We studied the activity and direction of partially purified sucrose synthase from taproots of different types of beet (sugar beet. fodder beet, mangolds. hybrids, and the wild species Beta trigyna W. et K.), The enzyme in different objects is shown to differ in the level of specific activity and direction of the reaction catalyzed. The course of the reaction in the direction of synthesis or splitting of sucrose changed during ontogenesis with no strict varietal specificity. The method of double immunodiffusion in agarose was used to conduct an immunochemical comparison of sucrose synthase from sugar beet taproots with the enzyme from taproots of beets with different sugar content and from starch-storing plants. No immunochemical differences were detected in sucrose synthase from the taproots of different types of beet. On the other hand, sucrose synthase isolated from plants that accumulate starch (potato tubers, com seeds) or inulin (Jerusalem artichoke tubers) as storage products was only partially identical in the immunochemical sense to the enzyme from sugar beet taproots.
We used the amperometric method to study the rate of photosynthetic O2 evolution by suspensions of freshly isolated protoplasts from pea leaves at saturating (0.1 mM) and superoptimal (0.6 mM) concentrations of CO2 in the medium. The dependency of pbotosynthesis on acidity of the medium was studied under conditions where constant CO2 concentration in the solution was maintained by varying the concentration of added KHCO3. The maximum photosynthetic rate was observed at pH values of 6.0 to 8.0. The extent of inhibition of photosynthesis by superoptimal concentrations of CO2 likewise did not depend on pH values within these limits. The inhibitor of carbonic anhydrase acetazolamide removed inhibition of photosynthesis by CO2 excess at pH 7.2, but did not act at pH 6.5 and 8.0. Addition of acetazolamide did not affect inhibition of pbotosynthesis by formiate. Undamaged protoplasts manifested carbonic anhydrase activity ranging from 3 to 11% of total activity of the protoplast lyzate obtained by lowering osmotic pressure of the medium. Successive washings of the carbonic anhydrase of undamaged protoplasts to constant activity showed that the enzyme is located in the plasmalemma of protoplasts.
In compiling genome libraries of different organisms (prokaryotes, algae, and higher plants), we compared DNA cloning efficiency in two types of vector systems-lambdaL47 and a cosmid of original construction. Universality is demonstrated for the new cosmid vector, which can be used to create a library of the "difficultly cloning" wheat genome. The obtained cosmid libraries of wheat and the alga Dunaliella salina Theod. were screened, size of the inserts of cloned DNA was determined, and clones were selected that hybridize with a fragment of the gene of the small subunit of pea RuBPCase and a spinach chloroplast gene.
Manifested in suppression of variable chlorophyll fluorescence (F(v)), inactivation of PSII during heterotrophic growth of the unicellular green alga Chlorella vulgaris Bejer. on glucose is strengthened during inhibition of glycolysis by iodoacetate, during blocking of the oxidative metabolism of glycolytic products under anaerobic conditions, and with decline in energy requirements of the cell under conditions of nitrogen deficit. Recovery of F(v) in heterotrophically growing culture occurs in the presence of dinitrophenol, which accelerates oxidation of glucose metabolites in the mitochondria, as well as after addition of exogenous ATP or during illumination of the cells. It is concluded that inactivation of PSII in Chlorella under conditions of heterotrophic growth and in autotrophic culture during nitrogen deficit is initiated by accumulation of metabolites general to glycolysis and the Calvin cycle.
We investigated the role of zinc, sulfhydryl groups, and other groups in functioning of carbonic anhydrase in leaves of chick-pea (Cicer arietinum L.). It is demonstrated that there are 64 sulfhydryl groups and no S-S bonds in the molecule of the enzyme. The enzyme possesses both accessible sulfhydryl groups and ones that are screened and submerged in the molecule. The obtained data indicate that at least one sulfhydryl group of each monomer enters the active site of the enzyme and takes part in manifestation of catalytic activity. In addition to this, sulfhydryl groups play a significant role in maintaining the native structure of this enzyme. The region of zinc binding is in a poorly accessible region of the molecule of plant carbonic anhydrase and is firmly bound to the apoenzyme. Histidine is involved in binding of zinc with the apoenzyme. The presence of zinc in the molecule is a necessary condition for manifestation of catalytic activity. Irreversible conformational changes in the molecule leading to inactivation of the enzyme occur when zinc is removed.
