T. I. Trunova

Chilling Tolerance of Potato Plants Transformed with a Yeast-Derived Invertase Gene under the Control of the B33 Patatin Promoter

Tolerance to chilling was compared under in vitro conditions in potato plants (Solanum tuberosum L., cv. Désirée) transformed with a yeast-derived invertase gene under the control of the B33 class 1 tuber-specific promoter (the B33-inv plants) and potato plants transformed only with a reporter gene (the control plants). The expression of the inserted yeast invertase gene was proved by following the acid and alkaline invertase activities and sugar contents in the leaves under the optimum temperature (22°C). The total activities of acid and alkaline invertases in the B33-inv plants exceeded those in the control plants by the factors of 2–3 and 1.3, respectively. In the B33-inv plants, the activity of acid invertase twice exceeded that of the alkaline invertase, whereas the difference equaled 12% in the control plants. The contents of sucrose and glucose increased in the B33-inv plants by 21 and 13%, respectively, as compared to the control. Chilling at +3 and –1°C for 1, 3, and 6 h did not affect the rate of lipid peroxidation, as measured by the content of malonic dialdehyde (MDA) in the leaves of the genotypes under study. Only the longer exposures (24 h at +3 and –1°C and 7 days at +5°C) produced a significant decline in the MDA content in the B33-invplants, as compared to the control. Following short freezing (20 min at –9°C), the content of MDA increased by 50% in the leaves of the control plants, while in the B33-inv plants, cold-treated and control plants did not differ in the MDA content. The authors presume that the potato plants transformed with the yeast invertase gene acquire a higher tolerance to low temperatures as compared to the control plants, apparently due to the changes in sugar ratio produced by the foreign invertase.

Insertion of cyanobacterial desA gene coding for Δ12-acyl-lipid desaturase increases potato plant resistance to oxidative stress induced by hypothermia

The role of Δ12-acyl-lipid desaturase in plant resistance to hypothermia-induced oxidative stress was investigated. This study focused on modulation of free-radical processes occurring at low temperature in leaf cells of potato plants (Solanum tuberosum L., cv. Desnitsa) transformed with the gene for Δ12-acyl-lipid desaturase from the cyanobacterium Synechocystis sp. PCC 6803. Nontransformed plants of the same cultivar were used as a control material. The plants were grown in vitro on Murashige and Skoog agarized medium containing 2% sucrose. During hypothermia the rate of superoxide anion generation and hydrogen peroxide concentration decreased significantly. In addition, the content of both primary products (conjugated dienes and trienes) and secondary products (malonic dialdehyde) of lipid peroxidation was lower in the transformed plant leaves than in leaves of wild-type plants. It is supposed that the insertion into the plant genome of Δ12-acyl-lipid desaturase stabilizes the composition and physical properties of biomembranes by promoting polyunsaturation of fatty acids, which averts the accelerated generation of O2·−, — and suppresses lipid peroxidation during hypothermia. These changes improved cold resistance of potato plants, which was evident from the less severe injury of leaf blades in cold-treated transgenic plants, as compared to that in the wild-type line. The activity of superoxide dismutase, a key enzyme of the antioxidant defense system was lower in leaves of transformed plants than in leaves of wild-type plants. A comparatively low activity of superoxide dismutase in transgenic plants implies that these plants experience less severe thermal and oxidative stress upon cooling and can cope with the cold without considerable increase in the enzyme activity. It is concluded that the insertion of the desA gene encoding Δ12-acyl-lipid desaturase into cold-resistant potato plants improves plant resistance to cold-induced oxidative stress by decreasing the rate of intracellular free-radical processes.

The Effects of Cold Acclimation of Winter Wheat Plants on Changes in CO2 Exchange and Phenolic Compound Formation

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.

Lipid Composition of Tomato Leaves as Related to Plant Cold Tolerance

We studied the effects of a low nonlethal temperature (6°C) on the content and composition of polar lipids and their fatty acids in tomato (Lycopersicon esculentum Mill., cv. Sibirskie skorospelye) leaves. We demonstrated that chilling resulted in a decrease in the content of total polar lipids per 1 mg protein. The content of lipids in chloroplast membranes (monogalactosyldiacylglycerols, digalactosyldiacylglycerols, sulfoquinovosyldiacylglycerols, and phosphatidylglycerols) changed less substantially than the content of phospholipids in other cell organelles and in the cytoplasm. Neutral lipids comprised only 1% of total lipids, and their content also decreased after chilling. The relative amounts of unsaturated and saturated fatty acids in polar lipids were practically unchanged. The conclusion was drawn that the maintenance of a high level of chloroplast membrane lipids under low temperatures could play an important role in the survival of cold-tolerant plants.

