Molecular Mechanism Of Reduced Vigor During Seed Aging In Eucommia Ulmoides

Eucommia ulmoides is a famous and precious woody medicine tree species and an important source of rubber in China,it belongs to family Eucommiaceae and genus Eucommia.It was found in the laboratory earlier that the vigor of E.ulmoides seed will decline after harvesting.However,the underlying mechanism still remain unknown.In this study,the seeds of‘Hua-Zhong No.15’E.ulmoides purchased from November 2020（stored in the seed bank for 3 months）were used as materials to accelerate the seed aging process through artificial aging method,and to explore the internal reasons for the decreased seed vigor.The results are as follows:1.Effects of different artificial aging treatment methods on the germination of E.ulmoides seedsIn this study,that chosed two artificial aging methods,high temperature（40℃）,high humidity（95%relative humidity）and hot water（58±1）℃,and analyzed the changes of seed vigor.The results showed that E.ulmoides seed were treated at high temperature and high humidity for 2,4,6,8 and 10 d,respectively,and the seed vigor decreased significantly compared with the control（Equilibrium at room temperature for 2 d only）.However,（58±1）℃hot water treatment had not change significantly.Therefore,high temperature and high humidity artificial treatment were used in this study to explore the aging of E.ulmoides.2.Effect of high temperature and high humidity aging treatment on membrane permeability of E.ulmoides seedsThe relative electrical conductivity of E.ulmoides seed aged for 10 days at high temperature and high humidity was 31.28%higher than that of the control（34.67%）,and absolute conductivity was 75.89%higher than the control 155.03μs/cm.MDA content increased by 58.18%compared with 50.22 nmol·g^-1,indicating that aging treatment increased cell membrane permeability and lipid peroxidation.3.Effects of high temperature and high humidity aging treatment on protective enzyme activity and osmotic regulatory substance content of E.ulmoides seedsThe protective enzyme activity and osmotic regulation substance content of E.ulmoides seeds were determined after high temperature and high humidity aging.The results showed that the protective enzyme activities decreased rapidly after 10 days of treatment,SOD activity decreased by 51.94%compared with the control 218.52 U·g^-1,POD activity decreased by 67.56%compared with the control 165.31 U·g^-1.CAT activity decreased by 30.73%compared with the control 387.30 U·g^-1,and APX activity decreased by 76.98%compared with the control 111.65 Umin·g^-1 after 6 days of treatment.The contents of soluble protein and soluble sugar decreased rapidly,the soluble protein content decreased by 59.16%compared with the control 16.55 mg·g^-1,and the soluble sugar content decreased by 52.00%compared with the control 38.47mg·g^-1.And proline content increased,when dealing with 10 d,compared with the reference 25.54 mg·g^-1 rose 91.11%,seed after aging treatment,respond to adversity stress related substance content increased,but with the processing time is extended,cell membrane permeability increased,integrity is impaired,extravasation,seed protective enzyme system destruction,lead to seeds aging.4.Effects of high temperature and high humidity aging treatment on gene expression related to E.ulmoides seeds physiology and membrane structureAfter high temperature and high humidity treatment,the expression of seven genes related to physiological and membrane structure of Eu PLD,Eu LOX and Eu LEA were up-regulated,which were 1.41,1.57 and 1.25 times of that of the control,respectively.The expression levels of Eu SOD,Eu ARF,Eu GA2ox and Eu PHB were down-regulated,which were 0.21,0.33,0.59 and 0.46 times of control,respectively.The results indicated that the stress resistance of seeds was enhanced to stabilize the structure of cell membrane,but the activity of PLD in cells affected the lipid composition of cell membrane and destroyed the integrity of cell membrane.LOX,a key enzyme of lipid peroxidation,catalyzed the degradation of stored lipid in seeds,which aggravated the degree of lipid peroxidation and led to the decrease of seed vigor.5.Effects of high temperature and high humidity aging treatment on fatty acid content and iodine value of E.ulmoides seedsThe fatty acid content and iodine value of E.ulmoides seeds were determined after high temperature and high humidity treatment.A total of 18 fatty acids were detected,including 9 unsaturated fatty acids,such as palmitic acid,oleic acid,linoleic acid,linolenic acid and cis-11-eicosenoic acid,and their contents all decreased compared with the control group,among which oleic acid,linolenic acid and linolenic acid decreased the most by 142.00 mg/100g,6.50 mg/100g,541.30 mg/100g,respectively.The results showed that the acid rancidity of unsaturated fatty acids was oxidized by aging treatment,leading to a decrease in the content and iodine value of unsaturated fatty acids.6.Effect of storage method on the viability of E.ulmoides seedsSeeds（purchased in November 2021）were stored at low temperature/vacuum（LT/V）,low temperature/nylon bag（LT/N）,room temperature/vacuum（RT/V）and room temperature/nylon bag（RT/N）to determine the change of seed vigor.The results showed that the activity indexes of the four storage methods all decreased.After 4 months of storage,germination rate decreased by 29.11%,33.44%,37.3%and44.67%compared with 79.67%of the control,germination potential decreased by17.67%,24.00%,23.44%and 31.00%compared with 49.89%of the control,respectively.Germination index decreased by 8.40,10.65,11.25,12.27 compared with control 19.83,vigor index decreased by 1.16,1.27,1.25,1.35 compared with control 2.02,respectively.The LT/V treatment had the smallest decrease in seed vigor index,which was beneficial to keep seed vigor.In this study,it was found that under high temperature and high humidity treatment,the vigor of E.ulmoides seeds decreased,which was due to the increase of conductivity and malondialdehyde content,and the destruction of cell membrane integrity.The seeds stabilized cell membrane structure by regulating proline content and improving Eu LEA gene expression in response to stress.However,with the prolonged treatment time,the physiological metabolism of seeds became dysregulated.Cut down the content of soluble protein and soluble sugar and protective enzyme activity decline is reduced,the degradation of storage material and respiratory metabolism speeds up,ros,a decrease in the ability of the mitochondria structure could be destroyed,seed germination may also with the decrease of the required material content,cell membrane integrity damage,increase cell oxidation degree and unsaturated fat oxidation,rancidity effect,Reduced seed vigor.In the process of storage,low temperature vacuum packaging can be adopted to delay the aging of seeds.