Peribacteroid membrane (PBM) vesicles isolated from root nodules of yellow lupine exhibit Mg2+-dependent H+-ATPase activity with a pH optimum of 5.4-5.7, as was demonstrated spectrophotometrically using the pH probe acridine orange. Proton transport and ATP hydrolysis were simultaneously inhibited by orthovanadate and nitrate at optimal pH values, which suggests the presence of two H+-ATPases -of the plasmalemma and the tonoplast types-on the PBM. In addition to this, activity of Mg2+,Ca2+-ATPase with broad substrate specificity and a pH optimum of 6.2-6.4 was clarified on PBM vesicles. The Mg2+-dependent ATPase activity was more sensitive to molybdate, nitrate, and vanadate, whereas Ca2+-dependent activity was more sensitive to fluoride and Cu2+ ions. The rate of CaATP2- was 1.5 times higher than that of MgATp2- hydrolysis, the apparent K(m) constituting 0.12 and 0.22 mM, respectively. The obtained data are discussed in the light of current ideas about heterogeneity of ATPases and genesis of the PBM.
The mathematical model of photosynthesis previously proposed by A. E. Fridlyand [8] is analyzed in the present paper. It is demonstrated that this model ignores certain characteristics of the photosynthesis of C4 plants. A different equation is suggested to describe carboxylation efficiency in C4 plants. It is also demonstrated that C3 plants with a CO2-concentrating mechanism can be discovered experimentally, and that they do not differ fundamentally from C3 plants without such a mechanism. However, all C3 plants differ fundamentally from C4 Plants in that the latter are characterized by minimal CO2 conductance from sheath cell to mesophyll cell.
We studied the relationship between resistance (cryo- and osmo-) and structurally damaging plasmalemma injuries in cells of three strains of Dioscorea deltoidea Wall. (IFR D-1, IFR DM-0.5, and IFR DM-8). The decisive role in plasmalemma injuries during freezing of Dioscorea cells is played by the osmotic stress associated with their severe dehydration. Plasmalemma injuries increase linearly in cells of all strains with reduction of temperature to -30-degrees-C (IFR DM-0.5 and IFR DM-8) and -40-degrees (IFR D-1), and with increase in osmolality of solutions of osmotic agents to 16.12. At lower temperatures, a pool of cells resistant to ultralow temperatures remains in heterogeneous cell populations, the number of such cells being two to three times greater in the mutant strains DM-0.5 and DM-8 than in strain D-1. The given difference is possibly associated with differences in the osmotic potential and structure of the plasmalemma in the studied cultures. Five types of osmotic injuries to the plasmalemma are presumed to exist in the investigated strains of Dioscorea in the presence of dimethyl sulfoxide (a cryoprotector).
Electron-microscopic morphometric studies of effective and ineffective nodules of yellow lupine (Lupinus luteus L.) and common sainfoin (Onobrychis sativa Lam.) were carried out to clarify new criteria of effectiveness of the nitrogen-fixing symbiotic system in root nodules of legumes. Optimal ratios are determined for values of the main compartments of the infected cell (bacteroids, PBS, and plant cell cytosol and cellular organelles) in the process of ontogenesis under conditions of effective symbiosis, and changes of these ratios are ascertained in ineffective nodules. Both during senescence of the cell and in ineffective symbiosis, the share of the PBS and bacteroids increases due to shrinkage of the plant cell cytosol and cellular organelles, which indicates sharp contraction of space occupied by the eukaryote in the infected cell. This is accompanied by decrease in overall metabolic activity of the cell and decline in the rate of utilization of photoassimilates, a situation that leads to accumulation of storage substances in bacteroids, the PBS, and plastids. Decline in content of bacteroid-containing tissue in nodules was also observed. Clarified on the basis of mathematical analysis, regularities governing internal organization of the nodule during its ontogenesis in effective and ineffective symbiosis made it possible to propose new criteria for estimating the effectiveness of symbiosis.
We studied the effect of the herbicide glyphosate on the hormonal system of com seedlings (Zea mays L.). The level of free and bound auxins (IAA) in seedlings and total content of IAA in the endosperm decline under the influence of glyphosate. Glyphosate also caused a decline in the level of endogenous ABA. Changes in the level of phytohormones in the presence of the herbicide occurred on a background of inhibition of the growth of plants treated with glyphosate.
