A. G. Shugaev

Effect of drought on chlorophyll content and antioxidant enzyme activities in leaves of three wheat cultivars varying in productivity

Seedlings of three wheat varieties (Triticum aestivum L.)—highly productive cv. Ballada, moderately productive cv. Belchanka, and low productive cv. Beltskaya—were exposed to progressive soil drought (cessation of watering for 3, 5, and 7 days) and then analyzed for chlorophyll content and activities of ferredoxin-NADP+ oxidoreductase (FNR) and antioxidant enzymes, namely, glutathione reductase (GR) and ascorbate peroxidase (AscP). In addition, the proline content, and the extent of lipid peroxidation were examined. In the first period of water limitation, the water loss from leaves was slight for all wheat cultivars, which is characteristic of drought-resistant varieties. After 7-day drought the leaf water content decreased by 5.2–6.8%. The total chlorophyll content expressed per unit dry weight increased insignificantly during the first two periods of drought but decreased by 13–15% later on. This decrease was not accompanied by changes in chlorophyll a/b ratio. The plant dehydration did not induce significant changes in FNR activity. Activities of GR and AscP in leaves of wheat cultivars Ballada and Belchanka increased on the 3rd and 5th days of drought. Owing to the coordinated increase in GR and AscP activities, the lipid peroxidation rate remained at nearly the control level observed in water-sufficient plants. When the dehydration period was prolonged to 7 days, activities of GR and AscP in wheat cultivars reduced in parallel with the increase in malonic dialdehyde (MDA) content, indicating that the antioxidant enzyme defense system was weakened and lipid peroxidation enhanced. Unlike Ballada and Belchanka, the wheat cv. Beltskaya did not exhibit the increase in GR and AscP activities during progressive soil drought. The increase in MDA content by 16% in this cultivar was only observed after a 7-day drought period. The proline content in leaves of all wheat cultivars increased substantially during drought treatment. Thus, in wheat cultivars examined, different responses of the defense systems were mobilized to implement plant protection against water stress. The activities of antioxidant enzyme defense system depended on wheat cultivar, duration of drought, and the stage of leaf development.

The Changes in the Pathways of Mitochondrial Oxidation at the Initial Period of Tuber Development

The rate of oxygen consumption and the participation of the mitochondrial oxidases cytochrome oxidase (COX) and the alternative KCN-resistant mitochondrial oxidase (AOX) were determined in nondifferentiated stolons and small newly developed tubers. High rates of about 300 μl O2/(g fr wt h) and low sensitivity to cyanide were characteristic of stolon respiration. The AOX activity comprised the major part of the latter (60%). As tubers developed, their respiration rate declined and the proportion of mitochondrial oxidases changed: COX became the major terminal oxidase, while the AOX input dropped to 15% of the total oxygen consumption. The AOX input correlated with the total monosaccharide content in stolons and tubers. These data are in line with the concept that the alternative pathway of mitochondrial oxidation serves as a mechanism of energy overflow by which to utilize excess carbohydrates that the cell can neither store nor utilize.

The inhibition of mitochondrial metabolic activity in etiolated pea seedlings under water stress

In the present work, we studied the influence of water (osmotic) stress on mitochondrial metabolic activity in etiolated pea (Pisum sativum L.) seedlings. Three-day-old pea seedlings were subjected to stress by placing their roots in 0.6 M mannitol for 48 h. Epicotyl growth was severely suppressed, and tissue water content was decreased. We revealed the negative influence of the water stress on mitochondrial metabolic activity of seedlings, which effect was retained also after organelle isolation. In particular, in the mitochondria of stressed seedlings, the rate of oxidation of malate and other respiratory substrates (in state 3) was severely decreased, as well as respiratory control ratio. The rate of proline oxidation was reduced most seriously (by 70%). The efficiency of oxidative phosphorylation, according to the ADP/O ratio was not changed or was increased as compared to mitochondria in control plants. Activation of CN-resistant oxidase and other alternative pathways of electron transport in the mitochondrial electron-transport chain in stressed plants were not observed. In the epicotyl tissues under water stress, no MDA was accumulated and proline accumulation was insignificant. The role of mitochondria in adaptation responses of young seedlings is discussed.

