Molecular Mechanism Of PIWRKY47 Transcription Factor Regulating The High-Temperature Stress Of Paeonia Lactiflora

In China,herbaceous peony(Paeonia lactiflora Pall.)is a well-known and traditional flower.It is frequently planted in China’s northern areas because it thrives in cold,cool and dry environments.With the promotion and application of herbaceous peony in gardens and the rapid development of the cut flower industry in recent years,a large number of herbaceous peony have been introduced in the middle and lower reaches of the Yangtze River and southern regions of China.These regions experience prolonged,strong summer high temperature,which frequently causes herbaceous peony’ stems and leaves to burn and wilt,turn yellow,and sear on the edges.Additionally,it results in inadequate underground root growth,which has a significant negative impact on the ornamental effect of plants and flowering the following year.WRKY transcription factors play a crucial role in plant response to high-temperature stress,but there has been no report on the study of WRKY transcription factors regulating the high-temperature tolerance of herbaceous peony.This study used PIWRKY47 as the research object in order to investigate the molecular mechanism of herbaceous peony WRKY transcription factor response to high-temperature stress,based on the prior findings of the research group.Firstly,virus induced gene silencing(VIGS)and tobacco genetic transformation techniques were used to determine whether P1WRKY47 has the function of responding to high-temperature stress;Secondly,based on literature reports and transcriptome sequencing(RNA-seq)results,the downstream target genes of PlWRKY47 were screened,and the expression characteristics and functional verification of the target gene were studied;In addition,we found regulatory proteins interacting with PlWRKY47,explored the regulatory effect of protein interaction on downstream target gene,and further carried out expression characteristics and functional verification research on interacting protein genes.The main results findings are as follows:(1)Herbaceous peony PlWRKY47 positively regulates the high-temperature tolerance of plants.The full-length sequence of PIWRKY47 gene was cloned by RACE technology,with a total of 1935 bp.The open reading frame(ORF)was 1524 bp,encoding 507 amino acids and belonging to the WRKY Ⅱb subfamily protein.High temperature can induce the expression of PlWRKY47,and the expression level of PlWRKY47 gene in herbaceous peony varieties with strong high-temperature resistance is higher than that in varieties with weak high-temperature resistance.PlWRKY47 is located in the nucleus and has transcriptional activation activity.Silencing the PlWRKY47 gene via VIGS in herbaceous peony under high-temperature stress,which also damages the cell membrane system,inhibites photosynthetic capacity,accumulates a lot of reactive oxygen species(ROS),and decreases antioxidant enzyme activity.Tobacco plants overexpressing the PIWRKY47 gene can maintain a good growth status compared to wild-type(WT)tobacco.Furthermore,they exhibit a more complete cell membrane system,stronger photosynthetic ability,a reduced level of ROS accumulation,and significantly increased antioxidant enzyme activity.(2)Herbaceous peony PlWRKY47 can directly activate the expression of the target gene PlGAPC2.The control(WT)treated with high temperature and the herbaceous peony plant(TRV-L1)with transient silencing of the PlWRKY47 gene were subjected to RNA-seq.Based on sequencing results and previous literature reports,a candidate target gene PlGAPC2 for PlWRKY47 was selected,and its expression was induced by high temperature and localized in the cytoplasm.The PlGAPC2 promoter sequence contains 5 W-boxes elements and 1 STRE thermally induced cis-acting element.The interaction between PlWRKY47 and PlGAPC2 promoter was verified by yeast single hybrid and double luciferase reporter system.Further functional verification of the PlGAPC2 gene found that high-temperature stress led to the deterioration of the growth status of the herbaceous peony plants that transiently silenced the PlGAPC2 gene,the damage of the cell membrane system,the inhibition of photosynthetic capacity,the accumulation of a large number of ROS,and the reduction of antioxidant enzyme activity.Under high-temperature stress,tobacco plants overexpressing the PlGAPC2 gene maintained better growth status compared to the WT tobacco plants,with a more intact cell membrane system,stronger photosynthetic ability,less ROS accumulation,increased antioxidative enzyme activity,and maintained cellular redox homeostasis.(3)The interaction between herbaceous peony PlWRKY47 and both its own and PlWRKY72 enhances the targeted regulation of PIGAPC2 gene expression.Based on previous literature findings and the previous transcriptome data,two candidate interacting proteins were screened,namely PlWRKY47 itself and PlWRKY72.The ORF of PlWRKY72 is 1797 bp,encoding 598 amino acids and belonging to the WRKY Ⅱb subfamily.The yeast two hybrid,bimolecular fluorescence complementation and dual luciferase reporter system were further used to verify that PlWRKY47 interacted with PlWRKY47 itself and PlWRKY72.Then,dual luciferase reporter system was used to detect that PlWRKY47 could enhance the expression regulation of PlGAPC2 gene by interacting with PlWRKY47 itself and PlWRKY72.(4)Herbaceous peony PlWRKY72 can improve the plant’s ability to withstand high temperatures.The expression of PlWRKY72 is induced by high temperature,localized in the nucleus,and has transcriptional activation activity.Compared with the control,herbaceous peony plants with transiently silenced PlWRKY72 gene exhibited leaf wilting and drooping after high-temperature stress,significantly increased relative electrical conductivity(REC)and malondialdehyde(MDA)content,increased ROS accumulation level,weakened photosynthetic ability,and decreased antioxidant enzyme activity.Compared with the WT,tobacco plants overexpressing the PlWRKY72 gene maintained better growth status after high-temperature stress,with a more complete cell membrane system,reduced ROS accumulation level,enhanced photosynthetic ability,and increased antioxidant enzyme activity.