Identification And Functional Study Of TmMYB3,a Transcription Factor Specific To Taxus Media

Numerous transcription factors(TFs)have been reported to play crucial roles in the transcriptional regulation of various developmental processes.MYB family is the most common and the most widely studied plant TFs.R2R3-MYB is a large subfamily,members of which are involved in the regulation of secondary metabolism in plants.Basing on metabolomic and proteomic analyses,differences in accumulation of taxoids as well as taxol biosynthetic pathway-related enzymes among different stem tissues were also well investigated.According to microsection observation,four major tissues across the stem of T.media,including cortex,phloem,xylem,and pith,were separated by simply peeling.Differences in the metabolomes and proteomes between the four stem tissues were analyzed.Principal components analysis(PCA)showed that the difference between the epidermis and the phloem is minimal.Metabolomic analysis showed that the phloem contains the highest level of metabolites including several precursors,intermediate products,final products of taxol biosynthesis.Furthermore,many catalytic enzymes involved in the taxol biosynthesis pathway were also highly accumulated in the phloem.Most taxoids showed varying levels in these four stem tissues.Specifically,both of PTX and 10-DAB accumulated to the highest levels in the phloem,BAC predominately accumulated in the pith,and DAP greatly accumulated in the cortex and phloem tissues.The xylem contained the lowest levels of these four taxoids.Based on omics data,phloem specific expressed protein Tm MYB3 was cloned from the T.media and its positive biological features,including subcellular localization,multiple sequence alignment and phylogenetic tree,have been analyzed by bioinformatics.Partial promoter sequences of five paclitaxel biosynthesis-related genes were cloned.Several classic MYB recognition elements were found in the promoters regions of TBT,DBTNBT and TS.Further more,the electrophoretic mobility shift assay(EMSA)indicated that Tm MYB3 was involved in paclitaxel biosynthesis by binding to the promoters of DBTNBT,TBT and TS.Tm MYB3 can regulate the expression of TS and TBT promoters,but DBTNBT promoter.Although most of the intermediate metabolites involved in paclitaxel biosynthesis might be produced in the phloem,several intermediate metabolites accumulated in different stem tissues.Interestingly,taxa-4(5),11(12)-diene,the downstream product of TS,maybe transferred to the xylem,and 10-deacetyl-2-debenzoylbaccatin III,the upstream product of TBT,was migrated to the pith and cortex.Taken together,our results revealed that Tm MYB3 possibly was a positive regulator involved in paclitaxel biosynthesis by activating the expression of TBT and TS.Moreover,possible movements of the TS and TBT-related products among different stem tissues were predicted.Differences in the taxoid composition of different stem tissues suggested that the whole stem of T.media has potential for biotechnological applications.Phloem-specific Tm MYB3 plays a important role in the transcriptional regulation of paclitaxel biosynthesis.