Regulatory Peptides in Plants.

Many different peptides regulating cell differentiation, growth, and development are found in plants. Peptides participate in regulation of plant ontogenesis starting from pollination, pollen tube growth, and the very early stages of embryogenesis, including formation of embryo and endosperm. They direct differentiation of meristematic stem cells, formation of tissues and individual organs, take part in regulation of aging, fruit maturation, and abscission of plant parts associated with apoptosis. Biological activity of peptides is observed at very low concentrations, and it has mainly signal nature and hormonal character. "Mature" peptides appear mainly due to processing of protein precursors with (or without) additional enzymatic modifications. Plant peptides differ in origin, structure, and functional properties. Their specific action is due to binding with respective receptors and interactions with various proteins and other factors. Peptides can also regulate physiological functions by direct peptide-protein interactions. Peptide action is coordinated with the action of known phytohormones (auxins, cytokinins, and others); thus, peptides control phytohormonal signal pathways.

Endonucleases and apoptosis in animals.

Endonucleases are the main instruments of obligatory DNA degradation in apoptosis. Many endonucleases have marked processive action; initially they split DNA in chromatin into very large domains, and then they perform in it internucleosomal fragmentation of DNA followed by its hydrolysis to small fragments (oligonucleotides). During apoptosis, DNA of chromatin is attacked by many nucleases that are different in activity, specificity, and order of action. The activity of every endonuclease is regulated in the cell through its own regulatory mechanism (metal ions and other effectors, possibly also S-adenosylmethionine). Apoptosis is impossible without endonucleases as far as it leads to accumulation of unnecessary (defective) DNA, disorders in cell differentiation, embryogenesis, the organism's development, and is accompanied by various severe diseases. The interpretation of the structure and functions of endonucleases and of the nature and action of their modulating effectors is important not only for elucidation of mechanisms of apoptosis, but also for regulation and control of programmed cell death, cell differentiation, and development of organisms.

Apoptosis in wheat seedlings grown under normal daylight.

Apoptosis was observed in the coleoptile and initial leaf in 5-8-day-old wheat seedlings grown under normal daylight. Apoptosis is an obligatory event in early wheat plant ontogenesis, and it is characterized by cytoplasmic structural reorganization and fragmentation, in particular, with the appearance in vacuoles of specific vesicles containing intact organelles, chromatin condensation and margination in the nucleus, and internucleosomal fragmentation of nuclear DNA. The earliest signs of programmed cell death (PCD) were observed in the cytoplasm, but the elements of apoptotic degradation in the nucleus appeared later. Nuclear DNA fragmentation was detected after chromatin condensation and the appearance in vacuoles of specific vesicles containing mitochondria. Two PCD varieties were observed in the initial leaf of 5-day-old seedlings grown under normal daylight: a proper apoptosis and vacuolar collapse. On the contrary, PCD in coleoptiles under various growing (light) conditions and in the initial leaf of etiolated seedlings is only a classical plant apoptosis. Therefore, various tissue-specific and light-dependent PCD forms do exist in plants. Amounts of O2*- and H2O2 evolved by seedlings grown under normal daylight are less than that evolved by etiolated seedlings. The amount of H2O2 formed in the presence of sodium salicylate or azide by seedlings grown under normal daylight was increased. Contrary to etiolated seedlings, the antioxidant BHT (ionol) did not inhibit O2*- formation and apoptosis and it had no influence on ontogenesis in the seedlings grown under normal daylight. Thus, in plants grown under the normal light regime the powerful system controlling the balance between formation and inactivation of reactive oxygen species (ROS) does exist and it effectively functions. This system is responsible for maintenance of cell homeostasis, and it regulates the crucial ROS level controlling plant growth and development. In etiolated plants, this system seems to be absent, or it is much less effective.

Apoptosis in plants: specific features of plant apoptotic cells and effect of various factors and agents.

