The colligative effects of endogenous highly concentrated sugar solutions on frost-resistant plants subjected to negative temperatures are reviewed. These effects prevent cell dehydration and formation of intercellular ice; they decrease the protoplast freezing point and neutralize toxic products. Several protective activities of plant sugars occurring under hypothermia are discussed. These are structural stabilization of membranes, lipids and proteins, deactivation of reactive oxygen species, participation in signaling, and involvement in metabolism as both energy sources and precursors for synthesis of different protective compounds.

The qualitative composition and changes in the total fatty acid (FA) content in leaves of wild-type potato ( Solanum tuberosum L., cv. Desnitsa) plants and plants transformed with the desA gene of Δ12-acyl-lipid desaturase from Synechocystis sp. PCC 6803 have been studied under cold hardening conditions (6 days at 5°С). During cold adaptation, plants of both types increase their cold resistance, but transformed plants significantly exceed control ones in relation to this parameter. Following cold hardening, the content of total etherified FA in leaves of control plants increases by almost 25% due to the decrease in the content of saturated FA and almost 30% increase in the content of polyunsaturated FA (PUFA); the growth of the PUFA content is provided mainly by linoleic (C 18:2 ) and linolenic (C 18:3 ) acids. Nonhardened leaves of transformed plants are characterized by the same FA content (especially C 18:3 ) as the leaves of hardened control plants. Due to this fact, increase in the total FA content in hardened transformants is not so significant as in the control; at the same time, the content of linoleic (C 18:2 ) acid increases by 30% (the calculated activity index for ∆12-desaturases also slightly increased). The growth of a hexadecatrienic (С 16:3 ) acid content in transformed plants probably results from a low-temperature activation of potato ω3-desaturases. The obtained data allow one to suppose that the higher resistance of transformed plants compared to control ones is connected with a constitutively increased PUFA content and determines more efficient cold hardening of such plants.

Arabidopsis thaliana Heynh . (L.) plants of the Columbia ecotype (Col-0) and its ethylene-insensitive etr1-1 (ethylene resistant 1) and ein2-1 (ethylene insensitive 2) mutants were studied. The plants were compared in respect to their cold tolerance conferred by cold acclimation. The tolerance to negative temperature increased in all the three genotypes after 5-day cooling of the plants at 2°C. Meanwhile, a quantitative difference was observed between them: the Col-0 plants survived better than the mutants, and the electrolyte leakage from the tissues reached 50% at higher (by 1°C and more) temperature in the hardened mutants than in the Col-0. Only the mutants manifested the increased lipid peroxidation over the hardening period; this indicates ROS production and general oxidative stress. From this point, we speculated that the tested Arabidopsis genotypes are different in the efficiency of their antioxidant systems. For testing, the total activities of the chief antioxidant enzymes superoxide dismutase (SOD) and catalase, including their isoenzymes, were monitored in the course of hardening. The total SOD activity was found to be lower in the mutants than in the Col-0 both at the normal growing temperature and over the whole time of the cold hardening. The isozyme analysis revealed the link of the reduced total SOD activity of the mutants with the reduced activity of their Cu/Zn-SOD isoforms at 22°C and at the beginning of the hardening. This relationship was supported by the lower relative content of the CSD1 and CSD2 gene transcripts of Cu/Zn-SOD in the mutants in comparison with the Col-0. The decrease in the total catalase activity was also observed and would be ascribed to the lowered activity of the CAT2 isoform. The reported results evidence to the principal feasibility of the ethylene signaling control of the Cu/Zn-SOD and catalase activities associated with the cold acclimation of Arabidopsis .

Activities of enzymes decomposing hydrogen peroxide (H 2 O 2 ) under long exposure to hardening low temperatures and the effect of Δ12-acyl-lipid desaturase on these processes were studied on potato ( Solanum tuberosum L., cv. Desnitsa), which typically represents cold-tolerant plants. We compared nontransformed plants (control) and the line transformed with the construction carrying the target desA gene of the mentioned desaturase from cyanobacterium Synechocystis sp. PCC ( desA-licBM3 plants). The plants were hardened at 5°C for six days under illumination of 50 μmol/(m 2 s). The hardening was found to favor plant tolerance to the subsequent frost, and the desA-licBM3 plants exceed the controls in this property. Of the studied H 2 O 2 -scavenging enzymes, soluble type III peroxidases (guaiacol peroxidases) displayed the most activity, and type I peroxidase (ascorbate peroxidase) was the least active in the two potato lines over the hardening period. The activity of catalase increased twofold in the control and fourfold in the transformed plants in the first day of the hardening. However, the doubled catalase activity did not appear to compensate the H 2 O 2 accumulation over this period. The recorded rise in catalase activity in the desA-licBM3 plants, together with the high activity of guaiacol peroxidases, favored lowering the hydrogen peroxide level in comparison with the initial values. For the first time, electrophoresis revealed two catalase isoforms, CAT1 and CAT2, in leaves of both potato lines. The significance of CAT1 was greater than that of CAT2 in the total catalase activity during the hardening period. It is concluded that, under the long-term cold hardening of potato plants, the content of hydrogen peroxide is determined by highly active guaiacol peroxidases and Class I catalase exerting energy-independent H 2 O 2 decomposing. In this case, in the transformants that are rich in membrane lipids, where polyunsaturated fatty acids predominate, the activity of H 2 O 2 -scavenging enzymes increased significantly more than in the control, which is why the hardening of the transformants is more effective.

