[LIGHT-DEPENDENT SYNTHESIS OF CELL MEMBRANES IN THE Brc-1 MUTANT OF CHLAMYDOMONAS REINHARDTII].

The structural organization of cells of the Brc-1 mutant of the unicellular green algae Chlamydomonas reinhardtii grown in the light and in the dark has been studied. The Brc-1 mutant contains the brc-1 mutation in the nucleus gene LTS3. In the light, all membrane structures in mutant cells form normally and are well developed. In the dark under heterotrophic conditions, the mutant cells grew and divided well, however, all its cell membranes: plasmalemma, tonoplast, mitochondrial membranes, membranes of the nucleus shell and chloroplast, thylakoids, and the membranes of dictiosomes of the Golgi apparatus were not detected. In the dark under heterotrophic conditions, mutant cells well grow and divide. It were shown that a short-term (1-10 min) exposure of Brc-1 mutant cells to light leads to the restoration of all above-mentioned membrane structures. Possible reasons for the alterations of membrane structures are discussed.

[Ultrastructural organization and composition of carotenoids in the eyespot in the mutant Chlamydomonas reinhardtii].

Biogenesis of the ultrastructure of the eyespot in the chloroplasts of unicellular green algae Chlamydomonas reinhardtii has been studied. We have found that the development of the structure of the eyespot correlates with the accumulation of carotenoids. Depending on their accumulation, the eyespots form from 1 to 4 lines of lipid-carotenoid globules. It has been shown that only carotenes are accumulated in the globules of the eyespots. We first have found that the composition of carotenes in the eyespots of the mutants may vary due to the changes in their composition in the membranes of chloroplasts.

[Biogenesis and possible modification of carotenoid composition in the Eyespot of Chlamydomonas reinhardtii mutants].

Biogenesis of the eyespot ultrastructure in the chloroplasts of the mutants of unicellular green algae Chlamydomonas renihardtii was studied. Development of the eyespot ultrastructure was found to correlate with carotenoid accumulation. Depending on their content, the eyespot formed 1 to 5 layers of lipid-carotenoid globules. Accumulation of carotene in the eyespot globules was shown. It was found that carotene composition in the eyespot of the mutants could vary depending on their composition in the chloroplast membranes.

[Structural-functional organization of the cells of Brc-1 mutant Chlamydomonas reinhardtii, supplying protoporphyrin IX in the dark].

The structural-functional characteristics of the cells of wild type CC-124 and Brc-1 mutant of the unicellular green algae Chlamydomonas reinhardtii while growing in the dark and light were studied. It has been shown that the cells of the wild type in heterotrophic and mixotrophic growth conditions had a well developed structure and high functional activity due to the ability of the cells to synthesize chlorophyll both in the light and in the dark. The cells of Brc-1 mutant lost their ability to synthesize chlorophyll in the dark and the cells' color was orange due to brc-1 mutation in the nuclear gene LTS3 that regulated the activity of Mg-chelatase enzyme. In the dark the mutant cells accumulated protoporphyrin IX and had a weakly developed structure with low functional activity. It has been ascertained that due to high content of protoporphyrin IX even a short-term exposure of the cells of Brc-1 mutant to the light was accompanied by very strong destructive changes in all the membranes in a cell: plasmalemma, chloroplast, mitochondrion, shells of the nucleus and vacuoles. The reasons of these significant damages of the membrane components and O2-gas exchange in the cells of Brc-1 mutant are discussed.

[Isolation and physiological and biochemical characterization of the Chlamydomonas reinhardtii C-41 mutant, a superproducer of ξ-carotene].

The composition of the carotenes and xanthophylls of Chlamydomonas reinhardtii Dang. C-41, a mutant of a unicellular green alga and a superproducer of ξ-carotene, was studied. The light-harvesting complexes and a complex of the PS-II reaction center were established to be disrupted in the C-41 mutant. However, the mutant retained a high (up to 46%) photosynthetic activity and the capacity to accumulate chlorophylls and carotenoids (up to 50%). The composition of carotenes was studied, and it was shown that, in contrast to wild-type K(+) cells, which accumulate up to 95% of ß13-carotene and 5% α-carotene, cells of the C-41 mutant contained 43% ß13-carotene, 19% ß3-zeacarotene, and 38% ξ-carotene. The high level of C-41 mutant biomass accumulation made it possible to recommend the mutant as a superproducer of ξ-carotene in phytobiotechnology.

