A. V. Usatov

Intraspecific genetic polymorphism of Russian sturgeon Acipenser gueldenstaedtii

Three populations (Azov, Caspian, and Black Sea) of Russian sturgeon Acipenser queldenstaedtii were tested for polymorphism at nuclear (RAPD and microsatellites) and mitochondrial (PCR identification of two mitotypes) markers. In addition, morphometric analysis of he representatives of Azov population was carried out. According to the morphological characters, the Black Sea population occupied an intermediate position between the Caspian and Azov populations, reflecting the phylogeography of this species. In agreement with the morphometric data, genetic distances (the data of STR analysis) also placed the Black Sea population between the Caspian and Azov populations (FST = 0.058 and 0.043). The genetic distance between the Azov and Caspian population was somewhat higher (FST = 0.070). The highest allelic polymorphism at four microsatellite loci was found observed in Caspian population, while the lowest polymorphism was in the Sea of Azov. RAPD analysis distinguished high polymorphism within the populations, although it was not feasible for interpopulation analysis. Using the method differentiating the “baerii-like” and typical “gueldenstaedtii” mitotypes, the absence of the “baerii-like” marker in the Black Sea population was demonstrated. The frequency of this marker in Caspian and Azov populations constituted 31.1 and 1.8%, respectively. Possible evolutionary reasons for the interpopulation differences observed are discussed.

Mechanisms of salt stress tolerance development in barley plants under the influence of 5-aminolevulinic acid

Growing barley (Hordeum vulgare L.) plants for 7 days on NaCl solutions (20–200 mM) decreased chlorophyll (Chl) a and b content with respect to that in untreated control plants. The content of free proline and the plant ability to synthesize 5-aminolevulinic acid (ALA) started to increase in parallel at salt concentrations of 20–50 mM. The maximum amount of ALA accumulated in plants grown at 100 mM NaCl was twofold higher than in control plants grown on fresh water. In this case the proline content increased 2.8-fold. On further increase in salt concentration, the rate of ALA accumulation decreased, approaching control values at 150 mM NaCl; even lower rates were observed at 200 mM NaCl. The reduced ability to synthesize ALA was accompanied by an increase in proline content. The albino tissue of plants treated at the seed stage with the antibiotic streptomycin lost its ability to synthesize ALA needed for Chl formation. The proline content in the albino tissue was tenfold higher than in control green plants and was 30-fold higher when the plants were grown on solutions with 100 mM NaCl. No effect of NaCl on ALA-dehydratase activity was noted. As NaCl concentration was raised, there occurred the decrease in magnesium chelatase activity, accumulation of reactive oxygen species (ROS), the increase in ascorbate peroxidase activity, and a slight decrease in lipid peroxidation level. Growing plants in the presence of 150 mM NaCl and 10 or 60 mg/l exogenous ALA led to the increase in proline content (by a factor of 1.8 and 4.2, respectively) and to the decrease in ROS content, in comparison with plants grown on salt solutions without ALA. Furthermore, in the presence of exogenous ALA, the parameters of seedling growth became similar to those of NaCl-untreated plants. The role of ALA in plants as an antistress agent is considered. ALA is supposed to confer tolerance to salt stress by taking part in Chl and heme biosynthesis and also through functioning as a plant growth regulator. A hypothesis is put forward that the impairment of ALA-synthesizing ability may redirect metabolic conversions of glutamic acid from Chl and heme synthesis to the proline synthesis pathway, which would stimulate proline biosynthesis and improve salt tolerance.

Comparative Analysis of Thermotolerance of Sunflower Chlorophyll Mutants

The influence of high temperatures on sunflower chlorophyll mutants was studied. The tolerance criteria were the level of chromosome aberrations and the mitotic index in the root apical meristem of seedlings, the level of nucleus-free cells in the epidermis of cotyledonous leaves, and the intensity of chlorophyll accumulation after the action of heat shock (HS). In addition, the frequency of plants with an altered content of pigments in M1 and M2 was analyzed. The results indicated that the plastomic mutant en-chlorina-5 is more tolerant to temperature stress as compared to other sunflower lines.

