Vol 66, No 5 (2019)

Certain aspects of Ca-dependent regulation of symbiosomes functioning mediated by the operation of Ca²⁺-translocating ATPase on the symbiosome membrane (SM) were considered. As follows from the results recently obtained by us, the mechanism underlying the action of this calcium pump exhibits a certain functional feature caused by that transfer by it of calcium ion through the SM is closely coupled with transmembrane translocation through the same membrane but in opposite direction of H⁺ ions. The data here outlined show that such the cation-exchanged mechanism of the Ca²⁺-ATPase operation can putatively lead to corresponding modulation or maintenance of both ionic and redox homeostasis of the symbiosome space (SS) around bacteroids at a proper level and thereby to the regulation of symbiosomes functioning. It is suggested that based on the same mechanism inherent function of the enzyme in question is associated with its involvement in symbiotic partner cells signaling as well. All these potential activities of the Ca²⁺-ATPase lead to the important question whether it is the factor determining substantial functions of symbiosomes. Although it is evident that research an answer to this question is a complex enough experimental problem, its solution may give significant contribution to elucidating mechanisms of Ca-dependent regulation of the processes occurring in mature symbiosomes.

CRISPR/Cas genome editing of plants is realized in three basic variants, including knockout mutations as indels, insertion of alien DNA fragments, and base editing via deamination of nitrogenous bases. The most important stages of the CRISPR/Cas-based genome editing are the choice of a target site, design of guide RNAs, creation of genetically engineered constructions, and delivery of CRISPR/Cas components into a plant cell. Rapid developments in the field of plant genome editing with the use of CRISPR/Cas systems requires more detailed consideration of the last stage, so this review is dedicated to the description of the main ways to deliver CRISPR/Cas components into cells of higher plants. In the first studies on the genome editing of different plant species, these components were delivered to the target site mainly by Agrobacterium tumefaciens. This approach supposes integration of T-DNA into a genome and a stable expression of CRISPR/Cas components or their transient expression in the case of agroinfiltration. Another widespread approach included the use of plant viruses as delivery platforms; in this case, viruses were used mainly for production of an increased amount of guide RNAs that significantly improved the efficiency of genome editing. Another approach provides for the use of another bacterium, A. rhizogenes, as a platform for delivery of CRISPR/Cas components. This bacterium induces hairy root formation that may be an indirect confirmation of successful genome editing and assist in the selection of genetically modified forms. Other common ways to obtain genetically edited plants are the biolistic delivery of genetically engineered constructions into explants and various protoplast transformation technologies. The review also considers some issues transgenic and GM status of CRISPR/Cas-edited plants to transgenic and GM plants. There are a number of cases in which new organisms created by a CRISPR/Cas genome editing without any introduction of alien DNA were not considered as transgenic ones; it is quite possible that such plants will not fall under Russian legislation prohibiting GMO cultivation.

The data concerning morphometric characterization of chloroplasts belonging to two groups of halophytes distinguished by their salt tolerance—“highly salt-tolerant” (Climacoptera aralocaspica L., Salicornia europaea L., and Suaeda arcuata (Bange)) and “moderately salt-tolerant” (Kochia prostrata (Schrad) L. and K. scoparia L.)—are presented. Plants of the first and second groups were grown for 2.5 months at soil salinization within limits 0–1000 and 0–600 mmol Na⁺/dm³, respectively. Morphometric parameters (linear sizes, proportion of stromal inclusions of starch and plastoglobules) of mesophyll chloroplasts of two groups were maintained at a constant level within corresponding range of soil salinization practically coinciding with limit of their stable photosynthetic activity. Based on these results, it is suggested that chloroplasts provide energy and assimilates to the processes of ionic and water homeostasis realized at cellular and overcellular levels within the organism’s response norm and, thereby, contribute to forming the system response of a plant to soil salinization.

