This lecture presents classical information and new data on the molecular events of the "basic" (core) cell cycle (CC) of plants. The impact of water deficit, CO2, light, and temperature on CC is briefly examined. Data on the regulation of cell proliferation by auxins, cytokinins, abscisic acid, gibberellins, brassinosteroids, and ethylene are presented. Commonality and peculiarities of the effect of phytohormones on CC in various organs and tissues are discussed.

The review examines the principles in formation of cell culture and tissue collections of higher plants in vitro as a variant of genetic collections, the modern state and perspective of using collections in studies of the peculiarities and patterns of synthesis of biologically active substances of higher plants, the important role of collections in biotechnological production of substances for medicinal preparations, and methods for maintaining the stability of cell cultures during long-term cultivation. Particular attention is paid to the retrospective and current state of the All-Russia Collection of Cell Cultures of Higher Plants, the founder of which was Russian Academy of Sciences Corresponding Member R.G. Butenko.

The involvement of endocytosis in the Na+ ion uptake from the external medium by the cells of suspension culture derived from A. thaliana (Col-0) leaves was investigated. Na+ ion uptake by endocytic structures occurred following the addition of NaCl at the final concentration of 100 mM to the incubation medium. The presence of Na+ in membranous structures was recorded using fluorescence microscopy by colocalization of FM4-64, a marker of endocytosis structures, and Asante NaTRIUM Green-2 TMA+ salt (ANG-2 TMA), a membrane impermeable probe for sodium ions, that enabled the detection of Na+ absorbed by the cells via endocytosis but not through ion channels or transporters of the plasma membrane. Following a 1.5-h incubation of the cells in the presence of NaCl, FM4-64 and ANG-2 TMA, fluorescence of the probes was colocalized in structures with sizes ranging from 800 to 3000 nm. It was shown by electron microscopy that NaCl added to the cell incubation medium stimulated vesiculation and vacuolization of the cytoplasm, formation of plasma membrane invaginations, as well as fusion of microvacuoles with each other. The size of the structures, in which the colocalization of the two probes was detected by fluorescent microscopy, matched the size of the microvacuoles revealed by the electron microscopy. The obtained results indicate the capture of sodium ions contained in the apoplast by endocytosis invaginations, their subsequent internalization by the cells, and transfer into microvacuoles.

Effect of nitric oxide (NO) on phosphorylation of soluble proteins in the cell culture of wild-type Arabidopsis thaliana (L.) Heynh. (ecoptype Columbia, Col-0) was studied. Among the identified proteins whose phosphorylation was affected by the NO donor treatment, the enzymes of primary metabolism (glyceraldehyde-3-phosphate dehydrogenase, enolase) and regulatory proteins (14-3-3-like protein GF14ω, protein-disulfide isomerase-like protein, chaperonin-60α) were detected. The results clarify possible mechanisms of NO action on primary metabolism, cell cycle, and stress-induced responses of cultured plant cells.

The efficiency of transient gene expression in plants credibly demonstrated characteristics of gene functions in numerous studies. Two key strategies of transient expression became favorites among researchers: protoplast transfection and agroinfiltration. Each of them, alongside the advantages, has its own constraints. In this work, an easy, rapid, and reliable system for characterization of the signal sequences and determinations of target protein localization in a plant cell is proposed and tested. This system—called the AgI–PrI—implies production of protoplasts from plant tissues after agroinfiltration. Reliability of the proposed system for transient gene expression has been proved using characterized signal sequences in Nicotiana benthamiana cells. The corresponding protocol is less expensive and depends to a lesser degree on the professional skills in the area of protoplast isolation and transfection; furthermore, it may be applicable to other plant species with either available efficient methods of agroinfiltration and protoplast isolation or with the potential for one of the protocols to be supplemented. Thus, the AgI–PrI technique makes it possible to combine the advantages of two widely used methods for the transient gene expression in plants—agroinfiltration and protoplast isolation and transfection—and concurrently avoids their critical points.

Ethylene, being one of five classical plant phytohormones is involved in regulation of numerous physiological processes. There are contradictory data about the effect of ethylene on the cell growth and division; although it is accepted that in culture flasks, the content of ethylene rises to a few tens of μL/L and production of ethylene is associated with the periods of active growth of the cells in vitro. We revealed a strong correlation (r = 0.96) between ethylene production and specific rate of dry weight accumulation in suspension cell cultures of Ajuga turkestanica, heterotrophic and mixotrophic strains of Arabidopsis thaliana, Beta vulgaris, Euonymus maximoviczianus, Medicago sativa, Panax ginseng, and Triticum timopheevii. In heterotrophic cell culture of A. thaliana, the peaks and general shape of the curves describing dynamics of ethylene production, the number of S-phase cells, and specific rate of increase in cell number coincided in log phase and in the phase of growth deceleration. Pretreatment of subculture inoculum with 100 μL/L ethylene caused doubling of S-phase cell number after 3-h-long culturing in fresh nutrient medium. It was found that exogenous ethylene affects the number of S-phase cells only when the level of endogenously produced ethylene is low.

The work considers the possibility of reducing the decomposition temperature of MgCO3 in dolomite rock, provides the results of studies of the effect of various additives and enhancers on the decomposition of magnesium and calcium components of dolomite. Chlorides additives are the most promising for dolomite rocks roast intensification. They allow shifting the MgCO3 decomposition to lower temperatures, without exerting a significant influence on the decomposition of CaCO3. Introduction of additives-enhancers is found to be an effective method of controlling the properties of dolomite MOC during roasting, producing high-strength dolomite magnesium oxychloride cements with change in volume during solidification.