Apoptosis is an integral part of plant ontogenesis; it is controlled by cellular oxidative status, phytohormones, and DNA methylation. In wheat plants apoptosis appears at early stages of development in coleoptile and initial leaf of 5- to 6-day-old seedlings. Distinct ultrastructural features of apoptosis observed are (1). compaction and vacuolization of cytoplasm in the apoptotic cell, (2). specific fragmentation of cytoplasm and appearance in the vacuole of unique single-membrane vesicles containing active organelles, (3). cessation of nuclear DNA synthesis, (4). condensation and margination of chromatin in the nucleus, (5). internucleosomal fragmentation of nuclear DNA, and (6). intensive synthesis of mitochondrial DNA in vacuolar vesicles. Peroxides, abscisic acid, ethylene releaser ethrel, and DNA methylation inhibitor 5-azacytidine induce and stimulate apoptosis. Modulation of the reactive oxygen species (ROS) level in seedling by antioxidants and peroxides results in tissue-specific changes in the target date for the appearance and the intensity of apoptosis. Antioxidant butylated hydroxytoluene (BHT) reduces the amount of ROS and prevents apoptosis in etiolated seedlings, prolongs coleoptile life span, and prevents the appearance of all apoptotic features mentioned. Besides, BHT induces large structural changes in the organization of all cellular organelles and the formation of new unusual membrane structures in the cytoplasm. BHT distorts mitosis and this results in the appearance of multiblade polyploid nuclei and multinuclear cells. In roots of etiolated wheat seedlings, BHT induces differentiation of plastids with the formation of chloro(chromo)plasts. Therefore, ROS controlled by BHT seems to regulate mitosis, trigger apoptosis, and control plastid differentiation and the organization of various cellular structures formed by endocytoplasmic reticulum.

[Effect of the antioxidant ionol on proteolytic apparatus of aging coleoptiles of wheat seedlings grown in light]. / Vliianie antioksidanta ionola na proteolitcheskii apparat stareiushchikh koleoptilei vyrashchivaemykh na svetu prorostkov pshenitsy.

The dynamics of changes in total proteolytic activity and activities of various groups of proteases in the coleoptiles of 3- to 12-day-old wheat seedlings grown in light with and without antioxidant BHT (2,6-di-tert-butyl-4-methylphenol) was studied. It was established that the specialized proteases that easily hydrolyze specific synthetic substrates and the enzymes actively hydrolyzing histone H1 dominate in young coleoptiles of 3- to 4-day-old seedlings. Proteases that degrade equally well the majority of the studied substrates are accumulated in the cells of old coleoptiles of 11- to 12-day-old seedlings. Under the effect of BHT, the plants grown in light (in comparison with etiolated seedlings) demonstrated a somewhat changed dynamics of proteolytic activity in young coleoptiles and the disappearance of proteases active toward histone H1. An inhibitory analysis revealed a relative domination of cysteine proteases in young coleoptiles at the initial development stage of seedlings, whereas the fraction of serine proteases markedly increased in old coleoptiles. We presume that the revealed quantitative and qualitative changes in the proteolytic apparatus of the coleoptile cells induced by BHT may be largely responsible for the retardant and geroprotective effect of this antioxidant in plants.

Effect of ethylene producer ethrel and antioxidant ionol (BHT) on the proteolytic apparatus in coleoptiles of wheat seedlings during apoptosis.

It was established that total proteolytic activity in etiolated wheat seedlings changes in ontogenesis in cycles: peaks of proteolytic activity correspond to the 3rd, 5th, and 8th days of seedling growth, respectively. The maximum of proteolytic activity preceded the maximum of nuclease activity, which may be due to activation of nucleases by proteolytic enzymes. According to inhibitory analysis the cysteine and serine proteases play the main role in apoptosis in wheat coleoptiles. Growing of seedlings in the presence of ethrel stimulated apoptosis in the coleoptile, and it increased (almost 6-fold) the proteolytic activity in its cells. On the other hand, the antioxidant ionol (BHT) suppressed the induction of proteases, particularly at the second stage of coleoptile development, and it slowed down the increase in the nuclease activity after 6th day of the seedling life. It is suggested that phytohormones and antioxidants participate in regulation of apoptosis in the ageing coleoptile, directly or indirectly effecting the proteolytic apparatus in the coleoptile cells.

Apoptosis in the initial leaf of etiolated wheat seedlings: influence of the antioxidant ionol (BHT) and peroxides.

Apoptosis was observed in the initial leaf of 5-8-day-old etiolated wheat seedlings. A condensation of cytoplasm in apoptotic cells, formation of myelin-like structures, specific fragmentation of cytoplasm, appearance in vacuoles of specific vesicles containing subcellular organelles, condensation and margination of chromatin in the nucleus, and internucleosomal fragmentation of nuclear DNA are ultrastructural features of apoptosis in the initial wheat leaf. Single-membrane vesicles detected in vacuoles of the leaf cells resemble in appearance the vacuolar vesicles in the coleoptile apoptotic cells described earlier (Bakeeva, L. E., et al. (1999) FEBS Lett., 457, 122-125); they contain preferentially plastids but not mitochondria as was observed in coleoptile. The vacuolar vesicles are specific for the apoptotic plant cells. Thus, apoptosis in various tissues is an obligatory element of plant (wheat) growth and development even in the early stages of ontogenesis. Contrary to strong geroprotecting action in coleoptile, the known antioxidant BHT (ionol, 2.27 x 10(-4) M) does not prevent in the leaf cells the apoptotic internucleosomal DNA fragmentation and appearance of specific vacuolar vesicles containing subcellular organelles. Therefore, the antioxidant action on apoptosis in plants is tissue specific. Peroxides (H2O2, cumene hydroperoxide) stimulated apoptosis (internucleosomal DNA fragmentation) in coleoptile and induced it in an initial leaf when apoptosis in a control seedling leaf was not yet detected. Thus, apoptosis that is programmed in plant ontogenesis and controlled by reactive oxygen species (ROS) can be modulated by anti- and prooxidants.

