Twelve genes for the potential serine-threonine protein kinases (STPKs) have been annotated in the genome of Synechocystis sp. PCC 6803. Based on similarities and distinctive domain organization, they were divided into two clusters: serine/threonine-protein N2-like kinases (PKN2-type) and "activity of bc1 complex" kinases (ABC1-type). While the activity of the PKN2-type kinases have been demonstrated, no ABC1-type kinases activity have hitherto been reported. In this study, a recombinant protein previ-ously annotated as a potential STPK of ABC1-type (SpkH, Sll00 05) was expressed and purified to ho-mogeneity. We demonstrated SpkH phosphorylating activity and substrate preference for casein in in vitro assays using [g-32P]ATP. Detailed analyses of activity showed that Mn2+ had the strongest acti-vation effect. The activity of SpkH was significantly inhibited by heparin and spermine, but not by staurosporine. By means of semi-quantitative mass-spectrometric detection of phosphopeptides, we identified a consensus motif recognized by this kinase -X1X2pSX3E. Thus, we first report here that SpkH of Synechocystis represents a true active serine protein kinase, which shares the properties of casein kinases according to its substrate specificity and sensitivity to some activity effectors. & COPY; 2023 Elsevier B.V. and Societe Francaise de Biochimie et Biologie Moleculaire (SFBBM). All rights reserved.

Twelve genes for the potential serine-threonine protein kinases (STPKs) have been annotated in the genome of Synechocystis sp. PCC 6803. Based on similarities and distinctive domain organization, they were divided into two clusters: serine/threonine-protein N2-like kinases (PKN2-type) and “activity of bc1 complex” kinases (ABC1-type). While the activity of the PKN2-type kinases have been demonstrated, no ABC1-type kinases activity have hitherto been reported. In this study, a recombinant protein previously annotated as a potential STPK of ABC1-type (SpkH, Sll0005) was expressed and purified to homogeneity. We demonstrated SpkH phosphorylating activity and substrate preference for casein in in vitro assays using [γ-32P]ATP. Detailed analyses of activity showed that Mn2+ had the strongest activation effect. The activity of SpkH was significantly inhibited by heparin and spermine, but not by staurosporine. By means of semi-quantitative mass-spectrometric detection of phosphopeptides, we identified a consensus motif recognized by this kinase – X1X2pSX3E. Thus, we first report here that SpkH of Synechocystis represents a true active serine protein kinase, which shares the properties of casein kinases according to its substrate specificity and sensitivity to some activity effectors.

Recent studies indicate direct links between molecular cell cycle and cell differentiation machineries. Ethylene and abscisic acid (ABA) are known to affect cell division and differentiation, but the mechanisms of such effects are poorly understood. As ethylene and ABA signaling routes may interact, we examined their involvement in cell division and differentiation in cell tissue cultures derived from several Arabidopsis thaliana plants: wild type (Col-0), and ethylene-insensitive mutants etr1-1, ctr1-1, and ein2-1. We designed an experimental setup to analyze the growth-related parameters and molecular mechanisms in proliferating cells upon short exposure to ABA. Here, we provide evidence for the ethylene–ABA signaling pathways’ interaction in the regulation of cell division and differentiation as follows: (1) when the ethylene signal transduction pathway is functionally active (Col-0), the cells actively proliferate, and exogenous ABA performs its function as an inhibitor of DNA synthesis and division; (2) if the ethylene signal is not perceived (etr1-1), then, in addition to cell differentiation (tracheary elements formation), cell death can occur. The addition of exogenous ABA can rescue the cells via increasing proliferation; (3) if the ethylene signal is perceived, but not transduced (ein2-1), then cell differentiation takes place—the latter is enhanced by exogenous ABA while cell proliferation is reduced; (4) when the signal transduction pathway is constitutively active, the cells begin to exit the cell cycle and proceed to endo-reduplication (ctr1-1). In this case, the addition of exogenous ABA promotes reactivation of cell division.

Here, for the first time, we report the presence of highly active extracellular carbonic anhydrase (CA) of alpha-class in cyanobacterial cells. The enzyme activity was confirmed both in vivo in intact cells and in vitro, using the recombinant protein. CA activity in intact cells of Cyanothece sp. ATCC 51142 reached similar to 0.6 Wilbur-Anderson units (WAU) per 1 mg of total cell protein, and it was inhibited by a specific CAs inhibitor, ethoxyzolamide. The genes cce_4328 (ecaA) and cce_0871 (ecaB), encoding two potential extracellular CAs of Cyanothece have been cloned, and the corresponding proteins EcaA and EcaB, representing CAs of alpha- and beta-class, respectively, have been heterologously expressed in Escherichia coli. High specific activity (similar to 1.1 x 10(4) WAU per 1 mg of target protein) was detected for the recombinant EcaA only. The presence of EcaA in the outer cellular layers of Cyanothece was confirmed by immunological analysis with antibodies raised against the recombinant protein. The absence of redox regulation of EcaA activity indicates that this protein does not possess a disulfide bond essential for some alpha-class CAs. The content and activity of EcaA in a fraction of periplasmic proteins was higher in Cyanothece cells grown at ambient concentration of CO2 (0.04%) compared to those grown at an elevated CO2 concentration (1.7%). At the same time, the level of ecaA gene mRNA varied insignificantly in response to changes in CO2 supply. Our results indicate that EcaA is responsible for CA activity of intact Cyanothece cells and point to its possible physiological role under low-CO2 conditions. (C) 2019 Elsevier B.V. and Societe Francaise de Biochimie et Biologie Moleculaire (SFBBM). All rights reserved.

