Functional Identification And The Regulatory Mechanism Of ZjPRX3 In Lignin Of Jujube Endocarp

Jujube(Ziziphus jujuba Mill.)belongs to the family Rhamnaceae.It is a traditional fruit and the tree species originally in China.Jujube is defined as a stone like fruit,and the high degree of lignification of its endocarp is one of the main characteristics of the fruit.In the process of long-term domestication and cultivation,jujube has produced a large number of natural variation types,stoneless jujube and soft stone jujube with degenerated or softened stone appeared in production.Stoneless fruits are easy to consume and have great development prospects in both fresh food and processing.The Z.jujuba cv.‘Wuhexiaozao’(WH)is a typical representative of stoneless jujube,with a small and thin endocarp,an inconspicuous stone layer,and an unformed fruit stone.It only has a soft and edible membrane or cavity.Therefore,WH as an excellent cultivar for studying the formation mechanism of jujube endocarp traits,analyzing the metabolic and molecular regulatory mechanisms of its formation process,has important value for molecular breeding of stoneless jujube traits,and will promote the improvement of jujube fruit quality and breeding process.In this study,WH was selected as the main material,as well as Z.jujuba cv.‘Jinsixiaozao’(JS),which has a similar phenological period and origin,as the control material.The changes in growth parameters of jujube fruit at different development stages were studied,and the dynamic accumulation process of lignin during the formation period of jujube endocarp was observed.The key formation period of jujube endocarp lignification was determined.By combining transcriptomics,metabolomics conjoint analysis and gene expression analysis,we found that class III peroxidases(ZjPRX3)may be involved in the lignin accumulation in the process of jujube endocarp lignification.By utilizing techniques such as bioinformatics and molecular biology,revealed the molecular mechanism by which specific MYBs binding motifs(binding motifs)were obtained in the ZjPRX3 promoter region of WH fruit due to base mutations,which can bind to the transcription factor Zj MYB12 and inhibit its expression,resulting in abnormal lignin accumulation.Systematically elucidated the molecular mechanism of the difference between jujube with stone and stoneless fruit,which is of great significance for the exploration and utilization of stoneless jujube germplasm and genetic improvement.The main findings are as follows:1.Insufficient lignin accumulation leads to abnormal development of the WHThe growth patterns of WH and JS fruits are basically the same at each stage of development.From YF to E,the fruit size of the two varieties of jujube fruits increases rapidly,but after the WM,the fruit size tends to be stable,showing an obvious ‘fast-slow’ growth characteristics.In addition to the vertical diameter index of the fruit,the fruit weight and lateral diameter of WH are smaller than those of JS,and the growth rate is also significantly lower than that of JS.It was observed by phloroglucinol staining that the lignification of jujube endocarp was deposited from inside to outside.At the E stage,the endocarp began to turn red,and at the WM stage,the whole endocarp turned red.The lignin deposition in the WM stage of the fruit had basically ended.The lignification of jujube endocarp also mainly occurred at the fruit E stage(30 to 50 days after flowering).During the YF stage and early stage of E,there was no significant difference in lignin content between the two varieties of jujube.However,in the middle and late stage of E,the lignin content in the endocarp rapidly increased,at the WM and full red fruits,the lignin content tends to stabilize,and the difference in lignin content between the two no longer increases.During the entire process of lignin accumulation in the endocarp of jujube,the staining range and degree of lignin in WH were lower than those in JS,and the lignin accumulation was also significantly lower than that in JS.The insufficient accumulation of lignin in the endocarp is the major reason for the abnormal formation of WH.2.ZjCCR3,ZjCAD1,and ZjPRX3 are candidate genes for lignin accumulation during the development of jujube endocarpThe metabolomics results showed that 406 metabolites were detected in the endocarp of jujube,including 33 phenylpropane compounds,of which most of the phenylpropane compounds differentially accumulated were significantly reduced in WH,especially the key intermediates for lignin monomer synthesis,such as ρ-Coumaric acid,ρ-Coumarin,Coniferyl aldehyde,Coniferyl alcohol;transcriptomics analysis showed that differentially expressed genes were significantly enriched in phenylpropane biosynthesis pathways related to lignin synthesis.Further screening analysis was conducted using the correlation between differential genes and differential metabolites,that there are 5 types of catalytic enzyme genes differentially expressed in the lignin synthesis pathway,including 5 CADs,2 CCRs,5 F5 Hs,5 HCTs,and 8 PRXs.By combining gene expression analysis and q PCR validation screening,the ZjCCR3,ZjCAD1,and ZjPRX3 were ultimately identified as candidate genes for regulating lignin accumulation in jujube endocarp lignification.3.ZjPRX3 is a key core gene responsible for lignin accumulation during the development of jujube endocarpGene expression analysis showed that ZjCCR3,ZjCAD1,and ZjPRX3 were highly expressed in lignified stems,bearing branch,and endocarp tissues.ZjCAD1 and ZjPRX3 were highly expressed during endocarp formation stage,and the expression levels of these three genes in JS were significantly higher than those in WH.After transient overexpression and silencing of ZjCCR3,ZjCAD1,and ZjPRX3 in jujube fruit,it was found that ZjCCR3 and ZjCAD1 cannot promote or reduce the accumulation of lignin in jujube fruit,while ZjPRX3 can significantly increase or decrease the content of lignin in fruit.By constructing a plant overexpression vector of ZjPRX3 and using Agrobacterium mediated transformation to transform the root tissue of jujube,’Micro Tom’ tomato,and tobacco,overexpression of ZjPRX3 significantly increased the lignin content in jujube roots compared to the control plant.Paraffin tissue sectioning observation showed that ZjPRX3-OE overexpression of root phloem cell walls thickened and arranged tightly,with most secondary cell walls stained red and lignin significantly deposited.The overexpression of ZjPRX3 in tomato and tobacco also promoted the accumulation of lignin.These results proved that ZjPRX3 is the core structural gene in the process of jujube endocarp lignification,which can promote the accumulation of lignin.4.Screened and determined that the inhibitor Zj MYB12 can directly interact with the ZjPRX3 promoter and inhibit its expressionZjPRX3 and its promoter sequence were cloned from WH and JS,respectively.It was found that its coding region sequence was consistent with the homologous sequence in the genome of Z.jujuba cv.‘Junzao’,Z.jujuba cv.‘Dongzao’,and there was no mutation.Twelve SNPs variation sites were found in the promoter region sequence.After prediction and analysis of the cis-acting element of ZjPRX3 promoter in WH and JS,the results showed that a MYBs binding primitive TGGTTAG(promoter upstream sequence 1001 bp to 1007bp)was formed at the 11 th SNP position of ZjPRX3 gene promoter in WH due to base mutation.After the homologous cluster analysis of transcriptome expression profile and differentially expressed MYBs transcription factors,an inhibitory MYB transcription factor Zj MYB12 was screened.Through GUS activity assay,Dual-luciferase reporting experiment(LUC),Yeast 1hybridization(Y1H)experiments,it has been proven that Zj MYB12 can bind to the ZjPRX3 gene promoter in WH and inhibit its expression,but cannot bind to the ZjPRX3 gene promoter in JS.Electrophoretic mobility shift assay(EMSA)experiment showed that Zj MYB12 could recognize MYB binding element formed by base mutation on ZjPRX3 promoter sequence in WH.The above finding suggest that the inhibitor Zj MYB12 inhibits the aggregation of lignin monomers by binding to the ZjPRX3 gene promoter in WH,resulting in abnormal accumulation of lignin in the endocarp of WH,and the occurrence of stoneless or cracking stone.