Mechanisms Of Exogenous 5-aminolevulinic Acid (ALA) On Alleviating Salt Stress In Cucumber Seedlings

In the protected cultivation,the salinization was caused by factors like increasing of cultivation years,improper or excessive application of chemical fertilizer,high temperature/high humidity in greenhouse and lack of rain water leaching.Accordingly,salinization in protected cultivation can bring a series of problems to agricultural production such as the obstacle of vegetable growth.At present,the problem of soil salinization has become an important factor of restricting the sustainable development of agriculture.Therefore,issues of improving the salt tolerance of crops have become a focus of agricultural science researches.In recent years,the application of 5-aminolevulinic acid（ALA）has become a rapid and effective method in increasing the yield of crops and enhancing crop resistance to abiotic stresses.However,the mechanism of ALA improving plant tolerance to salt stress is not clear.In this study,exogenous application of ALA was combined with NaCl stress.By studying plant growth morphology,antioxidant scavenging system,photosynthetic pigment biosynthesis and Na⁺transport distribution,the physiological and molecular mechanisms of exogenous ALA in alleviating salt stress in cucumber seedlings was discussed in order to provide some technical guidance and theoretical basis for cucumber cultivation under salinized conditions.The main results are as follows:1.The suitable concentration of exogenous ALA(25 mg L^-1)promoted the morphological growth of cucumber seedlings under moderate NaCl stress(50 mmol L^-1),including plant height and leaf area of seedlings,the growth inhibition of cucumber seedlings under salt stress was broken.However,the application of ALA to plants had a dose effect,specifically,the suitable concentration promoted plant growth,but the relative high concentration of ALA(50 mg L^-1)inhibited plant growth.Under moderate salt stress,exogenous application of ALA significantly increased the fresh weight and dry weight of cucumber seedlings,and also promote the formation of root morphology（including total root length,number of root tip,root volume and root surface area）of cucumber seedlings.In addition,exogenous ALA also inhibited the apoptosis of apical elongation region induced by salt stress,increased the number of living cells and enhanced the root activity of seedlings.2.Under NaCl stress,exogenous ALA increased the activities of ascorbic acid peroxidase（APX）,monodehydroascorbic acid reductase（MDHAR）,dehydroascorbic acid reductase（DHAR）and glutathione reductase（GR）involved in AsA-GSH cycle.Meanwhile,the contents of AsA and GSH were increased by exogenous ALA,as well as the ratio of AsA/DHA and GSH/GSSG.Therefore,the scavenging efficiency of H₂O₂ increased and the content of MDA decreased in cucumber seedlings.Moreover,the activity of ascorbic acid oxidase（AAO）,located in the cytoplasm,was also promoted by exogenous ALA,which indicated that ALA enhanced the extracellular buffering effect on oxidative stress.3.Exogenous ALA enhanced the chlorophyll fluorescence parameters of cucumber seedlings under salt stress conditions,including PSⅡactual photochemical efficiency（Y（II））,non-regulated energy dissipation of the quantum yield（Y（NO））,regulated energy dissipation of the quantum yield（Y（NPQ））and photochemical quenching coefficient（qL）.Besides,the photosynthetic gas exchange parameters of plants were also enhanced,such as net photosynthetic rate（Pn）,stomatal conductance（gs）,intercellular CO₂ concentration（Ci）and transpiration rate（Tr）.The study on ALA metabolic pathway found that ALA application significantly promoted the contents of endogenous ALA,uroporphyrinogenⅢ（UroⅢ）,protoporphyrinⅨ（ProtoⅨ）,Mg-protoporphyrinⅨ（Mg-ProtoⅨ）,protochlorophyllide（Pchlide）and chlorophyll a compared with the plants suffered salt stress.On the contrary,the content of heme increased under salt condition and decreased in treatments with ALA.Study on the relative expression levels of genes encoding key enzymes involved in ALA metabolic pathway showed that the relative expression levels of HEMA1,CHLH,POR and CAO were up-regulated in treatment with exogenous ALA.However,the expression level of HEMH gene was down regulated in exogenous ALA treatment.Besides,exogenous ALA treatment repaired the damage of ultrastructure of chloroplast caused by salt stress.Exogenous ALA restored the swollen chloroplast to its typical structure and formed an ordered thylakoid and granulocyte lamella.The degradation of thylakoid was reduced and the number of starch granules increased.4.The absorption and distribution of Na⁺in the root cells of cucumber were improved by exogenous ALA.Exogenous application of ALA reduced the uptake of Na⁺by root cells and alleviated the ion toxicity.In addition,exogenous ALA up-regulated the protein expression of plasma membrane H⁺-ATP enzyme（HA3）,tonoplast H⁺-ATP enzyme（VHA-A）,plasma membrane Na⁺/H⁺antiporter（SOS1）and tonoplast Na⁺/H⁺antiporter（NHX1）.Moreover,exogenous ALA treatment up-regulated the gene expressions of SOS1 and HA3 under NaCl stress condition;while the expression level of NHX1 and VHA-A had no significant difference compared with seedlings under salt stress.Finally,exogenous ALA also repaired the damage of the ultrastructure of the root tip cells caused by salt stress.Exogenous application ALA increased the number of mitochondria in root cells and decreased the ion deposition phenomenon in cytoplasm.