The influence of ammonium and nitrate of nutrient medium on ultrastructural organization and photosynthesis of callus cells in 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.     

Isolation and physiological and biochemical characterization of the Chlamydomonas reinhardtii C-41 mutant,a superproducer of ζ-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 ζ-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 β-carotene and 5% α-carotene, cells of the C-41 mutant contained 43% β-carotene, 19% β-zeacarotene, and 38% ζ-carotene. The high level of C-41 mutant biomass accumulation made it possible to recommend the mutant as a superproducer of ζ-carotene in phytobiotechnology.     

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 2.0 at 20°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 1 min 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.Translated from Izvestiya Akademii Nauk, Seriya Biologicheskaya, No. 2, 2005, pp. 155–161.Original Russian Text Copyright © 2005 by Stakhov, Ladygin, Stakhova.     

Reduction of the chloroplast membrane system caused by disorders in early stages of chlorophyll biosynthesis

A chlorophyll-deficient xantha mutant of cotton (Gossypium hirsutum L.) was examined with respect to development and structural organization of the chloroplast membrane system as affected by disruption of early stages of chlorophyll biosynthesis in the light. The analysis of early chlorophyll precursors showed that the mutant is unable to synthesize 5-aminolevulinic acid (5-ALA) in the light. The disorders in early stages of chlorophyll biosynthesis arrested the development of chloroplast membrane system at the stage of vesicles and single thylakoids. The accumulation of 2–5% chlorophyll in the mutant was related to the formation of light-harvesting chlorophyll-a/b-protein complexes I and II, whereas pigment-protein complexes composing reaction centers of photosystem I and photosystem II were lacking. It is concluded that the chloroplast membrane system in the mutant with impaired 5-ALA synthesis is incapable of development and is even reduced upon long-term growing under light.     

Chloroplast ultrastructure,photosynthetic apparatus activities and production of steviol glycosides in <Emphasis Type="Italic">Stevia rebaudiana in vivo</Emphasis> and <Emphasis Type="Italic">in vitro</Emphasis>

The accumulation of steviol glycosides (SGs) in cells of Stevia rebaudiana Bertoni both in vivo and in vitro was related to the extent of the development of the membrane system of chloroplasts and the content of photosynthetic pigments. Chloroplasts of the in vitro plants, unlike those of the intact plants, had poorly developed membrane system. The callus cells grown in the light contained proplastids of almost round shape and their thylakoid system was represented by short thylakoids sometimes forming a little number of grana consisting of 2–3 thylakoids. In cells of the etiolated in vitro regenerants and the callus culture grown in the dark, only proplastids practically lacking the membrane system were observed. All the chloroplasts having developed thylakoids and forming at least a little number of grana were equipped with photochemically active reaction centers of photosystems 1 and 2. Leaves of in vivo plants accumulated greater amount of the pigments than leaves of the in vitro plants. In both the callus culture grown in the light and the etiolated in vitro regenerants, the content of the pigments was one order of magnitude lower than that in leaves of the intact plants. The callus tissue grown in the dark contained merely trace amounts of the pigments. Leaves of the intact and the in vitro plants did not exhibit any significant differences in photosynthetic O₂ evolution rate. However, photosynthetic O₂ evolution rate in the callus cells was much lower than that in the differentiated plant cells. The in vitro cell cultures containing merely proplastids did not practically produce SGs. However, after transferring these cultures in the light, both the formation of chloroplasts and the production of SGs in them were detected.     

The Transformation of the Unicellular Alga Chlamydomonas reinhardtii by Electroporation

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 genehpt as the selective marker and the Tn₅ transposon of Escherichia coli under the control of the virus SV40 early gene promoter. Under optimal conditions (10⁶ mid-exponential cells/ml; electric field strength 1 kV/cm; and pulse length 2 ms), the transformation yielded 10³ Hyg^R transformants per 10⁶ 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 ofCh. reinhardtii with heterologous genes are discussed.     

Transformation of Chlamydomonas reinhardtii CW-15 with the Hygromycin Phosphotransferase Gene as a Selectable Marker

To transform Chlamydomonas reinhardtiiDang. cells, plasmid pCTVHyg was constructed with the use of theEscherichia colihygromycin phosphotransferase gene (hpt) controlled by the SV40 early promoter. Cells of the CW-15 mutant strain were transformed by electroporation, with the yield reaching 10³ hygromycin-resistant (Hyg^R) clones per 10⁶ recipient cells. The exogenous DNA integrated in the Ch. reinhardtii nuclear genome showed stable transmission for approximately 350 cell generations, while hygromycin resistance was expressed as an unstable character. Codon usage was compared for the hptgene and Ch. reinhardtiinuclear genes. The results testified that codon usage bias, which is characteristic of Ch. reinhardtii, is not the major factor affecting foreign gene expression. The advantages of the selective system for studying Ch. reinhardtii transformation with heterologous genes are discussed.