Effect Of Ethylene Responsive Factor PhERF2 On Waterlogging Tolerance Of Petunia Hybrida

As the perennial herbaceous plant belonging to the family of Solanaceae,petunia(Petunia×hybrida)is an important garden landscape flower with large market demand.However,considering that this plant must be kept away from water dampness,the current breeding of petunia shall focus on the goal of improving flower stress resistance.Based on the materials of Ph ERF2 six transgenic lines as well as the wild type of petunia,their waterlogging tolerance was evaluated after waterlogging stress treatment,and related physiological and biochemical indicators were determined in this paper.Afterwards,two materials with significant difference in waterlogging tolerance were selected for RNA-Seq study,so that the 6788 differentially expressed genes were screened.In this way,the downstream candidate genes(Sn RK2,ABF,EBF1/2,ERF1/2,ADH1,LDH,PDC4,CAT3,SODA,ACO1,ACS3)regulated by Ph ERF2 were obtained through GO classification and KEGG enrichment analysis,which laid foundation for the breeding of petunia waterlogging molecules.The main findings are as follows:1.A petunia waterlogging tolerance evaluation system was established.By means of pot plant flooding method,the phenotypic changes of plants during the flooding process were taken as evaluation indexes,and a waterlogging tolerance evaluation system was established based on the grade score criteria.According to the comprehensive identification of waterlogging tolerance of Ph ERF2 six transgenic lines as well as the wild type of petunia,it was preliminarily recognized that Ph ERF2-overexpressing lines were waterlogging tolerant.2.The root aerenchyma,root vigor,chlorophyll content,MDA content and soluble protein content of seven petunia materials were studied.During the entire flooding treatment,the root vigor of all plants showed a continuous downward trend,of which RNAi silencing lines presented a greater decline than Ph ERF2-overexpressing lines.After 48 h of flooding treatment,the chlorophyll and soluble protein content of Ph ERF2-overexpressing lines were significantly higher than that of RNAi silencing lines and wild type.Except for 12 h,the MDA content in both RNAi silencing lines and wild type were significantly higher than that in Ph ERF2-overexpressing lines,indicating that both wild type and RNAi silencing lines suffer from serious oxidative damage.At 24 h of flooding treatment,DNA in the root of Ph ERF2-overexpressing line I was concentrated to a moon-shaped nucleus,which has the typical characteristics of PCD,indicating that Ph ERF2 may avoid hypoxia stress and promoteplant waterlogging resistance by promoting the formation of root aeration tissue.3.The photosynthetic physiological indexes of seven petunia materials were measured.Under the waterlogging stress,Pn,Gs,Tr and Ci of Ph ERF2-overexpressing lines all decreased compared to the control group,indicating that the net photosynthetic rate decrease of Ph ERF2-overexpressing lines was mainly affected by stomatal limiting factors;Pn,Gs and Tr of RNAi silencing lines generally showed a downward trend,while Ci declined at 3h of treatment and then gradually increased,indicating that the net photosynthetic rate of RNAi silencing lines was mainly affected by non-stomatal limiting factors,and severely damaged by waterlogging.4.The leaf antioxidant enzyme activity and root anaerobic respiratory enzyme activity of seven petunia materials were measured.During the whole treatment period,the antioxidant enzyme activity of Ph ERF2-overexpressing lines was generally higher than that of RNAi silencing lines and wild type,indicating that Ph ERF2 may improve the plant's water tolerance by enhancing its ability to scavenge reactive oxygen species;the ADH and LDH activity in RNAi silencing lines and wild type was significantly higher than that in Ph ERF2-overexpressing lines,indicating that Ph ERF2 may reduce the accumulation of toxic substances such as lactic acid and ethanol in plants by regulating the anaerobic respiration pathway and improve plant tolerance.5.By selecting Ph ERF2-overexpressing line I and Ph ERF2-RNAi line 4B with significant difference in waterlogging tolerance as materials,and adopting wild type(WT)as a control,the roots were taken at 0h and 3h of waterlogging treatment for RNA-Seq analysis.According to the results,115.49 G of Clean Data was obtained in total;the effective data amount of each sample was distributed from 6.04 to 6.66G;Q30 bases were distributed from81.34 to 91.55%;the average GC content was 43.99%.The genome comparison of each sample was obtained by comparing reads to the reference genome,and the comparison rate was 92.16?94.49%.The expression analysis of protein-coding genes was conducted in accordance with the comparison results.Differential screening was performed based on the expression indexes of protein-coding genes in different samples,and nine differential groups were created.When the screening conditions were set to p<0.05 and FC>2,the detected quantity of differential protein-coding genes was 2589,2119,3728,1000,3793,1881,1500,5288 and 3187,respectively.6.Differentially expressed genes in the comparison group 3h-4B-VS-3h-WT and the comparison group 3h-I-VS-3h-WT were subjected to GO and KEGG enrichment analyses.The GO classification results showed that DEGs in both of the comparison groups were mainly enriched in metabolic process,cellular process,and single-organism process functional groups in Biological Process;cell,cell part,and membrane functional groups in Cellular Component;binding,catalytic activity,and transporter activity functional groups in Molecular Function.KEGG analysis found that DEGs in both comparison groups weresignificantly enriched in Ko04075,the plant hormone signal transduction metabolic pathway.7.The downstream candidate genes regulated by Ph ERF2 were selected from four aspects:(1)Plant hormone signal transduction metabolic pathways: Sn RK2,ABF,EBF1/2 and ERF1/2;(2)Anaerobic respiratory pathways: ADH1,LDH,PDC4,etc.;Antioxidant Enzyme system: CAT3,SODA,etc.;Ethylene and its signal pathway: ACO1,ACS3,etc.Therefore,it is concluded that by activating or inhibiting the expression of the above candidate genes at the transcription level,Ph ERF2 can regulate the abscisic acid signal transduction pathway,the ethylene metabolism pathway,the glycolysis/gluconeogenesis pathway,and the FOXO signaling pathway,thereby promoting plant photosynthesis,delaying plant senescence,reducing the toxic effect of anaerobic respiration products on cells,improving plant antioxidant capacity,so as to improve plants' waterlogging tolerance.It is necessary to emphatically screen out 1-2 important differentially expressed candidate genes for functional verification,complete the construction of inter-gene co-expression networks,and analyze the molecular mechanism of Ph ERF2 on petunia response to waterlogging stress.