Effect Of HY5-MIR858-MYBL2 Regulating Pathway On Anthocyanin Biosynthesis In Arabidopsis Thaliana

As a crucial environmental factor,light plays a cardinal role in controlling plant growth and development.Plants balance their physiological metabolism in order to accommodate light signal fluctuations.Anthocyanins are a class of plant secondary metabolites whose biosynthesis depends on the light signal.Thus,revealing the complex regulatory mode of anthocyanin biosynthesis can not only help us clearly understand the nature of biological phenomena,but also provide molecular basis for genetic improvement of anthocyanin related agricultural products.Currently,studies on the light signaling pathway were extensive and in detail,which serves as a solid theoretical foundation for future investigations.Nonetheless,knowledge about light-induced anthocyanin biosynthesis is limited and the regulatory mechanism involving microRNAs is not thoroughly elucidated.In this study,we expound an important molecular mechanism governing light-induced anthocyanin biosynthesis by means of bioinformatics,genetics and molecular biology in the model plant Arabidopsis thaliana,including the following aspects:(1)Throught cultivating wild-type and hy5 mutant seedlings under different light intensity conditions,we found that HY5 promotes the ability of seedlings to response to light intensity and induces anthocyanin accumulation;(2)RT-qPCR analysis demonstrated that HY5 affects expression of anthocyanin biosynthesis inhibitor gene MYBL2.We next designed proMYBL2:GUS construct and transformed it into Arabidopsis thaliana using Agrobacterium-mediated transgenic technology.The positive plants were crossed with hy5 mutant and produced proMYBL2:GUS/hy5.GUS staining results indicate that GUS activities were weaker in hy5 mutant than that in wild type.Therefore,HY5 can negatively control MYBL2 expression at the transcriptional level;(3)We conducted Chromatin Immunoprecipitation(ChIP)experiments and found HY5 could bind to two G-boxes in the MIR858 promoter.RT-qPCR showed that miR858 expression level in wild type is higher than that in hy5 mutant,indicating that HY5 promotes miR858 transcription.GUS staining was further performed and the results demonstrated that GUS activities depend on a functional HY5 and the G-boxes,confirming that HY5 plays a positive role in regulating MIR858;(4)By designing miR858 overexpression and silencing vector,we gained functionally transgenic plants.Mature miR858 was upregulated significantly in overexpression plants while downregulated significantly in silenced plants.After anthocyanin phenotypic observation,we concluded that miR858 accelerates anthocyanin accumulation.Further RT-qPCR analysis manifests that two key genes of the anthocyanin biosynthetic pathway,LDOX and DFR,have a higher expression level in overexpression plants,indicating that miR85 8 is a positive regulator in the anthocyanin metabolic process;(5)Through bioinformatics analysis we found that miR858 may target mRNA of MYBL2.5’ RACE analysis revealed that miR858 does not cleave the MYBL2 mRNA.RT-qPCR results suggest that altered mature miR858 levels do not affect on mRNA level of MYBL2;(6)GFP-MYBL2 fusion proteins were introduced to the miR858 overexpression and silencing plants via genetic crosses.Using detected GFP fluorescence to reflect MYBL2 protein levels,we realized that fluorescence of the GFP fusion protein exhibited opposite expression pattern against that of miR858.In further experiments based on the REN-LUC dual luciferase reporter system expressed in Nicotiana benthamiana protoplasts,we found that miR858 may negatively affect the chemiluminescence between MYBL2-LUC fusion protein and substrate;(7)Based on various genetic materials,through observation of the anthocyanins phenotype and quantitative analysis,we determined the genetics relationships of HY5,miR858,MYBL2 in the anthocyanin pathway.From the above results,we firstly discovered the HY5-miR858-MYBL2 loop is critically involved in the light-induced anthocyanin accumulation.We clarified the respective roles and interaction of the three genes.We proposed a HY5-MYBL2 seesaw model to explain "up" and "down" status of anthocyanin biosynthesis in the light and dark conditions.Our results enrich understanding of the anthocyanin metabolic regulatory networks and lay the foundation for optimizing agricultural production of this important plant pigment.