Response To High Temperature Stress And Its Chemicial Regulation Mechanisms In Herbaceous Peony (Paeonia Lactiflora Pall.)

Herbaceous peony(Paeonia lactiflora Pall.)is a traditional Chinese flower,which is adaptable to cool and dry environment but not adaptable to high temperature in summer.As an excellent ornamental flower with wide applications for landscape,P.lactiflora has been introduced to south region of the Yangzi River on large scale.However,the extreme high temperature in long summer greatly restricted the application of P.lactiflora in the southern region.Therefore,how to alleviate the high temperature stress injury on P.lactiflora is an urgent problem to be solved.It is of great theoretical and practical significance Study on the response mechanism to high temperature stress and the chemical regulatory measures could provide theoretical and practical basis for introduction of P.lactiflora.It is very important to alleviate the stress injury of peony and improve the difficulty of peony in the south.In this paper,firstly,the effects of high temperature stress on P.lactiflora were analyzed;secondly,the effects of paclobutrazol(PP333)and trehalose to alleviate the high temperature stress injury to P.lactiflora were studied;thirdly,the response of P.lactiflora to high temperature stress was studied based on the transcriptome and small RNA sequencing.The main results are as follows:(1)The net photosynthetic rate(Pn),stomatal conductance(Gs),intercellular C02 concentration(Ci),transpiration rate(Tr),water use efficiency(WUE),the maximal fluorescence yield(Fm),Fw/Fm,quantum yield of PSII(Y(II))and the relative electron transport rate(ETR)of P.lactiflora were decreased by the high temperature stress.While the minimal fluorescence yield(Fo),the quantum yield of regulated non-photochemical energy loss(Y(NPQ))and the quantum yield of non-regulated non-photochemical energy loss(Y(NO))were increased.High temperature stress also caused the closure of leaf stomatal,the damage to stomata guard cells,the compression or broken of cells and vacuoles,the disintegration of organelles,and the emerging of starch granules.(2)PP333 treatment could reduce the electrolyte leakage rate and the degree of membrane lipid peroxidation,restrain the production of superoxide anion(O2·-)and the hydrogen peroxide(H2O2),and increase activities of catalase(CAT)and superoxide dismutase(SOD)in P.lactiflora leaves under high temperature stress.Meanwhile,the Pn,GS,Tr,Ci,WUE,Fw/Fm and Y(II)were increase and the Y(NO)and Y(NPQ)were decreased.PP333 could not only reduce the injury to leaf cell structure,but also improve the expression levels of thermo-tolerance-related genes,especially HSP70-1,HSP70-2 and HSP20.(3)Trehalose treatment can also reduce the electrolyte leakage rate and the membrane lipid peroxidation of P.lactiflora leaves under high temperature stress.Meanwhile,it could inhibit the generation and accumulation of O2·-and H2O2,increase the activity of CAT and SOD,as well as improve Pn,Ci,WUE,Fv/Fm and Y(?).Trehalose could not only reduce the injury to leaf cell structure,but also improve the expression levels of thermo-tolerance-related genes,especially HSP40-1,HSP40-2 and POD.(4)The thermo-tolerance of 3 P.lactiflora cultivars were identified:'Zifengyu' was the thermo-tolerant cultivar and ' Hongy anzhenghui' was the thermos-sensitive cultivar.Subsequently,their transcriptomes were sequenced by RNA sequencing(RNA-seq)technology.In total,81,599 unigenes were obtained,and 34,940 unigenes had been annotated.Moreover,through digital gene expression(DGE)analysis of thermo-tolerant 'Zifengyu' and moderately thermo-tolerant 'Hongy anzhenghui',161 heat stress response genes had been screened involving heat shock protein genes(HSPs),plant hormone signal transduction related genes,fatty acid synthesis genes,reactive oxygen specie(ROS)-scavenging genes and secondary metabolites related genes.And the effectively and timely response of these genes to HS could endow thermo-tolerance to 'Zifengyu'.Among these genes,11 key thermo-tolerant related genes,whose expressions were all significantly up-regulated in 'Zifengyu' and 'Hongyanzhenghui' during development and the former possessed higher levels,could be regarded as the candidate genes.These genes including isoprene synthase gene(ISPS),2 peroxidase genes(POD),3-oxoacyl-acyl carrier protein reductase gene(FabG),3 transcription factor genes(bHLH,NAC and WRKY),HSP20 and 3 HSP70.(5)We chose a thermo-tolerant cultivar 'Zifengyu' and a moderately thermo-tolerant cultivar 'Hongyanzhenghui' under the natural long summer heat to investigate heat responsive miRNAs by miRNA sequencing.In total,24,008,974 and 21,981,360 raw reads were generated from 'Zifengyu' and ' Hongy anzhenghui',respectively,as well as 45,016,796 total sRNAs and 6,297,111 unique sRNAs were obtained.Then 271 conserved and 9 novel miRNAs were identified in 'Zifengyu',while 236 conserved and 11 novel miRNAs were identified in'Hongyanzhenghui'.Among them,71 known miRNAs with 272 potential target genes and 3 novel miRNAs with 4 potential target genes were significantly differential-expressed.Expression analysis of the 7 candidate miRNAs previously reported to be regulated by heat stress and their target genes were performed.It showed that miR172c-3p,miR395a,miR397a,miR408-5p and miR827 were up-regulated by heat stress,and expressed much higher in thermo-tolerant cultivar'Zifengyu',suggesting that they might be involved in heat stress response at the post-transcriptional level.These results could provide a better understanding about the thermos-tolerant mechanism of P.lactiflora on post-transcriptional level.