Developmental Changes in the NAD Content in Sugar-Beet Root Mitochondria and Their Effect on the Oxidative Activity of These Organelles

The NAD content was determined in mitochondria isolated from sugar-beet roots at various stages of plant development. A high NAD content (7.6 ± 0.9 nmol/mg mitochondrial protein) was observed in the mitochondria of actively growing roots of 80–95-day-old plants, but it decreased ca. twofold by the end of the first year of plant development, before the roots were harvested for storage. The mitochondria isolated from roots stored at low temperature for two to three months and those after five to eight days of regrowth during the second year of plant development manifested an even lower NAD content (2.2 ± 0.4 and 2.0 ± 0.5 nmol/mg protein, respectively). A drastic decrease in the NAD content in mitochondria from stored roots did not result from the impairment of the inner membrane of these organelles and was evidently regulatory in its nature. The effect of developmental changes in the intramitochondrial NAD content on the malate oxidation pattern was studied. In the mitochondria of stored roots, the low NAD content limited the rate of malate oxidation in state 3, because the addition to the reaction mixture of exogenous NAD, which can be transported to the mitochondrial matrix, promoted malate oxidation by 30–50%. Rotenone inhibited malate oxidation in the stored-root mitochondria by more than 70%; in this case, the rate of rotenone-resistant malate oxidation in these organelles increased by several times in the presence of exogenous NAD. In the mitochondria of the growing root, exo-genous NAD did not affect the rate of malate oxidation, and rotenone inhibited it only by 25–35%. The analysis of the data obtained here and the published evidence suggests the existence of a universal mechanism of respiration control and the regulation of the functional activity of plant mitochondria. This mechanism acts through a change in the NAD content in the organelle matrix. This NAD can be used in the course of plant development, e.g., during the transition of sugar-beet-root cells in the dormant state, when the respiration rate must decline.

Effect of water deficit on respiration of conducting bundles in leaf petioles of sugar beet

Isolated fibrovascular bundles from source leaf petioles of sugar beet (Beta vulgaris L.) and hog-weed (Heracleum sosnovskyi L.) were used to study the influence of long-term drought on the oxygen uptake rate and activities of mitochondrial oxidases, i.e., cytochrome oxidase and salicylhydroxamic acid-sensitive alternative oxidase (AO). Under normal soil moisture content (70% of full water-retaining capacity, WRC), the oxygen uptake by sugar beet conducting bundles was characterized by a high rate (> 700 μl O2/(g fr wt h)) and by distinct cytochrome oxidase-dependent manner of terminal oxidation (up to 80% inhibition of respiration in the presence of 0.5 mM KCN). After long-term water deficit (40% of WRC), the bundle respiration proceeded at nearly the same rate but featured an elevated resistance to cyanide. At early drought stage (10 days), a decrease in the activity of cytochrome-mediated oxidation pathway was largely counterbalanced by activation of mitochondrial AO, whereas long-term dehydration of plants was accompanied by activation of additional oxidative systems insensitive to both KCN and SHAM. Similar but even more pronounced changes in activities of terminal oxidases were discovered in conducting bundles of wild-grown hogweed plants exposed to long-term natural drought. It is supposed that the suppression of cytochrome-mediated oxidation coupled with ATP synthesis in the cells of sugar beet source leaves impedes the translocation of assimilates and their accumulation in the taproot, which represents an important factor of drastic decrease in the yield of this agricultural crop under conditions of water deficit.

Effect of exogenous glucose on electron flow to photosystem I and respiration in cyanobacterial cells

The effects of exogenous glucose on the rates of alternative pathways of photosystem II (PSII)-independent electron flow to PSI and of dark respiration in Synechocystis sp. 6803 cells were studied. The presence of glucose was shown to accelerate the electron flow to P700+, the PSI primary electron donor oxidized with Far-red light (FRL), which excites specifically only PSI. An increase in the glucose concentration was accompanied by a further activation of electron flow to PSI, which was supported by the dark donation of reducing equivalents to the electron transport chain. An increase in the external glucose concentration resulted also in the disappearance of lag-phase in the kinetics of P700+ reduction, which was observed in the cells incubated without glucose after FRL switching off. A similarity of nonphotochemical processes of electron transfer to PSI in cyanobacteria and higher plants was supposed, basing on the earlier observed fact of the occurrence of such lagphase in higher plants and its dependence on the exhausting of stromal reductants in the light. Acceleration of dark electron flow to PSI in the presence of glucose, a major respiratory substrate, may indicate the coupling between nonphotochemical processes in the photosynthetic and respiratory chains of electron transport in cyanobacterial cells. A close correlation between photosynthesis and respiration in cyanobacterial cells is also confirmed by a sharp acceleration of respiration with an increase in the glucose concentration in medium.

