| Plants often suffer from a variety of recurring environmental stress during its growth and development,but it doesn’t die or tend to extinction.On the contrary,plants have memory effect to environmental stress.When they are subjected to repeated environmental stress,they can quickly and better activate the response and adaptation mechanism to biotic stress,thus realizing long-term stable reproduction.However,there are many relative studies on model plants and main crops rather than medicinal plants.In addition,medicinal plants have long life cycle,its experimental conditions are not easy to control,and the metabolites are complex and difficult to obtain.Sorbus pohuashanensis has many chemical constituents such as flavonoids,terpenoids.biphenyl,etc.Sorbus pohuashanensis suspension cell(SPSC)with single system and fast growth rate has been established At the same time,several compounds and differentially expressed genes from SPSC induced by Yeast extract(YE)have been preliminarily screened.Therefore,in this study,YE was used as an elicitor to simulate biotic stress,and repeated biotic stress is applied to SPSC.The effects of stress memory are explored on physiological and biochemical levels,secondary metabolites and transcriptome levels when SPSC suffers from repeated biotic stress.The main research results are as follows:1.In order to establish the memory effect of SPSC on repeated biological stress induced by YE.In this study,the growth level of SPSC under repeated biotic stress was higher than that under single biotic stress at 1-4 days after the second treatment by giving SPSC single biotic stress and repeated biotic stress respectively.2.In order to explore the effect of memory effect on the physiological and biochemical functions of SPSCs under repeated biological stress,this study carried out studies from the aspects of cell water content,some substances in cell wall,damage degree of cell membrane and antioxidant system.The results were as follows:(1)At 1-5 days after the second treatment,the water content of the repeated stress group was higher than that of the single stress group.(2)At 48 h after the second treatment,the cellulose content of the repeated stress group was significantly higher than that of the single stress group,and at 24-48 h after the second treatment,the lignin content of the repeated stress group was higher than that of the single stress group,however there was no significant correlation between the pectin in the two treatment groups.(3)At 24-48 h after the second treatment,the O2-content of repeated stress group was lower than that of single stress group,and at 12-48 h after the second treatment,the H2O2 content of repeated stress group was lower than that of single stress group.At the same time,at 6-48 h after the second treatment,the activities of CAT,SOD and POD in repeated stress group were always higher than those in single stress group.In addition,at 12-48 h after the second treatment,the proline content in the repeated stress group was always higher than that in the single stress group,however,there was no significant correlation between carotenoids and GSH in the two treatment groups.(4)At 48 h after the second treatment,repeated stress group of electrolyte leakage rate is lower than the single stress,at 24-48 h after the second treatment,repeated stress cell vitality than single stress group,in addition to this 6-48 h after the second treatment,although the malondialdehyde content of repeated stress group always higher than that of single stress group,But the difference in levels between the two groups is getting smaller.3.In order to explore the influence of memory effect on the secondary metabolites of SPSC under repeated biological stress,UPLC-Q-TOF-MS technology was used in this study to analyze the secondary metabolites of SPSC under different treatment groups,and it was found that memory effect would change the overall accumulation of secondary metabolites of SPSC when it was under repeated biological stress.The secondary metabolite content was further analyzed according to some reference compounds that had been separated and obtained in the early stage,which found that after the second treatment group repeated stress of biphenyl compounds,the content of organic acid compounds,triterpene compound basic group is higher than the single stress,further analysis of the data found that,at 6-48 h,the memory effect always promotes the accumulation of biphenyl compounds noraucuparin,noraucuparin-5-O-β-D-Glu and 2’-hydroxyaucuparin-2’-O-β-D-Glu in SPSC under repeated biological stress.At the same time Memory effect promoted the part under most of the time point of the accumulation of organic compounds,in addition