Ethylene is known to influence the cell cycle (CC) via poorly characterized roles whilst nitric oxide (NO) has well-established roles in the animal CC but analogous role(s) have not been reported for plants. As NO and ethylene signaling events often interact we examined their role in CC in cultured cells derived from Arabidopsis thaliana wild-type (Col-0) plants and from ethylene-insensitive mutant ein2-1 plants. Both NO and ethylene were produced mainly during the first 5 days of the sub-cultivation period corresponding to the period of active cell division. However, in ein2-1 cells, ethylene generation was significantly reduced while NO levels were increased. With application of a range of concentrations of the NO donor, sodium nitroprusside (SNP) (between 20 and 500 mu M) ethylene production was significantly diminished in Col-0 but unchanged in ein2-1 cells. Flow cytometry assays showed that in Col-0 cells treatments with 5 and 10 mu M SNP concentrations led to an increase in S-phase cell number indicating the stimulation of G1/S transition. However, at >= 20 mu M SNP CC progression was restrained at G1/S transition. In the mutant ein2-1 strain, the index of S-phase cells was not altered at 5-10 mu M SNP but decreased dramatically at higher SNP concentrations. Concomitantly, 5 mu M SNP induced transcription of genes encoding CDKA; 1 and CYCD3; 1 in Col-0 cells whereas transcription of CDKs and CYCs were not significantly altered in ein2-1 cells at any SNP concentrations examined. Hence, it is appears that EIN2 is required for full responses at each SNP concentration. In ein2-1 cells, greater amounts of NO, reactive oxygen species, and the tyrosine-nitrating peroxynitrite radical were detected, possibly indicating NO-dependent post-translational protein modifications which could stop CC. Thus, we suggest that in Arabidopsis cultured cells NO affects CC progression as a concentration-dependent modulator with a dependency on EIN2 for both ethylene production and a NO/ethylene regulatory function.

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.

Life strategy of plants depends on successful seed germination in the available environment, and sufficient soil water is the most important external factor. Taking into account a broad spectrum of roles played by water in seed viability and its maintenance during germination, the review embraces early germination events in seeds different in their water status. Two seed types are compared, namely orthodox and recalcitrant seeds, in terms of water content in the embryonic axes, vacuole biogenesis, and participation of water channels in membrane water transport. Mature orthodox seeds desiccate to low water content and remain viable during storage, whereas mature recalcitrant seeds are shed while well hydrated but die during desiccation and cannot be stored. In orthodox Vicia faba minor air-dry seeds remaining viable at 8–10% water content in embryonic axes, the vacuoles in hypocotyl are preserved as protein storage vacuoles, then restored to vacuoles in imbibing seeds in the course of protein mobilization. However, in newly produced meristematic root cells, the vacuoles are formed de novo from provacuoles. In recalcitrant Aesculus hippocastanum seeds, embryonic axes have a water content of 63–64% at shedding and they lack protein storage vacuoles but preserve vacuoles preformed in maturing seeds. Independent of the vacuolar biogenetic patterns, their further trend is similar; they expand and fuse, thus producing an osmotic compartment, which precedes and becomes an obligatory step for the initiation of cell elongation. Prior to this, water moves in imbibing seeds through the membranes by diffusion, although the aquaporins forming water channels are present. In both seed types, water channels are opened and actively participate in water transport only after growth initiation. Aquaporin gene expression and their composition change in broad bean embryonic axes after growth initiation. This is the way how a mass water flow into growing seedling cells is achieved, independent of differences in seed water content and vacuole biogenesis patterns.

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.

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.

Ответ на стрессы у одноклеточной цианобактерии Synechocystis осуществляется при помощи нескольких регуляторных систем, например, двухкомпонентных, а также посредством отрицательной сверхспирализации геномной ДНК. В этой работе исследовано участие серин-треониновых протеинкиназ (СТПК) в реакции клеток цианобактерии Synechocystis на холодовой стресс. Скрининг коллекции мутантов по генам СТПК выявил группу из четырех протеинкиназ: SpkB, SpkD, SpkE, SpkG – как возможных регуляторов транскрипции при действии низкой температуры. Результаты исследования протеома бактерии Synechocystis, мутантной по SpkE, свидетельствуют о вкладе этой протеинкиназы в формирование белкового профиля. Реакция фосфорилирования in vitro рекомбинантного белка SpkE подтвердила, что это активная протеинкиназа с явным предпочтением основных белков в качестве субстратов.

Stress responses of the unicellular cyanobacterium Synechocystis involve several regulatory systems, including two-component ones, and negative supercoiling of genomic DNA. The role of serine/threonine protein kinases (STPKs) in the cold response was studied in Synechocystis. A screening of a collection of STPK mutants identified four enzymes-SpkB, SpkD, SpkE, and SpkG-as possible transcriptional regulators at lower temperatures. A proteome analysis in a SpkE Synechocystis mutant implicated SpkE in the formation of the protein pattern. In vitro phosphorylation assays of recombinant SpkE confirmed that the STPK was functionally active and utilized basic proteins as preferable substrates.