Regulatory Mechanism Of Anthocyanin Biosynthesis In Monocot Freesia Hybrida

The emergency of anthocyanin biosynthetic pathway has played vital roles in plant development.In the past several decades,an anthocyanin-promoting ternary protein complex(designated as MBW complex),composed of R2R3-MYB,basic helix–loop–helix(bHLH)and WD40 repeats(WDRs)has been well characterized in plants.However,divergent regulatory mechanisms were found in dicot and monocot plants represented by Arabidopsis thaliana and Zea mays,respectively.Presently,a whelming proportion of MBWs have been well elucidated in core dicot plants,whereas they are studied randomly in monocot plants,which are of crucial importance to decipher their functional diversities among angiosperms during evolution.Freesia hybrida,a monocotyledonous genus of herbaceous perennial flowering plants in the family Iridaceae,is grown universally as a cut flower plant with a wide range of flower colors including white,yellow,red,blue,purple and bicolors.In our previous studies,five kinds of anthocyanin aglycons,i.e.delphinidin,petunidin,malvinidin,peonidin,and cyanidin,were detected in F.hybrida"Red River~?".A strategy combining transcriptomic data,expression profiles,gain-of-function experiments and transient protoplast transfection assays was undertaken to identify F.hybrida anthocyanin regulators.Among the candidate genes isolated,five were similar to AtPAP1,AtTT8 and AtTTG1(FhPAP1L1/FhPAP1L2,FhTT8L/FhGL3L and FhTTG1L,respectively)and two encoded putative negative MYB regulators(FhMYB4L and FhCPCL).FhPAP1L1/FhPAP1L2,FhTT8L/FhGL3L and FhTTG1L were found to positively regulate anthocyanin biosynthesis when heterogeneously expressed in Arabidopsis or tobacco respectively.In addition,they interacted and activated the anthocyanin biosynthetic gene promoters when co-expressed in Arabidopsis leaf protoplasts.Further experiments revealed sets of the MBW complexes involved in anthocyanin biosynthesis were organized in a hierarchical and partially redundant manner in F.hybrida.Moreover,R2R3-MYB FhMYB4L and R3-MYB FhCPCL were deemed as negative regulators as both of them could inhibit anthocyanin biosynthesis.Genetic and molecular evidences showed that FhMYB4L functioned as part of the MBW complex by interacting with bHLH proteins and repressed transcription through its C-terminal EAR motif,which changed the positive MBW complex into negative one.Comparatively,FhCPCL functioned as negative regulators by competing bHLH regulators with anthocyanin-promoting MYBs,which titrated the formation of anthocyanin-promoting MBW complex.Actually,the precise regulatory cascade and feedback regulation in the control of anthocyanin biosynthesis in Freesia flowers were much similar like that in core eudicot plants,Arabidopsis and petunia.However,more and more evidences exhibited there supported the finds that divergent regulatory manners were evolved after the division of monocots and dicots.For example,FhPAP1L1 or FhPAP1L2 was sufficient to activate EBGs(early biosynthetic genes)as well as AtTT8,and further interacted with AtTT8 to regulate the expression of LBGs(late biosynthetic genes),whereas in Arabidopsis,the EBGs were activated by co-activator independent and functionally redundant R2R3-MYB regulatory genes(MYB11,MYB12,MYB111).Moreover,AtPAP1 could not activate AtTT8 or LBGs independently.In conclusion,it can be deduced that the regulatory mechanisms between monocots and dicots were divergent in many aspects,and the finds will benefit for the understanding of the genetic basis of the regulatory mechanism accounting for flower pigmentation in monocot plant.