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Laboratory of Cell Regulation
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Dmitry A. LOS
Professor
losda[at]ippras.ru |
Anna Zorina
PhD |
Elena Kupriyanova
PhD |
Kirill Mironov
PhD |
Galina Novikova
DSc |
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| Alexander Starikov |
Anna Kostistyna |
Pavel Leusenko |
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Membrane Fluidity and Gene Expression
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Poikilothermic organism often face experience changes in environmental parameters, and their survival directly depends on their adaptation abilities. Changes in ambient temperature lead to changes in membrane fluidity. These alterations are necessary to switch on the response of cells to cold stress and for the following cold acclimation. The molecular mechanisms responsible for the perception of changes in membrane fluidity are not fully characterized. The application of site-directed mutagenesis that allows genetic changes in membrane fluidity, and analysis of expression of the entire genome with DNA microarrays (NIBB, Japan) have lead to significant progress in understanding the mechanisms of regulation of membrane fluidity, namely, to identification of the sensor, which might perceive the changes in membrane fluidity (Vigh et al., 1993; Murata & Los, 1997; Los & Murata, 2000; 2004; Los et al., 2008). We are also studying the role of polyunsaturated fatty acids of membrane lipids in regulation of expression of the genes in the cyanobacterium Synechocystis (Inaba et al., 2003; Los & Murata, 2004; Los & Zinchenko, 2009; Mironov et al., 2012a, 2012b).
Fatty acids desaturases are enzymes that form double bonds between carbon atoms in the chains of fatty acids. The formation of double bonds, in turn, provides an increase in membrane lipid fluidity at low temperatures (Los & Murata 1998; 2004). We cloned several genes for fatty acid desaturases from different organisms, including cyanobacteria (Synechocystis sp., Synechococcus vulcanus, Gloeobacter violaceus, Prochlorothrix hollandica) and microalgae (Dunaliella salina), are characterized by their specificity and expression profiles.
The desaturase genes from cyanobacteria have been successfully expressed in higher plants (tobacco, different varieties of potatoes). Increasing the degree of unsaturation of the fatty acids of membrane lipids resulting in increased cold resistance and cold hardiness of plants (Kiseleva etal. 2000; Orlova et al. 2003; Maali et al. 2010). |
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Sensors and transducers of stress signals in cyanobacteria
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| In cyanobacteria, histidine kinases, response regulators, serine-threonine protein kinases and sigma factors of RNA polymerase, are the primary candidates for the role of sensor proteins that percieve and transmit the signals about changes in the environment. The genome of Synechocystis carries 44 genes for histidine kinases (Hiks), and 3 additional hik genes are located in the plasmids. Potential response regulators are encoded by 42 genes in the chromosome and by 3 genes in plasmids. In addition to two-component regulatory systems, there are 12 genes for eukaryotic-type serine-threonine protein kinases, and 58 genes that encode DNA-binding transcription factors. Almost all of these genes are mutated and their functional role in stress responses have been studied. Thus, the low temperature sensor have been identified (Suzuki et al. 2000), as well as salt and hyperosmotic stress (Paithoonrangsarid et al. 2004; Shoumskaya et al. 2005) sensor. The two-component system for the deficiency in manganese (Yamaguchi et al. 2002) yfve been discovered. The key role of serine-threonine kinase SpkA was demonstrated for cell motility (Panichkin et al. 2006). The studies on the functions of other serine-threonine protein kinases in Synechocytis are under way (Zorina et al. 2011a). The important role of changes in genomic DNA supercoiling in the regulation of stress response genes (Los 2004; Prakash et al. 2009) had been revealed. The research results are summarized in a review articles (Los et al. 2010; Zorina et al. 2011b) and in a book «Sensor systems of cyanobacteria» (Los 2010) published in Russian. |
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Ionic and water channels of cyanobacteria: the roles in perception and transduction of stress signals
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The role of mechanosensory ion channel MscL in stress responses in the freshwater cyanobacterium Synechocystis have been defined. Sharp drop in temperature or ionophore valinomycin in the presence of potassium ions cause rapid depolarization of the cytoplasmic membrane of Synechocystis. MscL functions as the verapamil/amiloride-sensitive calcium channel required for the release of calcium ions under stress conditions that cause membrane depolarization (Nazarenko et al. 2003).
