[THE EFFECT OF ACID RAIN ON ULTRASTRUCTURE AND FUNCTIONAL PARAMETERS OF PHOTOSYNTHETIC APPARATUS OF PEA LEAVES].

The effects of simulated acid rain (SAR) on the ultrastructure and functional parameters of the photosynthetic apparatus were studied using 14-day-old pea leaves as test system. Pea plants were sprayed with an aqueous solution containing NaNO3(0.2 mM) and Na2SO4(0.2 mM) (pH 5.6, a control variant), or with the same solution, which was acidified to pH 2.5 (acid variant). Functional characteristics were determined by chlorophyll fluorescence analysis. Acid rain application caused reduction in the efficiency of the photosynthetic electron transport by 25%, which was accompanied by an increase by 85% in the quantum yield of thermal dissipation of excess light quanta. Ultrastructural changes in chloroplast were registered by transmission electron microscopy (TEM) after two days of the SAR-treatment of pea leaves. In this case, the changes in the structure of grana, heterogeneity of thylakoids packaging in granum, namely, the increase of intra-thylakoid gaps and thickness of granal thylakoids compared to the control were found. The migration of protein complexes in thylakoid membranes of chloroplasts isolated from leaves treated with SAR was suppressed. It was shown also that carbonic anhydrase activity was inhibited in chloroplast preparations isolated from SAR-treated pea leaves. We proposed a hypothesis on the possible inactivation of thylakoid carbonic anhydrase under SAR and its involvement in the inhibition of photochemical activity of chloroplasts. The data obtained allows to suggest that acid rains negatively affect the photosynthetic apparatus disrupting the membrane system of chloroplast.

Biological effects due to weak magnetic field on plants.

Throughout the evolution process, Earth's magnetic field (MF, about 50 microT) was a natural component of the environment for living organisms. Biological objects, flying on planned long-term interplanetary missions, would experience much weaker magnetic fields, since galactic MF is known to be 0.1-1 nT. However, the role of weak magnetic fields and their influence on functioning of biological organisms are still insufficiently understood, and is actively studied. Numerous experiments with seedlings of different plant species placed in weak magnetic field have shown that the growth of their primary roots is inhibited during early germination stages in comparison with control. The proliferative activity and cell reproduction in meristem of plant roots are reduced in weak magnetic field. Cell reproductive cycle slows down due to the expansion of G1 phase in many plant species (and of G2 phase in flax and lentil roots), while other phases of cell cycle remain relatively stable. In plant cells exposed to weak magnetic field, the functional activity of genome at early pre-replicate period is shown to decrease. Weak magnetic field causes intensification of protein synthesis and disintegration in plant roots. At ultrastructural level, changes in distribution of condensed chromatin and nucleolus compactization in nuclei, noticeable accumulation of lipid bodies, development of a lytic compartment (vacuoles, cytosegresomes and paramural bodies), and reduction of phytoferritin in plastids in meristem cells were observed in pea roots exposed to weak magnetic field. Mitochondria were found to be very sensitive to weak magnetic field: their size and relative volume in cells increase, matrix becomes electron-transparent, and cristae reduce. Cytochemical studies indicate that cells of plant roots exposed to weak magnetic field show Ca2+ over-saturation in all organelles and in cytoplasm unlike the control ones. The data presented suggest that prolonged exposures of plants to weak magnetic field may cause different biological effects at the cellular, tissue and organ levels. They may be functionally related to systems that regulate plant metabolism including the intracellular Ca2+ homeostasis. However, our understanding of very complex fundamental mechanisms and sites of interactions between weak magnetic fields and biological systems is still incomplete and still deserve strong research efforts.

Changes in calcium signalling, gravitropism, and statocyte ultrastructure in pea roots induced by calcium channel blockers.

