in china , maize ( zea mays l. ) is the most important cereal crop , and the production of this crop is affected by soil salinity . thus , many salinity - tolerant crops , such as maize and rice , have been developed using transgenic technology . researchers have found that the microbial communities in the rhizosphere are influenced by the plant . questions have been raised about whether antibiotic resistance genes , as selective markers , can transfer from genetically modified gm plants to indigenous microbes in the soil rhizospheres . another question is whether certain gm plants differentially affect soil microbial communities compared to non - gm plants [ 2 , 3 ] . previous studies have shown that gm plants , including gm maize , potato , soybean , rice , and triticale , are equivalent to non - gm crops in terms of nutrition and are safe as food or feed . the effects of bacillus thuringiensis ( bt ) transgenic cotton (  mech 162  ) and non - bt plants of the same cultivar on the ecology of many organisms in the soil were evaluated over three years in a subtropical environment . the authors concluded that the bt cotton mech 162  did not have any negative effects on the organisms or biochemical characteristics of the soil . the bacterial communities in the rhizosphere were studied using gm and non - gm maize in another study . plant growth can promote rhizobacterial multiplication associated with both gm and non - gm plants , which indicates the mutually beneficial relationship between rhizobacteria and maize . no significant differences in the isolated rhizospheres were found during plant growth in gm or non - gm plants . using transgenic , salinity - tolerant suv3 and pdh45 rice , the communication between rhizobacteria and rice was studied , and no significant effect was found [ 3 , 7 ] . however , there have been few reports of the influence of gm and non - gm maize on rhizosphere soils . the bcwrky1 gene was cloned from boea crassifolia hemsl , and it encodes a 444-amino acid wrky - like protein containing two conserved domains : wrkygqk and c2h2 motifs . the full - length bcwrky1 cdna was 1,803  bp , and its expression could be induced by abiotic stresses , including soil salinity , low temperature , and drought . in addition , bcwrky1 transcription was accompanied by changes in plant hormones , including abscisic acid ( aba ) , salicylic acid ( sa ) , and jasmonic acid ( ja ) . then , the boea crassifolia dna helicase of bcwrky1 was overexpressed under salt stress in maize . the nacl stress - tolerant phenotype appeared even when plants were irrigated continuously with 150200  mm nacl , with no effect on their yield . furthermore , other salt - induced genes , including gmwrky54 , tawrky2 , and hvwrky38 , in arabidopsis thaliana also promoted strong nacl stress - tolerant phenotypes [ 1012 ] . in this study , gm maize ( wl-73 ) plants overexpressing the bcwrky1 gene and control non - gm maize lh1037 were used to evaluate the effects of wl-73 growth on the microbial populations in saline or nonsaline soils in harbin , china . bcwrky1-transgenic maize , which carries a kanamycin resistance gene , was compared to non - gm lh1037 maize to determine its effects on rhizosphere soil in terms of enzyme activities ( including dehydrogenase , alkaline phosphatase , urease , and sucrase activities ) , physicochemical properties , and microbial populations . the line wl-73 was derived from the maize inbred line lh1037 transformed with the vector pcambia 3300-ubi - wrky1 ( figure 1 ) . seeds were obtained from t0 antisaline transgenic maize selected by saline stress and self - crossed to t6 ; wl-73 plants overexpressing bcwrky1 were able to survive under 300  mm nacl stress . the seeds of wl-73 and its receptor line lh1037 ( wt ) were grown in a three - row field with simulated saline - alkaline soil derived from a natural saline - alkaline field in heilongjiang province , china . the saline soil contained 29.42  gkg organic matter , 0.31  gkg total n , 127.27  mgkg available n ( an ) , 23.54  mgkg available p ( ap ) , and 178.91  mgkg available k ( k2o ) ( ak ) and had a ph of 8.65 . the nonsaline control soil contained 58.02  gkg organic matter , 0.31  gkg total n , 119.26  mgkg an , 26.01  mgkg ap , and 267.14  mgkg ak and had a ph of 7.67 . the seeds were salt and mock treated following di 's method with modifications . wt and wl-73 seeds were germinated in sterilized vermiculite in a greenhouse with a humidity of 4050% at 22c and a light cycle of 16  h light/8  h darkness . in addition , 