[Disturbed structure and function of chloroplasts during blocked biosynthesis of 5'-aminolevulinic acid in the light].

Xantha-702 mutant of cotton (Gossypium hirsutum L.) proved to have blocked synthesis of 5'-aminolevulinic acid in the light. Accordingly, mutant leaves accumulated 2-5% chlorophyll of baseline. Mutant plants demonstrated disturbed production of pigment-protein complexes of photosystems I (PSI) and II (PSII) and generation of the chloroplast membrane system blocked at the early stages, largely, at the stages of vesicles and single short thylakoid. The functional activity of the PSI and PSII reaction centers was close to zero. Only the chlorophyll a/b light-harvesting complexes of PSI and PSII with the chlorophyll fluorescence peaks at 728 and 681 nm, respectively, were produced in the xantha-702 mutant. We propose that the genetic block of 5-aminolevunilic acid biosynthesis in the light in the xantha-702 mutant disturbs the formation and activity of the complexes of the reaction centers of PS-I and PS-II and inhibits the development of the whole membrane system of chloroplasts.

[Structural-functional organization of chloroplasts in leaves of xantha-702 mutant of Gossypium hirsutum L].

For cotton mutant xantha (Gossypium hirsutum L.), it has been established that synthesis of 5-aminolevulinic acid was blocked in the light. In the light this mutant accumulates chlorophyll by 30 times lower as compared to the parent type. In mutant xantha, a very few pigment-protein complexes of PS-I and PS-II are formed in chloroplasts, and formation of membrane system in these is blocked at the early stages, in most cases, at the stage of bubbles and single short thylakoids. Functional activity of reaction centers of PS-I and PS-II is close to zero. Only light-harvesting chlorophyll-a/b protein complexes of the two photosystems are formed in mutant xantha plastid membranes with maximum chlorophyll fluorescence at 728 and 681 nm, respectively. It has been concluded that in mutant xantha genetic block of 5-aminolevulinic acid biosynthesis in the light disturbs the formation and functioning of the complexes of reaction centers of PS-I and PS-II, hindering the development of the whole membrane system in chloroplasts, causing a sharp decrease in productivity.

[Spectral characteristics and the structure of chloroplasts upon blocking the early stages of chlorophyll biosynthesis].

The cotton mutant xantha (Gossypium hirsutum L.) with the blocked synthesis of 5-aminolevulinic acid in the light has been shown to accumulate chlorophyll 30 times less than the parent type. In chloroplasts of the mutant xantha, the formation of the membrane system is blocked at the earliest stages, mainly at the stage of bubbles and single short thylakoids. Only light-harvesting chlorophyll-a/b-protein complexes I and II with chlorophyll fluorescence maxima at 728 and 681 nm, respectively, are formed in plastid membranes of the mutant. It has been concluded that the genetic block of chlorophyll biosynthesis in the mutant xantha disturbs the formation and functioning of the complexes in reaction centers of PS-I and PS-II, inhibiting the development of the whole membrane system of chloroplasts at the stage of bubbles and single thylakoids.

[The current concepts of functional role of carotenoids in the eukaryotic chloroplasts].

Current state of knowledge of functional role of carotenoids in algal and higher plant chloroplasts is reviewed. Basic functions of carotenoids are shown to be light-protective, light-absorbing, and structural, as well as participating in photochemical processes of photosystems I and II. Such xanthophylls as neoxanthin, fucoxanthin, peridinin and alloxanthin, which have allenic or acetylenic bond, mostly function as light-absorbers. They transmit absorbed energy to chlorophyll b. Other xanthophylls occupying certain strictly specified loci in light-absorbing chlorophyll-a/b-protein complexes of photosystems have either structural function (lutein) or light-protective function (zeaxanthin, antheraxanthin, violaxanthin). Carotenoids of xanthophyll cycles preserve chlorophylls and lipids of photosynthetic membranes from photodestruction at overlighting in the presence of oxygen. In eukaryotic chloroplasts, three types of xanthophyll cycles were found: violaxanthin, lutein-5,6-epoxide, and diadinoxanthin. The similarities and dissimilarities between epoxidation and de-epoxidation reactions of these cycles are discussed in detail in the present work. The pattern of occurrence of xanthophyll cycles among higher plants and freshwater and marine algae is outlined.

[Time course of the content of folates and free amino acids in leaves of Pisum sativum L. after irradiation with ultraviolet C].