За последние годы получены результаты, позволяющие утверждать, что оксид азота (NO) – внутриклеточная сигнальная молекула, при помощи которой регулируются физиологические процессы на всех этапах жизненного цикла растений. Между тем, некоторые крайне важные аспекты биологии NO далеки от понимания. Так, существуют различные точки зрения в вопросе образования и утилизации NO у растений. Не до конца изучены механизмы восприятия и пути передачи сигнала NO, а также пока нет сведений о том, как обеспечивается специфичность, необходимая для координированного включения ответов на NO. Ответы на сформулированные вопросы представляется целесообразным искать, основываясь на знаниях, полученных при изучении особенностей функционирования NO у животных. Такой сравнительный анализ позволит выявить аналогии и подчеркнуть различия в современном понимании роли NO у растений. Рассмотрению этих аспектов посвящена данная лекция.

Research performed over the last few years identified nitric oxide (NO) as an intracellular signaling molecule involved in regulation of plant physiological processes at all stages of the life cycle. Nevertheless, some extremely important aspects of NO biology are still far from being clarified. There exist different points of view on NO formation and utilization in plants. The mechanisms of perception and transduction of the NO signal are not yet fully understood, and the origin of specificity underlying coordinated activation of responses to NO remains unresolved. It is reasonable to expect that the deep knowledge of NO functioning in animals may provide some keys to these questions. Such a comparative analysis is a way to reveal similarities and emphasize the differences in the current understanding of the NO role in plants. The present lecture highlights these aspects of NO functioning.

Этилен – один из пяти классических фитогормонов растений – участвует в регуляции многих физиологических процессов. Однако существуют противоречивые сведения о влиянии этилена на рост и деление клеток, хотя показано, что в культуральных сосудах содержание этилена увеличивается до нескольких десятков мкл/л, а продукция этилена связана с периодами активного роста клеток in vitro. Нами выявлена существенная корреляция (r = 0.96) между продукцией этилена и удельной скоростью увеличения сухого веса в суспензионных культурах клеток Ajuga turkestanica, гетеротрофного и миксотрофного штаммов Arabidopsis thaliana, Beta vulgaris, Euonymus maximoviczianus, Medicago sativa, Panax ginseng, Triticum timopheevii. Для гетеротрофной культуры клеток A. thaliana в логарифмической фазе и в фазе замедления роста показано совпадение максимумов и общего хода кривых, отражающих процессы динамики продукции этилена, доли S-фазных клеток и удельной скорости роста числа клеток. Предобработка исходного инокулята клеток 100 мкл/л этилена приводила через 3 ч их культивирования в свежей питательной среде к удвоению количества S-фазных клеток. Установлено, что экзогенный этилен влияет на количество S-фазных клеток лишь тогда, когда продукция эндогенного этилена мала.

Определены уровни гетерогенности и генетической изменчивости клеток в суспензии Arabidopsis thaliana, культивируемой in vitro более 7 лет. Показана значительная гетерогенность суспензии по размеру клеток. С помощью цитофотометрического анализа количества ядерной ДНК выявлена миксоплоидность суспензионной культуры. С использованием ДНК-маркеров (шести RAPD- и четырех ISSR-праймеров и их внутригрупповых комбинаций) показана низкая степень вариабельности ДНК. Генетические отличия полученных из суспензионной культуры клонов от растения инбредной линии Col-0 составили 1.5%. Различия между клонами были выявлены только с одной парой праймеров (M3+M7). Отмечено, что вариации количества ДНК в клетках суспензионной культуры не сопровождаются существенными изменениями последовательности ДНК.

Beginning with the pioneering work of Salic and Mitchison (2008), the application of thymidine analogue 5-ethynyl-2′-deoxyuridine (EdU) for the detection of cells replicating DNA is actively expanding. Being incorporated into DNA, this nucleoside after click reaction of azide-alkyne cycloaddition with azides of fluorochromes can be easily detected by fluorescence. Recently, protocols of EdU application in combination with click reaction adapted for plant cells appeared, and they are help for a monitoring S-period of the cell cycle in the root meristems and in vitro cultured cells with the help of a microscope and flow cytometer. In this work, we focused some details of developed methods and their modifications and also recommended new protocols. In particular, we suggested combining EdU incorporation into the cells replicating DNA with subsequent isolation of protoplasts from them and their preparation for the microscopic analysis and flow cytometry. In addition, the method of determination of EdU phosphorylation dynamics in the cells in vivo is suggested.

Contradictory data about ethylene influence on cell growth and division prompted us to investigate cytophysiological characteristics of suspension cultures of Arabidopsis thaliana of wild type Col-0 and ert1-1 mutant carrying a point mutation in the site of ethylene binding by the ETR1 receptor. Some cytophysiological characteristics of the etr1-1 cultivated cells differed from those of Col-0: the growth rate of mutant cells was less and cell sizes were smaller, the culture was committed to the formation of tracheary elements (TE), had a pronounced modal class of nuclei (54%) with the amount of DNA 8C and a tendency to expand the ploidy toward 32C. Despite the absence of ethylene perception by the ETR1 receptor, the cell culture of mutant responded to treatment with ethylene by growth acceleration, an increase in cell viability and in the number of cells in the S-phase of the cell cycle. The inhibitor of ethylene binding to receptors, 1-methylcyclopropene, suppressed growth and viability of the cells of both genotypes. In the etr1-1 cell culture, the inhibitor reduced the number of S-phase nuclei and activated TE formation. All data obtained indicate that ethylene perception and transduction of ethylene signal are required for the maintenance of cell viability and active in vitro growth. It is supposed that the functional activity of the ETR1 receptor is necessary for optimal cell expansion, whereas other receptors are responsible for cell proliferation.