Study On The Mechanism Of Leaf Coloration Of Acer Rubrum Based On Multi-omics Technology And Analysis Of Gene Function Of ArMyB89

The role of color tree species in landscaping has become increasingly prominent,Acer rubrum is playing a more and more important role in greening and landscape design because of straight dry type and gorgeous leaf color.The lack of complete genome sequence of A.rubrum restricts the genetic basis of this species.The mechanism of pigment and gene regulation affecting leaf discoloration of A.rubrum is still unclear.Previous studies showed that the accumulation of anthocyanins was the main reason for the redness of A.rubrum leaves.Although the function of some MYB transcription factors in anthocyanin synthesis has been confirmed,most of the studies on MYB transcription factors in woody plant are still in the stage of transcription sequencing and pattern expression analysis,and there are few reports on their biological functions.In this study,a complete genome sketch of A.rubrum was obtained by whole genome sequencing technology,and the molecular mechanism of leaf discoloration of A.rubrum was deeply explored by using the combined analysis technology of genomics,transcriptomics and metabolomics.The regulatory relationship between ArMYB89 and anthocyanin accumulation of A.rubrum was clarified,which the possible mechanism of ArMYB89 regulating anthocyanin synthesis was revealed by combining with functional research experiments.The main results are as follows:1.In this study,a relatively complete genome sketch of A.rubrum was obtained by whole genome sequencing.We sequenced the A.rubrum genome using Oxford Nanopore Technology(ONT)and Hi-C technology,the A.rubrum genome was 1.7 Gb in size with contig N50 of 547.18 Kb.A total of 39 pseudochromosomes were generated with a 99.61%(1.69 Gb)genome by taking advantage of Hi-C technology.The proportion of integrated tandem repeats(TRs)and other repeat sequence is 69.85%,most of them were transposable element(TE).A total of 64644 genes were predicted,with an average gene length of3801.1bp,and 7259 non-coding RNAs were annotated;62927 genes could be annotated into the functional database,accounting for 97.34% of the total number of genes in the genome of A.rubrum;A.rubrum was closely related to A.yangbiense,it was estimated that A.rubrum diverged from A.yangbiense about 6.34 million years ago.2.The leaf color of A.rubrum is closely related to the synthesis and accumulation of anthocyanins,chlorophyll and carotenoids.Green leaves,red leaves and yellow leaves of different branches of A.rubrum with special leaf color were used as experimental materials.In transcriptome sequencing,14009,20824 and 13620 differentially expressed genes were up-regulated and 14527,22193 and 13490 differentially expressed genes were downregulated in red leaves-green leaves group,yellow leaves-green leaves group and red leavesyellow leaves group,respectively.In metabolomic analysis,in positive ion mode,706,537 and 192 differentially accumulated metabolites were up-regulated and 671,1256 and 906 differentially expressed genes were down-regulated in red leaf green leaf group,yellow leaf green leaf group and red leaf yellow leaf group respectively;in the negative ion mode,355,141 and 70 differentially accumulated metabolites were up-regulated and 434,558 and 607 differentially accumulated metabolites were down-regulated in red leaves-green leaves group,yellow leaves-green leaves group and red leaves-yellow leaves group,respectively.The enrichment of KEGG pathway in transcriptome and metabolome indicated that the change of leaf color in A.rubrum was affected by the synthesis and accumulation of anthocyanins,chlorophyll and carotenoids.3.The accumulation of anthocyanins was the main reason for the red leaf of A.rubrum,and its content and proportion were higher than the other two pigments.The formation of green leaves is due to the accumulation of chlorophyll,while the formation of yellow leaves is due to the degradation of chlorophyll,the appearance of carotenoids and the accumulation of anthocyanins.In the biosynthesis of anthocyanins in A.rubrum,CHS genes positively regulate the production of anthocyanins and their derivatives.Cyanidin-3-(6''-acetylgalactoside)and cyanidin-3-arabinoside are the decisive factors for red or yellow leaves of A.rubrum.In the process of chlorophyll metabolism,chlorophyll a is the main pigment.Ar POR positively regulates the synthesis of chlorophyll a,while Ar NOL2 reversely regulates the production of chlorophyll a.The expression of Ar LUT5-3 and Ar LUT5-4 in red and yellow leaves was down-regulated during carotenoid biosynthesis.Under their positive regulation of ?-carotene,carotenoid biosynthesis changed from ?-branching to ?-branching during leaf senescence.4.Based on the whole genome analysis of MYB gene family in A.rubrum,a MYB transcription factor named ArMYB89 was cloned and its function was studied.Our research identified 393 MYB transcription factors in the A.rubrum genome,and these Ar MYB members were unevenly distributed across 34 chromosomes.Collinearity analysis showed that there were 16 tandem repeat gene pairs and 247 fragment repeat gene pairs in MYB family.Quantitative real-time PCR analysis revealed that the ArMYB89 transcription level was significantly up-regulated in red leaves in contrast to green leaves.Subcellular localization experiments indicated that ArMYB89 was localized on the nucleus.Further experiments revealed that ArMYB89 could interact with Ar SGT1 in vitro and in vivo.The overexpression of ArMYB89 in tobacco might enhance the anthocyanin content of transgenic plants.In conclusion,on the basis of obtaining the genome sequence of A.rubrum,combined with transcriptome and metabonomic analysis,it was clear that the formation of green leaves of A.rubrum was due to the accumulation of chlorophyll,and the main reason for red leaves was the accumulation of anthocyanins,while the reason for yellow leaves was the joint action of chlorophyll,carotenoids and anthocyanins.The whole genome analysis of MYB gene family that regulates anthocyanin synthesis in A.rubrum showed that the expression level of ArMYB89,located in the nucleus,in red leaves was significantly higher than that in green leaves.Overexpression of this gene could increase the anthocyanin content of transgenic plants,and ArMYB89 protein could interact with Ar SGT1 both in vivo and in vitro.This study is of great significance in understanding the mechanism of leaf discoloration and MYB transcription factor in Acer.It can provide specific gene resources for the study of Acer species directed improvement,and has important theoretical significance and application value for the innovation of Acer species germplasm resources and the enrichment of color leaf species resources.