the cultivation of soybean is increasing in the world due to its widespread use in the food and feed industry . abiotic stresses , such as drought , salinity , and extreme temperatures are serious threats to agriculture and the natural status of the environment . as a consequence of global heating , increased salinization of arable land is expected to have devastating effects . recently , it has been reported that salinity provoked in olive leaves an imbalance between reactive oxygen species ( ros ) production and antioxidant defenses , with the induction of oxidative stress . to counteract the toxicity of ros , defense systems that scavenge cellular ros have been developed in plants to cope with oxidative stress via the nonenzymatic and enzymatic systems . antioxidants ( ros scavengers ) include enzymes such as cat and sod , as well as nonenzyme molecules such as ascorbate , glutathione , carotenoids , and anthocyanins . among the various genes encoding proteins that possess antioxidant characteristics , there has been a growing interest over the last years in the heme oxygenase system ( ho , ec 1.14.99.3 ) , the family of enzymes that control the initial and rate - limiting step in heme catabolism . heme oxygenase catalyses the oxidation of heme to biliverdin ix ( bv ) , co , and fe . afterwards , bv is reduced to bilirubin through the action of bv reductase . both bv and bilirubin have strong antioxidant properties in vitro as well as in vivo   and play an important role against oxidative damage . while heme degradation is a common feature of all hos , this heat shock protein actually has a great variety of roles in the cell , due in part to the diversity of the reaction products . in mammals , it has been demonstrated that one of the three known isoforms , ho-1 , is induced by an array of pro - oxidant compounds [ 58 ] . the release of iron and the subsequent induction of iron - sequestering proteins such as ferritin have also been proposed as an important function of hos in counteracting oxidative stress [ 9 , 10 ] . in plants , recent reports have demonstrated the presence in soybean leaves and nodules of one ho closely related to the ho-1 of mammalian cells , on the basis of its antioxidant behavior under cd and uv - b treatment [ 11 , 12 ] . salt stress is one of the most important abiotic stresses that adversely affect natural productivity and causes significant crop loss worldwide . two major components have been identified in this phenomenon , osmotic stress and ion toxicity . higher plants have multiple protective mechanisms against salt stress including ion homeostasis , osmolyte biosynthesis , ros scavenging , water transport , and transducers of long - distance response coordination . it is generally accepted that many stresses , including salinity , induce an overproduction of ros , such as h2o2 , o2 , and hydroxyl radicals , and these species are thought to be responsible for the oxidative damage associated with plant stress . to counteract the toxicity of ros , defence systems that scavenge cellular ros have been developed in plants to cope with oxidative stress via the nonenzymatic and enzymatic systems [ 15 , 16 ] . with the aim of obtaining a better comprehension of the in vivo effect of salinity on oxidative stress defense system , we have analyzed oxidative stress parameters , the behavior of the well - known antioxidant enzymes cat and sod , as well as the response of ho-1 to different nacl concentrations . surface sterilized glycine max . l. seeds ( a6445rg ) were germinated directly in plastic pots containing vermiculite in controlled environmental chambers , with a photoperiod of 16 hours , photon flux density of 175 mol m s , and a day / night regime of 25/20c , and were simultaneously inoculated with bradyrhizobium japonicum strain e 109 ( inta , castelar ) . after this , they were watered daily with a nutrient solution   during the first 5 days and then with an n - free nutrient solution . after four weeks , plants were treated with nutrient solution devoid of salt ( control ) or containing 50 , 100 , and 200 mm of nacl . when the effect of znppix was investigated , plants were treated with 22 m znppix added to nacl solutions during 2 days before the end of the experiment . they were washed in deionized water to remove surface added electrolytes and placed in petri dishes with 15 ml of deionized water at 25c for 3 hours . electrical conductivity in the bathing solution was determined ( c1 ) . then , the samples were heated at 80c