Molecular Network Analysis Of ZmbZIP54 And ZmbZIP107 Transcription Factors Regulating Lead Tolerance In Maize Seedlings

Maize(Zea mays L.)is an important food crop,feed crop and industrial material in the world,which is cultivated almost all over the world.With the rapid development of heavy industry and the aggravation of environmental pollution,heavy metal pollution has become one of the most serious ecological and environmental problems in the world.Lead(Pb)is a highly toxic heavy metal pollutant,which has negative impact on the growth and development of plants and physiological functions.It enters the human body through the food chain,air and water and causes irreversible damage to health.Therefore,it is of great significance to identify the genes related to lead tolerance in maize and further analyze its genetic and molecular mechanisms for breeding maize varieties with low accumulation or high enrichment of Pb,and promoting the safe production of maize and the bio-remediation of Pb-contaminated soil.Plant basic leucine zipper(b ZIP)transcription factors are widely distributed and relatively conserved gene family in eukaryotes,whose members are involved in regulating plant growth,development and physiological processes,and play an important role in in abiotic stress such as cold,drought,salt and heat resistance.So far,there are few reports on the molecular mechanism of b ZIP gene regulating Pb tolerance in maize.In our previous study,we found that the ZmbZIP54 and ZmbZIP107 could enhance the tolerance of maize seedlings to Pb stress.Therefore,this study attempted to deeply explore the regulatory network and tolerance mechanism of these two b ZIP transcription factors in maize seedling response to Pb stress.Based on candidate gene association analysis,phenotypic identification of transgenic overexpressed lines and mutant lines,and heterologous expression,ZmbZIP54 and ZmbZIP107 can enhance seedling tolerance to Pb stress and reduce the uptake of Pb by roots.Subsequently,the distribution,morphology and tolerance mechanism of Pb ions in cells were identified by green fluorescence probe,transmission electron microscopy(TEM),energy dispersive spectroscopy(EDS)and chemical morphometry.Finally,yeast two-hybrid(Y2H),Bimolecular fluorescence complementation(Bi FC),yeast one-hybrid(Y1H),Luciferase assay(LUC)and transcriptome analysis were used to construct a molecular network for regulating lead tolerance in maize by b ZIP transcription factor.The main results are as follows:(1)The promoter region and gene body of ZmbZIP54 in 272 maize inbred lines were analyzed for the root lead tolerance coefficient(RW-LTC)phenotype candidate genes.Combined with promoter LUC activity verification,it was found that the 5’-UTR variation of ZmbZIP54 affected its expression level under Pb stress and led to the difference of Pb tolerance among different maize lines.It was demonstrated that salicylic acid(SA)signal promoted lead absorption and inhibited root development under lead stress by inhibiting ZmbZIP54 expression in maize roots.(2)The heterologous expression of ZmbZIP54 in yeast mutant(Δycf1)enhanced the tolerance of yeast cells under Pb stress.Pb content of Δycf1-ZmbZIP54 was significantly reduced by 25.7% and 42.3% compared with WT-p YES2 and Δycf1-p YES2,respectively.In addition,subcellular localization of maize protoplasts showed that ZmbZIP54 was located in the nucleus.The results of q RT-PCR showed that ZmbZIP54 was expressed in all tissues of maize,and the highest expression level was found in the root of three-leaf stage.Meanwhile,the expression of ZmbZIP54 was induced by Pb stress,and the expression level of root showed a trend of first increasing and then decreasing with the increase of stress time.