V. N. Popov

The Effect of Tobacco Plant Transformation with a Gene for Acyl-Lipid Δ9-Desaturase from Synechococcus vulcanus on Plant Chilling Tolerance

Tobacco plants with the introduced desC gene for acyl-lipid Δ9-desaturase from the thermophilic cyanobacterium Synechococcus vulcanus were cultivated on agar-solidified Murashige and Skoog nutrient medium supplemented with ferulic acid and antibiotics at 22°C and a 16-h photoperiod. Control plants were transformed with an empty pGA482 vector. The analysis of fatty acids (FAs) showed that, in transgenic plants, the level of 16:0 and 18:0 FAs decreased substantially, whereas the levels of di- and trienoic FAs increased. Transformed plants were more cold-tolerant. The tolerance to chilling was evaluated from electrolyte leakage from tissues damaged by cold treatments and from the accumulation of a product of lipid peroxidation, malondialdehyde. It was concluded that acyl-lipid Δ9-desaturase was actively expressed in transgenic tobacco plants and converted stearic acid into oleic acid, thus producing a substrate for further synthesis of di- and trienoic FAs. An increased proportion of polyunsaturated FAs in membrane lipids resulted in improved tobacco plant tolerance to chilling.

Involvement of cell-wall invertase in low-temperature hardening of tobacco plants

Specific features of low-temperature hardening (6 days at 8°C) of cold-sensitive tobacco plants (Nicotiana tabacum, cv. Samsun) related to changes in the cell-wall invertase activity were studied. During cold hardening, oppositely directed changes in this enzyme activity occurred in tobacco leaves and roots. In the leaves, cell-wall invertase was activated (approximately by 30%), the content of sugars increased (approximately by 25%), and the content of sucrose, the main transport form of sugars, in the apoplast reduced by three times; all these changes indicate that assimilate outflow from leaves to roots was inhibited. In contrast, in the root system, enzyme activity was decreased almost twice and the content of sugars in them was essentially unchanged. It is suggested that a strategy of low-temperature adaptation of cold-sensitive tobacco plants aimed at creating the high cold tolerance of aboveground parts, even at the expense of the root system, which, under conditions of native vegetation, is not practically exposed to damaging low temperatures.

Specific features of oxidative stress in the chilled tobacco plants following transformation with the desC gene for acyl-lipid Δ9-desaturase from Synechococcus vulcanus

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.

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.

Changes in the content and composition of lipid fatty acids in tobacco leaves and roots at low-temperature hardening

Changes in the fatty acid (FA) composition of leaf and root lipids of heat-loving tobacco (Nicotiana tabacum L., cv. Samsun) plants during low-temperature hardening (8°C for 6 days) were studied. Hardening could improve leaf but not root cold tolerance. As this took place, the relative content of polyunsaturated (18:2n-6 and 18:3n-3) FAs increased and the proportion of saturated and monounsaturated FAs decreased. In contrast, in the roots hardening slightly increased the concentration of saturated FAs (16:0 and 18:0) and reduced the level of unsaturated FAs (18:1n-9, 18:2n-6, and 18:3n-3). At the same time, root lipids contained much C20–24 FAs, and their content increased during hardening. It was suggested that an increased FA saturation and elevated proportion of C20–24 FAs in the root lipids resulting in the lower membrane fluidity could be a reason for incapability of heat-loving tobacco plant roots of hardening and plant death at the lowtemperature stress.

Lipid peroxidation during low-temperature adaptation of cold-sensitive tobacco leaves and roots

We studied low-temperature adaptation of cold-sensitive tobacco plants in relation to peroxidation of lipids (POL) in their leaves and roots. Experiments were performed with tobacco plants (Nicotiana tabacum L., cv. Samsun). Cold hardening (6 days at 8°C) exerted principally different action on tobacco leaves and roots. In the leaves, the contents of dienoic conjugates and MDA was reduced, and tissue cold tolerance, even to below zero temperatures, was improved. In contrast, in the roots, POL was activated and root cold tolerance decreased. It is suggested that an incapability of the tobacco root system to adapt to low temperature was a limiting factor determining the low potential of this and other cold-sensitive plants to hypothermia.

Chloroplast ultrastructure in leaves of tobacco plants with the introduced gene for the acyl-lipid Δ9-desaturase from Synechococcus vulcanus at normal and low temperature

Ultrastructural changes in chloroplasts of tobacco plants (Nicotiana tabacum L.) with the introduced desC gene for the acyl-lipid Δ9-desaturase from the thermophilic cyanobacterium Synechococcus vulcanus were investigated during plant acclimation to cold. Control plants were transformed with an empty pGA482 binary vector. At optimum growth temperature, a decreased number of grana and thylakoids and an increased number of plastoglobules and their larger area were observed in transgenic plants when compared to control ones. In control plants, acclimation to cold (6 days at 10°C) resulted in the larger areas of chloroplasts and grana. These changes indicated starting cold-induced injuries manifested in swelling of the stroma and a slight decrease in the total number of thylakoids in the chloroplast. In contrast, transgenic plants responded to cold by reducing the chloroplast, granal, and plastoglobule areas. Meantime, the number of thylakoids per granum increased noticeably. The total number of thylakoids in the chloroplast increased from 123 to 203. It was concluded that expression of the acyl-lipid Δ9-desaturase gene in tobacco plants provided for the formation of the cell ultrastructure similar to one characteristic of cold-tolerant plants.

