N. V. Astakhova

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.

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.

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.

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.

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.

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.

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.

Changes in chloroplast ultrastructure of tobacco plants in the course of protection from oxidative stress under hypothermia

Changes of ultrastructural organization of tobacco (Nicotiana tabacum L. cv. Samsun) chloroplasts associated with plant protection from oxidative stress during hypothermia were studied. It was found that the chilling hardening (6 days at 8°C) was accompanied by the significant reduction in the number of grana in a chloroplast simultaneously with area reduction of a granum that led to 30% decrease in the total area of grana in tobacco chloroplasts. In the course of tobacco plant hardening, approximately twofold decrease in generation rate of superoxide anion radical and 30% decrease in content of hydrogen peroxide occurred, which indicates retardation of oxidative processes in plant cells during the cold exposure. It is suggested that the ultrastructural changes in chloroplast organization that were found may prevent an overreduction of an electron-transport chain under hypothermia, when the ability of the Calvin cycle to utilize ATP and NADP·H is significantly reduced. The balanced work of components of light and dark photosynthetic phases may prevent the excessive generation of reactive oxygen species and render formation of tobacco plant tolerance to hypothermia.

Activities of antioxidant enzymes of Arabidopsis thaliana plants during cold hardening to hypothermia

Changes in activities of the enzymes performing direct antioxidant functions were studied in 7–8-week-old plants Arabidopsis thaliana Heinh (L.) of Columbia (Col-0) ecotype. It was found that 5-day cold hardening at 2°C increased plant cold resistance to the subsequent stronger cooling. Under these conditions, the marked changes occurred in activities of superoxide dismutase and III type (guaiacol) peroxidses but not in that of catalase. The total peroxidase activity exceeded the catalase activity before cold hardening. Therefore, peroxidases are able to decompose more H2O2 than catalases and appear to make the dominant contribution to the protection from the cold damage.

Changes in the chloroplast ultrastructure in potato leaves during long-term micropropagation in vitro

362 The method of in vitro micropropagation of potato plants by cuttings is widely applied for rapid and largescale propagation of cultivars free of viral and viroid infections and also for production of genetically modified forms by bacterial transformation. Cultured in vitro plants are kept under strictly controlled conditions, e.g. irradiance, photoperiod, temperature, humidity, and other factors. Therefore, they are a convenient model for studying physiological processes and the effects of various stressors. In our laboratory, investigations of the effects of the yeast invertase gene insertion into the potato ( Solanum tuberosum L., cv. Desiree) genome on plant carbohydrate metabolism and tolerance to hypothermia are performing during several years. In these experiments, we used plants grown in vitro in the light on agar-solidified Murashige and Skoog nutrient medium [1] containing 2% sucrose. The plants with the greatest growth rate and well-developed morphological traits were used for cutting performed every five weeks. Taking into amount that in vitro potato plants subjected to micropropagation for a long time period on artificial nutrient media grow predominantly heterotrophically [2], we can conclude that we performed directed selection of plants with better heterotrophic properties. This selection occurred after morphological traits and the growth rate under definite conditions of plant growing, i.e., predominantly heterotrophic nutrition. Photrophic and heterotrophic nutrition types are known to compete; therefore, such a selection results in the adverse effect to plant photosynthesis; and this is clearly manifested already in 10–15 generations [2]. This effect is related to the limitation of air CO 2 diffusion through the cotton stopper. This amount of CO 2 is capable of providing only 5–6% of photosynthetic demands of tube potato plants, which could grow only due to nutrient media enriched in organic compounds (mainly in sucrose), i.e., heterotrophically. Tsoglin et al. [3] believe that, keeping in mind a low level of gas exchange between environment and the tube, plants used for photosynthesis mainly CO 2 released at heterotrophic growth, whereas oxygen released by the plants, in its turn, could favor heterotrophic growth. In this connection, the main task of this work was the investigation of the effects of long-term in vitro micropropagation on the potato photosynthetic apparatus, in particular on the chloroplast ultrastructure. To this end, we compared our earlier data concerning chloroplast ultrastructure and morphometry [4] with the results of electron-microscopic analysis of chloroplasts obtained after 45 cutting cycles of one and same potato genotypes differing in carbohydrate metabolism. Experiments were performed with potato ( S. tuberosum L.) cv. Desire plants transformed with the inv gene of yeast invertase under the control of tuber-specific B33 class I patatin promoter and with the proteinase inhibitor II leader peptide sequence providing for enzyme apoplastic localization (further designated as B33inv plants). Potato plants transformed with the marker GUS gene under the control of 35S CaMV promoter were used as control (further designated as 35SGUS plants). GUS gene encodes β -glucurinidase, which has no functions in the higher plants and thus does not influence carbohydrate metabolism. The plants studied harbored also the nptII gene of kanamycin resistance under the control of the 35S CaMV promoter. Transgene expression in plants was confirmed by PCR analysis [5] and by direct determination of the reporter GUS gene activity [6]. Plants were selected from the collection of clones obtained as a result of collaboration of the Max Planck Institute of Molecular Plant Physiology (Golm, Germany) and Chailakhyan Laboratory of Signaling Pathways Controlling Development (Timiryazev Institute of Plant Physiology, RAS, Moscow, Russia). Plants were in vitro propagated by single-node cuttings grown in the phytotron chamber (Timiryazev Institute of Plant Physiology, RAS) at 22 ° C and 16-h illumination from white-light luminescence tubes (an illuminance of 4 klx) during 5 weeks in tubes on agarGENERAL BIOLOGY