(3)The results of Pb ion green fluorescence probe detection showed that Pb was mainly accumulated in meristem and elongation regions of root tips,and the fluorescence intensity of the OE lines was reduced by 47.57%-77.97% compared with wild type.Compared with wild type,the fluorescence intensity in the root tips of the zmbzip54 was increased by 42.61-41.73%.TEM and EDS results showed that in the OE lines,Pb was mainly attached to the cell wall in the form of acicular sediments or existed in the intercellular space in the form of complexes,and only a small amount of Pb entered the cell interior.In the zmbzip54 lines,Pb enters into the cytoplasm through the cell wall,forms complexes with the cytoplasmic substances and accumulates in organelles and cytoplasms,and causes plasmolysis of maize root cells.Permanent plasmolysis may cause water loss and death of plant tissues.6,5,5 and 5 peaks of Pb were detected in the root tips sediments of zmbzip54,W22,OE and B104 lines,respectively.The peak value of Pb in the zmbzip54 was significantly higher than that in the W22.The peak value of Pb in the OE lines was significantly lower.The results of different chemical morphology of Pb showed that the main components of Pb in the B104 and OE lines were lead oxalate extracted from hydrochloric acid and insoluble lead phosphate extracted from acetic acid,accounting for 50%-61.7% and 12.9%-25%of the total,respectively.However,it is mainly the Pb extracted from hydrochloric acid and the Pb extracted from ethanol in the form of inorganic substance which is easy to transfer and more toxic in the W22 and zmbzip54 lines,accounting for 42.7%-47.5%and 23%-25.7% of the total amount,respectively.(4)The ZmbZIP54 can bind to the promoter of ZmPRP1 and promote its transcription through LUC,Y1 H and maize protoplast LUC activity detection.Tissue expression patterns showed that ZmPRP1 expression was the highest in maize root,induced by Pb treatment,and was up-regulated gradually with the increase of Pb treatment time.The transformation of ZmPRP1 into the protoplasts of zmbzip54 showed that the number of undamaged protoplasts in a unit area was significantly higher in the transformants than that in the mutant under Pb treatment.These results suggested that overexpressing ZmPRP1 improved the tolerance of the mutant zmbzip54 to Pb tolerance.Y2 H and Bi FC indicated that ZmFdx5 could interact with ZmbZIP54 in the nucleus,which inhibited the transcriptional promotion of ZmbZIP54 on ZmPRP1.In all the genetic materials,the expression of ZmFdx5 in maize roots under control condition was much higher than that under Pb condition,indicating that ZmFdx5 and ZmbZIP54 mainly occurred under control condition.(5)Transcriptome analysis of ZmbZIP54 overexpression,mutant and wild-type lines under Pb stress showed that ZmbZIP54 regulates downstream abiotic stress response pathways and plant hormone signaling pathways.Combined with RNA-Seq and gene functional annotation,a protein encoding heavy metal transport was identified.It was verified by Y1 H and LUC that ZmbZIP54 binds to the G-box element on the promoter of ZmNRAMP6 and inhibits its transcriptional expression.Thereby regulating the absorption and transport of Pb in plants.(6)A molecular network regulating Pb tolerance in maize seedlings was constructed with ZmbZIP54 as the central gene.Our study showed that ZmbZIP54 protein binds to the ZmPRP1 promoter and promotes its transcription,and ZmPRP1 mediates Pb tolerance in maize by altering the balance of the redox systems.As a protein interacting with ZmbZIP54,ZmFdx5 acts as a switch that controls the regulation of ZmPRP1 expression by ZmbZIP54.Under normal conditions,ZmFdx5 represses ZmPRP1 expression by interacting with ZmbZIP54,contributing to energy saving mechanisms in maize.Under Pb stress,ZmFdx5 detaches from ZmbZIP54,thus activating ZmPRP1 transcription to initiate maize tolerance to Pb.ZmbZIP54 binds to the ZmNRAMP6 promoter and inhibits its expression.It effectively inhibited the transport of Pb ions to the shoots,and protected the aboveground parts from the Pb stress to a certain extent.(7)Subcellular localization of tobacco leaves indicated that ZmbZIP107 protein was located in the nucleus.The expression pattern of ZmbZIP107 showed that ZmbZIP107 was highly expressed in the root tissues of maize trileaf stage.The heterologous expression of ZmbZIP107 in rice enhances Pb tolerance in seedlings,and the traits of TRL,TSA,BSH,PRL,MNR and SHL in OE lines were significantly increased by 7.3%-21.9% compared with the wild type under Pb stress.Compared with the wild type,the Pb content in the roots of OE lines decreased by 42.3% and 41.4%,but there was no significant difference of Pb content in the shoots.These suggested that increased ZmbZIP107 expression in rice transgenic lines effectively inhibited Pb absorption by the roots,and alleviating the toxic effect of Pb on seedlings.