| The arbuscular mycorrhizal (AM) fungal and bacterial community structures in therhizospere of the dominant plant Sophora viciifolia Hance. grown at Qiandongshan lead andzinc mine were investigated using PCR-DGGE. Using the technologies of transmissionelectron microscope (TEM), energy dispersive X-ray spectroscopy (EDS) and qutitativereverse transcription polymerase chain reaction (qRT-PCR), we discussed the mechnisms ofAM fungi improving S. viciifolia’s Pb resistance ability in the aspects of ultrastructurallocalization of Pb in AM, Pb chelation by glomalin related soil protein (GRSP) and the S.viciifolia phytochelatin synthase gene expression pattern impacted by AM fungi under Pbthreat. The main results are as follows:1. The bacterial community structure in the rhizospere of S. viciifolia grown at fiveincreasing Pb concentration sitesIn the five sites with increasing soil Pb concentration, the diversity of bacteria declinedwith increasing Pb concentration, the band number in DGGE profile decreased from22to13.Bacterial community H′was negatively correlated with the total and available Pbconcentrations in the soil. RDA analysis revealed that the available nitrogen, the available Pb,the available P affected the bacterial community mostly, with eigenvalues of21.5%,17%and16.5%, respectively. Among the fifteen species cloned from the gel, eleven wereAcinetobacter, Serratia and Sphingomonas in Proteobacteria; three were Aerococcus inFirmicutes and the other one was Actinobacteria in Actinobacteria.2. The AM fungi community structure in the rhizospere of S. viciifolia grown at fiveincreasing Pb concentration sites and the heavy metal concentration chelated by glomalinrelated soil proteinAM root colonization decreased from30.5%to9.6%with the increase of the Pbconcentration in soil. The band number in the DGGE gel decreased from31to13. The H′ofthe AM fungal community showed a significant negative correlation with the available Pbconcentration The total soil Pb concentration, the available P, the available N and pH affectedthe AM fungi community structure mostly. The dominant AM fungi is Glomus in Glomeraceae and Diversispora in Diversisporaceae, among the thirteen sequences clonedfrom the gel, ten were Glomeraceae; two were Diversisporaceae and the other one was non-AM fungi.Total GRSP (2.9~6.8mg/g dry soil) increased with the increase of the Pb concentration(57.5~6295.2mg/kg) in soil. The amount of Pb bound to GRSP varied from3.3to172.5mg/kg, which positively correlated with total and available soil Pb concentration, thus reducingthe bioavailability of Pb.3. The influence of AM fungi inoculation on the growth and Pb uptake of S. viciifoliaand the Pb ultrastructure localizationThe seedlings of S. viciifolia were inoculated with Glomus mosseae under different Pbappication levels, results showed low Pbconcentration (50μg/g) promoted the growth of S.viciifolia seedlings, but higher (500and1000μg/g) inhibited. Compared with non-inoculationtreatment, G. mosseae inoculation decreased both the Pb concentrations of aboveground andbelowground, the Pb concentrations of aboveground and belowground of the mycorrhizal S.viciifolia were118.48and47.49μg/g when exposed to1000μg/g Pb, decreased by61.0%and15.2%. The root length, root forks, root tips, root surface and root volumn of mycorrhizalS. viciifolia were higher than corresponding non-mycorrhizal plants. Compared with non-mycorrhizal plant, these parameters of mycorrhizal plants increased by220%、219%、157%、225%'278%when exposed to1000μg/g Pb(NO3)2. The ratio of root length whosediameters were between0~0.2mm to the total root length significantly increased with theincreasing Pb additions, and G. mosseae inoculation significantly reduced the ratio. Under Pbstress, G. mosseae increased the ratios of root length with0.4~0.6,0.6~0.8and0.8~1.0mmdiameters to the total root length, indicating that AM fungi inoculation thicken the roots with0.4~1.0mm diameter under Pb additions.The combination data of TEM and EDS indicated that Pb can deposited not only in plantcells, but also the cell walls and vacuoles of the AM fungi intracellular hyphae, whichrevealling that the subcellular level mechanism of AM fungi alleviated the Pb toxicity to host.4. Cloning the genes of SvPCS1and SvActinThe full length of SvPCS1cDNA is2098bp, containing a1503bp length ORF, coding501amino acids, predicted molecular weight is55.46kDa and the isoelectric point is7.13.The deduced amino acids of SvPCS1showed84.63%homology with LjPCS1(Lotusjaponicas, AAT80342), and85.43%with GmPCS1(Glycine max, AAL78384). The fulllength of SvActin cDNA is1679bp, including an1134bp length ORF, coding378aminoacids, predicted molecular weight is41.65kDa and the isoelectric point is5.05. Thehomology of SvActin with RcActin (Ricinus communis, AAR15174) and TpActin (Trifolium pretense, AAQ74875) reached98.67%and98.42%, respectively. The NCBI gene bankaccessing numbers of SvPCS1and SvActin are JQ780609and JQ780610, respectively.5. The influence of AM fungi inoculation on SvPCS1expressionThe response of SvPCS1expression, chlorophyll fluorescence parameters and phyto-chelatins (PCs) to G. mosseae inoculation under Pb stress (0,50and200μM Pb(NO3)2) atdifferent durations (1,3and7day) were studied. The chlorophyll fluorescence parametersFv/Fm, Fv/Fo, qP and Y(Ⅱ) of non-mycorrhizal S. viciifolia decreased and NPQ rose with theincreasing Pb concentration, all the parameters of mycorrhizal S. viciifolia showed the samechange patterns with non-mycorrhizal, but smaller change, indicating that mycorrhizalsymbiosis alleviated the Pb toxicity to plants. When S. viciifolia was exposed to0,50and200μM Pb for1and3d, G. mosseae inoculation promoted the expression of SvPCS1andsynthesis of PCs; when the duration increased to7d, SvPCS1expressions and PCs productionexposed to0and50μM Pb were still higher than controls, but lower in the S. viciifoliaexposed to200μM Pb, indicating that G. mosseae inoculation promoted SvPCS1expressionand SvPCs production under Pb threat for a certain period of time. |
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