Transgenic Bt Rice Bt-SY63and Its Related Microbial Diversity Responses To Elevated O₃

Elevated tropospheric ozone (O3) causes severe damage to crop productionincluding rice (Oryza sativa L.) in the worldwide scale. Security certificates for BtShanyou63, a transgenic insect-resistant rice, was officially awarded in our countryin2009.Therefore, monitoring changes in Bt Shanyou63’ morphology,structure,physiological characteristic and environmental microbial under the projected hightropospheric ozone should be done as soon as possible before its commercial release,and it is of realistic significance and value in practice for assessing the potentialecologic risk of transgenic rice under rapidly elevated tropospheric ozoneconcentration in China.Using a free-air concentration enrichment (FACE) system which was built byChina and Japan, simulating tropospheric O3concentration in the eastern part of ourcountry by2030, we investigated the changes in leaf morphology and ultrastructureof two rice varieties grown in plastic pots, transgenic Bt Shanyou63(Bt-SY63,carrying a fusion gene of cry1Ab and cry1Ac) and its non-transgenic counterpart(SY63), in elevated O3(E-O3) versus ambient O3(A-O3) at heading stage, latefilling stage and maturing stage. The soluble protein and soluble sugar of leaves,stems and roots of the two rice varieties responses to elevated O3were studied attillering stage, heading stage and late filling stage. The changes in the numbers ofcultivable microorganisms from the rhizosphere soil and leaf surface, the number oftotal bacteria in the rhizosphere soil, the structural diversity and the functionaldiversity of bacteria community from soil and leaf surface under E-O3versus A-O3were analyzed and the relative quantity of the mRNA express of Bt gene, theconcentration of Bt protein and their relationship between Bt gene and Bt protein inrice leaf under elevated O3versus A-O3were also studied. The results show that:(1) Leaf histophysiology responses to elevated O3in Bt-SY63and SY63riceThe results showed that the leaf length and leaf area of Bt-SY63under A-O3were significantly smaller than those of SY63at the same O3concentration, and thecell wall thickness of the mesophyll of Bt-SY63under A-O3was significantlysmaller than that of SY63at the same O3concentration. Compared with thatobserved under A-O3, the leaf thickness of Bt-SY63increased, whereas that of SY63decreased under E-O3. E-O3had no significant effects on leaf length, leaf width, leafarea, stomatal length, stomatal density and cell wall thickness for both Bt-SY63andSY63. The changes observed in chloroplasts of rice leaves suggest that E-O3accelerated the leaf senescence of the two genotypes rice. Bt-SY63is more sensitive to E-O3than SY63with the former coupled with greater damage.E-O3significantly increased the soluble protein contents of the stem and root ofBt-SY63, whereas no significant increase was found in the soluble protein content ofthe leaf of Bt-SY63relative to A-O3. No significant difference in the soluble proteincontents of the tissues mentioned above of SY63was found between the two O3concentration conditions. E-O3had no significant effect on the soluble sugar contentof the tissues mentioned above of both Bt-SY63and SY63.(2) The transcription of Bt gene and the express of Bt protein in Bt-SY63underelevated O3The results tested by ELISA showed that Bt protein contents of the stem androot of Bt-SY63under E-O3were significantly higher compared with A-O3, whichwas occured particularly at later growth stage, while there was no significantdifference in Bt protein contents of the leaf of Bt-SY63under E-O3relative to A-O3in2010. There were no significant changes in Bt protein contents of the leaf, stemand root of Bt-SY63under E-O3relative to A-O3in2011. The results tested byReal-time PCR showed that no significant difference was found on the relativequantity of the mRNA express of Bt gene extracted from the leaf of Bt-SY63underE-O3relative to A-O3in2011.The Bt gene of Bt-SY63in response to E-O3isconsistent with the response of Bt protein content itself under E-O3.(3) Effects of elevated O3on the population and diversity of rice soil microbialcommunity in Bt-SY63and SY63The cultivable bacteria number and actinomyces number in the soil rhizosphereof Bt-SY63significantly increased under E-O3versus A-O3, whereas no significantchanges were found on fungi number and anaerobic bacteria number under E-O3versus A-O3. The cultivable bacteria number in the soil rhizosphere of SY63significantly discreased under E-O3versus A-O3, whereas no significant changeswere found on fungi number, anaerobic bacteria number and actinomyces numberunder E-O3versus A-O3.The numbers of total rice soil microorganisms weredetermined using Real-time quantity PCR. The results showed that the number oftotal bacteria in Bt-SY63rice soil significantly increased under E-O3compared withA-O3, whereas no significant change was found on the number of total bacteria