Survival in Winter Wheat and Barley Grown in the Field.

Low temperatures represent a crucial environmental factor determining winter survival (WS) of barley and wheat winter-type varieties. In laboratory experiments, low temperatures induce an active plant acclimation response, which is associated with an enhanced accumulation of several stress-inducible proteins including dehydrins. Here, dehydrin accumulations in sampled wheat (WCS120 protein family, or WCS120 and WDHN13 transcripts) and barley (DHN5 protein) varieties grown in two locations for two winters were compared with the variety WS evaluated by a provocation wooden-box test. A high correlation between dehydrin transcripts or protein relative accumulation and variety WS score was found only in samples taken prior vernalization fulfillment, when high tolerant varieties accumulated dehydrins earlier and to higher level than less tolerant varieties, and the plants have not yet been vernalized. After vernalization fulfillment, the correlation was weak, and the apical development indicated that plants reached double ridge (DR) in barley or stayed before DR in wheat. Dehydrin proteins and transcripts can be thus used as reliable markers of wheat or barley variety winter hardiness in the field conditions; however, only at the beginning of winter, when the plants have not yet finished vernalization. In wheat, a higher correlation was obtained for the total amount of dehydrins than for the individual dehydrin proteins. HIGHLIGHTS: -More tolerant winter-type wheat and barley plants reveal higher threshold induction temperatures for dehydrin accumulation in comparison to less tolerant varieties. Thus, more tolerant winter cereals have higher dehydrin levels than the less tolerant ones upon the same ambient temperature in November samplings.-A significant correlation between dehydrin transcript/protein accumulation and winter survival was found in both winter wheat and winter barley plants in the field conditions, but only prior to vernalization fulfillment.

WCS120 protein family and frost tolerance during cold acclimation, deacclimation and reacclimation of winter wheat.

We studied how long-term cold acclimation of winter wheat (variety Mironovskaya 808), interrupted by deacclimation and then followed by reacclimation, affected the levels of cold-induced WCS120 proteins, dry-weight content, and frost tolerance in leaves. Two experiments were performed: (1) plants undergoing long-term cold acclimation (up to 112days) were quickly deacclimated (for 5days), and then reacclimated again to cold; (2) plants vernalized for varying periods of time in an early stage of their development were, after a longer deacclimation of about 14days, exposed for the same time period to cold. Five members of the WCS120 protein family were detected and quantified by image analysis in protein gel blots (in the first experiment); as well as in two-dimensional electrophoresis gels (in the second experiment). In both experiments, partially vernalized plants, after reacclimation, re-established their frost tolerance to levels similar to plants having had the same duration of cold treatment, but without deacclimation. On the other hand, these partially and fully vernalized plants reaccumulated WCS120 proteins to lower levels than plants that were not deacclimated. Further, using a mathematical model (the peak four-parameter Weibull equation), the same type of response curve was observed during plant cold treatment not only for the level of frost tolerance, but also for dry-weight content and accumulation of WCS120 proteins, with the maximum values reached at about the same time as vernalization saturation.

Proteomic and physiological approach reveals drought-induced changes in rapeseeds: Water-saver and water-spender strategy.

The cultivar-dependent differences in Brassica napus L. seed yield are significantly affected by drought stress. Here, the response of leaf proteome to long-term drought (28days) was studied in cultivars (cvs): Californium (C), Cadeli (D), Navajo (N), and Viking (V). Analysis of twenty-four 2-D DIGE gels revealed 134 spots quantitatively changed at least 2-fold; from these, 79 proteins were significantly identified by MALDI-TOF/TOF. According to the differences in water use, the cultivars may be assigned to two categories: water-savers or water-spenders. In the water-savers group (cvs C+D), proteins related to nitrogen assimilation, ATP and redox homeostasis were increased under stress, while in the water-spenders category (cvs N+V), carbohydrate/energy, photosynthesis, stress related and rRNA processing proteins were increased upon stress. Taking all data together, we indicated cv C as a drought-adaptable water-saver, cv D as a medium-adaptable water-saver, cv N as a drought-adaptable water-spender, and cv V as a low-adaptable drought sensitive water-spender rapeseed. Proteomic data help to evaluate the impact of drought and the extent of genotype-based adaptability and contribute to the understanding of their plasticity. These results provide new insights into the provenience-based drought acclimation/adaptation strategy of contrasting winter rapeseeds and link data at gasometric, biochemical, and proteome level. SIGNIFICANCE: Soil moisture deficit is a real problem for every crop. The data in this study demonstrates for the first time that in stem-prolongation phase cultivars respond to progressive drought in different ways and at different levels. Analysis of physiological and proteomic data showed two different water regime-related strategies: water-savers and spenders. However, not only water uptake rate itself, but also individual protein abundances, gasometric and biochemical parameters together with final biomass accumulation after stress explained genotype-based responses. Interestingly, under a mixed climate profile, both water-use patterns (savers or spenders) can be appropriate for drought adaptation. These data suggest, than complete "acclimation image" of rapeseeds in stem-prolongation phase under drought could be reached only if these characteristics are taken, explained and understood together.

