Arbuscular Mycorrhizal Fungi Regulating Water Transport And Drought Tolerance Related Gene Expression Of Populus × Canadesis ‘Neva'

Arbuscular mycorrhizal fungi(AMF)can improve the drought tolerance of plants,but the mechanism of AMF to improve drought tolerance of plants is not yet fully understood.In this study,the effects of Rhizophagus irregularis on the embolism vulnerability,the absorption of nutrient elements,and the allocation of nutrient elements to root,stem and leaf of Populus×canadensis‘Neva'under drought stress were studied.Transcriptome sequencing technology was used to screen the differentially expressed genes of phosphate transporter and aquaporin between the inoculation treatment and the control,and the effect of AMF on the relative expression of phosphate transporter and aquaporin was analyzed by fluorescence real-time quantitative PCR.A split-root system was used to study the regulation effect of AMF on photosynthesis,active oxygen homeostasis,the expression of phosphate transporter and aquaporin genes of Populus×canadensis‘Neva'.The main results are as follows:(1)Effect of AMF on embolism vulnerability of Populus×canadensis‘Neva'under drought stressUnder well-watered,mildly stressed,and severely stressed,inoculation of Populus×canadensis‘Neva'with R.irregularis increased the relative water content of the leaves by4.02%,3.30%and 3.93%,increased the specific leaf area by 25.96%,15.04%and 18.71%,increased the stomatal conductance by 83.33%,27.27%,and 26.67%,and increased the net photosynthetic rate by 36.21%,32.58%,and 37.70%.Compared with no-inoculation treatment,the P₅₀ value of Populus×canadensis‘Neva'inoculated with R.irregularis under mildly stressed increased by 12.62%,indicating that embolism became more vulnerable;the P₅₀ value of Populus×canadensis‘Neva'inoculated with R.irregularis under severe drought stress decreased by 10.60%,indicating that embolism vulnerability decreased.The results of the study showed that the inoculation of R.irregularis changed the water management behavior of Populus×canadensis‘Neva'from isohydrous behavior to non-isohydrous behavior by adjusting the relative water content of the leaves,specific leaf area,stomatal conductance,and net photosynthetic rate,and stabilized the embolism vulnerability of the xylem.(2)Effects of AMF on nutrient uptake and distribution of Populus×canadensis‘Neva'under drought stressInoculation with AMF increased P concentration in leaves and roots of Populus×canadensis‘Neva'under different water regimes.Under drought stress,AMF inoculation increased Ca concentration in leaves and decreased Ca concentration in stems.Under drought stress,AMF inoculation increased the contents of phosphorus,calcium,and manganese in leaves and the contents of phosphorus and copper in roots of Populus×canadensis‘Neva'.Under severe drought stress,inoculation with AMF reduced the distribution of nitrogen,phosphorus,potassium,and magnesium in leaves and the distribution of potassium,calcium,manganese,and zinc in roots.Inoculation of AMF increased the distribution of copper in roots under different water regimes.Principal component analysis showed that under well-watered and severely stressed,inoculation with AMF had a significant effect on the nutrient absorption and distribution of Populus×canadensis‘Neva'.The results showed that compared with non-mycorrhizal Populus×canadensis‘Neva',the nutrient absorption and nutrient allocation of mycorrhizal Populus×canadensis‘Neva'were enhanced and improved,resulting in faster growth,lower biomass loss and higher drought tolerance during drought stress.