organisms ' adaptation to environmental stress has become a subject of great interest over the last decades [ 15 ] . like other organisms
, budding yeast saccharomyces cerevisiae has developed several strategies to survive stressful changes in their environment .
clearly , yeast cells respond rapidly and modify their internal systems to prevent dramatic events . depending on the intensity and type of stress ,
it is widely believed that cell exposure to mild stress results in the acquisition of cellular resistance to further lethal stress , what is called  adaptive response  or
the phenomenon has been observed in various organisms : from bacteria to humans . in many cases , an exposure to mild stress develops tolerance not only to higher doses of the same stressor but also to stress caused by other factors .
cross - adaptation  [ 6 , 7 ] , suggests the existence of complex mechanisms , which sense and respond to different kinds of stress .
the literature includes data on s. cerevisiae general response , pre - adaptation , and cross - adaptation to extreme temperatures , osmotic shock , and oxidative stress [ 2 , 6 , 812 ] .
there is information on the increased resistance to severe stress in yeast preexposed to mild sublethal stress , which requires the global - stress transcription factors msn2/4p to regulate induction of the so - called environmental stress response genes [ 6 , 11 ] .
activation of msn2/4p , in particular , is an important way to induce antioxidant defense against hydrogen peroxide [ 13 , 14 ] . yap1p
transcriptional regulatory protein also mediates an adaptive response of yeast to h2o2-induced stress [ 4 , 9 , 1315 ] .
sublethal hormetic concentrations of hydrogen peroxide are believed to induce a protective response with increased resistance to subsequent lethal stress in yeast cells [ 6 , 7 , 10 , 16 ] . however , unlike msn2/4p , the potential role of yap1p in the cross - adaptation phenomenon is poorly investigated .
yap1p is found to be localized in the cytoplasm under nonstressful conditions , but upon exposure to hydrogen peroxide it rapidly translocates to the nucleus and changes the expression of target genes [ 1719 ] .
although yap1p was earlier suggested to play a minor role in the regulation of gene expression under ethanol stress , like h2o2 , ethanol was recently found to trigger yap1 nuclear accumulation and activate some antioxidant enzymes in s. cerevisiae . in the present study , we have shown that low hormetic concentrations of hydrogen peroxide as well as ethanol stimulated yeast colony growth .
therefore , we evaluated the role of one substance in cell cross - adaptation to the other substance as well as some weak organic acid preservatives , and potential role of yap1p in yeast cross - adaptation by hormetic concentrations of hydrogen peroxide and ethanol to stressful conditions .
the saccharomyces cerevisiae strains used in this study are yph250 ( wild type : mat a trp1-1 his3-200 lys2 - 801 leu2-1 ade2 - 101 ura3 - 52 ) and its isogenic derivative yap1 ( yph250 yap1::his3 ) described earlier . the strains were kindly provided by professor yoshiharu inoue ( kyoto university , japan ) .
yeast cells were grown in erlenmeyer flasks containing ypd liquid medium ( 1% yeast extract , 2% peptone , and 2% glucose ) in a volume that respected the ratio 1  :  5 regarding media volume to flask volume .
, 28c , and ph 7.0 for 24  h to late exponential phase ( a600 ~ 0.8 - 0.9 ) .
the experimental culture after growth under the conditions mentioned above was split into three groups : one exposed to stress , another preadapted by low concentrations of hydrogen peroxide or ethanol and then exposed to stress , and the last one serving as a control to which none of the abovementioned substances was added . for stress induction
, aliquots of experimental culture were incubated at 28c with 50  mm h2o2 for 30  min , 15% or 20% ethanol for 60  min , and 200  mm acetic or 100  mm propionic acid for 120  min . at low ph , acetic acid ( pka 4.75 ) and propionic acid ( pka 4.88 )
exist mainly in the undissociated state , in which they enter the cell rather easily . for acid stress ,
the ph value of ypd medium was adjusted to 3.0 with hcl in order to reach maximum penetration of the acids into cells [ 24 , 25 ] . under stress induced by hydrogen peroxide or ethanol
the ph value of ypd medium was 7.0 . to study the preadaptation effect on cell survival under stress , aliquots of experimental culture were preincubated with 0.05 , 0.25 , and 0.5  mm h2o2 or 1% , 2.5% , and 5% ethanol at 28c and ph 7.0 for 30 or 60 minutes , respectively .
