MYB Transcription Factors Regulate Flavonoid-mediated Stress Response In Rosa Rugosa

Rosa rugosa is an important ornamental and economic plant which is widely cultivated around the world.However,roses are often subjected to various biotic and abiotic stresses during the growth process,which seriously affects their ornamental and economic values.MYB transcription factors(TFs)and flavonoids play pivotal roles in plant stress response.It has important practical value to study the regulation mechanism of MYB TFs in response to stresses for the improvement of roses stress resistance and genetic improvement.In this study,R.rugosa was used as the experimental material to isolate MYB TFs in response to abiotic stress,and their functions and molecular regulatory mechanisms were further studied.The major findings are as follows:(1)Isolation and identification of stress response-related MYB TFs in R.rugosa:firstly,combining with the Arabidopsis thaliana TAIR database,the strawberry transcriptome database and our assembled R.rugosa transcriptome database,18 MYB candidate genes were preliminary selected,which possible correlation with stress response.Secondly,expressional levels of these 18 MYB candidate genes in wounded rose petals and leaves were analyzed,and then two candidate genes were chosen.Phylogenetic tree analysis indicated that the two candidate genes belonged to PA1-and TT2-type proanthocyanidin(PA)activators,respectively.Thirdly,expressional levels of these two candidate genes in oxidative stress-treated rose leaves were analyzed,and it was confirmed that these two candidate genes were closely related to the stress response.Therefore,these two candidate genes were selected for further study,and they were named as RrMYB5 and RrMYB10,respectively.RrMYB5 belongs to the PA 1-type PA activator and RrMYB10 belongs to the TT2-type PA activator.(2)RrMYB5 and RrMYB10 interact and activate the expression of key structural genes in flavonoid biosynthesis pathway:yeast two-hybrid assays and split-luciferase complementation assays show that there is an interaction between RrMYB5 and RrMYB 10.In addition,both RrMYB5 and RrMYB10 interact with AtEGL3,a bHLH TF,to form a MBW transcriptional complex.In vivo dual-luciferase reporter assays showed that RrMYB5 and RrMYB10 activate each othe's expression,and they also regulate the expression of RrDFR,RrLAR,and RrANR.(3)RrMYB5 and RrMYB10 regulate the biosynthesis of flavonoids in plants:over-expression of RrMYB5 and RrMYB10 genes in R.rugosa and tobacco through Agrobacterium tumefaciens-mediated genetic transformation system.The results showed that the content of PAs in transgenic R.rugosa and tobacco was significantly increased,and the expression level of key enzyme genes of flavonoid biosynthesis was also significantly increased.These results indicate that RrMYB5 and RrMYB10 transcription factors positively regulate flavonoid synthesis in plants.(4)Transcriptome analysis of partial organ tissues of RrMYBl0,RrDFR and RrANR transgenic tobacco plants:RNA-seq analyses of transgenic and control tobacco plants using second-generation sequencing technology,the results indicated that the expression level of antioxidant-related genes in transgenic tobacco plants(especially PODs)was significantly upregulated,and the expression level of ABA signal transduction-related genes(especially PP2Cs)was significantly downregulated.Taken together,over-expression of RrMYB10,RrDFR,and RrANR enhance plant tolerance to oxidative stress not only by promoting the accumulation of flavonoids but also by regulating the expression of antioxidant enzymes and hormone signaling related genes.(5)Overexpression of RrMYB5 and RrMYB10 increases the tolerance to oxidative stress in transgenic R.rugosa and tobacco plants:comprehensive analysis of plant tolerance to oxidative stress in transgenic tobacco(RrMYB5,RrMYB10,RrDFR,and RrANR)and transgenic R.rugosa(RrMYB5 and RrMYB10)showed that the stress-treated transgenic plants with lower ROS levels and higher activities of antioxidant enzymes than control.The antioxidant capacities of transgenic plants were significantly enhanced.In a word,RrMYB5 and RrMYB10 significantly enhance the plant's resistance to stress by increasing the plant ROS scavenging abilities.In summary,the MYB transcription factors(RrMYB5 and RrMYB10)induced by wounding and oxidative stress in R.rugosa promote the accumulation of flavonoids by regulating the expression of flavonoid-related genes,thereby improving the scavenging abilities of ROS,in turn,enhancing the plant stress resistance.Our study provides new insights into the regulatory mechanisms of flavonoid biosynthesis by MYB TFs and their essential physiological functions in plant responses to wounding and oxidative stresses.