Activities of antioxidant enzymes in mitochondria of growing and dormant sugar beet roots

In mitochondria isolated from growing (70–85 days) and dormant (stored for 8–12 weeks) sugar beet (Beta vulgaris L.) roots, activities of superoxide dismutase (SOD) and enzymes of the ascorbate-glutathione cycle were determined. The activity of SOD, the enzyme involved in superoxide detoxification, was much higher in mitochondria of the growing root, whereas activities of ascorbate peroxidase (APO) and glutathione reductase (GR), key enzymes of the ascorbate-glutathione cycle involved in the hydrogen peroxide degradation, increased substantially in mitochondria of dormant storage roots. Catalase (CAT) activity was detected in the fraction of root mitochondria purified in the sucrose density gradient, which activity was inhibited by cyanide by 85–90% and much weaker, by aminotriazol (by 30–35%). Submitochondrial localization of APO and CAT was analyzed using proteinase K. It was established that a substrate-binding APO center is localized on the external side of the inner membrane, whereas CAT is localized in the mitochondrial matrix. A possible role of mitochondria as ROS (hydrogen peroxide) acceptors in the cells of storage parenchyma of the stored root is discussed.

Run-on transcription as a method for the analysis of mitochondrial genome expression

The main stages of the run-on transcription method as applied to the quantification of mitochondrial gene transcription are described. As distinct from widely applied methods of northern hybridization and PCR after reverse transcription, this method permits studying the effects of exogenous and endogenous factors on the rate of mitochondrial gene transcription. The main method stages are (1) isolation of intact organelles; (2) preparation of nylon membranes with gene-specific fragments of DNA; (3) transcription in the lysates of these organelles in the presence of 32P-labeled RNA precursors; and (4) hybridization of newly synthesized labeled transcripts with DNA fragments of tested mitochondrial genes loaded on the nylon membrane.

Involvement of the energy-dissipating systems in modulating the energetic efficiency of respiration in mitochondria from etiolated winter wheat seedlings

Effects of cyanide-resistant alternative oxidase (AOX) and modulators of plant uncoupling mitochondrial proteins (PUMP) on respiration rate and generation of transmembrane electric potential (ΔΨ) were investigated during oxidation of various substrates by isolated mitochondria from etiolated coleoptiles of winter wheat (Triticum aestivum L.). Oxidative phosphorylation in wheat mitochondria during malate and succinate oxidation was quite effective (it was characterized by high respiratory control ratio as defined by Chance, high ADP/O ratio, and rapid ATP synthesis). Nevertheless, the effectiveness of oxidative phosphorylation was substantially modulated by operation of energy-dissipating systems. The application of safranin dye revealed the partial dissipation of ΔΨ during inhibition of cytochrome-mediated malate oxidation by cyanide and antimycin A and demonstrated the operation of AOX-dependent compensatory mechanism for ΔΨ generation. The complex I of mitochondrial electron transport chain was shown to play the dominant role in ΔΨ generation and ATP synthesis during AOX functioning upon inhibition of electron transport through the cytochrome pathway. Effects of linoleic acid (PUMP activator) at physiologically low concentrations (4–10 μM) on respiration and ΔΨ generation in mitochondria were examined. The uncoupling effect of linoleic acid was shown in activation of the State 4 respiration, as well as in ΔΨ dissipation; this effect was eliminated in the presence of BSA but was insensitive to purine nucleotides. The uncoupling effect of linoleic acid was accompanied by reversible inhibition of AOX activity. The results are discussed with regard to possible physiological role of mitochondrial energy-dissipating systems in regulation of energy transduction in plant cells under stress conditions.

Metabolic activity of plant mitochondria in hypertonic sucrose solutions

This study deals with effects of hypertonic sucrose solutions on respiration and oxidative phosphorylation of intact mitochondria isolated from sugar beet (Beta vulgaris L.) taproots and etiolated pea (Pisum sativum L.) seedlings. Mitochondria from plants, like those of animals, showed a trend to inhibition of oxidative phosphorylation in hypertonic sucrose solutions. The increase in sucrose concentration from 0.5 to 1.0 M suppressed malate oxidation in the presence of glutamate in state 3 by a factor of 2.5–3.5 and diminished the respiratory control ratio by a factor of 1.5–2.0. Plant mitochondria turned out remarkably resistant to osmotic stress; they retained significant respiratory control and high ADP/O ratios in a hypertonic 1 M sucrose solution. Although the origin of the observed phenomenon remains unresolved and warrants further studies, it is evident that elevated resistance of plant mitochondria to osmotic stress might be significant for energy supply under extreme environmental conditions (upon drought and salinity) when the plant organism experiences dehydration with a concomitant increase in the cytoplasmic osmolarity.