Effects Of Exogenous Brassinolide On Root Proteomics And Cell Walls In Cucumber Seedlings Under Ca?NO₃?₂ Stress

The NO3-and Ca2+ accumulation are the main characteristics of soil secondary salinization,the problem of soil salinization severely limits the development of vegetable horticulture in China,especially cucumber.The application of exogenous hormones is an effective measure to alleviate the stress injury of vegetable crops.Brassinosteroid has been widely used in plant growth and development,and stress defense.Previous studies showed that exogenous 24-epibrassinolid(EBL)could alleviate the inhibition of root growth of cucumber seedlings under Ca(NO3)2 stress,but the exactmechanism is unknown.For this purpose,this study was based on the salt sensitive type of cucumber variety "Jin you 4" as the test material,to study the effect of exogenous EBL on the proteome and cell wall of cucumber seedlings under 80 mmol·L-1 stress,to investigate the effect of EBL on the function location and life process of cucumber seedlings under salt stress.The results obtained are listed as followings:1.Under Ca(NO3)2 stress,cucumber seedling roots growth was significantly inhibited and more lateral roots produced to adapt to salt stress.However,exogenous EBL significantly alleviated the restrictions on the root growth by salt stress.Under Ca(NO3)2 stress,the Ca2+ ion content of cucumber seedling roots was significantly increased,while content of K+ and K+/Ca2+ ratio were significantly down regulated.Ca2+ content of cucumber seedling roots under salt stress was decreased while K+ content and the ratio of K+/Ca2+ increased with the application of exogenous EBL.Ca(NO3)2 stress significantly increased the contents of osmotic adjustment substance in cucumber seedling roots(soluble sugar,proline and soluble protein),as well as the relative electrolyte leakage.Exogenous EBL treatment reduced soluble protein and soluble sugar of cucumber seedlings under salt stress,but had little effect on proline content and relative electrolyte leakage.These showed that exogenous EBL could increase the salt tolerance of cucumber seedlings by adjusting the content of osmotic substances.2.About 15 differential bands were found in SDS-PAGE map,which exhibited soluble protein of four treatments cucumber seedling roots.A two-dimensional electrophoresis gel coupled with MALDI-TOF/TOF MS was performed to investigate the effects of exogenous 24-epibrassinolide(EBL)on proteomic changes in cucumber seedling roots under Ca(NO3)2 stress.A total of 80 differentially accumulated protein spots in response to stress and/or exogenous EBL were identified and grouped into different categories of biological processes according to Gene Ontology,including metabolism,energy,cell structure,defense,transcription and translation,protein modification and degradation and miscellaneous proteins.Under Ca(NO3)2 stress,proteins related to nitrogen metabolism and lignin biosynthesis were induced,while those related to cytoskeleton organization and cell-wall neutral sugar metabolism were inhibited.However,the accumulation of abundant proteins involved in protein modification and degradation,defence mechanisms against antioxidation and detoxification,and lignin biosynthesis by exogenous EBL might play important roles in salt tolerance.These results suggested that exogenous EBL can operate cucumber root proteome under Ca(NO3)2 stress,then enhance cucumber tolerance to Ca(NO3)2 stress,in turn,promote the growth of cucumber seedlings.3.EBL could significantly alter the root cell wall complex under Ca(NO3)2 stress,and contents of pectin and hemicellulose uronic acid reached the highest value.Ca(NO3)2 had no significant effect on the total sugar content in cell wall of cucumber seedling roots.3 days later,Ca(NO3)2 significantly increased the content of lignin,while exogenous EBL can make lignin content in roots of seedlings under salt stress maintained at 25 mg-g-1.The expression levels of PAL?COMT788?COMT109 and LAC525 in stressed plants were significantly higher than those in control.At the same time,the expression of PAL?COMT109?COMT290?CCoAOMT121?LAC525 and LAC947in roots of plants under salt stress were maintained at low levels by exogenous EBL.7 days after treatment,Ca(NO3)2 promoted the differentiation of the root of cucumber seedlings and the accumulation of lignin in the primary xylem,while exogenous EBL made the root cell wall lignin mainly deposited in the casparian strips.Under Ca(NO3)2 stress,exogenous EBL increased the level of the transcription of glycosyltransferase and ?-D-galactosidase,after which the control and lower levels were maintained,however MKK4/5 on the contrary.Also,Ca(NO3)2 can induce BR signal of cucumber root,and EBL can regulate the BR signal transduction.It is possible to resist the active transport of Ca2+ and NO3-by promoting the thickening of the secondary cell wall of casparian strips,and/or enhance intercellular signaling and the water transport between cells,to maintain the growth of cucumber seedlings.