| Anthocyanins that accumulated in swollen root peels of Tsuda' turnip are induced by light.UV-A is reported to be the most effective wavelength involved in generating anthocyanin accumulation in swollen root peel,of which regulatory mechanism remains unclear.In this study,phenotypic analysis,gene expression and genetic analysis were performed to characterize the anthocyanin deficient-mutant g120w.Using the F2 population,we mapped the candidate gene BrLETM2 by combining the BSA-Seq with CAPS marker linkage analysis and verified its function by co-segregation analysis and genetic transformation experiment.On the other hand,transcriptome analysis was performed to identify the target genes of BrLETM2 and signaling pathways that regulated by BrLETM2,revealing the function of BrLETM2 on light-induced anthocyanin biosynthesis.The main results of this study are as follows:1.Phenotypic characterization and genetic analysis of g120w mutantWe isolated an anthocyanin deficient mutant g120w from the T-DNA library and compared phenotypic variations of g120w with wild type in leaf,flower and swollen root.The swollen root peel and margins of leaf in g120w accumulated less anthocyanins than that of in the wild type.The RT-qPCR results of anthocyanin biosynthetic genes showed that expression levels of structural genes BrCHS1,BrDFR,BrANS1 and regulatory genes BrPAP1.3 and BrTT8 in g120w was significantly decreased,indicating that the candidate gene promotes anthocyanin biosynthesis by activating MBW complex.Genetic analysis showed that a single recessive gene controlled the anthocyanin deficiency phenotype of g120w.2.Identification of candidate gene in g120w by BSA-seqWe used purple peel and white peel progenys in the F2 population as materials for genome resequencing and mapped the g120w locus at a region of 19.00 Mb to 21.86 Mb on chromosome A07 by using the BSA-seq method.Finally,we performed fine mapping of g120w locus using white peel individuals and flanked the candidate region between the markers CAPS5474 and 4119INDEL,which only contains one candidate gene Bra004118.The Bra004118 from g120w includes a seven-amino-acid variation and one amino acid insertion in the first exon.Phylogenetic tree analysis showed that Bra004118 is more closely to AtLETM2 that encodes a calcium-binding EF-hand family protein.Expression pattern analysis showed that the highest expression level of BrLETM2 was in the flowers,followed by root peel,cotyledon and silique.The lowest BrLETM2 expression levels were established for the leaves and hypocotyls.3.Functional analysis of BrLETM2 on anthocyanin biosynthesis by using RNA-seq analysis and ectopic expression of BrLETM2 in Arabidopsis.To understanding how BrLETM2 regulates anthocyanin biosynthesis,transcriptome from light-exposed swollen root peel of wild type and g120w were analyzed.A total of 3268 genes were differentially expressed(DEGs)in root peel of wild type and g120w,of which 1728 were upregulated and 1540 were downregulated.GO analysis of DEGs showed that the upregulated DEGs were mainly enriched in response to light stimulation,anthocyanin biosynthesis,positive regulation of reactive oxygen species(ROS)biosynthesis process,carboxylic acid decomposition,in response to red and far-red light,and hydrogen peroxide metabolism,etc.The down-regulated DEGs were mainly enriched in disease resistance,plant immunity,negative regulation of cytokinin,glutathione metabolism and so on.The expression levels of ROS responsive genes and ROS levels were significantly decreased in g120w,which was consistent with the expression pattern of BrLETM2,suggesting that BrLETM2 is involved in the activation of ROS responsive genes.In transgenic Arabidopsis plants,the BrLETM2WT-OE seedlings accumulated more anthocyanins than both BrLETM2mu-OE and wild type seedlings under high sucrose(6%)treatment.The anthocyanin biosynthetic genes AtCHS,AtDFR,AtLDOX and AtPAP1 were expressed higher in BrLETM2WT-OE seedlings than those of in BrLETM2mu-OE and wild type,but not AtTT8,indicating that BrLETM2 promotes anthocyanin accumulation in Arabidopsis seedlings by activation of AtPAP1.4.BrLETM2 protein modulates anthocyanin accumulation by promoting ROS production in 'Tsuda' turnip seedlings under UV-A irradiation.To validate BrLETM2 and ROS roles in anthocyanin biosynthesis under UV-A irradiation,we performed treatment in seedlings of g120w and wild-type:dark treatment,24 h UV-A treatment and 24 h UV-A+DPI(NADPH oxidase inhibitor)treatment.Then,anthocyanin contents,ROS levels,expression levels of ROS responsive genes BrCAT2,BrPOD and anthocyanin biosynthetic gene of seedlings were measured and analyzed after treatment,respectively.The results showed that anthocyanin levels of DPI-treated seedlings were robustly reduced after 24 h UV-A treatment than H2O-treated seedlings,indicating that ROS participates in the UV-A induced anthocyanin accumulation.Meanwhile,the anthocyanin level in the hypocotyl of g120w was significantly lower than in the wild type.Furthermore,ROS components O2-and H2O2 levels in different treatment seedlings were detected using NBT and DAB staining,respectively.The results showed that O2-and H2O2 levels in seedlings of wild type were increased under UV-A irradiation and inhibited by the pretreated DPI.The O2-and H2O2 levels in hypocotyls in gl20w were significantly lower than those of in wild type,suggesting that BrLETM2 is involved in ROS formation during UV-A irradiation,which promotes anthocyanin accumulation in the hypocotyl of 'Tsuda' turnip seedlings.The expression level of BrLETM2 and ROS-related genes were induced by UV-A in a time-dependent and intensity-dependent pattern.Taken together,the results suggest that BrLETM2 is involved in the regulation of ROS levels to promote UV-A-induced anthocyanin accumulation in turnip,which provides new insights into the mechanism of short-wavelength light on anthocyanin biosynthesis. |
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