Resistant Functions Of Mulberry Polyphenol Oxidase Genes

Mulberry(Morus alba L.)is an important economic crop.It is used not only as a food fed for silkworm but also as a traditional Chinese medicine.The previous studies showed that mulberry is rich in flavonoids,alkaloids,polysaccharides,and other active ingredients.It has anti-inflammatory,lipid-lowering,and hypoglycemic effects.In addition,the root system of mulberry is relatively developed,which can grow in arid,saline alkali and other poor soil.It can be used as an ecological tree species for windbreak and sand fixation,afforestation of barren mountains and wasteland,etc.Mulberry has the characteristics of adapting to bad environment and resisting adversity.So the mechanism of mulberry’s resistance to adversity has been widely studied.Studies have shown that polyphenol oxidase is an important defensive enzyme.So far,many species(such as tomato,eggplant,apple,pear,grape,and strawberry)of polyphenol oxidase genes have been cloned,and their functions have been reported.However,the polyphenol oxidase genes in mulberry trees have not been cloned,and their functions are unclear.Therefore,it is particularly important to clone and study the anti-stress functions and mechanism of polyphenol oxidase gene in mulberry.This will provide useful information for improving the tolerance of mulberry trees and other plants.In this study,five polyphenol oxidase genes in the genome of M.notabilis C.k.Schn(M.notabilis)were identified.The function of polyphenol oxidases in mulberry were explored by enzyme activity analysis combined with the antimicrobial capacity analysis of polyphenol oxidase products.In addition,the antimicrobial mechanism of mulberry polyphenol oxidase 1 product,Mn PPO1,was discussed.In order to learn more about the regulation mechanism of the mulberry polyphenol oxidase,the upstream regulatory transcription factor Mn MYB3R1 of Mn PPO1 gene was identified using DNA pull down-MS.Moreover,the interaction between the promoter region of the Mn PPO1 gene and the transcription factor Mn MYB3R1 was verified using the yeast single hybrid,luciferase reporter system,and chromatin immunoprecipitation.Expression analysis and functional analysis using the transgenic tobacco lines demonstrated that Mn MYB3R1 was involved in the regulation of polyphenol oxidase genes in plant against drought.The main findings of this study are as follows:1.Cloning and functional study of polyphenol oxidase genes from mulberryFive polyphenol oxidase genes were identified in the M.notabilis genome,named Mn PPO1,Mn PPO2,Mn PPO3,Mn PPO4,and Mn PPO5.The amino acid sequence of mulberry polyphenol oxidases were predicted by NCBI conserved domain analysis.The results indicated that the mulberry polyphenol oxidases belong to the tyrosinase superfamily and all have intact type III copper ion protein binding sites(Cu A and Cu B).The subcellular localization,signal peptide,transit peptide,and transmembrane were predicted online.The results showed that the mulberry polyphenol oxidases did not contain transmembrane region and signal peptide,but contained chloroplast transit peptides and located in the chloroplast.The transcriptional levels of five PPO genes in the leaves of M.notabilis after stress treatment were analyzed by q PCR.When the plants were subjected to biotic(pathogenic bacteria treatmentor)or abiotic stress(drought treatment),the expression levels of the five Mn PPO genes were changed.The expression level of Mn PPO1 peaked at 2 days of drought stress and then decreased.The expression levels of Mn PPO2,Mn PPO3,Mn PPO4,and Mn PPO5 peaked at 4 days of drought stress,and then decreased.After drought stress,the up-regulation of Mn PPO1 gene was the most obvious among the five polyphenol oxidase genes in mulberry.The expression level of Mn PPO3 reached the highest value when treated with Botrytis cinerea(B.cinerea)for 8 h,while the other four polyphenol oxidase genes reached the highest expression level when treated with B.cinerea for 24 h.After the treatment with gray mold,the up-regulation of Mn PPO1 gene expression was the highest.The expression pattern analysis of five polyphenol oxidase genes in mulberry trees under stress indicated that five polyphenol oxidase genes of mulberry were involved in the process of