for 2 hours and the conductivity was read again in the bathing solution ( c2 ) . relative ion leakage was expressed as a percentage of the total conductivity after heating at 80c ( relative ion leakage % = c1/c2  100 ) . lipid peroxidation was measured as the amount of tbars determined by the thiobarbituric acid ( tba ) reaction as described by heath and packer . fresh control and treated leaves ( 0.3 g ) were homogenized in 3 ml of 20% ( w / v ) trichloroacetic acid ( tca ) . the homogenate was centrifuged at 3500      g for 20 minutes . to 1 ml of the aliquot of the supernatant , 1 ml of 20% tca containing 0.5% ( w / v ) tba and 100 l 4% butylated hydroxytoluene ( bht ) in ethanol was added . the mixture was heated at 95c for 30 minutes and then quickly cooled on ice . leaves ( 0.5 g of fresh weight ) were homogenized with 96% ethanol ( 1:30 w / v ) . after centrifugation , the absorbance was measured in the supernatant at 665 , 649 , and 654 nm as described by wintermans and de mots . leaves from control and treated plants were excised and immersed in a 1% solution of 3,3-diamino benzidine ( dab ) in tris hcl buffer ( ph 6.5 ) , vacuum - infiltrated for 5 minutes , and then incubated at room temperature for 16 hours in the absence of light . leaves were illuminated until appearance of brown spots characteristic of the reaction of dab with h2o2 . h2o2 deposits were determined by scanning spots from leaf pictures and the numbers of pixels were quantified using the public domain nih image program ( developed at the us national institutes of health ) . the results were expressed as percentage of spots area versus total leaf area ( ( spot area / total leaf area )      100 ) in order to compensate the differences in leaves size . leaves from control and treated plants were excised and immersed in a 0.1% solution of nitroblue tetrazolium ( nbt ) in k - phosphate buffer ( ph 6.4 ) , containing 10 mm na - azide , and were vacuum - infiltrated for 5 minutes and illuminated until the appearance of dark spots , characteristic of blue formazan precipitate . extracts for determination of catalase ( cat , ec 1.11.1.6 ) and superoxide dismutase ( sod , ec 1.15.1.1 ) activities were prepared from 0.3 g of leaves homogenized under ice - cold conditions in 3 ml of extraction buffer , containing 50 mm phosphate buffer ( ph 7.4 ) , 1 mm edta , 1 g pvp , and 0.5% ( v / v ) g for 20 minutes and the supernatant fraction was used for the assays . cat activity was determined in the homogenates by measuring the decrease in absorption at 240 nm in a reaction medium containing 50 mm potassium phosphate buffer ( ph 7.2 ) and 2 mm h2o2 . the pseudo - first - order reaction constant ( k = k. [ cat ] ) of the decrease in h2o2 absorption was determined and the cat content in pmol mg protein was calculated using k = 4.7  10   ms ) . total sod activity was assayed by the inhibition of the photochemical reduction of nbt , as described by becana et al . . the reaction mixture consisted of 50150 l of enzyme extract and 3.5 ml o2 generating solution which contained 14.3 mm methionine , 82.5 m nbt , and 2.2 m riboflavin . extracts were brought to a final volume of 0.3 ml with 50 mm k - phosphate ( ph 7.8 ) and 0.1 mm na2edta . test tubes were shaken and placed 30 cm from light bank consisting of six 15-w fluorescent lamps . the reaction was allowed to run for 10 minutes and stopped by switching the light off . blanks and controls were run the same way but without illumination and enzyme , respectively . one unit of sod was defined as amount of enzyme which produced a 50% inhibition of nbt reduction under the assay conditions . leaves ( 0.3 g ) were homogenized in a potter - elvehejm homogenizer using 1.2 ml of ice - cold 0.25 m sucrose solution containing 1 mm phenylmethyl sulfonyl fluoride , 0.2 mm edta , and 50 mm potassium phosphate buffer ( ph 7.4 ) . homogenates were centrifuged at 20 000      g for 20 minutes and chloroplasts were used for activity determination . the assays ( 1 ml final volume ) contained 250 l of extract ( 0.5 mg protein ) , 10 m hemin , 0.15 mg ml bovine serum albumin , 50 g ml ( 4.2 m ) spinach ( spinacia oleracea ) ferredoxin ( sigma chemical co. ) , 0.025 units ml spinach ferredoxin - nadp reductase ( sigma chemical co. ) . the reaction was started by adding nadph to a final concentration of 100 m , samples were incubated at 37c for 30 minutes and bv formation was calculated using the absorbance change at 650 nm . the concentration of bv was estimated using a molar