to this,at 6 h,only 3 triterpenoid compounds showed higher content in memory group than in non-memory group,and to the 12 h,the number rose to 7,to 24 h,however,the number reduced to 4,and at 48 h,This number went down to zero.4.In order to explore the influence of memory effect on the transcriptome levels of SPSC under repeated biological stress,transcriptome sequencing was conducted on the cell samples collected at 0 h,4 h,6 h,12 h,24 h and 48 h after the second treatment.The results were as follows:(1)Phenylpropanoid biosynthesis pathway is the most significant metabolic pathways both in single stress and in repeated stress,and the difference of metabolic pathways between the two treatment groups had no obvious difference.(2)Compared with the single stress group,the number of up-regulated genes was higher in the repeated stress group at 4-24 h after the second treatment than that in the down-regulated genes.(3)In this study,80 genes were screened out,including 14 citrate synthase genes(CS),7 cisaconitase genes(ACO),30 iso-citrate dehydrogenase genes(IDH),4 fumarate hydratase genes(FH),and 25 malate dehydrogenase genes(MDH).Further analysis showed that the expression levels of 69 genes were increased in the repeated stress group,of which 20 genes were mainly reflected in the early stage and 49 genes were mainly reflected in the late stage.In the single stress group,a total of 65 genes showed increased expression levels,of which 16 genes were mainly reflected in the prophase and 49 genes were mainly reflected in the late phase.Annotation to(3)In biphenyl compounds biosynthesis pathway 119 key enzyme genes were obtained,including 58 UDP-glycosyl transferase gene and 61 other related enzyme gene(OMT,BIS,PKS,B4H,PAL),further analyzing the data be repeated stress of the communist party of China has 33 set of genes as metabolism of biphenyl compounds accumulated trend,there were 14 UDP-glycotransferase candidate genes and 19 other related enzymes,respectively.The expression levels of 40 genes in the single stress group were consistent with the accumulation trend of biphenyl compounds in the metabolome,including 22 UDP-glycotransferase candidate genes and 18 other related enzymes candidate genes,respectively.(4)article 15 key enzyme genes were obtained in triterpene compounds biosynthesis pathway,including 2 squalene synthase(SQS),13 squalene epoxidase(SE),repeated stress group after eight genes have been raised in trend of the cut after seven genes have been cut the change trend of increase,and all genes in single stress group had no obvious change trend.The main conclusions are as follows:1.In this study,SPSC growth level under repeated stress was found to be higher than that under single stress,which established the phenomenon of biotic stress memory induced by YE.2.By studying the effects of memory effect on the physiological and biochemical functions of SPSCs under repeated biological stress,it was found that memory effect could improve the accumulation of water content,some substances in the cell wall,antioxidant enzymes and some antioxidant substances in SPSCs under repeated biological stress.At the same time,memory effect can enhance the scavenging effect of reactive oxygen free radicals in SPSC after repeated biotic stress after a period of time.In addition,the memory effect can relatively reduce the damage of SPSC cell membrane after a period of repeated biotic stress.It is concluded that memory effect can improve the physiological and biochemical functions of SPSC under repeated biological stress.3.By studying the memory effect on SPSC metabolic levels,it found that under repeated stress and single stress,SPSC secondary metabolites appeared more obvious change,then carries on the quantitative analysis of some compounds have been obtained,determine the memory effect for the promotion of biphenyl compounds accumulate strongest,and then organic compounds,Triterpenes are the weakest.4.By studying the effect of memory effect on SPSC transcriptome level under repeated biotic stress,we found that the differential expressed gene metabolism pathways were not obvious compared with single stress.Then it was speculated that memory effect might have specific selectivity on the number and types of gene expression by analyzing the genes of the tricarboxylic acid cycle,biphenyl biosynthesis pathway and key enzyme genes of triterpenoid biosynthesis pathway in the repeated stress and the single stress.In addition,memory effect may be more inclined to up-regulate the expression of some genes under repeated stress and single stress to make SPSC better adaptation to repeated biotic stress induced by YE. |
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