Combined Effect Of Elevated CO₂ And Endophytic Bacterium On Plant Growth Cd Accumulation And Microbial Community Structure Of Hyperaccumulator Sedum Alfredii

Phytoextraction is the most widely studied and promising method for phytoremediation of heavy metal contaminated soil at present.Due to the long time and low efficiency of phytoextraction,it is difficult to promote the application in large scale,so bioaugmentation is a research hotpot currently.Endophytes can colonize in plants,which directly affect the physiological characteristics of plants,promote plants growth and heavy metal uptake and accumulation.Elevated CO2(eCO2)affect plant photosynthesis,root morphology,soil properities and microbial community,thus influence plants growth and heavy metal uptake and accumulation.To date,most studies on the effect of eCO2 or endophytic bacterium on plant growth and metal accumulation were conducted with C3 or C4 species,few studies about the combined effect of eCO2 and endophytic bacterium on heavy metal uptake and accumulation and microbial community are reported,especially for crassulacean acid metabolism(CAM)plants.Hydroponic experiment and rhizobox experiment were conducted to study the combined effect of elevated CO2 and endophytic bacterium on plant growth,Cd accumulation and microbial community structure of hyperaccumulator Sedum alfredii by in situ analysis,molecular biological technique and microbiology technology.The main results are as follows:1.A hydroponic experiment was carried out to study the effect of endophytic bacterium Buttiauxella sp.SaSR13(SaSR13),which isolated from the root of hyperaccumulator Sedum alfredii,on plant growth and cadmium(Cd)accumulation in S.alfredii.Laser scanning confocal microscopic(LSCM)images showed that SaSR13 was mainly colonized in the root elongation and mature zones.The inoculation with SaSR13 to Cd-treated plants significantly enhanced plant growth(by 39%and 42%for shoot and root biomass,respectively),chlorophyll contents(by 38%)and Cd concentration in the shoot and root(by 32%and 22%,respectively).SaSR13 stimulated the development of roots(increased root length,surface area and root tips number)due to an increase in the indole-3-acid(IAA)concentrations and a decrease in the concentrations of superoxide anion(O2·-)in plants grown under Cd stress.Furthermore,inoculation with SaSR13 enhanced the release of root exudates,especially malic acid and oxalic acid,which might have facilitated the uptake of Cd by S.alfredii.It is suggested that inoculation with endophytic bacterium SaSR13 is a promising bioaugmentation method to enhance the Cd phytoextraction efficiency by S.alfredii.In addition,we also compared the characteristics of Buttiauxella sp.SaSR13 with Sphingomonas sp.SaMR12.It was found that both SaSR13 and SaMR12 have strong colonization ability in the root of Sedumiaff alfredii,but SaMR12 has higher cadmium tolerance,larger auxin secretion and cadmium concentrations in the root and shoot.Therefore,SaMR12 was selected for the following study on the interaction of elevated CO2 and endophytes inoculation.2.A hydroponics experiment was conducted to investigate the combined effect of elevated CO2(eCO2)and inoculation with endophyte SaMR12(ES)on the photosynthetic characteristics and cadmium(Cd)accumulation in hyperaccumulator Sedum alfredii.The results showed that eCO2×ES interaction promoted the growth of S.alfredii.Shoot and root biomass were increased by 121.6%and 242.9%,respectively,as compared with plants grown in ambient CO2(aCO2).The interaction of eCO2 and ES significantly(P<0.05)increased chlorophyll content(53.2%),Pn(111.6%),Pnmax(59.8%),AQY(65.1%)and Lsp(28.8%),but reduced Gs,Tr,Rd and Lcp.The promotion of photosynthetic efficiency was due to higher activities of Ca2+-ATPase and Mg2+-ATPase and over-expression of two genes related to photosystem(SaPsbS and SaLhcb2),PS II activities were significantly(P<0.05)enhanced with Fv/Fm and O(II)increased by 12.3%and 13.0%,respectively,compared with plants grown in aCO2.In addition,Cd uptake in shoot and root of S.alfredii grown at eCO2xES treatment was improved by 57.4%and 61.6%,due to increased expression of SaHMA2 and SaCAX2 genes related to Cd transport.Our results suggest that eCO2×ES can promote the growth of S.alfredii due to increased photosynthetic efficiency and improved Cd accumulation,and showed great potential of improving the phytoextraction of Cd by S.alfredii.3.A hydroponic experiment showed that inoculation with endophytes could promote the development of root hair and elevated CO2 had a significant effect on the formation of lateral root.In addition,elevated CO2 significantly ehnaced NO content and the interaction of elevated CO2 and endophytes inoculation could increase enthylene content.NO and ethylene as plant signaling molecules,are involved in the formation of plant lateral root and root hair.Both hydroponics experiment and rhizobox experiment showed that the interaction of elevated CO2 and endophytes inoculation could promote the root growth and Cd accumulation of Sedum alfredii.The pH of rhizosphere soil was significantly decreased under the treatment of elevated CO2,but no significant effect was found on the pH of bulk soil.Soil enzyme activity and microbial biomass carbon were affected by the interaction of elevated CO2 and endophytes inoculation.The results of 16S rRNA and ITS gene amplicon sequencing showed that a-diversity of bacterial communities in different compartments could be enhanced under the interaction of elevated CO2 and endophytes inoculation.However,elevated CO2 or inoculation with endophytes decreased the richness and diversity index of bacterial community in phyllosphere.The study showed that the compartment was the largest source of bacterial community variation,followed by inoculation with endophytes.The greatest variation source of bacterial community in phyllosphere is the treatment of inoculation with endophytes,but the greatest variation source of soil fungal community is the concentration of CO2.PICRUSt analysis predicted the higher relative abundance of functional genes associated with amino acid metabolism in bulk and rhizosphere soil and higher relative abundance of genes related to genetic information processing in the endosphere.In addition,interaction of elevated CO2 and endophytes inoculation resulted in higher relative abundance of genes related to genetic information processing and other functional genes in phyllosphere.The study indicated that the interaction of elevated CO2 and endophytes inoculation affected the root development and the microbial community,thus affected the uptake and accumulation of cadmium in hyperaccumulator Sedum alfredii.