Effect Of Extracellular ATP On The Cold Resistance Of Brassica Campestris L.

Low temperature is an important environmental factor which affects plant growth and the crop yield.It not only causes a decrease in the crop yield,but even causes the death of plant in severe cases.At present,the problem of low temperature disaster has become a crucial factor restricting the development of agriculture.Improving the cold resistance of crop has become the focus of agricultural science researches.Many plants can activate metabolic mechanisms of body to increase resistance to low temperature by sensing changes of low temperature.Low temperature stress reduces the integrity of plasma membrane and results in intracellular solutes,including ATP,releasing into the extracellular environment.The extracellular ATP（eATP）becomes a critical signal molecule and plays a significant role in the regulation of defense responses after the intracellular ATP diffused into extracellular space through active transport or passive transport.The signal of ATP is involved in the regulation of a variety of physiological processes,and the concentration of ATP determines different physiological responses.Researches have shown that with the participation of eATP signal,plants respond to lots of environmental stresses such as pathogen invasion,mechanical damage,high salt stress and so on.However,there are few researches both home and abroad on whether eATP participates in the response of plants to low temperature stress,and the mechanism of improving the cold resistance of plants is still unclear.In this experiment,Brassica campestris Longyou 7 was selected as the research material,to alleviate the physiological and molecular mechanisms in low temperature stress of rape seedlings from these aspects such as the damage of membrane system,active oxygen metabolism,antioxidant system,osmotic adjustment system and photosynthetic system,by adding extracellular ATP to handle the rape seedlings under the low temperature stress,in order to provide a theoretical basis for planting rape under low temperature conditions.The main results of the research are as follows:1.In order to clarify the suitable concentration of extracellular ATP for Brassica campestris Longyou 7,this experiment choosed the influence of the four concentrations of ATP(10μmol·L^-1,25μmol·L^-1,50μmol·L^-11 and 100μmol·L^-1)on the biochemical growth of rape seedlings under 4℃.The results showed that 25μmol·L^-1ATP could significantly increase photosynthetic pigment contents and permeability regulating substances included soluble protein and soluble sugar of rape seedlings,and alleviated membrane peroxidation damage.25μmol·L^-11 ATP also could reduce the relative electrical conductivity and malondialdehyde content caused by low temperature in order to promote the growth of rape seedlings.In this study,the alleviation effect on the low temperature stress to rape seedlings of 25μmol·L^-1ATP was the best.2.Under low temperature stress,ROS accumulated gradually in rape seedlings.Proline and soluble sugar contents in rapes were both higher than those in the controlled group,indicating that the permeability regulators could alleviate the oxidative damage.Antioxidant enzymes SOD,POD,CAT,and APX enzymatic activities could increase to remove redundant ROS from cells caused by low temperature stress.Low temperature stress might cause the decrease of actual photochemical quantum yield Y（II）,the maximum quantum efficiency of PSⅡphotochemistry at illumination Fv’/Fm’under light adaptation,photochemical quenchin（qP）,apparent electron transport efficiency（ETR）in chlorophyⅡfluorescence parameters,and increase the quantum yield of regulated energy dissipation of Y（NPQ）and the non-photochemical quenching NPQ.After low temperature and ATP composite processing,the accumulation of H₂O₂ and·OH increased,but O₂^·?accumulation decreased compared with low temperature condition.Proline,soluble sugar contents and antioxidant enzymes SOD,POD,CAT and APX activities were all significantly elevated.All these could alleviate the changes in chlorophyll fluorescence parameters,and also could reduce MDA content to ease the degree of membrane lipid peroxidation.These figures indicated that extracellular ATP could improve the cold resistance of rape seedlings effectively.3.Compared with the low temperature and ATP composite group,the rape seedlings in another group which first pretreated by inhibitors included DPI（NADPH oxidase inhibitors）,DMTU（H₂O₂ scavenger）,U0126（MAPKK specificity blockers）and EGTA(Ca²⁺chelating agent)then make a composite processing had decreased permeability regulating substance contents,serious membrane lipid injury,antioxidant enzyme activity.These results indicated that NADPH oxidase,H₂O₂,MAPK cascade pathway and Ca²⁺were all involved in ATP-induced cold resistance of rape seedlings.4.Compared with low temperature treatment,low temperature and ATP composite processing induced an increase in H₂O₂ contents and the activity of Ca²⁺-ATPase.The pretreatment of extracellular CaCl₂ could promote the increase of H₂O₂ contents under the low temperature stress.On the contrary,the pretreatment of extracellular H₂O₂ also increased the activity of Ca²⁺-ATPase under low temperature stress.When DMTU（H₂O₂scavenging agent）was used to pretreat rape seedlings,the activity of Ca²⁺-ATPase decreased under low temperature stress.When the EGTA(Ca²⁺chelating agent)was used to pretreat rape seedlings,H₂O₂ contents decreased.Interestingly,adding ATP in DMTU/EGTA and low temperature composite group could significantly alleviate the inhibition by DMTU and EGTA.Through the study on the relative expression of key enzyme genes in the metabolic pathway,this research suggested that the relative expression quantities of respiratory burst oxidase genes RBOHD,RBOHF,calcium dependent protein kinase genes CPK4 and CPK5 were all up-regulated in ATP process.It indicated that Ca²⁺and H₂O₂ interacted with each other under low temperature stress,while Ca²⁺played a role together with H₂O₂ in activating the signaling pathways related to the cold resistance of rape seedlings.All of these results showed that extracellular application of 25μmol·L^-11 ATP could significantly improve the cold resistance of rape seedlings and alleviate the damnification caused by low temperature stress.Moreover,NADPH oxidase,MAPK cascade pathway,H₂O₂ and Ca²⁺were all involved in the ATP-induced cold resistance process of rape seedlings.Extracellular ATP could induce the transcriptional levels of RBOH genes and CPK genes of rape seedlings under low temperature stress,and promote the increase of H₂O₂ and Ca²⁺,which could further affect other physiological and biochemical reactions of rape seedlings,there by enhancing the cold resistance of rape seedlings.