Characterization Of 24-Epibrassinolide In Cucumber Response To Low-Temperature Stresses

24-Epibrassinolide（EBR）is a plant growth-promoting steroid hormone,play essential roles in wide range of physiological and developmental processes under both normal and stress conditions.An accumulating body of suggestion indicates that EBR take part in regulation of functional reactions to several abiotic stresses in plant.The functional mechanism of EBR induce stress resistance are not completely elucidated.In present study investigates the physiological and biochemical mechanisms of EBR in cucumber under temperature stresses（low-temperature and suboptimal root zone temperature）.To investigated the role of EBR in low-temperature stress tolerance,the cucumber seedlings were treated with EBR and exposed to low-temperature.The treatments were,NT（normal temperature,26℃/18℃ day/night）,and three low-temperature（12℃/8℃ day/night）treatments:CK（low-temperature stress）;EBR（low-temperature and 0.1μM EBR）;and BZR（low-temperature and 4μM BZR（Brassinazole）,a specific EBR biosynthesis inhibitor）.The results indicated that low-temperature stress proportionately decreased cucumber seedling growth and the strong seedling index,chlorophyll（Chl）content,photosynthetic capacity,and antioxidant enzyme activities,while increasing reactive oxygen species（ROS）and MDA（Malondialdehyde）contents,hormone levels,and EBR biosynthesis gene expression level.However,EBR treatments significantly enhanced cucumber seedling growth and the strong seedling index,chlorophyll content,photosynthetic capacity,activities of antioxidant enzymes,the cell membrane stability,and endogenous hormones（IAA,ABA,ZR and EBR）,and upregulated EBR biosynthesis gene expression level,while decreasing ROS and the MDA content.Based on these results,it can be concluded that exogenous EBR regulates endogenous hormones by activating at the transcript level EBR biosynthetic genes,which increases antioxidant enzyme capacity levels and reduces the overproduction of ROS and MDA,protecting chlorophyll and photosynthetic machinery,thus improving cucumber seedling growth.The experiment was designed,to investigate the role of EBR in suboptimal root zone temperature（RZT）.The cucumber seedlings were exposed to suboptimal RZT（14±2℃）and treated with 0.1μM EBR（EBR group）,4μM BZR,or untreated（LT group）.The NT group（control,normal RZT）was exposed to 22±2℃.The results showed that LT and BZR（Brassinazole,Specific EBR biosynthesis inhibitor）significantly decline cucumber seedling;growth,chlorophyll contents,photosynthetic capacity,antioxidant enzyme activities,nutrients and hormones accumulation.Exogenous EBR significantly enhanced cucumber seedling growth,chlorophyll contents,photosynthetic capacity,nutrient contents,activities of antioxidant enzymes and reduced the contents of reactive oxygen species（ROS）;H2O2,?-and MDA.However,LT induced JA and ZR contents,while EBR treatments O2 enhanced ABA,IAA,GA3,EBR and iPA contents.The transcript levels of EBR-biosynthesis（CsDWF1,CsDWF2,CsDWF3,CsDWF4,CsDWF5,CsDWF6 and CsCYP85A2）genes are reduced by LT,while reported significantly higher in EBR treated seedling.These results suggest that EBR could alleviate the harmful effect of suboptimal RZT by regulating EBR-biosynthesis genes expression,levels of endogenous hormones and activate antioxidant enzymes to reduce the ROS production,and protect chlorophyll and photosynthetic machinery,thus leading to improved plant growth.Further we,investigate the mechanism of EBR in nitrate and ammonium flux rate,N metabolism,and transcript levels of NRT1 family genes in cucumber seedling suboptimal root zone temperature（RZT）.The cucumber seedlings were exposed to suboptimal RZT（14±2℃）and treated with 0.1μM EBR（EBR group）,4μM BZR,or untreated（LT group）.The NT group（control,normal RZT）was exposed to 22±2℃.The results showed that suboptimal RZT（LT）adversely affected on bleeding rate;root activity;enzyme activities of nitrate reductase（NR）,nitrite reductase（NiR）,glutamine synthetase（GS）,and glutamate synthase（GOGAT）;nitrate（NO3-）influx rate;ammonium（NH4+）efflux rate;and transcript levels of nitrate transporter（NRT1）encoding genes.However,exogenous EBR reduced the harmful effects and increased endogenous EBR contents,bleeding rate,root activity,enzyme activities（NR,NiR,GS,GOGAT）,NH 4+and NO ₃-flux rates and contents,and N accumulation.EBR-treated seedlings also upregulated the transcript levels of nitrate transporters（CsNRT1.1,CsNRT1.2A,CsNRT1.2B,CsNRT1.2C,CsNRT1.3,CsNRT1.4A,CsNRT1.5B,CsNRT1.5C,CsNRT1.9,and CsNRT1.10）,and downregulated CsNRT1.5A and CsNRT1.8.LT treatment upregulated the expression level of CsNRT1.5A,while exogenous BZR application downregulated the expression levels of NRT1 genes.These results indicate that exogenous application of EBR alleviated the harmful effects of suboptimal RZT through changes in N metabolism,NH 4+and NO 3-flux rates,and NRT1 gene expression,leading to improved cucumber seedling growth.This study provides the first evidence of the potent roles of EBR in the response to suboptimal RZT in cucumber,which can be used to improve vegetable production.The results of this study demonstrated that EBR has a great potential to alleviate the harmful effects of temperature stresses.The information generated in this study could improve our understanding temperature stress tolerance of cucumber plants and would be useful for greenhouse and protected vegetation production system management.