absorption coefficient at 650 nm of 6.25 mm cm in 0.1 m hepes - naoh buffer ( ph 7.2 ) . one unit of the enzyme forms 1 nmol of biliverdin in 30 minutes under assay conditions . forty g of protein from leaf homogenates were subjected to sodium dodecyl sulfate ( sds)-poliacrylamide gel electrophoresis using a 12% acrylamide resolving gel ( mini protean ii system , biorad , hertz , uk ) , according to laemmli . separated proteins were then transferred to nitrocellulose membranes and nonspecific binding of antibodies was blocked with 3% nonfat dried milk in pbs , ph 7.4 for 1 hour at room temperature . membranes were then incubated overnight at 4c with primary antibodies raised against arabidopsis thaliana ho-1 ,   diluted 1:2000 in tris - nacl buffer plus 1% nonfat milk . immune complexes were detected using peroxidase - conjugated goat antirabbit inmunoglobulin g and visualized with the enhanced chemiluminescence western blotting procedure ( ecl , amersham pharmacia biotech , uppsala , sweden ) . the films were scanned ( fotodyne incorporated , wiss , usa ) and analyzed using gel - pro analyzer 3.1 software ( media cybernetics , md , usa ) . total rna was extracted from soybean leaves by use of trizol reagent ( gibco brl ) . four micrograms of total rna were treated with rnase - free dnase i ( promega , calif , usa ) and then 1.0 g was reverse transcribed into cdna using random hexamers and m - mlv superscript ii rt ( invitrogen , calif , usa ) . pcr reactions were carried out using glycine max ho-1 and 18s specific primers , as previously described . in addition , cat gene expression was assessed by use of a primer pair specific to glycine max cat cdna ( sense primer , 5-ctgctggaaactatcctgagtg-3 ; antisense primer , 5-attgacctcttcatccctgtg-3 ) . the pcr profile was set at 94c for 1 minute and then 29 cycles at 94c for 0.5 minute , 54c for 1minute , and 72c for 1 minute , with a final extension at 72c for 7 minutes . cycling conditions were as follows : 94c for 1 minute , then 33 cycles at 94c denaturing for 0.5 minute , 55c annealing for 1 minute , and 72c extension for 1 minute , and then a final step of 72c for 7 minutes . each primer set was amplified using an optimized number of pcr cycles to ensure the linearity requirement for semiquantitative rt - pcr analysis . the amplified transcripts were visualized on 1.5% agarose gels with the use of ethidium bromide . gels were then scanned ( fotodyne incorporated , wiss , usa ) and analyzed using gel - pro analyzer 3.1 software ( media cybernetics , md , usa ) . protein concentration was evaluated by the method of bradford   using bovine serum albumin as a standard . differences among treatments were analyzed by one - way anova , taking p < .05 as significant according to turkey 's multiple range test . oxidative stress is the result of excessive production of oxidant species and/or depletion of intracellular antioxidant defenses , leading to an imbalance in the redox status of the cell . therefore , tbars formation in plants exposed to adverse environmental conditions is a reliable indicator of tissue free radical formation . as shown in table 1 , a 22 and 37% increase occurred in plants treated with 100 and 200 mm nacl , resp . , whereas no difference respect to controls were observed in 50 nacl - treated plants . loss of membrane integrity during stress was studied by measuring the leakage of electrolytes from soybean leaves ( table 1 ) . salinity stress induced a maximum relative injury ( 192% ) , when soybean plants were subjected to 200 mm nacl and 53% with 100 mm nacl , whereas plants treated with 50 mm nacl revealed no differences with respect to controls . reduction of chlorophyll content can be used as a marker to measure the effect of salinity stress on the integrity of chloroplasts . table 1 shows that this parameter decreased under 100 and 200 mm nacl treatment ( 24 and 58% , resp . ) , while plants subjected to 50 mm did not differ from controls . in order to assess the oxidative damage caused by salt stress , accumulation of h2o2 and o2 were performed in vivo by histochemical methods ( figure 1(a ) ) . as shown in figure 1(b ) , ( c ) , treatment with 100 mm nacl produced 15% and 18% h2o2   and o2 spots area versus total leaf area , respectively , while 200 mm salt treatment markedly increased both reactive species oxygen ( 45% for h2o2 and 40% for o2 ) . the lower nacl concentration ( 50 mm ) did not produce neither h2o2 nor o2 in soybean leaves ( figure 1 ) . in order to get further insight into