EffEct of prEparations mEthyurE and ivinE on Са2+-atpases activity in plasma and vacuolar mEmbranE of corn sEEdling roots undEr salt strEss conditions

Ca2+-atPases regulate the functioning of Ca2+-dependent signaling pathway SOS which provides removal of Na+ from the cytoplasm of cells via Na+/H+-antiporters in saline conditions. The influence of synthetic preparations Methyure and Ivine on the Ca2+-atPase activity was investigated. It was shown that exposition of corn seedlings in the presence of 0.1 M NaCl rather enhanced hydrolytic than transport activity of Ca2+-atPases in plasma and vacuolar membrane of root cells. It was found that seed treatment with such preparations, especially Methyure, caused intensification of the both activities of Ca2+-atPases, mainly in vacuolar membrane. the results indicate than salt protective activity of preparations, especially Methyure, is associated with increased Ca2+-atPase activity, which regulates the functioning of Na+/H+-antiporters.


EffEct of prEparations mEthyurE and ivinE
on Са 2+ -atpases activity in plasma and vacuolar mEmbranE of corn sEEdling roots undEr salt strEss conditions M. V. RUdNytSka, t. a. PalladINa kholodny Institute of Botany, National academy of Sciences of Ukraine, kyiv e-mail: tatiana_ palladina@ukr.netCa 2+ -atPases regulate the functioning of Ca 2+ -dependent signaling pathway SOS which provides removal of Na + from the cytoplasm of cells via Na + /H + -antiporters in saline conditions.The influence of synthetic preparations Methyure and Ivine on the Ca 2+ -atPase activity was investigated.It was shown that exposition of corn seedlings in the presence of 0.1 M NaCl rather enhanced hydrolytic than transport activity of Ca 2+ -atPases in plasma and vacuolar membrane of root cells.It was found that seed treatment with such preparations, especially Methyure, caused intensification of the both activities of Ca 2+ -atPases, mainly in vacuolar membrane.the results indicate than salt protective activity of preparations, especially Methyure, is associated with increased Ca 2+ -atPase activity, which regulates the functioning of Na + /H + -antiporters.k e y w o r d s: Zea mays l., plasma membrane, vacuolar membrane, salt stress, hydrolytic and transport activities of Са 2+ -atPase, Methyure, Ivine.
S alted medium is one of the most negative abio tic factors of permanent action for plants; they create a serious problem for farming which is intensified because of the global warming [1].Its negative effect may be reduced using the methods of chemical melioration of soils with introduction of calcium [2].Calcium plays a regulatory role as a secondary messenger in activation mechanisms of numerous signal paths in plant organisms, including the salt stress conditions, activating the processes of sodium removal from cell cytoplasm [3,7].
The salt stress in plant organisms is multicom ponent; it causes the disturbance of osmotic and ionic homeostasis and is accompanied by appearance of secondary oxidizing stress [1].Plant organisms radi cally differ from animal ones in their attitude to Na + that is redundant and even toxic element for them; it gets into cell cytoplasm through potentialdepen dent potassium channels of the plasma membrane [4].Plants adaptation to salinity conditions consists in non-admission of high concentration of Na + in cy toplasm by its removal from cells to the outer and vacuolar space with the help of Na + /H + antiporters, which work in the plasma and vacuolar membrane.Their function is regulated with the help of salt over ly sensitive system (SOSsystem) that consists of the chain SOS3-SOS2-SOS1, where SOS1 is Na + /H + antiporter, and SOS3 і SOS2 -regu latory proteinases.The increase of Ca 2+ concentration in cytoplasm activates signals transduction through SOSsystem that results in removal of Na + [5].
Calcium homeostasis in cytoplasm under sa linity conditions is maintained by Ca 2+ -АТРases and Ca 2+ /Н + antiporters, which function in various cell membrane structures, the latter being included at high concentrations [6].Ca 2+ -АТРases of the plasma and vacuolar membrane play the important role in formation of cell response to different stimuli, in cluding salting conditions characterized by con tinuous action.They belong to P-type of ATPases, which form intermediate phosphorylated product and are characterized by high affinity, transferring Ca 2+ in nanomolar concentrations.Ca 2+ -ATPase of plant membrane is represented by two subtypes: Ca 2+ -АТРase ІІА and Ca 2+ -АТРase ІІВ, the latter displaying sensiti vity to calmodulin.They are simi lar to Ca 2+ -АТРase of animal cells as to their struc ture and membrane localization [6][7][8][9][10][11][12].
Our previous investigations have revealed salt protective effect of synthetic preparations Methyure (6methyl2mercapto4hydroxypyrimidine) and to a less extent Ivine (N-oxide-2,6-dimethyl pyridine) that determines the fitness of the former for agro practice on salted soils [13].The work is aimed at finding out the mechanism of these preparations ef fect on the function of Са 2+ -АТРases of the plasma and vacuolar membrane in the presence of Na + .

