[Chemical signaling in plant microspore cells].

Chemical signal transduction from the cell surface to organelles was studied in unicellular vegetative (Equisetum arvense) and generative (Hippeastrum hybridum pollen) microspores of plants. Neurotransmitters acetylcholine, dopamine, and serotonin, their agonists and antagonists, Na+, K+, and Ca2+ channel blockers, as well as forskolin and theophylline (agents increasing the intracellular level of cyclic adenosine monophosphate) were used as chemical signals. Both types of microspores exposed to neurotransmitters, their agonists, forskolin, and theophylline demonstrated growth activation, while neurotransmitter antagonists and ion channel blockers inhibited this process. No stimulating effects of neurotransmitters were observed for cells pretreated with the antagonists and ion channel blockers. Pretreatment with ion channel blockers and then by anticontractile agents (cytochalasin B or colchicine) either had no effect or increased the inhibition of microspore growth. Pathways of chemical signal transduction from the cell surface to organelles are discussed.

[Contractile proteins in chemical signal transduction in plant microspores].

Involvement of contractile components in chemical signal transduction from the cell surface to the organelles was studied using unicellular systems. Neurotransmitters dopamine and serotonin as well as active forms of oxygen hydrogen peroxide and tert-butyl peroxide were used as chemical signals. Experiments were carried out on vegetative microspores of field horsetail Equisetum arvense and generative microspores (pollen) of amaryllis Hippeastrum hybridum treated with cytochalasin B (an inhibitor of actin polymerization in microfilaments), colchicine, and vinblastine (inhibitors of tubulin polymerization in microtubules). Both types of thus treated microspores demonstrated suppressed development, particularly, for cytochalasin B treatment. At the same time, an increased typical blue fluorescence of certain cell regions (along the cell wall and around nuclei and chloroplasts) where the corresponding contractile proteins could reside was observed. In contrast to anticontractile agents, dopamine, serotonin B, and the peroxides stimulated microspore germination. Microspore pretreatment with cytochalasin B and colchicine followed by the treatment with serotonin, dopamine, or the peroxides decreased the germination rate. Involvement of actin and tubulin in chemical signal transduction from the cell surface to the nucleus is proposed.

[Reactive oxygen forms and luminescence of intact microspore cells]. / Aktivnye formy kisloroda i liuminestsentsiia intaktnykh kletok mikrospor.

The participation of reactive oxygen species (ROS) in luminescence (chemiluminescence and autofluorescence induced by ultraviolet light of 360-380 nm) was analyzed. Microspores, the pollen (male gametophyte) of Hippeastrum hybridum, Philadelphus grandiflorus, and Betula verrucosa and vegetative microspores of the spore-breeding plant Equisetum arvense served as models. It was found that the addition of the chemiluminescent probe lucigenin, which luminesces in the presence of superoxide anionradicals, leads to intensive chemiluminescence of microspores. No emission was observed in the absence of lucigenin and in the presence of the dye luminol as a chemiluminescent probe. The emission decreased significantly if superoxide dismutase, an enzyme of the superoxide anionradical dismutation during which this radical disappeared, was added before the dye addition. The autofluorescence intensity of microspores decreased in the presence of both superoxide dismutase and peroxidase, an enzyme destroying hydrogen peroxide and organic peroxides. The most significant effect was noted after the addition of peroxidase, which indicates a greater contribution of peroxides to this type of emission. The fumigation with ozone, which increases the amount of ROS on the cell surface, enhanced the intensity of the chemiluminescence of microspores with lucigenin, but decreased the intensity of the autofluorescence of microspores. Exogenous peroxides (hydrogen peroxide and tert-butylhydroperoxide) stimulated the autofluorescence of pollen and vegetative spores in a concentration-dependent manner. It was shown that the formation of ROS contributes to the luminescence of plant microspores, which reflects their functional state.

[Investigation of the pollen chromatin state of Hippeastrum hibridum at the early stages of development with fluorescent dye Hoechst 33342]. / Issledovanie sostoianiia khromatina pyl'tsevogo zerna Hippeastrum hibridum na nachal'nykh stadiiakh razvitiia s pomoshch'iu fluorestsentnogo krasitelia Khekhsta 33342.