The effect of Fusarium culmorum infection and deoxynivalenol (DON) application on proteome response in barley cultivars Chevron and Pedant.

Fusarium head blight (FHB) disease adversely affects grain quality and final yield in small-grain cereals including barley. In the present study, the effect of an artificial infection with Fusarium culmorum and an application of deoxynivalenol (DON) on barley spikes of cultivars Chevron and Pedant during flowering was investigated at grain mid-dough stage (BBCH 73) 10days after pathogen inoculation (10 dai). Proteomic analysis using a two-dimensional differential gel electrophoresis (2D-DIGE) technique coupled with LC-MS/MS investigated 98 protein spots revealing quantitative or qualitative differences between the experimental variants. Protein functional annotation of 93 identified protein spots revealed that most affected functional groups represent storage proteins (globulins, hordeins), followed by proteins involved in carbohydrate metabolism (α-amylase inhibitor, ß-amylase, glycolytic enzymes), amino acid metabolism (aminotransferases), defence response (chitinase, xylanase inhibitor, serpins, SGT1, universal stress protein USP), protein folding (chaperones, chaperonins), redox metabolism (ascorbate-glutathione cycle), and proteasome-dependent protein degradation. The obtained results indicate adverse effects of infection on plant proteome as well as an active plant response to pathogen as shown by enhanced levels of several inhibitors of pathogen-produced degradation enzymes (α-amylase inhibitor, xylanase inhibitor, serpins), chaperones, and other stress-related proteins (SGT1, USP). Genotypic differences were found in hordein abundance between Chevron and Pedant.

Complex phytohormone responses during the cold acclimation of two wheat cultivars differing in cold tolerance, winter Samanta and spring Sandra.

Hormonal changes accompanying the cold stress (4°C) response that are related to the level of frost tolerance (FT; measured as LT50) and the content of the most abundant dehydrin, WCS120, were compared in the leaves and crowns of the winter wheat (Triticum aestivum L.) cv. Samanta and the spring wheat cv. Sandra. The characteristic feature of the alarm phase (1 day) response was a rapid elevation of abscisic acid (ABA) and an increase of protective proteins (dehydrin WCS120). This response was faster and stronger in winter wheat, where it coincided with the downregulation of bioactive cytokinins and auxin as well as enhanced deactivation of gibberellins, indicating rapid suppression of growth. Next, the ethylene precursor aminocyclopropane carboxylic acid was quickly upregulated. After 3-7 days of cold exposure, plant adaptation to the low temperature was correlated with a decrease in ABA and elevation of growth-promoting hormones (cytokinins, auxin and gibberellins). The content of other stress hormones, i.e., salicylic acid and jasmonic acid, also began to increase. After prolonged cold exposure (21 days), a resistance phase occurred. The winter cultivar exhibited substantially enhanced FT, which was associated with a decline in bioactive cytokinins and auxin. The inability of the spring cultivar to further increase its FT was correlated with maintenance of a relatively higher cytokinin and auxin content, which was achieved during the acclimation period.

Plant proteome changes under abiotic stress--contribution of proteomics studies to understanding plant stress response.

Plant acclimation to stress is associated with profound changes in proteome composition. Since proteins are directly involved in plant stress response, proteomics studies can significantly contribute to unravel the possible relationships between protein abundance and plant stress acclimation. In this review, proteomics studies dealing with plant response to a broad range of abiotic stress factors--cold, heat, drought, waterlogging, salinity, ozone treatment, hypoxia and anoxia, herbicide treatments, inadequate or excessive light conditions, disbalances in mineral nutrition, enhanced concentrations of heavy metals, radioactivity and mechanical wounding are discussed. Most studies have been carried out on model plants Arabidopsis thaliana and rice due to large protein sequence databases available; however, the variety of plant species used for proteomics analyses is rapidly increasing. Protein response pathways shared by different plant species under various stress conditions (glycolytic pathway, enzymes of ascorbate-glutathione cycle, accumulation of LEA proteins) as well as pathways unique to a given stress are discussed. Results from proteomics studies are interpreted with respect to physiological factors determining plant stress response. In conclusion, examples of application of proteomics studies in search for protein markers underlying phenotypic variation in physiological parameters associated with plant stress tolerance are given.