Structural and Functional Characteristics of the Mutant Plastids from Extranuclear Variegated Forms of Sunflower

The electron-microscopic analysis of extranuclear variegated forms of sunflower demonstrated that plastids from white leaf areas are practically devoid of inner membrane structures. The mutants and the initial (wild-type) green line were compared by the contents of chlorophyll (Chl), carotenoids, and 70S ribosomes and by the activity of Rubisco. A mutant Var10 line was used to demonstrate that the primary characteristic manifestations of Chl deficiency include the synchronous retardation of the synthesis of a specific Chl precursor, 5-aminolevulinate, a decrease in the Chl a/b ratio to the level below that in the wild type, and the reduction of photosystem (PS) I Chl luminance. The progress of photodestructive processes in the mutant aggravated the listed disturbances; as a result, PSII complexes and light-harvesting complexes gradually degraded. The manifestation of the trait of Chl-deficiency notably varied depending on plant growth conditions (primarily, temperature and illumination regimes). The dependence of this manifestation on light and temperature was observed only at the early stage of development of pigment-containing tissues. When plants terminated this stage under low irradiation, they did not become Chl-deficient, and when their leaves were subsequently transferred to the conditions promoting the maximum pigment anomaly, the destructive processes did not advance in the mutant tissue. The authors assume that the plastom mutations under study impair the control over the expression of the structural plastogenes that determine the synthesis of pigment and protein components of the photosynthetic apparatus, rather than directly damage to the primary structures of plastogenes.

Role of nitrogen metabolism in the development of salt tolerance in barley plants

The 7- to 8-day-old barley (Hordeum vulgare L.) seedlings grown in KNO3 solutions (1-40 mM) were characterized by the substrate activation of nitrate reductase (NR) in the apical leaf segments (1–2 cm in length), as well as by stimulated growth, broadened leaf blades, and by vigorously developed system of shortened roots. When the seedlings were grown in the presence of 20 mM KNO3, the ability of leaf segments to generate superoxide anion radical remained at the level typical of control plants grown in water. The content of 5-aminolevulinic acid (ALA) in plants grown in the presence of 20 mM KNO3 was 2.2–2.4 times higher than in control plants. The plants grown in the presence of nitrate had an elevated content of chlorophylls a and b, heme, and protein (by 42%). At the same time, the proline content was almost twofold lower than in control plants, which was due to substantial reduction (by 40%) in activity of Δ1-pyrroline-5-carboxylate synthetase (P5CS). It is concluded that the substrate activation of NR by KNO3 under normal growth conditions results in predominant utilization of glutamic acid (the primary product of inorganic nitrogen assimilation) for biosynthesis of tetrapyrroles and protein amino acids at the expense of inhibition of proline synthesis. When barley seedlings were grown in 150 mM NaCl solution, the plant growth and the root system development were suppressed to the levels of 63 ± 6% and 61 ± 11% of the control values, respectively. In the apical leaf tissues of plants adapted to NaCl, there was a slight decrease in the total NR activity (by 10%), a significant reduction in protein content (by 32%), and a parallel increase in the content of ALA (by a factor of 4.3), chlorophylls, heme, carotenoids, proline (2.2-fold) and P5CS (1.6-fold) with respect to the control values. It is proposed that the accumulation of ALA and proline under salinity-induced suppression of nitrogen assimilation results from the predominant allocation of glutamate for biosyntheses of ALA and proline at the expense of inhibition of growth-related processes requiring intense protein synthesis. The substrate activation of NR by KNO3 under salinity conditions was associated with prevailing allocation of the assimilated nitrogen for synthesis of proline and protein amino acids, which reinforced plant cell protection against salinity and stimulated plant growth.