The decreased cold resistance of chilling-sensitive plants is related to suppressed CO2 assimilation in leaves and sugar accumulation in roots

Tomato (Lycopersicon esculentum L., cv. Sibirskii skorospelyi) and cucumber (Cucumis sativus L., cv. Konkurent) plants were grown in a soil culture in a greenhouse at an average daily temperature of 20°C and ambient illumination until the development of five and eight true leaves, respectively. During the subsequent three days, some plants were kept in a climatic chamber at 6°C in the light, whereas other plants remained in a greenhouse (control). The cold-resistance of cucumber leaves and roots, as assayed from the electrolyte leakage, was reduced after cold exposure stronger than cold-resistance of tomato organs. The ratio photosynthesis/dark respiration was lower in cucumber than in tomato leaves at all measurement temperatures. The concentrations of sugars (sucrose + glucose + fructose) increased in chilled tomato roots but decreased in cucumber roots. Cold exposure changed the activities of various invertase forms (soluble and insoluble acidic and alkaline invertases). The total invertase activity and the ratio of mono- to disaccharides increased. The lower cucumber cold-resistance is related to the higher sensitivity of its photosynthetic apparatus to chilling and, as a consequence, insufficient root supply with sugars.

Effect of the desA gene encoding Δ12 acyl-lipid desaturase on the chloroplast structure and tolerance to hypothermia of potato plants

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.

Processes hindering activation of lipid peroxidation in cold-tolerant plants under hypothermia

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 (O2·− 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.

Ultrastructural Organization of Chloroplasts of the Leaves of Potato Plants Transformed with the Yeast Invertase Gene at Normal and Low Temperature

According to the reaction induced by exposure to low temperature, plants fall into the following groups: frost-resistant, cold-tolerant, and cold-sensitive. Frostresistant plants are able to withstand the formation of extracellular ice. Cold-tolerant plants are tolerant to low temperatures at which ice is not formed, and coldsensitive plants are not tolerant to low positive temperatures. Depending on specific genetic features and characteristics of temperature stress, the plants of these groups during the adaptation period produce cells of specific ultrastructure. It was shown that, in cells of frost-adapted plants of winter wheat and rye [1, 2] and wintering trees and shrub plants [3] exposed to hypothermia, there was cytoplasm proliferation, a decrease in the vacuole volume, an increase in the vacuole electron density, an increase in the number of cell elements, and an increase in the number of plastoglobules. This prevented intracellular ice formation and caused an increase in the plant resistance to the formation of extracellular ice. Exposure of cold-sensitive plants to low positive temperatures induces destructive changes in the cell ultrastructure: swelling of chloroplast stroma [4], rupture of chloroplast envelope and lamellae [5], a decrease in the number of ribosomes, and failure in the tonoplast intactness [6]. The changes in the structure of cells of cold-tolerant tomato plants (cultivar Sibirskii Skorospelyi) induced by long-term exposure to low positive temperatures were mainly manifested as the formation of xeromorphous structure [7]. The following changes in the ultrastructure of plant cells were observed after the exposure at 6 ° C: a decrease in the areas of the cell, cytoplasm, chloroplast, grana, and starch grains; a decrease in the section count of chloroplasts, mitochondria, starch grains, and granal structures; and a decrease in the number of thylakoids per grana. It was suggested that these changes in the ultrastructure of tomato cells and chloroplasts should be regarded as manifestation of adaptive or protection responses rather than cell damage.

Changes in the activity of superoxide dismutase isoforms in the course of low-temperature adaptation in potato plants of wild type and transformed with Δ12-acyl-lipid desaturase gene

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.

Activity of Lectin-Like Proteins of the Cell Walls and the Outer Organelle Membranes as Related to Endogenous Ligands in Cold-Adapted Seedlings of Winter Wheat

The hemagglutinating activity (HA) of lectin-like components in the cell walls and the outer organelle membranes was studied in freezing-tolerant winter wheat (Triticum aestivum L., cv. Mironovskaya 808) plants in the course of hardening at 2°C, in parallel with the effects of endogenous ligands from the soluble fraction on HA. Low hardening temperature divergently changed HA of the lectin-like components in the cell walls, the outer membranes of nuclei, plastids, and mitochondria, and the microsomal membranes: HA increased in the cell walls, nuclei, and plastids and decreased in the mitochondria and microsomal membranes. Under hardening conditions, with plant growth slowed down, HA of the lectin-like proteins from the outer organelle membranes was inhibited in the presence of the soluble fraction components (soluble ligands); such inhibition was not observed in the case of actively growing nonhardened seedlings. The authors put forward a hypothesis that the lectin-like proteins from both peripheral (cell walls) and intracellular (outer organelle membranes) compartments are essential for developing freezing tolerance. HA of the cell walls and the outer membranes of nuclei and plastids enhanced by hardening manifested positive correlation with freezing tolerance and negative correlation with the growth rate. In contrast, HA of the outer membranes of mitochondria and microsomes was positively related to plant growth and negatively, to freezing tolerance. As negative and positive effectors of membrane-dependent processes, the lectin-like components of the outer organelle membranes seem to control membrane functional activities in the course of cold adaptation.