| Clematis fruticosa is an important soil retaining shrub which widely distributes in the sunny slopes of Daqingshan Mountain,eapecially in the arid and low-nutrient soil.It showed extremely strong vitality and played important roles in ecological protections of the sunny slopes.To discuss the influences of arbuscular mycorrhizal fungi(AMF)on the survival of C.fruticosa in the sunny slopes of Daqingshan Mountains,we systematically evaluated the community compositions of AMF and the relationships among the season,elevation,soil factors and AMF diversity in the rhizosphere of C.fruticosa based on Illumina MiSeq sequencing.Then,the dominant AMF species were screened by the method of spore morphological identification,and C.fruticosa were artificial inoculatedwith these dominant species.The growth,physiological and biochemical indexes weremeasured under well water and drought stress conditions,and the best drought resistance of AMF-C.fruticosa was selected based on these paramters.In addition,RNA-Seq technology was ulitied to analyze the different expressed unigenes of mycorrhizal seedings from different water conditions.Finally,the response mechanisms of mycorrhizal seedings to drought stress at the levels of physiology,biochemistry and unigenes were explained.The results were as followed:(1)A total of 247842 AMF sequences and 174 operational taxonomic units(OTUs)were obtained by Illumina MiSeq sequencing from 54 soil samples,belonging to Glomus,uncultured Glomeromycota,Diversispora,Rhizophagus,Claroideoglomus,Funneliformis,Scutellospora,Paraglomus,Ambispora,Septoglomus and Archaeospora,and Glomus was the daominant genus.(2)Seaonal variations significantly affected the colonization rate,arbuscular abundance,AM fungal richness,Chao 1 index,Shannon-Wiener index and Simpson index of AMF.Altitudinal variations had significant influences on the spore density,arbuscular abundance,and richness and Shannon-Wiener index of AMF.The interaction of season and elevation signidicantly affected the Shannon-Wiener index,while did not influence other indexes.In addition,AMF community composition significantly influenced by seasonal and altitudinal variations,but did not affected by the interaction of season and elevation.Specifically,AMF community composition in spring and 1600 m was significant different from the other seasons and elevations,but not significant different between summer vs autumn and 1400 m vs 1200 m.(3)Linear regression analysis indicated that season,elevation,soil available N,available P and soil water content significantly influenced the AM fungal richness and Shannon-Wiener index.Structural equation model(SEM)showed that season and elevation all directly affected the AM fungal comunity and Shannon-Wiener index,and they also could indirectly influence the AM fungal community and Shannon-Wiener index by the soil factors including available N,available P and soil moisture content.Soil available P was the primary factor that influenced the AMF community and Shannon-Wiener index,and compared with elevation,season was more likely to strongly affect AM fungal community and diversity.(4)A total of 3 dominant AMF species were obtanined from 54 soil samples based on the method of spore morphology identification,they were Funneliformis mosseae,Rhizophagus intraradices and Acaulospora mellea.(5)Colonization rate,growth,physiological and biochemical indexes were determined after the C.fruticosa were colonizated with 3 AMF species under well water and drought stress conditions,the results showed that the C.fruticosa incolated with R.intraradices had the highest colonization rate,arbuscular abundance,plant height,aboveground biomass,underground biomass,net photosynthetic rate,stomatal conductance,activities of SOD?CAT and POD,and content of total N and P than other treatments,indicating that R.intraradices-C.fruticosa had the best drought resistance.(6)The influences of well water and drought stress conditions on mycorrhizal seedlings were firstly analyzed at the levels of growth,physiology and biochemistry.Compared with well water conditions,drought stress significantly decreased the colonization rate,arbuscular abundance,aboveground biomass,net photosynthetic rate,stomatal conductance,total N and total P content,but significantly improved the content of proline and mavolonic dialdehyde,and the activities of SOD,CAT and POD.(7)RNA-Seq was used to evaluate the mycorrhizal seedlings responsed to different water conditions.105203 unigenes were generated after quality control and Trinity mosaic.Compared with the water well treatment,a total of 762 different expressed unigenes(DEGs)were found in drought stress treatment,including 370 up-regulated and 392 down-regulated DEGs.(8)The enriched analysis results of these DEGs related KEGG pathway to further explain the mycorrhizal seedlings response to drought stress,including ? The drought stress decreased the stability of PS ? and PS ? systems,and the expression of ribulose-bisphosphate carboxylase and fructose-bisphosphate aldolase in mycorrhizal seedlings for maintaing a relatively low rate of photosynthesis and carbon fixation in photosynthetic organisms.? Tryptophan synthesis promoted auxin formation;the up-regulated expression of lycopene epsilon-cyclase and 9-cis-epoxycarotenoid dioxygenase enhanced the carotenoid biosynthesis,and the down-regulated expression of abscisic acid 8'-hydroxylase inhibited the ABA degradation,this could improved the content of ABA;the up-regulated expression of 12-oxophytodienoic acid reductase could catalyze alpha-Linolenic acid to synthesis jasmonic acid;the biosynthesis of these hormones combined impact on the mycorrhizal seedlings,making it better response to drought stress.? The up-regulated expression of glutamate 5-kinase improved the formation of glutamate,and it could enhance the content of proline;the down-regulated expression of beta-glucosidase reduced the hydrolysis of sucrose;the up-regulated expression of beta-amylase significantly ehanced the hydrolysis of starch into maltose;it could improve the content of osmotic adjustment substances,and then maintain a suitable osmotic pressure of C.fruticosa under drought stress conditions.? The drought stress induced the biosynthesis of antioxidants including ascorbate,glutathione,isoquinoline alkaloid,ubiquinone and other terpenoid-quinone,and then the disadvantageous effects from active oxygen were decreased.? the up-regulated expression of mitogen-activated protein kinase kinase could activate the MAPK,and it was benefical for seedlings survived under poor water conditions. |
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