Mechanism Of Arbuscular Mycorrhizal Fungi Enhancing The Salt Tolerance Of Casuarina Glauca Sieb.ex Spreng.

Casuarina species are widely used as coastal shelterbelts,but the high salt content and lack of nutrients in coastal sandy land led to the inhibition of Casuarina growth.Arbuscular mycorrhiza（AM）fungi can form mycorrhiza with Casuarina glauca Sieb.ex Spreng.under salt stress,thereby enhancing the salt tolerance and growth of C.glauca.However,the related mechanism is still unclear.This study analyzed the effects of AM fungi（Rhizophagus irregularis）on the growth,photosynthesis,ion distribution,osmotic regulation,and antioxidant capacity of C.glauca under 600 mM NaCl stress.Transcriptome sequencing technology was used to explore the effect of AM fungi on the gene expression of C.glauca.Full-length gene cloning technology and quantitative real-time PCR（qRT-PCR）analysis were used to study the effects of AM fungi on the Na⁺and Cl^-transporter genes in C.glauca.The mechanism of AM fungi enhancing the salt tolerance of C.glauca was revealed from the physiological,biochemical,and molecular levels.The main conclusions are as follows:1.Effects of AM fungus on the growth and photosynthesis of C.glauca under NaCl stressUnder 600 mM NaCl stress,AM fungus increased the fresh weight of shoots,the fresh weight of roots,ground diameter,and height by 368.96%,265.14%,76.79%,and 116.38%.600 mM NaCl stress increased the mycorrhizal dependence of the shoots and roots by 39.41%and 13.02%.While the salt tolerance index of shoots and roots in mycorrhizal C.glauca increased by 35.09%and 9.13%.This means that 600 mM NaCl stress not only increased the degree of influence of AM fungus on C.glauca,but also increased the degree of dependence of C.glauca on AM fungus.In conclusion,AM fungus promoted the growth and improved the salt tolerance of C.glauca.Under 600 mM NaCl stress,AM fungus significantly enhanced the photosynthetic capacity of C.glauca,increased net photosynthetic rate（Pn）,stomatal conductance（Gs）,intercellular CO₂ concentration（Ci）,and transpiration rate（Tr）by 48.68%,39.74%,13.79%,and41.26%.The maximum fluorescence efficiency（Fv/Fm）,actual photochemical quantum yield（ФPSII）,and electron transfer rate（ETR）were increased by 8.79%,32.16%,and 32.21%.The increase of Gs and Tr contributed to the binding of CO₂ to carboxylation sites on chloroplasts,while the increase of Ci increased the concentration of photosynthetic substrates.These two factors were favorable for CO₂ fixation,thereby consuming excess energy in photosynthetic cells,alleviating the energy dynamic imbalance,reducing the transfer of electrons to O₂,the production of reactive oxygen species（ROS）,and the damage caused by 600 mM NaCl stress.Higher ETR andФPSII could be used to buffer the loss of photosynthetically active leaf surface area.These effects were finally manifested in the promotion of the photosynthesis of C.glauca under NaCl stress.2.Effects of AM fungus on the salt tolerance of C.glauca under NaCl stressUnder 600 mM NaCl stress,the Na⁺and Cl^-transfer factor（TF）of mycorrhizal C.glauca were increased by 28.02%and 22.63%.AM fungus increased the Na⁺content in shoots and roots by 306.28%and 233.55%,and the Cl^-content in shoots and roots by 329.30%and260.06%.The Na⁺and Cl^-contents in the soil were reduced by 37.47%and 18.10%.Under600 mM NaCl stress,AM fungus increased the K⁺content of shoots and roots by 391.09%and 337.90%,and the proline concentration of shoots and roots by 22.55%and 38.63%.The activity of peroxidase（POD）in shoots and superoxide dismutase（SOD）in roots were increased by 10.91%and 62.76%.The Na⁺/K⁺in shoots and roots were reduced by 7.72%and23.79%,and the malonaldehyde（MDA）concentration in shoots and roots were reduced by38.54%and 24.06%.It indicated that AM fungus promoted cells to scavenge ROS,reduced the osmotic potential,relived the Na⁺and Cl^-stress,enhanced salt tolerance of C.glauca.3.Analysis of differentially expressed genes（DEGs）in the roots of mycorrhizal C.glauca under NaCl stressMycorrhizal C.glauca up-regulated high-affinity K⁺transporter gene 5（CgHAK5）,K⁺channel gene 3（CgKAT3）and stelar K⁺outward rectifier gene（CgSKOR）to absorb K⁺and balance Na⁺/K⁺.AM fungus promoted the absorption of water by up-regulating plasma membrane intrinsic protein gene 1-2（CgPIP1-2）,promoted the synthesis of POD and SOD by up-regulating peroxidase gene 64（CgPER64）,cytochrome c peroxidase gene（CgCPER）,CgPER53,and germin-like protein gene 10（CgGLP10）.AM fungus alleviated the damage to C.glauca’s roots by up-regulating genes（CgMYB46,CgNAC43,CgWRKY1,and CgWRKY19）related to transcription factors（MYB,NAC,and WRKY）under 600 mM NaCl stress.Results showed that AM fungus enhanced 600 mM NaCl tolerance of C.glauca’s roots by participating in the regulation of ion transport,antioxidant enzyme activity,carbohydrate metabolism,cell wall synthesis,and transcription factors.4.Effects of AM fungus on the expression of Na⁺and Cl^-transporter genes in C.glauca under NaCl stressUnder 600 mM NaCl stress,AM fungus regulated the expression of Na⁺efflux-related gene CgNHX7（Na⁺/H⁺exchanger gene 7）,reducing the expression of CgNHX7 in the shoots by 35.50%,weakening the efflux ability of Na⁺;increasing the expression of CgNHX7 in the roots by 50.68%,enhancing the efflux ability of Na⁺.The upregulation of CgNHX1 and CgNHX2-1 induced by AM fungus alleviated the inhibitory effect of Na⁺stress through Na⁺compartmentalization in vacuoles and K⁺homeostasis.AM fungus promoted the transfer of Cl^-from cytoplasm to vacuoles in mesophyll by up-regulating CgCLCG,promoted the Cl^-compartmentalization by up-regulating CgCLCD and CgCLCF,thereby improving the tolerance of mycorrhizal C.glauca to 600 mM NaCl stress and promoting its growth.The different coping mechanisms of Na⁺and Cl^-stress in C.glauca were clarified.The salt accumulation approach to Na⁺,which transferred Na⁺from roots to shoots,a process related to CgNHX7;the mechanism of Cl^-tolerance changed from salt accumulation to salt exclusion,and Cl^-no longer transferred to shoots,but began to accumulate in roots.