We studded the influence of the expression of SUC2 gene encoding invertase of the Saccharomyces cerevisiae (apoplastic version of enzyme localization) on morphological, physiological and biochemical traits of transformed potato plants (Solanum tuberosum L., cv.Désirée).It was shown that invertase activity increased the sugars content in the apoplast, leaves and roots of the transformed plants and led to changes in their morphometric and physiological parameters.Our data indicate higher resistance of transformed plants to severe low temperature conditions.

Аннотация.Изучали изменения общей активности каталазы, осуществляющей энергонезависимое разложение пероксида водорода (Н 2 О 2 ) и ее изоформ при низкотемпературном закаливании картофеля, а также влияние ∆12-ацил-липидной десатуразы цианобактерий на эти процессы.В листьях трансформированных и контрольных растений выявлены две изоформы каталазы КАТ1 и КАТ2, из которых вклад КАТ1 в суммарную активность фермента был существенно выше, чем КАТ2.В процессе закаливания активность

Alterations in the ultrastructure of chloroplasts and the content of sugars during low-temperature hardening (5°C, 6 days) of potato plants (Solanum tuberosum L., cv. Desnitsa) and a transformed line with the desA gene of Δ12 acyl-lipid desaturase Synechocystis sp. RCC 6803 were studied. It was noted that during hardening, sugars were accumulated in the leaves of both lines, but with greater speed in the leaves of the transformants. In control plants after hardening, an increase in the total number of thylakoid membranes and in the number of plastoglobules per chloroplast were obtained accompanied by a decrease in the chloroplast cross-sectional area and starch grain area. It was found that chloroplasts of transformed plants, which were different from the control plants in the higher absolute content of membrane lipids and the amount of polyunsaturated fatty acids, corresponded to the hardened plants before cold exposure by some ultrastructural parameters. In the early period of low-temperature hardening, the transformed plants were more adaptable, according to the data on the sugar content.

Introduction: Invertase is the key enzyme of carbohydrate metabolism, which catalyzes the sucrose hydrolysis. In this study, we investigated a functional role of the apoplastic invertase (cell-wall invertase) on sugars distribution in vegetative organs and cell compartments of potato plants in vitro.Methods: Our study was carried out with potato (S. tuberosum L., cv. Desiree) plants and the line which expressed the suc2 gene of S. cerevisiae under control of the tuberspecific patatin B33-promoter of class I with an N-end-connected potato proteinase II inhibitor signal peptide, which provides apoplastic localization of yeast invertase. The suc2 gene expression were shown using RT-PCR. The results of Ds-Na-PAGE of apoplast proteins from leaves and MALDI-TOF MS analysis indicate the presence of the yeast invertase in the apoplastic space of the transgenic potato plants. The content of fructose and sucrose was determined according to Roe. The glucose content was determined by the glucose oxidase method.Results: The integrated target suc2 gene encodes the invertase of yeast with an N-endconnected potato proteinase II inhibitor signal peptide, which provides apoplastic localization of foreign invertase.Conclusions: Transformation of potato plants resulted in accumulation of fructose in the apoplast, sucrose and glucose in the leaves, and especially, glucose in roots and microtubers. It is indicative of regulatory function of cell-wall invertase that could be found some application in medical, biological and pharmaceutical engineering.

Involvement of apoplastic invertase in the formation of resistance of cold-tolerant plants to hypothermia was established for Solanum tuberosum L. cv. Désirée and a transformant of the same cultivar expressing the yeast suc2 gene encoding apoplastic invertase. The dependence of the formation of increased constitutive and stress-induced cold-tolerance on the activity of apoplastic invertase and, consequently, on the level of intracellular sugars in the roots and leaves was demonstrated.

the essential cell-wall invertase activity in the leaves of transformed plants indicates significant changes in the cellular carbohydrate metabolism and regulatory function of this enzyme. The activity of yeast invertase changed the composition of intracellular sugars in the leaves of the transformed potato plant. The total content of sugars (sucrose, glucose, fructose) in the leaves and apoplast was higher in the transformants, in comparison by WTplants. Our data indicate higher constitutive resistance of transformants to severe hypothermia conditions compared to WT-plants. This fact allows us to consider cell-wall invertase as a enzyme of carbohydrate metabolism playing an important regulatory role in the metabolic signaling upon forming increased plant resistance to low temperature. Thus, the potato line with the integrated SUC2 gene is a convenient tool to study the role of the apoplastic invertase and the products of its activity during growth, development and formation constitutive resistance to hypothermia.

We studied the involvement of apoplastic sugars (glucose, fructose, and sucrose) and the cell-wall invertase (CWI) in the formation of the tolerance of cold-resistant potato plants (Solanum tuberosum L., cv Désirée) to hypothermia. The activity of CW1 and the content in the cell and the apoplast substrate (sucrose) and the reaction products of this enzyme (glucose and fructose) have a significant influence on the formation of the tolerance of cold-resistant potato plants to hypothermia.

Some properties and activity of extracellular invertase in the Saccharomyces cerevisiae yeasts encoded by the suc2 gene in heterologous expression were described. It was shown that the target suc2 gene is actively expressed in the genome of the transformed potato plants and S. cerevisiae invertase synthesized by this gene is transported into the apoplast due to the signal peptide of the proteinase II inhibitor. This enzyme is present in the apoplast in a soluble form and absorbed into the cell wall.