Functional Study Of Key Structural Modification Enzymes CtUGTs Involved In Safflower Flavonoid Glycosides Biosynthesis

Studying the biosynthesis of active ingredients in medicinal plants is of great significance for the efficient regulation and high-quality breeding of traditional Chinese medicine,as well as for revealing the mechanism of authentic medicinal herb formation.It also provides conditions for achieving efficient and manageable heterologous production.The post-modification enzymes（UGTs）involved in the biosynthesis of major medicinal components such as Hydroxysafflor yellow A（HSYA）in safflower have not been fully characterized.Therefore,exploring the function of post-modification enzymes in safflower,elucidating the flavonoids biosynthetic pathway have long been the focus of research.The study used two genetically stable safflower germplasm resources with extreme phenotypes,namely the safflower yellow line（ZHH0119）featured with yellow flower and HSYA,and the safflower white line（Xin Honghua 7）with white flower and no HSYA or other chalcone glycosides.BSA-seq was employed for the first time to screen molecular markers associated with flower color phenotypes in different safflower lines.Additionally,transcriptomic and metabolomic profiling were conducted to analyze the accumulation levels and expression patterns of flavonoid related genes and derivatives in the two different lines and at different flowering stages.Target CtUGTs which involved in the flavonoid glycosides biosynthesis were screened out by integrated omics analysis.Based on analysis above,the bioinformatic features of target CtUGTs were characterized,and their in vitro functions were validated through heterologous expression.Enzymatic properties of target CtUGTs were determined meanwhile the interaction mechanism between CtUGT and various structure types ligands were analyzed through molecular docking simulation and site-directed mutagenesis experiment.The in vivo expression characteristics of CtUGT were determined through subcellular localization and q RT-PCR,furthermore,CtUGTs overexpressed transgenic safflowers were constructed to study its modification role in flavonoid biosynthesis in safflower.Main research results are as follows:1.Two safflower lines mentioned above were selected as parent plants,after reciprocal crossing and selfing,resulting in the 4^th filial generation of two lines with stable phenotypes.By constructing extreme gene pools of parent and 4^th filial generation plants with yellow and white colors,whole-genome resequencing was performed using the BSA-seq method.Alignment analysis with the reference genome revealed a sequencing coverage ranging from0.9375 to 0.9933（10×）across all 12 chromosomes.Through employing Single Nucleotide Polymorphism（SNP）detection and filtering based on parental SNP information,a total of2202977 high-quality SNP loci were identified in the safflower yellow line,while 1373482SNP loci were identified in the safflower white line.Different genetic regions associated with safflower color traits were located on five chromosomes based onΔ（SNP-index）:chromosome 2（11.4-31.2 Mb）,chromosome 8（12.5-19.9 Mb）,chromosome 9（10.6-16.4Mb）,chromosome 10（0.002-0.083 Mb）and chromosome 12（58.5-62.1 Mb）.1923 gene information which containing SNP loci were obtained by function annotation and a total of6 CtPALs,3 CtC4Hs,2 Ct4CLs,1 CtCHSs,32 CtUGTs,70 CtCYPs and 466 transcription factors related to flavonoid compound biosynthesis were identified through reference genome alignment and gene annotation.2.Transcriptome analysis based on the safflower reference genome was conducted on the 2^nd filial generation of two lines and at different flowering stages（Y＿I:3 days before flowering,Y＿II:on the day of flowering,Y＿III:the first day of flowering,Y＿IV:3 days after flowering）.32915 expressed genes were detected,including 27876 known genes and 5039novel genes.Additionally,77240 expressed transcripts were identified,consisting of 45331known transcripts and 31909 novel transcripts.Gene annotation related to flavonoid biosynthesis including 169 CtUGTs was completed through database alignment.Using white safflower line as control,a total of 1 CtPAL,5 CtC4Hs,4 CtCHSs,3 CtCHIs,3 CtFLSs,48CtUGTs,51 CtCYPs and 75 transcription factors were identified as significantly upregulated in the 2^nd filial generation of safflower yellow line’s gene pool and individual plants.Based on transcriptomic WGCNA analysis,a total of 52 CtUGT and 101 CtCYP were identified as being associated with the yellow phenotype of safflower at different flowering stages.STEM analysis revealed 17 upregulated CtUGT and 25 upregulated CtCYP in the yellow line compared with white line.3.Through non-targeted and targeted metabolomic determination,the accumulation levels and expression patterns of flavonoid derivatives were profiled in the 2^nd filial generation of two safflower lines.Non-targeted metabolomic testing identified 130flavonoid compounds,among which 41 differentially expressed flavonoid compounds were found between two lines,with chalcone glycosides and quercetin glycosides accumulated at higher levels in yellow line,while Kaempferol derivatives were more abundant in white line.Targeted metabolomic analysis was performed to determine the 13 target metabolites content,including Phenylalanine,HSYA,Nicotiflorin,Naringenin,Kaempferol,Astragalin,Quercetin,Isoquercetin,Rutin,Apigenin,Luteolin,Eriodictyol and Scutellarin.It showed that the accumulation of HSYA gradually increased in yellow line,with small difference in content between Y＿II and Y＿III and a significant upward trend from Y＿III to Y＿IV.Safflower white line,which did not contain HSYA,had significantly higher Nicotiflorin and Astragalin content than yellow line at each stage.4.By Pearson correlation analysis,the relationship between differentially expressed genes which were upregulated in yellow line and targeted metabolites involved in chalcone glycoside