(3)Cloning and expression analysis of phosphate transporter genes from Populus×canadensis‘Neva'Between inoculation and non-inoculation treatments,10 differentially expressed phosphate transporter genes were screened out by transcriptome sequencing analysis.Use transcriptome data to screen and clone 4 phosphate transporter genes of Pc PT3,Pc PT4,Pc PT5,and Pc PT9 from Populus×canadensis‘Neva'.Pc PT3,Pc PT4,Pc PT5,and Pc PT9are stable hydrophobic transmembrane proteins located on the plasma membrane of the cell.All four proteins belonged to the MFS and all?-type proteins.Compared with the amino acid sequences of Populus trichocarpa and Populus euphratica,the full-length similarity was 93.96%?100.00%,and had similar evolutionary pattern with Populus trichocarpa and Populus euphratica.4 phosphate transporters belong to the PHT1 family and have the characteristic sequence GGDYPXSAXXXSX of the phosphorus transporter genes.Among them,Pc PT3 is specifically induced by AMF.Real-time fluorescence quantitative PCR analysis showed that AMF inoculation significantly upregulated the relative expression of Pc PT3 and Pc PT9 in roots of Populus×canadensis‘Neva',thus affecting the absorption of phosphorus.(4)Cloning and expression analysis of aquaporin genes from Populus×canadensis‘Neva'Between inoculation and non-inoculation treatments,28 differentially expressed aquaporin genes were screened.Use transcriptome data to screen and clone 10 aquaporin genes(Pc PIP1-1,Pc PIP1-2,Pc PIP1-3,Pc PIP2-1,Pc PIP2-2,Pc PIP2-3,Pc PIP2-4,Pc PIP2-5,Pc TIP1-1,and Pc TIP1-2).The 10 aquaporins are all stable hydrophobic transmembrane proteins,belonging to the full?-type protein,MIP(major intrinsic protein)family,with the highly conserved sequence of MIP family proteins NPA and the signal sequence of MIP family proteins SGXHXNPAVT.The full-length similarity with the amino acid sequence of Populus trichocarpa and Populus euphratica is 94.22%?100%.The aquaporin of Populus×canadensis‘Neva'had high homology with Populus trichocarpa and Populus euphratica,indicating that the aquaporin was highly conserved and had a similar evolutionary pattern with Populus trichocarpa and Populus euphratica.Real-time fluorescence quantitative PCR analysis showed that AMF inoculation significantly up-regulated the relative expressions of Pc PIP1-3 and Pc TIP1-1 in roots of Populus×canadensis‘Neva',but had no significant effect on the expressions of the other 8 genes,indicating that Pc PIP1-3 and Pc TIP1-1 played an important role in water absorption and transportation.(5)Effect of AMF on the drought tolerance of Populus×canadensis‘Neva'under the condition of split-root systemThe results of split-root system culture showed that AMF inoculation increased the net photosynthetic rate and stomatal conductance of leaves,increased the activity of POD,CAT and APX,reduced the contents of H₂O₂ and MDA,and the effect of AMF in double-rooted chambers was greater than that of single-rooted chambers,indicating that AMF on Populus×canadensis‘Neva'leaf photosynthesis and active oxygen scavenging have a cumulative effect.Inoculation with AMF increased the activity of APX in roots,decreased the activity of POD and CAT,and increased the content of soluble protein in roots,indicating that AMF had a partial effect on root ROS scavenging.Under well-watered,inoculation with AMF up-regulated the expression of Pc PT3,Pc PT4,Pc PT5,Pc PIP1-3,Pc PIP2-1,Pc PIP2-3,Pc TIP1-1,and Pc TIP1-2 in roots.Under drought stress,inoculation with AMF up-regulated the expression of Pc PT3,Pc PT4,Pc PT5,Pc PIP2-3,Pc PIP2-5,and Pc TIP1-1 in roots,and down-regulated the expression of Pc PT9,Pc PIP1-1,Pc PIP1-3,and Pc PIP2-1 in roots,indicating that AMF could affect the water and nutrient absorption of Populus×canadensis‘Neva'by locally regulating the expression of aquaporin and phosphate transporter genes.This study had shown that AMF can improve the drought tolerance of Populus×canadensis‘Neva'through the accumulation of carbon assimilation and reactive oxygen species(ROS)clearance in leaves,and local regulation of root ROS clearance,the expression of phosphate transporter and aquaporin.