Salt Tolerance Mechanism Of Alfalfa Under Non-uniform Salt Stress

Soil salinity is one of the most harmful environmental factors that limit both plant growth and yield.Due to interactions among different environmental factors,soil salinity is rarely spatially uniform in field conditions,and varies greatly in plant rhizosphere,even within a single plant.Studies have indicated that compared with uniform salt stress,non-uniform salinity can alleviate the damage caused by salt stress to plants.However,the salt tolerance mechanisms of plant under non-uniform salt stress remain largely unknown.In this study,a split-root system was established to divide the root of alfalfa into two parts which were under uniform and non-uniform salt stress,and then inoculated with arbuscular mycorrhizal fungi（AMF）.The physiological and biochemical indexes of plant performance,ion regulation,oxidative damage and antioxidant enzyme activities,and transcriptome analysis were recorded.The main aims of our study were to investigate the evaluation of salt tolerance in different alfalfa varieties under non-uniform salt stress;effects of different concentrations of non-uniform salt stress on growth characteristics of alfalfa;physiological regulation mechanism of alfalfa or alfalfa-AMF under non-uniform salt stress;and the response of alfalfa at transcriptome level under non-uniform salt stress.The results will build an important theoretical basis for establishment and management,and breeding of salt-tolerance varieties of alfalfa.The main findings are as follows:（1）Both uniform and non-uniform salt stress significantly decreased the growth rate,above/below-ground biomass,the water uptake,and chlorophyll contents of all the six alfalfa varieties,while the membrane permeability,contents of malondialdehyde（MDA）and proline（Pro）increased at high salt stress.Six alfalfa varieties showed different responses to salt stress.The salt tolerant sequence of cultivar at uniform salt stress of 100/100 and 200/200 mmol·L^-1 NaCl was Zhongmu No.1,WL354HQ,WL343HQ,WL353LH,Algonquin,WL298HQ,but at the non-uniform salt stress of 0/200 mmol·L^-1 NaCl was Zhongmu No.1,WL354HQ,WL298HQ,WL343HQ,WL353LH,Algonquin.Zhongmu No.1 appeared to be the most tolerant cultivar to different salinity,whereas Algonquin had a lower salt tolerance.WL298HQ under uniform and non-uniform salt stress varied greatly,other cultivars showed a moderate salt tolerance.（2）Salt stress can inhibit the growth of alfalfa,however when the part of root system was under no-or low-saline condition,the plant with a higher K+/Na+,increased the chlorophyll contents and decreased the degree of membrane lipid peroxidation compared with the uniform high salt stress treatments.The no-or low-saline part showed the compensated water uptake and compensatory growth of the roots,which promote the growth of plant,increase shoot and root biomass.Within a concentration range(0～200 mmol·L^-1 NaCl),the more difference between two sides of the roots,the relief effects were more obvious.（3）Non-uniform root zone salinity alleviated the damage caused by high salt stress and improved the growth of both alfalfa cultivars,associated with the lower leaf Na+concentrations,higher leaf K+concentrations,and higher activities of SOD,CAT,and POD compared with high uniform salt stress.We found that the decreased Na+ accumulation and increased K+concentration by ion recycling between shoot and root,as well as increased Na+efflux and decreased K+efflux in the no-or low-saline part of roots might play an important role in the alleviation of salt damage.In addition,osmotic adjustment and antioxidant defense were not localized in the plants,and that there was a functional equilibrium within the root system under non-uniform salt stress.The salt-tolerant cultivar Zhongmu No.1 exhibited greater Na+tolerance and efflux and better antioxidant defense capacity for high salt tolerance.（4）AMF inoculation ameliorated negative effects by enhancing the dry weight,plant growth rate,photosynthesis,activities of antioxidant enzymes including CAT,POD,SOD,ions and nutritive equilibrium,which might be the result of lower level lipid peroxidation and H₂O₂ contents under both uniform and non-uniform salinity treatments,especially with the whole root system inoculated AMF.Under non-uniform salinity,the oxidative defense was restricted to that compartment at the high-saline root side inoculated AMF,and it is indicated that AMF play the key role to alleviate the damage by salt stress.On the contrary,both root compartments have a systemically oxidative defense at the non-saline root side inoculated,and the functional equilibrium within root system was more important to enhance salt tolerance.In addition,the P uptake and ions regulation was more effective in the AMF root side no matter under uniform and non-uniform salinity.（5）We conducted transcriptomic analysis on the leaves and roots of alfalfa grown under uniform and non-uniform salinity treatments.A total of 233,742 unigenes were obtained from the assembled cDNA libraries.There were 98 and 710 unigenes identified as significantly differentially expressed genes（DEGs）in the leaves of alfalfa under non-uniform and uniform salt treatment,respectively.Furthermore,there were 5178 DEGs in the roots under uniform salt stress.There were 273 DEGs in the non-saline side and 4616 in the high-saline side roots under non-uniform salt stress.The Kyoto Encyclopedia of Genes and Genomes（KEGG）pathway enrichment analysis of the DEGs in roots revealed both sides of the non-uniform salinity were enriched in pathways related to "phenylpropanoid biosynthesis" and "linoleic acid metabolism",and "MAPK signaling pathway-plant" was also indicated as a key pathway in the high-saline roots.We also combined a set of important salt-response genes and found that roots of alfalfa from the non-saline side developed more roots with increased water uptake by altering the expression of aquaporins and genes related to growth regulation.This might be a key mechanism involved in alleviating the damage caused by non-uniform salinity.In addition,hormone signal transduction and the antioxidant defense probably play important roles in increasing resistance to non-uniform salt stress on both sides of the roots.In conclusion,alfalfa mainly relied on the compensatory growth and compensated water uptake form no-or low-saline roots,and the balance of ion regulation and antioxidant defense function at the whole plant level to alleviate salt damage under non-uniform salt stress.The mechanism of symbiotic AMF in root system of both sides was different under non-uniform salt stress.In addition,through the analysis of the transcriptome level,it was found that"phenylpropanoid biosynthesis","linoleic acid metabolism" and "MAPK signaling pathway-plant" pathways play important roles in the regulation of alfalfa under non-uniform salinity.