| Anthocyanins are secondary metabolites that contributes to plant pigmentation and protects plant in response to various biotic and abiotic stresses.The anthocyanins of swollen root peel in Tsuda’ turnip are induced by short wavelengh light,especially the UV-A and combination of blue+UV-B light,of which the mechanism is remained unclear.In this study,we used EMS-mutagenized mutants as materials and identified the defective gene of white mutant w68 by the combination of MutMap,linkage analysis and transcriptome.The candidate gene encodes a R2R3 MYB transcription factor BrPAP1a.Then,using protein-protein interaction and transcriptional activation experiments to further investigate the regulatory mechanism of BrPAP1a on light induced anthocyanin accumulation and function of the highly conserved amino acids in the R3 domain.The main results were presented as follows:1.Genetic analysis and gene expression analysis of EMS mutantsThe anthocyanin contents and expression level of anthocyanin biosynthesis genes in swollen root peel of mutants were determined-The result showed that the anthocyanin contents of white mutant w9,w68 and w204 were significantly decreased at aboveground parts,and expression levels of anthocyanin biosynthetic genes and regulatory genes were significantly reduced.The red mutant r21 showed significantly higher levels of anthocyanin contents at both aboveground and underground parts of peels than those of in wild type,which was consistent with expression levels of anthocyanin biosynthesis related genes.Genetic analysis of w9,w68,w204 and r21 mutants showed that,proportions of F2 plants between wild-type and Mutant-type in w9,w68 and w204 mutants fit the 3:1 ratio that for one-locus segregation,but not in r21.2.Map-based cloning of w68 locus and functional validationBy using combination of MutMap analysis with linkage analysis,the w68 locus was mapped to a region of～500 kb on chromosome A07.According to functional annotation and gene expression analysis,we found that BraA07003293 encodes an R2R3-MYB transcription factor BrPAP1a,which expression pattern was correlated with anthocyanin accumulation.In w68 mutant,BraA07003293(BrPAP1a)contains a G-to-A transition in the third exon,resulting in a single amino acid change(G94S).Sequence alignment of the deduced amino acid sequences of BrPAP1a with other R2R3 MYB factors revealed that residue G94 is located in the turn region of the R3 domain,which is highly conserved in R2R3 MYB proteins among species,indicating the importance of the highly conserved amino acids in maintaining the function of BrPAP1a.Complementary analysis in transgenic Arabidopsis lines indicated that BrPAP1a is a positive regulator of anthocyanin accumulation,while G94S mutation led to loss-of-function BrPAP1a.3.The regulatory mechanism of BrPAP1a on light-induced anthocyanin biosynthesisWe used light and dark parts of swollen root peel in wild type and w68 to perform transcriptome analysis,and obtained 975 up-regulated differentially expressed genes(DEGs)and 443 down-regulated DEGs,respectively.The results of GO and KEGG analysis showed that these up-regulated DEGs were involved in regulation of flavonoid biosynthetic process,response to high light or UV-B,defense response to fungus and bacterium,photosynthesis,response to far-red light and glucose metabolic process and so on.Among the anthocyanin-related transcription factors,the expression of bHLH transcription factor BrTT8 was significantly decreased.The transcriptional activation,Dual-luciferase and EMSA experiments confirmed that BrPAP1a can directly bind the MYB recognition elements(MRE)in the BrTT8 promoter to activate BrTT8 expression,while G94S substitution caused diminished DNA-binding activity and trans-activation ability of BrPAPla.According to 3D model analysis,we observed a conformational change of R91 in the R3 domain caused by G94S substitution that probabley impaires the interaction with bHLH protein and stability of MBW complex.Using the protein-protein interaction analysis,including yeast two-hybrid assay and LUC complementation imaging assay,we found that G94S mutation caused significantly reduced interaction of BrPAP1a with BrTT8,and abolished the interaction of BrPAP1a with BrEGL3.Meanwhile,the protein-protein interaction analysis of other MBW components showed that BrPAP1a plays a critical role in formation and stabilization of BrPAP1a-BrEGL3-BrTTG1 complex by bridging both BrEGL3 and BrTTGl and then activates BrTT8 expression.Taken together,our study showed that BrPAP1a is a positive regulator of anthocyanin accumulation in ’Tsuda’ turnip.BrPAP1a interacts with BrTT8,BrEGL3 and BrTTG1 to form MBW complex and activates regulatory factor BrTT8 and biosynthetic genes such as BrCHS,BrDFR expression.This study provides new insights into regulatory mechanism of light induced anthocyanin accumulation in Brassica rapa and reveals that the highly conserved turn region in R3 domain plays crucial roles in stabilizing the spatial conformation and activity of MYB protein. |