The effect of Ca2+ channel blockers (D600 and nicardipine) were investigated in our experiments on 5-day seedlings of pea. Nicardipine had more inhibiting effect on root elongation than D600. The Ca2+ channel blockers (CCB) depressed gravitropic response in roots. In root statocytes, the destruction of the polar arrangement of cell organelles and other changes were induced by 10(-5) M D600 or nicardipine treatment for 12 h. At ultrastructural level, there were observed a lack of polarity, pronounced vacuolization, and changes in dictyosome structure in treated statocytes. Cytochemical study indicated that Ca2+ ions were concentrated in the intracellular organelles and cell walls in statocytes treated with CCB similar to untreated control. The data suggest that the effects of the CCB that demonstrated the correlation between the loss of polarity in statocytes and altered root gravitropism may be functionally related to systems that regulate Ca2+ homeostasis, particularly Ca2+ channels.

Lithium-induced changes in gravicurvature, statocyte ultrastructure and calcium balance of pea roots.

Calcium signaling has been implicated in plant graviperception. In order to investigate the role of intracellular calcium in the process, I used lithium ions (LiCl), which suppress inositol 1,4,5-trisphosphate (IP3) cycling and signaling by inhibiting inositol-1-phosphatase. After 4 h of gravistimulation, no curvature was observed in 81% of the roots of 5-day Pisum sativum seedlings pretreated with 5 mM LiCl. Structural features of statocyte ultrastructure in these roots were the following: loss of a cellular polarity, appearance of amyloplast clusters, condensed mitochondria, local dilations in a perinuclear space, increases in a relative volume of vacuoles. The intensity of a cytochemical reaction (pyroantimonate staining which detected Ca2+ ions) was moderate: the Ca2+ pyroantimonate deposits were observed in all organelles. There were few granules of this precipitate in a hyaloplasm of the statocytes. Mitochondria and vacuoles were found to contain more granules of the precipitate compared with the controls. Additionally, Ca(2+)-ATPase activity in the statocytes of pea roots pretreated with LiCl was approximately the same as in control roots. Data obtained by using inhibitor of inositol signaling suggest that the observed effects of LiCl on root gravicurvature and ultrastructure of root statocytes were due to effects on Ca2+ homeostasis, particularly on IP3-mediated release of intracellular Ca2+ which can be inhibited by inositol depletion. The work demonstrates the key role played by second messengers (Ca2+ and IP3) in a gravity perception and response.

Gravicurvature loss, changes in ultrastructure and calcium balance of pea root statocytes treated with EGTA.

Calcium is known to play a key role in the regulation of a wide variety of cellular events in plants, in particular as a second messenger in the transduction of gravity signals in statocytes. In order to investigate the role of extracellular calcium in gravity perception, many investigators have used ethylene glycol-bis-(aminoethyl ether)-N,N'-tetraacetic acid (EGTA), whose main property is the ability to form stable water-soluble complex compounds with divalent metals, particularly with Ca2+ ions, that do not penetrate across plasma membrane into a cell.

Ultrastructure and calcium balance in meristem cells of pea roots exposed to extremely low magnetic fields.

Investigations of low magnetic field (LMF) effects on biological systems have attracted attention of biologists due to planned space flights to other planets where the field intensity does not exceed 10(-5) Oe. Pea (Pisum sativum L.) seeds were grown in an environment of LMF 3 days. In meristem cells of roots exposed to LMF, one could observe such ultrastructural peculiarities as a noticeable accumulation of lipid bodies, development of a lytic compartment (vacuoles, cytosegresomes and paramural bodies), and reduction of phytoferritin in plastids. Mitochondria were the most sensitive organelle to LMF application. Their size and relative volume in cells increased, matrix was electron-transparent, and cristae reduced. Because of the significant role of calcium signalling in plant responses to different environmental factors, calcium participation in LMF effects was investigated using a pyroantimonate method to identify the localization of free calcium ions. The intensity of cytochemical reaction in root cells after LMF application was strong. The Ca2+ pyroantimonate deposits were observed both in all organelles and in a hyaloplasm of the cells. Data obtained suggest that the observed LMF effects on ultrastructure of root cells were due to disruptions in different metabolic systems including effects on Ca2+ homeostasis.

Calcium balance in pea root statocytes under both clinorotation and Ca2+ channel blockers' influence.

