Identification And Functional Analysis Of Bomyb51 From Broccoli (Brassica Oleracea Var. Italica)

Glucosinolates are sulfur and nitrogen containing secondary metabolites derived from amino acids and specifically present in the order Brassicales.Based on the structure of precursor amino acids,glucosinolates are generally classified into three major groups,aliphatic,indolic and aromatic.Hydrolyzed by thioglucosidase(myrosinase),these metabolites produce breakdown products with diverse biological activities.Broccoli(Brassica oleracea var.italica)is a popular vegetable with high nutritive value and anti-cancer properties which has informed an increased interest in recent years.The most effective cancer-preventive compound in broccoli is sulforaphane.The indolic glucosinolates and their corresponding hydrolysis products have also been implicated in a variety of anticarcinogenic mechanisms and are essential for effective defense responses to a wide range of pathogens and herbivores.In model plant Arabidopsis,the indolic glucosinolate metabolic pathway has been identified.The MYB transcription factor family members MYB51,MYB34 and MYB122 play important roles in the regulation of indolic glucosinolate biosynthesis.MYB51 is the key regulator that predominantly controls indolic glucosinolates in the shoot while MYB34 plays a dominant role in regulating indolic glucosinolates in roots.Overexpressing MYB51 significantly promotes the expression of indolic glucosinolate biosynthetic pathway genes,leading to increased accumulation of indolic glucosinolates.The mechanisms by which indolic glucosinolates involve in defense against pathogens have gradually been discovered,these compounds are found to be required in pathogen triggered callose deposition in the cell wall and attenuation pathogen induced programmed cell death.Recently,based on genomic analysis and transcriptome assembly glucosinolate metabolism pathway and the regulators have been successfully identified in some Brassica vegetables.Several studies about the identification and expression analysis of glucosinolate biosynthetic genes in Brassica plants have also been reported.However,in the popular and nutritious vegetable broccoli,the physiological roles and molecular characteristics of MYB51,the gene encoding key regulator of indolic glucosinolate biosynthesis,remains unclear.In this study,we report the isolation and identification of an MYB51 gene from broccoli designated as BoMYB51.Gene expression in indolic glucosinolate biosynthetic pathway and the content of glucosinolates,callose deposition induced by Flg22(Flagellin22)and tolerence of salt stress in seedlings were investigated in the 35S::BoMYB51 transgenic Arabidopsis.The expression patterns of BoMYB51 in response to exogenous hormones and stress treatments were analyzed in detail by expressing BoMYB51 promoter driven GUS(?-glucuronidase)in Arabidopsis and Real-time PCR analysis in broccoli.Our study provides information on molecular characteristics of BoMYB51 and possible physiological processes BoMYB51 may involve.Our research attempts to deepen the understanding of the mechanism of indolic glucosinolate regulation in Brassica vegetables,provide theoretical data for the study of cruciferous indolic glucosinolates,and lay a theoretical foundation for the improvement of disease resistant varieties of broccoli.The main results were as follows:(1)Identification and bioinformatics analysis of BoMYB51According to our previous study of RNA-seq analysis of broccoli transcriptome,the unigene MYB51 was used as reference sequence to perform RACE(Rapid-amplification of cDNA Ends)PCR and the full-length cDNA was amplified from ‘Qingxiu' broccoli cultivar.The obtained full-length cDNA was 1641 bp long with a 355-bp 5' untranslated region,a 323-bp 3' untranslated region and a 963-bp CDS(Coding sequence)encoding 320 amino acids.Combined with the available genomic sequence of broccoli in NCBI,the structure of full length gene was constructed,and the gene was designated as BoMYB51(GenBank accession No.MF693155).(2)Overexpressing BoMYB51 in ArabidopsisPlant expression vector 35S::BoMYB51 was constructed and transferred into Arabidopsis.Overexpression of BoMYB51 was confirmed by RT-PCR(Reverse transcription-PCR)analysis and five transgenic lines were confirmed.The expression transcript levels of the genes involved in indolic glucosinolate metabolism were detected by Real-time PCR in wild-type and 35S::BoMYB51 Arabidopsis plants respectively.The expression level of the genes responsible for core structure biosynthesis,TSB1,CYP79B2,CYP79B3,CYP83B1 and SOT16 were significantly upregulated in 35S::BoMYB51.Genes involved in structure modification CYP81F2 and IGMT2 were not affected.TGG1 and PEN2,the genes encoding myrosinase(glucosinolate-degrading enzyme)and transcription factor genes regulating indolic glucosinolates MYB34 and MYB122 were significantly downregulated in 35S::BoMYB51.Glucosinolates were detected in leaves of wild-type and 35S::BoMYB51 Arabidopsis plants by HPLC(High Performance Liquid Chromatography).In 35S::BoMYB51 indolic glucosinolate contents were significantly increased,most aliphatic glucosinolates were not affected except 4MSOB(4-methylsulfinylbutyl-Glucosinolate),which was significantly decreased compared to wild-type.These results demonstrated that BoMYB51 positively regulated indolic glucosinolate production via activating the biosynthesis pathway genes.A callose-response assay was used to detect callose deposition induced by Flg22 in wild-type and 35S::BoMYB51 Arabidopsis.Under normal condition,enhanced callose deposition was observed in 35S::BoMYB51 compared to wild-type.Callose deposition was significantly induced by Flg22 both in wild-type and 35S::BoMYB51 and was more heavily induced in 35S::BoMYB51.The results demonstrated that overexpression of BoMYB51 promoted defense response against pathogen attack by enhancing Flg22 induced callose deposition.(3)Spatial expression pattern of BoMYB51 promoter driven-GUSPlant expression vector ProBoMYB51::GUS was constructed and transferred into Arabidopsis.BoMYB51 promoter was detected by GUS signal.In seedlings,GUS activity was observed in the vascular tissues of cotyledon,hypocotyl and root.In adult plants,strong GUS staining was detected in mid veins of rosette leaves,petioles,stem,inflorescence and upper part of young siliques,showed that BoMYB51 expression was widely detected in both vegetative and reproductive organs.GUS expression responsed differently to different hormones and stresses,SA(Salicylic acid)and MeJA(Methyl jasmonate)strongly induced GUS expression,the effect of ABA(Abscisic acid)and ET(Ethylene)was not significant,the GUS expression in response to GA(Gibberellin acid)varied in different tissues.Wounding systemic induced GUS expression,Flg22,NaCl and UV(Ultraviolet)treatment induced GUS expression.The activity of BoMYB51 promotor was not only induced by hormones,but also by biotic and abiotic stresses.(4)Expression profiles of BoMYB51 in broccoliTranscript level of BoMYB51 was measured by Real-time PCR in broccoli,BoMYB51 was widely expressed in all different organs,and the expression profile was consistent with the GUS activity under the control of BoMYB51 promoter in Arabidopsis.SA,MeJA,Flg22,NaCl,wounding and UV treatments induced BoMYB51 expression significantly in broccoli.In response to GA,cotyledons and true leaves were detected separately,the expression level of BoMYB51 was largely repressed in the emerging true leaves and enhanced in cotyledons,as was observed in ProBoMYB51::GUS Arabidopsis.However,in response to ABA,ET and mannitol,BoMYB51 expression was significantly up-regulated in broccoli,which was not observed in Arabidopsis expressing GUS driven by BoMYB51 promotor.In summary,BoMYB51 responds to multiple hormone signaling pathways and may play a role in abiotic stresses.