MYB21 Interacts With MYC2 To Control The Expression Of Terpene Synthase Genes In Flowers Of F. Hybrida And A. Thaliana

The evolution of plants has gone through a long process from aquatic to terrestrial,from simple to complex.About 100 million years ago,angiosperms emerged and quickly developed into the most widely distributed and diverse plants on the planet.The emergence of floral organs is the key to the adaptability of angiosperms to the terrestrial environment.In addition to the showy colors,flowers can also attract different kinds of pollinators by releasing complex volatile organic compounds,ensuring the smooth reproduction process and greatly promoting the prosperity of angiosperms.In nature,volatile organic compounds can be divided into terpenes,benzenoid aromatics and aliphatic derivatives.Terpenoids are the most important components,which not only play an important role in attracting pollinators,but also exhibited multiple biological functions of regulating plant development and plant resistance.The biosynthesis and transcriptional regulation of plant terpenoid have received extensive attention from botanists and molecular biologists.After decades of research,the synthesis pathway of plant terpenoid has been elucidated.As a key in the synthesis pathway,the cloning and functional analysis of terpene synthase genes have been the focus of scientists,while the transcriptional regulation of terpene synthase genes has lagged behind.In this study,Herbaceous flowers of the iris family,Freesia hybrida,were used as research materials,and a series of molecular biological techniques such as volatile component detection,transcriptome sequencing,and protoplast transfection were employed to preliminary analyze the synthetic regulation of linalool.Then we further explored the conservative property of the new discovered regulatory mechanism in Arabidopsis.The main findings are as follows:Solid phase headspace microextraction combined with gas chromatography-mass spectrometry was used to analyze the components of the freesia volatile cocktails.The results showed that different kinds of freesias could release the most amount linalool,which suggested that the linalool synthase gene might be relatively conserved.Using the freesia transcriptome and digital expression profiling database,we cloned 56 transcription factors that potentially regulate the expression of linalool synthase genes.Through protoplast transient transfection experiments and EMSA,we finally confirmed that FhMYB21L1 and FhMYB21L2 directly regulate FhTPS1 expression.In addition,we found that FhMYC2 could interact with FhMYB21 Ls and negatively regulated FhTPS1 expression by inhibiting the binding of FhMYB21 Ls to FhTPS1 promoter.However,the expression levels of FhMYB21L1 and FhMYB21L2 were significantly higher than those of FhMYC2 during the flowering period.Therefore,the release of linalool in the volatiles of freesia flowers was impressive.In Arabidopsis,AtMYB21 could positively regulate the expression of AtTPS14,AtTPS11 and AtTPS21;while AtMYC2 could only positively regulate the expression of AtTPS11 and AtTPS21,rather than AtTPS14;AtMYC2 could interact with AtMYB21 and inhibited the activate effect of AtMYB21 on AtTPS14.AtMYC2,and its homologous AtMYC3,AtMYC4 and AtMYC5 showed high levels of expression during the flowering period of Arabidopsis.Therefore,caryophyllene was highly released in the volatiles of arabidopsis flowers.This study revealed the transcriptional regulation mechanism of linalool synthesis in freesia.For the first time,the positive regulatory function of MYB21 on monoterpene linalool and the negative regulation function of MYC2 on linalool synthesis were discovered,and their synergistic relationship in regulating the synthesis of volatile terpenoids was expounded in detail.This research has laid a certain theoretical foundation for revealing the transcriptional regulation model of plant volatile terpenoid synthase genes and flower improvement.