The work was carried out on the pollen of Hippeastrum hibridum, whose size (ca 55 mcm) permits to follow both the pollen tube formation and development of the vegetative nucleus and generative cell within the first two hours before the growing point arising. Using fluorescent dye Hoechst 33342 chromatin state alterations accompanying changes in the pollen physiological state were investigated. The maximum fluorescent intensity was observed in 30 min of staining and reflected the maximum chromatin functional activity. Histograms of pollen distribution according to fluorescent intensity differ considerably with doses of irradiation (500, 1000, 2000, 3000 and 4000 Gy). Besides, a germinative index of fluorescence intensity was calculated. A comparative analysis of these data has shown that the lowest decrease in fluorescent intensity observed at 500, 3000 and 4000 Gy, was accompanied by different germinative indices. At 500 Gy, the index was 2.5 times lower than in the control, but at 4000 G no germination was not noted. The process of pollen grain development is supposed to be more intensive and faster at 500 Gy, than in the control. At 4000 Gy, a decrease in the functional pollen activity is accompanied by decrease or inhibition of chromatin functional activity.

[Autofluorescence of intact Equisetum arvense L. spores during their development]. / Avtofluorestsentsiia intaktnykh spor khvoshcha Equisetum arvense L. v protsesse razvitiia.

The autofluorescence of horsetail Equisetum arvense spores excited with UV-light of 360-380 nm was studied by microspectrofluorimetry during their development from an individual cell to the formation of a multicellular thallus with the generative organs. The investigation involved the registration of the fluorescence spectra of individual intact developing cells and the measurement of the ratio of cell fluorescence intensities in the blue and red regions of the spectrum. Dry blue-fluorescing microspores showed the maxima at 460 and 530 nm and a small maximum at 680 nm. Thirty minutes after moistening in water, red-fluorescing cells arose among blue-fluorescing microspores, indicating the onset of development. Red fluorescence with a maximum at 680 nm enhanced as cells put off their cover, which brightly fluoresced in the blue region of the spectrum with the main maximum at 460 nm. By estimating the ratio of autofluorescence intensities in the blue region of the spectrum to red lightening of microspores at the first stages of development up to 24 h (in particular, their first division, the formation of nonfluorescencing rhizoid, etc.), nonviable (only blue-lightening) cells were distinguished from viable cells, in which red fluorescence began to prevail. After 25-40 days of development, the gametophyte fluorescing mainly at 680 nm formed male organs, antheridia, with blue-green-fluorescing spermatozoids. Then female generative organs archegonia with the egg cell appeared, which fluoresced blue, whereas the surrounding cells fluoresced red. It was supposed that the lightening in the blue and green regions of the spectrum is due to the presence of phenols, terpenoids, and azulenes, whereas the emission in the red region is associated with the presence of chlorophyll and azulenes. The observation of autofluorescence makes it possible to easily distinguish generative cells without additional staining.

[Functions of neuromediators in plants]. / Funktsii neiromediatornykh veshchestv u rastenii.

In plants, neurotransmitters play a major biological role in chemo-signaling, as regulating agents of growth and development, membrane permeability, etc. In the plant cells, there are also main elements of cholinergic and aminergic systems including the enzymes as well as functional analogues of cholino- and aminoreceptors. It is important that the systems should be taken into consideration in fertilizing and chemical interaction of the plant cells. Acetylcholine and cholinesterase were shown to be secreted in male sexual cells (the pollen). Depression of cholinesterase on the gene level correlates with male sterility. Preliminary treatment of a female gametophyte with antagonists of acetylcholine and histamine, prior to pollination, blocked the normal process of fertilizing. Cholinesterase was also found in secrets released from the pollen surface and pistil.

[An enzyme from the fungus Aspergillus niger that hydrolyzes choline ethers]. / Ferment iz griba Aspergillus niger, gidrolizuiushchii kholinovye éfiry.

