Physiological And Molecular Mechanism Of 24-epibrassinolide Induced Cold Tolerance In Brassica Rapa L.

Low temperature is one of the abiotic stresses that limit the geographical distribution of crops and seriously affect the growth,development,quality and yield of crops.The global crop losses caused by low temperature disasters are as high as hundreds of billions of dollars every year.Brassica rapa L.is an important oil crop and winter mulch crop in northern China.As an overwintering crop,its yield is seriously limited by low temperature stress.Therefore,studying the cold tolerance mechanism of Brassica rapa L.and using plant hormones to improve the cold tolerance of Brassica rapa L.is very important to improve oil yield and economic benefits.As a new type of plant hormone,brassinolide is widely involved in the process of plant growth and stress response.At present,studies have reported the role of BRs in regulating plant development and its tolerance to abiotic stress,but there are few reports on BRs alleviating cold tolerance of Brassica rapa L.seedlings.In this study,the Brassica rapa L.variety"Longyou 8"was used as the experimental material,24-epibrassinolide?EBL?was sprayed on the leaves to study the effects of EBL on the growth phenotype,membrane lipid damage,osmotic regulation,photosynthetic capacity and antioxidant capacity of Brassica rapa L.seedlings under low temperature stress,and the effect of EBL on Brassica rapa L.transcriptome under low temperature stress was analyzed by RNA-seq technique.The role of Ca²⁺in the regulation of cold tolerance of Brassica rapa L.by EBL was studied by adding Ca²⁺chelator?EGTA?and Ca²⁺inhibitor?LaCl₃?.The physiological and molecular mechanisms of cold tolerance induced by EBL in Brassica rapa L.were analyzed from the aspects of gene,physiology and biochemistry,which provided a new theoretical basis for the regulation mechanism of EBL in alleviating low temperature stress.The main experimental results are as follows:?1?Compared with low temperature stress alone,EBL pretreatment could reduce the wilting degree,MDA content,relative electrical conductivity,Y?NPQ?,NPQ,Y?NO?,ROS content,DHA content and H₂O₂ content of stomatal guard cells of Brassica rapa L.seedlings under low temperature stress.It promoted the increase of proline content,soluble protein content,photosynthetic pigment content,Fv'/Fm',ETR,Y?II?,qP,stomatal pore size,pore size ratio,CAT activity,POD activity,AsA content and AsA/DHA,as well as the up-regulation of CPK4,CPK5,ICE,COR and APX gene expression.The results showed that EBL pretreatment could alleviate the damage of Brassica rapa L.seedlings caused by low temperature stress and enhance the cold tolerance of Brassica rapa L..?2?Using RNA-seq technique,11335 differentially expressed?DEGs?,were identified in CK vs EL group,of which 5570 were up-regulated and 5765 were down-regulated.3913 DEGs,were identified in;CK vs L group,of which 1826 were up-regulated and 2087 were down-regulated.In L vs EL group,116 DEGs,were identified,of which 70 were up-regulated and 46 were down-regulated.GO functional annotations show that these DEGs are mainly involved in cell process,stimulus response and metabolism,as well as membrane,antioxidant activity,signal transduction and so on.The analysis of KEGG metabolites showed that DEGs was significantly enriched in glucose metabolism pathway,as well as in energy metabolism,signal transduction,membrane transport and other pathways.Real-time fluorescence quantitative PCR was used to verify10 genes with different expression in the transcriptome,and the results were highly correlated with the results of RNA-seq.?3?In order to study the difference of gene expression in the regulation of cold tolerance of Brassica rapa L.seedlings by EBL,DEGs in L vs EL comparison group was further analyzed,and some genes related to photosynthesis,biofilm system osmotic regulation,calcium signal,redox,transcription factor and post-transcriptional regulation,as well as some proteins and protein kinases were screened.EBL can induce the up-regulation of most genes related to photosynthesis,osmotic regulation of biofilm system and redox,and cause calcium signal transduction pathway.The results showed that EBL could alleviate the low temperature stress of rapeseed by inducing calcium signal transduction pathway,improving photosynthetic capacity,antioxidant capacity,material and energy transport capacity,osmotic regulation capacity and so on.?4?Compared with EBL+4?,EBL+LaCl₃+4?and EBL+EGTA+4?treatment could significantly increase MDA content,relative electrical conductivity,Y?NPQ?,NPQ,Y?NO?,ROS content,DHA content and H₂O₂ content of stomatal guard cells.It promoted the decrease of proline content,soluble protein content,Fv'/Fm',ETR,Y?II?,qP,stomatal pore size,pore size ratio,AsA content and AsA/DHA,and down-regulated the gene expression of CPK4,CPK5,ICE,COR and APX.The results showed that Ca²⁺was involved in the regulation of cold tolerance induced by EBL in Brassica rapa L..