The Effects Of Artificial Aging Treatment On Brassica Napus L. Seed Lipidome

The rapeseed(Brassica napus L.)is one of the most important oil crops in China,which has an oil content that ranges between 27.4% and 52.7%.The high content lipids in seeds are more susceptible to peroxidation during storage,which causes a rapid decline in seed vigor.In this study,“Zheyou 18” rapeseed was selected as experimental material.Seeds were aged to different germination rate level that included 80%,49% and 5% by high temperature treatment.We used UPLC-QTOF-MS/MS method to investigate the lipidomic changes in artificially aged raspeseed,detected the changes in cellular redox state,gene expression and also observed the ultra-structure and integrity of plasma membrane,in order to gain a better understanding of the mechanism of oil seed aging.Main results are as follows:(1)Artificial aging treatment affected lipid components in rapeseed.In artificially aged seeds,although the total lipid content changed slightly,the glycerolipids content increased slightly and the phospholipid content decreased rapidly.Compared with the control(99%),when seed vigor declined to 80%,49% and 5%,the phospholipid relative content was respectively reduced by 9.11%,25.95% and 27.76%,while the relative content of diacylglycerol was respectively increased by 3.28%,5.37%,9.31%.Meanwhile,fatty acid content decreased gradually in aged seeds.(2)Artificial aging effected phospholipid composition.Compared with the control,the relative contents of Phosphatidylcholine(PC),Phosphatidylethanolamine(PE),Phosphatidic acid(PA)and Phosphatidylserine(PS)were dramatically reduced,whereas there was contrary variation tendency for the relative content of Phosphatidylinositol(PI)and Phosphatidylglycerol(PG).Along with the loss of seed vigor,PC content was declined from 3.84% to 2.75%.PC continually decreased during the aging process,however,several PC,such as PC(46),increased with seed aging,which indicated that there were differences in the effects of artificial aging on phospholipids with different carbon chain lengths.The unsaturation index of total phospholipid decreased significantly with seed aging.This reduction tendency was much more significant in PA,PC,PE and PG with highest number of unsaturated bond.Changes in membrane lipid content(as PC)implied that artificial aging adversely affected phospholipids composition in the cell membrane system,and the decrease in phospholipid unsaturation may be associated with lipid peroxidation.(3)Artificial aging influenced specific expression of key genes involved in phospholipid metabolism.In aging process,increased expression of phospholipase genes such as phospholipase C(PLC)and phosphatidic acid phosphatase(PAP)might lead to the decomposition of phospholipid into DG.But PC and PE were catalyzed into PA by Phospholipase D(PLD)which was only slightly up-regulated.DG was transformed into PE and PC by ethanolamine/choline phosphate transferase(CEPT)which was up-regulated expression.Combined with changes in phospholipid content,these results manifested that artificial aging leads to an imbalance in the gene expression of phospholipid metabolism in seeds.(4)ROS accumulation and lipid peroxidation may be one important reason of seed aging.DAB dyeing reflected that H2O2 accumulation ascended gradually.With aging treatment deepening,GSH content was ultimately decreased by 69.8%,and EGSSG/2GSH was raised from-184.97 m V to-166.06 m V,thus making the cellular environment being oxidation state.Meanwhile,compared with control,MDA content increased nearly 50% in low vigor seed.These results illuminated that ROS excessive accumulation broke redox homeostasis,resulted in the oxidation of cell membrane lipids,dropped the degree of membrane lipids unsaturation,and destroyed stability and mobility of membrane system,which caused the loss of seed vigor.(5)Artificial aging caused the damage of cell membrane structure.The results of membrane fluorescence labeling FM4-64 showed consistent plasma membrane staining and no intracellular fluorescence accumulation,which indicated integrative plasma membrane structure in control seeds.Whereas the intracellular fluorescence intensity was enhanced after artificial aging treatment.Cell membrane discontinuities were observed in the aged seed cells by using electron microscopy.Significant increasing in relative electrolyte leakage further showed that cell membrane integrity and permeability was broken and lots of soluble substances leaked out in aged seeds.(6)Lipid candidates as indicators for seed vigor loss.we used OPLS-DA model to screen the lipidome data and selected 9 lipids which correlate with seed vigor significantly.These lipids including six Phospholipids(mainly PC),one Glycerolipid and two Fatty acyls.We believe that we can work on these lipids to develop early-warning techniques for seed vigor loss in the future.In conclusion,in this study we found that ROS excessive accumulation in artificially aged “Zheyou 18” seed(Brassica napus L.)broke cellular redox homeostasis,resulted in lipid peroxidation,affected the expression of phospholipid genes,reduced phospholipid content,aggravated cell membrane injuring and destroyed osmosis and stability of membrane.Finally,the function in membrane system was disordered,and seed vigor was reduced.