Molecular Cloning And Functional Analysis Of The Gene RAP In A Fragaria Vesca Mutant With Reduced Anthocyanins

Cultivated strawberry(Fragaria ×ananassa Duch)is a perennial herbaceous species that belongs to Rosacea family.The fruit is widely appreciated by consumers worldwide for its bright red color,taste and nutrition.The fruit contains abundant flavonoid compounds including anthocyanins.Anthocyanins are water-soluble and give rise to the bright red color of foliage and fruit in strawberry.Anthocyanins are synthesized in the endoplasmic reticulum and stored in the vacuole.Anthocyanins are synthesized by a well-studied pathway with a series of enzymes and regulatory genes,but little is known about how anthocyanins are transported from the ER to the vacuole.We obtained a mutant with reduced anthocyanins in petioles from ENU mutagenized population of YW5AF7,a whitefruited variety of the wild strawberry Fragaria vesca.In this study,the mutant and three F.vesca accessions were utilized in our research: YW5AF7,H4 and red-fruited Ruegen.We cloned the causative gene and analyzed its functions through a combination of genetic analysis,whole genome re-sequencing,phylogenetic analysis,transient gene expression,stable transformation,and expression analysis.We first report the main transport gene in anthocyanin synthesis pathway,which has important theoretical significance and application value.The main results are as follows:1.A mutant produced as a result of an ENU chemical mutagenesis was identified to accumulate very little anthocyanin in the leaf petioles and named as Reduced Anthocyanins in Petioles(rap).The epidermis and some cortex cells especially around the vasculatures in petioles display a bright red color in YW5AF7,while the red pigmentation is greatly reduced in the rap mutant.Compared with the wild type YW5AF7,total anthocyanin content was remarkably reduced in the petioles of rap,consistent with the phenotype observed.The rap mutant was backcrossed with YW5AF7.In the F2 population,the ratio of wild-type to mutant plants is close to 3:1,indicating that rap is caused by a single gene with a recessive mutation.Through whole genome resequencing,we identified the primary candidate gene called gene31672 that has a SNP of C to T at the second exon resulting in a premature stop codon.To confirm this result,the SNP was individually examined in 50 rap mutants found in the F2 population by PCR-amplification and Sanger sequencing,all of which were homozygous,confirming that gene31672 is linked with the phenotype.Besides,we found that the expression level of RAP is greatly reduced in the petioles of rap probably as a result of nonsense-mediated decay.2.There are 7 other homologs of RAP in F.vesca belonging to the phi subfamily of GST family.These homologs have different expression patterns.RAP is most abundantly expressed in ripening fruit of Ruegen.Analysis of GUS activity in the RAPpro::GUS transgenic lines showed that RAP was specifically expressed in the epidermal cells of petiole and around the vascular bundles.Overexpressing RAP into the Arabidopsis homolog mutant tt19-7 restored anthocyanin accumulation in both stems and leaves,suggesting that RAP is the ortholog of TT19 in Arabidopsis.3.The red-fruited Ruegen was crossed with rap.In the F2 population,only one plant with the genotype of MYB10+;rap-was identified out of 66 green petioles,indicating that MYB10 and RAP is linked to each other.The anthocyanin content in fruit receptacle of MYB10+;rap-is very low,while the achenes of MYB10+;rap-also accumulate much less anthocyanin than that of Ruegen,indicating that RAP is a important regulator of fruit coloration and acts downstream of MYB10.q RT-PCR revealed that expression level of RAP is nicely correlated with MYB10.HPLC analysis showed that each of the anthocyanin compounds of MYB10+;rap-is much lower than that of MYB10+;RAP+,suggesting that RAP is responsible for the transport of all of these compounds.4.Transient assay revealed that overexpression of RAP together with MYB10 can rescued the fruit coloration of rap mutant,but none of the RAP-Like genes gave the red color to the same extent as RAP when their expression levels are comparable,indicating that RAP and its homologs have different anthocyanin binding capability.Using transient expression assay in tobacco leaves,we found that RAP,RAP-L1 and RAP-L3-5 were localized in the cytosol and nuclei,while RAP-L2 was mainly localized in chloroplast.Together,besides transcriptional control,protein sequences and subcellular localization underlie functional divergence of RAP and its homologs.Besides,both N-and C-terminals are essential for the binding capacity according to the domain swap experiment.5.Overexpressing RAP into rap rescued the coloration in petioles,resulted in coloration in root tips and pistils that do not contain anthocyanins in wild type strawberry,and produced red fruit that looks different from the fruit in Ruegen and does not rely on MYB10.Transient knock-down of RAP reduced the fruit coloration in cultivated strawberry.Taken together,RAP can be used as one promising candidate gene during strawberry breeding for fruit color.