0.5x hoagland 's nutrient solution with 300  mmol nacl was applied to the salt treatment plants daily for 7 days , while the same solution without nacl was applied to the control plants at the same frequency . both the salt - treated and control plants in the experiment were then watered with 0.5x hoagland 's nutrient solution every 3 days to prevent excessive nacl accumulation in the vermiculite . leaves of the salt - treated seedlings were collected , and dna and rna were isolated . the ctab method was used to isolate genomic dna from the two youngest leaves of each plant . total rna was isolated using trizol following the manufacturer 's protocol ( tiangen biotech , beijing , china ) under the requirement of 100  mg of young seeding leaves per ml of trizol . in addition , the membrane integrity parameters of the plants were determined by detecting superoxide dismutase ( sod ) and peroxidase ( pod ) activity , proline ( pro ) and malondialdehyde ( mda ) content , relative electrical conductivity ( rec ) , and chlorophyll content in leaves following the methods of arnon   and bates et al . . wl-73 and wt maize plants were grown in saline or conventional soil in triplicate from 2012 to 2014 at the transgenic experiment station of northeast agricultural university , harbin , heilongjiang , china ( longitude 12673 , latitude 4575 ) . soil samples were isolated from the rhizosphere of wl-73 and wt at the v3 ( the three lowest leaves have a visible collar ) , v9 ( nine leaves have collars present ) , r1 ( silking ) , and r6 ( physiological maturity ) stages . after removing the surface leaves , three soil samples from each plot the soil volumes between 0 and 20  cm in depth were extracted using a soil auger with a 4  cm diameter , and the bulk soil on the root was shaken off . the soil from the root was stripped using a sterilizing brush and constituted the rhizosphere soil samples . we mixed three rhizosphere soil samples from each plot into one sample and then divided this sample into two . another sample was air - dried at room temperature , homogenized by sieving through a 2  mm mesh , and stored at 4c until analysis . soil sucrase activity was measured using the 3,5-dinitrosalicylic acid method [ 21 , 22 ] . the physicochemical characteristics of soil and nutrient constituents , including soil type , ph , electrical conductivity ( ec ) ( mscm ) , organic carbon ( oc ) ( % ) , an ( kgha ) , ap ( kgha ) , and ak ( kgha ) , were determined . a 50  g soil sample was suspended in 100  ml of distilled , deionized water and stirred for 1  h at 100  rpm on a rotary shaker . the ec was recorded using a conductivity meter against 0.01  n kcl , and the ph was measured . the available carbon , nitrogen , phosphorus , and potassium contents in the soils were determined following standard methods [ 2427 ] . the calcium ( ca ) , sodium ( na ) , and magnesium ( mg ) ion concentrations were determined using an atomic absorption method to determine the sodium adsorption ratio ( sar ) . the sar was then calculated using the following formula : ( 1)sar = na+1/2ca2++mg2 + . to obtain isolated colonies , serial dilutions ( 10 dilution ) prepared from 1  g soil samples colonies were then selected , diluted , and spread onto plates containing beef extract peptone agar to detect bacteria , gause 's agar to detect actinomycetes , and rose bengal agar to detect fungi . three replicates of the inoculated agar plates were incubated at 30c , 28c , or 28c for 3 d for bacteria , 3 d for actinomycetes , and 5 d for fungi , after which the number of various types of colonies was recorded . the total populations of bacteria , actinomycetes , and fungi in each petri dish were counted as colony forming units ( cfu)g dry soil . the block treatments were the four growth stages ( v3 , v9 , r1 , and r6 ) , the two maize materials ( wl-73 and wt ) , and the two soil types ( saline and nonsaline ) . all of the experiments were performed with three biological replicates over three years from 2012 to 2014 . an analysis of variance ( anova ) was performed on treatment means using a generalized linear mixed model ( glmm ) , including treatment and sample time , in sas 9.1 ( copyright 2008 , sas institute , cary , nc ) . the bcwrky1 gene was cloned from boea crassifolia , which has the ability to tolerate salt stress . wl-73 transgenic plants overexpressing the bcwrky1 gene were successfully generated by agrobacterium - mediated transformation with the binary vector pcambia3300-ubi - bcwrky1 ( figure 1 ) introduced into the inbred line