The water channel AqpZ is encoded by a single gene in the genome of Synechocystis. AqpZ is used for rapid release of water from the cytoplasm of cells under hyperosmotic and salt stress. Mutants deficient in this channel can not quickly change the cell volume. The latter ability is important for the expression of genes required for cells to acclimatize to new habitat conditions (Shapiguzov et al. 2005). |
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Phytohormone interactions in the regulation of cellular processes and MAPK cascades in relation to phytohormone signal transduction
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We are currently focused on phytohormonal signaling in intact plants and in plant cell cultures. Our particular interest is ethylene, because it plays a pivotal role in plant development. The chemical simplicity of the gaseous hormone ethylene contradicts somehow with the complexity of the physiological reactions it regulates. The use of Arabidopsis ethylene response mutants has led to the elucidation of ethylene signal transduction chain, which includes five partially functionally redundant receptors and the putative MAPKKK (CTR1). The ethylene receptors and CTR1 operate as negative regulators. The physical interaction between the receptors and CTR1 in the absence of ethylene keeps downstream signaling components (EIN2 and EIN3) inactive. Our findings have substantially changed this view. We propose several regulatory modules in addition to the basic signaling pathways. These modules reveal new mechanisms to modulate ethylene signaling. Thus, the pathway of ethylene signal transduction appears to be more complex than it was considered before. There is no doubt that the presently identified components play their roles in a scene, they do not cover the whole scenario.
Recently, we have studied actively proliferating Arabidopsis cells of wild type and of three ethylene-insensitive mutants, etr1-1, ctr1-1 and ein2- 1. In this system, ABA affects ethylene biosynthesis, mitotic activity, synthesis of nuclear DNA, growth and differentiation of the cells. The analysis of MAPK gene expression and studies of phosphoproteome allowed us to conclude that in addition to generally accepted view of the mutual influence of ethylene and ABA on the synthesis of each other, the interaction between ABA and ethylene signaling pathways exists with the commonly shared component – MPK6. Thus, in biological models, where different external factors cause the enhancement in ethylene biosynthesis, the activation (switching on) of the ABA signal transduction pathway is required for correct DNA synthesis, maintenance of mitotic activity and subsequent cell proliferation. |
Carbonic anhydrases of the relict cyanobacteria
The basis for modern biosphere was formed about 2 billion years ago, when prokaryotes were predominant and the only organisms on Earth. Ancient cyanobacteria converted the early reducing atmosphere into an oxygen atmosphere by binding of large amounts of CO2 in a form of carbonates and by releasing of O2 during photosynthesis. Participation of cyano-bacterial community in the binding of atmospheric CO2 in the early stages of Earth’s history is confirmed by the presence in the sedimentary rocks of stromatolites — layered deposits of limestone, which are composed of lithified cyano-bacterial communities similar to the modern benthic mats.
At present, the formation of stromatolites, with the participation of cyano-bacterial community is a small-scale process, geomicrobiological mechanisms of which are poorly understood. So far it is unclear whether any physiological processes were taking part in the mineralization of cyano-bacterial community, or this mineralization is the consequence of only chemical sedimentation. We have suggested that the enzyme carbonic anhydrase (CA) may play the key role in the mechanism of biomineralization, which regulates the equilibrium in forms of inorganic carbon, including bicarbonate that participats in natural calcium deposition (Kupriyanova et al., 2007).
The biological role of the CA, which is defined as catalysis of hydration-dehydration of carbon dioxide, is very diverse. This enzyme is involved in such fundamental processes as photosynthesis, respiration, transport of inorganic carbon compounds and ions, regulation of acid-base balance. According to modern taxonomy of CAs, they are divided into three main classes (alpha, beta and gamma) with no significant homology in their amino acid sequences. It is suggested that these classes of CAs evolved independently from each other.
Currently, CAs have been found in all groups of living organisms, where they are involved in a number of physiological processes, including deposition of calcium carbonate. Since prokaryotes are characterized by extracellular precipitation of calcium carbonate, which is controlled by pH of the environment, it is likely that CAs are localized in the outer layers of cyanobacteria, where they may participate in stabilization of pH and salinity around the cells. Thus, the enzymatic activity of the SC could also contribute to the formation of Precambrian stromatolites (Kupriyanova et al., 2007; 2011).
The microbial communities of extreme habitats, where no higher organisms are present, may serve as very convenient model systems for studying the procell of stromatolite formation. These are the thermophilic communities of hot springs, halophils of saline lagoons, alkalophils of soda lakes. Such communities are called relict ecosystems and they are considered to be the analogous to the ancient ecosystems.
Also, the research of CA properties of relict organisms may be of interest for understanding the evolution of three classes of CAs (Kupriyanova et al., 2003, 2004; Kupriyanova & Pronina, 2011).
