Studies On Physiological And Molecular Mechanisms Of Poplar In Respond To Partial Submergence

Floodplains and beach wetlands are frequently affected by flooding or waterlogging,and they are intermediate type between water and land.Most of those flood-affected lands are fecund and productive,and have ecological protecting fucntion.However,waterlogged lands are always submerged by flooding for 1-3 months in summer,leading to low yielding of crops.Plantation is a key solution to floodplain utilization and ecological protection.It has advantages in water and soil conservation,biodiversity protection,water purification,etc.Owing to the characteristics of fast growth and good flood tolerance,poplars are served as major afforestation tree species in flood-prone areas.However,flooding stress still negatively affects its survive and growth in these regions.Thus,it is important to uncover the mechanisms involved in its flood tolerance,which may facilitate developing poplar clones with improved flood tolerance.In order to uncover the mechanism of flood tolerance in poplar and find key genes participated in flood response regulation,44 full-sib family clones originated from Populus deltoides cv.Lux ex.I-69/55(flood-tolerant)×P.simonii(flood-susceptible)were adopted as the materials to flooding treatment.Form the 44 clones,LS1(the most tolerant to flooding)and LS2(the most susceptive to flooding)are selected to study the mechanisms contribute to their differences in flood tolerance on morphology,anatomy,metabolism and molecular transcriptional regulation systematically.The aim is to uncover the molecular and physiological mechanism of flood tolerance in poplar,find and clone candidate genes related to flood-tolerant regulation,and implement genetic transformation.The major resuls are as follow:1.After 50-d flooding treatment and 7-d recovery,only a few poplar clones survived.Significant differences were observed in survival rate,survival duration and growth among 44 full-sib clones.The most flood-tolerant clone,LS68,and the most flood-susceptive clone,LS81,were selected by comprehensive analysis on their survival rate,survival duration and growth parameters.LS68 was less inhibitied on growth,survived longer and had higher survival rate under flooding treatment.While,LS81exhibited characteristics of flood-susceptive species,which were severve growth inhibition and rapid death,and no LS81 plant survived through flooding stress in this study.2.To explore the response of ecophysiological parameters,anatomy and ultrastructure to flooding stress,LS68(redefined as LS1)and LS81(redefined as LS2)were compared for waterlogging effects on them.Seedlings were subjected for 15 days to flooding followed by a three-day drainage and recovery stage.Results showed that flooding stress affected the characteristics mentioned above of leaves and roots adversely in all flooded plants.As compared with LS1(flood-tolerant),LS2(flood-susceptive)displayed clearly more severe destructions during hypoxia and slower recovery ability after drainage,indicated by morphology,biomass accumulation,gas exchange,chlorophyll fluorescence,relative membrane permeability and malonaldehyde contents of roots,as well as the over-all root metabolism parameter dehydrogenase activity.In conformity with the results mentioned above,earlier and more pronounced destructions in anatomy and ultrastructure of leaves and roots were also investigated in LS2,including palisade cells deformation in leaves,cortical parenchyma cells lysis and decomposition of nucleus and most organelles in roots.Our result showed that stable intercellular and intracellular structure helped flood-tolerant clones perform better than flood-suspective clones.Roots suffered more severe and earlier destructions than leaves under flooding treatment.Rapid aerenchyma formation followed by large-scale cortical parenchyma cells lysis in roots is harmful to plants because of strong loss of normal root activities and irregular cortical parenchyma cells arrangement.3.LS1 and LS2 were subjected to root hypoxia,and by using transcript and metabolite profiling,we concluded that there were three characteristics that might contribute to the differences in flood tolerance between LS1 and LS2.First,fermentation was initiated through lactic dehydrogenation in LS1 root under flooding,and subsequently dominated by alcohol fermentation.However,lactic dehydrogenase was persistently active in flooded LS2.Second,we listed 13 differentially expressed genes(with higher transcript levels in LS2 under flooding)associated with energy and O₂consumption processes under soil flooding,including sucrose degradation,amino acid metabolism,beta-oxidation of fatty acids,and sulfur assimilation.These processes might contribute to better energy-/O₂-saving abilities and behaviors in flood-tolerant LS1 than in flood-susceptive LS2 under hypoxic stress.Third,LS1 possessed increased reactive oxygen species scavenging abilities compared to LS2 under edaphic flooding.Remarkably high glutathione content(163.1%of the control),coupled with increased levels of POPTR₀001s34980(encoding L-aspartate oxidase)and POPTR₀016s12540(encoding acyl-CoA oxidase)transcripts,caused flooded LS2 to show dramatically higher reactive oxygen species contents(e.g.,H₂O₂)than the control on the 1st day of flooding,.Additionally,a great number of transcription factor genes differentially expressed in flooding treated LS1 and LS2,including 3 AP2/ERF family menbers Pt.ERF87,Pt.RAP7and Pt.DREB16.These 3 genes were defined as candidate gene,which might be keys to flood-tolerant regulation of poplar.4.The relative transcript levels of 3 candidate genes(Pt.ERF87,Pt.RAP7 and Pt.DREB16)in partial submerged LS1 and LS2 were determined by qRT-PCR assay,and the result was similar to the RNA-seq data.Phylogenetic trees of 3 candidate genes were constructed with AP2/ERF family genes in Arabidopsis.Pt.RAP7 might participate in glycolysis and fatty acid metabolism regulation,and Pt.DREB16 might respond to water stress in poplar.Thereafter,over expression and RNAi vector of the 3 candidate genes were constracted using Gateway method,and genetic transformation on agrobacterim C58and Populus×euramericana‘Nanlin 895'were implemented.Totally,we obtained 35positive individuals from poplar transformation,and relative expression levels of candidate genes in 24 from 35 individuals were significantly up-or down-regulated compared with the wide types.This work provided essential plant material for further functional analysis of the 3 candidate genes.