| Wheat（Triticum aestivum L.）is a major cereal growing throughout the world,and its grain yield is significantly restrained by salt stress.In our previous works,EsMYB90 gene identified from halophytic Eutrema salsugineum played a critical regulating role in promoting the biosynthesis of many flavonoid compounds in transgenic tobacco.In this study,we generated the Ubi:3×Flag-EsMYB90 construct to ectopically express EsMYB90 in transgenic wheat plants,and the function and regulation mechanism of EsMYB90 in flavonoids biosynthesis and salt stress tolerance in transgenic wheats were researched.The main study results are as follows:1.Construction of Ubi:3×Flag-EsMYB90 overexpression vector and obtain of transgenic wheat plantsThe pLGYOE3-Ubi:3×Flag-EsMYB90 overexpression vector was constructed,and introduced into wheat by Agrobacterium-mediated transformation.The ectopic EsMYB90 transgenic wheats were obtained by the Basta screening and PCR molecular identification.The obtained T1generation transgenic wheats were further reproducted to get the homozygous transgenic plants.2.EsMYB90 gene significantly increased flavonoids contents in leaf sheaths of transgenic wheats The leaf sheath tissues of EsMYB90 transgenic wheat showed an obvious purple-red color with higher anthocyanin and total flavonoid accumulation than that in the wild type.The contents of anthocyanins and total flavonoids in leaf sheaths of TL30 transgenic lines were 103 and 86 times higher than those in wild type,respectively.Therefore,it is suggested that EsMYB90 play a key role in regulating flavonoid biosynthesis and accumulation in transgenic wheat lines.3.EsMYB90 gene markedly up-regulated the expression of flavonoids biosynthesis genes in transgenic wheatsUsing RNA-seq and real-time quatitative PCR,we found that EsMYB90 significantly up-regulated the expression of flavonoid biosynthesis genes such as TaPAL,TaCHS,TaF3H,TaFLS,TaDFR,TaLDOX and TaUFGT in leaf sheaths of transgenic wheats.Thus,it is inferred that EsMYB90 gene promote the expression of flavonoid biosynthesis genes,to enhance biosynthesis and accumulation of flavonoids in the transgenic wheats.4.EsMYB90 remarkably enhanced the antioxidant capacity of transgenic wheats under salt stressUnder 200 mM NaCl treatment,the EsMYB90 transgenic lines were observed to have asignificant increase in root length and fresh weight when compared with the wild type（WT）wheats.Also,the activities of antioxidant enzymes GST and POD were significantly elevated,while the contents of malondialdehyde（MDA）and hydrogen peroxide（H2O2） were remarkably decreased in transgenic wheat plants.Therefore,it is suggested that EsMYB90 remarkedly enhanced the activities of antioxidant enzymes such as GST and POD to reduce the extent of oxidative damage in the transgenic wheats.5.EsMYB90 gene obviously enhanced expression of antioxidant related genes in response to salt stressRNA-seq analysis revealed that differentially expression genes（DEGs）in leaf sheaths oftransgenic wheats under salt stress were significantly enriched in the GO terms of oxidoreductase activity,antioxidant activity,and response to oxidative stress.Importantly,EsMYB90outstandingly up-regulated expression of flavonoid biosynthesis genes TaANS2 and TaDFR1,as well as antioxidantive enzyme genes TaGSTs and TaPODs.6.Identification of EsMYB90 protein binding motifs and its regulation of downstream antioxidant genes in transgenic wheat using DAP-seqDual luciferase assay revealed that EsMYB90 significantly activated expression of TaANS2and TaDFR1 in transgenic wheat plants.DAP-seq analysis demonstrated that EsMYB90 may directly bind to cis-elements in the promoter regions of cytochrome P450 TaCYP76M5 and NAD（P）H-quinone oxidoreductase subunit gene TaNQO5.Also,the binding sites of EsMYB90were found to exist in the adjacent intergenic regions of phenylalanine lyase TaPAL,glutathione transferase TaGST-BZ2 and peroxidase TaPOD genes.The dual luciferase assay and DAP-seq indicated that EsMYB90 may play an important role in the biosynthesis of non-enzymatic antioxidants（flavonoids and antitoxins）as well as enzymatic antioxidant reaction.Using MEME-Ch IP software,the EsMYB90 transgenic wheats based on the DAP-seq were revealed,that the downstream binding conserved motifs of EsMYB90 transcription factor（TF）are CACCTACCGCW and AAAARTRGAAAAGGA.Given the motif（CACCTACCGCW）was highly homologous with the binding motifs of At MYB4 and At MYB111 that involved in flavonoid biosynthesis and salt tolerance in Arabidopsis,it is inferred that EsMYB90 could be associated with modulation of flavonoid biosynthesis and salt tolerance in transgenic wheats via regulating the expression of enzymatic and non enzymatic antioxidant related genes in transgenic plants.Taken together,the results showed that（1）EsMYB90 significantly enhanced the flavonoid content and antioxidant enzyme activity of transgenic wheat plants via promoting expression of the genes involved in biosynthesis of flavonoids and antioxidant enzymes,to display an obvious purple-red color phenotype;（2）Under salt stress,EsMYB90 transgenic plants showed a significantly increased root length and fresh weight,as well as a significantly enhanced antioxidative capacity compared to wild-type plants;（3）Analysis of EsMYB90 TF conserved binding motifs in EsMYB90 transgenic wheats revealed that EsMYB90 could be involved in regulating the expression of flavonoid biosynthesis and salt tolerance related genes.This study systematically demonstrated the functions of EsMYB90 gene in enhancing antioxidant capacity and salt tolerance,and analyzed its possible regulatory mechanisms in transgenic wheats.This study provides an important potential wheat germplasm resource possessing a higher antioxidant capacity under salt stress,indicating that EsMYB90 is an ideal candidate gene for the genetic improvement of crops. |