control cells were incubated under the same conditions but without hydrogen peroxide , ethanol , acetate , or propionate . in preliminary experiments ,
it was shown that yeast colony growth was virtually the same at ph  3.0 and 7.0 [ 7 , 26 ] .
thus , control cells for acetate- and propionate - induced stress were incubated in ypd medium at ph  3.0 without organic acids .
yeast colony growth was analyzed by plating in triplicate on ypd agar after proper dilution .
the plates were incubated at 28c for 3 days and the colony forming units ( cfu ) were counted .
yeast colony growth was expressed as percentage of total amount of respective control cells plating on ypd agar .
experimental data are expressed as the mean value of 46 independent experiments  the standard error of the mean ( sem ) , and statistical testing used student 's t - test .
the biphasic - dose response to hydrogen peroxide is the phenomenon of particular interest in hormesis research [ 8 , 2831 ] . in the case of budding yeast , special attention
is focused on the concentrations of hydrogen peroxide ( 0.4  mm ) found to stimulate yeast colony growth by about 30% ( stimulatory / hormetic zone ) . at the same time
, hydrogen peroxide has been shown to induce cell toxicity at concentrations higher than 0.5  mm .
earlier , the nonlinear dependence of s. cerevisiae rz53 - 6 survival on different concentrations of hydrogen peroxide was demonstrated by davies and colleagues . in the present study ,
we observed nonlinear dependence of s. cerevisiae yph250 viability on different levels of h2o2 ( figure 1 ) . since h2o2 low concentrations ( 0.5  mm ) inhibited significantly the colony growth of yap1 mutant derived from yph250 wild type ( figure 1 ) , we suggested yap1p involvement in hormetic effect by low doses of hydrogen peroxide in the parental strain . in different animal models ,
the dose - response curve for ethanol is also biphasic showing an inhibitory effect at high doses but a stimulatory effect at low doses [ 32 , 33 ] .
we observed non - linear dose response to ethanol in s. cerevisiae yph250 wild strain ( figure 2 ) .
yeast ability to form colonies in cultures treated with 1% ethanol was decreased by 30% comparing to untreated control cells .
cells exposed to 2.5% ethanol demonstrated percent colony growth that exceeded the original control value by about 20% . at the higher concentrations used ( 5% ) ,
figure 2 demonstrates also the influence of different concentrations of ethanol on the reproductive ability of yap1 mutant isogenic derivative of yph250 . generally , the effect is somewhat similar to that obtained for yph250 parental strain .
it should be noted that 2.5% ethanol increased the parameter comparing with that found for cells exposed to 1% ethanol , but colony growth of the yap1 cells treated with 2.5% ethanol did not exceed the initial control value
. we also supposed that yap1 regulatory protein was involved in some way in the stimulation of yeast colony growth under stress induced by 2.5% ethanol ( figure 2 ) .
the yap1 transcription factor controls the expression of over 150 genes in the response of s. cerevisiae to hydrogen peroxide [ 3436 ] .
since most of them were found to play an important role in yeast survival under h2o2-induced stress , we expected that yap1p was involved in hormetic effect by hydrogen peroxide . at the same time , there are somewhat controversial data regarding yap1p activation by ethanol .
earlier yap1p was suggested to play a minor role in regulation of gene expression under ethanol stress in s. cerevisiae ; however , ethanol was recently found to trigger yap1 nuclear accumulation and to activate some antioxidant enzymes in the yeast . since both hydrogen peroxide and ethanol at low concentrations were found to stimulate the colony growth of s. cerevisiae wild type , next we evaluated the role of one substance in cell cross - adaptation to the other substance as well as some weak organic acid preservatives .  