The effect of irradiation with UV-C on the time course of the content of total folates and free amino acids in leaves of pea (Pisum sativum L.) cultivar Neistoshchimyi was studied. It was shown that photolysis of folates is a rapid response to exposure to ultraviolet, as a result of which the plant produces a stable compound, pterin-6-carboxylic acid, with a relative fluorescence quantum yield approximately 2.0 at 20 degrees C (total value, 0.58). Presumably, this compound may be involved in the pterin-mediated photosensitization of singlet oxygen production. The kinetics of changes in the composition of free amino acids after exposure to UV-C has been studied. Exposure to UV-C for 0.5 and 1min induced utilization of free amino acids, suggesting activation of the synthesis of hormones and alkaloids that may facilitate resistance to the stressor. Greater doses as a result of exposure to radiation for 10 and 40 min decreased the content of free hydrophobic amino acids. This phenomenon could be due to the formation of covalent cross-links in membranes, which decrease the accessibility of hydrophobic amino acids. It is concluded that the changes in the qualitative and quantitative composition of free amino acids in leaves of irradiated plants were due to glycolysis.

[Photochemical activity, spectral properties, and structure of chloroplasts in leaves of Pisum sativum L. under iron deficit and root anaerobiosis].

A combined effect of iron deficit and root anaerobiosis on the biochemical composition, functional activity, and structure of chloroplasts in pea leaves was studied. These factors are shown to affect the chlorophyll accumulation, causing leaf chlorosis. Iron deficit makes itself evident in the chlorosis of top leaves. In the case of root anaerobiosis, chlorosis damages lower plant layers. The destructive effects are summarized under the influence of both factors. The light-harvesting complexes of photosystems are reduced to a greater degree under iron deficit; under root anaerobiosis, complexes of reaction centers of photosystem I and II are reduced. Nevertheless, even under the combined effect of these factors, all pigment-protein complexes and their functional activities are preserved in yellow leaves. The ultrastructure of chloroplasts is gradually reduced in the course of developing chlorosis. In the begging, intergranal sites of thylakoids are destroyed, which is typical for iron deficit, then granal sites are broken. However, even in yellow and almost white leaves, small thylakoids capable of forming stacking and small grana of 2-3 thylakoids are preserved. The destructive effects are summarized due to different mechanisms of action of iron deficit and root anaerobiosis on the structure and function of leaves under their combined effect.

[Changes in the biochemical composition, structure, and function of pea leaf chloroplasts in iron deficiency and root anoxia]. / Izmenenie biokhimicheskogo sostava, struktury i funktsii khloroplastov v list'iakh gorokha pri defitsite zheleza i anaérobioze kornei.

A combined effect of iron deficiency and root anoxia on the biochemical composition, function, and structure of pea leaf chloroplasts was studied. It was found that the chlorosis of apical leaves in response to iron deficiency was determined by the reduction of light-harvesting complexes I and II. Under root anoxia, complexes of the reaction centers of photosystems I and II degraded first. Weak activity was preserved even in yellow and white leaves under the effect of both factors. The ultrastructure of leaf chloroplasts gradually degraded. Initially, intergranal thylakoid sites were reduced, and the longitudinal orientation of grana was disturbed. However, yellow and white leaves still retained small thylakoids and grana. It is concluded that the degrading effects of iron deficiency and root anoxia on the complex composition and leaf chloroplast structure and function are additive because of their autonomous mechanisms.

[An efficient way for obtaining transformants of Chlamydomonas reinhardtii by electroporation]. / Effektivnyi sposob polucheniia transformantov Chlamydomonas reinhardtii putem élektroporatsii.

The cells of mutant CW-15, which does not form the cell wall, were transformed by electroporation. It was found that the transformation was optimal at a suspension density of 10(6) cells/ml in the middle of the logarithmic growth phase at a field intensity of 1 kW/cm and pulse duration 2 ms. It was shown that, under these conditions, the efficiency of transformation reached 10(3) HygR-clones per 10(6) recipient cells. Exogenic DNA integrated into the genome of the nucleus of Ch. reinhardtii was inherited constantly for more than 350 generations; however, the tolerance to hygromycin appeared as an unstable feature. The advantages of using mutant CW-15 and the selective system to study the transformation of Ch. reinhardtii by heterologous genes are discussed.

[Photosystem damage and spatial architecture of thylakoids in chloroplasts of pea chlorophyll mutants]. / Narushenie fotosistem i prostranstvennaia arkhitektura tilakoidov v khloroplastakh khlorofil'nykh mutantov gorokha.

