The role of fatty acid ω3 acyl-lipid desaturases in low-temperature hardening (7 days at 3°C) of potato plants (Solanum tuberosum L., cv. Yubilei Zhukova) was studied. It was found that transcriptions of the three genes of ω3 acyl-lipid desaturases present in the potato genome significantly differed in their response to low temperature. The content of the FAD3 gene transcripts dramatically fell in the first day of cooling and was not restored until the end of the hardening period. The FAD7 gene was constitutively expressed through almost the entire adaptation period. The level of the FAD8 gene transcripts sharply increased in the first day of the hardening. The total fraction of trienoic fatty acids, which are synthesized by ω3 acyl-lipid desaturases, rose by 3% during the hardening, which is a relatively large value for potato. The hardened plants, in comparison with the unhardened ones, manifested a higher net photosynthesis/dark respiration ratio, higher (2.5-fold) concentration of soluble sugars, and stronger resistance to negative (–2 or –3°C) temperatures. It is suggested that the augmentation of the trienoic FA fraction, which are mainly localized in the thylakoid membranes of the potato chloroplasts, sustains the photosynthesis under low temperatures. Therefore, the maintenance of photosynthetic activity during the low-temperature acclimation supplies the plant with photoassimilates that are the principal sources of energy and metabolites necessary for the establishment of potato plant resistance to hypothermia.

Changes in the activity of superoxide dismutase (SOD), ascorbate peroxidase (APX), and catalase (CAT) for the plantsNicotiana tabacumL. andSecale cerealeL. during low-temperature hardening have been investigated. It was determined that a transient increase in the content of MDA occurs in tobacco plants at the beginning of the hardening period with subsequent activation of SOD, APX, and CAT. Unlike tobacco, winter rye has an MDA content and activity of SOD, APX, and CAT throughout the hardening period that remains at the level of unhardened plants. In tobacco cells, the majority of SOD activity was detected in chloroplasts (90%), while other cellular structures accounted for only 10% of the total activity identified in PAGE. In winter rye cells, SOD activity was distributed evenly between chloroplasts and other cellular compartments (46% in chloroplasts and 54% outside of chloroplasts). The distribution of APX and CAT activity in tobacco and winter rye cells was identical: all APX was concentrated in chloroplasts, while all catalase activity was manifested outside of chloroplasts. It is concluded that tobacco and winter rye plants showed different antioxidant defense strategies under conditions of low hardening temperatures. Tobacco plants provided protection against oxidative damage during low-temperature hardening by increasing activity of SOD, APX, and CAT. Winter rye plants were able to avoid the development of oxidative stress during hardening due to uniform distribution of SOD activity in cells, maintaining the constitutive activity of SOD, APX, and CAT, and, probably, due to the accumulation of nonenzymatic antioxidants.

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.

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.

We investigated the changes in the total activity of superoxide dismutase (SOD) and the role of its isoforms in hardening potato (Solanum tuberosum L., cv. Desnitsa) plants of wild type and transformed with desA gene of Δ12-acyl-lipid desaturase from Synechocystis sp. PCC 6803. Hydroponically grown 8-week-old plants were exposed for six days to hardening temperature of 5°C. Before chilling, the total SOD activity in the transformed plants was somewhat greater than in the control plants. By the first day of hardening, SOD activity in both potato genotypes rose almost 1.5 times; however, the absolute value of SOD activity was considerably greater in the transformed plants. Subsequently, the total SOD activity in both genotypes decreased and by the end of the 6th day, it almost returned to the initial level. Electrophoretic and inhibitor analyses of potato plants revealed three types of SOD with one isoform of Mn-SOD, four isoforms of Fe-SOD, and two isoforms of Cu/Zn-SOD. In both genotypes, Fe-SOD3 manifested the greatest activity before chilling and in the course of hardening. Such changes in SOD activity corresponded to the rate of generation of superoxide anion radical and elevation of the content of products of peroxide oxidation of lipids (POL). Our data suggest that in the course of hardening of cold-resistant potato plants, the total SOD activity changed mostly due to Fe-SOD3 and to some extent as a result of elevated Cu/Zn-SOD2 activity, which was particularly evident at the beginning of hardening and more pronounced in the transformed plants. We assume that such temporal pattern is related to a greater rate of superoxide anion generation in the transformed plants as compared with control plants.

The effects of potato ( Solanum tuberosum L., cv. Desnitsa) plant transformation with the desA gene encoding Δ12-acyl-lipid desaturase from Synechocystis sp. PCC 6803 on the regulation of free-radical processes in relation to plant tolerance to hypothermia are considered. It was shown that the content of polyunsaturated fatty acids (PUFA) in transformed plants was higher than in wild-type ones. In particular, the content of linoleic acid in transformants was higher by 35% and the content of linolenic acid was by 41% higher than in untransformed plants. In addition, transformation induced an increase in the absolute content of C 16 -PUFA and on the whole resulted in a marked accumulation of membrane lipids. As judged from the values of the damage index and the ratio of photosynthesis to respiration in wild-type and transformed plants under cold treatment, these changes in lipid metabolism favored the protection of coupling membranes, thus preventing plants against free-radical oxidation under low-temperature stress. As a result, the intensity of oxidative stress in transformed plants was much lower than in wild-type ones, whereas antioxidant enzymes (superoxide dismutase, catalase, peroxidase) were not substantially activated under 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 reasons why the rate of lipid peroxidation (POL) associated with a long-term action of low above-zero temperature (5°C, 6 days) on 6-week-old plants of two potato (Solanum tuberosum L.) cultivars (cold-tolerant cv. Desnitsa and less tolerant cv. Desiree) did not rise were investigated. Upon a long-term action of low hardening temperatures on the plants of both cultivars, there was an equilibrium between the rate of generation of superoxide anion (O 2 ·− and activity of superoxide dismutase (SOD), which inactivated it with the formation of H2O2. Among the enzymes breaking up hydrogen peroxide, the highest activity was observed for guaiacol peroxidases, which was an order of magnitude greater than the activity of catalase. In potato cultivars, POL processes were not considerably activated; however, activities of antioxidant enzymes (SOD, catalase, and guaiacol peroxidases) in cold-tolerant cv. Desnitsa and less tolerant cv. Desiree differed. It was concluded that, upon a long-term action of hardening temperatures, cold-tolerant plants could sup-press POL processes. Moreover, a test for tolerance to damaging temperature (−3°C, 18 h) showed that detected preservation of the prooxidant/antioxidant equilibrium not only maintained vital activities at low above-zero temperatures but also elevated tolerance to short-term frosts, with this adaptability being cultivar-specific.