Reorganization of chloroplast ultrastructure associated with low-temperature hardening of Arabidopsis plants

When following low-temperature acclimation (5 days at 2°C) of cold-resistant plants of Arabidopsis (Arabidopsis thaliana Heynh. (L.), ecotype Columbia) in relation to the changes in chloroplast ultrastructure, we registered the high efficiency of hardening and the ability of hardened plants to lower a threshold of frost damage by about 3°C. During hardening, the area of grana in the chloroplasts more than doubled, with considerably increased numbers of thylakoids per granum and thylakoids per chloroplast. The rate of apparent photosynthesis decreased to lesser extent than the rate of dark respiration, as a result the content of soluble sugars increased fourfold, ensuring an adaptive reorganization of metabolism, which enabled the hardened plants to survive even at below-zero temperatures (up to −7°C). The authors conclude that a considerable increase in the number of thylakoids in the chloroplasts helps maintain photosynthesis at low above-zero temperatures and is a prerequisite for the accumulation of soluble sugars in Arabidopsis leaves.

CO2 exchange as related to sugar accumulation and invertase activity during winter wheat cold hardening.

Plant frost resistance depends on the balance between key metabolic processes occurring at low temperatures, especially on the type of carbohydrate metabolism [1]. One of plant adaptive responses to low temperature is the accumulation of such water-soluble carbohydrates as sucrose, glucose, fructose, raffinose, stachyose, fructosans, and oligosaccharides. According to the published data [2–6], an improved plant tolerance to low temperatures is related to the total amount of water-soluble carbohydrates rather than to some particular carbohydrate species. The role of carbohydrates in the improving plant resistance to low temperatures is known to be determined by their cryoprotective effects on cell membranes and by their involvement in metabolic processes as an energy source and precursors for the synthesis of other protectants. In addition, soluble carbohydrates serve osmoregulators reducing the rate of extracellular ice formation. They are also storage compounds providing for spring leaf regrowth. It was reported that sugars can directly modify the plasmalemma, facilitating its homeoviscosity adaptation [7]. However, the detail pattern of changes in the carbohydrate metabolism during autumnal cold hardening of winter plants is not well elucidated. It is not known whether the ratio between various carbohydrate species changes, whether simple or compound water-soluble sugar forms accumulate, what is the role of various invertases in these processes. It is also important to establish, whether cold hardening is the result of enhanced carbohydrate catabolism (cleavage of polymeric sugar into simpler forms), their enhanced synthesis with the accumulation of polymeric sugars, or both. In this work, we attempted to answer these questions by studying winter wheat ( Triticum aestivum L.) freezing resistance and the rates of photosynthesis and respiration at temperatures of plant active growth and cold hardening. Experiments were performed with widely spread and frost resistant wheat cv. Mironovskaya 808. Plants were grown in soil culture for 45–60 days under natural conditions until the phenophase of tillering. Some plants, which partially passed through the first stage of cold hardening at around-zero temperatures, were transferred to the greenhouse with a controlled temperature of 18 ± 3°ë (control plants), whereas other plants were transferred to unheated greenhouse with a naturally decreasing temperature, where plants completed hardening (hardened plants). 14 days after plant transfer to different temperature conditions, we started measuring experiments.

Changes in the content of fatty acid desaturases gene transcripts for Arabidopsis plants under adaptation to hypothermia

Changes in the content of acyl-lipid desaturases gene transcripts in Arabidopsis (Arabidopsis thaliana Heynh. (L.), ecotype Columbia) plants subjected to low temperature hardening for 5 days at 2°C were investigated. In the course of the experiments with hourly dynamics of plant chilling, it was established that the genes under study by their responses to cold action can be separated into two groups. The genes of the first group represented by ADS2, FAD2, and FAD7 are characterized by the amount of their transcripts that rapidly increase even for the first 2–4 h of cold exposition, while the same value for the genes FAD3, FAD6, and FAD8 belonging to the second group remained unchanged under the same conditions. The experiments with the above hardening dynamics provided evidence for consecutive character of genes ADS2, FAD2, and FAD7 transcript accumulation, where the process for ADS2 was sharply intensified even for the first days of hardening, while that for other two genes achieved maximal intensity only on the fourth day of hardening. These results, taken together, lead to the conclusion that the agreeing in time increase in the transcript content of genes ADS2, FAD2, and FAD7 encoding Δ9-, Δ12-, and ω3-desaturases resulted in the change of FAs composition of chloroplast lipids. Increasing portion in them of triene FAs during hardening time provided the maintenance of chloroplast membranes in a native state allowing the Arabidopsis plant to successfully adapt to hypothermia.