The effect of chlorophyll-protein complexes on the ultrastructure of chloroplasts was studied in the leaves of pea, the parent cultivar Torsdag and mutants chlorotica 2004 and 2014. The mutants were shown to accumulate 80 and 55% of chlorophyll, relative to the control, while the composition of the synthesized photosystem complexes was the same as in the parent cultivar Torsdag. The size of the light-harvesting antenna was similar to the control in the 2014 mutant but considerably increased (by 30%) in the 2004 mutant. These changes were due to a proportional decrease in the number of all complexes (by 40-45%) in the 2014 mutant. At the same time, the number of reaction center complexes of photosystem I (PS I) decreased by 50% while that of photosystem II (PS II) remained virtually constant in the 2004 mutant. A proportional decrease in the number of the PS I and PS II complexes in the chlorotica 2014 mutant was accompanied by a partial reduction of the entire chloroplast membrane system against the background of normal development of both granal and intergranal sites of thylakoids. Conversely, the loss of PS I reaction centers led mainly to the reduction of the intergranal sites of thylakoids in chloroplasts. This effect is attributed to the prevalence of PS I complexes in the intergranal thylakoids.

[Pigment-protein complexes nd the number of the reaction photosystem centers in pea chlorophyll mutants]. / Pigment-belkovye kompleksy i chislo reaktsionnykh tsentrov fotosistem u khlorofil'nykh mutantov gorokha.

We studied fluorescent and absorption properties of the chloroplasts and pigment-protein complexes isolated by gel electrophoresis from the leaves of pea, the initial cultivar Torsdag and mutants chlorotica 2004 and 2014. Specific maxima of fluorescence and chlorophyll forms in individual complexes have been determined from the absorption and fluorescence spectra of the chloroplast chlorophyll and their secondary derivatives at 23 and -196 degrees C. Chlorotica 2004 mutant proved to have an increased intensity of a long-wave band at both 23 degrees C (745 nm) and -196 degrees C (728 nm) of the light-harvesting complex I. At the same time, this mutant featured a decreased accumulation of chlorophyll forms at 690, 697, and 708 nm forming the nearest-neighbor antenna of PSI reaction center. No spectral differences have been revealed between chlorotica 2014 mutant and the initial cultivar. Gel electrophoresis demonstrated synthesis of all chlorophyll-protein complexes in both mutants. At the same time, analysis of photochemical activity of PSI and PSII reaction centers and evaluation of the light-harvesting antenna as well as the number of reaction centers of the photosystems suggest that chlorotica 2004 mutant has 1.7 times less PSI reaction centers due to a mutation-disturbed chlorophyll a-protein complex of PSI. The primary effect of chlorotica 2014 mutation remains unclear. The proportional changes in the photosystem complexes in this mutant suggest that they are secondary and result from a 50% decrease in chlorophyll content.

[The transformation of the unicellular alga Chlamydomonas reinhardtii by electroporation]. / Transformatsiia odnokletochnoi vodorosli Chlamydomonas reinhardtii putem électroporatsii.

The cell wall-lacking mutant CW-15 of the unicellular green alga Chlamydomonas reinhardtii was transformed by electroporation using plasmid pCTVHyg, which was constructed with the hygromycin phosphotransferase gene hpt as the selective marker and the Tn5 transposon of Escherichia coli under the control of the virus SV40 early gene promoter. Under optimal conditions (10(6) mid-exponential cells/ml; electric field strength 1 kV/cm; and pulse length 2 ms), the transformation yielded 10(3) HygR transformants per 10(6) recipient cells. The exogenous DNA integrated into the nuclear genome of Ch. reinhardtii was persistently inherited through more than 350 cell generations. The advantages of this system for the transformation of Ch. reinhardtii with heterologous genes are discussed.

[The influence of ammonium and nitrate of nutrient medium on ultrastructural organization and photosynthesis of callus cells in Glycine max L]. / Vliianie ammoniia i nitrata pitatel'noi sredy na ul'trastrukturnuiu organizatsiiu i fotosintez kletok kallusa Glycine max L.

We studied the influence of exogenic ammonium on the functional activity and ultrastructural organization of cells of the mixotrophic soybean callus (Glycine max L.). Ammonium available in the nutrient medium increased the chlorophyll content, accelerating the rate of photosynthetic O2 evolution per unit of biomass. The presence of ammonium in the medium promoted formation of the protein-synthesizing system, which manifested itself as increased numbers of ribosomes, and thylakoids of chloroplasts, and higher electron density of the stroma in mitochondria and cytoplasm of mixotrophic cells. It has been concluded that the use of ammonium may lead to activation of protein synthesis, thus rising photosynthetic activity and favouring formation and development of membrane structures in chloroplasts.