We have previously demonstrated that space flight and clinorotation conditions increase cytoplasmic Ca2+ level in pea root statocytes. A rise in [Ca2+]i may be a serious problem for plants in microgravity environment. It is hypothesized that involvement of Ca2+ channel blockers in the growth medium may rescue a plant from abundance of Ca2+ ions. Indeed, combination of clinorotation (2 rpm, 5 days) and any Ca2+ channel blocker (1 micromole D600 or nicardipine, 12 hr) causes decreasing the Ca2+ concentration in pea root statocytes in comparison with clinorotation alone. Redistribution of Ca(2+)-ATPase activities observed under clinorotation comes to normal after D600 application whereas following by nicardipine action the pattern of the cytochemical staining is intermediate between those in stationary control and under clinorotation. Our data support the hypothesis that Ca2+ channel blockers may act as protectors for plants against rise in [Ca2+]i. The role for Ca2+ channels in graviperception and in microgravity effects as well as ways for stabilization of Ca2+ balance in plant cells in space flights are discussed.

Free and membrane-bound calcium in microgravity and microgravity effects at the membrane level.

The changes of [Ca2+]i controlled is known to play a key regulatory role in numerous cellular processes especially associated with membranes. Previous studies from our laboratory have demonstrated an increase in calcium level in root cells of pea seedlings grown aboard orbital station "Salyut 6". These results: 1) indicate that observed Ca(2+)-binding sites of membranes also consist in proteins and phospholipids; 2) suggest that such effects of space flight in membrane Ca-binding might be due to the enhancement of Ca2+ influx through membranes. In model presented, I propose that Ca(2+)-activated channels in plasma membrane in response to microgravity allow the movement of Ca2+ into the root cells, causing a rise in cytoplasmic free Ca2+ levels. The latter, in its turn, may induce the inhibition of a Ca2+ efflux by Ca(2+)-activated ATPases and through a Ca2+/H+ antiport. It is possible that increased cytosolic levels of Ca2+ ions have stimulated hydrolysis and turnover of phosphatidylinositols, with a consequent elevation of cytosolic [Ca2+]i. Plant cell can response to such a Ca2+ rise by an enhancement of membranous Ca(2+)-binding activities to rescue thus a cell from an abundance of a cytotoxin. A Ca(2+)-induced phase separation of membranous lipids assists to appear the structure nonstable zones with high energy level at the boundary of microdomains which are rich by some phospholipid components; there is mixing of molecules of the membranes contacted in these zones, the first stage of membranous fusion, which was found in plants exposed to microgravity. These results support the hypothesis that a target for microgravity effect is the flux mechanism of Ca2+ to plant cell.

Root-hair response to vector-free gravity: role of Ca2+ ions.

In plants, apical growth is demonstrated by a variety of cells, including root hairs (RH) which are tubular outgrowths of root epidermal cells. They are likely to be involved in uptake of nutrients and water, anchorage of plants, maintenance of contact between roots and soil, and root exudation. Over the last years, it has become clear that calcium is involved in various processes which result in tip growth. Previous studies from our laboratory have demonstrated an increase in calcium level in root cells of pea seedlings grown under conditions of space flight and clinorotation. On the basis of these data, we have suggested that such effects of microgravity and clinorotation might be due to the enhancement of Ca2+ influx into hyaloplasm evidently through Ca2+ channels. In this regard, it was interesting to examine the effects of clinorotation and Ca2+ channel blockers (verapamil and nifedipine) on orientation and structure of growing RH that might be an appropriate model system being sensitive to calcium for studying the gravitational effects at cellular level.

The function of calcium in plant graviperception.