figure 3 shows the effect of yeast pretreatment with low h2o2 concentrations on its reproductive ability under stress induced by ethanol , acetate , and propionate .
as seen in the figure , all the stressors significantly inhibited colony growth of s. cerevisiae yph250 ( by 35% , 57% , 23% , and 32% in response to 15% or 20% ethanol and 200  mm acetate or 100  mm propionate , resp . ) .
yeast pretreatment with hormetic doses of hydrogen peroxide markedly increased reproductive ability under the stressful conditions .
for example , the highest colony growth ( 134% and 118% comparing to control untreated cells ) was observed in yeast preincubation with 0.25  mm h2o2 and then stressed by 15% or 20% ethanol . in the case of weak organic acids , the highest reproductive ability was observed in yeast pre - treated with 0.5  mm h2o2 ( 118% and 113% for acetate- and propionate - exposed cells , resp . ) .
hydrogen peroxide is known to affect expression of a variety of genes involved in signal transduction , transcriptional regulation , antioxidant defence , and protein , carbohydrates , or lipid metabolism in different organisms [ 9 , 1214 , 29 , 3539 ] .
that is why at low hormetic concentrations , h2o2 may act as an important signal molecule stimulating various defensive mechanisms and enhancing survival of yeast cells under lethal stress .
it should be noted that , comparing to the wild type , yap1 mutant demonstrated significantly higher sensitivity to all the types of stressors and no cross - adaptation by low concentrations of hydrogen peroxide to any stressors used ( figure 4 ) .
the latter confirms an important role of yap1p in cross - adaptation effect by hormetic doses of hydrogen peroxide in parental yph250 strain .
earlier it was suggested that depending on strain genotype , ethanol either provided or did not provide yeast protection against environmental stress . despite the fact that yeast cells demonstrated non - linear dose response to ethanol ( figure 2 ) , we did not observe any stimulatory effect by ethanol preadaptation on colony growth of yeast under stressful conditions ( figure 5 ) .
as seen , 50  mm h2o2 , 200  mm acetate , and 100  mm propionate inhibited s. cerevisiae yph250 ability to form colonies by 33% , 76% , and 69% , respectively , and preexposure of yeast by ethanol did not change the parameter . as seen in figure 6 , yap1 mutant demonstrated much higher sensitivity to all the types of stressors comparing to wild type as well as no cross - adaptation by ethanol to any stressors used .
this study demonstrates that , unlike ethanol , hormetic concentrations of hydrogen peroxide cause cross - resistance of s. cerevisiae yph250 to different stresses .
the regulatory protein yap1 plays an important role in the hormetic effects caused by low concentrations of either hydrogen peroxide or ethanol , and it is involved in the yeast cross - adaptation to stressful conditions by low sublethal doses of hydrogen peroxide .
 the biphasic - dose response of microorganisms to hydrogen peroxide is a phenomenon of particular interest in hormesis research . in different animal models , 
 the dose - response curve for ethanol is also nonlinear showing an inhibitory effect at high doses but a stimulatory effect at low doses . in this study 
 , we observed the hormetic - dose response to ethanol in budding yeast s. cerevisiae . 
 cross - protection is a phenomenon in which exposure to mild stress results in the acquisition of cellular resistance to lethal stress induced by different factors . 
 since both hydrogen peroxide and ethanol at low concentrations were found to stimulate yeast colony growth , we evaluated the role of one substance in cell cross - adaptation to the other substance as well as some weak organic acid preservatives . 
 this study demonstrates that , unlike ethanol , hydrogen peroxide at hormetic concentrations causes cross - resistance of s. cerevisiae to different stresses . 
 the regulatory protein yap1 plays an important role in the hormetic effects by low concentrations of either hydrogen peroxide or ethanol , and it is involved in the yeast cross - adaptation by low sublethal doses of hydrogen peroxide . 
1. Introduction
2. Materials and Methods
3. Results and Discussion
4. Conclusion