materials and methods
Seeds were wetted during a day in 10 7 M water solutions of synthetic preparations (Methyure and Ivine).Corn seedlings (hybrid Ostrech SV) were grown in water culture on Hogland medium under the conditions of 16-hour light day at 24 °С and il lumination 50 W/m 2 .They were exposed at a week age in 0.1 М NaCl during 1 and 10 days.Membrane preparations were isolated from roots using a cen trifuge Optima TM L90K Beckman Coulter.The fraction of plasma membrane was obtained by the phase separation method [14], while the fraction of vacuolar membrane -in the stepped gradient of sac charose [15].Purity of the obtained fractions and availability of membrane additives were determined by the activity of marker enzymes [16], a role of vesicles oriented outwards -by the effect of Triton X-100 on ATP hydrolysis [14], integrity of vesicleswith the help of electron microscopy [17].Protein content in membrane fractions was determined by the standard method [18].
All the experiments were performed in five bio logical and three analytical repeats, and reliability of the data obtained was determined following Stu dent's criterion.

results and discussion
The research was performed on preparations of plasma and vacuolar membrane represented by closed vesicles equal in size.Some role of plasma membrane (PM) with outward orientation was 72%, and vacuolar -15%.Composition of PM prepara tions was as follows: plasma membrane -83%, vacuolar membrane -8%, mitochondrial and Golgi apparatus 4-5% each.Composition of vacuolar mem brane preparations was: vacuo lar membrane -85%, plasma membrane -6.4%,mitochondrial and Golgi apparatus -4.3% each.
Under control conditions the hydrolytic and transport activity of Са 2+ -АТPase of PM had to change during 10 days, while a 1day salt exposure caused their intensification to 40%, which continues during 10 days (Table 1).
Hydrolytic and transport activity of Са 2+ АТPase almost did not change, the 1-day exposure in the presence of 0.1 М NaCl resulted in the 22% Here and in Tabl.23 # P < 0.05 is probable in respect of control without salt exposure decrease of transport activity, and only 2% decrease of hydrolytic activity, while prolongation of salt ex posure to 10 days intensified hydrolytic activity by 48%, and transport activity only by 11% (Table 2).

T a b l e 1. Effect of the term of salt exposure on activity of Са
The obtained results demonstrate a higher ac tivity of Са 2+ -АТpase in plasma membrane compared with vacuolar one on condition of salt exposure that evidences for its important functional role.Such an effect of salt exposure on activity of Ca 2+ -АТPases in these membrane was also shown on other cultures.The given Ca 2+ -АТPases play great role in ecologic plasticity of plants that was demonstrated at the early varieties of rice, tomatoes, radish and corn [6][7][8][9].
A comparison of Methyure and Ivine effect on activity of Ca 2+ -АТPase of plasma membrane has shown that under control conditions the preparations something increased the both activities, which grew during the exposure (Table 3).Salt exposure caused further increase in, especially, hydrolytic activity,

Variant
Term the effect of Ivine was lower than that of Methyure that explains a stronger salt-protective effect of the latter preparation (Table 4).
Investigations of the effect of these preparations on Ca 2+ -АТPase of vacuolar membrane have shown that in control conditions the both preparations, es pecially Methyure and, to a lesser extent, Ivine in tensified essentially hydrolytic and to a lesser extent transport activity of Ca 2+ -АТPase, which almost did not change with time (Table 5).
The preparation, especially Methyure, intensi fied to a higher extent the both activities, especially hydrolytic one, in the presence of NaCl (Table 6).Thus, the salt protective effect of the above prepara tions may be connected with intensification of Ca 2+ АТPase activity, especially in vacuolar membrane.This results in stabilization of functioning of Ca dependent SOS-systems by accumulation of Са 2+ in vacuolar space.Са 2+ /Н + antiporters also function in plasma and vacuolar membrane; they take part in the main tenance of cell Ca homeostasis under the effect of salt stress, which causes a considerable increase in calcium level in cytoplasm, the Са 2+ /Н + antiporter of vacuolar membrane being the more mighty [5,6].Joint work of Са 2+ -ATPases and Са 2+ /Н + antiporters in these membrane maintains functioning of Са 2+ / Н + -antiporters which remove Na + from cell cyto plasm.

T a b l e 3. Effect of Methyure and Ivine on activity of Ca
We have shown the capacity of synthetic bioac tive preparations to intensify activity of Ca 2+ -ATPase in the plasma and vacuolar membrane that favors Na + /Н + antiporters functioning in them.Under these conditions a higher activity of Са 2+ -АТPase in vacuo lar membrane provides longterm functioning of its Na + /Н + antiporter, which accumulates sodium in vacuolar space, supporting osmotic pressure in cells.Thus, the use of the above preparations, espe cially Methyure, favors intensification of functioning of Ca 2+ -dependent paths of Na + level normalization in cell cytoplasm, opposing the salt stress formation in them.

Table 6 #
2+-АТPase of plasma membrane from the root cells of 8-and 17-days corn seedlings under the absence of salt exposure (M ± m; n = 5) Effect of Methyur and Ivine on activity of Ca 2+ -АТPase of plasma membrane from the root cells of 8-and 17-days corn seedlings exposed in the presence of 0.1 M NaCl (M ± m; n = 5) P < 0.05 is probable in respect of control without salt exposure; * P < 0.05 is probable in respect of control with salt exposure T a b l e 5. Effect of Methyure and Ivine on Ca 2+ -АТPase activity of vacuolar membrane from root cells of 8and 17-day corn seedlings without salt exposure (M ± m; n = 5) # P < 0.05 is probable in respect of control without salt exposure T a b l e 6.Effect of Methyure and Ivine on Ca 2+ -АТPase activity of vacuolar membrane from root cells of 8and 17-day corn seedlings exposed in the presence of 0.1 M NaCl (M ± m; n = 5)