lh1037 . the 1308-bp bcwrky1 pcr product was amplified from wl-73 transgenic plants with bcwrky1 gene - specific primers ( figure 2(a ) ) . the transcription of the bcwrky1 gene in plant leaves was detected by rt - pcr ( table 1 , figure 2(b ) ) . as expected , pcr and rt - pcr bands characteristic of bcwrky1 were detected in wl-73 but not in wt plants . when we treated maize plants with 300  mm nacl solution for 7 days , the wl-73 plants were 5.3  cm taller and 60% heavier ( fresh weight ) than wt plants ( table 2 ) . wt seedlings became almost entirely yellow on the 7th day after salt stress ( figure 3 ) . the membrane integrity of the plants was measured in terms of parameters such as sod , pod , pro , mda content , rec , and chlorophyll content following salt stress ( figure 4 ) . the sod , pod , pro , mda , and rec of wl-73 plants were significantly lower than those of wt plants ( p < 0.01 ) , while the chlorophyll content of wl-73 plants was higher than that of wt plants under the 300  mm nacl treatment ( table 2 ) . however , no significant difference was found in these values between wl-73 and wt plants under control conditions . these results suggest that the membranes of wl-73 plants were less damaged than those of the wt plant . our results suggest that bcwrky1 enhanced the tolerance of wl-73 plants to salinity stress via membrane stabilization and reduced rec and mda contents compared with wt plants under salt stress ( figure 4 , table 2 ) . the bcwrky1 gene has been analyzed with other stress - related genes in transgenic plants , where it enhances the tolerance to salt and drought stress [ 1012 ] . therefore , increasing the expression level of bcwrky1 via transgenic technology should be critical for engineering crop plants with improved tolerance under multiple environmental stresses . the effects of wl-73 maize compared to control maize on rhizosphere soil enzyme activity , including the activities of alkaline phosphatase , urease , dehydrogenase , and sucrase , were studied in saline or control soil environments at four maize growth stages ( v3 , v9 , r1 , and r6 ) from 2012 to 2014 . these four enzymes are the main enzymes in soil and significantly affect the growth and yield of maize . dehydrogenase is the main oxidoreductase , and sucrose is the major hydrolase enzyme . during the three years of this study , the alkaline phosphatase , urease , dehydrogenase , and sucrase activities in the rhizosphere soil of wl-73 and wt plants were not different ( p > 0.05 ) in the two soil environments ( saline or nonsaline ) or at any of the four growth stages ( v3 , v9 , r1 , and r6 ) . the anova results showed that the dehydrogenase and alkaline phosphatase activities in the rhizosphere soil of wl-73 and wt maize were similar , with values ranging from 34.84 to 39.04  g  pnp  gh in saline soil and from 40.74 to 57.44  g  pnp  gh in control soil for wl-73 and from 34.74 to 41.85  g  pnp  gh in saline soil and from 40.62 to 59.26  g  pnp  gh in control soil for wt maize ( tables 3 and 4 ; figures 5(a)5(d ) ) . however , the activities of these four soil enzymes were significantly different between wl-73 and wt plants in some soil environments , years , and growth stages . for example , there were significant differences in alkaline phosphatase activity in saline soil at r6 in 2013 ( p = 0.03 ) ; in urease activity in control soil at r1 in 2012 and in saline soil at v9 in 2013 ( p < 0.05 ) ; in dehydrogenase activity in control soil at v9 in 2013 ; and in sucrase activity in saline soil at v9 in 2014 ( table 3 , figures 5(a)5(d ) ) . however , no consistent trends in enzyme activity were detected in the two soil environments , over the three - year study , or in the four growth stages analyzed here . these results agree with those of previous studies in other regions and for a variety of crops [ 2932 ] . soil enzymes play an important role in maintaining soil ecology , physicochemical properties , fertility , and health [ 33 , 34 ] . the overexpression of pdh45 and suv3 in transgenic rice has no adverse effect on rhizosphere soil or its microflora [ 3 , 7 ] . in other studies of transgenic crops , the only consistent significant differences in soil enzymes and physicochemical properties between transgenic and nontransgenic plants were due to seasons and crop varieties . there were no significant differences in the enzyme activities of rhizospheric microbes from soils in which bt or non - bt cotton was grown . the results of the present study indicated few significant differences in