Regulation Mechanism Of Tobacco Flavonoid Biosynthesis And Their Function In Salt Resistance

Flavonoids are important secondary metabolites in plants and have important roles in plant growth,the resistance to oxidative damage caused by ROS during plant growth and abiotic stresses due to their antioxidant activity,and beneficial for human health.In our previous research,we found that NtCHS1 gene take important roles in biosynthesis of tobacco flavonoid.It make sense to discover and investigate the function and regulation mechnasim of NtCHS1 gene.In order to recover the regulation mechanism of NtCHS1 gene,we further investigate the regulation factors in following sections:(1)We employed Yeast one-hybrid library system to identify the candidate regulators of NtCHS1 gene in transcription level.The promoter sequence of NtCHS1 gene that start from intiation code "ATG" up to+1500 bp length,was combined with pHIS bait vector.The screening result showed that there are more than 300 transcrioption factors regulate the promoter region of NtCHS1 gene,which was classified into 26 types and including AP2,MYB,bHLH,WRKY,etc.(2)The results showed that NtMYB4 might directly binding to the promoter of NtCHS1 gene.High amino acide sequence homologuous between NtMYB4 and AtMYB4 and other sub 4 group of R2R3-MYB members was observed,which contain the EAR motif pdLNLD/ElxiG/S and ZINCF motif CX1-2CX7-12CX2C,which the typical repressor domain of sub-group 4 R2R3-MYB members.The transsent GAL4-GUS assay and subcellular location reults showed that NtMYB4 is a nuclear localized transcription repressor in tobacco.(3)The expression of NtMYB4 was suppressed by 200 mM NaCl treatment.Overexpression of NtMYB4 dramatically reduced the salt tolerance in transgenic tobacco plants,which indicated the candidate function of NtMYB4 in salt resistance.NtMYB4 repressed the promoter activity of NtCHS1 and negatively regulated its expression.Rutin accumulation was significantly decreased in NtMYB4 overexpressing transgenic plants and NtCHS1 RNAi silenced transgenic plants.Moreover,high H2O2 and O2-contents were detected in both types of rutin-reduced transgenic plants under high salt stress.In addition,exogenous rutin supplementation effectively scavenged ROS(H2O2 and O2-)and improved the salt tolerance of the rutin-reduced transgenic plants.In contrast,NtCHS1 gene overexpressing plants had increased rutin accumulation,lower H2O2 and O2-contents,and higher tolerance to salinity.The results suggested that tobacco NtMYB4 acts as a salinity response repressor and negatively regulates NtCHS1 gene expression,which results in the reduced flavonoid accumulation and weakened ROS-scavenging ability under salt stress.Meanwhile,our results reveal the mechanism of flavonoid how to response the salnity.The expression of NtMYB4 was down-regulated by salt,as a result reduced its repression to NtCHS1 gene.The expression of NtCHS1 gene was activated and cause the accumulation of rutin,the incrase the ROS scaveng ability and maintained a balance ROS level in plant.(4)In order to discover the mechanism of NtMYB4 how to response the abiotic stress and biotic stress,we use the Yeast two-hybrid library system to identify the candidate transcription factors may interact with NtMYB4 in transcription level.Totally,we screened 8 transcription factors might interact with NtMYB4,including C2H2-like zinc finger protein,AtbHLH036,HAT14,SPL12,HB-7,MYB60 and AGL75.Futher investigation will be focus on their interaction and the mechanism.