The acetylthiocholine-hydrolyzing enzymatic activity inhibited by the neostigmine and partly physostigmine has been found in extracts from mycelium of fungus Aspergillus niger. The enzyme has been isolated and 15-20 fold purified. The cholinesterase activity of the protein (Kmu 7.10-7 M) is comparable with known for analogous enzymes from higher plants, for its inhibition high concentrations of substrate (greater than 10-3M) are required. The enzyme hydrolyzes acetylthiocholine with rate approximately 1.5 times higher than butyrylthiocholine. Molecular mass of native protein is approximately 600 kDa, subunits -63 and 44 kDa.

[Photochemical activity of pea mutants with damaged photosystems]. / Issledovanie fotokhimicheskoi aktivnosti mutantov gorokha s narusheniem fotosistem.

The interrelationship between biochemical photosynthetic reactions and mutations was investigated, using five nuclear recessive lethal pea mutants with damaged photosystems II (1, 19) and I (5, 21, 22). Based on the data from photoreduction of NADP+, light-induced redox conversions of exogenous and endogenous plastocyanines in isolated chloroplasts, light-induced redox conversions of cytochrome f and absorbance changes at 520 nm in chloroplasts and leaves, the possible existence of alternative pathways of electron transfer and the pleiotropic effects of mutations, are discussed. A correlation between the structural damages of chloroplasts and the photochemical activity of mutants was found. The existence of cyclic electron transport around photosystem I involving cytochrome f and plastocyanine in mutants 1 and 19 and of an electron transfer pathway from H2O to NADP+ without plastocyanine in mutant 22 is postulated.

[Membrane-active inhibitors of electron transport in chloroplasts]. / Membranoaktivnye ingibitory élektronnogo transporta v khloroplastakh.

The effects of polyene antibiotic amphotericin B and the organophosphorus insecticide chlorophos (trichlorofon) on the electron transport in isolated pea chloroplasts were compared; both compounds were found to inhibit the electron transport. The concentration dependence of the inhibitory effects on the photoreactions in chloroplasts was established. Affer washing of the chloroplasts from amphotericin B admixtures the photoreduction of NADP+ was decreased by 50%, the photooxidation of cytochrome f in situ was affected comparatively little, while the photoreduction of plastocyanin in situ was decreased by 60%. The supernatant after amphotericin B washing contained plastocyanin. An addition of chlorophos to the medium caused a 70% inhibition of NADP+ photoreduction and a 40% inhibition of cytochrome f photooxidation and of plastocyanin photoreduction. It was assumed that the active site of amphotericin B lies in the plastocyanin region, that of chlorophos--at the site of NADP+ photoreduction in photosystem I. Experiments with isolated pea plastocyanin and ferredoxin and cytochrome C553 of Chlorella demonstrated that neither of the inhibitors interacts with the prosthetic groups or with he protein part of these electron carriers. Presumably the mechanism of action of amphotericin B and chlorophos on the photosynthetic electron transport consists in a destruction of chloroplast membrane structures. Using membrane-active inhibitors, the alternative pathways of electron transfer through cytochrome f and plastocyanin were established.

[The defect in cytochrome c553 in non-photosynthetic mutant cells of Chlamydomonas reinhardii]. / O povrezhdenii tsitokhroma c553 v kletkakh nefotosinteziruiushchikh mutantov Chlamydotonan reinhardii.

The light electron transport in non-photosynthetic mutants of Chlamydomonas reinhardii A-36 and A-68 with a decreased content of cytochrome c553 was studied. The light-induced changes of absorption at 520, 559, 563 nm were shown to occur in the films of intact cells and in cell fragments. However, no maximum of the "light minus dark" difference absorption spectra of mutants belonging to cytochrome c553 was revealed. The electron transport from water to NADP+ or ferricyanide in the mutants was completely blocked; however, it was partly restored by an addition of exogenous cytochrome c553 to the cell fragments. A conclusion on the genetic damage of cytochrome c553 in mutants A-36 and A-68 was made. The localization of the cytochrome in the electron transport chain between the photosystems was demonstrated.