Most studies related to the functions of miR165/166 have used Arabidopsis as the experimental material. However, Nicotiana benthamiana Domin is a more ideal research material as its typical stem structure. Additionally, most of the relevant research has focused on the functions of HD-ZIP III genes. As an upstream regulatory factor, the function of miR165/166 needs to be defined more directly. So far, the function of miR165/166 in N. benthamiana has not yet been examined. The Agrobacterium-mediated genetic transformation method was used to transform LaMIR166a from Larix leptolepis overexpression vector into N. benthamiana leaves, and three transgenic lines were obtained after molecular detection. Observation on the one-month seedlings revealed that overexpression of LaMIR166a markedly enhanced the biomass accumulation of N. benthamiana plants. Additionally, growth parameters including root length and basal diameter, lateral root density, stem ground diameter and stem internode length of transgenic plants were significantly higher than in wild type (WT) plants. Microscopic observation of stem slices and root squashes showed that overexpression of LaMIR166a significantly promoted xylem formation and development, and the growth of root hairs, which showed a bent phenotype. The results of the present study suggest that up-regulation of miR166a promotes the growth and development of N. benthamiana plants.

Phosphoenolpyruvate carboxylase (PEPC) is the primary acceptor of carbon dioxide in C₄ photosynthesis. Three isozymes of PEPC: PEPC1, PEPC2, and PEPC3, in the single cell, C₄ plant Bienertiasinuspersici Akhani were determined in this study. Differential expressions of these isoforms were observed at different stages of leaf development. Amino acid sequence alignment revealed that PEPC1, PEPC2, and PEPC3 had characteristics of C₃-C₄ PEPC type, C₄ PEPC, and C₃ PEPC, respectively. Gene expression profiles showed that PEPC3 was highly expressed in young leaves while PEPC1 and PEPC2 were more expressed in intermediate and mature leaves, respectively. Using immunoblotting, we also assessed tissue-specific expression and post-translational modifications (PTMs) of PEPC in B. sinuspersici. During leaf maturation, phosphorylation at Ser 11 and monoubiquitination at Lys 629 were highly increased. The phosphorylation pattern on PEPC was diurnally regulated but monoubiquitination was not. Moreover, dimorphic chloroplasts from B. sinuspersici chlorenchyma cell of mature leaf were isolated to study spatial localization of PEPC by PTM. Monoubiquitinated PEPC were specifically interacting with peripheral chloroplasts. These results indicated that phosphorylation and monoubiquitination on PEPC were mutually exclusive as well as affecting spatial localization in single cell C₄ plants.

Flax (Linum usitatissimum L.), as an important commercial crops, is a rich source of fatty acids, protein, dietary fiber and lignans. Flax plant has an effective anticancer activity due to lignan contents. Nanoparticles have recently been used as efficient non-biologic elicitors to improve the biosynthesis of secondary metabolites. In this study, the effects of different concentration of ZnO (0, 30, 60, and 120 mg/L) and TiO₂ (0, 50, 100, and 150 mg/L) nanoparticles at different time (0, 24, 48, and 72 h) were investigated on enzyme activities and production of secondary metabolites in cell suspension cultures of flax. The results indicated that the highest activity of phenylalanine ammonia lyase (PAL) was observed in 30 mg/L nano-ZnO treatment at 48 h, whereas the effect of nano-TiO₂ on PAL enzyme activity was not statistically significant. According to the results, the highest activity of CAD (cinnamyl alcohol dehydrogenase) was observed in 60 mg/L concentration of nano-ZnO at different intervals. The use of 150 mg/L nano-TiO₂ led to increased activity of CAD. The maximum content of total phenol was detected at 150 mg/L nano-TiO₂. Different concentrations of nano-TiO₂, caused to an increase in total lignan at all intervals. The highest amount of total phenol and lignans was observed in 30 and 60 mg/L ZnO. In the present study, we were observed different effects of nanoparticle on enzymes activity and secondary metabolite production in cell suspension cultures of flax plant, depending on concentration and type of nanoparticles.