plant stress resistance.Mulberry polyphenol oxidase proteins were produced by prokaryotic expression system.Five mulberry polyphenol oxidases have tyrosinase activities and all of them have monophenolase and bisphenolase activities.Since chlorogenic acid is the most suitable substrate for mulberry polyphenol oxidases,it was used as a substrate to investigate the inhibitory effect of mulberry polyphenol oxidases on the growth of microorganisms.The results showed that the products of five mulberry polyphenol oxidases had inhibitory effects on Escherichia coli,Staphylococcus aureus,P s e u d o m o n a s a e r u g i n o s a,S c l e ro t i n i a s c l e ro t i o r u m,B o t r y t i s c i n e re a.The products of Mn PPO1 showed the best inhibitory effects,while that of Mn PPO2 had the weakest inhibitory effects.2.Antimicrobial mechanism of MnPPO1 productSince the Mn PPO1 product has the strongest antimicrobial ability,the anti-microbial mechanism of mulberry polyphenol oxidase product was studied by using the reaction product of Mn PPO1 and chlorogenic acid.In this study,macro-molecular infiltration was used to assess cell membrane integrity.When the Mn PPO1 product was applied to E.coil,P.aeruginosa,and S.aureus,the absorbance of the product group was found to be higher than that of the control group.An increase in absorbance in the treated supernatant indicates bacterial cell wall and/or membrane damage.The experimental results showed that the soluble protein content in the product group was lower than that in the control group.The drug activity against the cell wall is usually investigated by examining the activity of cell wall-associated hydrolases such as chitinase and β-1,3-glucanase.In this study,the activitvies of chitinase and β-1,3-glucanase were measured by double antibody sandwich assay.The chitinase and β-1,3-glucanase activities in the Mn PPO1 product-added group were significantly increased compared with the control group.The results indicated that the Mn PPO1 product can increase the activity of hydrolase associated with the fungal cell wall,resulting in cell wall degradation.In addition,the subcellular structure changes of the test bacteria were examined by transmission electron microscopy.The results showed that untreated E.coli and S.aureus cells showed intact structure.However,when the Mn PPO1 reaction product was applied to the test cells for 8 hours,its subcellular structure was significantly changed.The cell membrane and cell wall of most bacterial cells were damaged,and the cytoplasm of the cells cannot be uniformly distributed.Moreover,some cells have cytoplasmic deletions and cells have been vacuolated.3.Analysis of upstream transcriptional regulatory factors of MnPPO1 geneA 2001 bp(-2000 to +1,+1 translation initiation site)region upstream of the Mn PPO1 gene was cloned and a drought-associated MYB transcription factor binding site,MSA element,Salicylic acid(SA),and ethylene(ET)response elements were identified in this promoter region.Subsequently,a 1,268 bp upstream of the Mn PPO1 gene promoter region containing MSA was selected as template and a biotin-labeled promoter probe was synthesized by PCR for DNA pull down followed by mass spectrometry to identify the protein captured by biotin affinity.Mass spectrometry results indicated that the biotin-labeled treatment identified a MYB transcription factor of the R1R2R3 type,Mn MYB3R1,whereas this protein was not identified in the control(non-biotinylated group),suggesting that Mn MYB3R1 may be an upstream regulatory transcription factor of the Mn PPO1 gene.The analysis results of yeast single-hybrid assay,luciferase reporter assay,and chromatin immunoprecipitation demonstrated that Mn MYB3R1 transcription factor was bound to the MSA element in promoter region of the Mn PPO1 gene.Overexpression of Mn MYB3R1 gene in tobacco and drought stress response experiments proved that Mn MYB3R1 transcription factor regulated the expression of PPO and participated in the plant drought stress response.The results indicated that the overexpression of Mn MYB3R1 gene increased the polyphenol oxidase activity and enhanced the tolerance of tobacco against drought,suggesting that Mn MYB3R1 transcription factor positively regulated PPO and involved in plant response to drought stress.