The fundamental question of gravitational biology is how do plants perceive a gravity. Recent experimental results have demonstrated that Ca second-messenger system has an essential role in induction of graviresponsiveness. Our data, that stimuli of various nature cause a rise of hyaloplasm Ca level revealed by means of pyroantimonate method, as well as complete inhibition of the gravitropism in roots of pea seedlings, provide indirect but consistent evidence of this role of Ca ions. A possible explanation for these results is that they may be due to an unbalanced and undirectional influx of Ca ions in statocytes from cell walls or from intracellular Ca stores, while in the presence of the Earths 1 g vector, this process occurs directionally, along this vector. It is possible that a target for the gravity stimulus is the flux mechanism of Ca to statocytes, including participation of the phosphatidylinositol system and calmodulin. The data that have become available from space flight experiments will be reviewed and an attempt will be made to compare these results with ground-based observations.

Identification of RNA replicase subunits responsible for initiation of RNA synthesis of tick-borne encephalitis virus by affinity labelling.

Porcine embryo kidney cells infected by tick-borne encephalitis virus (TBEV) were fractionated into nuclear, membrane, and cytoplasmic fractions. To identify proteins involved in the initiation of RNA replication at different stages of infection a highly specific affinity labelling technique was used. In samples of the nuclear fraction taken from cells 45 h after infection (late stage), affinity labelling with aldehyde-containing derivatives of ATP and elongation of this label with [alpha-32P]GTP identified a polypeptide with a molecular mass of about 69 kDa. By means of affinity labelling with aldehyde-containing analogues of GMP, GDP, and GTP as initiation substrates and [alpha-32P]ATP as the elongation substrate, a polypeptide of 100 kDa was selectively modified in the nuclear fraction of cells at the early stages of infection (8 h). These proteins were immunostained with TBEV-specific antibodies, and were identified as the nonstructural TBEV proteins NS3 and NS5, respectively. It was concluded that NS3 and NS5 take part in the initiation of TBEV genome replication at the late and early stages of infection, respectively.

Mapping of the region of the tick-borne encephalitis virus replicase adjacent to initiating substrate binding center.

Affinity labelling with aldehyde-containing analogs of initiation substrates of nuclear fraction of tick-borne encephalitis virus (TBEV) infected cells results in a labelling of a single polypeptide with a molecular mass of 68 kDa which was immunologically identified as TBEV NS3 protein. A single-hit hydroxylamine hydrolysis, using limited and long-term CNBr cleavages allowed one to identify Lys1800 and/or Lys1803 as the label attachment sites. These amino acid residues are situated in the proximity of the 'B'-site of NTP-binding motif of viral RNA replicase.

The role of calcium ions in cytological effects of hypogravity.

Electron-cytochemical and biochemical methods made it possible to reveal certain differences in ATPase activity stimulation by calcium ions in root apex cells of pea seedlings and moss protonema Funaria hygrometrica grown under stationary and slow clinostatic (2 rev/min) conditions. It was showed that under clinostatic conditions in comparison with the control variant the ATPase activity decreases in plasmalemma. The protein content in the plasmalemma fraction was also twice as low under these conditions. The root apex cells of the pea seedlings grown under spaceflight conditions were found to contain high concentrations of membrane-bound calcium. The data obtained are discussed in relation to problems of possible mechanisms of disturbance in calcium balance and the system of active calcium ion transport through plasmalemma under hypogravity.

Biological effects of weightlessness and clinostatic conditions registered in cells of root meristem and cap of higher plants.

Research in cellular reproduction, differentiation and vital activity, i.e. processes underlying the development and functioning of organisms, plants included, is essential for solving fundamental and applied problems of space biology. Detailed anatomical analysis of roots of higher plants grown on board the Salyut 6 orbital research station show that under conditions of weightlessness for defined duration mitosis, cytokinesis and tissue differentiation in plant vegetative organs occur essentially normally. At the same time, certain rearrangements in the structural organization of cellular organelles--mainly the plastid apparatus, mitochondria, Golgi apparatus and nucleus--are established in the root meristem and cap of the experimental plants. This is evidence for considerable changes in cellular metabolism. The structural changes in the subcellular level arising under spaceflight conditions are partially absent in clinostat experiments designed to simulate weightlessness. Various clinostatic conditions have different influences on the cell structural and functional organization than does space flight. It is suggested that alterations of cellular metabolism under weightlessness and clinostatic conditions occur within existing genetic programs.