Two inbred lines of spring maize (Zea mays L.), CML 32 (stress tolerant) and LM 11 (stress susceptible) were taken to study the effect of salicylic acid (SA) on their carbohydrate status, under heat stress. Heat stress increased the content of total soluble sugars and sucrose in the shoots and residual mass of CML 32 seedlings. On the other hand, the shoots and roots of LM 11 seedlings showed reduced sucrose content. Application of SA increased the contents of total soluble sugars and sucrose in the roots and shoots of both the inbred lines. Heat stress reduced the activities of acid and neutral invertases in the shoots of CML 32 seedings that corresponded with their increased sucrose content. Foliar spray of SA increased acid invertase but decreased neutral invertase activities in shoots of both the inbred lines that might help in maintaining sucrose levels and energy needs for better seedling growth under stress conditions. Heat stress induced CML 32 seedlings to exhibit stress tolerance by increasing amylase activity in their residual masses that led to their reduced starch content. SA application reduced the amylolytic activity of the residual mass of CML 32 seedlings that corresponded with their higher starch content. SA application led to increased sucrose synthase (synthesis) and sucrose phosphate synthase activities in the roots of both the inbred lines and the shoots of CML 32 seedlings. It also decreased sucrose synthase (cleavage) activity in CML 32 roots and LM 11 shoots, under heat stress leading to optimization of their sucrose levels.

The aim of the study was an evaluation of the effect of exogenous application of 2-hydroxybenzoic acid on the performance of photosynthetic apparatus of two basket willow (Salix viminalis L.) varieties growing in elevated concentrations of cadmium salts and an evaluation of the suitability of fluorescence method to characterize the physiological state of the examined willow genotypes under stress conditions caused by an increased content of cadmium ions. The influence of cadmium ions at a concentration of 50 mg/dm³ and salicylic acid at a concentration of 138 mg/dm³ on the physiological reaction of basket willow of Bjor and Tora varieties cultivated in hydroponics was investigated. The applied dose of salicylic acid effectively eliminated the influence of cadmium, activating the synthesis of chlorophyll a and b and carotenoids as well as increasing the intensity of the determined fluorescence processes and indicators. The use of the fluorescent method to characterize the physiological state of the examined willow genotypes under the conditions of stress caused by an increased content of cadmium ions allows a better understanding of the immune system’s response to these metal ions.

This study investigated whether vanillic acid (VA) mitigated dehydration stress responses in blueberry (Vaccinium corymbosum L.), and analyzed potential mechanisms mediating this activity. We pretreated 2‑year-old blueberry plants with 40 μM VA for two days, and then induced dehydration stress by irrigating with nutrient solution containing 10% (w/v) polyethylene glycol 6000 for two days. VA pretreatment increased the transcript levels of genes encoding eight antioxidant enzymes in leaves, including iron superoxide dismutase, chloroplast copper/zinc superoxide dismutase, cytoplasmic copper/zinc superoxide dismutase, catalase, guaiacol peroxidase, glutathione peroxidase, glutathione reductase, and dehydroascorbate reductase. These increased transcript levels were consistent with enhanced activities of superoxide dismutase and glutathione peroxidase and elevated contents of reduced glutathione and ascorbate. Subjecting the V--A‑treated blueberry to dehydration stress further enhanced expression levels of these genes, compared with the control plants subjected only to dehydration stress, increased the contents of endogenous VA, proline, and soluble sugars, enhanced the relative water content and osmotic potential, and reduced the levels of superoxide anion, hydrogen peroxide, and malondialdehyde. So pretreatment of blueberry with VA reduces lipid peroxidation and mitigates dehydration stress by enhancing the endogenous VA content, activating the expression of antioxidant enzyme genes, and increasing the levels of proline and soluble sugars.

The preparative technique for water extraction of goat willow (Salix caprea L.) growing leaves was elaborated in order to stimulate Scots pine (Pinus sylvestris L.) seed germination by the extracts (patent no. 2662999). It was shown that the effects of the extract were accounted for by its biological activity, which considerably varied depending on the time of day for extract sampling. The maximal increase in gibberellin-like activity (equivalent to 3.4–3.6 µM GA₃) and marked decrease in ABA-like activity (equivalent to 0.15‒0.30 µM ABA) were recorded in the willow leaf extracts harvested in the morning (8:00 a.m.) or evening (8:00 p.m.) in South Karelia (60° N) in mid-May. Application of the extracts to pine seeds afforded the maximal effects on their germination. Thus, the second-class seeds displayed qualitative characteristics of the first-class seeds (germination exceeding 80%) after the treatment. It is concluded that the introduction of the method into practice will allow utilization of the accessible, widespread plant raw material for production of the effective natural preparation, which is capable of improving sowing qualities of Scots pine seeds.