Function And Characterization Of PuHSFA4a In Regulation Of Zn Toxicity In Populus

Zinc(Zn)is an essential micronutrient for plants,being involved in several cellular functions as a component of many enzymes and proteins.The increase in heavy metal concentration in natural and agricultural ecosystems has become a widespread problem.And heavy metal Zn excess in soil is highly toxic to plants.Plants use specific pathways to regulate Zn homeostasis and cope with excess Zn;these pathways are generally tightly regulated by a specific series of genes.However,the transcription factors involved in excess Zn tolerance are poorly understood.1.Here,we characterized PuHSFA4a,a transcription factor(TF)that acts as a positive regulator of excess Zn tolerance in Populus ussuriensis.qRT-PCR and GUS activity suggested that PuHSFA4a functions in root specifically in response to high Zn stress.The subcellular localization showed that PuHSFA4a-GFP fusion protein was observed in the nucleus and the cytoplasm.The transactivation activity of PuHSFA4a showed that PuHSFA4a acts as a transcriptional activator and that the region between 214 AA and 278 AA—which contains the AHA 1 domain—was required for transcription activation.2.We used overexpression and repression lines to clarify the targets of PuHSFA4a.PuHSFA4a transcription is specifically induced in roots by high Zn.Overexpression of PuHSFA4a specifically conferred excess Zn tolerance and suppression of PuHSFAa increased excess Zn sensitivity in P.ussuriensis by regulating reactive oxygen species(ROS)scavenging.PuHSFA4a played a key role in the excess zinc regulation mechanism.3.To identify genes regulated by PuHSFA4a in response to Zn-induced stress in roots,we performed RNA-seq experiments.The result showed that differentially expressed genes(DEGs)between PuHSFA4a-OE and WT roots in pathways related to:abiotic stress responses and lateral root formation.4.ChIP-PCR,ChIP-qPCR,Yeast one-hybrid(Y1H),luciferase reporter assay(LUC)and electrophoretic mobility shift assay(EMSA)showed that PuHSFA4a coordinately activates genes related to abiotic stress responses and root development and directly binds to the promoter regions of PuGST and PuPLA2.5.PuGSTU17 overexpression significantly increased GST activity and reduced reactive oxygen species levels in roots while PuGSTU17-RNAi lines exhibited opposite phenotype.The PuPLA2overexpression promoted root growth under high Zn stress.Taken together,we provide evidence that PuHSFA4a coordinately activates antioxidant scavenging and root development-related genes and directly targets PuGST and PuPLA2 to positively regulate excess Zn tolerance in P.ussuriensis.Our study shows that HSFs participate in high zinc stress pathway at first time and expand physiological and molecular mechanisms of excess Zn response.Our study provides theoretical basis and reference in remediation of heavy metal contaminated soil.It is significant to nurture some plants against heavy metal,and has some good performances.