inSY63rice soil between E-O3and A-O3.The diversity of soil bacteria in the two ozone concentration conditions wasanalyzed by PCR-DGGE method. The results showed that there were significantchanges of the structural diversity of soil bacteria community in Bt-SY63, in whichShannon index and Evenness index significantly increased at the tillering stage,while the two indices mentioned above were significantly discreased at headingstage and late filling stage under E-O3relative to A-O3. No significant differenceswere found on the Simpson index in SY63between E-O3and A-O3throughout cropduration. It was similar for the three indice mentioned above in Bt-SY63and SY63 rice, only those of SY63were within a smaller range than Bt-SY63. Similary ofDGGE profiles was analyzed under the two ozone concentration conditions. Theresults showed that E-O3changed the flora composition the of soil bacteriacommunity structure in both Bt-SY63and SY63(later growth stage) compared withA-O3. Some new kinds of bacteria appeared under E-O3relative to A-O3, such asuncultured Verrucomicrobia bacterium, uncultured Acidobacteria bacterium,uncultured Sulfurimonas, uncultured Bacteroidales bacterium and unculturedBacteroidales bacterium.(4) Effects of elevated O3on the metabolic functional diversity of rice soilmicrobial community in Bt-SY63and SY63The metabolic functional diversities of soil microbial community under E-O3versus A-O3were determined using Biolog-ECO plate. The results showed thatmetabolic activities of Bt-SY63and SY63tended to decrease under E-O3relative toA-O3, but the differences were not significant compared with respective A-O3. Therewere also significant differences on the changes of metabolic functional indice ofsoil microorganisims in Bt-SY63and SY63under E-O3compared with respectiveA-O3. The edaphon’s ability of utilizing substrates of some specific carbon sourcesin both Bt-SY63and SY63(late filling stage) rice soil changed under E-O3versusA-O3. High ability of utilizing substrates of Carbohydrates and their derivants inBt-SY63rice soil under A-O3was converted into high ability of utilizing substratesof Metabolic mediates and secondary metabolites under E-O3. High ability ofutilizing substrates of amino acid substrates and their derivants, metabolic mediatesand secondary metabolites and fat acid and lipid in SY63rice soil under A-O3wasconverted into high ability of utilizing substrates of carbohydrates and theirderivants under E-O3.(5) Effects of elevated O3on the population and diversity of leaf surfacemicrobial community in Bt-SY63and SY63riceThe cultivable bacteria number on the leaf surface of SY63significantlyincreased under E-O3versus A-O3in2010, whereas no significant change was foundon the same parameter of SY63under E-O3versus A-O3in2011. No significantdifferences were observed on the above parameter of Bt-SY63under E-O3versusA-O3in2010and2011.There were no significant changes in cultivable fungi numberand actinomyces number on the leaf surface of both SY63and Bt-SY63under E-O3compared with respective A-O3.The diversity of leaf surface bacteria in the two ozone concentration conditionswas analyzed by PCR-DGGE method. The results of similary of DGGE profilesshowed that no significant differences were found on bacteria community structuresfrom leaf surface in both Bt-SY63and SY63under E-O3compared with respectiveA-O3. Compared with E-O3, different growth stages was the major excuse causeddifferent bacteria community structures in both Bt-SY63and SY63. (6) Effects of elevated O3on the metabolic functional diversity of leaf surfacemicrobial community in Bt-SY63and SY63riceThe metabolic functional diversities of leaf surface microbial community underE-O3versus A-O3were determined using Biolog-ECO plate. The results showed thatmetabolic activities of microbial community from the leaf surface of Bt-SY63andSY63tended to increase under E-O3relative to A-O3, which was significantparticularly at late filling stage. Significant changes were also occurred easily atlater growth stages on the leaf surface edaphon’s ability of utilizing substrates ofsome specific carbon sources in both Bt-SY63and SY63under E-O3versus A-O3.The kinds of utilizing substrates were different accoding to different growth stagesevery year.There were also no significant differences on the changes of metabolicfunctional indice of leaf surface microorganisms in Bt-SY63and SY63under E-O3compared with respective A-O3.In conclusion, different responses to elevated O3in transgenic Bt-SY63rice andconventional SY63rice were observed on leaf morphology, ultrastructure, solubleprotein, cultivable microbial number from soil and leaf surface, the number of totalbacteria including uncultured bacterium in the rhizosphere soil, the structuraldiversity and the metabolic functional diversity, which indicates that it is possiblefor facing different environmental risk in transgenic rice and conventional rice underthe projected high tropospheric ozone.