We studied the intracellular distribution of metabolites and transport of radioactive photosynthetic products into the central vacuole of isolated protoplasts of sugar beet (Beta vulgaris L.) mesophyll. It is demonstrated that C-14-sucrose enters the central vacuole during the first minutes of photosynthesis, and more than 50% of assimilates formed de novo accumulates in it over the course of the next 40 min. Dominant in the composition of radioactive photosynthetic products accumulated by the vacuole are C-14-sugars, malate, and alanine, which account for 70, 12, and 10% of radioactivity, respectively. Entry of phosphorylated compounds into the vacuole was not detected. A significant portion of sucrose transported into the vacuole undergoes hydrolysis to monosaccharides. The equilibrium relationship of the starting disaccharide and products of its hydrolysis corresponds to 10 and 90% of the total mass of sugars of the vacuolar pool. The central vacuole houses 90% of acid invertase activity of the protoplast. The vacuolar pool is shown to be significantly dominant over the cytoplasmic pool in regard to monosaccharides, malate, alanine, and valine, while sucrose is evenly distributed between these two pools. It is hypothesized that deposition of sucrose and other photosynthetic products in the central vacuole of the protoplast prevents the possibility of osmotic damage to the cytoplasmic compartment under conditions of excessive accumulation of assimilates and promotes maintenance of the level of metabolism with decline of carbon nutrition.
We used the method of mathematical experiment planning (a 2(3) scheme) to study the influence of environmental factors separately or in combination on the photosynthetic rate and distribution of C-14 among products of photosynthetic carbon metabolism in the cotton (Gossypium hirsutum L.) leaf Increase of light intensity during cultivation accelerated photosynthesis and stimulated incorporation of C-14 into phosphoglyceric acid (PGA), sugar diphosphate (SDP), fructose monophosphate (FMP), and malate, but suppressed incorporation of C-14 into sucrose, glucose monophosphate (GMP), and glycerate. Temperature increase by itself and in any combination with other factors at the upper level suppressed photosynthesis. Elevated temperature increased accumulation of the label in PGA, sucrose, and malate, but lowered it in GMP, alanine, glycine, and serine. Growing plants at enhanced CO2 concentration led to acceleration of photosynthesis and increase of the share of C-14 in SDP, GMP, and malate, but decrease of it in sucrose, alanine, glycine, and serine. Very perceptible effects of interaction are discernible in different combinations of factors. All three factors at the upper level appreciably induced activity of phosphoenolpyruvate carboxylase (PEPCase) in cotton leaves.
Heating strawberry plants of the Alexandria cultivar at 42-degrees-C for a period of 3 h leads to increase of the photosynthetic rate, raises the primary thermostability of photosynthesis, and stimulates the ability to repair heat injury caused by heating at 49-degrees for 30 min. Following return of the plants to normal temperature conditions (18/14-degrees, day/night), the primary thermostability of photosynthesis and photosynthetic rate declined to the starting level in approximately 12 days. However, elevated reparability was preserved throughout this period. After 3-h heating at 42-degrees, heat shock granules (HSG) were detected in the cytoplasm, chloroplasts, and mitochondria of mesophyll cells, and perichromatin granules were detected in the nucleoplasm. Heat shock granules were lacking in the cells at 24 h after hardening heating, when thermostability was highest. Stronger beating (49-degrees for a period of 30 min) caused appearance of HSG only in solitary cells.
We conducted a histological and electron microscope study on callus cultures of Tatar [Fagopyrum tataricum (L.) Gaertn.] and common (F esculentum Moench) buckwheat that in the course of prolonged cultivation (for more than 1.5 years) preserve definite morphology and regenerative capacity (ability to form roots, buds, and embryoids). Tissue specificity and different degrees of differentiation are clarified in calluses of wild and common buckwheat. It is demonstrated that callus cultures of Tatar buckwheat with thickenings or F-type calluses are complex systems composed of soft callus tissue, thickenings, and embryoids at different stages of development. Regeneration of proembryogenic cellular complexes (PECC) in such cultures is realized on a callus-producing medium as a result of several renewal cycles. A medium lacking 2,4-D is required for induction of the proembryogenic state and subsequent embryoid formation in densely globular calluses of common buckwheat or D-type calluses. It is established that regeneration of plants is possible through organogenesis or embryoidogenesis in tissue culture of common buckwheat, but through embryoidogenesis or unfinished embryoidogenesis in calluses of Tatar buckwheat.
We studied the qualitative and quantitative composition, accumulation, and location of steroid glycosides in four strains of cell culture of Dioscorea deltoidea Wall. Reardless of activity of endogenous beta-glucosidases, all strains are characterized by the presence of steroid glycosides only in the furostanol form. The qualitative composition of steroid glycosides is the same for all four strains and is represented by four compounds: deltoside, protodioscin (aglycone diosgeinin), and their 25S analogs (aglycone yamogenin). The strains differ significantly in quantitative content of saponins, strain DM-0.5 being a superproducer in regard to these compounds (up to 12% of cell dry mass). The content of steroid glycosides is analyzed in cells of strain DM-0.5 and protoplasts obtained from it. These compounds are shown to be located in protoplasts and the free space of cells. their quantitative distribution varying with culture age.