Response of Mutant Sunflower Lines to Heat Shock

The effects of heat shock (HS) (40°C for 1 h) on the level of malondialdehyde (MDA), the terminal product of lipid peroxidation, superoxide dismutase (SOD) activity, catalase activity, and total peroxidase activity (TPA) were studied in root meristems and chloroplasts of several sunflower (Helianthus annuusL.) lines that carried nuclear or plastome chlorophyll mutations. HS either lowered or did not affect the MDA level in the root meristem and in the chloroplasts from the first true leaf, as compared to the untreated plants. In both treatments, the root and leaf enzyme activities varied in the sunflower lines. In the root meristem, catalase was the most sensitive to HS, whereas, in the chloroplasts from HS-treated sunflower lines, HS activated either TPA or SOD.

Study of Chloroplast DNA Polymorphism in the Sunflower (Helianthus L.)

The polymorphism of microsatellite loci of chloroplast genome in six Helianthus species and 46 lines of cultivated sunflower H. annuus (17 CMS lines and 29 Rf-lines) were studied. The differences between species are confined to four SSR loci. Within cultivated forms of the sunflower H. annuus, the polymorphism is absent. A comparative analysis was performed on sequences of the cpDNA inbred line 3629, line 398941 of the wild sunflower, and the American line HA383 H. annuus. As a result, 52 polymorphic loci represented by 27 SSR and 25 SNP were found; they can be used for genotyping of H. annuus samples, including cultural varieties: twelve polymorphic positions, of which eight are SSR and four are SNP.

Responses of Sunflower Chlorophyll Mutants to Increased Temperature and Oxidative Burst

A tolerance of nuclear, plastome, and mitochondrial mutants of sunflower (Helianthus annuus L.) to extreme factors, increased temperature and oxidative stress, which was simulated by hyperbaric oxygenation was studied. In model experiments, activities of superoxide dismutase and catalase were assayed; cytogenetic analysis was used for the assessments of proliferative activity of cells in the root meristem; in field experiments, seedling emergence and growth at the stage of 3–4 leaf pair were evaluated. Most tolerant to increased temperature and oxygen pressure were a plastome en:chlorina-5 mutant and a partial revertant pr6-en:chlorina-7 with a changed structure of mitochondrial DNA.

Activity of the System for Chlorophyll Biosynthesis and Structural and Functional Organization of Chloroplasts in a Plastome en:chlorina-5 Sunflower Mutant

Chlorophyll (Chl) deficiency in leaves of a plastome sunflower (Helianthus annuus L.) en:chlorina-5 mutant is due to the formation of smaller chloroplasts with a markedly reduced membrane system, as compared to the parent 3629 line. Abnormalities in the structure of the photosynthetic apparatus in the mutant can be mainly attributed to changes in the formation of photosystem I and its light-harvesting complexes. Chl deficiency in en:chlorina-5 correlated with its lower capability of synthesizing the first specific Chl precursor, 5-aminolevulinic acid (ALA) in the light. Light-independent stages of Chl biosynthesis in the mutant had the same efficiency as in leaves of the parent line. ALA formation in darkness and its conversion into protochlorophyllide did not depend on the extent of photosynthetic membrane development and photosynthetic activity.

Modification of the Effect of Nitrosomethylurea on Sunflower Seedlings by Heat Shock

Separate and combined action of nitrosomethylurea (NMU) and heat shock (HS) on germination of seeds of the inbred line 3629 and derived nuclear and plastome mutant lines, n-chlorina-1 and en-chlorina-5, respectively, were studied. NMU at a concentration of 0.015%, as well as HS (40°C), had no effect on the spontaneous level of chromosome aberrations in root meristem of sunflower seedlings. However, at a concentration of 0.03%, the mutagen considerably increased the frequency of chromosome rearrangements. Pretreatment with heat for 30 min enhanced the cytogenetic effect of NMU (0.03%) on line 3629 seedlings. In the nuclear mutant, the reaction of root meristematic cells did not depend on the additional heat treatment. The combined action of HS and NMU at early germination stages had no effect on the survival of line 3629 plants and plastome mutant en-chlorina-5; the survival of n-chlorina-1 plants decreased. The highest frequency of M1plants with chlorophyllic abnormalities (green revertants, lethals, and variegated forms) was observed in n-chlorina-1 line.