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Suleyman I. Allakhverdiev
Professor
suleyman.allakhverdiev[at]gmail.com |
Vladimir S. Bedbenov
PhD |
Natalia A. ronina
DSc, Professor |
Roman A. Voloshin
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Margarita V. Rodionova
PhD Student
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Vladimir D. Kreslavski
D.Sci |
David A. Gabrielyan
PhD |
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Stress physiology of photosynthesis and molecular mechanisms of stress resistance of the photosynthetic apparatus
A new type of PSII repair have been discovered and described in the dark (without de novo synthesis of the D1 protein ) after photoinactivation in intact Synechocystis cells at low temperatures (0°- 10° C). Salt stress and reactive oxygen species (H2O2, 1O2) inhibit the de novo synthesis of D1 protein under conditions of photoinactivation due to inhibition of transcription and translation of the psbA gene, which encods the precursor of the D1 protein.
It was shown that glycine betaine protects not only oxygen evolution in the PSII particles in vitro, but also other reactions associated with electron transport, including the P680 photooxidation and photoreduction of pheophytin under conditions of thermo- and photoinactivation. Organisms that can synthesize glycine betaine, are characterized by high resistance of PSII to thermal and photoinactivation, as shown in the in vivo model system of Synechococcus transformed with the codA gene for choline oxidase, which is responsible for the synthesis of glycine betaine.
A model of inactivation of PSII under salt and hyperosmotic stress has been proposed. The existence of two phases (fast reversible and slow irreversible) of inactivation of PSII under salt stress, and the existance of only one fast reversible phase of inactivation under osmotic stress have been postulated. The reversible phase is caused by the osmotic effect of salt or osmolytes, whereas an irreversible phase appears due to the action of Na+ that destroy the apparatus, which is necessary for the restoration of the photosystems after salt stress. Salt and hyperosmotic stresses affect the expression of different sets of genes required for adaptation of Synechocystis. Salt stress activates slr1390 and slr1604 genes for metalloprotease FtsH, which participates in degradation of the damaged D1 protein in PSII, and for the protease CtpA, which catalyzes the cleavage of the C-terminal part from the precursor of the D1 protein. Induction of stpA gene family does not happen under hyperosmotic stress. The essential role of water channels in acclimation of the PSII to hyperosmotic stress have been demonstrated.
The important role of the unsaturated fatty acids in resistance of PSII to salt stress have been determined. Cells that can synthesize polyunsaturated FAs are more resistant to NaCl . Mutant cells of Synechocystis, defective in desA and desD genes, for ?12-and ?6-desaturase, has no polyunsaturated FAs and, consequently, is characterized by hypersensitivity to salt stress. The increase in the content of unsaturated fatty acids in the membranes of Synechococcus, transformed with the gene for the ?12-desaturase, greatly enhances the stability of PSII to salt stress.
Carbonic anhydrases and CO2-concentrating mechanisms
Low concentration of CO2 in the atmosphere limits the photosynthetic efficiency of plants, and especially aquatic algae, because of the slow rate of diffusion of CO2 in water. Earlier, it was shown that adaptation of algae to low content of CO2 is associated with the induction of CO2-concentrating mechanism (CCM) in photosynthetic microorganisms, different from the previously known C4 or CAM photosynthesis. This mechanism involves the induction of the enzyme carbonic anhydrase (CA) and active transport of inorganic carbon (Ci) into the cell. These studies were complemented by studies on the localization of CA in cells of Chlamydomonas reinhardtii, which showed that one form of CA is localized in the chlorophyll-protein complex of PSII and oriented into the lumen. These data are consistent with the proposed hypothesis on the involvement of thylakoid CA in concentration of molecular form of CO2 in the stroma by catalytic conversion of bicarbonate into CO2 in the lumen, followed by its transfer into the stroma along with the concentration gradient. The mutant of C. reinhardtii, CIA-3, deficient in the activity of PSII-CA is unable to grow at a low concentration of CO2, although it can accumulate Ci cells. We also demonstrate the importance of PSII-CA for the functioning of the CCM in microalgae. Western blotting demonstrated the presence of PSII-CA in Dunaliella salina, spinach, and Arabidopsis. Studies of the kinetic characteristics of this enzyme in D. salinademostrated that this CA functions at acidic pH of ??lumen. The activity of PSII and of electron transport chain of chloroplast is inhibited by specific inhibitors of the CA. The obtained results suggest the interaction of CCM and O2-evolving systems of the chloroplast (Sinetova et al. 2012).
Biotechnology of microalgae and cyanobacteria
We developed and constructed photobioreactors for intensive cultivation of different strains of microalgae and cyanobacteria. The technology of biomass production enriched with stable isotopes was developed. A number of algal strains have been selected for controlled production of b-carotene, phycoerythrin, phycocyanin, long-chain polyunsaturated fatty acids, and steroid compounds. The industrial technology have been developed for production of Spirulina enriched with biogenic iodine, selenium, and zinc — essential microelements for nutrition. |
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