[Pigment composition and photosynthetic activity of pea chlorophyll mutants]. / Sostav pigmentov i fotosinteticheskaia sposobnost' khlorofil'nykh mutantov gorokha.

Pea chlorophyll mutants chlorotica 2004 and 2014 have been studied. The mutants differ from the initial form (pea cultivar Torsdag) in stem and leaf color (light green in the mutant 2004 and yellow-green in the mutant 2014), relative chlorophyll content (approximately 80 and 50%, respectively), and the composition of carotenoids: the mutant 2004 contains a significantly smaller amount of carotene but accumulates more lutein and violaxanthine; in the mutant 2014, the contents of all carotenoids are decreased proportionally to the decrease in chlorophyll content. It is shown that the rates of CO2 assimilation and oxygen production in the mutant chlorotica 2004 and 2014 plants are reduced. The quantum efficiency of photosynthesis in the mutants is 29-30% lower than in the control plants; in their hybrids, however, it is 1.5-2 higher. It is proposed that both the greater role of dark respiration in gas exchange and the reduced photosynthetic activity in chlorotica mutants are responsible for the decreased phytomass increment in these plants. On the basis of these results, the conclusion is drawn that the mutations chlorotica 2004 and 2014 affect the genes controlling the formation and functioning of various components of the photosynthetic apparatus.

[Pigment accumulation and functional activity of chloroplasts in common Pisum sativum L. mutants with low chlorophyll level (chlorotica)]. / Nakoplenie pigmentov i funktsional'naia aktivnost' khloroplastov u nizkokhlorofil'nykh mutantov (chlorotica) posevnogo gorokha Pisum sativum L.

Pea mutants chlorotica 2004 and 2014 with a low content of chlorophyll were studied. The mutant 2004 has light green leaves and stem, and the mutant 2014 has yellow green leaves and stem. They accumulate approximately 80 and 50% chlorophylls of the parent form of pea Torsdag cv. The content of carotene in carotenoids of the mutant 2004 was much lower, and the accumulation of lutein and violaxanthine was increased. The accumulation of all carotenoids in the mutant 2014 decreased almost proportionally to a decrease in the chlorophyll content. The rate of CO2 evolution in mutant chlorotica 2004 and 2014 was established to be lower. The quantum efficiency of photosynthesis in the mutants was 29-30% lower as compared to the control, and in hybrid plants it was 1.5-2-fold higher. It is assumed that the increase in the activity of the night-time respiration in gas exchange of chlorotica mutants and the drop of photosynthesis lead to a decrease in biomass increment. The results obtained allow us to conclude that the mutation of chlorotica 2004 and 2014 affects the genes controlling the formation and functioning of different components of the photosynthetic apparatus.

[Ultrastructure organization of chloroplasts in chlorotica mutants of Pisum sativum L]. / Ul'trastrukturnaia organizatsiia khloroplastov chlorotica-mutantov Pisum sativum L.

A study was made of chlorophyll-protein complexes of photosystems, and of ultrastructural organization of chloroplasts in pea leaves of the primary cultivar Torsdag and of its mutants, chlorotica 2004 and 2014. It has been shown that mutants accumulated 80 and 55% chlorophyll, respectively, and were able to synthesize all four types of photosystem complexes. The value of the light-harvesting antenna in mutant 2014 was close to the control one, and in mutant 2004 it increased significantly (by 30%). These changes were caused by a proportional decrease (40-50%) in any complexes in mutant 2014, whereas the number of PS-I reaction centre complexes, decreased by 50% in mutant 2004 at nearly complete storage of PS-I reaction centre complexes, decreased by 50% in mutant 2004 at nearly complete storage of PS-II complexes. The proportional decrease of PS-I and PS-II complexes in mutant chlorotica 2014 was followed by partial reduction of the entire membrane system in chloroplasts, but with a normal development of both granal and intergranal thylakoids. On the contrary, the loss of PS-I reaction centre complexes in mutant chlorotica 2004 leads to reduction of unstacked sites of thylakoids in chloroplasts. It is concluded that this effect may be associated with localization of PS-I complexes mainly in unstacked sites of thylakoids.