A. N. Deryabin

Lipid fatty acid composition of potato plants transformed with the Δ12-desaturase gene from cyanobacterium

Potato (Solanum tuberosum L.) plants were transformed with the desA gene encoding Δ12 acyl-lipid desaturase in the cyanobacterium Synechocystis sp. PCC 6803. To evaluate the efficiency of this gene expression in the plant, its sequence was translationally fused with the sequence of the reporter gene encoding thermostable lichenase. A comparison of native and hybrid gene expression showed that lichenase retained its activity and thermostability within the hybrid protein, whereas desaturase retained its capability of inserting the double bond in fatty acid (FA) chains and, thus, to modify their composition in membrane lipids. In most transformed plants, shoots contained higher amounts of polyunsaturated FAs, linoleic and linolenic (by 39–73 and 12–41%, respectively). The total absolute content of unsaturated FAs was also higher in transformants by 20–42% as compared to wild-type plants. When transformed plants were severely cooled (to −7°C), the rate of their membrane lipid peroxidation was not enhanced, whereas in wild-type plants, it increased substantially (by 25%) under such conditions. These results could indicate a higher tolerance of transformed plants to low temperatures and the oxidative stress induced by hypothermia.

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.

Effect of sugars on the development of oxidative stress induced by hypothermia in potato plants expressing yeast invertase gene

The influence of sugars on the development of oxidative stress induced by hypothermia was investigated in the leaves of two genotypes of potato (Solanum tuberosum L.) grown in vitro on the Murashige and Skoog medium supplemented with 2% sucrose. We used wild-type plants of potato, cv. Désirée, and potato plants expressing a yeast invertase gene under the control of the B33 class I patatin promoter and carrying a sequence of proteinase inhibitor II leader peptide for the apoplastic enzyme localization. At temperature of 22°C optimal for growth, expression of the yeast invertase gene in the leaves of transformed plants brought about a modification in the carbohydrate metabolism manifested in the activation of acid forms of invertase and accumulation of intracellular sugars (predominantly of sucrose because of its resynthesis). The exposure of plants to light under prolonged hypothermia (5°C, 6 days) activated all the forms of invertase (predominantly of acid invertase) and induced accumulation of sugars. In the leaves of potato expressing the yeast invertase gene, these processes were more intense. Under chilling, superoxide dismutase activity and the rate of lipid peroxidation in the leaves of investigated potato genotypes depended on the level of accumulated intracellular sugars. It was concluded that sugars play an important role as stabilizers of cellular membranes and scavengers of reactive oxygen species decelerating the processes of free radical oxidation of biomolecules upon the development of oxidative stress induced by hypothermia.

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.

Characteristics of oxidative stress in potato plants with modified carbohydrate metabolism

Effects of sugars on the development of hypothermia-induced oxidative stress were studied in leaves of two potato genotypes (Solanum tuberosum L., cv. Désirée): with normal carbohydrate metabolism and a genotype with increased sugar content modified by insertion of yeast-derived invertase gene. It was found that generation of proceeds more actively in transformed plants than in control plants. On the contrary H2O2 concentration and the catalese and peroxidase activities were lower. At the same time, the activities of superoxide dismutase were similar in plants of both genotypes. A short-term incubation of plants at −7°C confirmed that a higher freezing tolerance of transformed plants was due to low-molecular-weight components of antioxidant protection system rather than to enzymatic component. Literature data and experimental results suggest that the protective effect of sugars is caused by their ability to scavenge ROS nonspecifically under stress conditions

The changes in invertase activity and the content of sugars in the course of adaptation of potato plants to hypothermia

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.

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.

Characteristics of extracellular invertase of Saccharomyces cerevisiae in heterologous expression of the suc2 Gene in Solanum tuberosum plants

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.

CO2 exchange and structural organization of chloroplasts under hypothermia in potato plants transformed with a gene for yeast invertase

Growth, CO2 exchange, and the ultrastructure of chloroplasts were investigated in the leaves of potato plants (Solanum tuberosum L., cv. Désirée) of wild type and transformed with a gene for yeast invertase under the control of patatin class I B33 promoter (for apoplastic enzyme) grown in vitro on the Murashige and Skoog medium supplemented with 2% sucrose. At a temperature of 22°C optimal for growth, the transformed plants differed from the plants of wild type in retarded growth and a lower rate of photosynthesis as calculated per plant. On a leaf dry weight basis, photosynthesis of transformed plants was higher than in control plants. Under hypothermia (5°C), dark respiration and especially photosynthesis of transformed plants turned out to be more intense than in control material. After a prolonged exposure to low temperature (6 days at 5°C), in the plants of both genotypes, the ultrastructure of chloroplasts changed. Absolute areas of sections of chloroplasts and starch grains rose, and the area of plastoglobules decreased; in transformed plants, these changes were more pronounced. By some ultrastructural characteristics: a reduction in the cold of relative total area of sections of starch grains and plastoglobules (in percents of the chloroplast section area) and in the number of granal thylakoids (per a chloroplast section area), transformed plants turned out to be more cold resistant than wild-type plants. The obtained results are discussed in connection with changes in source-sink relations in transformed potato plants. These changes modify the balance between photosynthesis and retarded efflux of assimilates, causing an increase in the intracellular level of sugars and a rise in the tolerance to chilling.

Alternative pathways of photosystem I-dependent electron transport in two genetically different potato cultivars in vitro

Alternative pathways of electron transport involving photosystem I (PSI) only were studied in leaves of potato plants (Solanum tuberosum L., cv. Desiree), modified by yeast invertase gene, controlled by tuber-specific class I patatin B33 promoter with proteinase II signal peptide for apoplastic localization of the enzyme. Nontransformed (wild-type) potato cultivar Desiree was used as a source of control plants. Phototrophic cultures grown in vitro on the sucrose-free Murashige and Skoog medium, as well as plants grown on the medium with 4% sucrose were examined. Various PSI-dependent alternative pathways of electron transport were discriminated by quantitative analysis of kinetic curves of dark reduction of P700+, the primary electron donor of PSI, oxidized by far-red light known to excite selectively PSI. In potato plants with two different genotypes, four exponentially decaying kinetic components were found, which suggests the existence of multiple alternative routes for electron input to PSI. Inhibitor analysis (with diuron and antimycin A) allowed identification of each route. A minor ultra-fast component originated from weak residual excitation of PSII by far-red light and represented electron flow from PSII to PSI. Ferredoxin-dependent cyclic electron flow around PSI accounted for the middle component, and two slower components were assigned to donation of electrons to PSI from reductants localized in the chloroplast stroma. The rates of all components were somewhat higher in leaves of the transformed plants than in the wild-type plants. However, relative contributions of separate components to the kinetics of dark P700+ reduction in leaves of both potato genotypes were similar. Growing plants on the medium with sucrose dramatically increased the amplitude of absorbance change at 830 nm in the transformed (but not in wild type) plants, which indicated a drastic increase in P700 concentration in their leaves.