the effect all salt - stress on oxidative stress parameters , cat and sod activities and gene expression on the other hand , under 50 mm nacl , there was no increase in cat activity respect to controls , but a significant inhibition ( 32% ) was observed in the presence of 200 mm nacl . rt - pcr analysis revealed that these activities are positively correlated with gene expression ( figure 4 ) . sod activity ( figure 2(b ) ) and gene expression ( figure 4 ) were increased under 100 mm nacl ( 30% ) , and no differences were observed respect to controls under 50 mm salt . treatment with 200 mm nacl caused a diminution in enzyme activity ( 35% ) as well as in gene expression ( 20% ) . as shown in figure 2(c ) , under 50 mm nacl there was no increase in the ho activity respect to controls . concentration of 100 mm nacl was capable of inducing ho-1 activity ( 7 fold ) , while 200 mm salt produced a decrease in the enzyme activity . when protein amount ( figure 3 ) and gene expression ( figure 4 ) were assayed , a positive correlation with enzyme activity was found . to determine the involvement of ho-1 in the defense system against oxidative damage caused by salinity , the effect of znppix , a well - known ho-1 strong inhibitor , on tbars , ion leakage , chlorophyll content , and ho activity was assayed . simultaneous treatment with 100 mm nacl and znppix significantly enhanced tbars levels ( 49% ) and ion leakage ( 130% ) when compared with the values obtained when only 100 mm nacl was added . however , the diminution produced in the chlorophyll content by this saline concentration was not altered by znppix addition , and therefore , remained similar to that found with 100 mm salt alone . treatment with znppix alone did not modify ion leakage , tbars levels , and chlorophyll content , compared to controls . this study was undertaken to evaluate in soybean leaves , the in vivo response of ho-1 to salinity . salt stress disrupts plant ion homeostasis , resulting in excess toxic na in the cytoplasm and a deficiency of essential ions such as k . drought , salinity , and extreme temperatures are often interconnected , and may induce similar cellular damage by means of ros formation [ 29 , 30 ] . for example , drought and/or salinization are manifested primarily as osmotic stress , resulting in the disruption of homeostasis and ion distribution in the cell [ 31 , 32 ] . oxidative stress , which frequently accompanies high temperature , salinity , or drought stress , may cause denaturing of functional and structural proteins . as a consequence , these diverse environmental stresses often activate similar cell signaling pathways [ 3336 ] and cellular responses , such as the production of stress proteins and upregulation of antioxidants [ 37 , 38 ] . in order to establish whether oxidative stress occurred under our experimental conditions , .   demonstrated that peroxidation of membrane lipids is an indicator of membrane damage under salt stress . from our experiments , it can be concluded that 200 mm nacl is effective in triggering membrane damage and leakage . on the other hand , this result is in agreement with those from kahn   and abdelkader et al .   that postulated that plants grown either under conditions of increased salinity or treated with salt solutions have a reduced chlorophyll accumulation . a number of antioxidant responses have been studied in bacteria , and it has been established that ros are directly sensed by key regulatory molecules that activate the expression of genes encoding antioxidant proteins at transcription level [ 42 , 43 ] . superoxide dismutase , which catalyses the dismutation of o2 to o2 and h2o2 , is one of the major cellular defense enzymes that perform a vital role in protecting cells against the toxic effects of o2 . catalase converts harmful metabolic byproducts and , under normal physiological conditions , it controls the h2o2 concentration so that it does not reach toxic levels that could bring about oxidative damage inside peroxisomes and the surrounding cytoplasm . the results presented in this study showed that soybean leaves sod gene was induced by 100 mm nacl , no induction was observed under 50 mm , and a downregulation occurred in plants treated with 200 mm nacl . the same behavior was observed when cat gene expression and activity were analyzed . according to our results , it can be suggested that under 50 mm nacl the antioxidant defense system can cope with ros damage , under 100 mm nacl sod and cat are induced in an attempt