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Changes in invertase activities were studied upon hardening of chilling-sensitive (Nicotiana tabacum L.) and cold-resistant (Arabidopsis thaliana Heynh. (L.)) plants to hypothermia. In tobacco plants, the activity of cytoplasmic and vacuolar invertases was found to decrease by 20% during hardening, while the activity of cell wall invertase increased almost twofold. In arabidopsis, the activity of all three types of invertases increased more than twofold during hardening. The hardened tobacco plants showed a 20% increase in sugar content, while the relative content of sucrose and hexoses (fructose and glucose) in sugars did not change after hardening and equaled approximately 50% each. During hardening of arabidopsis plants, the sugar content increased 2.5 fold, whereas the proportion of sucrose in the total sugar content decreased (from 44 to 24%) and the proportion of hexoses increased (from 56 to 76%). It was proposed that the increase in activity of cell wall invertase in both plant species suppressed the outflow of assimilates and facilitated the accumulation of photosynthates in mesophyll cells of tobacco and arabidopsis leaves. The decrease in activity of cytoplasmic and vacuolar invertases in N. tabacum restricted the formation of hexoses in cells and reduced the efficiency of cold hardening in tobacco plants. More than a twofold increase in the content of soluble carbohydrates in arabidopsis was mainly caused by hexose accumulation, which was due to the increased activity of cytoplasmic and vacuolar invertases and ensured high efficiency of A. thaliana hardening to hypothermia.

Chloroplast ultrastructural changes were investigated in chilling-sensitive (cucumber, Cucumis sativus L.) and cold-tolerant (winter rye, Secale cereale L.) plants during low-temperature hardening. Cold hardening was found to shorten the total length of thylakoid membranes in chloroplasts of both species by 20–30%. The reduction in total membrane length in cucumber was due to the decline in membrane length of both grana and stroma thylakoids, whereas the membrane length of grana thylakoids in winter rye was reduced without changes in length of stroma thylakoid membranes. In cucumber plants, the chloroplast section area decreased during hardening in parallel with more than threefold increase in starch grain area. In winter rye, the chloroplast area increased after hardening, while starch grains were completely absent. Both S. cereale and C. sativus retained photosynthetic activity at hardening temperatures, although photosynthetic rates decreased by 2–3 times compared to those in untreated plants. The content of soluble sugars in leaves increased by 20% in cucumber and increased more than threefold in winter rye. The accumulation of starch in cucumber chloroplasts provides evidence that a significant part of photosynthetic products was removed from the osmotic pool, which limited the efficiency of hardening in C. sativus plants. In winter rye plants, the thylakoid system rearrangement and the lack of starch synthesis in chloroplasts caused a severalfold increase in the content of intracellular soluble sugars, which ensured highly efficient low-temperature hardening of these plants.

The freezing tolerance of Arabidopsis thaliana (L.) Heynh. was studied in relation to functioning of the ethylene signaling pathway. Constitutive freezing tolerance was compared in wild-type plants (ecotype Col-0) and ethylene-insensitive mutants etr1-1 and ein2-1. For the first time it was established that the ethylene-insensitive mutants had a 25-30% lower net photosynthesis rate, a decreased content of soluble sugars, and, as a result, a lower freezing tolerance. Our work provides evidence that the perception and transduction of ethylene signal are necessary for constitutive tolerance of Arabidopsis to low temperature.

Changes in activities of the enzymes performing direct antioxidant functions were studied in 7–8-week-old plants Arabidopsis thaliana Heinh (L.) of Columbia (Col-0) ecotype. It was found that 5-day cold hardening at 2°C increased plant cold resistance to the subsequent stronger cooling. Under these conditions, the marked changes occurred in activities of superoxide dismutase and III type (guaiacol) peroxidses but not in that of catalase. The total peroxidase activity exceeded the catalase activity before cold hardening. Therefore, peroxidases are able to decompose more H2O2 than catalases and appear to make the dominant contribution to the protection from the cold damage.

Changes of ultrastructural organization of tobacco (Nicotiana tabacum L. cv. Samsun) chloroplasts associated with plant protection from oxidative stress during hypothermia were studied. It was found that the chilling hardening (6 days at 8°C) was accompanied by the significant reduction in the number of grana in a chloroplast simultaneously with area reduction of a granum that led to 30% decrease in the total area of grana in tobacco chloroplasts. In the course of tobacco plant hardening, approximately twofold decrease in generation rate of superoxide anion radical and 30% decrease in content of hydrogen peroxide occurred, which indicates retardation of oxidative processes in plant cells during the cold exposure. It is suggested that the ultrastructural changes in chloroplast organization that were found may prevent an overreduction of an electron-transport chain under hypothermia, when the ability of the Calvin cycle to utilize ATP and NADP·H is significantly reduced. The balanced work of components of light and dark photosynthetic phases may prevent the excessive generation of reactive oxygen species and render formation of tobacco plant tolerance to hypothermia.