Effect of the antioxidant ionol (BHT) on growth and development of etiolated wheat seedlings: control of apoptosis, cell division, organelle ultrastructure, and plastid differentiation.

Ionol (BHT), a compound having antioxidant activity, at concentrations in the range 1-50 mg/liter (0.45 x 10(-5)-2.27 x 10(-4) M), inhibits growth of etiolated wheat seedlings, changes the morphology of their organs, prolongs the coleoptile life span, and prevents the appearance of specific features of aging and apoptosis in plants. In particular, BHT prevents the age-dependent decrease in total DNA content, apoptotic internucleosomal fragmentation of nuclear DNA, appearance in the cell vacuole of specific vesicles with active mitochondria intensively producing mtDNA, and formation of heavy mitochondrial DNA rho = 1.718 g/cm3) in coleoptiles of etiolated wheat seedlings. BHT induces large structural changes in the organization of all cellular organelles (nucleus, mitochondria, plastids, Golgi apparatus, endocytoplasmic reticulum) and the formation of new unusual membrane structures in the cytoplasm. BHT distorts the division of nuclei and cells, and this results in the appearance of multi-bladed polyploid nuclei and multinuclear cells. In roots of etiolated wheat seedlings, BHT induces intensive synthesis of pigments, presumably carotenoids, and the differentiation of plastids with formation of chloro- or chromoplasts. The observed multiple effects of BHT are due to its antioxidative properties (the structural BHT analog 3,5-di-tert-butyltoluene is physiologically inert; it has no effect similar to that of BHT). Therefore, the reactive oxygen species (ROS) controlled by BHT seem to trigger apoptosis and the structural reorganization of the cytoplasm in the apoptotic cell with formation of specific vacuolar vesicles that contain active mitochondria intensively producing mtDNA. Thus, the inactivation of ROS by BHT may be responsible for the observed changes in the structure of all the mentioned cellular organelles. This corresponds to the idea that ROS control apoptosis and mitosis including formation of cell wall, and they are powerful secondary messengers that regulate differentiation of plastids and the Golgi apparatus in plants.

Subcellular reorganization of mitochondria producing heavy DNA in aging wheat coleoptiles.

Unusual closed membrane vesicles containing one or more mitochondria were isolated from homogenates of aging wheat coleoptiles. Very similar (or the same) bodies were shown to exist in situ in vacuoles of undividing cells in the apical part of intact senescent coleoptiles. Vesicles isolated from coleoptile homogenate free of nuclei by 10 min centrifugation at 1700 x g and traditional mitochondria (sedimented at between 4300 x g and 17,400 x g) are similar in respiration rate, composition and content of cytochromes and sensitivity to respiration inhibitors. However, vesicles contain about 2-fold more Ca2+ ions than free mitochondria do. The specific feature of vesicles containing mitochondria in aging coleoptiles is an intensive synthesis of heavy (rho = 1.718 g/cm3) mitochondrial DNA (H-mtDNA). Thus, aging in plants is accompanied by an increased selective H-mtDNA production and change in subcellular organization of mitochondria.

[Cytokinins do not noticeably change the methylation of adenine residues of DNA in cultured Escherichia coli B]. / Tsitokininy zametno ne izmeniaiut metilirovanie adeninovykh ostatkov DNK v kul'ture kletok Escherichia coli B.

6-Benzylaminopurine (6-BAP) (1 mg/ml) does not influence the growth of E. coli B cell cultures or the number of [8-14C] labeled N6-methyladenine (m6A) residues in the total DNA [(100.m6A/(A x m6A) = 1.7]. The growth of bacterial cells in the presence of adenine or cytokinins (6-BAP, kinetin, zeatin) (1 mg/ml) was unaccompanied by significant changes in the intracellular content of plasmid pBR 322. The mode of restriction by endonuclease Cfu I hydrolyzing the Gm6ATC site of plasmids pBR 322 from E. coli B cells grown in the presence of adenine or one of the above-mentioned cytokinins is identical. These plasmids also have identical restriction products Mbo I or Sau 3AI. Thus, the cytokinins under study do not markedly affect the methylation of adenine residues in total DNA of E. coli B cell cultures and the GATC sequence in plasmids pBR 322 isolated from these cells.

