Mechanisms Of Reactive Oxygen Species In Brassica Napus Growth And Development And Signal Transduction Under Cold Stress

Brassica napus L.is an important oil crop in China,but it is difficult to overwinter in the north of latitude 35^obecause of its poor cold resistance.Therefore,the new B.napus type16VHNTS309 was developed by crossing winter B.napus with Brassica rapa L.Longyou 7with strong cold resistance.The aim of this study was to clarify the mechanism of reactive oxygen(ROS)in regulating the growth and development of 16VHNTS309 and its response to cold stress based on physiological,biochemical,cellular and molecular studies.1)Using female parent Longyou 7 as a genomic probe,GISH results showed that a genomic signal was detected on 30 chromosomes of 16VHNTS309(2n=38),which was mainly distributed in the centromeres,satellite and short-arm translocations,suggesting that the cold resistance of 16VHNTS309 was related to the infiltration of a gene(small fragment or large fragment)into strong cold-resistant B.napus.2)The hypocotyls and cotyledons of B.napus 16VHNTS309 were used as explants to construct the regeneration system of B.napus by adding different concentrations of 2,4-D,6-BA,NAA and Ag NO₃into MS medium.The results showed that cotyledons and hypocotyls were precultured on MS+1 mg/l and 1.5 mg/l 2,4-D medium respectively for 7days,and then buds were induced on MS+3.0 mg/l 6-BA+0.2 mg/l 2,4-D+3 mg/l Ag NO₃medium.In the end,regenerated plantlets of B.napus can be obtained on MS+0.2 mg/l NAA rooting medium.3)In order to adapt to the cold stress environment,16VHNTS309 showed some phenotypic characters such as creeping growth,leaf color changing to yellow-green or purple,etc.The activities of SOD,CAT and POD and the accumulation of osmotic regulators(Pro,soluble protein and soluble sugar)of 16VHNTS309 were significantly higher than that of Tianyou2238,this is also effective to a certain extent to eliminate the accumulation of ROS in the body and reduce the damage to cells.The results also showed that the mesophyll cell ultrastructure of the weak cold-resistant cultivar Tianyou 2238changed significantly under cold stress,including expansion and dispersion of cell membrane,unclear outline,condensation of nuclear Chromatin,destruction of mitochondria and chloroplast structure,etc.4)Both 16VHNTS309 and Tianyou2238 accumulate a small amount of ROS during the normal metabolic process.However,ROS is released rapidly after low temperature stress,which is not only an important signal of local immune response,but also an important signal of cell-to-cell communication.However,the accumulation of ROS(H₂O₂and O₂^-)in the cells of 16VHNTS309 was significantly lower than that of Tianyou2238.The results of O₂^-sub-cellular localization showed that O₂^-diffused to surrounding cells,which indicated that ROS signal transduction was a dynamic process.O₂^-cell localization and DPI validation further strengthened the idea that chloroplasts,mitochondria and plasma membrane NADPH oxidase were involved in ROS formation in B.napus cells.The ROS production mechanism was different in different tissues and different tissues of the same site.This also provides strong evidence for the NADPH enzyme-mediated"ROS wave"signaling mechanism.The results also showed that the vascular tissue system of B.napus could synthesize redox messenger,amplify signal and transfer system,and it was a long distance signal transmission channel between different tissues and organs of ROS,which could realize cold stress response of B.napus.5)Appropriate ROS(H₂O₂and O^-₂)are also the key molecules necessary for the growth and development of B.napus.The results showed that O₂^-signal was detected in root meristem,shoot meristem,leaf primordia,leaf margin and callus,which indicated that O^-₂was actively involved in regulating cell division.When DPI was added to seedlings,Calli and seeds to decrease endogenous ROS production and inhibit the growth and development of 16VHNTS309.However,after the application of 0.6%H₂O₂,the endogenous ROS increased significantly and the germination rate of seeds reached 67.5%,which indicated that exogenous H₂O₂could effectively alleviate the inhibition of DPI,and further confirmed that the appropriate amount of ROS played a key role in regulating the growth and development of B.napus seeds.The results also showed that Bn UBP1 gene was negatively correlated with the accumulation of O₂^-signal in B.napus.The silent expression of Bn UPB1 gene could regulate the accumulation of O₂^-and enhance the ability of cell division.This study supports the previous research that UPB1 plays an important role in regulating the balance between H₂O₂and O^-₂.6)The results showed that 0.3%-0.6%H₂O₂was the optimum concentration range for seed germination of B.napus.High concentration of H₂O₂(0.7%-1.3%)decreased the ability of eliminating ROS,and more ROS(H₂O₂and O^-₂)were produced in B.napus,which inhibited the growth and development of B.napus.The results showed that 1.4%-1.5%H₂O₂was the semi-lethal concentration of H₂O₂for the growth and development of B.napus,but when H₂O₂concentration was obove 2.1%,the seeds of B.napus did not germinate and the contents of O^-₂,SOD,POD and CAT decreased to the lowest value,which indicated that high concentration of exogenous H₂O₂could induce cell death,enhance cell permeability and accumulate high concentration of H₂O₂in the cells,which was consistent with DAB staining and H₂O₂content measurement.7)Based on the transcriptome data GO and Kegg analysis,57 pathways in16VHNTS309 showed significant changes(q Value<0.05),while 9 pathways in 2238showed significant changes(q Value<0.05),which may be related to the difference of cold resistance in B.napus.In 16VHNTS309,the metabolism pathway of vitamin B6,peroxisome,autophagy and sulfur,which are related to ROS production and clearance,plays an important role in the process of stress tolerance.It also indicates that the variety with strong cold resistance has high ROS clearance ability and less ROS accumulation in cells.It has been proved that ROS plays an important role in the growth and cold stress signal transduction of B.napus,and there is a"ROS wave"dynamic signal transmission mechanism between cells.The Ca²⁺,MAPK cascades,transcription factors(WRKY),ABA and H₂S of 16VHNTS309 showed significant changes after cold stress,this further suggests that there are significant interactions between RO and key molecules such as Ca²⁺,MAPK and transcription factors(WRKY),ABA,H₂S,which regulate the expression of cold tolerance gene in 16VHNTS309,the 16VHNTS309 showed strong cold resistance.