Effects of the desA gene from the cyanobacterium Synechocystis sp. encoding Δ12 acyl-lipid desaturase and increasing the level of unsaturated fatty acids (linoleic acid (18:2) primarily) in membrane lipids, which was inserted into potato (Solanum tuberosum L., cv. Desnitsa) plants, on chloroplast ultrastructure and plant tolerance to low temperatures were studied. The main attention was focused on modifications in the chloroplast structure and their possible relation to potato plant tolerance to oxidative and low-temperature stresses under the influence to their transformation with the Δ12 acyl-lipid desaturase gene from cyanobacterium (desA-licBM3-plants). Morphometric analysis showed that, in comparison with wild-type (WT) plants, in desA-licBM3-plants the number of grana in chloroplasts increased substantially. The total number of thylakoids in transformant chloroplasts was almost twice higher than in WT plants. The number of plastoglobules per chloroplast of transformed plants increased by 25%. A marked increase in the number of grana, total number of thylakoids, and the number of plastoglobules in chloroplasts of desA-licBM3-plants indicates their more intense lipid metabolism, as compared with WT plants, and this resulted in the conservation of some part of lipids in plastoglobules. In addition, the expression of heterological desA gene encoding Δ12 acyl-lipid desaturase positively influenced stabilization of not only structure but also functioning of chloroplast membranes, thus preventing a transfer of electrons from the ETR to oxygen and subsequent ROS generation at hypothermia. This was confirmed by the analysis of the rate of superoxide anion generation in tested genotypes.

The content of saccharides and phenolic compounds (including flavonoids and lignin) and the activity of L-phenylalanine ammonia-lyase (PAL, EC 4.3.1.5) were determined in leaves and tillering nodes of winter wheat Triticum aestivum L.) cultivars Inna and Moskovskaya 39, differing in the level of frost resistance. These parameters were determined in three groups of plants--control, hardened, and treated with synthetic antioxidants (ambiol and amerol 2000). Cold hardening increased accumulation of primary and secondary metabolites in tissues but decreased the enzyme activity. Treatment with antioxidants also increased the content of saccharides and phenolic compounds (primarily flavonoids) and PAL activity. These changes were more pronounced in cultivar Inna, which is less frost resistant than Moskovskaya 39.

The pattern of changes in the activity of various forms of invertase (acid soluble, alkaline, and acid insoluble) and the content of sugars (glucose, fructose, and sucrose) in the course of plant adaptation to prolonged (6 days) hypothermia (5°C) was investigated in the leaves of potato plants (Solanum tuberosum L., cv. Desiree) produced in vitro. We used the wild-type plants as a control and transformed plants with carbohydrate metabolism modified by inserting the yeast gene for invertase (apoplastic enzyme). In the course of adaptation to hypothermia, the activity of acid invertase was shown to rise and the content of sucrose and glucose to increase in the leaves of both genotypes. The greatest activity of acid invertases by the third day of cold acclimation corresponded to the peak level of sugars; in transformed plants, these characteristics exceeded those in the control plants. The transformed plants were more cold resistant than the control plants as suggested by the lack of disturbance of ion permeability of their membranes. It was concluded that owing to accumulation of low-molecular carbohydrates in the course of cold acclimation caused by activation of acid invertase cold resistant plants better adapt to temperature drop.

Tobacco plants (Nicotiana tabacum L.) transformed with the desC gene for acyl-lipid Δ9-desaturase from a thermophilic cyanobacterium Synechococcus vulcanus were cultivated on the agarized Murashige and Skoog medium at 22°C and a 16-h photoperiod. Tobacco plants transformed with an empty binary vector pGA482 served as the control. The investigations showed that, in contrast to the control, transgenic plants maintained a higher activity of antioxidant enzymes during 2-h incubation at 2°C; as a result, these plants resisted more efficiently the accumulation of reactive oxygen species and reduced the rate of the lipid peroxidation. The activity of antioxidant enzymes in the transformed plants is apparently related to the operation of the introduced desC gene for acyl-lipid Δ9-desaturase because the enhanced activity of the latter increased the relative content of polyunsaturated FAs in membrane lipids and in this way promoted the liquid state of membranes during the chilling period. These changes helped preserve the cellular homeostasis and thereby maintain the steady synthesis of antioxidant enzymes at hypothermic conditions; as a result, cold resistance of transformed tobacco plants increased.

We studied CO2 exchange and phenolic compound production in various organs of unhardened and hardened winter wheat (Triticum aestivum L.) plants. The rates of CO2 assimilation at saturating illumination (photosynthesis) and CO2 evolution in darkness (respiration) declined substantially at the autumnal decrease of ambient temperature. However, because of a higher cold resistance of photosynthesis, the ratio of photosynthesis to respiration rates increased 1.5-fold. These gas exchange changes were accompanied by the accumulation of total soluble phenolics in leaves and a polymeric phenolic compound lignin in roots. We did not observe any changes in the production of either soluble or polymeric (lignin) phenolics in crowns.

Tolerance to chilling was compared in potato plants ( Solanum tuberosum L., cv. Desiree) transformed with a yeast-derived invertase gene under the control of the B33 class 1 tuber-specific promoter and with the proteinase inhibitor II leader peptide sequence providing for the apoplastic enzyme localization (the B33 -inv plants) and potato plants transformed only with a reporter gene (the control plants) under in vitro conditions. The total activities of acid and alkaline invertases and contents of sugars in the B33 -inv plants exceeded those in the control plants. The exposures at low temperature produced a significant less lipid peroxidation activity in the B33- inv plants, as compared to the control. The authors presume that the B33 -inv plants acquire a higher tolerance to low temperatures apparently due to the changes in sugar ratio produced by the yeast invertase.

Cycloheximide in the concentration inhibiting protein synthesis and frost resistance of plants proved to prevent formation of cell and chloroplast structures specific for the plants adapted to frost. Important role of structural rearrangement of the cells during hardening preventing intracellular ice nucleation is proposed.