[5-Methylcytosine in pyrimidine sequences of plant and animal DNA: specificity of methylation]. / 5-Metiltsitozin v pirimidinovykh posledovatel'nostiakh DNK rastenii i zhivotnykh: spetsifichnost' metilirovaniia.

A method for two-dimensional DEAE-TLC fractionation and a subsequent quantitative spectrophotometric determination of the 5-methylcytosine containing pyrimidine oligodeoxyribonucleotides isolated from plant and animal DNA is proposed. Using this method, the distribution of 5-methylcytosine among pyrimidine oligodeoxyribonucleotides in various plant and animal DNA was studied. It was found that the methylation patterns of DNA in higher plants and animals are quite different, but they are similar among various plants or among different animal DNAs. More than half of the 5-methylcytosine residues in animal DNAs are localized in the monopyrimidine fraction (Pu-C-Pu); however, in plant DNAs there are two types of heavily methylated sequences (Pu-C-Pu and Pu-(CT)-Pu). It is suggested that methylation of plant DNAs occurs in two different sites possessing the second order symmetry with axis lined across the dinucleotide CpG or complementary AT base pair. Possible involvement of eucaryotic DNA-methylases in replicative and reparative DNA synthesis and in hormonal regulation of DNA methylation at and after DNA synthesis in plants and animals as well as a possible role of DNA methylation in regulation of gene activity in tissues and cells are discussed.

[Replication and methylation of DNA in cells of tobacco suspension culture and the effect of auxin]. / Replikatsiia i metilirovanie DNK v kletkakh suspenzionnoi kul'tury tabaka i vliianie auksina.

After 2 min of incubation of tobacco cell culture in a medium with [3H] -- thymidine the bulk of radioactivity of newly synthesized DNA is found in short (about 5S) fragments, whereas after a prolonged incubation of the cells, i. e. 5--60 min--in long replication fragments as well. Hence DNA replication in tobacco cells occurs discretely via formation and cross-linking of Okazaki fragments. At high cell concentrations in the medium the linking of 5S fragments is suppressed. It was shown that the Okazaki fragments and other fragments of DNA replication are subjected to methylation, the DNA methylation occurring immediately after the onset of replication. The level of methylation of the 4--6S fragments is two times less than that of the linked ones; therefore replicative methylation occurs in at least two steps: at first the Okazaki fragments undergo methylation and once they are linked, an additional methylation of DNA takes place. Auxin (2,4-dichlorophenoxyacetic acid) at concentration of 5 mg per 1 of medium does not affect the ratio of the replication fragments and methylation of the Okazaki fragments, but completely inhibits the second step of replicative methylation of DNA, i. e. methylation of the linked fragments. Phytohormones can probably control the transcription of newly synthesized DNA via regulation of methylation.

[Quantitative spectrophotometric determination of non-identified nucleotides in DNA]. / Kolichestvennoe spektrofotometricheskoi opredelenie neizvestnykh nukleotidov v DNK.

A method for calculation of molar extinction coefficients (epsilon) and for quantitative spectrophotometric determination of non-identified nucleotide analogs from nucleic acids of some bacteriophages is proposed. The method is based on spectral properties of known and unknown nucleotides and of their dinucleotide isolated from the DNA studied.

Structure of the surface of spores and morphological-physiological properties of individual strains of Bacillus megaterium.

The structure of the surface of spores of 10 strains of Bacillus megaterium was investigated by the method of carbon replicas. They were separated into three groups according to the peculiarities of the structural organization of the surface of the spores. The combination of other morphological and physiological-biochemical characteristics of these strains was characteristic of the species Bac. megaterium. Strains with differences in the structure of the spore surface also showed similarities in other peculiarities of carbohydrate and nitrogen metabolism studied in the work, as well as in the primary structure of DNA. The date obtained lead to the conclusion that structural differences in the surface of the spores cannot be considered a sufficient basis for isolating the bacteria studied into separate taxonomic groups.

[Nucleotide makeup of the DNA of thermophilic bacteria of the genus Thermus]. / Nukleotidnyi sostav DNK termofil'nykh bakterii roda Thermus.

The nucleotide composition of DNA was determined in extreme thermophilic and obligate thermophilic nonsporeforming bacteria belonging to a new genus Thermus. The GC content (in mol%) in the DNA of exteme thermophilic bacteria varied from 65.3 to 70.8 per cent depending on the strain. The amount of GC (in mol% in the DNA of obligate thermophilic and extreme thermophilic bacteria of the Thermus genus was higher than that in the DNA of sporeforming obligate thermophilic Bac. coagulans. Bac. circulans and Bac. stearothermophilus.