[Determination of cytochromes in chloroplasts of Chlamydomonas reinhardii mutants]. / Opredelenie tsitokhromov v khloroplastakh mutantov Chlamydomonas reinhardii.

A method for qualitative determination of cytochromes of the wild-type and mutant strains of Chalmydomonas reinhardii was developed. The effect of different techniques of cell disruption (ultrasound, Triton X-100, acetone, etc) on detection of cytochrome maxima in the oxidized minus reduced difference spectra was studied on a comparative basis. The development of the stable difference spectrum of the disrupted cell suspension was shown to be a function of incubation time in the presence of an oxidizing or a reducing agent. Cytochromes of the wild-type and 10 nonphotosynthesizing mutants were determined. Four mutants with lesions in cytochromes B559 and C553 were detected. Mutants with lesions in the reaction center of photosystem 2 were found to have a substantially reduced content of cytochrome B559, whereas those with normal photosystems--of cytochrome C553.

[Effect of dibromothymoquinone on photosynthetic electron transport]. / O deistvii dibromtimokhinona na fotosinteticheskii elektronnyi transport.

The effect of dibromothymoquinone on photosynthetic electron transport in pea dependent on concentration was studied. Dibromothymoquinone inhibited general electron transport from water to NADP+ in isolated chloroplasts and ethiochloroplasts and the electron transfer via plastoquinone and cytochrome f in the leaves and isolated plastids. At all concentrations studied dibromothymoquinone significantly affected the absorption changes at 590 nm in the ethiochloroplasts associated with plastocyanine photoreactions. Possible location of electron carriers in the photosynthetic electron transport chain is discussed.

[Effect of phloridzin on photosynthetic electron transport]. / Deistvie floridzina na fotosinteticheskii elektronnyi transport.

Phloridzin (2',4',6',4-tetraoxyhydrochalcon-2'-glucoside) was used to study the localization of synthesis of ATP in the electrontransporting chain of photosynthesis. It was shown that phloridzin inhibits the rate of photoreduction of NADP+ by isolated pea chloroplasts by 40%, electron transport via cytochrome f by 100% and via plastocyanin--by 50%. The "crossover" experiments demonstrated that phloridzin inhibits ADP-induced photoreduction of cytochrome f, having no effect on plastocyanin under identical conditions. It is assumed that the site of ATP synthesis is localized on the reduced site of cytochrome f, while the carrier itself is located in the electron transporting chain coupled to phosphorylation. It is possible that only part of the plastocyanin molecules are located in the phosphorylating pathway of electron transport.

[Photosynthetic electron transfer at the level of cytochrome f and plastocyanin]. / Fotosinteticheskii perenos elektronov na urovne tsitokhromaF i plastotsianina.

Light-induced redox conversions of cytochrome f and plastocyanin in situ and electron transporst from H2O to NADP+ were studied by a dual-wave differential spectrophotometry under identical conditions and subsequently compared. The analysis in red and far red light, treatment by inhibitors, e. g. diurone and dibromothymoquinone, and the analysis of photoreactions during the greening of etiolated seedlings demonstrated that cytochrome f functions only in the non-cyclic chain of electron transport, whereas plastocyanin--both in the non-cyclic and in the cyclic electron transport chains. The jositions of cytochrome f and plastocyanin in various electron-transport chains are proposed.

[Light-induced absorption changes at 590 nm in isolated pea chloroplasts and their relation to plastocyanin]. / Svetoindutsiruemye izmeneniia absorbtsii pri 590 nm v izolirovannykh gorokha i ikh sviaz' s plastotsianinom.

The light-induced decrease in absorption with the minimum at 590-595 nm has been found in chloroplasts of etiolated pea seedlings by the method of dual-wavelength difference spectrophotometry. It has been shown that this effect is caused by photoreduction of the electron carrier with the absorption maximum of its oxidized form at 590 nm. Photoreduction of the carrier has been observed after excitation both by the short-wave (646 nm) and long-wave (709 nm) red light, although the latter is less effective. It has been suggested that the absorption changes at 590 nm are caused by light-induced redox conversions of plastocyanin bound to chloroplast membrane.