the alkaline phosphatase , urease , dehydrogenase , and sucrase activities in the rhizosphere soil between wl-73 and wt plants , as in the five studies cited above . the physicochemical properties , including the ph , sar , ec , an , ak , ap , and oc , of rhizosphere soil from wl-73 and wt maize plants are shown in figures 6(a)6(g ) . there were no overall significant differences between wl-73 and wt plants for most of the physicochemical properties ( p > 0.05 ) at four maize growth periods ( r1 , r9 , v1 , and v6 ) in saline and control soil environments from 2012 to 2014 ( table 5 ) . however , there were significant differences between wl-73 and wt plants ( p < 0.05 ) for nine combinations of factors and very significant differences between the genotypes for three combinations of factors . there were significant differences in specific parameters under some conditions , including ph in saline soil at v9 in 2013 ; ph in control soil at r1 in 2014 ; sar in saline soil at v9 in 2012 and 2014 ; an in saline soil at v9 in 2013 and in saline soil at r6 in 2014 ; ak in control soil at v9 in 2013 ; ap in saline soil at r1 in 2013 ; and organic matter in saline soil at v9 in 2013 . there were very significant differences in sar in control soil at v9 in 2014 ; ak in saline soil at v9 in 2013 ; and ak in saline soil at r6 in 2013 ( table 3 ; figures 6(a)6(g ) ) . however , no overall consistent trends in physicochemical properties were detected in the two soil environments , over the three - year study , or in the four growth stages . the number of total actinomycetes , bacteria , and fungi per gram of dry rhizosphere soil from wl-73 and wt plants over the crop developmental cycle is shown in figure 7 . no significant differences were found in the total number of bacteria , fungi , and actinomycetes between the two maize rhizosphere soils at any of the plant growth stages , except for actinomycetes in saline soil at v9 and in control soil at r6 in 2014 ( table 3 ) . the total number of each of the three kinds of microbes in the rhizosphere soil peaked at the v1 stage . subsequently , the populations of all three types of microorganisms decreased at the v6 stage ( figures 7(a)7(c ) ) . soil microbial analysis is a common method used to detect the effect of exogenous chemicals or environmental pollutants on soil fertility and crop yields similarly , monitoring soil microbial populations in response to transgenic plants will reveal the risks of exogenous genes in soil . investigations of the microbial populations of rhizosphere soil found that bt maize had no direct effect on soil ecology . both the number and the diversity of microorganisms exhibit only significant seasonal variation , with no long - term effect on the cultivation of cry1ac - transgenic cotton . no significant effects were found on the populations of various soil microorganisms with the growth of transgenic insect - resistant maize , bt maize , and cotton compared to nontransgenic plants under field conditions [ 30 , 31 , 3537 ] . there was no adverse effect on soil enzymatic activities or rhizosphere microbial communities by the cultivation of transgenic plants , such as mcm6 transgenic tobacco , pdh45 transgenic rice , and suv3-overexpressing transgenic rice [ 3 , 7 , 37 ] . in our study , significant variation was detected in actinomycete populations in saline soil at v9 and in control soil at r6 in 2014 ( p > 0.05 ) . however , we did not find a significant effect on enzyme activities , physicochemical properties , or populations of soil microbes due to the long - term cultivation of wl-73 compared to wt . our results are consistent with previous studies showing that the long - term cultivation of salt - tolerant gm plants has no effect on soil microbial populations . the effects that we observed were due to particular individual plants , techniques , exogenously expressed proteins , or environmental conditions . in the present study , the minor significant differences in the rhizosphere soil between transgenic and nontransgenic maize plants were not as large as the effects associated with plant growth stages . these results indicated that the effects of bcwrky1 maize wl-73 on rhizosphere soil ecology are within the variation expected in conventional agriculture . the long - term planting ( 3 years ) of wl-73 plants had no detectable effects on the enzymatic activities , physicochemical properties , or microbial populations of the rhizosphere soil compared with the wt at any of four maize growth stages ( v3 , v9 , r1 , and r6 ) .