biosynthesis was demonstrated.A total of 16 CtUGTs（r>0.90）showed significant positive correlations with chalcone glycoside biosynthesis in yellow line and were selected for further study.Subsequently,nine full-length CtUGTs（CtUGT50-CtUGT58）were successfully cloned,and their potential roles in chalcone,flavanol,and flavonoid glycoside biosynthesis in safflower yellow line were preliminarily deduced.5.Phylogenetic tree analysis revealed that the nine target CtUGTs originated from UGT71,74,76,87,708 families.Among them,the UGT708 family contained several reported C-glycosyltransferases,suggesting that CtUGT55,which belonging to this family,might participate in the biosynthesis of glycosylated compounds in safflower yellow line.Additionally,several CtUGTs exhibited higher homology（75.68%-87.26%）with UGTs from Cynara cardunculus,which was consistent with the safflower genome evolutionary analysis.6.CtUGT50 was proved glycosylation activity towards Quercetin and Luteolin,with glycosylation occurring at 3-OH or 7-OH,indicating that CtUGT50 might involve in the Isoquercetin and Luteolin-7-O-glucoside biosynthesis of safflower.Two UGT71 family glycosyltransferases,CtUGT51 and CtUGT54,with substrate promiscuity were identified and their activities were found to be similar in vitro.They might participate in the biosynthesis of flavonols and flavones monoglucosides in safflower.Furthermore,CtUGT51and CtUGT54 exhibited glycosyltransferase activity towards polyphenolic compound Resveratrol while with different catalytic products,indicating that they owned distinct catalytic mechanism for smaller molecular compounds.CtUGT53,CtUGT56 and CtUGT57could only catalyze Kaempferol to produce Astragalin.CtUGT55,which belonged to UGT708 family,could catalyze the glycosylated reaction of 2-Hydroxynaringenin.7.The first chalcone-specific glycosyltransferase,CtUGT52,was identified and found to possess significant substrate promiscuity and catalytic promiscuity.The prokaryotic-expressed CtUGT52 could utilize UDP-glucose as donor,catalyzing the formation of monoglucosides from chalcone compounds such as Naringenin chalcone and Phloretin,as well as synthesizing monoglucosides and diglucosides from flavonols（Kaempferol,Quercetin）and flavones（Apigenin,Luteolin）,and monoglucosides from dihydroflavonoid（Naringenin）.Moreover,CtUGT52 transient expressed by tobacco could also catalyze Pinocembrin chalcone to form its monoglucoside.It was revealed that CtUGT52 might be involved in the biosynthesis of chalcone glycosides and other various flavonoid glycosides in yellow safflower cultivar.In addition,the same glycosyltransferase expressed by Escherichia coli and tobacco showed different catalytic activities,indicating that CtUGT might form different tertiary structures in different hosts,leading to functional difference.8.Enzyme property study revealed that the optimal conditions of CtUGT52 catalyzed Phloretin and Kaempferol,two substrates with different structural types,were similar（37℃,p H 7.4）,multiple glycosylation products could be detected after 15 minutes reaction,and glycoside products yield significantly increased within 3-5 hours.Additionally,CtUGT52could catalyze the conversion of Kaempferol monoglycosides into diglycosides,providing a new synthetic method for obtaining flavonoid diglycosides.9.Autodock Vina simulation was used to study the interaction mechanism of CtUGT52with Phloretin,Kaempferol,and Luteolin as substrates,and UDP-glucose as glycosyl donor.Site-directed mutagenesis experiment confirmed that S²⁷⁷and Q³³⁵ were critical binding sites for CtUGT52 with Phloretin,while Q³³⁵ and E³⁵⁸ were critical binding sites for CtUGT52with Kaempferol,and P¹⁶² and E²¹³ were critical binding sites for CtUGT52 with Luteolin.10.Based on subcellular localization in tobacco,CtUGT52 was inferred primarily distributed in the nucleus and cytoplasmic,indicating that its glycosylated modification function could occur throughout the entire cell.CtUGT52 expression characteristic analysis using q RT-PCR revealed that its expression level was low in Y＿I and Y＿II phases,and sharply increased in Y＿III phase,further increased in Y＿IV phase,showing a consistent expression trend with HSYA.11.The Agrobacterium-mediated transformation method was successfully used to construct and screen CtUGT52 overexpressed plants.Through quantitative detection,it showed that HSYA content in CtUGT52 overexpressed safflower had significant increased2.01 to 3.81 times than wild-type safflower.Astragalin was observed increasing up to 2.25-fold.The content of Isoquercetin did not show a significant soar.The important precursor for the flavonoids biosynthesis,Phenylalanine,showed no significant difference between CtUGT52 overexpressed and wild-type plants.These confirmed that CtUGT52 can promote glycosylation of Naringenin chalcone and Kaempferol in vivo,resulting in an increased accumulation of chalcone and flavonol glycosides in safflower.In summary,the main genomic regions associated with safflower color traits were located on chromosomes 2,8,9,10 and 12.It was inferred that CtUGT50,CtUGT51,CtUGT52 and CtUGT54 were involved in the biosynthesis of flavonol and flavonoid glycosides in safflower,while CtUGT53,CtUGT56 and CtUGT57 might participate in the biosynthesis of Astragalin.CtUGT52 was the first glycosyltransferase from safflower that lead to the formation of naringenin chalcone glycosides with significant substrate promiscuity and catalytic promiscuity,which could also catalyze the glycosylation of various chalcones,flavonols and flavones.Based on expression characteristics,in vitro and in vivo functional validation,CtUGT52 was revealed as an important glycosyltransferase involved in the biosynthesis of chalcone glycosides and other flavonoid glycosides in safflower.These experiment provided data support for a deeper understanding of synthesis mechanism and industrial production of the unique flavonoid glycosides in safflower.