Molecular Mechanism Of Anthocyanin Biosynthesis And Regulation In Actinidia Arguta

Kiwifruit is perennial deciduous vine that originates from China.It belongs to the family Actinidiaceae,genus Actinidia,which is rich in germplasm resources presenting 75 taxa,including 54 species and 21 varieties.There are various fruit characteristics,such as delicious fruit with much juice,rich in vitamin C and minerals,high nutritional value,etc.Fruit color,as the most intuitive character among fruit traits,affects the appearance quality of fruit and purchase intention of consumers,directly determining its commercial value.Fruit color can be mainly divided into three categories including green,yellow and red.The specific metabolic processes corresponding to three typed color are chlorophyll,carotenoid and anthocyanin metabolism,respectively.The synthesis and accumulation of anthocyanin lead to the formation of red kiwifruit.Therefore,study on molecular mechanism of anthocyanin biosynthesis is of great importance for genetic improvement of red kiwifruit breeding.Studies on anthocyanin synthesis in kiwifruit have been reported,but the regulatory mechanism remains unclear.Besides,most of studies focused on partially red-typed Actinidia chinense,rarely in A.arguta.In this study,A.arguta fruits with different colors were used as experimental materials to perform metabolome analysis and small RNA sequencing.The key regulatory factors controlling anthocyanin biosynthesis were screened and their functions were verified,and the mechanism of anthocyanin biosynthesis and regulation was explored.The main results were showed below:1.Screening of coloration-related metabolites based on metabolome analysis.Through metabolome analysis for all-red-typed A.arguta ‘HB’ and all-green-typed A.arguta ‘YF-1’,a total of 28,837 metabolites were identified,of which 13,715 were successfully assigned and annotated to the level-one and level-two databases.There were 3,785 up-regulated metabolites in comparison of before and after fruit coloration,of which 41 metabolites belonged to flavonoids,with a total of 7 metabolites presenting significant differences,among which 4 anthocyanin-related metabolites including dihydromyricetin,cyanidin,pelargonidin,delphinidin might play a key role in the kiwifruit color-change period.Association analysis of transcriptome and metabolome and gene expression profile validation showed that F3 H,LDOX and UFGT might be the key structural genes involved in anthocyanin biosynthesis.2.Mining of key regulator based on small RNA sequencing.A.arguta ‘HB’ at three developmental stages were investigated by small RNAs high-throughput sequencing.A total of 1,063 mi RNAs,including 482 conserved mi RNAs corresponding to 526 pre-mi RNAs and 581 novel mi RNAs corresponding to 619 pre-mi RNAs,were identified and grouped into 46 mi RNA families.A total of 321 mi RNAs with differential expressions during ‘HB’ fruit coloring process were used to predict target genes,which were assigned to GO and KEGG databases to obtain corresponding functional information.The annotated data showed that 814 target genes were assigned into 25 biological processes,15 cell component and 10 molecular function items in GO classification,and 207 target genes were assigned into 18 items in KEGG classification.This will provide basic information and data reference for subsequent mi RNA screening.According to the combined analysis of gene expression level,correlation of gene expression and anthocyanin component content,and sequence alignment between mi RNA and target genes,a highly expressed mi R858 in green fruit-staged and the target gene Aa MYBC1 were screened as candidates possibly participating in regulation of anthocyanin biosynthesis in A.arguta.3.Functional verification of regulators.mi R858 and its target Aa MYBC1 were located in the 1.2 Mb of the 13 th chromosome and 0.8 Mb of the 9th chromosome based on genomic location,respectively.The target site is the region of 289-309 bp in coding sequence of Aa MYBC1.A.arguta ‘HB’ fruit was used for Aa MYBC1 cloning of the complete c DNA,the length of which was 798 bp encoding 265 aa.Functional protein association network prediction showed there might be interaction between Aa MYBC1 and Aab HLH42 transcription factors(TFs).The two recombinant vectors,Aa MYBC1-p GBKT7 and Aab HLH42-p GADT7,were constructed to perform yeast two-hybrid(Y2H)assay,which showed Aa MYBC1 TF indeed interacted with Aab HLH42 TF.Subcellular localization analysis indicated that Aa MYBC1 TF targeted to nucleus,and Aab HLH42 TF located to nucleus and cytoplasm,which suggested those two TFs both have the general characteristics of TF.Through constructions of expression vectors for mi R858 from,Aa MYBC1 target site,and mutant of Aa MYBC1 target site and co-transformation into Nicotiana benthamiana assays,we proved the targeting relations between mi R858 and Aa MYBC1.4.Overexpression of mi R858 was carried out in off-tree A.arguta ‘HB’ core.Compared to control,the core of mi R858 overexpression could not present red color,and anthocyanin was hardly detected.Expression of Aa MYBC1 and late biosynthetic genes(LBGs)involved in anthocyanin biosynthesis significantly decreased,indicating mi R858 inhibits synthesis and accumulation of anthocyanin by repression of Aa MYBC1 expression,thus suppresses fruit coloration.Virus-induced gene silencing system was used for silencing Aa MYBC1 in on-tree A.arguta ‘HB’ core.Compared to control,the redness and anthocyanin content in silencing core were lightener and lower,respectively.Expression of Aa MYBC1 and late biosynthetic genes(LBGs)involved in anthocyanin biosynthesis significantly decreased,indicating silencing Aa MYBC1 could repress anthocyanin biosynthesis,and thus disturb fruit coloration.However,the red coloration could not be completely suppressed.5.The difference between red and green A.arguta fruits might be attributed to the regulation of differential expression of mi R858.No light and high temperature could induce mi R858 expression,and thus repress fruit coloration by experiments of bagging and temperature treatments in A.arguta fruits.The mi R858-mediated regulatory model of anthocyanin biosynthesis response to environmental factors in A.arguta might be described as follows: under normal circumstances,mi R858 present low expression or no expression.Aa MYBC1 and Aab HLH42 TFs normally interact with each other to form a protein complex by recruiting Aa WD40 TF to activate the expression of LBGs,leading to normal anthocyanin accumulation,thus lead to fruit normal coloration.When the fruits under stress conditions,such as the fruits subjected to harmful environmental factors, including very high temperature and no light,mi R858 is induced to high expression.A reduction in the Aa MYBC1 levels weakens the stabilization of the MBW complex,thereby hindering the expression of LBGs and leading to a decrease in anthocyanin content,finally fruits presented abnormal coloration.Using the multi-omics approaches,we systematically revealed the molecular mechanism of anthocyanin biosynthesis,preliminarily explored possible reason of the difference between red and green A.arguta fruits,and established mi R858-mediated regulatory network of anthocyanin biosynthesis in A.arguta,which provide not only novel insight into clarifying kiwifruit coloration but the theoretical basis for genetic improvement of red traits,being of great significance for molecular breeding to guide production.