Expression of dehydrins in wheat and barley under different temperatures.

The review summarizes recent knowledge on the expression of cold-inducible dehydrins with a special attention to Wcs120 and Dhn5 genes in wheat and barley plants under different temperatures. When plants are exposed to cold, dehydrins start accumulating both in freezing-tolerant and freezing-susceptible plants; however, their accumulation correlates with plant acquired frost tolerance (FT). During a long-term cold acclimation (CA), dehydrin accumulation is significantly affected by Vrn1/Fr1 locus and the expression of the major vernalization gene VRN1, respectively. A different dynamics of dehydrin transcripts and proteins during CA is also observed. Transcripts reach their maximum within the first week of CA while proteins gradually accumulate until vernalization. Vernalization is associated with a significant decrease in dehydrin accumulation while the decrease of acquired FT is delayed. Studies carried out on plants grown at moderately cold temperatures (9-20 °C) have shown that both dehydrin transcripts and proteins can be detected even at these temperatures and that plants with different FT levels can be distinguished according to dehydrin accumulation without any exposure to severe cold. In conclusion, the potential use of these results in the breeding programmes aimed at the enhancement of wheat and barley FT is discussed.

Expression of dehydrin 5 during the development of frost tolerance in barley (Hordeum vulgare).

The Dhn5 gene is the major cold-inducible dehydrin gene in barley. This study deals with the relationship between Dhn5 gene expression and its protein product accumulation, and the development of frost tolerance (FT) upon cold acclimation (CA) in 10 barley cultivars of different growth habits and geographical origins. The activation of Dhn5 gene expression was determined by quantitative reverse transcription-polymerase chain reaction (qRT-PCR), the accumulation of DHN5 protein was evaluated by protein gel blot analysis using a specific anti-dehydrin antibody, and the acquired level of FT was determined by a direct frost test. During the first 2 weeks of CA, there was a rapid increase in Dhn5 gene expression, DHN5 protein accumulation and FT in all cultivars examined. After 2 weeks of CA, differences in DHN5 accumulation and in FT measured as lethal temperature (LT(50)) were observed between the cultivars belonging to different growth habits. Specifically, intermediate (I) and winter (W) cultivars showed a higher level of DHN5 accumulation and FT than the spring (S) cultivars, which exhibited a lower level of accumulated DHN5 and FT. (Intermediate cultivars do not have vernalization requirement, but they are able to induce a relatively high level of FT upon CA.) In contrast, no differences between the cultivars belonging to different growth habits in Dhn5 mRNA accumulation were found. After 3 weeks of CA, the differences in accumulated DHN5 and FT between the individual growth habits became evident due to different developmental regulation of FT. The amount of accumulated DHN5 corresponded well with the level of FT of individual cultivars. We conclude that the amount of accumulated DHN5 after a certain period of CA differed according to the growth habits of cultivars and can be used as a marker for determination of FT in barley.

Relationships among vernalization, shoot apex development and frost tolerance in wheat.

BACKGROUND AND AIMS: Frost tolerance of wheat depends primarily upon a strong vernalization requirement, delaying the transition to the reproductive phase. The aim of the present study was to learn how saturation of the vernalization requirement and apical development stage are related to frost tolerance in wheat. METHODS: 'Mironovskaya 808', a winter variety with a long vernalization requirement, and 'Leguan', a spring variety without a vernalization requirement, were acclimated at 2 degrees C at different stages of development. Plant development (morphological stage of the shoot apex), vernalization requirement (days to heading) and frost tolerance (survival of the plants exposed to freezing conditions) were evaluated. KEY RESULTS: 'Mironovskaya 808' increased its frost tolerance more rapidly; it reached a higher level of tolerance and after a longer duration of acclimation at 2 degrees C than was found in 'Leguan'. The frost tolerance of 'Mironovskaya 808' decreased and its ability to re-acclimate a high tolerance was lost after saturation of its vernalization requirement, but before its shoot apex had reached the double-ridge stage. The frost tolerance of 'Leguan' decreased after the plants had reached the floret initiation stage. CONCLUSIONS: The results support the hypothesis that genes for vernalization requirement act as a master switch regulating the duration of low temperature induced frost tolerance. In winter wheat, due to a longer vegetative phase, frost tolerance is maintained for a longer time and at a higher level than in spring wheat. After the saturation of vernalization requirement, winter wheat (as in spring wheat) established only a low level of frost tolerance.