to protect the cell against this insult , and under 200 mm nacl ros production overwhelms the antioxidant defense system of the cell . the oxidative stress response and its relationship to heat shock phenomena are being intensely investigated in plant tissues [ 45 , 46 ] . to survive under environmental stress conditions , plants undergo a process of stress acclimation , which may require changes in the flow of metabolites , suppression of pathways involved in the excessive production of ros , and the induction of various defense genes such as heat shock proteins and ros scavenging enzymes [ 30 , 47 , 48 ] . the downregulation of sod and cat genes as well as the inhibition of enzyme activities could be attributed to ros - induced alterations , such as transductional modifications , dna damage , protein fragmentation , and increased susceptibility to proteolysis and cross - linking reactions . the involvement of h2o2 as a leading ros in cd - induced oxidative stress generation has been reported in tobacco cell cultures   and both h2o2 and o2 have been shown to act directly or indirectly in signal transduction of defense responses . studies carried out in rat liver suggested that ho-1 induction occurs when steady - state levels of the ros are increased and defenses ( e.g. , cat , sod ) are decreased . to the best of our knowledge , no information is still available on the behavior of ho-1 of plant tissues subjected to salinity . if we compare the expression profile of cat , sod , and ho , it is clear that these enzymes respond to the same signal in the same direction . liberation of fe , a potent oxidant that may exacerbate oxidative stress , takes place as a consequence of heme degradation . it is interesting to note that ferritins play an essential role in iron homeostasis by sequestering iron in a bioavailable and nontoxic form . in plants , such accumulation leads to a subsequent ferritin protein synthesis and fe storage . during the last five years , it has been established that the ho-1 induction could alleviate the oxidative damage caused by cd or uv - b radiation in soybean plants [ 11 , 12 ] . the effect elicited by znppix was not due to a pro - oxidant action because , when it was employed alone , the parameters associated with the oxidative stress , tbars levels , ion leakage , and chlorophyll content , were not modified . however , these results imply that the pro - oxidant effect of znppix when it was administered together with 100 mm nacl was due to the lack of ho-1 activity resulting from the presence of the inhibitor , indicating the role that this heat - shock enzyme plays in the attenuating salinity - induced damage . taken together , the present results allow us to conclude that under salinity ho-1 gene expression , protein amount and activity are enhanced in an attempt to protect leaves tissues against this insult . nevertheless , when the increase in h2o2 and o2 formation overwhelms the defense capacity of the cell , ho-1 gene is downregulated as it occurs with sod and cat . inhibition of ho-1 activity demonstrated that this enzyme plays a leading role in the defense mechanism against salinity , and it could be considered as essential component of enzymatic antioxidant defense system in plant tissues . further studies on the comparison of antioxidant enzyme profiles between salinity - tolerant and sensitive genotypes are necessary to determine if , like many newly discovered genes , ho may serve as a candidate for modifying plant salinity tolerance .
plants are frequently subjected to different kinds of stress , such as salinity and , like other organisms , they have evolved strategies for preventing and repairing cellular damage caused by salt stress . 
 glycine max l. plants were subjected to different nacl concentrations ( 0200 mm ) for 10 days . 
 treatments with 100 and 200 mm nacl induced ion leakage and lipid peroxidation augmentation , loss in chlorophyll content , and accumulation of 
                   o2 and h2o2 . 
 however , 50 mm nacl did not modify these parameters , which remains similar to control values . 
 catalase , superoxide dismutase , and heme oxygenase ( ho-1 ) activities and gene expressions were increased under 100 mm nacl , while no differences were observed with respect to controls under 50 mm salt . 
 treatment with 200 mm nacl caused a diminution in the enzyme activities and gene expressions . 
 results here reported let us conclude that ho also plays a leading role in the defense mechanisms against salinity .