The Mechanism Of ZmPRX1 Gene Regulating Drought Stress Resistance In Maize Seedling Stage

Global warming and water shortage caused by climate change pose serious and urgent challenges to global food security.One third of the world’s arable land is faced with water shortage,while the remainder is often affected by periodic or unpredictable droughts,which often lead to crop yield reduction or even death.Drought stress is multi-dimensional,which leads to alterations in plant morphology,physiology,biochemistry and molecular events.In response,plants evolve various stress adaptation mechanisms in the process of natural selection,among which root structure modification is one of the key factors to obtain sufficient water and nutrients under drought conditions.Maize(Zea mays L.),as an important feed,food and energy crop in the world,has become one of the largest crops in the world.However,drought is one of the main abiotic factors restricting maize production.Therefore,understanding the drought resistance mechanism and mining drought tolerance genes in maize,as well as further applying it to improve drought tolerance is the key approach to stabilize maize production.In a preliminary study,a guaiacol peroxidase 1 gene(ZmPRX1)was discovered,through combined transcriptome and proteome analysis,to be significantly upregulated in response to drought stress in maize.In this study,Arabidopsis(Arabidopsis thaliana),maize EMS mutants and maize overexpression materials were used to verify the functions of ZmPRX1 gene in maize growth and development,lignin accumulation and drought tolerance.Then,the transcription factor ZmWRKY86 was screened by yeast one-hybrid and double luciferase reporting experiments,and the expression of ZmPRX1 target gene regulated by ZmWRKY86 was analyzed.The main results are as follows:1.ZmPRX1 gene contains three exons and two introns,encodes a polypeptide of 367 amino acids,and coding sequence(CDS)length of 1104 bp.ZmPRX1 is localized in the cell wall,and the expression level in roots at seedling stage was significantly higher than that in stems and leaves.There are several cis-acting elements related to stress resistance in the promoter of ZmPRX1 gene,which are involved in the response of roots to drought stress at the seedling stage.These include abscisic acid response elements(ABRE),Dehydration responsive element(DRE),drought-induced MYB binding site(MBS),MYB recognition element(MYBs),MYC binding element(MYCs),TC-rich,jasmonic acid response element(CGTCA-motif),W box and as-1.2.Overexpression of ZmPRX1 gene increased drought resistance of Arabidopsis at the seedling stage.Under drought conditions,the survival rate of Arabidopsis wild-type WT(Col-0)was 4.3%,while the overexpression materials L1,L11 and L12 of ZmPRX1 gene had a survival rate of 84.0%.Compared with WT(Col-0),the malonaldehyde(MDA)content of L1,L11 and L12 decreased by 22.4-25.3%,guaiacol peroxidase(POD)and superoxide dismutase(SOD)activity increased by 35.8-41.0%and 13.0-18.6%,respectively.3.ZmPRX1 gene regulates root development and plant height of maize.Under normal conditions,compared with maize wild type WT(B73),root length(RL),total root length(TRL)and radicles length of maize mutant zmprx1 decreased by 17.9%,39.3%and 84.2%,respectively,and the total root projection area(TRPA),total root surface area(TRSA)and total root volume(TRV)decreased by 32.4%,32.4%and 23.4%,respectively.Root fresh weight(RFW)and root dry weight(RDW)decreased by 40.3%and 24.9%,respectively.In addition,the plant height of zmprx1 was 25.4%lower than that of WT(B73),Maize overexpression material ZmPRX1-OE showed the opposite phenotype.Compared to maize wild type WT(B104),the root length(RL)and radicles length of ZmPRX1-OE increased by 20.0%-35.0%and 19.3%-38.3%,respectively,and the total root projection area(TRPA),total root surface area(TRSA)and total root volume(TRV)increased by 19.0%-28.7%,19.0%-28.7%and 25.8%-35.9%,respectively.Root fresh weight(RFW)and root dry weight(RDW)also increased significantly,increasing by 10.5%-27.6%and 20.9%-37.4%,respectively.The observation of aboveground parts showed that the plant height of ZmPRX1-OE was 5.0%-8.1%higher than that of WT(B104).4.ZmPRX1 gene regulates drought tolerance of maize at the seedling stage.Under drought conditions,the survival rate of zmprx1 was 12.0%,which was significantly lower than that of WT(B73).Compared with WT(B73),the relative conductivity and MDA content of zmprx1 roots increased by 28.4%and 30.1%,respectively,and POD,SOD and CAT activities decreased by 38.8%,16.5%and 46.6%,respectively.After drought treatment,the proline(PRO)content in WT(B73)roots increased rapidly,but zmprx1 did not show significant change.In addition,the leaf water loss rate of zmprx1 was significantly higher than that of WT(B73),indicating that the loss of function of ZmPRX1 gene increased the sensitivity of maize to drought at the seedling stage.ZmPRX1-OE showed the opposite phenotype,that is,after drought treatment,compared with 12.0%survival rate of WT(B104),the survival rate of ZmPRX1-OE was up to 60.0%.Compared with WT(B104),ZmPRX1-OE roots had higher PRO content and antioxidant enzyme activity,lower electrical conductivity and MDA content.In addition,the water loss rate of ZmPRX1-OE was lower than that of WT(B104).Under normal conditions,the lignin content in the roots of zmprx1 was reduced by 10.6%compared with WT(B73).Conversely,the lignin content in the roots of ZmPRX1-OE seedling increased by 9.8%-13.4%compared to WT(B104).5.ZmPRX1 is negatively regulated by transcription factor ZmWRKY86.Yeast single-hybrid and diluciferase reporter experiments showed that the stress-associated transcription factor ZmWRKY86 could bind to the promoter of ZmPRXl and negatively regulate the expression of ZmPRX1.It was observed(by confocal microscope)that ZmWRKY86 was located in the nucleus,and the expression of ZmWRKY86 gene in maize root decreased continuously under drought condition.In summary,ZmPRX1 is negatively regulated by the transcription factor ZmWRKY86,and ZmPRX1 is involved in root development and accumulation of lignin in maize and affects the drought tolerance of maize.This study provides new insights into the growth and development of maize roots,the accumulation of lignin,and the response mechanism of maize to drought stress.